WO2020027859A1 - Nutritional phytonutrient compositions and methods of use - Google Patents

Nutritional phytonutrient compositions and methods of use Download PDF

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WO2020027859A1
WO2020027859A1 PCT/US2018/057647 US2018057647W WO2020027859A1 WO 2020027859 A1 WO2020027859 A1 WO 2020027859A1 US 2018057647 W US2018057647 W US 2018057647W WO 2020027859 A1 WO2020027859 A1 WO 2020027859A1
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composition
weight
rhob
mammal
egcg
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PCT/US2018/057647
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French (fr)
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Chunyu Zhang
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Sanulife Inc.
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/174—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/50—Feeding-stuffs specially adapted for particular animals for rodents
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins

Definitions

  • the present disclosure generally relates to nutritional compositions and methods of use, and in particular, to nutritional phytonutrient compositions and methods of using the nutritional phytonutrient compositions for preventing cancer, attenuating cancer and/or slowing the aging process.
  • Cancer usually refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer has become a significant threat to the life and health of human and non-human animals, especially to those diagnosed with advanced cancer. Even if the malignant tumor is removed or the cancer is currently under control, there is still a possibility that the cancer would return in a few years (e.g., in 2 years or in 5 years). Thus, there is a need to prevent cancer from happening or reoccurring. Moreover, cancer is more common in older people than in young people. In fact, aging is considered not only as an important risk factor for cancer, but also for a number of other diseases such as but not limited to cardiovascular diseases. In addition, aging is associated with the decline of various body functions such as but not limited to physical strength, stamina, memory, and general metabolism. Therefore, slowing the aging process may not only help preventing or attenuating cancer, but also improve general health and body functions.
  • composition s components are understood and clarified.
  • a nutritional phytonutrient composition may include ceramide, epigallocatechin gallate (EGCG), and curcumin.
  • the composition may be used for preventing or attenuating cancer in a mammal.
  • the composition may be used for slowing aging process in a mammal.
  • the composition may be used for preventing or attenuating cancer in a mammal or for slowing aging process in the mammal, by maintaining, increasing or restoring RhoB level in the mammal.
  • the ceramide may be 2% to 10% of the composition by weight.
  • the ceramide may be 4% to 6% of the composition by weight.
  • the ceramide may be about 4.9% of the composition by weight.
  • the ceramide may be extracted from rice.
  • the EGCG in the composition may be in the form of green tea extract.
  • the great tea extract contains about 95% EGCG by weight.
  • the EGCG may be 5% to 20% of the composition by weight.
  • the EGCG may be 10% to 15% of the composition by weight.
  • the EGCG may be about 12.2 of the composition by weight.
  • the curcumin may be 15% to 35% of the composition by weight.
  • the curcumin may be 22% to 27% of the composition by weight.
  • the curcumin may be about 24.4% of the composition by weight.
  • the curcumin may be cavacurmin.
  • the composition may further include astaxanthin.
  • the astaxanthin may be 1 % to 3.5% of the astaxanthin
  • composition by weight.
  • the astaxanthin may be 2% to 3% of the composition by weight.
  • the astaxanthin may be about 2.4% of the
  • composition by weight.
  • the composition may further include a cell protection agent.
  • the cell protection agent may include Vitamin E.
  • the Vitamin E may be 1 % to 3% of the composition by weight.
  • the Vitamin E may be about 2.2% of the composition by weight.
  • the composition may further include an emulsifier.
  • the emulsifier may include lecithin.
  • the lecithin may be extracted from sunflower or soybean.
  • the lecithin may be 15% to 35% of the composition by weight.
  • the lecithin may be about 24% to 25% of the composition by weight.
  • the composition may further comprise a flow agent.
  • the flow agent may include rice hull powder.
  • the rice hull powder may be 15% to 35% of the composition by weight.
  • the rice hull powder may be about 24% to 25% of the composition by weight.
  • the composition may further include a silica supplement.
  • the silica supplement may include bamboo extract.
  • the bamboo extract may be 2% to 10% of the composition by weight.
  • the bamboo extract may be about 4.9% of the composition by weight.
  • the ceramide may be 4% to 6% of the composition by weight; the EGCG may be 10% to 15% of the composition by weight; and the curcumin may be 22% to 27% of the composition by weight.
  • the ceramide may be about 4.9% of the composition by weight; the EGCG may be about 12.2% of the composition by weight; and the curcumin may be about 24.4% of the composition by weight.
  • the ceramide may be 4% to 6% of the composition by weight; the EGCG may be 10% to 15% of the composition by weight; and the curcumin may be 22% to 27% of the composition by weight.
  • the composition may further comprise astaxanthin, which may be 2% to 3% of the composition by weight;
  • the composition may further comprise Vitamin E, which may be 1 % to 3% of the composition by weight;
  • the composition may further comprise lecithin, which may be 15% to 35% of the composition by weight;
  • the composition may further comprise rice hull powder, which may be 15% to 35% of the composition by weight; and
  • the composition may further comprise bamboo extract, which may be 2% to 10% of the composition by weight.
  • the ceramide may be about 4.9% of the composition by weight; the EGCG may be about 12.2 of the composition by weight; and the curcumin may be about 24.4% of the composition by weight, the astaxanthin may be about 2.4% of the composition by weight; the Vitamin E may be about 2.2% of the composition by weight; the lecithin may be about 24% to 25% of the composition by weight; the rice hull powder may be about 24% to 25% of the composition by weight; and the bamboo extract may be about 4.9% of the composition by weight.
  • the composition may be provided in a unit dosage form.
  • each dosage may include 10 mg to 30 mg ceramide.
  • each dosage may include about 20 mg ceramide.
  • each dosage may include 40 mg to 60 mg EGCG.
  • each dosage may include about 50 mg EGCG.
  • each dosage may include 90 mg to 110 mg curcumin.
  • each dosage may include about 100 mg curcumin.
  • each dosage may include 5 mg to 15 mg astaxanthin.
  • each dosage may include about 10 mg astaxanthin.
  • each dosage may include 15 IU to 25 IU Vitamin E.
  • each dosage may include about 10 IU Vitamin E in the form of D-alpha tocopheryl acetate.
  • each dosage may include 80 mg to 120 mg lecithin.
  • each dosage may include about 100 mg lecithin.
  • each dosage may include 80 mg to 120 mg rice hull powder.
  • each dosage may include about 100 mg rice hull pow
  • each dosage may include 10 mg to 30 mg bamboo extract.
  • each dosage may include about 20 mg bamboo extract.
  • FIG. 1 A is a representative picture illustrating immunoblotting results of RhoB protein in two young mice at the age of 2 months and their parents at the age of 12 months according to some embodiments of the present disclosure
  • FIG. 1 B is an analytical graph illustrating immunoblotting results of RhoB and p-Akt-S473 according to some embodiments of the present disclosure
  • FIG. 2 is an analytical graph illustrating unsupervised clustering of gene expression microarray data in h157 cells treated with PI3K/Akt/mTOR pathway inhibitors according to some embodiments of the present disclosure
  • FIG. 3 is a representative picture illustrating immunoblotting analysis results of RhoB induction 2h post LBAI treatment in H157 cells according to some embodiments of the present disclosure
  • FIG. 4 is an analytical graph illustrating identification results of the
  • FIG. 5 is a representative picture illustrating chromatin immunoprecipitation analysis results of transcription factors C/EBRb, NFYA and SP1 , as well as RNA polymerase II (RPII) binding to endogenous RhoB promoter hRB3 region in A549 cells exposed to LBAI according to some embodiments of the present disclosure;
  • FIG. 6A is an analytical graph illustrating luciferase analysis in A549 cells co transfected with hRB3 and C/EBRb plasmid or O/EBRb- ⁇ q ⁇ b ⁇ siRNA duplexes according to some embodiments of the present disclosure
  • FIG. 6B is an analytical graph illustrating results of LBAI induction of hRB4 luciferase activity according to some embodiments of the present disclosure
  • FIG. 6C is an analytical graph illustrating luciferase analysis in A549 cells co- transfected with hRB3 and C/EBRb plasmid or C/EBPp-targeting siRNA duplexes according to some embodiments of the present disclosure
  • FIG. 6D is an analytical graph illustrating immunoblotting analysis results of the full-length isoforms and N-terminal truncated isoforms of C/EBRb and C/EBRa in H 157 cells at 6h post LBAIs (10 mM) treatment according to some embodiments of the present disclosure;
  • FIG. 7A is an combination of representative pictures illustrating
  • FIG. 7B is an analytical graph illustrating luciferase assay results and immunoblotting analysis in A549 cells at 48h post co-transfection with hRB and FIA- tagged Akt1 (PKBa), Akt2 (RKBb) or C-terminal truncated Akt3 (PKB 1 ) constructs according to some embodiments of the present disclosure;
  • FIG. 7C is an analytical graph illustrating luciferase assay results and immunoblotting analysis in A549 cells at 72h post transfection with hRB and Non- Target (NT) or PTEN siRNA duplexes according to some embodiments of the present disclosure
  • FIG. 7D is an analytical graph illustrating Dual luciferase assay results in isogenic K562 cell lines (C10, C11 and C22) with various levels of constitutively active myr-Akt1 at 48h post co-transfection with hRB and CMV-hRluc constructs according to some embodiments of the present disclosure;
  • FIG. 7E is a combination of representative pictures illustrating RT-PCR analysis results of RhoB transcript in Rh30 cells under high serum (20% FBS) condition at 24h post treatment with different doses (0, 10 and 30 pM) of PIA5 according to some embodiments of the present disclosure;
  • FIG. 7F is a combination of representative pictures illustrating
  • FIG. 8A is a combination of representative pictures illustrating results of Rh30 and RD cells treated with 10 mM of LY294002 or LBAI in 0.1 % FBS RPMI1640 for 6h according to some embodiments of the present disclosure;
  • FIG. 8B is an analytical graph illustrating MTS assay results of Rh30 and RD cells treated with 10 mM of LY294002 or LBAI in 0.1 % FBS RPMI 1640 for 72h according to some embodiments of the present disclosure
  • FIG. 9A is an analytical graph illustrating luciferase assay results in elF2a wild type MEF (S/S) or Ser51 mutation MEF (A/A) cells co-transfected with hRB and CMV-hRluc constructs at 6h post LBAIs treatment according to some embodiments of the present disclosure;
  • FIG. 9B is a combination of representative pictures illustrating r
  • FIG. 9C is a combination of representative pictures illustrating immunoblotting analysis results of p-S51 elF2a in H157 cells pretreated (1 h) with 5 mM of Purine (Control) and 2-Aminopurine at 6h post LBAIs treatment according to some
  • FIG. 9D is an analytical graph illustrating MTS assay results in H157 cells pretreated (1 h) with 5 mM of Purine and 2-Aminopurine at 18h post LBAIs treatment according to some embodiments of the present disclosure
  • FIG. 9E is an analytical graph illustrating Apoptosis analysis in H157 cells pretreated (1 h) with 5 mM of Purine and 2-Aminopurine at 18h post LBAIs treatment according to some embodiments of the present disclosure
  • FIG. 10A is an analytical graph illustrating Immunoblotting analysis results of PKR and PACT in H157 cells stably transfected with empty vector (pRS) or shRNA constructs for Non-target (shNT), PKR (shPKR) or PACT (shPACT) at 6h post LBAIs treatment, and MTS assay at 24 h post LBAIs treatment according to some embodiments of the present disclosure;
  • FIG. 10B is a combination of representative pictures illustrating
  • FIG. 10C is an analytical graph illustrating MTS assay results of A549 cells stably transfected with shPKR or shPACT were treated with LBAI for 6h
  • FIG. 10D is an analytical graphillustrating MTS assay results in U251 cells stably transfected with shNT, shPKR and shPACT at 24, 48 and 72 h post LBAI treatment in full serum condition (5% FBS) as well as effects of LBAI in H322, A549 and H157 non-small cell lung cancer cells according to some embodiments of the present disclosure;
  • FIG. 11 A is a combination of representative pictures illustrating
  • FIG. 11 B is a schematic combination of representative pictures diagram illustrating Immunoprecipitation (IP) and immunoblotting (IB) analysis results in H157 cells at 6h post LBAI treatment according to some embodiments of the present disclosure;
  • FIG. 12A is an analytical graph illustrating stained H1 155 cells transfected with pcDNA3 CMV empty vector, pcDNA3-HA-RhoB and pcDNA3-HA-RhoB-N19 and flow cytometric analysis results 48 h post transfection according to some embodiments of the present disclosure;
  • FIG. 12B is a combination of representative pictures illustrating results of RhoB specific cellular effects on H2882 cells and photographs of RhoB-null H2882 cells co-transfected with pmaxGFP and pcDNA3 CMV empty vector, pcDNA3-HA-RhoB or pcDNA3-HA, RhoB-N19 according to some embodiments of the present disclosure;
  • FIG. 13A is a combination of representative pictures of forced expression of RhoB in Rh 30 cells according to some embodiments of the present disclosure
  • FIG. 13B is a combination of representative pictures of reconstituted C/EBRb in O/EBRb-/- transformed MEF cells according to some embodiments of the present disclosure
  • FIG. 13C is a combination of representative pictures of restored PTEN in PTEN-null H157 cells according to some embodiments of the present disclosure.
  • FIG. 13D is a combination of representative pictures illustrating
  • FIG. 13E is an analytical graph illustrating prevention of RhoB induction by PIA23 using RhoB siRNA according to some embodiments of the present disclosure
  • FIG. 14A is a combination of marked pictures illustrating quantification of D- Luciferin signal changes in 3 mice carrying FI157-hRB3-DS (Left) and Rh30-hRB3- DS (Right) cells when they were treated with Vehicle only according to some embodiments of the present disclosure;
  • FIG. 14B is a combination of marked pictures illustrating quantification of D- Luciferin signal changes in the same 3 mice cells when they were treated with PIA5 according to some embodiments of the present disclosure
  • FIG. 14C is a combination of marked representative pictures illustrating quantification of D-Luciferin or coelenterazine signal changes in two pairs of mice when they were treated with Vehicle or PIA5 according to some embodiments of the present disclosure
  • FIG. 14D is an analytical graph illustrating administration regimen (5 mice for Vehicle and 5 mice for PIA5) and body weights of the mice during treatment according to some embodiments of the present disclosure
  • FIG. 14E is a schematic diagram illustrating representative combination of representative pictures embodiments of the present disclosure.
  • FIG. 14F is a illustrating Tumor growth curves of H157 and Rh30 xenografts in Vehicle and PIA5 28 treated groups according to some embodiments of the present disclosure
  • FIG. 15A is an analytical graph illustrating fluorescent micrographs of H157 cells stained with an anti-ceramide antibody (red), according to some embodiments of the present disclosure
  • FIG. 15B is an analytical graph illustrating RhoB promoter activity according to some embodiments of the present disclosure.
  • FIG. 15C is a combination of representative pictures illustrating LAP accumulation and RhoB induction according to some embodiments of the present disclosure
  • FIG. 15D is an analytical graph illustrating exogenous C2-Ceramide (10 mM), a cell-permeable analog of naturally occurring ceramides, transactivated RhoB promoter activity according to some embodiments of the present disclosure
  • FIG. 15E is a combination of representative pictures illustrating the inhibition effect of the exogenous C2-Ceramide inhibited Akt/mTOR and activated PKR/elF2a pathways according to some embodiments of the present disclosure
  • FIG. 15F is an analytical graph illustrating a viability of the F1157 cells according to some embodiments of the present disclosure.
  • FIG. 15G is an analytical graphillustrating the blocking effect of the
  • FIG. 16 is a combination of representative pictures illustrating
  • FIG. 17 is an analytical graph illustrating the male C57/BL6 mice (at the age of 12 months) fed with standard AIN-93 diet, or EGCG and Curcumin containing AIN- 93 diet for 10 months according to some embodiments of the present disclosure
  • FIG 18A is an analytical graph illustrating the tumor multiplicity according to some embodiments of the present disclosure.
  • FIG. 18B is an analytical graph of size in left-single lung according to some embodiments of the present disclosure.
  • FIG. 19 is an analytical graph illustrating interactions between RhoB and EGFR, Ras, PI3K/Akt/mTOR, MYC, and FID AC;
  • FIG. 20 is an analytical graph illustrating translation of RhoB is epigenetically downregulated by miRNA-19/21/223.
  • FIG. 21 is an analytical graph illustrating the potential roles of tumor suppressor RhoB in cancer and aging.
  • ceramide refers to one or more types of
  • the ceramide may include but is not limited to C2-ceramide (N-(Acetyl)- sphing-4-enine), C6-ceramide (N-Hexanoylsphingosine), C12-ceramide (N- (Dodecanoyl)-sphing-4-enine), C16-ceramide (N-(hexadecanoyl)-sphing-4-enine), C24-ceramide (N-(tetracosanoyl)-sphing-4-enine), or the like, or an analog thereof (e.g., a salt having a similar structure that is safe to be administered to a mammal), or a combination thereof.
  • C2-ceramide N-(Acetyl)- sphing-4-enine
  • C6-ceramide N-Hexanoylsphingosine
  • C12-ceramide N- (Dodecanoyl)-sphing-4-enine)
  • C16-ceramide N-(
  • Exemplary analogs of C6-ceramide may include but are not limited to C6-Nbd-ceramide, C6-pyridinium-ceramide, C6-NBD lactosyl ceramide, or the like, or any analog thereof, or any combination thereof.
  • ceramide has a structure of:
  • the term“epigallocatechin gallate”, also abbreviated as “EGCG”, refers to one or more types of substance, each of which includes the ester of epigallocatechin and gallic acid in the structure.
  • the EGCG may include but is not limited to Epigallocatechin 3-gallate and/or an analog thereof.
  • Exemplary analogs of the epigallocatechin 3-gallate may include but are not limited to methylated EGCG, a D-ring analog of the epigallocatechin 3-gallate, EGCG 4- palmitate, EGCG 4-stearate, or the like, or any analog thereof, or any combination thereof.
  • EGCG has the following structure:
  • curcumin refers to one or more types of substance, each of which has a structure that is at least partly similar to
  • the curcumin may include but is not limited to
  • curcumin may include but are not limited to bisdiferuloylmethane, curcumin glucuronide, curcumin sulfate, monodemethoxycurcumin, tetrahydrocurcumin, curcumin pyrazole, or the like, or any analog thereof, or any combination thereof.
  • curcumin has the following structure:
  • a nutritional phytonutrient composition provided according to some embodiments of the present disclosure may include one or more natural ingredients derived from plants, such as vegetables, fruits, soybeans, grains, nuts, or the like, or any combination thereof.
  • the terms“nutritional phytonutrient composition” and“the composition” are used interchangeably herein.
  • the ingredients of the composition may include ceramide, EGCG, or curcumin, or any combination thereof.
  • the ceramide may be extracted from rice, rice bran, rice germ, wheat, konjac, sweet potatoes, soybeans, spinach, or the like, or any combination thereof.
  • the EGCG may be extracted from green tea, seaweed, aloe vera, grains, apples, blackberries, carob flour, or the like, or any combination thereof.
  • the EGCG in the composition may be present in the form of green tea extract.
  • the curcumin may be extracted from turmeric, mango ginger, or the like, or any combination thereof.
  • the composition provided according to some embodiments of the present disclosure may be used to prevent or attenuate cancer, or slow the aging process of a mammal.
  • the mammal may include a human or a non-human mammal.
  • the non-human mammal may include monkeys, orangutans, tigers, cats, dogs, rabbits, ferrets, pigs; gerbils, hamsters, chinchillas, rats, mice, guinea pigs, hedgehogs, sugar gliders, chinchillas, chipmunks, squirrels, or the like, or any combination thereof.
  • the cancer may include but is not limited to non-small cell lung cancer (NSCLC),
  • rhabdomyosarcoma breast cancer, bladder cancer, colorectal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer,, melanoma, oral and oropharyngeal cancer, uterine cancer, or the like, or any combination thereof.
  • the composition provided according to some embodiments of the present disclosure may prevent or attenuate cancer or slow the aging process via a RhoB-dependent mechanism.
  • RhoB refers to a member of the Rho GTPase family. RhoB may inhibit function of the epidermal growth factor receptor (EGFR)-Ras signaling pathway and function of the PI3K/Akt/mTOR signaling pathway.
  • EGFR epidermal growth factor receptor
  • PI3K refers to phosphatidylinositol 3-kinase
  • mTOR refers to the mammalian target of rapamycin.
  • RhoB may facilitate Myc protein turnover in cancer cells.
  • the Myc proteins may serve as transcription factors to activate the expression of may pro-proliferation genes.
  • RhoB may inhibit the proliferation of the cancer cells, the invasion and metastasis of the tumor, and/or promoting the apoptotic death of the cancer cells.
  • RhoB may also help keep oncogenic Myc at a relatively low level, which may extend mammalian healthy lifespan and reduce cancer incidence with age.
  • the increase of RhoB in normal cells may slow down cell cycle, stabilize genome, help repair damaged DNA, and promote the activation of nuclear factor erythrold 2-related factor2 (Nrf2).
  • Nrf2 nuclear factor erythrold 2-related factor2
  • the Nrf2 may contribute to cytoprotective responses and the extension of lifespan.
  • RhoB may help slow the aging process and extend the healthspan.
  • RhoB may be inhibited and/or decreased during tumorigenesis and aging process.
  • embodiments of the present disclosure may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal.
  • the ingredients in the composition according to some embodiments may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal.
  • embodiments of the present disclosure may maintain, increase or restore RhoB activity in certain cells, tissues, or organs of the mammal.
  • the ingredients in the composition according to some embodiments of the present disclosure may maintain, increase or restore RhoB expression level in certain cells, tissues, or organs of the mammal.
  • the ceramide, EGCG and curcumin in the composition provided according to some embodiments of the present disclosure may transactivate RhoB promotor, inhibit function of the
  • PKR protein kinase R
  • elF2a eukaryotic translation initiation factor
  • Rho family of GTPases may be characterized for its role in the regulation of cellular actin that forms the cytoskeleton mediated functions of motion and adhesion as well as cell-cycle progression, regulation of transcription factors, and protein trafficking.
  • Rho GTPases may act as a critical component of intracellular signaling pathways.
  • RhoA, RhoB, and RhoC, as members of the Rho GTPase family may have distinct functional roles.
  • RhoB localizes in endosomes where it regulates their intracellular movements and can be differentially prenylated by a farnesyl or geranylgeranyl group.
  • RhoB localizes in endosomes where it regulates their intracellular movements and can be differentially prenylated by a farnesyl or geranylgeranyl group.
  • RhoB has been shown to be significantly down regulated in cancer specimens from many tissue types, thereby allowing opportunities for developing therapies targeted at key points in regulation of RhoB as well as interplay in intracellular signaling pathways.
  • the functional implication of RhoB in tumorigenesis is critical in the context of aged associated decreases in expression found primarily in muscle and lung tissue. Further analysis of RhoB unique cellular functions, characterization of its role in tumorigenesis, and exploration of the association between age and RhoB loss, may open avenues for elucidating and potentially treating elusive pathways.
  • RhoB may be suppressed by oncogenic signaling.
  • EGFR may reduce RhoB promoter activity via Ras signaling.
  • Epidermal growth factor receptor (EGFR) is a member of the erbB family of receptor tyrosine kinases that serves as an interface between the extracellular environment and intracellular signal transduction moderating cell growth, differentiation, survival, and progression through the cell cycle.
  • EGFR may include an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain, and binds primarily to EGF and TGF-a. Once bound, the receptor may be activated resulting in the phosphorylation of the tyrosine kinase domain and
  • EGFR may be altered through various mechanisms such as gain-of-function mutations, EGFR gene gain, overexpression of ligands and receptors, etc.
  • increased EGFR expression may promote tumorigenesis through the down-regulation of RhoB (as shown in FIG. 19).
  • RhoB may inhibit RhoB promoter activity in cancer cells heavily depending on the presence of Ras, another oncogene, suggesting that EGFR suppresses RhoB promoter activity through Ras pathway.
  • RhoB RhoB promoter activity in cancer cells heavily depending on the presence of Ras, another oncogene, suggesting that EGFR suppresses RhoB promoter activity through Ras pathway.
  • the Ras subfamily may include Fl-Ras, N-Ras, and K-Ras monomeric GTPases, and mediate signal transduction between cell surface growth receptors and intracellular signaling pathways.
  • the Ras proteins may activate once bound to GTP, a process that is catalyzed by guanine nucleotide exchange factors (GEFs), inactivate through GTP hydrolysis which is catalyzed by GTPase-activating proteins (GAPs) (as shown in FIG. 19). Oncogenic mutations of the three Ras genes occur in codons 12, 13, or 61.
  • mutated K-Ras may be observed in cancers of the pancreas, esophagus, cardia and distal stomach, stomach, biliary tract, bile duct, ampulla, gallbladder, colon, lung cancer, or the like, or any
  • K-Ras mutations may be associated with a poor prognosis in individuals with colorectal cancer and non-small-cell lung cancer, virtually in all individuals with early-stage NSCLC and adenocarcinoma.
  • K-Ras may decrease the promoter transcriptional activity of RhoB in a dose-dependent manner in NIFI3T3 cells and suppressed RhoB protein levels in various types of cancer cells from pancreatic, cervical, and lung tumors.
  • oncogenic K-Ras demonstrated some degree of anticancer drug resistance through effectively blocking the induction of RhoB protein levels and promoter site activity by 5-fluorouracil.
  • Ectopic expression of RhoB was found to inhibit K-Ras transformation of NIFI3T3 cells, further lending to the concept that RhoB suppression is required for some oncogenes to transform cells.
  • Phosphatidylinositol 3-kinase is one of the main effector pathways of Ras, regulating cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and metabolism.
  • PI3K may be necessary for Ras-induced transformation in vitro, and more importantly, mice with mutations in the PI3K catalytic subunit p110a that block its ability to interact with Ras are highly resistant to endogenous oncogenic K-Ras induced lung tumorigenesis and Fl-Ras induced skin carcinogenesis.
  • Ras may downregulate RhoB expression by PI3K- and Akt- but not Mek- dependent mechanism. Genetic and pharmacological blockade of PI3K/Akt may result in upregulation of RhoB expression. The importance of the downregulation of RhoB in oncogenesis is supported by demonstrating that RhoB antagonizes
  • RhoB Ras/PI3K/Akt malignancy. Ectopic expression of RhoB, but not the close relative RhoA, may inhibit Ras, PI3K, and Akt induction of transformation, migration, and invasion and may induce apoptosis and anoikis. Finally, RhoB may inhibit melanoma metastasis to the lung in a mouse model. These studies identify suppression of RhoB as a mechanism by which the Ras/PI3K/Akt pathway induces tumor survival, transformation, invasion, and metastasis (as shown in FIG. 19).
  • RhoB gene may be not genetically mutated or altered in various tumors and carcinoma. Consequently, it was then proposed that RhoB expression is controlled by epigenetic events. Nucleosomes, a complex of core histones wrapped by chromosomal DNA, may contribute to the stability of chromatin structure and the repression of genetic transcription in eukaryotes. Depending on the acetylation status of the histone amino termini that extend from the nucleosome core, this activity can be modified and is dynamically coordinated by Flistone
  • HATs Acetyltransferases
  • HDACs Histone Deacetyltransferases
  • HDAC inhibitors may target histone deacetylases and serve as powerful antitumor agents that induce differentiation and apoptosis through transcriptional modulation (as shown in FIG. 19).
  • RhoB may be further controlled by MicroRNAs (miRNAs), 18-24 base-pair non-coding small RNAs that function in post- transcriptional regulation of gene expression.
  • miRNAs MicroRNAs
  • genes encoding miRNAs may be transcribed into pri-miRNA by RNA Polymerase II and subsequently processed by Drosha to form pre-miRNA.
  • pre-miRNAs may be then further processed by the cytoplasmic enzyme complexes Dicer and RISC. Once fully processed, miRNA may then bind to the 3’ untranslated region (3’-UTR) of target mRNA, leading to destabilization of the mRNA and thus decreased mRNA translation.
  • RhoB transcript plays a regulatory role in RhoB expression. Translation of mRNA with the RhoB 3’-UTR may decrease expression of reporter transcripts. Therefore, regulation of the 3’-UTR of RhoB, which may be facilitated by miRNA, may in turn regulate expression of RhoB, as shown in FIG. 20.
  • RhoB may decrease during tumorigenesis and aging.
  • the RhoB mRNA was decreased from immortalization stage.
  • HDAC1 regulates RhoB promoter activity through an inverted CCAAT element within the RhoB promoter.
  • Levels of HDAC1 binding to CCAAT boxes may change with age. There was no association between HDAC1 and the CCAAT elements in young tissue ( ⁇ 4 weeks), but the binding increased as the mice aged. Therefore, RhoB is gradually decreased during aging process in important tissues including lung and muscle by epigenetic mechanism. Given that age is the biggest risk for cancer, a decrease in RhoB from aged tissues may propose the possibility that RhoB loss leads to increased cancer rates with age.
  • RhoB is required for apoptosis in cells transformed by DNA-damaging agents, its loss increases DSB- mediated genomic instability and tumor progression and promotes tumorigenesis. RhoB appears to function as a suppressor or negative modifier in cancer
  • RhoB mRNA and protein from aged mice might increase the occurrence of cancer in pulmonary tissue, as was explained in a human non-small lung carcinoma cell line.
  • RhoB may be used as a prognostic marker of NSCLC aggression.
  • IHC and RT-qPCR to compare RhoB in control patients and patients with known advanced lepidic adenocarcinoma, it may be found that patients with more aggressive forms of lepidic adenocarcinoma had greater losses of RhoB expression.
  • RhoB expression can be utilized to determine prognosis of NSCLC.
  • the deceased expression of RhoB may mediate higher malignancy potential in clear cell renal cell carcinoma.
  • restoring RhoB levels may be used in treating cancer.
  • Restored RhoB in ovarian adenocarcinoma cells with undetectable levels of RhoB may suppress tumor growth.
  • Recombinant adenovirus transduced with RhoB cDNA may activate apoptosis in vitro, whereas injections of Ad-RhoB in nude mice with ovarian cancer xenografts may demonstrate suppression of tumor growth in vivo, thereby illustrating that restoration of RhoB may prove useful in cancer therapy.
  • RhoB restoration may contribute to a longer and healthier lifespan.
  • genetically down-regulating mTOR expression or pharmacologically inhibited mTOR by Rapamycin was shown to produce a profound increase in lifespan.
  • Transgenic mice carrying additional copies of PTEN, the negative regulator of PI3K/AKT/mTOR pathway, may live longer and have lower incidence of cancer relative to normal.
  • RhoB may be potently and selectively inducted by LBAIs (lipid-based inhibitors).
  • LAP isoform of C/EBRb may be responsible for RhoB transactivation by LBAIs.
  • LBAI may rapidly activate RhoB transcription by increasing levels of the transcriptionally active C/EBRb isoform LAP.
  • the increase of LAP protein upon LBAIs treatment may be through translation alteration of C/EBRb from LIP to LAP.
  • PACT-mediated PKR activation may switch C/EBRb isoform translation from LIP to LAP.
  • the inhibitory phosphorylation of elF2a by PACT-mediated PKR activation may be the major mechanism for LBAIs-mediated RhoB induction and cytotoxicity.
  • RhoB restoration in Akt highly active or RhoB-null cancer cells may be cytotoxic.
  • Akt has a central role in the phosphoinositide-3- kinase (PI3K)/Akt signaling pathway that controls cellular processes integral in the development of cancer including growth, metabolism and survival.
  • PI3K phosphoinositide-3- kinase
  • inhibitors may be developed as cancer therapeutics.
  • Akt is activated in response to the lipid products of class I PI3K, PI3,4P2 (PIP2) and PI3,4,5 P3 (PIP3).
  • PIP2 and PIP3 attract Akt to the plasma membrane through binding to the pleckstrin homology (PH) domain of Akt. This may promote a conformational change, allowing it to be phosphorylated on T308 by PDK-1 and at S473 by the rictor/mTOR complex. Once phosphorylated, Akt may dissociate from the plasma membrane and moves to various cellular compartments where it phosphorylates downstream substrates.
  • PH pleckstrin homology
  • LBAIs such as phosphatidylinositol ether lipid analogs and the alkylphospholipids perifosine and miltefosine may be designed to interfere with the PH domain of Akt.
  • LBAIs may selectively target metabolically active, proliferating tumor cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional
  • the dramatically increased expression of the tumor suppressor RhoB may contribute to enhanced cytotoxicity beyond PI3K/Akt/mTOR pathway inhibition.
  • Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of tumor suppressor PTEN.
  • Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by LBAI.
  • the C/EBPbeta full-length isoform LAP may be responsible for transcriptional induction through its binding site within the RhoB proximal promoter.
  • LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity.
  • RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice.
  • LBAIs administration increased luciferase activity and decreased the growth of human tumor xenografts.
  • Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy.
  • Anticancer phospholipids that inhibit Akt such as LBAIs promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. Short-term incubation with these compounds may induce rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceram ide.
  • Pretreatment with sphingomyelinase inhibitors may block ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to perifosine and PIAs in non-small cell lung cancer cell lines.
  • exogenous Ceramides C2, C6 or C12-Ceramide
  • ceramide may mediate and mimic bioeffects of LBAIs. Treatment of LBAIs on H157 cells may cause ceramide accumulation,
  • RhoB promoter activation LAP isoform of C/EBRb translation and RhoB protein induction. All these bioeffects may be prevented by pretreatment of neutral sphingomyelinase specific inhibitor GW4869, which blocks the generation of ceramides from sphingomyelins in plasma membrane. Exogenous Ceramide itself may transactivate RhoB promotor, inhibit Akt/mTOR and activate PKR/elF2a pathways, downregulate protein translation activity, which resulted in RhoB induction by switching translation of C/EBRb LAP isoform in cancer cells with high Akt activity. Therefore, the viability decreased by LBAIs and the sub-G1 DNA content (apoptosis biomarker) increased by LBAIs can be blocked by pretreatment of GW4869.
  • the ingredients in the composition according to some embodiments, the ingredients in the composition according to some embodiments, the ingredients in the composition according to some embodiments, the ingredients in the composition according to some embodiments, the ingredients in the composition according
  • embodiments of the present disclosure may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal.
  • the ingredients in the composition according to some embodiments may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal.
  • embodiments of the present disclosure may maintain, increase or restore RhoB activity in certain cells, tissues, or organs of the mammal.
  • the ingredients in the composition according to some embodiments of the present disclosure may maintain, increase or restore RhoB expression level in certain cells, tissues, or organs of the mammal.
  • the ceramide, EGCG and curcumin in the composition provided according to some embodiments of the present disclosure may transactivate RhoB promotor, inhibit function of the
  • AKt/mTOR signaling pathways and activate PKR/elF2a pathways which may result in RhoB induction (e.g., as described in connection with Examples 11 and 12).
  • the composition provided according to some embodiments of the present disclosure may be a parenteral formulation (e.g., a solid form, a liquid form, or a lyophilized form) and administered to a mammal via parenteral administration.
  • parenteral administration may include but is not limited to a subcutaneous-injection administration, an intramuscular- injection administration, an intravenous-injection administration, or the like, or any combination thereof.
  • the composition may be any combination thereof.
  • the composition for injection may be formulated as power, a solution, a suspension, or an emulsion, or the like.
  • the solution, the suspension or the emulsion may include dissolved or dispersed ingredients (i.e. , the ceramide, the EGCG, and/or the curcumin) and other pharmaceutically injectable ingredients (e.g., glucose and/or sodium chloride).
  • the composition formulated as powder may be dissolved or dispersed in a saline solution (e.g., containing sodium chloride) to obtain a solution or a suspension for injection.
  • the composition may be orally administered to a mammal.
  • the composition for oral administration may be used as a dietary supplement, a food, a drink, a food additive, a drink additive, a drug, or the like, or any combination thereof.
  • the composition for oral administration may be used as a dietary supplement, a food, a drink, a food additive, a drink additive, a drug, or the like, or any combination thereof.
  • composition for the oral administration may be present in the form of, for example, a tablet, granules, powder, a liquid, micellas, a suspension, an emulsion, or the like, or any combination thereof.
  • the composition for the oral administration may further include an acceptable carrier.
  • the carrier may include a coating layer, a capsule, a microcapsule, a nanocapsule, or the like, or any combination thereof. It should be noted that the carrier may need to be nontoxic and may not have any significant impact on the activity of the key
  • the carrier may provide protection for the key ingredients against some undesired conditions, such as oxidation, the decomposition or inactivation of the key ingredients. For instance, enzymes or relatively low-pFI in the stomach may cause the decomposition or inactivation of the key ingredients.
  • the carrier may help maintain or increase the efficacy of the composition by protecting the key ingredients in the composition.
  • the carrier may be used for controlled release of the key ingredients.
  • the controlled release may include but is not limited to slow release, sustained release, targeted release, or the like.
  • the carrier may include hydrogel capsules, microcapsules or nanocapsules made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, or the like, or any combination thereof.
  • the composition may be added to a food or a drink as a food additive or drink additive.
  • the composition may be added to dairy products (e.g., milk, cheese, yoghourt, or milk powder), canned fruits, canned vegetables.
  • the composition may be administered to a mammal via topical administration.
  • the composition may be applied to the skin to prevent or attenuate cancer (e.g., skin cancer or lymphoma), or slow the aging process of the skin.
  • the ceramide in the composition may help preserve moisture for improved skin health and appearance.
  • the composition for topical administration provided according to some embodiments of the present disclosure may be formulated as powder, granules, nanoparticles, cream, a liquid, a suspension, an emulsion, or the like.
  • the composition may be formulated as or added into skin care products and/or drugs for external use (e.g., an ointment).
  • the ceramide may be present in the composition at a weight percentage of 2% to 10%. In some embodiments, the ceramide may be present in the composition at a weight percentage of 4% to 6%. For example, the ceramide may be present in the composition at a weight percentage of about 4.9%, 4.8%, 4.7%, 4.5%, 5.0%, 5.2%, or 5.5%, etc.
  • the ceramide may be extracted from rice, rice bran, rice germ, wheat, konjac, sweet potatoes, soybeans, spinach, or the like, or any combination thereof. In some embodiments, the ceramide may be extracted merely from rice. In some embodiments, the ceramide in the composition may be Ceramide-PCD ® .
  • the Ceramide-PCD ® from rice may be a food-grade powder used in capsules, tablets, liquids, and/or other forms.
  • the EGCG may be present in the composition at a weight percentage of 5% to 20%. In some embodiments, the EGCG may be present in the composition at a weight percentage of 10% to 15%. For instance, the EGCG may be present in the composition at a weight percentage of about 14.1 %, 13%, 12.2%, 11 %, or 10.5%, etc. In some embodiments, the EGCG may be extracted from green tea, seaweed, aloe vera, grains, apples, blackberries, carob flour, or the like, or any combination thereof. In some embodiments, the EGCG may be present in the composition in the form of the green tea extract and the weight percentage of EGCG in the green tea extract may be 85% to 98%, such as 96%, 95%, 94%, 90%, or 88%, etc.
  • the curcumin may be present in the composition at a weight percentage of 15% to 35%. In some embodiments, the curcumin may be present in the composition at a weight percentage of 22% to 27%. For example, the curcumin may be present in the composition at a weight percentage of about 26%, 25%, 24.4%, 23.5%, or 22.5%, etc. In some embodiments, the curcumin may be extracted from turmeric, mango ginger, or the like, or any combination thereof.
  • the curcumin in the composition may be CAVACURMIN ® .
  • CAVACURMIN ® is a highly bioavailable curcumin powder that may be more efficiently absorbed compared to many of the existing leading commercial curcumin supplement products.
  • the ceramide may be present in the composition at the weight percentage of 4% to 6%; the EGCG may be present in the composition at the weight percentage of 10% to 15%; and the curcumin may be present in the composition at the weight percentage of 22% to 27%.
  • the ceramide may be present in the composition at the weight percentage of about 4.9%; the EGCG may be present in the composition at the weight percentage of about 12.2%; and the curcumin may be present in the composition at the weight percentage of about 24.4%.
  • the composition may be provided in a unit dosage form.
  • the unit dosage form may include but is not limited to a tablet, a capsule, a package of granules at a predetermined weight, a package of powder at a
  • the weight of each dosage may be 350-500 mg.
  • the weight of each dosage may be 400 mg.
  • each dosage of the composition may include 10 mg to 30 mg ceramide. In some embodiments, each dosage of the composition may include about 20 mg ceramide.
  • each dosage of the composition may include 40 mg to 60 mg EGCG. In some embodiments, each dosage of the composition may include about 50 mg EGCG.
  • each dosage of the composition may include 90 mg to 110 mg curcumin. In some embodiments, each dosage of the composition may include about 100 mg curcumin.
  • each dosage of the composition may include 10 mg to 30 mg ceramide, 40 mg to 60 mg EGCG, and 90 mg to 110 mg curcumin. In some embodiments, each dosage of the composition may include 20 mg ceramide, 50 mg EGCG, and 100 mg curcumin.
  • the composition when the composition is intended for injective administration, the composition may further include other pharmaceutically injectable ingredients.
  • possible pharmaceutically injectable ingredients may include agents that promote the dissolution, dispersion, stability of the key
  • the pharmaceutically injectable ingredients may include an acetic acid-sodium acetate buffer, lecithin, gelatin, sodium bisulfate, glucose, mineral salt, or the like, or any combination thereof.
  • the adoption of the pharmaceutically injectable ingredients may need to be safe for injection and may not significantly reduce the efficacy or the stability of the key ingredients in the composition (i.e. , the ceramide, the EGCG, and/or the curcumin).
  • the weight percentage of glucose in the composition may be 2% to 25%. In some embodiments, the weight percentage of glucose in the composition may be 4% to 10%.
  • the weight percentage of glucose in the composition may be 5%, 8%, 10%, 15%, etc.
  • the mineral salt may include sodium chloride, potassium chloride, and/or other pharmaceutically acceptable mineral salts. The mineral salt may help maintain the normal osmotic pressure for cells. Merely by way of example, the weight percentage of sodium chloride in the composition may be 0.9%.
  • the volume of each dosage may be 2 ml_, 5ml_, 100 ml_, 250 ml_, 500 ml_, etc.
  • the weight of the key ingredients in each dosage may be determined according to the weight percentage or the weight percentage range of the key ingredients.
  • the composition when used for oral administration, may further include additional agents such as an antioxidant, a cell protection agent, an emulsifier, a flow agent, a silica supplement, a micronutrient supplement, a flavoring agent, a coloring agent, a buffer solution, or the like, or any combination thereof.
  • additional agents such as an antioxidant, a cell protection agent, an emulsifier, a flow agent, a silica supplement, a micronutrient supplement, a flavoring agent, a coloring agent, a buffer solution, or the like, or any combination thereof.
  • the antioxidant derived from plants may include but is not limited to Vitamin C, Vitamin E, b-carotene, astaxanthin, taurin, hypotaurine, glutathione, rosmarinic acid, or the like, or any combination thereof.
  • the antioxidant in the composition may include astaxanthin.
  • the astaxanthin may combat visible signs of aging, improve muscle endurance and recovery, increases lipid metabolism, reduces eye fatigue, improve blood flow rate and vascular health, and improve gastric health.
  • the astaxanthin in the composition may be provided by astaxanthin 2% powder (i.e, AstaREAL ® P2AF).
  • the AstaREAL ® P2AF may be a powderized astaxanthin with relatively high stability and flow properties extracted using supercritical fluid extraction.
  • supercritical carbon dioxide (CO2) may be used as the extracting solvent.
  • the astaxanthin may be present in the composition at a weight percentage of 1 % to 3.5%.
  • the astaxanthin may be present in the composition at a weight percentage of 2% to 3%.
  • the weight percentage of astaxanthin in the composition may be 2.8%, 2.5%, 2.4%, or 2.2%, etc.
  • the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 5 mg to 10 mg astaxanthin.
  • the composition may include about 10 mg astaxanthin.
  • the Vitamin E may serve as the antioxidant as well as the cell protection agent.
  • Vitamin E refers to a group of eight compounds that include four tocopherols and four tocotrienols.
  • the Vitamin E may help protect cell membranes against damages caused by free radicals and prevent the oxidation of low density lipoprotein cholesterols.
  • the Vitamin E in the composition may be D-alpha tocopheryl acetate.
  • the Vitamin E may be present in the composition at a weight percentage of 1 % to 3%. For instance, the weight percentage of Vitamin E in the composition may be 2.8%, 2.5%, 2.4%, 2.2%, or 2.0%, etc.
  • the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 15 IU (10.05 mg) to 25 IU (16.75 mg) Vitamin E.
  • the composition may include about 10 IU (6.7 mg) Vitamin E (1 IU Vitamin E may be the biological equivalent of about 0.67 mg D-alpha tocopheryl acetate).
  • the composition may further include an emulsifier.
  • emulsifiers derived from plants may include but are not limited to lecithin, vegetable gums, magnesium stearate, glycerides, or the like, or any combination thereof.
  • the emulsifier in the composition may include lecithin.
  • the lecithin may be extracted from sunflowers, soybeans, cauliflowers, oranges, peanuts, or the like, or any combination thereof.
  • the lecithin may be present in the composition at a weight percentage of 15% to 35%. In some embodiments, the lecithin may be present in the composition at a weight percentage of 24% to 25%. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 80 mg to 120 mg lecithin. For example, the composition may include about 100 mg lecithin.
  • a unit dosage form e.g., a tablet of 400mg
  • each dosage may include 80 mg to 120 mg lecithin.
  • the composition may include about 100 mg lecithin.
  • the composition may further include a flow agent.
  • the flow agent may be configured to facilitate the ingredients in the composition to move smoothly and prevent the ingredients from sticking to equipment used in the manufacturing process, thus improving the efficiency of manufacturing the
  • Exemplary flow agents derived from plants may include but are not limited to rice hull powder, magnesium stearate, stearic acid, or the like, or any combination thereof.
  • the flow agent in the composition may include rice hull powder.
  • the rice hull powder may be present in the composition at a weight percentage of 15% to 35%.
  • the rice hull powder may be present in the composition at a weight percentage of 24% to 25%.
  • the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 80 mg to 120 mg rice hull powder.
  • the composition may include about 100 mg rice hull powder.
  • the composition may further include a silica supplement.
  • Silica may be used to maintain the integrity and health of the skin, ligaments, tendons, bones, teeth, nails and some other tissues.
  • the silica may also be used to reduce wrinkles, maintain cardiovascular health, and support joints flexibility.
  • the silica supplement in the composition may be bamboo extract.
  • the bamboo extract may include 70% organic silica by weight.
  • the bamboo extract may be present in the composition at a weight percentage of 2% to 10%. In some embodiments, the bamboo extract may be present in the composition at a weight percentage of 4% to 5%, such as 4.5%, 4.8%, 4.9%, etc.
  • the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 10 mg to 30 mg organic bamboo extract. For example, the composition may include about 20 mg organic bamboo extract.
  • methods for preventing or attenuating cancer, or slowing aging process of a mammal may include administering an effective amount of the above-mentioned composition to the mammal.
  • the composition provided according to some embodiments of the present disclosure may be administered to a human.
  • the composition provided according to some embodiments of the present disclosure may be administered to a mammal livestock a companion animal (also referred to as a“pet”), a mammal of a protected species, or the like, or any combination thereof.
  • a mammal livestock may include a pig, a cow, a horse, a sheep, a goat, or the like, or any combination thereof.
  • the companion animal may include a cat, a dog, a rabbit, a ferret, a pig, a gerbil, a hamster, a chinchilla, a rat, a mouse, a guinea pig, a hedgehog, a sugar glider, a chinchilla, a chipmunk, a squirrel, or the like, or any combination thereof.
  • a mammal of a protected species may include a panda, a snub-nosed monkey, a south China tiger, an antelope, or the like, or any combination thereof.
  • the cancer may include but is not limited to non- small cell lung cancer (NSCLC), rhabdomyosarcoma, malignant glioblastoma tumor, metastatic mammary carcinoma, bladder cancer, colorectal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, or the like, or any combination thereof.
  • NSCLC non- small cell lung cancer
  • rhabdomyosarcoma malignant glioblastoma tumor
  • metastatic mammary carcinoma bladder cancer, colorectal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, or the like, or any combination thereof.
  • age may be a significant risk factor
  • the method of administering the effective amount of the composition to the mammal to prevent or attenuate cancer, or slow the aging process may be related to a RhoB-dependent mechanism.
  • the key ingredients (i.e. , the ceramide, the EGCG, and/or the curcumin) in the composition may prevent or attenuate cancer or slow the aging process by maintaining, increasing or restoring RhoB in the mammal.
  • the RhoB may inhibit the EGFR/Ras and PI3K/Akt/mTOR signaling and facilitates MYC turnover in cancer cells, and thus inhibiting the proliferation of the cancer cells, the invasion and metastasis of the tumor, and promotes the apoptotic death of the cancer cells.
  • RhoB may keep oncogenic MYC at a relatively low level, which may extend mammalian healthy lifespan and reduce cancer incidence with age.
  • the increase of RhoB in normal cells may slow down cell cycle, stabilize genome, help repair damaged DNA, and promote longevity transcription factor Nrf2 to slow down the aging process.
  • the composition may be administered to the mammal to prevent cancer by preventing the generation of a benign or malignant tumor, inhibiting the growth of a benign tumor, or preventing the transformation of a benign tumor to a malignant tumor, or the like, or any combination thereof.
  • the composition may be administered to the mammal to attenuate cancer by inhibiting the growth of the malignant tumor, the invasion and metastasis of the malignant tumor, or promote the apoptotic death of cancer cells, or the like, or any combination thereof.
  • the method for may include orally administering the effective amount of the composition to the mammal.
  • the method may include administering the effective amount of the composition to the mammal by a subcutaneous injection, an intramuscular injection, an intravenous injection, or the like, or any combination thereof.
  • the method may include administering the composition to the mammal by a combination of oral administration and injection.
  • the composition may be administered to the mammal every day, every two days, every three days, every week, etc.
  • the method of administering the effective amount of the composition to the mammal for attenuating cancer may be used to enhance the effects of a cancer therapy.
  • the composition may be administered to the mammal with cancer before, during or after the cancer therapy.
  • the cancer therapy may include a surgery for removing the tumor, a radiotherapy, a chemotherapy, an immunotherapy, an acupuncture therapy, or the like, or any combination thereof.
  • the chemotherapy may include administering a chemotherapy agent to the mammal diagnosed with cancer.
  • the chemotherapy agent may include paclitaxel, doxorubicin, epirubicin, fluorouracil, cyclophosphamide, methotrexate, or the like, or any combination thereof.
  • the immunotherapy may include a dendritic cell therapy, a chimeric antigen receptor (CAR-T) cell therapy, an antibody therapy, a cytokine therapy, or the like, or any combination thereof.
  • CAR-T chimeric antigen receptor
  • the effective amount of the composition may be 50 mg to 20 g in a day (24h). In some embodiments, the effective amount of the composition may be 100 mg to 5g in a day. In some embodiments, the effective amount of the composition may be 150 mg to 2g in a day.
  • the safe dosage of ceramide for an adult mammal may be less than or equal to 60mg/kg/day, where“kg” refers to the body weight of the mammal. For instance, for a 60 kg adult, the safe dosage of ceram ide may be less than or equal to 3600 mg in a day. In some embodiments, 10mg to 400 mg ceramide may be administered to the mammal in a day.
  • 20 mg to 350 mg ceramide may be administered to the mammal in a day.
  • 20 mg, 40mg, 80mg, 160mg or 350 mg of ceramide may be administered to the mammal in a day.
  • the safe dosage of EGCG for an adult mammal may be less than or equal to 800 mg in a day.
  • 20 mg to 700 mg EGCG may be administered to the mammal in a day.
  • 50 mg to 700 mg curcumin may be administered to the mammal in a day.
  • 75 mg to 600 mg curcumin may be administered to the mammal in a day.
  • RhoB gene expression during the aging process and carcinogenesis in A/J inbred mice were investigated. RhoB protein was detected by immunoblotting in the two young C57/BL6 mice at age of 2 months and their parents at age of 12 months. The results are shown in FIG. 1A. Among nine A/J mice, three at the age of two months received IP Vehicle 3x, three from 6 at age of 12 months received IP Vehicle 3x or NNK three times a day (or“3x”), followed by immunoblotting of RhoB and p-Akt-S473. The results are shown in FIG. 1 B.
  • RhoB protein was decreased in pulmonary tissue with age (12 months vs 2 months) and further decreased in lung adenocarcinoma induced by tobacco specific carcinogen NNK.
  • Example 2 - RhoB is potently and selectively induced by LBAIs
  • PIA23 was used as representative active PIAs to further compare 9
  • PI3K/Akt/mTOR pathway inhibitors two PI3K inhibitors including LY294002 and wortmannin; a PDK-1 inhibitor including OSU03012; five Akt inhibitors including PIA23, perifosine, miltefosine, API-2, DZ-50; and an mTOR inhibitor including rapamycin
  • Ly represents LY294002
  • Wt represents Wortmannin
  • microarray data which were validated by RT-PCR and immunoblotting, demonstrates that the induction of RhoB at both mRNA and protein levels was selective for LBAIs among the PI3K/Akt/mTOR pathway inhibitors tested, and did not appear to be directly related to inhibition of Akt or mTOR, because other inhibitors decreased phosphorylation of Akt and S6K but did not induce RhoB.
  • Example 3 - LAP isoform of C/EBRb is responsible for RhoB transactivation by
  • RhoB Rapid induction of RhoB relied on transcription-dependent de novo protein synthesis, because 1 h pretreatment of either a transcription blocker (actinomycin D) or a translation inhibitor (cyclohexamide) completely prevented RhoB protein accumulation.
  • RhoB transactivation by LBAIs By analyzing RhoB transactivation by LBAIs, a new transcription factor C/EBRb binding site CEBPB in the RhoB proximal promoter region was found, which was responsible for RhoB induction by LBAIs.
  • Example 4 Identification of the responsive RhoB promoter region to LBAIs
  • hRB, hRB2, hRB3 and hRB4 constructs were transfected into A549 cells and the cells were treated with 10 mM of PI3K inhibitor LY294002, LBAIs (PIA23, perifosine and miltefosine), as well as a DNA damaging agent cisplatin. Luciferase activity was measured after 6h. The results are shown in FIG. 4. LY294002 and cisplatin had little effect on luciferase activity of each construct.
  • hRB4 construct contains the elements responsive to LBAIs.
  • hRB4 contains an inverted CCAAT-NFY binding site NFY(-), a CCAAT/enhancer binding protein beta (C/EBP b ) binding site CEBPB, and 2 TATA box binding protein binding sites MTATA(+/-).
  • mutants of the NFY(-) and CEBPB sites were generated by deleting their core sequences TTGG and GAAA, respectively, and cells were treated with LBAI.
  • Deletion of NFY(-) in hRB3 and hRB4 or CEBPB in hRB4 completely abolished basal and induced activities.
  • Deletion of CEBPB in hRB3 decreased basal activity and eliminated the induced activity by LBAIs.
  • SP1/GC(-) and NFY demonstrated that although SP1/GC(-) and NFY sites coordinate with CEBPB and NFY(-) sites to optimize promoter activity, they are not required for CEBPB site-mediated transcriptional activation by LBAIs.
  • Chromatin immunoprecipitation (ChIP) assay was performed to confirm the association between transcription factors and the RhoB promoter in A549 cells.
  • LBAI increased immunoprecipitation of RhoB promoter DNA fragments by a C/EBP b antibody.
  • the results were shown in FIG. 5. This enhanced DNA association was specific for C/EBRb, as similar intensities of PCR products were observed between control DMSO and LBAIs treatments when NFYA and SP1 antibodies were used.
  • the recruitment of RNA polymerase II (RPII) was specifically increased at the RhoB promoter upon LBAIs treatment but not at the promoter of housekeeping gene GAPDH.
  • RPII RNA polymerase II
  • Akt/mTOR activation and elF2a inhibitory phosphorylation were tested with different levels of Akt/mTOR activation and elF2a inhibitory phosphorylation.
  • H322, A549 and H157 cells Akt/mTOR signaling and the inhibitory phosphorylation of elF2 a were inversely correlated.
  • the relative expression of C/EBRb isoforms was also correlated in that switching an increased LAP to LIP ratio was observed with increased basal levels of Akt activation.
  • Treatment with LBAI inhibited Akt/mTOR signaling increased the inhibitory phosphorylation of elF2 a, especially in the Akt highly active H 157 cells.
  • the cumulative effect of these disruptions may decrease translational factor activity, which could favor LAP isoform translation.
  • 2-hour treatment of LBAI increased LAP protein expression, which was associated with increased expression of RhoB but not RhoA or RhoC (FIG. 7A).
  • RhoB promoter activity was not significantly affected by PKByl , a splice variant of Akt3 lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic carboxyl-terminal domain, making the splice variant of Akt3 incapable of being activated to the same extent as PKB.
  • Dual luciferase assay in isogenic K562 cell lines (C10, C11 and C22) with various levels of constitutively active myr-Akt1 at 48h post co-transfection with hRB and CMV-hRluc constructs were conducted. The results are shown in FIG. 7C.
  • Akt activity determined the inhibitory strength on RhoB promoter, which was also supported by the results after silencing PTEN, a negative regulator of Akt signaling.
  • hRB and CMV-hRluc vectors were co- transfected into isogenic K562 cell lines stably expressing with different levels of constitutively active Akt, it was found that Akt inhibited hRB-driven Firefly luciferase activity in an activation- dependent manner, leaving CMV-driven Renilla luciferase unaffected (Fig. 7D).
  • LBAIs may reverse C/EBRb isoform translation by inhibiting Akt/mTOR signaling and promoting elF2 a inhibitory phosphorylation.
  • B MTS assay of Rh30 and RD cells treated with 10 pM of LY294002 or LBAI in 0.1 % FBS RPMI1640 for 72h.
  • LBAIs inhibited Akt/mTOR pathway signaling to a similar extent as other PI3K/Akt pathway inhibitors did, only LBAIs potently induced RhoB and killed human rhabdomyosarcoma cells Rh30 and RD cells with high levels of Akt activation.
  • a PI3K inhibitor, LY294002 did not show any visible cellular effects at 6h, and had no detectable apoptosis at 72h (Fig. 8B).
  • mice with a non- phosphorylatable Ser51 Ala mutant knock- in of the gene encoding elF2a A/A MEF
  • S/S MEF isogenic wild-type counterparts
  • RhoB promoter activity and endogenous expression was greatly attenuated in A/A MEF cells (Fig. 9A and FIG. 9B).
  • PKR is an elF2a kinase
  • 2-Aminopurine a chemical inhibitor of PKR. 2-Aminopurine but not purine prevented the increases in elF2a
  • Fig.9C phosphorylation
  • Fig.9D cell death
  • Fig.9E apoptosis
  • elF2a kinases seemed not to be involved in cellular killing by LBAIs, because knockout of the endoplasmic reticulum (ER)- resident protein kinase PERK or general control nonrepressed 2 GCN2 (data not shown) did not prevent cytotoxicity from LBAIs.
  • ER endoplasmic reticulum
  • GI50 the concentration of the anti- cancer drug that inhibits the growth of cancer cells by 50%.
  • Table 1 LBAIs drug sensitivity positively correlates with mRNA levels of molecular targets on the PACT-PKR pathway
  • RhoB was transfected into cancer cells with high levels of Akt activation and suppressed basal levels of RhoB expression.
  • ectopic expression of RhoB rather than an inert RhoB mutant (RhoB-N19) caused cell detachment, and significantly induced apoptosis (Fig. 12A).
  • RhoB or its mutant RhoB-N19 was co-transfected with GFP reporter into RhoB null FI2882 cells. The results showed that all GFP positive cells from
  • RhoB+GFP co- transfection group were round and dying with apoptotic
  • RhoB morphological features, whereas those from CMV+GFP or RhoB-N19+GFP group were alive with normally morphology (Fig.12B).
  • these cellular phenotypes of RhoB overexpression were mimicked by forced expression of PTEN in PTEN-null F1157 (Fig. 13C), which inhibited Akt activity, increased elF2a
  • RhoB restoration in Akt highly active or RhoB-null cancer cells is cytotoxic. Consistent with this, RhoB siRNA prevents apoptosis and rescues viability (Fig. 13E) in H 157 cells treated with PIA23 by blocking RhoB induction.
  • Example 10 - RhoB transactivation by LBAI can be monitored in vivo
  • RhoB promoter-driven firefly luciferase and CMV-driven Renilla luciferase constructs were created, and FI157-hRB3-DS and Rh30-hRB3-DS double stable cell lines were established for real-time gene expression analysis in a mouse xenograft model using an In Vivo Imaging System.
  • intraperitoneal administration of vehicle had no effects on the D-Luciferin signal compared to that before treatment (Fig. 14A)
  • intraperitoneal administration of PIA5 increased the D-Luciferin signal compared to that before treatment regardless of tumor size or location (Fig. 14B).
  • PIA5 is specific to RhoB promoter-driven firefly luciferase but not CMV-driven Renilla luciferase, because firefly luciferase substrate D-Luciferin signal was increased by PIA5, but Renilla luciferase substrate coelenterazine signal was not (Fig. 14C).
  • the antitumor activity of PIA5 was verified in this xenograft model (Figs. 14D-F). A similar induction of RhoB and antitumor activity in
  • RhoB activity in vivo and anti-cancer activity was also observed (data not shown).
  • perifosine was also observed.
  • H157 cells were stained with an anti-ceramide antibody (red), and the nuclei were blue (DAPI).
  • Cells were pre-treated with DMSO or 5 mM GW4869 for 2 h, followed by treatment with LBAIs for 6h.
  • Treatment of LBAIs on H 157 cells caused ceramide accumulation (FIG. 15A), RhoB promoter activation (Fig. 15B), LAP isoform of C/EBRb translation and RhoB protein induction (Fig. 15C).
  • Fig. 15A ceramide accumulation
  • Fig. 15B RhoB promoter activation
  • Fig. 15C LAP isoform of C/EBRb translation
  • RhoB protein induction Fig. 15C
  • Example 13 The diet with EGCG and Curcumin attenuates mice aging process
  • mice were fed with standard AIN- 93 diet (the control group, shown on the left portion of FIG. 17), or EGCG (66 PPM) and curcumin (66 PPM) containing AIN-93 diet (the treatment group, shown on the right portion of FIG. 17) for 10 months.
  • Results indicate that, compared to those who were fed using the control diet, these mice fed using EGCG and curcumin obviously aged slower.
  • C57/BL6 mice underwent alopecia, coarser fur, and fur greying under normal aging conditions. During the course of this experiment, it was observed that mice undergoing EGCG and curcumin treatment did not experience any of these characteristic phenotypic properties.
  • the fur on treated C57/BL6 mice were remarkably similar to the condition of the fur prior to the commencement of treatment, 10 months prior.
  • Example 14 The diet with EGCG and Curcumin prevents lung carcinogenesis
  • Example 15 A dietary supplement ROVIDIUM - a composition that includes ceramide, ECCG and Curcumin
  • a dietary supplement ROVIDIUM is formulated using the composition of ceramide, EGCG and curcumin as the core ingredients, as shown in Table 2.
  • the core ingredients of the ROVIDIUM may be obtained as commercial products.
  • the composition of ROVIDIUM is provided in a unit dosage form (e.g., 400-500 mg).
  • the Vitamin E D-Alpha Tocopheryl Acetate
  • the sunflower lecithin powder may be added as an emulsifier.
  • the AstaReal ® P2AF may be astaxanthin 2% powder and may be added to the composition as an antioxidant.
  • the Ceram ide-PCD ® , the green tea extract (95% EGCG), and the CAVACURMIN ® contained the key ingredients ceram ide, EGCG, and curcumin, respectively, which may be added to prevent or attenuate cancer, or slow aging process.
  • the organic rice hull powder may be added as a flow agent to facilitate the ingredients in the composition to move smoothly and prevent the ingredients from sticking to equipment used in the manufacturing process, thus improving the efficiency of manufacturing the composition.
  • the organic bamboo extract may include organic silica and may be added in the composition as a silica supplement to maintain the health of the skin, ligaments, tendons, bones, teeth, nails, and other tissues or functions thereof.
  • composition of the ROVIDIUM is merely provided for the purposes of illustration, and not intended to limit the scope of the present disclosure.
  • composition of the ROVIDIUM is merely provided for the purposes of illustration, and not intended to limit the scope of the present disclosure.
  • each dosage of the composition of ROVIDIUM may include 40 mg to 60 mg EGCG, 90 mg to 110 mg curcumin, and 10 mg to 30 mg ceram ide.
  • RhoB functions is differentially regulated by factors including EGFR, K-Ras, and PI3K/AKT/mTOR.
  • EGFR is a receptor tyrosine kinase that autophosphorylates upon binding ligands such as EGF and TGF-a.
  • the activated EGFR can then facilitate activation of RAS-GDP into RAS-GTP via GEFs.
  • RAS-GTP can lead to increased activity of the PI3K/AKT/mTOR pathway.
  • AKT then co-localizes near the nuclear membrane along with RhoB, where AKT becomes phosphorylated and downregulates RhoB.
  • RhoB can then inhibit or (in angiogenic states) enhance AKT activity, inhibit the EGFR receptor, antagonize Ras/PI3K/mTOR signaling, facilitating MYC turnover, and inhibit overall cell growth, proliferation, and survival.
  • RAS-GTP can also affect regulation of RhoB by means of cross-talk between RAF and AKT.
  • RAS-GTP can activate RAF, which may either upregulate or downregulate function of AKT, which is known to inhibit RhoB.
  • AKT also inhibits the function of RAF.
  • FIDAC1/6 Acetylation of chromatin by FIATs causes relaxation of the chromatin structure, allowing for transcriptional activation of RhoB.
  • deacetylation of chromatin by FIDAC1/6 creates a condensed structure that represses transcription of RhoB.
  • RhoB translation is downregulated by miRNA-19a, miRNA -21 , and miRNA -223 (TS1 and TS2) (as shown in part (A) and part (B) of FIG. 20).
  • Each miRNA inhibits translation of RhoB mRNA by binding specific target sites in the mRNA 3’-UTR (as shown in part (B) and part (C) of FIG. 20).
  • Each miRNA binds to known codon sequences in the 3’-UTR (as shown in part (C) and of FIG. 20).
  • Both miR-19a and miR-21 have only one binding site, whereas miR-223 has two separate target sites, TS1 and TS2 (as shown in part (D) and of FIG. 20).
  • Example 18 The potential roles of tumor suppressor RhoB in cancer and aging
  • RhoB gradually decreases during aging in important tissues including lung and muscle by epigenetic mechanism.
  • RhoB is down regulated by multiple mechanisms, including oncogenic signaling, epigenetic modification, microRNAs, pseudogenes.
  • Restoration of RhoB suppresses oncogenic signaling and antagonizes aging promoters.“CR” in FIG. 21 represents Caloric Restriction.

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Abstract

A nutritional phytonutrient composition for preventing or attenuating cancer, or for slowing aging process in a mammal, by maintaining, increasing or restoring RhoB level in the mammal is provided. The composition may include ceramide, epigallocatechin gallate (EGCG), and curcumin.

Description

NUTRITIONAL PHYTONUTRIENT COMPOSITIONS AND METHODS OF
USE
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application Ser. No.
62/712,190, filed on July 30, 2018, the contents of which are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present disclosure generally relates to nutritional compositions and methods of use, and in particular, to nutritional phytonutrient compositions and methods of using the nutritional phytonutrient compositions for preventing cancer, attenuating cancer and/or slowing the aging process.
BACKGROUND
[0003] Cancer usually refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Cancer has become a significant threat to the life and health of human and non-human animals, especially to those diagnosed with advanced cancer. Even if the malignant tumor is removed or the cancer is currently under control, there is still a possibility that the cancer would return in a few years (e.g., in 2 years or in 5 years). Thus, there is a need to prevent cancer from happening or reoccurring. Moreover, cancer is more common in older people than in young people. In fact, aging is considered not only as an important risk factor for cancer, but also for a number of other diseases such as but not limited to cardiovascular diseases. In addition, aging is associated with the decline of various body functions such as but not limited to physical strength, stamina, memory, and general metabolism. Therefore, slowing the aging process may not only help preventing or attenuating cancer, but also improve general health and body functions.
[0004] While some plant components and extracts have been reported to have an anti-aging effect and/or anti-cancer effects, the effectiveness and safety of such components and extracts have been unclear. In addition, even when positive effects are observed, due to lack of clarification to the mechanism, there is doubt as to the side effects. It is always desirable to provide safe and effective nutrientional phytonutrient composition and methods of using the nutritional phytonutrient compositions for preventing cancer, attenuating cancer and/or slowing the aging process. In addition, it is desirable to provide safe and effective nutritional phytonutrient compositions, wherein the mechanisms of the actions of the
composition’s components are understood and clarified.
SUMMARY
[0005] According to an aspect of the present disclosure, a nutritional phytonutrient composition is provided. The nutritional phytonutrient composition (also referred to as“the composition”) may include ceramide, epigallocatechin gallate (EGCG), and curcumin.
[0006] In some embodiments, the composition may be used for preventing or attenuating cancer in a mammal.
[0007] In some embodiments, the composition may be used for slowing aging process in a mammal.
[0008] In some embodiments, the composition may be used for preventing or attenuating cancer in a mammal or for slowing aging process in the mammal, by maintaining, increasing or restoring RhoB level in the mammal.
[0009] In some embodiments, the ceramide may be 2% to 10% of the composition by weight.
[0010] In some embodiments, the ceramide may be 4% to 6% of the composition by weight.
[0011] In some embodiments, the ceramide may be about 4.9% of the composition by weight.
[0012] In some embodiments, the ceramide may be extracted from rice.
[0013] In some embodiments, the EGCG in the composition may be in the form of green tea extract.
[0014] In some embodiments, the great tea extract contains about 95% EGCG by weight.
[0015] In some embodiments, the EGCG may be 5% to 20% of the composition by weight.
[0016] In some embodiments, the EGCG may be 10% to 15% of the composition by weight.
[0017] In some embodiments, the EGCG may be about 12.2 of the composition by weight.
[0018] In some embodiments, the curcumin may be 15% to 35% of the composition by weight.
[0019] In some embodiments, the curcumin may be 22% to 27% of the composition by weight.
[0020] In some embodiments, the curcumin may be about 24.4% of the composition by weight.
[0021] In some embodiments, the curcumin may be cavacurmin.
[0022] In some embodiments, the composition may further include astaxanthin.
[0023] In some embodiments, the astaxanthin may be 1 % to 3.5% of the
composition by weight.
[0024] In some embodiments, the astaxanthin may be 2% to 3% of the composition by weight.
[0025] In some embodiments, the astaxanthin may be about 2.4% of the
composition by weight.
[0026] In some embodiments, the composition may further include a cell protection agent.
[0027] In some embodiments, the cell protection agent may include Vitamin E.
[0028] In some embodiments, the Vitamin E may be 1 % to 3% of the composition by weight.
[0029] In some embodiments, the Vitamin E may be about 2.2% of the composition by weight.
[0030] In some embodiments, the composition may further include an emulsifier.
[0031] In some embodiments, the emulsifier may include lecithin.
[0032] In some embodiments, the lecithin may be extracted from sunflower or soybean.
[0033] In some embodiments, the lecithin may be 15% to 35% of the composition by weight.
[0034] In some embodiments, the lecithin may be about 24% to 25% of the composition by weight.
[0035] In some embodiments, the composition may further comprise a flow agent. [0036] In some embodiments, the flow agent may include rice hull powder.
[0037] In some embodiments, the rice hull powder may be 15% to 35% of the composition by weight.
[0038] In some embodiments, the rice hull powder may be about 24% to 25% of the composition by weight.
[0039] In some embodiments, the composition may further include a silica supplement.
[0040] In some embodiments, the silica supplement may include bamboo extract.
[0041] In some embodiments, the bamboo extract may be 2% to 10% of the composition by weight.
[0042] In some embodiments, the bamboo extract may be about 4.9% of the composition by weight.
[0043] In some embodiments, the ceramide may be 4% to 6% of the composition by weight; the EGCG may be 10% to 15% of the composition by weight; and the curcumin may be 22% to 27% of the composition by weight.
[0044] In some embodiments, the ceramide may be about 4.9% of the composition by weight; the EGCG may be about 12.2% of the composition by weight; and the curcumin may be about 24.4% of the composition by weight.
[0045] In some embodiments, the ceramide may be 4% to 6% of the composition by weight; the EGCG may be 10% to 15% of the composition by weight; and the curcumin may be 22% to 27% of the composition by weight.
[0046] In some embodiments, the composition may further comprise astaxanthin, which may be 2% to 3% of the composition by weight;
[0047] In some embodiments, the composition may further comprise Vitamin E, which may be 1 % to 3% of the composition by weight;
[0048] In some embodiments, the composition may further comprise lecithin, which may be 15% to 35% of the composition by weight;
[0049] In some embodiments, the composition may further comprise rice hull powder, which may be 15% to 35% of the composition by weight; and
[0050] In some embodiments, the composition may further comprise bamboo extract, which may be 2% to 10% of the composition by weight.
[0051] In some embodiments, the ceramide may be about 4.9% of the composition by weight; the EGCG may be about 12.2 of the composition by weight; and the curcumin may be about 24.4% of the composition by weight, the astaxanthin may be about 2.4% of the composition by weight; the Vitamin E may be about 2.2% of the composition by weight; the lecithin may be about 24% to 25% of the composition by weight; the rice hull powder may be about 24% to 25% of the composition by weight; and the bamboo extract may be about 4.9% of the composition by weight.
[0052] In some embodiments, the composition may be provided in a unit dosage form.
[0053] In some embodiments, each dosage may include 10 mg to 30 mg ceramide.
[0054] In some embodiments, each dosage may include about 20 mg ceramide.
[0055] In some embodiments, each dosage may include 40 mg to 60 mg EGCG.
[0056] In some embodiments, each dosage may include about 50 mg EGCG.
[0057] In some embodiments, each dosage may include 90 mg to 110 mg curcumin.
[0058] In some embodiments, each dosage may include about 100 mg curcumin.
[0059] In some embodiments, each dosage may include 5 mg to 15 mg astaxanthin.
[0060] In some embodiments, each dosage may include about 10 mg astaxanthin.
[0061] In some embodiments, each dosage may include 15 IU to 25 IU Vitamin E.
[0062] In some embodiments, each dosage may include about 10 IU Vitamin E in the form of D-alpha tocopheryl acetate.
[0063] In some embodiments, each dosage may include 80 mg to 120 mg lecithin.
[0064] In some embodiments, each dosage may include about 100 mg lecithin.
[0065] In some embodiments, each dosage may include 80 mg to 120 mg rice hull powder.
[0066] In some embodiments, each dosage may include about 100 mg rice hull pow
[0067] In some embodiments, each dosage may include 10 mg to 30 mg bamboo extract.
[0068] In some embodiments, each dosage may include about 20 mg bamboo extract.
[0069] Additional features will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the accompanying drawings or may be learned by production or operation of the examples. The features of the present disclosure may be realized and attained by practice or use of various aspects of the methodologies,
instrumentalities, and combinations set forth in the detailed examples discussed below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0070] The present disclosure is further described in terms of exemplary
embodiments. These exemplary embodiments are described in detail with reference to the drawings. It should be noted that the drawings are not to scale. These embodiments are non-limiting exemplary embodiments, in which like reference numerals represent similar structures throughout the several views of the drawings, and wherein:
[0071] FIG. 1 A is a representative picture illustrating immunoblotting results of RhoB protein in two young mice at the age of 2 months and their parents at the age of 12 months according to some embodiments of the present disclosure;
[0072] FIG. 1 B is an analytical graph illustrating immunoblotting results of RhoB and p-Akt-S473 according to some embodiments of the present disclosure;
[0073] FIG. 2 is an analytical graph illustrating unsupervised clustering of gene expression microarray data in h157 cells treated with PI3K/Akt/mTOR pathway inhibitors according to some embodiments of the present disclosure;
[0074] FIG. 3 is a representative picture illustrating immunoblotting analysis results of RhoB induction 2h post LBAI treatment in H157 cells according to some embodiments of the present disclosure;
[0075] FIG. 4 is an analytical graph illustrating identification results of the
responsive RhoB promoter region to LBAIs according to some embodiments of the present disclosure;
[0076] FIG. 5 is a representative picture illustrating chromatin immunoprecipitation analysis results of transcription factors C/EBRb, NFYA and SP1 , as well as RNA polymerase II (RPII) binding to endogenous RhoB promoter hRB3 region in A549 cells exposed to LBAI according to some embodiments of the present disclosure;
[0077] FIG. 6A is an analytical graph illustrating luciferase analysis in A549 cells co transfected with hRB3 and C/EBRb plasmid or O/EBRb-ίq^bί^ siRNA duplexes according to some embodiments of the present disclosure;
[0078] FIG. 6B is an analytical graph illustrating results of LBAI induction of hRB4 luciferase activity according to some embodiments of the present disclosure; [0079] FIG. 6C is an analytical graph illustrating luciferase analysis in A549 cells co- transfected with hRB3 and C/EBRb plasmid or C/EBPp-targeting siRNA duplexes according to some embodiments of the present disclosure;
[0080] FIG. 6D is an analytical graph illustrating immunoblotting analysis results of the full-length isoforms and N-terminal truncated isoforms of C/EBRb and C/EBRa in H 157 cells at 6h post LBAIs (10 mM) treatment according to some embodiments of the present disclosure;
[0081] FIG. 7A is an combination of representative pictures illustrating
immunoblotting analysis results of Akt/mTOR pathway signaling, elF2a inhibitory phosphorylation, C/EBRb isoforms and Rho family members, as well as effects of LBAI in H322, A549 and F1157 non-small cell lung cancer cells according to some embodiments of the present disclosure;
[0082] FIG. 7B is an analytical graph illustrating luciferase assay results and immunoblotting analysis in A549 cells at 48h post co-transfection with hRB and FIA- tagged Akt1 (PKBa), Akt2 (RKBb) or C-terminal truncated Akt3 (PKB 1 ) constructs according to some embodiments of the present disclosure;
[0083] FIG. 7C is an analytical graph illustrating luciferase assay results and immunoblotting analysis in A549 cells at 72h post transfection with hRB and Non- Target (NT) or PTEN siRNA duplexes according to some embodiments of the present disclosure;
[0084] FIG. 7D is an analytical graph illustrating Dual luciferase assay results in isogenic K562 cell lines (C10, C11 and C22) with various levels of constitutively active myr-Akt1 at 48h post co-transfection with hRB and CMV-hRluc constructs according to some embodiments of the present disclosure;
[0085] FIG. 7E is a combination of representative pictures illustrating RT-PCR analysis results of RhoB transcript in Rh30 cells under high serum (20% FBS) condition at 24h post treatment with different doses (0, 10 and 30 pM) of PIA5 according to some embodiments of the present disclosure;
[0086] FIG. 7F is a combination of representative pictures illustrating
immunoblotting analysis of Akt/mTOR pathway signaling, elF2a inhibitory
phosphorylation and PARP cleavage according to some embodiments of the present disclosure; [0087] FIG. 8A is a combination of representative pictures illustrating results of Rh30 and RD cells treated with 10 mM of LY294002 or LBAI in 0.1 % FBS RPMI1640 for 6h according to some embodiments of the present disclosure;
[0088] FIG. 8B is an analytical graph illustrating MTS assay results of Rh30 and RD cells treated with 10 mM of LY294002 or LBAI in 0.1 % FBS RPMI 1640 for 72h according to some embodiments of the present disclosure;
[0089] FIG. 9A is an analytical graph illustrating luciferase assay results in elF2a wild type MEF (S/S) or Ser51 mutation MEF (A/A) cells co-transfected with hRB and CMV-hRluc constructs at 6h post LBAIs treatment according to some embodiments of the present disclosure;
[0090] FIG. 9B is a combination of representative pictures illustrating r
immunoblotting analysis results of p-S51 elF2a, total elF2a and RhoB in elF2a wild type MEF (S/S) or Ser51 mutation MEF (A/A) cells at 6 h post LBAI treatment according to some embodiments of the present disclosure;
[0091] FIG. 9C is a combination of representative pictures illustrating immunoblotting analysis results of p-S51 elF2a in H157 cells pretreated (1 h) with 5 mM of Purine (Control) and 2-Aminopurine at 6h post LBAIs treatment according to some
embodiments of the present disclosure;
[0092] FIG. 9D is an analytical graph illustrating MTS assay results in H157 cells pretreated (1 h) with 5 mM of Purine and 2-Aminopurine at 18h post LBAIs treatment according to some embodiments of the present disclosure;
[0093] FIG. 9E is an analytical graph illustrating Apoptosis analysis in H157 cells pretreated (1 h) with 5 mM of Purine and 2-Aminopurine at 18h post LBAIs treatment according to some embodiments of the present disclosure;
[0094] FIG. 10A is an analytical graph illustrating Immunoblotting analysis results of PKR and PACT in H157 cells stably transfected with empty vector (pRS) or shRNA constructs for Non-target (shNT), PKR (shPKR) or PACT (shPACT) at 6h post LBAIs treatment, and MTS assay at 24 h post LBAIs treatment according to some embodiments of the present disclosure;
[0095] FIG. 10B is a combination of representative pictures illustrating
immunoblotting analysis results of PKR and PACT in U251 cells stably transfected with shNT, shPKR or shPACT at 6 h post LBAI treatment, and MTS assay at 18h post LBAIs treatment according to some embodiments of the present disclosure; [0096] FIG. 10C is an analytical graph illustrating MTS assay results of A549 cells stably transfected with shPKR or shPACT were treated with LBAI for 6h
(immunoblotting) and 72h according to some embodiments of the present disclosure;
[0097] FIG. 10D is an analytical graphillustrating MTS assay results in U251 cells stably transfected with shNT, shPKR and shPACT at 24, 48 and 72 h post LBAI treatment in full serum condition (5% FBS) as well as effects of LBAI in H322, A549 and H157 non-small cell lung cancer cells according to some embodiments of the present disclosure;
[0098] FIG. 11 A is a combination of representative pictures illustrating
immunoblotting analysis results of PKR, PACT, p-T446 PKR, LAP, LIP and RhoB in H157 cells stably transfected with shNT, shPKR or shPACT at 6h post LBAI treatment according to some embodiments of the present disclosure;
[0099] FIG. 11 B is a schematic combination of representative pictures diagram illustrating Immunoprecipitation (IP) and immunoblotting (IB) analysis results in H157 cells at 6h post LBAI treatment according to some embodiments of the present disclosure;
[0100] FIG. 12A is an analytical graph illustrating stained H1 155 cells transfected with pcDNA3 CMV empty vector, pcDNA3-HA-RhoB and pcDNA3-HA-RhoB-N19 and flow cytometric analysis results 48 h post transfection according to some embodiments of the present disclosure;
[0101] FIG. 12B is a combination of representative pictures illustrating results of RhoB specific cellular effects on H2882 cells and photographs of RhoB-null H2882 cells co-transfected with pmaxGFP and pcDNA3 CMV empty vector, pcDNA3-HA-RhoB or pcDNA3-HA, RhoB-N19 according to some embodiments of the present disclosure;
[0102] FIG. 13A is a combination of representative pictures of forced expression of RhoB in Rh 30 cells according to some embodiments of the present disclosure;
[0103] FIG. 13B is a combination of representative pictures of reconstituted C/EBRb in O/EBRb-/- transformed MEF cells according to some embodiments of the present disclosure;
[0104] FIG. 13C is a combination of representative pictures of restored PTEN in PTEN-null H157 cells according to some embodiments of the present disclosure;
[0105] FIG. 13D is a combination of representative pictures illustrating
immunoblotting analysis of PI3K/Akt and PKR/elF2a pathways, as well as RhoB induction by restoration of PTEN in H157 cells according to some embodiments of the present disclosure;
[0106] FIG. 13E is an analytical graph illustrating prevention of RhoB induction by PIA23 using RhoB siRNA according to some embodiments of the present disclosure;
[0107] FIG. 14A is a combination of marked pictures illustrating quantification of D- Luciferin signal changes in 3 mice carrying FI157-hRB3-DS (Left) and Rh30-hRB3- DS (Right) cells when they were treated with Vehicle only according to some embodiments of the present disclosure;
[0108] FIG. 14B is a combination of marked pictures illustrating quantification of D- Luciferin signal changes in the same 3 mice cells when they were treated with PIA5 according to some embodiments of the present disclosure;
[0109] FIG. 14C is a combination of marked representative pictures illustrating quantification of D-Luciferin or coelenterazine signal changes in two pairs of mice when they were treated with Vehicle or PIA5 according to some embodiments of the present disclosure;
[0110] FIG. 14D is an analytical graph illustrating administration regimen (5 mice for Vehicle and 5 mice for PIA5) and body weights of the mice during treatment according to some embodiments of the present disclosure;
[0111] FIG. 14E is a schematic diagram illustrating representative combination of representative pictures embodiments of the present disclosure;
[0112] FIG. 14F is a illustrating Tumor growth curves of H157 and Rh30 xenografts in Vehicle and PIA5 28 treated groups according to some embodiments of the present disclosure;
[0113] FIG. 15A is an analytical graph illustrating fluorescent micrographs of H157 cells stained with an anti-ceramide antibody (red), according to some embodiments of the present disclosure;
[0114] FIG. 15B is an analytical graph illustrating RhoB promoter activity according to some embodiments of the present disclosure;
[0115] FIG. 15C is a combination of representative pictures illustrating LAP accumulation and RhoB induction according to some embodiments of the present disclosure; [0116] FIG. 15D is an analytical graph illustrating exogenous C2-Ceramide (10 mM), a cell-permeable analog of naturally occurring ceramides, transactivated RhoB promoter activity according to some embodiments of the present disclosure;
[0117] FIG. 15E is a combination of representative pictures illustrating the inhibition effect of the exogenous C2-Ceramide inhibited Akt/mTOR and activated PKR/elF2a pathways according to some embodiments of the present disclosure;
[0118] FIG. 15F is an analytical graph illustrating a viability of the F1157 cells according to some embodiments of the present disclosure;
[0119] FIG. 15G is an analytical graphillustrating the blocking effect of the
pretreatment of GW4869 on the increased LBAIs according to some embodiments of the present disclosure;
[0120] FIG. 16 is a combination of representative pictures illustrating
immunoblotting analysis of phosphorylation inhibition of Akt/mTOR downstream substrates according to some embodiments of the present disclosure;
[0121] FIG. 17 is an analytical graph illustrating the male C57/BL6 mice (at the age of 12 months) fed with standard AIN-93 diet, or EGCG and Curcumin containing AIN- 93 diet for 10 months according to some embodiments of the present disclosure;
[0122] FIG 18A is an analytical graph illustrating the tumor multiplicity according to some embodiments of the present disclosure;
[0123] FIG. 18B is an analytical graph of size in left-single lung according to some embodiments of the present disclosure;
[0124] FIG. 19 is an analytical graph illustrating interactions between RhoB and EGFR, Ras, PI3K/Akt/mTOR, MYC, and FID AC;
[0125] FIG. 20 is an analytical graph illustrating translation of RhoB is epigenetically downregulated by miRNA-19/21/223; and,
[0126] FIG. 21 is an analytical graph illustrating the potential roles of tumor suppressor RhoB in cancer and aging.
DETAILED DESCRIPTION
[0127] The following description is presented to enable any person skilled in the art to make and use the present disclosure and is provided in the context of a particular application and its requirements. Various modifications to the disclosed
embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the present disclosure. Thus, the present disclosure is not limited to the embodiments shown, but is to be accorded the widest scope consistent with the claims.
[0128] The terminology used herein is to describe particular example embodiments only and is not intended to be limiting. As used herein, the singular forms“a,”“an,” and“the” may be intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms“comprises,” “comprising,”“includes,” and/or“including” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
[0129] These and other features, and characteristics of the present disclosure, as well as the methods of operation and functions of the related elements of structure and the combination of parts and economies of manufacture, may become more apparent upon consideration of the following description with reference to the accompanying drawing(s), all of which form a part of this specification. It is to be expressly understood, however, that the drawing(s) are for the purpose of illustration and description only and are not intended to limit the scope of the present disclosure. It is understood that the drawings are not to scale.
[0130] As used herein, the term“ceramide” refers to one or more types of
substances, each of which includes sphingosine and a fatty acid in the structure.
For instance, the ceramide may include but is not limited to C2-ceramide (N-(Acetyl)- sphing-4-enine), C6-ceramide (N-Hexanoylsphingosine), C12-ceramide (N- (Dodecanoyl)-sphing-4-enine), C16-ceramide (N-(hexadecanoyl)-sphing-4-enine), C24-ceramide (N-(tetracosanoyl)-sphing-4-enine), or the like, or an analog thereof (e.g., a salt having a similar structure that is safe to be administered to a mammal), or a combination thereof. Exemplary analogs of C6-ceramide may include but are not limited to C6-Nbd-ceramide, C6-pyridinium-ceramide, C6-NBD lactosyl ceramide, or the like, or any analog thereof, or any combination thereof.
[0131] In some embodiments, ceramide has a structure of:
Figure imgf000014_0001
where“R” can be any functional group.
[0132] As used herein, the term“epigallocatechin gallate”, also abbreviated as “EGCG”, refers to one or more types of substance, each of which includes the ester of epigallocatechin and gallic acid in the structure. For instance, the EGCG may include but is not limited to Epigallocatechin 3-gallate and/or an analog thereof.
Exemplary analogs of the epigallocatechin 3-gallate may include but are not limited to methylated EGCG, a D-ring analog of the epigallocatechin 3-gallate, EGCG 4- palmitate, EGCG 4-stearate, or the like, or any analog thereof, or any combination thereof.
[0133] In some embodiments, EGCG has the following structure:
Figure imgf000014_0002
[0134] As used herein, the term“curcumin” refers to one or more types of substance, each of which has a structure that is at least partly similar to
diferuloylmethane ((1 E,6E)-1 ,7-Bis(4-hydroxy-3-methoxyphenyl)hepta-1 ,6-diene-3,5- dione). For instance, the curcumin may include but is not limited to
diferuloylmethane and/or an analog thereof. Exemplary analogs of the curcumin may include but are not limited to bisdiferuloylmethane, curcumin glucuronide, curcumin sulfate, monodemethoxycurcumin, tetrahydrocurcumin, curcumin pyrazole, or the like, or any analog thereof, or any combination thereof.
[0135] In some embodiments, curcumin has the following structure:
Figure imgf000015_0001
[0136] In an aspect of the present disclosure, nutritional phytonutrient compositions are provided by the present disclosure. A nutritional phytonutrient composition provided according to some embodiments of the present disclosure may include one or more natural ingredients derived from plants, such as vegetables, fruits, soybeans, grains, nuts, or the like, or any combination thereof. For brevity, the terms“nutritional phytonutrient composition” and“the composition” are used interchangeably herein. In some embodiments, the ingredients of the composition may include ceramide, EGCG, or curcumin, or any combination thereof. For example, the ceramide may be extracted from rice, rice bran, rice germ, wheat, konjac, sweet potatoes, soybeans, spinach, or the like, or any combination thereof. As another example, the EGCG may be extracted from green tea, seaweed, aloe vera, grains, apples, blackberries, carob flour, or the like, or any combination thereof. In some embodiments, the EGCG in the composition may be present in the form of green tea extract. As yet another example, the curcumin may be extracted from turmeric, mango ginger, or the like, or any combination thereof.
[0137] In some embodiments, the composition provided according to some embodiments of the present disclosure may be used to prevent or attenuate cancer, or slow the aging process of a mammal. The mammal may include a human or a non-human mammal. For instance, the non-human mammal may include monkeys, orangutans, tigers, cats, dogs, rabbits, ferrets, pigs; gerbils, hamsters, chinchillas, rats, mice, guinea pigs, hedgehogs, sugar gliders, chinchillas, chipmunks, squirrels, or the like, or any combination thereof. In some embodiments, the cancer may include but is not limited to non-small cell lung cancer (NSCLC),
rhabdomyosarcoma, breast cancer, bladder cancer, colorectal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer,, melanoma, oral and oropharyngeal cancer, uterine cancer, or the like, or any combination thereof.
[0138] In some embodiments, the composition provided according to some embodiments of the present disclosure may prevent or attenuate cancer or slow the aging process via a RhoB-dependent mechanism. As used herein, the term“RhoB” refers to a member of the Rho GTPase family. RhoB may inhibit function of the epidermal growth factor receptor (EGFR)-Ras signaling pathway and function of the PI3K/Akt/mTOR signaling pathway. As used herein, the term“PI3K” refers to phosphatidylinositol 3-kinase, and the term“mTOR” refers to the mammalian target of rapamycin. The EGFR/Ras signaling pathway and the PI3K/Akt/mTOR signaling pathway may contribute to the cell growth, proliferation, and survival of the cancer cells. Moreover, RhoB may facilitate Myc protein turnover in cancer cells. The Myc proteins may serve as transcription factors to activate the expression of may pro-proliferation genes. Thus, RhoB may inhibit the proliferation of the cancer cells, the invasion and metastasis of the tumor, and/or promoting the apoptotic death of the cancer cells. In some embodiments, RhoB may also help keep oncogenic Myc at a relatively low level, which may extend mammalian healthy lifespan and reduce cancer incidence with age. In some embodiments, the increase of RhoB in normal cells may slow down cell cycle, stabilize genome, help repair damaged DNA, and promote the activation of nuclear factor erythrold 2-related factor2 (Nrf2). The Nrf2 may contribute to cytoprotective responses and the extension of lifespan. Thus, RhoB may help slow the aging process and extend the healthspan. Flowever, RhoB may be inhibited and/or decreased during tumorigenesis and aging process. In some embodiments, the ingredients in the composition according to some
embodiments of the present disclosure may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal. In some embodiments, the ingredients in the composition according to some
embodiments of the present disclosure may maintain, increase or restore RhoB activity in certain cells, tissues, or organs of the mammal. In some embodiments, the ingredients in the composition according to some embodiments of the present disclosure may maintain, increase or restore RhoB expression level in certain cells, tissues, or organs of the mammal. For instance, the ceramide, EGCG and curcumin in the composition provided according to some embodiments of the present disclosure may transactivate RhoB promotor, inhibit function of the
AKt/mTOR signaling pathways and activate PKR/elF2a pathways, which may result in RhoB induction (e.g., as described in connection with Examples 11 and 12). As used herein, the term“PKR” refers to protein kinase R, and the term“elF2a” refers to eukaryotic translation initiation factor. The inhibitory phosphorylation of elF2a by PKR activation may lead to RhoB induction.
[0139] More specifically, the Rho family of GTPases may be characterized for its role in the regulation of cellular actin that forms the cytoskeleton mediated functions of motion and adhesion as well as cell-cycle progression, regulation of transcription factors, and protein trafficking. As molecular switches regulated by the status of GTP, Rho GTPases may act as a critical component of intracellular signaling pathways. In some embodiments, RhoA, RhoB, and RhoC, as members of the Rho GTPase family, may have distinct functional roles. In comparison to RhoA and RhoC, RhoB localizes in endosomes where it regulates their intracellular movements and can be differentially prenylated by a farnesyl or geranylgeranyl group. The unique nature of RhoB is particularly highlighted in its role, again, in stark contrast to oncogenes RhoA and RhoC, as a putative tumor suppressor, and it is no surprise that it has been shown to be significantly down regulated in cancer specimens from many tissue types, thereby allowing opportunities for developing therapies targeted at key points in regulation of RhoB as well as interplay in intracellular signaling pathways. The functional implication of RhoB in tumorigenesis is critical in the context of aged associated decreases in expression found primarily in muscle and lung tissue. Further analysis of RhoB unique cellular functions, characterization of its role in tumorigenesis, and exploration of the association between age and RhoB loss, may open avenues for elucidating and potentially treating elusive pathways.
[0140] In some embodiments, RhoB may be suppressed by oncogenic signaling. EGFR may reduce RhoB promoter activity via Ras signaling. Epidermal growth factor receptor (EGFR) is a member of the erbB family of receptor tyrosine kinases that serves as an interface between the extracellular environment and intracellular signal transduction moderating cell growth, differentiation, survival, and progression through the cell cycle. EGFR may include an extracellular ligand-binding domain, a transmembrane lipophilic domain, and an intracellular tyrosine kinase domain, and binds primarily to EGF and TGF-a. Once bound, the receptor may be activated resulting in the phosphorylation of the tyrosine kinase domain and
homodimerization/heterodimerization between different receptors. This
autophosphorylation may recruit various intracellular signaling proteins and activates downstream signaling cascades, mainly the Ras/Raf/MAPK/ERK and the PI3K/Akt pathways (as shown in FIG. 19). In cancer cells, EGFR may be altered through various mechanisms such as gain-of-function mutations, EGFR gene gain, overexpression of ligands and receptors, etc. In some embodiments, increased EGFR expression may promote tumorigenesis through the down-regulation of RhoB (as shown in FIG. 19). EGFR may inhibit RhoB promoter activity in cancer cells heavily depending on the presence of Ras, another oncogene, suggesting that EGFR suppresses RhoB promoter activity through Ras pathway. There may be a sharp inhibition of transformation in EGFR-transfected NIFI3T3 cells through the ectopic expression of RhoB.
[0141] The Ras subfamily may include Fl-Ras, N-Ras, and K-Ras monomeric GTPases, and mediate signal transduction between cell surface growth receptors and intracellular signaling pathways. The Ras proteins may activate once bound to GTP, a process that is catalyzed by guanine nucleotide exchange factors (GEFs), inactivate through GTP hydrolysis which is catalyzed by GTPase-activating proteins (GAPs) (as shown in FIG. 19). Oncogenic mutations of the three Ras genes occur in codons 12, 13, or 61. These mutations may prevent the proteins from becoming inactive due to resistance of GAP-mediated GTP hydrolysis, allowing them to stimulate growth, differentiation, and survival uninhibited. While wild-type K-Ras may serve as a suppressor of oncogenic activity, mutated K-Ras may be observed in cancers of the pancreas, esophagus, cardia and distal stomach, stomach, biliary tract, bile duct, ampulla, gallbladder, colon, lung cancer, or the like, or any
combination thereof. K-Ras mutations may be associated with a poor prognosis in individuals with colorectal cancer and non-small-cell lung cancer, virtually in all individuals with early-stage NSCLC and adenocarcinoma.
[0142] Autophosphorylation of the tyrosine residing on EGFR may result in the activation of the Ras/Raf/MAPK/ERK pathway which may modulate cell growth and proliferation and is hyperactive in cancers. This intracellular cascade may begin once the phosphorylated tyrosine residues interact with Grb2, an adaptor protein, which recruits GEFs to initiate the formation of Ras-GTP, the active form (as shown in FIG. 19). In addition to being a critical mediator in the suppression of RhoB via EGFR and ErB2 transfection, K-Ras may decrease the promoter transcriptional activity of RhoB in a dose-dependent manner in NIFI3T3 cells and suppressed RhoB protein levels in various types of cancer cells from pancreatic, cervical, and lung tumors. Moreover, oncogenic K-Ras demonstrated some degree of anticancer drug resistance through effectively blocking the induction of RhoB protein levels and promoter site activity by 5-fluorouracil. Ectopic expression of RhoB was found to inhibit K-Ras transformation of NIFI3T3 cells, further lending to the concept that RhoB suppression is required for some oncogenes to transform cells.
Phosphatidylinositol 3-kinase (PI3K) is one of the main effector pathways of Ras, regulating cell growth, cell cycle entry, cell survival, cytoskeleton reorganization, and metabolism. PI3K may be necessary for Ras-induced transformation in vitro, and more importantly, mice with mutations in the PI3K catalytic subunit p110a that block its ability to interact with Ras are highly resistant to endogenous oncogenic K-Ras induced lung tumorigenesis and Fl-Ras induced skin carcinogenesis.
[0143] Ras may downregulate RhoB expression by PI3K- and Akt- but not Mek- dependent mechanism. Genetic and pharmacological blockade of PI3K/Akt may result in upregulation of RhoB expression. The importance of the downregulation of RhoB in oncogenesis is supported by demonstrating that RhoB antagonizes
Ras/PI3K/Akt malignancy. Ectopic expression of RhoB, but not the close relative RhoA, may inhibit Ras, PI3K, and Akt induction of transformation, migration, and invasion and may induce apoptosis and anoikis. Finally, RhoB may inhibit melanoma metastasis to the lung in a mouse model. These studies identify suppression of RhoB as a mechanism by which the Ras/PI3K/Akt pathway induces tumor survival, transformation, invasion, and metastasis (as shown in FIG. 19).
[0144] RhoB gene may be not genetically mutated or altered in various tumors and carcinoma. Consequently, it was then proposed that RhoB expression is controlled by epigenetic events. Nucleosomes, a complex of core histones wrapped by chromosomal DNA, may contribute to the stability of chromatin structure and the repression of genetic transcription in eukaryotes. Depending on the acetylation status of the histone amino termini that extend from the nucleosome core, this activity can be modified and is dynamically coordinated by Flistone
Acetyltransferases (HATs) and Histone Deacetyltransferases (HDACs). HDACs may be generally located in large multi-protein complexes that regulate a variety of genes. Normally, decreased levels of histone acetylation are linked to
transcriptional repression while increased levels of histone acetylation are linked with active transcription (as shown in FIG. 19). A large body of research investigating agents that upregulate RhoB through the reversal of this process, namely HDAC inhibitors, has resulted from the correlation of RhoB repression with deacetylation. HDAC inhibitors may target histone deacetylases and serve as powerful antitumor agents that induce differentiation and apoptosis through transcriptional modulation (as shown in FIG. 19).
[0145] In some embodiments, the expression of RhoB may be further controlled by MicroRNAs (miRNAs), 18-24 base-pair non-coding small RNAs that function in post- transcriptional regulation of gene expression. Briefly, genes encoding miRNAs may be transcribed into pri-miRNA by RNA Polymerase II and subsequently processed by Drosha to form pre-miRNA. These pre-miRNAs may be then further processed by the cytoplasmic enzyme complexes Dicer and RISC. Once fully processed, miRNA may then bind to the 3’ untranslated region (3’-UTR) of target mRNA, leading to destabilization of the mRNA and thus decreased mRNA translation.
[0146] The 3’-untranslated region (3’-UTR) of RhoB transcript plays a regulatory role in RhoB expression. Translation of mRNA with the RhoB 3’-UTR may decrease expression of reporter transcripts. Therefore, regulation of the 3’-UTR of RhoB, which may be facilitated by miRNA, may in turn regulate expression of RhoB, as shown in FIG. 20.
[0147] In some embodiments, it may be found that RhoB may decrease during tumorigenesis and aging. The RhoB mRNA was decreased from immortalization stage. HDAC1 regulates RhoB promoter activity through an inverted CCAAT element within the RhoB promoter. Levels of HDAC1 binding to CCAAT boxes may change with age. There was no association between HDAC1 and the CCAAT elements in young tissue (<4 weeks), but the binding increased as the mice aged. Therefore, RhoB is gradually decreased during aging process in important tissues including lung and muscle by epigenetic mechanism. Given that age is the biggest risk for cancer, a decrease in RhoB from aged tissues may propose the possibility that RhoB loss leads to increased cancer rates with age. As RhoB is required for apoptosis in cells transformed by DNA-damaging agents, its loss increases DSB- mediated genomic instability and tumor progression and promotes tumorigenesis. RhoB appears to function as a suppressor or negative modifier in cancer
progression. Thus, a reduction in RhoB mRNA and protein from aged mice might increase the occurrence of cancer in pulmonary tissue, as was explained in a human non-small lung carcinoma cell line.
[0148] Given the role of RhoB as a tumor suppressor, investigations have been conducted exploring RhoB for cancer prognosis and prevention. Loss of RhoB expression contributes to increased invasiveness of lung cancer through pathways, such as PI3K/AKT and Rad . In some embodiments, RhoB may be used as a prognostic marker of NSCLC aggression. By utilizing IHC and RT-qPCR to compare RhoB in control patients and patients with known advanced lepidic adenocarcinoma, it may be found that patients with more aggressive forms of lepidic adenocarcinoma had greater losses of RhoB expression. Furthermore, in studies of mice with inducible EGFRL858R and either RhoB+/+, RhoB+/-, and RhoB-/- genotypes, it may be found that the mice with the most aggressive tumors were RhoB-/-, followed by RhoB+/-. RhoB expression can be utilized to determine prognosis of NSCLC. In some embodiments, the deceased expression of RhoB may mediate higher malignancy potential in clear cell renal cell carcinoma.
[0149] In some embodiments, restoring RhoB levels may be used in treating cancer. Restored RhoB in ovarian adenocarcinoma cells with undetectable levels of RhoB may suppress tumor growth. Recombinant adenovirus transduced with RhoB cDNA may activate apoptosis in vitro, whereas injections of Ad-RhoB in nude mice with ovarian cancer xenografts may demonstrate suppression of tumor growth in vivo, thereby illustrating that restoration of RhoB may prove useful in cancer therapy.
RhoB restoration may contribute to a longer and healthier lifespan. In mammals, genetically down-regulating mTOR expression or pharmacologically inhibited mTOR by Rapamycin was shown to produce a profound increase in lifespan. Transgenic mice carrying additional copies of PTEN, the negative regulator of PI3K/AKT/mTOR pathway, may live longer and have lower incidence of cancer relative to normal.
Reduced expression of MYC may increase longevity and enhance healthspan without any apparent developmental tradeoffs. Therefore, RhoB restoration might benefit cancer prevention and healthspan by antagonizing RAS/PI3K/AKT/mTOR signaling and facilitating MYC turnover (as shown in Fig.21 ). [0150] In some embodiments, RhoB may be potently and selectively inducted by LBAIs (lipid-based inhibitors). LAP isoform of C/EBRb may be responsible for RhoB transactivation by LBAIs. LBAI may rapidly activate RhoB transcription by increasing levels of the transcriptionally active C/EBRb isoform LAP. The increase of LAP protein upon LBAIs treatment may be through translation alteration of C/EBRb from LIP to LAP. PACT-mediated PKR activation may switch C/EBRb isoform translation from LIP to LAP. The inhibitory phosphorylation of elF2a by PACT-mediated PKR activation may be the major mechanism for LBAIs-mediated RhoB induction and cytotoxicity.
[0151] In some embodiments, RhoB restoration in Akt highly active or RhoB-null cancer cells may be cytotoxic. Akt has a central role in the phosphoinositide-3- kinase (PI3K)/Akt signaling pathway that controls cellular processes integral in the development of cancer including growth, metabolism and survival. As Akt promotes therapeutic resistance and is active in a majority of human cancers, inhibitors may be developed as cancer therapeutics. Akt is activated in response to the lipid products of class I PI3K, PI3,4P2 (PIP2) and PI3,4,5 P3 (PIP3). Once synthesized, PIP2 and PIP3 attract Akt to the plasma membrane through binding to the pleckstrin homology (PH) domain of Akt. This may promote a conformational change, allowing it to be phosphorylated on T308 by PDK-1 and at S473 by the rictor/mTOR complex. Once phosphorylated, Akt may dissociate from the plasma membrane and moves to various cellular compartments where it phosphorylates downstream substrates.
[0152] Most Akt inhibitors in development target the ATP-binding region, whereas LBAIs such as phosphatidylinositol ether lipid analogs and the alkylphospholipids perifosine and miltefosine may be designed to interfere with the PH domain of Akt. LBAIs may selectively target metabolically active, proliferating tumor cells, inducing growth arrest and apoptosis, and are potent sensitizers of conventional
chemotherapy and radiotherapy. In some embodiments, the dramatically increased expression of the tumor suppressor RhoB may contribute to enhanced cytotoxicity beyond PI3K/Akt/mTOR pathway inhibition.
[0153] Activation of the PI3K/Akt/mTOR pathway in cancers can occur through loss of tumor suppressor PTEN. Transcriptional profiling of pathway inhibitors identified the tumor suppressor RhoB as a gene markedly upregulated by LBAI. The C/EBPbeta full-length isoform LAP may be responsible for transcriptional induction through its binding site within the RhoB proximal promoter. LBAI strongly transactivate RhoB by switching translation of C/EBPbeta from the truncated isoform LIP to LAP via PACT-mediated PKR activation in cancer cells with high Akt activity. Unlike PTEN commonly mutated, endogenous RhoB tumor-suppressive activity can be reconstituted by restoring its expression, which was noninvasively monitored by a RhoB promoter-driven luciferase reporter in living mice. LBAIs administration increased luciferase activity and decreased the growth of human tumor xenografts. Increased PKR activation by LBAI leads to more robust RhoB induction and cytotoxicity than other PI3K/Akt/mTOR axis inhibitors, revealing a novel strategy for cancer therapy.
[0154] Anticancer phospholipids that inhibit Akt such as LBAIs promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. Short-term incubation with these compounds may induce rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceram ide.
Pretreatment with sphingomyelinase inhibitors may block ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to perifosine and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous Ceramides (C2, C6 or C12-Ceramide) may also decrease active Akt and induced nanovesicle release.
[0155] In some embodiments, ceramide may mediate and mimic bioeffects of LBAIs. Treatment of LBAIs on H157 cells may cause ceramide accumulation,
RhoB promoter activation, LAP isoform of C/EBRb translation and RhoB protein induction. All these bioeffects may be prevented by pretreatment of neutral sphingomyelinase specific inhibitor GW4869, which blocks the generation of ceramides from sphingomyelins in plasma membrane. Exogenous Ceramide itself may transactivate RhoB promotor, inhibit Akt/mTOR and activate PKR/elF2a pathways, downregulate protein translation activity, which resulted in RhoB induction by switching translation of C/EBRb LAP isoform in cancer cells with high Akt activity. Therefore, the viability decreased by LBAIs and the sub-G1 DNA content (apoptosis biomarker) increased by LBAIs can be blocked by pretreatment of GW4869. In some embodiments, the ingredients in the composition according to some
embodiments of the present disclosure may prevent or attenuate cancer, or slow the aging process by maintaining, increasing or restoring RhoB in the mammal. In some embodiments, the ingredients in the composition according to some
embodiments of the present disclosure may maintain, increase or restore RhoB activity in certain cells, tissues, or organs of the mammal. In some embodiments, the ingredients in the composition according to some embodiments of the present disclosure may maintain, increase or restore RhoB expression level in certain cells, tissues, or organs of the mammal. For instance, the ceramide, EGCG and curcumin in the composition provided according to some embodiments of the present disclosure may transactivate RhoB promotor, inhibit function of the
AKt/mTOR signaling pathways and activate PKR/elF2a pathways, which may result in RhoB induction (e.g., as described in connection with Examples 11 and 12).
[0156] In some embodiments, the composition provided according to some embodiments of the present disclosure may be a parenteral formulation (e.g., a solid form, a liquid form, or a lyophilized form) and administered to a mammal via parenteral administration. For example, the parenteral administration may include but is not limited to a subcutaneous-injection administration, an intramuscular- injection administration, an intravenous-injection administration, or the like, or any combination thereof. In some embodiments, the composition may be
stereotactically injected into the tumor or a region close to a tumor. In some embodiments, the composition for injection may be formulated as power, a solution, a suspension, or an emulsion, or the like. The solution, the suspension or the emulsion may include dissolved or dispersed ingredients (i.e. , the ceramide, the EGCG, and/or the curcumin) and other pharmaceutically injectable ingredients (e.g., glucose and/or sodium chloride). Merely by way of example, the composition formulated as powder may be dissolved or dispersed in a saline solution (e.g., containing sodium chloride) to obtain a solution or a suspension for injection.
[0157] In some embodiments, the composition may be orally administered to a mammal. In some embodiments, the composition for oral administration may be used as a dietary supplement, a food, a drink, a food additive, a drink additive, a drug, or the like, or any combination thereof. In some embodiments, the
composition for the oral administration may be present in the form of, for example, a tablet, granules, powder, a liquid, micellas, a suspension, an emulsion, or the like, or any combination thereof. In some embodiments, the composition for the oral administration may further include an acceptable carrier. For instance, the carrier may include a coating layer, a capsule, a microcapsule, a nanocapsule, or the like, or any combination thereof. It should be noted that the carrier may need to be nontoxic and may not have any significant impact on the activity of the key
ingredients in the composition. In some embodiments, the carrier may provide protection for the key ingredients against some undesired conditions, such as oxidation, the decomposition or inactivation of the key ingredients. For instance, enzymes or relatively low-pFI in the stomach may cause the decomposition or inactivation of the key ingredients. The carrier may help maintain or increase the efficacy of the composition by protecting the key ingredients in the composition. In some embodiments, the carrier may be used for controlled release of the key ingredients. The controlled release may include but is not limited to slow release, sustained release, targeted release, or the like. For instance, the carrier may include hydrogel capsules, microcapsules or nanocapsules made of collagen, gelatin, chitosan, alginate, polyvinyl alcohol, or the like, or any combination thereof. {{You may want to add an administration method via topical formulations, mainly re cutaneous formulation, like cream, ointment, etc. Since a function of the ceramide is to preserve moisture for improved skin health and appearance.— added in the 2nd following paragraph}}
[0158] In some embodiments, the composition may be added to a food or a drink as a food additive or drink additive. For instance, the composition may be added to dairy products (e.g., milk, cheese, yoghourt, or milk powder), canned fruits, canned vegetables.
[0159] In some embodiments, the composition may be administered to a mammal via topical administration. For instance, the composition may be applied to the skin to prevent or attenuate cancer (e.g., skin cancer or lymphoma), or slow the aging process of the skin. Moreover, the ceramide in the composition may help preserve moisture for improved skin health and appearance. In some embodiments, the composition for topical administration provided according to some embodiments of the present disclosure may be formulated as powder, granules, nanoparticles, cream, a liquid, a suspension, an emulsion, or the like. In some embodiments, the composition may be formulated as or added into skin care products and/or drugs for external use (e.g., an ointment).
[0160] In some embodiments, the ceramide may be present in the composition at a weight percentage of 2% to 10%. In some embodiments, the ceramide may be present in the composition at a weight percentage of 4% to 6%. For example, the ceramide may be present in the composition at a weight percentage of about 4.9%, 4.8%, 4.7%, 4.5%, 5.0%, 5.2%, or 5.5%, etc. In some embodiments, the ceramide may be extracted from rice, rice bran, rice germ, wheat, konjac, sweet potatoes, soybeans, spinach, or the like, or any combination thereof. In some embodiments, the ceramide may be extracted merely from rice. In some embodiments, the ceramide in the composition may be Ceramide-PCD®. The Ceramide-PCD® from rice may be a food-grade powder used in capsules, tablets, liquids, and/or other forms.
[0161] In some embodiments, the EGCG may be present in the composition at a weight percentage of 5% to 20%. In some embodiments, the EGCG may be present in the composition at a weight percentage of 10% to 15%. For instance, the EGCG may be present in the composition at a weight percentage of about 14.1 %, 13%, 12.2%, 11 %, or 10.5%, etc. In some embodiments, the EGCG may be extracted from green tea, seaweed, aloe vera, grains, apples, blackberries, carob flour, or the like, or any combination thereof. In some embodiments, the EGCG may be present in the composition in the form of the green tea extract and the weight percentage of EGCG in the green tea extract may be 85% to 98%, such as 96%, 95%, 94%, 90%, or 88%, etc.
[0162] In some embodiments, the curcumin may be present in the composition at a weight percentage of 15% to 35%. In some embodiments, the curcumin may be present in the composition at a weight percentage of 22% to 27%. For example, the curcumin may be present in the composition at a weight percentage of about 26%, 25%, 24.4%, 23.5%, or 22.5%, etc. In some embodiments, the curcumin may be extracted from turmeric, mango ginger, or the like, or any combination thereof.
In some embodiments, the curcumin in the composition may be CAVACURMIN®. CAVACURMIN® is a highly bioavailable curcumin powder that may be more efficiently absorbed compared to many of the existing leading commercial curcumin supplement products. [0163] In some embodiments, the ceramide may be present in the composition at the weight percentage of 4% to 6%; the EGCG may be present in the composition at the weight percentage of 10% to 15%; and the curcumin may be present in the composition at the weight percentage of 22% to 27%.
[0164] In some embodiments, the ceramide may be present in the composition at the weight percentage of about 4.9%; the EGCG may be present in the composition at the weight percentage of about 12.2%; and the curcumin may be present in the composition at the weight percentage of about 24.4%.
[0165] In some embodiments, the composition may be provided in a unit dosage form. The unit dosage form may include but is not limited to a tablet, a capsule, a package of granules at a predetermined weight, a package of powder at a
predetermined weight, a bottle of a solution, a suspension, or an emulsion at a predetermined weight or volume, or the like. Merely by way of example, the weight of each dosage may be 350-500 mg. For instance, the weight of each dosage may be 400 mg.
[0166] In some embodiments, each dosage of the composition may include 10 mg to 30 mg ceramide. In some embodiments, each dosage of the composition may include about 20 mg ceramide.
[0167] In some embodiments, each dosage of the composition may include 40 mg to 60 mg EGCG. In some embodiments, each dosage of the composition may include about 50 mg EGCG.
[0168] In some embodiments, each dosage of the composition may include 90 mg to 110 mg curcumin. In some embodiments, each dosage of the composition may include about 100 mg curcumin.
[0169] In some embodiments, each dosage of the composition may include 10 mg to 30 mg ceramide, 40 mg to 60 mg EGCG, and 90 mg to 110 mg curcumin. In some embodiments, each dosage of the composition may include 20 mg ceramide, 50 mg EGCG, and 100 mg curcumin.
[0170] In some embodiments, when the composition is intended for injective administration, the composition may further include other pharmaceutically injectable ingredients. For example, possible pharmaceutically injectable ingredients may include agents that promote the dissolution, dispersion, stability of the key
ingredients, agents that maintain the osmotic pressure, nutrients, or the like, or any combination thereof. For instance, the pharmaceutically injectable ingredients may include an acetic acid-sodium acetate buffer, lecithin, gelatin, sodium bisulfate, glucose, mineral salt, or the like, or any combination thereof. The adoption of the pharmaceutically injectable ingredients may need to be safe for injection and may not significantly reduce the efficacy or the stability of the key ingredients in the composition (i.e. , the ceramide, the EGCG, and/or the curcumin). In some embodiments, the weight percentage of glucose in the composition may be 2% to 25%. In some embodiments, the weight percentage of glucose in the composition may be 4% to 10%. In some embodiments, the weight percentage of glucose in the composition may be 5%, 8%, 10%, 15%, etc. In some embodiments, the mineral salt may include sodium chloride, potassium chloride, and/or other pharmaceutically acceptable mineral salts. The mineral salt may help maintain the normal osmotic pressure for cells. Merely by way of example, the weight percentage of sodium chloride in the composition may be 0.9%. In some embodiments, when the composition is provided in a unit dosage form, the volume of each dosage may be 2 ml_, 5ml_, 100 ml_, 250 ml_, 500 ml_, etc. The weight of the key ingredients in each dosage may be determined according to the weight percentage or the weight percentage range of the key ingredients.
[0171] In some embodiments, when the composition is used for oral administration, the composition may further include additional agents such as an antioxidant, a cell protection agent, an emulsifier, a flow agent, a silica supplement, a micronutrient supplement, a flavoring agent, a coloring agent, a buffer solution, or the like, or any combination thereof.
[0172] In some embodiments, the antioxidant derived from plants may include but is not limited to Vitamin C, Vitamin E, b-carotene, astaxanthin, taurin, hypotaurine, glutathione, rosmarinic acid, or the like, or any combination thereof. In some embodiments, the antioxidant in the composition may include astaxanthin. The astaxanthin may combat visible signs of aging, improve muscle endurance and recovery, increases lipid metabolism, reduces eye fatigue, improve blood flow rate and vascular health, and improve gastric health. In some embodiments, the astaxanthin in the composition may be provided by astaxanthin 2% powder (i.e, AstaREAL® P2AF). The AstaREAL® P2AF may be a powderized astaxanthin with relatively high stability and flow properties extracted using supercritical fluid extraction. For instance, supercritical carbon dioxide (CO2) may be used as the extracting solvent. In some embodiments, the astaxanthin may be present in the composition at a weight percentage of 1 % to 3.5%. In some embodiments, the astaxanthin may be present in the composition at a weight percentage of 2% to 3%. For instance, the weight percentage of astaxanthin in the composition may be 2.8%, 2.5%, 2.4%, or 2.2%, etc. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 5 mg to 10 mg astaxanthin. For example, the composition may include about 10 mg astaxanthin.
[0173] In some embodiments, the Vitamin E may serve as the antioxidant as well as the cell protection agent. Vitamin E refers to a group of eight compounds that include four tocopherols and four tocotrienols. For instance, the Vitamin E may help protect cell membranes against damages caused by free radicals and prevent the oxidation of low density lipoprotein cholesterols. In some embodiments, the Vitamin E in the composition may be D-alpha tocopheryl acetate. In some embodiments, the Vitamin E may be present in the composition at a weight percentage of 1 % to 3%. For instance, the weight percentage of Vitamin E in the composition may be 2.8%, 2.5%, 2.4%, 2.2%, or 2.0%, etc. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 15 IU (10.05 mg) to 25 IU (16.75 mg) Vitamin E. For example, the composition may include about 10 IU (6.7 mg) Vitamin E (1 IU Vitamin E may be the biological equivalent of about 0.67 mg D-alpha tocopheryl acetate).
[0174] In some embodiments, the composition may further include an emulsifier. Exemplary emulsifiers derived from plants may include but are not limited to lecithin, vegetable gums, magnesium stearate, glycerides, or the like, or any combination thereof. In some embodiments, the emulsifier in the composition may include lecithin. In some embodiments, the lecithin may be extracted from sunflowers, soybeans, cauliflowers, oranges, peanuts, or the like, or any combination thereof.
In some embodiments, the lecithin may be present in the composition at a weight percentage of 15% to 35%. In some embodiments, the lecithin may be present in the composition at a weight percentage of 24% to 25%. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 80 mg to 120 mg lecithin. For example, the composition may include about 100 mg lecithin.
[0175] In some embodiments, the composition may further include a flow agent.
The flow agent may be configured to facilitate the ingredients in the composition to move smoothly and prevent the ingredients from sticking to equipment used in the manufacturing process, thus improving the efficiency of manufacturing the
composition. Exemplary flow agents derived from plants may include but are not limited to rice hull powder, magnesium stearate, stearic acid, or the like, or any combination thereof. In some embodiments, the flow agent in the composition may include rice hull powder. In some embodiments, the rice hull powder may be present in the composition at a weight percentage of 15% to 35%. In some embodiments, the rice hull powder may be present in the composition at a weight percentage of 24% to 25%. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 80 mg to 120 mg rice hull powder. For example, the composition may include about 100 mg rice hull powder.
[0176] In some embodiments, the composition may further include a silica supplement. Silica may be used to maintain the integrity and health of the skin, ligaments, tendons, bones, teeth, nails and some other tissues. The silica may also be used to reduce wrinkles, maintain cardiovascular health, and support joints flexibility. In some embodiments, the silica supplement in the composition may be bamboo extract. The bamboo extract may include 70% organic silica by weight.
In some embodiments, the bamboo extract may be present in the composition at a weight percentage of 2% to 10%. In some embodiments, the bamboo extract may be present in the composition at a weight percentage of 4% to 5%, such as 4.5%, 4.8%, 4.9%, etc. In some embodiments, the composition may be provided in a unit dosage form (e.g., a tablet of 400mg) and each dosage may include 10 mg to 30 mg organic bamboo extract. For example, the composition may include about 20 mg organic bamboo extract.
[0177] In another aspect of the present disclosure, methods for preventing or attenuating cancer, or slowing aging process of a mammal is provided. The method may include administering an effective amount of the above-mentioned composition to the mammal. In some embodiments, the composition provided according to some embodiments of the present disclosure may be administered to a human. In some embodiments, the composition provided according to some embodiments of the present disclosure may be administered to a mammal livestock a companion animal (also referred to as a“pet”), a mammal of a protected species, or the like, or any combination thereof. For example, a mammal livestock may include a pig, a cow, a horse, a sheep, a goat, or the like, or any combination thereof. As another example, the companion animal may include a cat, a dog, a rabbit, a ferret, a pig, a gerbil, a hamster, a chinchilla, a rat, a mouse, a guinea pig, a hedgehog, a sugar glider, a chinchilla, a chipmunk, a squirrel, or the like, or any combination thereof.
As yet another example, a mammal of a protected species may include a panda, a snub-nosed monkey, a south China tiger, an antelope, or the like, or any combination thereof. In some embodiments, the cancer may include but is not limited to non- small cell lung cancer (NSCLC), rhabdomyosarcoma, malignant glioblastoma tumor, metastatic mammary carcinoma, bladder cancer, colorectal cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, pancreatic cancer, prostate cancer, skin cancer, thyroid cancer, or the like, or any combination thereof. In some embodiments, age may be a significant risk factor for cancer incidence. Thus delaying the aging process may also benefit cancer prevention and healthy longevity.
[0178] In some embodiments, the method of administering the effective amount of the composition to the mammal to prevent or attenuate cancer, or slow the aging process may be related to a RhoB-dependent mechanism. For example, the key ingredients (i.e. , the ceramide, the EGCG, and/or the curcumin) in the composition may prevent or attenuate cancer or slow the aging process by maintaining, increasing or restoring RhoB in the mammal. The RhoB may inhibit the EGFR/Ras and PI3K/Akt/mTOR signaling and facilitates MYC turnover in cancer cells, and thus inhibiting the proliferation of the cancer cells, the invasion and metastasis of the tumor, and promotes the apoptotic death of the cancer cells. The increased RhoB may keep oncogenic MYC at a relatively low level, which may extend mammalian healthy lifespan and reduce cancer incidence with age. In some embodiments, the increase of RhoB in normal cells may slow down cell cycle, stabilize genome, help repair damaged DNA, and promote longevity transcription factor Nrf2 to slow down the aging process.
[0179] In some embodiments, the composition may be administered to the mammal to prevent cancer by preventing the generation of a benign or malignant tumor, inhibiting the growth of a benign tumor, or preventing the transformation of a benign tumor to a malignant tumor, or the like, or any combination thereof. In some embodiments, the composition may be administered to the mammal to attenuate cancer by inhibiting the growth of the malignant tumor, the invasion and metastasis of the malignant tumor, or promote the apoptotic death of cancer cells, or the like, or any combination thereof.
[0180] In some embodiments, the method for may include orally administering the effective amount of the composition to the mammal. In some embodiments the method may include administering the effective amount of the composition to the mammal by a subcutaneous injection, an intramuscular injection, an intravenous injection, or the like, or any combination thereof. In some embodiments, the method may include administering the composition to the mammal by a combination of oral administration and injection. In some embodiments, for example, the composition may be administered to the mammal every day, every two days, every three days, every week, etc.
[0181] In some embodiments, the method of administering the effective amount of the composition to the mammal for attenuating cancer may be used to enhance the effects of a cancer therapy. Specifically, the composition may be administered to the mammal with cancer before, during or after the cancer therapy. Merely by way of example, the cancer therapy may include a surgery for removing the tumor, a radiotherapy, a chemotherapy, an immunotherapy, an acupuncture therapy, or the like, or any combination thereof. For instance, the chemotherapy may include administering a chemotherapy agent to the mammal diagnosed with cancer. The chemotherapy agent may include paclitaxel, doxorubicin, epirubicin, fluorouracil, cyclophosphamide, methotrexate, or the like, or any combination thereof. As another example, the immunotherapy may include a dendritic cell therapy, a chimeric antigen receptor (CAR-T) cell therapy, an antibody therapy, a cytokine therapy, or the like, or any combination thereof.
[0182] In some embodiments, the effective amount of the composition may be 50 mg to 20 g in a day (24h). In some embodiments, the effective amount of the composition may be 100 mg to 5g in a day. In some embodiments, the effective amount of the composition may be 150 mg to 2g in a day. In some embodiments, the safe dosage of ceramide for an adult mammal may be less than or equal to 60mg/kg/day, where“kg” refers to the body weight of the mammal. For instance, for a 60 kg adult, the safe dosage of ceram ide may be less than or equal to 3600 mg in a day. In some embodiments, 10mg to 400 mg ceramide may be administered to the mammal in a day. In some embodiments, 20 mg to 350 mg ceramide may be administered to the mammal in a day. For example, 20 mg, 40mg, 80mg, 160mg or 350 mg of ceramide may be administered to the mammal in a day. In some embodiments, the safe dosage of EGCG for an adult mammal may be less than or equal to 800 mg in a day. In some embodiments, 20 mg to 700 mg EGCG may be administered to the mammal in a day. In some embodiments, 50 mg to 700 mg curcumin may be administered to the mammal in a day. In some embodiments, 75 mg to 600 mg curcumin may be administered to the mammal in a day.
[0183] The present disclosure is further described according to the following examples, which should not be construed as limiting the scope of the present disclosure.
EXAMPLES
Example 1 - RhoB gene expression decreases during lung carcinogenesis and aging process
[0184] RhoB gene expression during the aging process and carcinogenesis in A/J inbred mice (or A/J mice) were investigated. RhoB protein was detected by immunoblotting in the two young C57/BL6 mice at age of 2 months and their parents at age of 12 months. The results are shown in FIG. 1A. Among nine A/J mice, three at the age of two months received IP Vehicle 3x, three from 6 at age of 12 months received IP Vehicle 3x or NNK three times a day (or“3x”), followed by immunoblotting of RhoB and p-Akt-S473. The results are shown in FIG. 1 B.
Tobacco carcinogen NNK treated mice had large adenocarcinomas occupied a large percentage of lung volume. These results demonstrated that RhoB protein was decreased in pulmonary tissue with age (12 months vs 2 months) and further decreased in lung adenocarcinoma induced by tobacco specific carcinogen NNK.
In contrast, Akt activation was increased during pulmonary aging and further in lung tumorigenesis. Example 2 - RhoB is potently and selectively induced by LBAIs
[0185] PIA23 was used as representative active PIAs to further compare 9
PI3K/Akt/mTOR pathway inhibitors (two PI3K inhibitors including LY294002 and wortmannin; a PDK-1 inhibitor including OSU03012; five Akt inhibitors including PIA23, perifosine, miltefosine, API-2, DZ-50; and an mTOR inhibitor including rapamycin) using high-density oligonucleotide arrays representing 34,542 transcripts in H157 non-small cell lung cancer cells. The result are shown in FIG. 2. As shown in FIG. 2, Ly represents LY294002, Wt represents Wortmannin, Os
represents OSU-03012, Dz represents DZ-50, Pi represents PIA23, Pf represents Perifosine, Mf represents Miltefosine, Ap represents API-2, and Rp represents Rapamycin. The two-way unsupervised clustering reveals that LBAIs including PIA23, perifosine and miltefosine, showed very similar gene expression signatures. The microarray data, which were validated by RT-PCR and immunoblotting, demonstrates that the induction of RhoB at both mRNA and protein levels was selective for LBAIs among the PI3K/Akt/mTOR pathway inhibitors tested, and did not appear to be directly related to inhibition of Akt or mTOR, because other inhibitors decreased phosphorylation of Akt and S6K but did not induce RhoB.
Example 3 - LAP isoform of C/EBRb is responsible for RhoB transactivation by
LBAIs
[0186] Rapid induction of RhoB relied on transcription-dependent de novo protein synthesis, because 1 h pretreatment of either a transcription blocker (actinomycin D) or a translation inhibitor (cyclohexamide) completely prevented RhoB protein accumulation. Immunoblotting analysis of RhoB induction 2h post LBAI treatment without or with 1 h pretreatment with cycloheximide (1.4 pg/ml) or Actinomycin D (0.5 pg/ml) in H 157 cells was conducted. The results are shown in FIG. 3. As shown in FIG. 3, D represents DMSO, P represents PIA23, PF represents Perifosine, and MF represents Miltefosine. By analyzing RhoB transactivation by LBAIs, a new transcription factor C/EBRb binding site CEBPB in the RhoB proximal promoter region was found, which was responsible for RhoB induction by LBAIs. Example 4 - Identification of the responsive RhoB promoter region to LBAIs
[0187] To identify the promoter regions responsible for RhoB induction, hRB, hRB2, hRB3 and hRB4 constructs were transfected into A549 cells and the cells were treated with 10 mM of PI3K inhibitor LY294002, LBAIs (PIA23, perifosine and miltefosine), as well as a DNA damaging agent cisplatin. Luciferase activity was measured after 6h. The results are shown in FIG. 4. LY294002 and cisplatin had little effect on luciferase activity of each construct. By contrast, LBAIs significantly induced luciferase activity in all constructs except for the pGL3 vector, with an average 3~4-fold relative induction even in the shortest hRB4 construct. Cisplatin did not induce RhoB, suggesting that this is not a general response to DNA damage or cytotoxic insults. These results show that the hRB4 construct contains the elements responsive to LBAIs. hRB4 contains an inverted CCAAT-NFY binding site NFY(-), a CCAAT/enhancer binding protein beta (C/EBP b ) binding site CEBPB, and 2 TATA box binding protein binding sites MTATA(+/-). In order to determine which TFBS is responsible for LBAIs stimulation, mutants of the NFY(-) and CEBPB sites were generated by deleting their core sequences TTGG and GAAA, respectively, and cells were treated with LBAI. Deletion of NFY(-) in hRB3 and hRB4 or CEBPB in hRB4 completely abolished basal and induced activities. Deletion of CEBPB in hRB3 decreased basal activity and eliminated the induced activity by LBAIs. These results indicate that the CEBPB site is required for responsiveness to LBAIs in an NFY(-) site-dependent manner. Further evaluation of the upstream TFBSs
SP1/GC(-) and NFY demonstrated that although SP1/GC(-) and NFY sites coordinate with CEBPB and NFY(-) sites to optimize promoter activity, they are not required for CEBPB site-mediated transcriptional activation by LBAIs.
Example 5 - Binding of C/EBRb to the RhoB Promoter by LBAIs treatment
[0188] Chromatin immunoprecipitation (ChIP) assay was performed to confirm the association between transcription factors and the RhoB promoter in A549 cells.
Compared to DMSO treatment, LBAI increased immunoprecipitation of RhoB promoter DNA fragments by a C/EBP b antibody. The results were shown in FIG. 5. This enhanced DNA association was specific for C/EBRb, as similar intensities of PCR products were observed between control DMSO and LBAIs treatments when NFYA and SP1 antibodies were used. The recruitment of RNA polymerase II (RPII) was specifically increased at the RhoB promoter upon LBAIs treatment but not at the promoter of housekeeping gene GAPDH. Thus C/EBP pbinding to RhoB promoter is induced by LBAIs and is concurrent with RPII recruitment. Collectively, these experiments demonstrate that LBAI rapidly and specifically increase RhoB
transcriptional activity by enhancing C/EBRb binding to its cognate site on the RhoB promoter.
Example 6 - LBAIs induced transactivation of RhoB by the LAP isoform of
C/EBRb
[0189] Levels of C/EBRb were genetically manipulated to further verify the role of C/EBP b in RhoB transactivation. The Luciferase analysis results in A549 cells co transfected with hRB3 and C/EBRb plasmid or O/EBRb-ίq^bί^ siRNA duplexes are shown in FIG. 6A. Co-transfection of hRB3 with a C/EBRb construct or C/EBRb targeting siRNA duplexes in A549 cells caused a 9-fold increase or 40% decrease in luciferase activity, respectively. Relative induction folds after depletion of C/EBRb in A549 cells are shown in FIG. 6B. Depletion of C/EBRb in A549 cells prevented LBAI induction of hRB4 luciferase activity, directly demonstrating the importance of C/EBRb interaction with its binding site in RhoB proximal promoter region for RhoB transactivation by LBAI.
[0190] To determine which isoform of C/EBRb was responsible for RhoB promoter activation, LAP or LIP expression constructs were co-transfected with a hRB-driven luciferase vector in H 157 cells. Luciferase activity was induced by LAP but not LIP. The results were shown in FIG. 6C. LBAIs specifically increased LAP protein accompanied by RhoB induction, leaving LIP unaffected or slightly diminished. This isoform specific accumulation seems to be selective to C/EBRb, because both full- length and truncated forms of another C/EBP, C/EBRa, similarly increased upon LBAI treatment (as shown in FIG. 6D). These data show that LBAI rapidly activate RhoB transcription by increasing levels of the transcriptionally active C/EBRb isoform LAP. Example 7 - PACT-mediated PKR activation switches C/EBP b isoform
translation from LIP to LAP
[0191] Three NSCLC cell lines were tested with different levels of Akt/mTOR activation and elF2a inhibitory phosphorylation. In H322, A549 and H157 cells, Akt/mTOR signaling and the inhibitory phosphorylation of elF2 a were inversely correlated. The relative expression of C/EBRb isoforms was also correlated in that switching an increased LAP to LIP ratio was observed with increased basal levels of Akt activation. Treatment with LBAI inhibited Akt/mTOR signaling increased the inhibitory phosphorylation of elF2 a, especially in the Akt highly active H 157 cells. The cumulative effect of these disruptions may decrease translational factor activity, which could favor LAP isoform translation. Indeed, 2-hour treatment of LBAI increased LAP protein expression, which was associated with increased expression of RhoB but not RhoA or RhoC (FIG. 7A).
[0192] Luciferase assay and immunoblotting analysis in A549 cells at 48h post co- transfection with hRB and FIA-tagged Akt1 (PKBa), Akt2 (RKBb) or C-terminal truncated Akt3 (PKB 1 ) constructs were conducted. The results are shown in FIG. 7B. RhoB promoter activity was inhibited significantly by Akt1/PKB a ectopic expression, and to a lesser extent by AM2/RKBb. Flowever, RhoB promoter activity was not significantly affected by PKByl , a splice variant of Akt3 lacking the second regulatory phosphorylation site Ser-472 in the hydrophobic carboxyl-terminal domain, making the splice variant of Akt3 incapable of being activated to the same extent as PKB. Dual luciferase assay in isogenic K562 cell lines (C10, C11 and C22) with various levels of constitutively active myr-Akt1 at 48h post co-transfection with hRB and CMV-hRluc constructs were conducted. The results are shown in FIG. 7C. Akt activity determined the inhibitory strength on RhoB promoter, which was also supported by the results after silencing PTEN, a negative regulator of Akt signaling. When hRB and CMV-hRluc vectors were co- transfected into isogenic K562 cell lines stably expressing with different levels of constitutively active Akt, it was found that Akt inhibited hRB-driven Firefly luciferase activity in an activation- dependent manner, leaving CMV-driven Renilla luciferase unaffected (Fig. 7D).
[0193] RT-PCR analysis of RhoB transcript in Rh30 cells under high serum (20% FBS) condition at 24h post treatment with different doses (0, 10 and 30 mM) of PIA5. The pictures were taken 5 days post the treatment. Both Akt/mTOR pathway inhibition and elF2 a inhibitory phosphorylation correlated with RhoB induction by LBAIs, which in combination could induce apoptosis. Indeed, this was observed in highly refractory Rh30 cells, even in very high serum conditions (FIG. 7E). PIA5 inhibited Akt/mTOR downstream signaling, induced inhibitory phosphorylation of elF2 a and transactivation of RhoB, accompanied by apoptosis as shown by cellular morphology, PARP cleavage in a dose-dependent manner (Fig. 7F). Therefore, LBAIs may reverse C/EBRb isoform translation by inhibiting Akt/mTOR signaling and promoting elF2 a inhibitory phosphorylation. These disruptions lead to C/EBP b translational initiation switching to LAP from LIP, thereby increasing RhoB
transcription and causing cellular apoptosis in cancer cells.
Example 8 - RhoB induction and cytotoxicity by LBAIs due to PKR activation
[0194] Rh30 and RD cells treated with 10 mM of LY294002 or LBAI in 0.1 % FBS RPMI1640 for 6h. (B) MTS assay of Rh30 and RD cells treated with 10 pM of LY294002 or LBAI in 0.1 % FBS RPMI1640 for 72h. Although LBAIs inhibited Akt/mTOR pathway signaling to a similar extent as other PI3K/Akt pathway inhibitors did, only LBAIs potently induced RhoB and killed human rhabdomyosarcoma cells Rh30 and RD cells with high levels of Akt activation. In comparison, a PI3K inhibitor, LY294002, did not show any visible cellular effects at 6h, and had no detectable apoptosis at 72h (Fig. 8B).
[0195] To test whether the inhibitory phosphorylation of elF2a played a critical role in RhoB induction and induction of cytotoxicity by LBAIs, mouse embryonic fibroblasts (MEF) from mice with a non- phosphorylatable Ser51 Ala mutant knock- in of the gene encoding elF2a (A/A MEF) and their isogenic wild-type counterparts (S/S MEF) were transfected with RhoB promoter driven luciferase reporter construct and treated with LBAIs for 6h. RhoB promoter activity and endogenous expression was greatly attenuated in A/A MEF cells (Fig. 9A and FIG. 9B). The cytotoxicity of LBAIs was also prevented by elF2a A/A mutation (data not shown). Because PKR is an elF2a kinase, cells were treated with 2-Aminopurine, a chemical inhibitor of PKR. 2-Aminopurine but not purine prevented the increases in elF2a
phosphorylation (Fig.9C), cell death (Fig.9D) and apoptosis (Fig.9E) induced by LBAIs.
[0196] To verify the specific role of PKR and its signaling in this process, several cell lines (H157, U251 and A549) were created, in which the gene expression levels of PKR or its cellular activator PACT were stably knocked down using PKR or PACT shRNA constructs. Knockdown of PKR or PACT rescued these cancer cells from killing elicited by LBAIs (Figs. 10A, 10B and 10C) even in full serum conditions over 72h (Fig. 10D). Interestingly, other elF2a kinases seemed not to be involved in cellular killing by LBAIs, because knockout of the endoplasmic reticulum (ER)- resident protein kinase PERK or general control nonrepressed 2 GCN2 (data not shown) did not prevent cytotoxicity from LBAIs.
[0197] Knockdown of PKR or PACT blocked the effects of LBAIs, including PACT induced phosphorylation of PKR at Thr446, LAP accumulation, and RhoB induction (Fig. 11 A). PACT binding to PKR upon LBAIs stimulation was confirmed by
immunoprecipitation in H157 cells (Fig. 11 B).
[0198] The matrix COMPARE analysis was performed in the NCI60 cancer cell line panel by using the GI50 data of indicated compounds against the mRNA levels of molecular targets on the PACT-PKR pathway. GI50 = the concentration of the anti- cancer drug that inhibits the growth of cancer cells by 50%. The bold PCC numbers (0.25 or -0.25 PCC cutoff) indicate that the correlations are significant (p =¾ 0.05).
Consistent with the above experimental data, a matrix COMPARE analysis from the NCI60 cancer cell lines database indicates that the drug sensitivity of LBAIs positively correlates with mRNA levels of molecular targets on PACT-PKR
pathway (p =¾ 0.05 for both or one of them. Table 1 ).
[0199] Taken together, these data demonstrate that the inhibitory phosphorylation of elF2a by PACT-mediated PKR activation is the major mechanism for LBAIs- mediated RhoB induction and cytotoxicity.
Table 1 LBAIs drug sensitivity positively correlates with mRNA levels of molecular targets on the PACT-PKR pathway
Figure imgf000039_0001
Figure imgf000040_0001
Example 9 - Consequences of RhoB restoration in cancer cells with high Akt
Activity
[0200] RhoB was transfected into cancer cells with high levels of Akt activation and suppressed basal levels of RhoB expression. In H 1155 cells, ectopic expression of RhoB rather than an inert RhoB mutant (RhoB-N19) caused cell detachment, and significantly induced apoptosis (Fig. 12A). To specify the cellular effects induced by RhoB, RhoB or its mutant RhoB-N19 was co-transfected with GFP reporter into RhoB null FI2882 cells. The results showed that all GFP positive cells from
RhoB+GFP co- transfection group were round and dying with apoptotic
morphological features, whereas those from CMV+GFP or RhoB-N19+GFP group were alive with normally morphology (Fig.12B). Reconstitution of RhoB in Rh30 cells (Fig. 13A) and its transactivator C/EBRb in O/EBRb-/- MEF cells (Fig. 13B) induced cell death, as indicated by the floating cells. Intriguingly, these cellular phenotypes of RhoB overexpression were mimicked by forced expression of PTEN in PTEN-null F1157 (Fig. 13C), which inhibited Akt activity, increased elF2a
phosphorylation and subsequently induced RhoB (Fig. 13D), similar to LBAIs.
These data demonstrate that RhoB restoration in Akt highly active or RhoB-null cancer cells is cytotoxic. Consistent with this, RhoB siRNA prevents apoptosis and rescues viability (Fig. 13E) in H 157 cells treated with PIA23 by blocking RhoB induction.
Example 10 - RhoB transactivation by LBAI can be monitored in vivo
concomitantly with antitumor activity
[0201] RhoB promoter-driven firefly luciferase and CMV-driven Renilla luciferase constructs were created, and FI157-hRB3-DS and Rh30-hRB3-DS double stable cell lines were established for real-time gene expression analysis in a mouse xenograft model using an In Vivo Imaging System. Although intraperitoneal administration of vehicle had no effects on the D-Luciferin signal compared to that before treatment (Fig. 14A), intraperitoneal administration of PIA5 increased the D-Luciferin signal compared to that before treatment regardless of tumor size or location (Fig. 14B). Furthermore, the effect of PIA5 is specific to RhoB promoter-driven firefly luciferase but not CMV-driven Renilla luciferase, because firefly luciferase substrate D-Luciferin signal was increased by PIA5, but Renilla luciferase substrate coelenterazine signal was not (Fig. 14C). The antitumor activity of PIA5 was verified in this xenograft model (Figs. 14D-F). A similar induction of RhoB and antitumor activity in
glioblastoma U251 xenografts during PIA23 administration was also observed (data not shown). Likewise, induction of RhoB activity in vivo and anti-cancer activity was observed with perifosine (data not shown). These results show that the induction of RhoB can be noninvasively monitored in vivo, accompanied by an anti-tumor activity.
Example 11 - Ceramide mediated bioeffects of LBAI
[0202] H157 cells were stained with an anti-ceramide antibody (red), and the nuclei were blue (DAPI). Cells were pre-treated with DMSO or 5 mM GW4869 for 2 h, followed by treatment with LBAIs for 6h. Treatment of LBAIs on H 157 cells caused ceramide accumulation (FIG. 15A), RhoB promoter activation (Fig. 15B), LAP isoform of C/EBRb translation and RhoB protein induction (Fig. 15C). Interestingly, all these bioeffects were prevented by pretreatment of neutral sphingomyelinase specific inhibitor GW4869, which blocked the generation of ceramides from
sphingomyelins in plasma membrane. Exogenous ceramide did transactivate RhoB promotor (Fig. 15D), inhibit Akt/mTOR and activate PKR/elF2a pathways,
downregulate protein translation activity, which resulted in RhoB induction by switching translation of C/EBRb LAP isoform in cancer cells with high Akt activity (Fig. 15E). Therefore, the viability decreased by LBAIs (Fig. 15F) and the sub-G1 DNA content (apoptosis biomarker) increased by LBAIs (Fig. 15G) can be blocked by pretreatment of GW4869. Example 12 - The combination of EGCG and Curcumin increases RhoB in normal human skin cells
[0203] Whether the combination of EGCG and curcumin transactivates RhoB by inhibiting Akt/mTOR and activating PKR/elF2a pathway in Normal Human Skin Fibroblasts (NHSFs) just as LBAIs and ceramide do in PTEN-null or Akt highly active cancer cells was investigated. The results demonstrated that the combination of EGCG and Curcumin did inhibit phosphorylation of Akt/mTOR downstream substrates S6 at S235/236 sites and 4E-BP1 at S65 site, as well as activate phosphorylation of PKR downstream substrate elF2a at S51 site in NHSFs that were treated with the composition for 48 and 72 hours. As expected, the composition did induce RhoB (Fig. 16), accompanied by p21 increase.
Example 13 - The diet with EGCG and Curcumin attenuates mice aging process
[0204] The male C57/BL6 mice (at age of 12 months) were fed with standard AIN- 93 diet (the control group, shown on the left portion of FIG. 17), or EGCG (66 PPM) and curcumin (66 PPM) containing AIN-93 diet (the treatment group, shown on the right portion of FIG. 17) for 10 months. Results indicate that, compared to those who were fed using the control diet, these mice fed using EGCG and curcumin obviously aged slower. As mice age, C57/BL6 mice underwent alopecia, coarser fur, and fur greying under normal aging conditions. During the course of this experiment, it was observed that mice undergoing EGCG and curcumin treatment did not experience any of these characteristic phenotypic properties. The fur on treated C57/BL6 mice were remarkably similar to the condition of the fur prior to the commencement of treatment, 10 months prior.
Example 14 - The diet with EGCG and Curcumin prevents lung carcinogenesis
[0205] In order to test whether induction of RhoB by the combination of EGCG and curcumin prevents cancer, tobacco-specific carcinogen NNK and nicotine was used to induce lung carcinogenesis in A/J mice. At the age of 12 months, the protein level of RhoB in mouse lung has greatly decreased compared to that at the age of 2 months (Fig. 1 ). Fifteen mice were fed with standard AIN-93 diet as control group (G1 ), and thirty mice were fed with EGCG and Curcumin containing AIN-93 diet at high dose (G2, n=15) or low dose (G3, n=15) as treatment groups.
[0206] 15 weeks later, all the mice were killed to check the lung tumor multiplicity and size. The experimental results demonstrated that the incidence and size of tumor were significantly reduced by treatments in a dose-dependent manner, which were indicated by tumor multiplicity and burden in mouse left-single lung (Fig. 18).
Example 15 - A dietary supplement ROVIDIUM - a composition that includes ceramide, ECCG and Curcumin
[0207] To harness RhoB restoration for preventing cancer and fighting aging, a dietary supplement ROVIDIUM is formulated using the composition of ceramide, EGCG and curcumin as the core ingredients, as shown in Table 2.
Table 2 Sample ingredients of ROVIDIUM
Figure imgf000043_0001
[0208] As shown in Table 2, at least a part of the core ingredients of the ROVIDIUM may be obtained as commercial products. The composition of ROVIDIUM is provided in a unit dosage form (e.g., 400-500 mg). The Vitamin E (D-Alpha Tocopheryl Acetate) may be added to protect the cells against damages caused by free radicals and prevent the oxidation of low density lipoprotein cholesterols. The sunflower lecithin powder may be added as an emulsifier. The AstaReal®P2AF may be astaxanthin 2% powder and may be added to the composition as an antioxidant. The Ceram ide-PCD®, the green tea extract (95% EGCG), and the CAVACURMIN® contained the key ingredients ceram ide, EGCG, and curcumin, respectively, which may be added to prevent or attenuate cancer, or slow aging process. The organic rice hull powder may be added as a flow agent to facilitate the ingredients in the composition to move smoothly and prevent the ingredients from sticking to equipment used in the manufacturing process, thus improving the efficiency of manufacturing the composition. The organic bamboo extract may include organic silica and may be added in the composition as a silica supplement to maintain the health of the skin, ligaments, tendons, bones, teeth, nails, and other tissues or functions thereof.
[0209] It should be noted that the composition of the ROVIDIUM is merely provided for the purposes of illustration, and not intended to limit the scope of the present disclosure. For persons having ordinary skill in the art, multiple variations and modifications may be made under the teachings of the present disclosure.
Flowever, those variations and modifications do not depart from the scope of the present disclosure. For instance, each dosage of the composition of ROVIDIUM may include 40 mg to 60 mg EGCG, 90 mg to 110 mg curcumin, and 10 mg to 30 mg ceram ide.
Example 16 - Interactions between RhoB and EGFR, Ras, PI3K/Akt/mTOR,
MYC, and HD AC
[0210] As shown in FIG. 19, RhoB’s function is differentially regulated by factors including EGFR, K-Ras, and PI3K/AKT/mTOR. EGFR is a receptor tyrosine kinase that autophosphorylates upon binding ligands such as EGF and TGF-a. The activated EGFR can then facilitate activation of RAS-GDP into RAS-GTP via GEFs. RAS-GTP can lead to increased activity of the PI3K/AKT/mTOR pathway. AKT then co-localizes near the nuclear membrane along with RhoB, where AKT becomes phosphorylated and downregulates RhoB. Finally, RhoB can then inhibit or (in angiogenic states) enhance AKT activity, inhibit the EGFR receptor, antagonize Ras/PI3K/mTOR signaling, facilitating MYC turnover, and inhibit overall cell growth, proliferation, and survival.
[0211] Aside from the PI3K pathway, RAS-GTP can also affect regulation of RhoB by means of cross-talk between RAF and AKT. RAS-GTP can activate RAF, which may either upregulate or downregulate function of AKT, which is known to inhibit RhoB. Conversely, AKT also inhibits the function of RAF.
[0212] Furthermore, transcription of RhoB is tightly controlled by FIATs and
FIDAC1/6. Acetylation of chromatin by FIATs causes relaxation of the chromatin structure, allowing for transcriptional activation of RhoB. On the other hand, deacetylation of chromatin by FIDAC1/6 creates a condensed structure that represses transcription of RhoB.
Example 17 - Translation of RhoB is epigenetically downregulated by miRNA-
19/21/223
[0213] As shown in FIG. 20, RhoB translation is downregulated by miRNA-19a, miRNA -21 , and miRNA -223 (TS1 and TS2) (as shown in part (A) and part (B) of FIG. 20). Each miRNA inhibits translation of RhoB mRNA by binding specific target sites in the mRNA 3’-UTR (as shown in part (B) and part (C) of FIG. 20). Each miRNA binds to known codon sequences in the 3’-UTR (as shown in part (C) and of FIG. 20). Both miR-19a and miR-21 have only one binding site, whereas miR-223 has two separate target sites, TS1 and TS2 (as shown in part (D) and of FIG. 20). The target sites for miR-223 (TS1 ), miR-19a, miR-223 (TS2), and miR-21
respectively begin 625, 847, 1 ,261 , and 1 ,310 nucleotides downstream of the coding region.
Example 18 - The potential roles of tumor suppressor RhoB in cancer and aging
[0214] As shown in FIG. 21 , RhoB gradually decreases during aging in important tissues including lung and muscle by epigenetic mechanism. During carcinogenesis, RhoB is down regulated by multiple mechanisms, including oncogenic signaling, epigenetic modification, microRNAs, pseudogenes. Restoration of RhoB suppresses oncogenic signaling and antagonizes aging promoters.“CR” in FIG. 21 represents Caloric Restriction.
[0215] Flaving thus described the basic concepts, it may be rather apparent to those skilled in the art after reading this detailed disclosure that the foregoing detailed disclosure is intended to be presented by way of example only and is not limiting. Various alterations, improvements, and modifications may occur and are intended to those skilled in the art, though not expressly stated herein. These alterations, improvements, and modifications are intended to be suggested by this disclosure, and are within the spirit and scope of the exemplary embodiments of this disclosure.
[0216] Moreover, certain terminology has been used to describe embodiments of the present disclosure. For example, the terms“one embodiment,”“an
embodiment,” and“some embodiments” mean that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Therefore, it is emphasized and should be appreciated that two or more references to“an embodiment” or“one
embodiment” or“an alternative embodiment” in various portions of this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined as suitable in one or more embodiments of the present disclosure.
[0217] Similarly, it should be appreciated that in the foregoing description of embodiments of the present disclosure, various features are sometimes grouped together in a single embodiment, figure, or description thereof to streamline the disclosure aiding in the understanding of one or more of the various embodiments. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed subject matter requires more features than are expressly recited in each claim. Rather, claim subject matter lie in less than all features of a single foregoing disclosed embodiment.

Claims

WE CLAM:
1. A nutritional phytonutrient composition, comprising:
ceramide;
epigallocatechin gallate (EGCG); and
curcumin.
2. The composition of claim 1 , for preventing or attenuating cancer in a mammal.
3. The composition of claim 1 , for slowing aging process in a mammal.
4. The composition of claim 1 , for preventing or attenuating cancer in a mammal or for slowing aging process in the mammal, by maintaining, increasing or restoring RhoB level in the mammal.
5. The composition of claim 1 , wherein the ceramide is 2% to 10% of the composition by weight.
6. The composition of claim 5, wherein the ceramide is 4% to 6% of the composition by weight.
7. The composition of claim 6, wherein the ceramide is about 4.9% of the composition by weight.
8. The composition of claim 1 , wherein the ceramide is extracted from rice.
9. The composition of claim 1 , wherein the EGCG in the composition is in the form of green tea extract.
10. The composition of claim 9, wherein the great tea extract contains about 95% EGCG by weight.
11. The composition of claim 1 , wherein the EGCG is 5% to 20% of the composition by weight.
12. The composition of claim 11 , wherein the EGCG is 10% to 15% of the composition by weight.
13. The composition of claim 12, wherein the EGCG is about 12.2 of the composition by weight.
14. The composition of claim 1 , wherein the curcumin is 15% to 35% of the composition by weight.
15. The composition of claim 14, wherein the curcumin is 22% to 27% of the composition by weight.
16. The composition of claim 15, wherein the curcumin is about 24.4% of the composition by weight.
17. The composition of claim 14, wherein the curcumin is cavacurmin.
18. The composition of claim 1 , further comprising astaxanthin.
19. The composition of claim 18, wherein the astaxanthin is 1 % to 3.5% of the composition by weight.
20. The composition of claim 19, wherein the astaxanthin is 2% to 3% of the composition by weight.
21. The composition of claim 20, wherein the astaxanthin is about 2.4% of the composition by weight.
22. The composition of claim 1 , further comprising a cell protection agent.
23. The composition of claim 22, wherein the cell protection agent includes Vitamin E.
24. The composition of claim 23, wherein the Vitamin E is 1 % to 3% of the composition by weight.
25. The composition of claim 24, wherein the Vitamin E is about 2.2% of the composition by weight.
26. The composition of claim 1 , further comprising an emulsifier.
27. The composition of claim 26, wherein the emulsifier includes lecithin.
28. The composition of claim 27, wherein the lecithin is extracted from sunflower or soybean.
29. The composition of claim 27, wherein the lecithin is 15% to 35% of the composition by weight.
30. The composition of claim 29, wherein the lecithin is about 24% to 25% of the composition by weight.
31. The composition of claim 1 , further comprising a flow agent.
32. The composition of claim 31 , wherein the flow agent includes rice hull powder.
33. The composition of claim 32, wherein the rice hull powder is 15% to 35% of the composition by weight.
34. The composition of claim 33, wherein the rice hull powder is about 24% to 25% of the composition by weight.
35. The composition of claim 1 , further comprising a silica supplement.
36. The composition of claim 35, wherein the silica supplement includes bamboo extract.
37. The composition of claim 36, wherein the bamboo extract is 2% to 10% of the composition by weight.
38. The composition of claim 37, wherein the bamboo extract is about 4.9% of the composition by weight.
39. The composition of claim 1 , wherein:
the ceram ide is 4% to 6% of the composition by weight;
the EGCG is 10% to 15% of the composition by weight; and
the curcumin is 22% to 27% of the composition by weight.
40. The composition of claim 39, wherein:
the ceramide is about 4.9% of the composition by weight;
the EGCG is about 12.2% of the composition by weight; and
the curcumin is about 24.4% of the composition by weight.
41. The composition of claim 1 , wherein:
the ceramide is 4% to 6% of the composition by weight;
the EGCG is 10% to 15% of the composition by weight;
the curcumin is 22% to 27% of the composition by weight;
the composition further comprises astaxanthin, which is 2% to 3% of the composition by weight;
the composition further comprises Vitamin E, which is 1 % to 3% of the composition by weight;
the composition further comprises lecithin, which is 15% to 35% of the composition by weight;
the composition further comprises rice hull powder, which is 15% to 35% of the composition by weight; and
the composition further comprises bamboo extract, which is 2% to 10% of the composition by weight.
42. The composition of claim 41 , wherein:
the ceramide is about 4.9% of the composition by weight;
the EGCG is about 12.2 of the composition by weight; and
the curcumin is about 24.4% of the composition by weight.
the astaxanthin is about 2.4% of the composition by weight;
the Vitamin E is about 2.2% of the composition by weight;
the lecithin is about 24% to 25% of the composition by weight;
the rice hull powder is about 24% to 25% of the composition by weight; and the bamboo extract is about 4.9% of the composition by weight.
43. The composition of claim 1 , wherein the composition is provided in a unit dosage form.
44. The composition of claim 43, wherein each dosage includes 10 mg to 30 mg ceramide.
45. The composition of claim 43, wherein each dosage includes about 20 mg ceramide.
46. The composition of claim 43, wherein each dosage includes 40 mg to 60 mg EGCG.
47. The composition of claim 43, wherein each dosage includes about 50 mg EGCG.
48. The composition of claim 43, wherein each dosage includes 90 mg to 110 mg curcumin.
49. The composition of claim 43, wherein each dosage includes about 100 mg curcumin.
50. The composition of claim 43, wherein each dosage includes 5 mg to 15 mg astaxanthin.
51. The composition of claim 43, wherein each dosage includes about 10 mg astaxanthin.
52. The composition of claim 43, wherein each dosage includes 15 IU to 25 IU Vitamin E.
53. The composition of claim 43, wherein each dosage includes about 10 IU Vitamin E in the form of D-alpha tocopheryl acetate.
54. The composition of claim 43, wherein each dosage includes 80 mg to 120 mg lecithin.
55. The composition of claim 43, wherein each dosage includes about 100 mg lecithin.
56. The composition of claim 43, wherein each dosage includes 80 mg to 120 mg rice hull powder.
57. The composition of claim 43, wherein each dosage includes about 100 mg rice hull powder.
58. The composition of claim 43, wherein each dosage includes 10 mg to 30 mg bamboo extract.
59. The composition of claim 43, wherein each dosage includes about 20 mg bamboo extract.
60. A nutritional phytonutrient composition for preventing or attenuating cancer, or for slowing aging process in a mammal, by maintaining, increasing or restoring RhoB level in the mammal, the composition comprising: ceramide.
61. A nutritional phytonutrient composition for preventing or attenuating cancer, or for slowing aging process in a mammal, by maintaining, increasing or restoring RhoB level in the mammal, the composition comprising: epigallocatechin gallate (EGCG) and curcumin.
62. A method of preventing or attenuating cancer, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
63. A method of preventing or attenuating cancer by maintaining, increasing or restoring RhoB level, comprising administering an effective amount of the
composition of any of claims 1 -61 to the mammal.
64. A method of slowing aging process, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
65. A method of slowing aging process by maintaining, increasing or restoring RhoB level, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
66. The method of any of claims 62-65, wherein the mammal is a human.
67. The method of any of claims 62-65, wherein the mammal is a non-human.
68. The method of claims 67, wherein the mammal is a companion animal.
69. The method of claims 68, wherein the mammal is a companion animal selected from the group consisting of: cats, dogs, rabbits, ferrets, pigs; gerbils, hamsters, chinchillas, rats, mice, and guinea pigs.
70. The method of claims 68, wherein the mammal is a cat or a dog.
71. The method of any of claims 62-65, wherein the composition is administered orally.
72. A method of preventing or attenuating carcinogenesis, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
73. A method of preventing or attenuating lung carcinogenesis, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
74. A method of enhancing effects of immunotherapy on cancer, comprising administering an effective amount of the composition of any of claims 1 -61 to the mammal.
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