WO2015019193A2 - Dérivés acylés de phloridzine et d'isoquercétrine en tant qu'agents thérapeutiques anticancéreux et leurs procédés d'utilisation - Google Patents

Dérivés acylés de phloridzine et d'isoquercétrine en tant qu'agents thérapeutiques anticancéreux et leurs procédés d'utilisation Download PDF

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WO2015019193A2
WO2015019193A2 PCT/IB2014/002520 IB2014002520W WO2015019193A2 WO 2015019193 A2 WO2015019193 A2 WO 2015019193A2 IB 2014002520 W IB2014002520 W IB 2014002520W WO 2015019193 A2 WO2015019193 A2 WO 2015019193A2
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acid ester
phloridzin
isoquercetrin
cells
group
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PCT/IB2014/002520
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WO2015019193A3 (fr
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H.P. Vasantha RUPASINGHE
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Dalhousie University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
    • C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
    • C07H17/06—Benzopyran radicals
    • C07H17/065—Benzo[b]pyrans
    • C07H17/075—Benzo[b]pyran-2-ones

Definitions

  • the invention relates generally to the discovery and synthesis of novel derivatives of phloridzin and isoquercetrin that display strong anticarcinogenic effects in vitro.
  • compositions, methods, and kits for decreasing proliferation and viability of cancer cells and treating cancer in a subject include use of these novel derivatives are also disclosed.
  • TNBC triple negative breast cancer
  • ER estrogen receptor
  • PGR progesterone receptor
  • HER2 human epidermal growth factor receptor 2
  • Flavonoids are polyphenolic plant secondary metabolites which have been shown to have strong antioxidant, antiproliferative and other biological activities beneficial to human health by both epidemiological and in vitro studies.
  • oral administration of cranberry concentrate reduced the growth of N-butyl-N-(4-hydroxybutyl)-nitrosamine- (OH-BBN) induced urinary bladder tumor growth in female Fisher-344 rats.
  • NSCLC subcutaneously transplanted human non- small-cell lung cancer
  • flavonoids have shown antiangiogenic and antimetastatic properties in in vivo models as well.
  • KS-IMM kaposi's sarcoma cell
  • Isoquercetrin or quercetin 3-O-glucoside has been studied for its anticancerous and toxicological properties in various transformed cell lines making it a candidate for cancer therapeutics. Specifically, dietary supplementation of quercetin significantly suppressed growth of orthotopically transplanted MIA PaCa-2, human pancreatic cells in nude mice. However, poor bioavailability limits its biological effects in vivo due to low membrane permeability and hence, its application as a therapeutic agent is limited.
  • Phloridzin like quercetin 3-O-glucoside, is a naturally occurring flavonoid, however, it does not possess strong anti-cancer properties.
  • acylation of flavonoid glycosides such as, naringin (naringenin-7-O-rhamnoglucoside) and isoquercetrin has been done using carboxylic acids (palmitic, cinnamic and phenylpropionic (PPA) acids and hydroxylated derivatives of PPA) as acyl donors. This resulted in major mono-acylated products but also with minor mono- acylated products.
  • carboxylic acids palmitic, cinnamic and phenylpropionic (PPA) acids and hydroxylated derivatives of PPA
  • compositions, methods and kits for treating cancers such as leukemias, breast cancer and liver cancer.
  • an acylated flavonoid derived from a flavonoid selected from the group consisting of phloridzin, luteolin, isoquercetrin, and quercetin; and an acylating agent selected from the group consisting of long chain saturated or polyunsaturated fatty acids, wherein the fatty acids are selected from the group consisting of stearic, oleic, linoleic, alpha-linolenic, docosahexaenoic, eicosapentaenoic, palmitic, cinnamic, phenylpropionic, butyric, octanoic, and dodecanoic.
  • composition comprising a
  • an acylated flavonoid derived from a flavonoid selected from the group consisting of phloridzin, luteolin, isoquercetrin, and quercetin; and an acylating agent selected from the group consisting of long chain saturated or polyunsaturated fatty acids, wherein the fatty acids are selected from the group consisting of stearic, oleic, linoleic, alpha-linolenic, docosahexaenoic, eicosapentaenoic, palmitic, cinnamic, phenylpropionic, butyric, octanoic, and dodecanoic.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and an acylated flavonoid derived from a flavonoid selected from the group consisting of phloridzin, luteolin, isoquercetrin, and quercetin; and an acylating agent selected from the group consisting of long chain saturated or polyunsaturated fatty acids, wherein the fatty acids are selected from the group consisting of stearic, oleic, linoleic, alpha-linolenic, docosahexaenoic, eicosapentaenoic, palmitic, cinnamic, phenylpropionic, butyric, octanoic, and dodecanoic.
  • a method of treating cancer in a patient in need thereof comprising administering a therapeutically effective amount of a a pharmaceutical composition comprising a pharmaceutically acceptable carrier; and an acylated flavonoid derived from a flavonoid selected from the group consisting of phloridzin, luteolin, isoquercetrin, and quercetin; and an acylating agent selected from the group consisting of long chain saturated or poly-unsaturated fatty acids, wherein the fatty acids are selected from the group consisting of stearic, oleic, linoleic, alpha-linolenic, docosahexaenoic, eicosapentaenoic, palmitic, cinnamic, phenylpropionic, butyric, octanoic, and dodecanoic.
  • a kit for treating cancer in a subject the
  • FIG. 1 is a schematic illustration of acylation of phloridzin and isoquercetrin using enzymes.
  • FIG. 2a-c is a table illustrating structures and percent yield of acylated fatty acid derivatives of phloridzin and isoquercetrin.
  • FIG. 3 is a schematic illustration of sonochemical acylation of phloridzin and isoquercetrin using lipases.
  • FIG. 4a-h is a table of structures and percent yields of acylated fatty acid derivatives of phloridzin and isoquercetrin, as well as reaction times.
  • FIG. 5a-e is a series of bar graphs showing the metabolic inhibition of MDA-MB- 231 cells by PZ, DHA, and PZ-DHA at 3 hours (Fig. 5 A), 6 hours (Fig. 5B), 12 hours (Fig. 5C), 18 hours (Fig. 5D) and 24 hours of treatment.
  • FIG. 6 is a graph showing that low PZ-DHA concentrations selectively inhibits MDA-MB-231 triple negative breast cancer cells.
  • FIG. 7A is an illustration of the caliper measurements of tumors on various days and tumor volume.
  • FIG. 7B is an illustration of the weight of excised tumors after day 15.
  • FIG. 8 is a series of bar graphs showing the inhibition of phosphatase enzyme activity of MDA-MB-231 cells by PZ, DHA, and PZ-DHA at 3 hours (Fig. 8A), 6 hours (Fig.
  • FIG. 9 is an illustration of the mean luminescence measured in three independent experiments of MDA-MB-231 cells treated with various test compounds.
  • FIG. 10 is a table of EC50 values of three human cancer cell lines in relation to dose and exposure time of six fatty acid esters of phloridzin in comparison to parent flavonoid and fatty acids and two prescribed drugs.
  • FIG. 11 A-B is a series of graphs showing survival of HepG2 cells
  • EC50 values were calculated using Graphpad Prism 6 software.
  • FIG. 12A-B is a series of graphs showing survival of MDA-MB-231 cells (breast cancer) after incubation with six fatty acid esters of phloridzin in comparison to parent flavonoid and fatty acids and the prescribed drug doxorubicin hydrochloride at various doses and exposure times.
  • EC50 values were calculated using Graphpad Prism 6 software.
  • FIG. 13A-B is a series of graphs showing survival of THP-1 (leukemia) cells after incubation with six fatty acid esters of phloridzin in comparison to parent flavonoid and fatty acids and the prescribed drug doxorubicin hydrochloride at various doses and exposure times.
  • EC50 values were calculated using Graphpad Prism 6 software.
  • FIG. 14A-B is a pair of graphs showing antiproliferative effects of long chain fatty acid esters on HepG2 cells. The figure describes the percentage of viable HepG2 cells after treatment with long chain fatty acid esters of Q3G and chemotherapy drugs for 6 (A) and 24 hours (B).
  • FIG. 15A-B is a pair of graphs showing the effect of long chain fatty acid esters of Q3G on cytotoxicity in HepG2 cells.
  • the figure describes the percentage of LDH release from HepG2 cells after treatment with long chain fatty acid esters of Q3G and chemotherapy drugs (Sorafenib and Cisplatin) for 6 and 24 hours.
  • FIG. 16 is a graph showing the effect of long chain fatty acid esters of Q3G on viability of normal rat hepatocytes.
  • the figure describes the percentage of viable rat hepatocytes cells after treatment with long chain fatty acid esters of Q3G.
  • Cells (1 x 10 4 cells per well; 96-well plate) were treated with 100 ⁇ off long chain fatty acid esters of Q3G, precursor compounds (quercetin and Q3G) and chemotherapy drug (Sorafenib) for 24 hours. After treatment viable cell percentage was determined by MTS assay as described in materials and methods. Results are expressed relative to control (24-h incubation without test compounds).
  • FIG. 17 is a table listing the effects of OA-Q3G treatment on gene level expression in HepG2 cells.
  • compositions, methods and kits include acylated flavonoids, such as phloridzin and isoquercetrin derivatives, in amounts effective for decreasing proliferation and viability of cancer cells in a subject.
  • acylated flavonoids such as phloridzin and isoquercetrin derivatives
  • the compositions are administered to a subject (e.g., human) having cancer in an amount effective for treating cancer in the subject.
  • protein and “polypeptide” are used synonymously to mean any peptide-linked chain of amino acids, regardless of length or post-translational modification, e.g., glycosylation or phosphorylation.
  • gene is meant a nucleic acid molecule that codes for a particular protein, or in certain cases, a functional or structural RNA molecule.
  • nucleic acid or a “nucleic acid molecule” means a chain of two or more nucleotides such as RNA (ribonucleic acid) and DNA (deoxyribonucleic acid).
  • patient means a vertebrate animal, typically mammalian (e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.), subject to be treated and/or to obtain a biological sample from.
  • mammalian e.g., human, rodent, non-human primates, canine, bovine, ovine, equine, feline, etc.
  • modulation refer to the ability of an agent to either inhibit or enhance or maintain activity and/or function of a molecule.
  • an inhibitor of DNA topoisomerase II would down-regulate, decrease, reduce, suppress, or inactivate at least partially the activity and/or function of DNA topoisomerase II.
  • inhibiting means slowing or stopping the growth of. Up-regulation refers to a relative increase in function and/or activity.
  • isoquercetrin and “quercetin 3-O-glucoside,” referring to the flavonoid compound, are used interchangeably herein, and encompass naturally occurring as well as synthesized compounds.
  • isolated or biologically pure refer to material, which is substantially or essentially free from components which normally accompany it as found in its native state.
  • agent is meant a polypeptide, peptide, nucleic acid molecule, small molecule, or mimetic.
  • analog is meant an agent having structural or functional homology to a reference agent.
  • the term “derivative” means a compound derived or obtained from another and containing essential elements of the parent substance. [0046] By “modifies” is meant alters. An agent that modifies a cell, substrate, or cellular environment produces a biochemical alteration in a component (e.g., polypeptide, nucleotide, or molecular component) of the cell, substrate, or cellular environment.
  • a component e.g., polypeptide, nucleotide, or molecular component
  • diagnosis means identifying the presence or nature of a pathologic condition (e.g., cancer).
  • sample is used herein in its broadest sense.
  • a sample including polynucleotides, polypeptides, peptides, antibodies and the like may include a bodily fluid, a soluble fraction of a cell preparation or media in which cells were grown, a biopsy, genomic
  • DNA, RNA or cDNA a cell, a tissue, skin, hair and the like.
  • samples include saliva, serum, blood, biopsies, urine and plasma.
  • treatment is defined as the application or
  • Treatment can include, for example, reducing the number of cancer cells in a subject, eliminating cancer cells in a subject, reducing cancer cell viability and proliferation, eliminating a cancerous tumor or reducing the size of a cancerous tumor, etc.
  • safety and effective amount refers to the quantity of a component, which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the manner of this invention.
  • therapeutically effective amount is meant an amount of a composition of the present invention effective to yield the desired therapeutic response. For example, an amount effective to decrease proliferation and viability of cancer cells (e.g., to induce apoptosis of cancer cells) in a subject having cancer.
  • an amount effective to delay the growth of or to cause a cancer in a subject to shrink or prevent metastasis of the cancer in the subject.
  • a cancer e.g., breast cancer, liver cancer, leukemia
  • the specific safe and effective amount or therapeutically effective amount will vary with such factors as the particular condition being treated, the physical condition of the patient, the type of mammal or animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives.
  • Flavonoids are polyphenolic compounds found in nature which generally consist of two aromatic rings, connected through a three-carbon bridge that may be part of a six- membered heterocyclic pyran ring.
  • ROS reactive oxygen species
  • Flavonoids for use in the present invention can include natural and synthetic compounds.
  • flavonoids include anthoxanthins, flavanones, flavanonols, and flavans (catechins).
  • the anthoxanthins include flavones, such as luteolin, apigenin, and tangeritin; and flavonols (3 -hydroxy flavones), such as quercetin, kaempferol, myricetin, fisetin, galangin, isorhamnetin, pachypodol, rhamnazin, pyranoflavonols, and furanflavonols.
  • Flavanones include hesperetin, naringenin, eirodictyol, and homoeriodictyol.
  • Flavanonols include taxifolin (dihydroquercetin), and dihydrokaempferol.
  • Flavans can include flavan-4-ol; flavan-3,4-diols; and flavan-3-ols, such as catechin, gallocatechin, catechin-3-gallate, gallocatechin-3-gallate, epicatechin, theaflavin, thearubigin,
  • flavonoids useful in the present invention include phloridzin, luteolin, isoquercetrin, and quercetin.
  • Phloridzin phloretin 2'-0- glucoside
  • isoquercetrin quercetin 3-O-glucoside
  • acylation of flavonoids with a primary aliphatic hydroxyl group on the sugar moiety can occur to form acylated flavonoid derivatives.
  • the acylating agents used in forming the acylated flavonoid derivatives include short, medium, and long chain fatty acids, including long chain saturated or poly-unsaturated fatty acids.
  • Non-limiting examples of fatty acids useful in the acylation of the flavonoids of the invention include, stearic, oleic, linoleic, alpha-linolenic, docosahexaenoic, eicosapentaenoic, palmitic, cinnamic, phenylpropionic, butyric, octanoic, and dodecanoic.
  • the acylated flavonoid derivative can be an ester.
  • the acylated derivatives include, but are not limited to: Stearic acid ester of phloridzin, Oleic acid ester of phloridzin, Linoleic acid ester of phloridzin, alpha- linolenic acid ester of phloridzin, Docosahexaenoic acid ester of phloridzin, Eicosapentaenoic acid ester of phloridzin, Stearic acid ester of isoquercetrin, Oleic acid ester of isoquercetrin, Linoleic acid ester of isoquercetrin, alpha-linolenic acid ester of isoquercetrin,
  • any suitable acylated flavonoid derivative can be created using known flavonoids and acylating agents using known acylating mechanisms.
  • acylated phloridzin and isoquercetrin derivatives can be synthesized by any suitable method, e.g., according to the methods described in Ziaullah et al., Biorganic and Medicinal
  • any suitable acylated flavonoid derivatives that exhibit
  • the acylated flavonoid derivative can be used for decreasing cancer cell viability and proliferation and treating cancer, for example, breast cancer, liver cancer, and acute monocytic leukemia cells, in a subject.
  • the acylated flavonoid derivative can induce apoptosis of the cancer cells.
  • the acylated flavonoid derivatives of the present invention can activate caspase-3- family member activation and induce s-phase cell cycle arrest in cancer cells.
  • the derivatives can decrease proliferation and viability specifically of cancer cells compared to noncancerous cells.
  • a subject (patie nt) having cancer may have a tumor.
  • a tumor may be a solid or hematological tumor, benign or malignant (metastic or nonmeta static), such as, for example, breast, liver, prostate, cervical, ovarian, colon, brain, pancreatic, bladder esophagus, gut, head and neck, kidney, melanoma, stomach, testes, thyroid, uterine and lung cancers, leukemias and lymphomas, such as acute myelogenous leukemia, acute or chronic lymphocytic leukemia, Hodgkin's and non-Hodgkin lymphoma, and myelomas.
  • leukemias and lymphomas such as acute myelogenous leukemia, acute or chronic lymphocytic leukemia, Hodgkin's and non-Hodgkin lymphoma, and myelomas.
  • Persons of skill in the art will be able to determine by routine experimentation the types of tumors that are
  • compositions comprising the acylated flavonoid derivatives, such as the acylated phloridzin and acylated isoquercetrin derivatives, described herein can be used for decreasing proliferation and cancer cell viability; inducing apoptosis of the cancer cells; and treating cancer in a subject.
  • acylated flavonoid derivatives such as the acylated phloridzin and acylated isoquercetrin derivatives, described herein can be used for decreasing proliferation and cancer cell viability; inducing apoptosis of the cancer cells; and treating cancer in a subject.
  • the disclosed pharmaceutical composition can comprise a therapeutically effective amount of an acylated flavonoid derivative, such as an acylated phloridzin or isoquercetrin derivative, comprising a long chain saturated or poly-unsaturated fatty acid (e.g., one or more of stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, docosahexaenoic acid, and eicosapentaenoic acid), and optionally a pharmaceutically acceptable carrier.
  • an acylated flavonoid derivative such as an acylated phloridzin or isoquercetrin derivative
  • a long chain saturated or poly-unsaturated fatty acid e.g., one or more of stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, docosahexaenoic acid, and eicosapent
  • the pharmaceutical composition can further comprise at least one additional anticancer drug (e.g., a second anti-cancer drug).
  • additional anti-cancer drugs such as a chemotherapy agent or an antiangiogenic agent, include sorafenib, doxorubicin, methotrexate, vinblastine, vincristine, cladribine, fluorouracil, cytarabine, anthracyclines, cisplatin, cyclophosphamide, fludarabine, gemcitabine, aromatase inhibitors, irinotecan, navelbine, oxaliplatin, taxol, docetaxel, bevacizumab, pegaptanib, and
  • a combination therapy including a standard chemotherapy drug and an acylated flavonoid, such as phloridzin or isoquercetrin, derivative may be particularly therapeutic.
  • compositions described herein can be administered to a mammal (e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.) in an effective amount, that is, an amount capable of producing a desirable result in a treated subject (e.g., inhibiting growth of cancer cells, inducing apoptosis of cancer cells in the subject).
  • a mammal e.g., rodent, human, non-human primates, canine, bovine, ovine, equine, feline, etc.
  • Toxicity and therapeutic efficacy of the compositions utilized in methods of the invention can be determined by standard pharmaceutical procedures.
  • dosage for any one animal depends on many factors, including the subject's size, body surface area, body weight, age, the particular composition to be administered, time and route of administration, general health, the clinical symptoms of the cancer and other drugs being administered concurrently.
  • a pharmaceutical composition as described herein is typically administered at a dosage that can at least one of induce apoptosis of cancer cells, or reduces or eliminates cancer cell growth or proliferation.
  • the amount of acylated phloridzin derivatives used to demonstrate antiproliferative activity was from about 0.1 ⁇ to about 200 ⁇ , for example from about 10 ⁇ to about 150 ⁇ , and from about 50 ⁇ to about 100 ⁇ ; and the amount of acylated isoquercetrin derivatives used to demonstrate antiprolierative and cytotoxic activity was from about 0.1 ⁇ to about 200 ⁇ , for example from about 10 ⁇ to about 150 ⁇ , and from about 50 ⁇ to about 100 ⁇ .
  • Described herein are methods of decreasing proliferation and viability of cancer cells and treating cancer (e.g., liver cancer, breast cancer, leukemia, etc.) and/or disorders or symptoms thereof in a subject.
  • the methods include administering to the subject (e.g., a mammal such as a human) a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an acylated flavonoid derivative, such as an acylated phloridzin or isoquercetrin derivative, to decrease proliferation and viability of cancer cells in the subject.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a therapeutically effective amount of an acylated flavonoid derivative, such as an acylated phloridzin or isoquercetrin derivative
  • an amount of acylated phloridzin or isoquercetrin derivative sufficient to induce death (e.g., apoptosis) of cancer cells in the subject can be typically administered.
  • the acylated flavonoid derivative such as acylated phloridzin or isoquercetrin derivative, may activate caspase-3 family member activation and induce s-phase cell cycle arrest in the cancer cells.
  • the acylated phloridzin or isoquercetrin derivative decreases proliferation and viability specifically of cancer cells compared to noncancerous cells (i.e., the acylated phloridzin or isoquercetrin derivatives described herein display specificity for cancer cells and are not cytotoxic to noncancerous cells).
  • the pharmaceutical composition can be administered to a subject who is resistant to commonly used standard chemotherapy or other anti-cancer drugs.
  • a subject who is resistant to Sorafenib may respond to treatment with the disclosed acylated flavonoid derivatives, such as an acylated phloridzin or isoquercetrin derivative described herein.
  • the composition can further comprise or be co-administered with a therapeutically effective amount of at least one additional anti-cancer drugs or standard chemotherapy.
  • the therapeutic methods of the invention in general include administration of a therapeutically effective amount of the compositions described herein to a subject in need thereof, including a mammal, particularly a human.
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for cancer or a disorder or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, marker (as defined herein), family history, and the like).
  • the administration of the disclosed pharmaceutical composition comprising an acylated phloridzin or isoquercetrin derivative for the treatment of cancer may be by any suitable means that results in a concentration of the therapeutic that, (e.g., in some instances when combined with other components), is effective in ameliorating, reducing, or stabilizing a cancer.
  • the acylated phloridzin or isoquercetrin derivative may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% ,for example from about 5 -90%, from about 10-85%, and as a further example from about 15-80% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for local or systemic administration (e.g., parenteral, subcutaneously, intravenously, intramuscularly, or intraperitoneally).
  • parenteral subcutaneously, intravenously, intramuscularly, or intraperitoneally
  • the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically-acceptable buffer such as physiological saline.
  • Exemplary routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient.
  • the pharmaceutical compositions may be formulated according to
  • compositions as described herein may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • injection, infusion or implantation subcutaneous, intravenous, intramuscular, intraperitoneal, or the like
  • suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • the acylated flavonoid may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • the pharmaceutical composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing agents.
  • the pharmaceutical compositions described herein may be in a form suitable for sterile injection.
  • a parenterally acceptable liquid vehicle that may be employed are water, water adjusted to a suitable pH by addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer, 1,3-butanediol, Ringer's solution, and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
  • Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam- nine) and, poly(lactic acid).
  • Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.
  • Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g.,
  • Formulations for oral use include tablets containing the active ingredient(s) (e.g., acylated phloridzin or isoquercetrin derivative) in a mixture with non-toxic pharmaceutically acceptable excipients.
  • active ingredient(s) e.g., acylated phloridzin or isoquercetrin derivative
  • Such formulations are known to the skilled artisan.
  • Excipients may include but are not limited to, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate, carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants
  • the tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period.
  • the coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating).
  • the coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose,
  • hydroxypropylcellulose carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose).
  • a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.
  • Nasal and other mucosal spray formulations can include purified aqueous solutions of the active compounds with preservative agents and isotonic agents. Such formulations are preferably adjusted to a pH and isotonic state compatible with the nasal or other mucous membranes. Alternatively, they can be in the form of finely divided solid powders suspended in a gas carrier. Such formulations may be delivered by any suitable means or method, e.g., by nebulizer, atomizer, metered dose inhaler, or the like.
  • a pharmaceutical composition of the present invention can have immediate release, sustained release, delayed-onset release or any other release profile known to one skilled in the art.
  • Pharmaceutical compositions as described herein may be formulated to release the acylated flavonoid derivative substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations which include formulations that create a substantially constant concentration of the drug within the body over an extended period of time; formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic pattern); formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and formulations that target the site of a pathology.
  • controlled release formulations obviate the need for frequent dosing to sustain the enzyme activity at a therapeutic level.
  • composition as described herein may be administered in any convenient manner.
  • a composition as described herein may be administered in any convenient manner.
  • an effective amount of an acylated phloridzin derivative or acylated isoquercetrin derivative as described herein is administered in combination with radiation therapy.
  • Combinations are expected to be advantageously synergistic.
  • Therapeutic combinations that inhibit cancer (e.g., leukemia, liver cancer, breast cancer, etc.) cell growth and/or induce apoptosis of cancer cells are identified as useful in the methods described herein.
  • the invention provides a method of monitoring treatment progress.
  • the method includes the step of determining a level of changes in cancer cell viability parameters and/or cell cycle arrest and/or induction of apoptosis as diagnostic markers or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to cancer (e.g., liver cancer, breast cancer, leukemia, etc.) or a disorder or symptom thereof in which the subject has been administered a therapeutic amount of a composition as described herein.
  • the level of marker determined in the method can be compared to known levels of marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status.
  • a second level of marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy.
  • a pre-treatment level of marker in the subject is determined prior to beginning treatment according to the methods described herein; this pre- treatment level of marker can then be compared to the level of marker in the subject after the treatment commences, to determine the efficacy of the treatment.
  • kits for treating cancer e.g., liver cancer, leukemia, breast cancer, etc.
  • a typical kit includes a pharmaceutical composition comprising an acylated flavonoid, such as an acylated phloridzin derivative as described herein or an acylated isoquercetrin derivative as described herein, in a therapeutically effective amount for decreasing proliferation and viability of cancer cells (e.g., inducing apoptosis of cancer cells) in the subject, packaging, and instructions for use.
  • the composition may further comprise a pharmaceutically acceptable carrier in unit dosage form.
  • the kit can comprise a sterile container which contains the disclosed pharmaceutical composition; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • compositions, kits, and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable compositions, kits, and methods are described below. All publications, patent applications, and patents mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, will control. The particular embodiments discussed below are illustrative only and not intended to be limiting.
  • Phloridzin phloretin 2'-0-glucoside
  • isoquercetrin quercetin 3-0- glucoside
  • Phloridzin fatty esters inhibited DNA topoisomerases Ila activity that might induce GO/GI phase arrest, and apoptosis via activation of caspase-3in human hepatocellular carcinoma cells.
  • Described herein is the regioselective enzymatic acylation of two series of acylated derivatives of phloridzin and isoquercitrin with six different long chain saturated, mono- and poly-unsaturated fatty acids.
  • the biocatalytic synthesis was optimized to achieve 81 to 98% yields, using immobilized lipase B, from Candida antarctica (Novozym 435 ® ), in acetone at 45 C.
  • the synthesized esters were analyzed by NMR, 13 C NMR spectroscopy ing in vitro assays.
  • Phloridzin and Isoquercitrin (2-7 and 9-14) To flame dried 3 °A molecular sieves, in round bottom flask, was added phloridzin (0.500 g; 1.15 mmol), stearic acid (1.62 g, 5.72 mmol), Novozyme 435 ® (1.30 g). It was followed by the addition of dry acetone (5 ml) and the mixture was stirred and heated at 45 °C for 12-24 h. The progress of reaction was monitored by thin layer chromatography (TLC), followed by staining with anisaldehyde spray reagent and then heating at 110 °C.
  • TLC thin layer chromatography
  • the reaction was carried out in an ultrasonic bath of 20 kHz/1000 Watts (model 750D, VWR, West Chester, PA, USA) and exposed for 3.5-5 h under ultrasound irradiation alone (for 15-20 min each with 10 min interval in between) and for 2-3.5 h (for 15-20 min each with 10 min interval in between) under ultrasound irradiation coupled with stirring at 40-45 °C ( Figure 4a-h).
  • MDA-MB-231 is an adherent, epithelial like cell line derived from the metastatic site (pleural effusion) of human breast (mammary gland) tissue of 51 year old female Caucasian (MDA-MB-231 : ATCC catalogue number HTB 26, Manassas, VA, USA). It is an estrogen independent cell line which serves as a useful in vitro model for human breast adenocarcinoma studies and express epidermal growth factor (EOF) and transforming growth factor alpha (TGF-a).
  • EEF epidermal growth factor
  • TGF-a transforming growth factor alpha
  • the base medium for MDA-MB-231 cell line was Dulbecco's Modified Eagle's Medium (Cat no.
  • MTS assay The reduction in metabolic activity induced by phloretin (PT), doxorubicin (DOX) and docetaxel (DOC) was evaluated using MTS assay.
  • the assay was performed using commercially available MTS assay kit (Promega, Madison, WI, USA).
  • MDA-MB-231 cells 100 ⁇ were seeded in 96-well flat-bottom cell culture plates at a density of 5 x 10 3 cells per well and were incubated overnight to facilitate cell adhesion.
  • Adhered cells were treated with PT, phloridzin (PZ), docosahexaenoic acid (DHA), phloridzin docosahexaenoate (PZ- DHA), DOX and DOC (10, 50, 100, 150 and 200 ⁇ /L) and incubated for 3, 6, 12, 18 and 24 hr at 37 ° C. Vehicle controls were used for all the treatment concentrations separately and untreated control (cells treated with cDMEM without any treatment) was also used.
  • Treatment blank cDMEM with treatment
  • vehicle control cDMEM with vehicle
  • 20 ⁇ , of combined MTS/PMS reagent final concentrations of 333 ⁇ g/mL MTS and 25 ⁇ PMS (Sigma Aldrich, Oakville, ON)
  • 20 ⁇ , of combined MTS/PMS reagent final concentrations of 333 ⁇ g/mL MTS and 25 ⁇ PMS (Sigma Aldrich, Oakville, ON)
  • the absorbance of the colored product was measured at 490 nm using a micro-plate reader (BMG-LABTECH) (Ortenberg, Germany).
  • mice Six to eight weeks old NOD-SCID mice were acclimatized for 1 week. MDA-MB-231 cells for injection were mouse-pathogen tested and thawed from liquid nitrogen freshly. They were grown in T-75 and only cells in 3-4 flasks were harvested at a time. To maintain cells at its maximum viability, cells were maintained at ice cold temperature throughout the procedure preparing them for injection. Injections were prepared in 1 mL syringes (BD PrecisionGlideTM, Franklin Lakes, NJ, USA) and not more than 4 injections were prepared at a time. Cells (5 x 10 5 ) were subcutaneously injected into right hind flank in 100 ⁇ plain DMEM (without supplements) using 26G needles (BD
  • mice were administered every other day (day 1, 3, 5, 7 and 9) for nine days.
  • mice were monitored for one more week (day 11, 13 and 15) and their tumor sizes and body weights were recorded.
  • Tumor bearing mice were euthanized by isofluorane inhalation followed by C0 2 inhalation at day- 15 (at the end of experimental period) or earlier if they show any signs of tumor ulceration or distress. Euthanized mice were photographed and the tumors were excised.
  • Excised tumors were weighed, photographed and fixed in 10% v/v acetate buffered formalin (0.2 L, 37% formaldehyde, 1.8 L, distilled water and 46.1 g sodium acetate.3H 2 0) (Fisher Scientific, ON, Canada).
  • Acid phosphatase assay measures the metabolic activity of live cells in terms of cytosolic acid phosphatase activity by hydrolyzing the phosphatase substrate, p-nitrophenyl phosphatase at acidic pH levels. The hydrolyzed product, p-nitrophenol produces the yellow- colored end product, p-nitrophenolate under alkaline conditions which could be read at 405 nm in a microplate reader and the absorbance is directly proportional to viable cell count. In alkaline pH, the acid phosphatase activity is ceased marking the end point of reaction. Acid phosphatase assay was performed as described in Yang et al., (1996) with minor
  • MDA-MB-231 cells (in 100 ⁇ cDMEM) were seeded in 96-well flat-bottom cell culture plates at a density of 5 x 10 3 cells per well and were incubated overnight to promote cell adhesion.
  • Adhered cells were treated with PT, PZ, DHA, PZ-DHA, DOX and DOC (10, 50, 100, 150 and 200 ⁇ /L) and incubated for 3, 6, 12, 18 and 24 hr at 37 ° C. Vehicle controls were used for all the treatment concentrations separately and untreated control (cells treated with cDMEM without any treatment) was also included. At the end of the incubation period the plates were centrifuged at 405 x g for 10 minutes and supernatant was discarded.
  • Assay buffer 100 ⁇ (0.1 M sodium acetate; pH 5.5, 0.1 % v/v Triton X-100 and 4 mg/mL phosphatase substrate) was added to each well and incubated for 2 hr at 37 ° C. Ten microliter of IN NaOH was added to each well to cease the reaction and to develop the color of the end product.
  • the absorbance of the colored product was measured at 405 nm using a micro-plate reader (BMG-LABTECH) (Ortenberg, Germany) and percentage relative phosphatase activity was calculated as given in the following equation where ⁇ : absorbance of test compound treated cells; Ac: absorbance of vehicle treated cells; AB: absorbance of blanks. Blank (100 ⁇ L ⁇ of assay buffer with 10 ⁇ L ⁇ of IN NaOH) was used to compensate the background absorbance.
  • 7-AAD (7-Aminoactinomycin) assay was carried out to confirm the reduction in metabolic activity measured in MTS and acid phosphatase assays was due to the death of cells and not false positive results given by cell growth inhibition.
  • MDA-MB-231 cells were plated at a density of 1 x 10 5 cells per well in 6-well plates. The cells were incubated overnight at 37 ° C to support cell adhesion. Adhered cells were treated with 50 and 100 ⁇ /L of PT, PZ, DHA, PZ- DHA, vehicle or medium and incubated for 24 hr at 37 ° C.
  • cells were harvested using TrypLE express and combined with its respective media. Cells were centrifuged at 500 x g and resuspended in 1 x PBS. Cells were incubated with 5 ⁇ L ⁇ of 7-AAD viability staining solution (ebioscience Inc. San Diego, CA, USA) at room temperature. Flow cytometric analysis was performed using a FACS Calibur instrument (BD Bioscience, Mississauga, ON) on detector, FL3. Cells (1 x 10 4 ) were counted per sample and both live and dead cells were included in counts.
  • 7-AAD viability staining solution ebioscience Inc. San Diego, CA, USA
  • Loss of cell membrane integrity at the late apoptosis/necrosis could be measured by 7-AAD which could penetrate into the cells once membrane become permeable.
  • 7-AAD staining a promising cell death was detected on FL3 after incubating cells at 50 ⁇ and 100 ⁇ concentrations of the test compounds for 24 hr.
  • the percentage of dead cells noticed in PT, DHA, PZ-DHA but not PZ treated cultures was notably different from both vehicle and medium treated cells.
  • PT-, DHA- and PZ-DHA- induced cell death observed at 100 ⁇ and 50 ⁇ were comparable.
  • Apoptosis programmed cell death is characterized by a series of morphological and biochemical changes taking place in a cell and DNA fragmentation is considered a hallmark of apoptosis.
  • TUNEL staining was carried out using commercially available In Situ Cell Death Detection Kit, POD (Roche applied Science, Laval, QC, Canada) according to the manufacturer's instructions.
  • MDA-MB-231 cells were seeded in 6-well plates containing sterile cover slips, at a density of 4 x 10 5 cells per well and incubated overnight at 37 °C to induce cell adhesion.
  • Adhered cells were incubated with 100 ⁇ /L of PT, PZ, DHA, PZ-DHA, vehicle or medium and incubated for 3, 6, 12, 18, and 24 hours at 37 ° C.
  • DOX, DOC (100 ⁇ /L) and 1 ⁇ /L staurosporine were used as the positive control to induce DNA fragmentation.
  • MgCl 2 (2 mM ) in 1 x PBS was used as the washing buffer in all the steps.
  • PFA paraformaldehyde
  • Amplex Red Assay The Amplex Red assay was carried out in a cell-free environment with H2O2 as the positive control and with comparison to 100 ⁇ of EGCG in phenol red-free cDMEM. This assay was performed to assess whether test compounds (PT, PZ, DHA and PZ-DHA) reacts with sodium bicarbonate in cell culture medium to produce hydrogen peroxide (H2O2). Amplex Red assay was performed in a 96-well flat-bottom plate in quadruplicates and the final concentration of PT, PZ, DHA, PZ-DHA and positive control was kept at 100 ⁇ /L.
  • test compounds PT, PZ, DHA and PZ-DHA
  • Standard curve of H2O2 was generated using a standard series (0.1, 0.056, 0.032, 0.018, 0.01 and 0.006 mM) of H 2 0 2 .
  • 100 pIJwell of master mix at a final concentration of 25 ⁇ Amplex red and 0.005 U/mL HRP in phenol red-free cDMEM was added on top of the treatments and incubated for 2 and 24 hr at 37 ° C.
  • NAC is an antioxidant that scavengse the ROS produced in the cells which could be involved in the induction of cell apoptosis.
  • the cell death caused by test compounds was measured in Annexin-V-FLUOS/PI staining at 6 hr of post treatment. Apoptosis and late
  • apoptosis/necrosis was detected by analyzing cells at FL1 and FL2 for Annexin-V-FLUOS and PI, respectively. Incorporation of NAC in the culture medium did not alter the cytotoxicity induced by the test compounds. Hence, NAC failed to protect breast cancer cells from cell death induced by the test compounds. When the anticipated background indicated by DMSO vehicle control treated cells is considered, late apoptosis/necrosis noticed in PZ- treated cells was minimal, while all the other test compounds showed more substantial cell death on Annexin-V-FLUOS/PI staining.
  • caspase-3 Activity Activation of caspase 3 and 7 enzymes were tested using caspase-Glo® 3/7 assay kit (Promega, Madison, WI, USA).
  • Caspase (cysteine- dependent aspartate-directed protease) enzymes are the central components of apoptosis and caspase 3 is the most frequently activated protease in mammalian cell apoptosis. Because caspase 7 is comparable to caspase 3 in in vitro substrate preference (toward substrate Ac- DEVD-pNA) this assay measures activation of both caspase 3 and 7. The assay was performed in triplicates according to the manufacturer's instructions.
  • MDA-MB-231 cells (in 100 xL cDMEM) were seeded in 96-well, flat-bottom white- walled cell culture plates at a density of 5xl0 3 cells per well and were incubated overnight to activate cell adhesion.
  • Adhered cells were treated with 100 ⁇ /L of PT, PZ, DHA or PZ-DHA and incubated for 6 and 12 hr at 37 ° C.
  • Doxorubicin, docetaxel (100 ⁇ /L) and staurosporine (1 ⁇ /L) were used as positive controls. Vehicle controls and medium controls were also included in triplicates.
  • contents of Caspase-Glo® 3/7 buffer vial was transferred into Caspase-Glo® 3/7 substrate and mixed by swirling.
  • the 96-well plates incubated at 37 ° C were equilibrated to room temperature before adding Caspase-Glo® 3/7 assay buffer mixture.
  • DHA fatty acid showed less potency (EC50 of 119.9 ⁇ at 24h) which is 2.3 -fold higher than phloridzin DHA ester.
  • Phloretin the aglycone of phloridzin
  • the standard liver cancer drug sorafenib (Nexavar®) showed a similar effect to phloridzin esters with EC50 of 67.5 ⁇ at 3h, which further decrease to 10.7 ⁇ at 24h.
  • phloridzin esters parent compounds, phloretin showed similar effects in cell survival as HepG2 cells.
  • phloretin was effective with a lower EC50 of 17.3 ⁇ compared to HepG2 (EC50 97.20 ⁇ ) or MDA- MB-231 (EC50 104.7 ⁇ ).
  • Doxorubicin hydrochloride a standard drug for breast cancer and acute monocytic leukemia (a type of acute myeloid leukemia) also showed a similar effect in cell viability as of phloridzin esters.
  • Phloridzin fatty acid esters exhibited much stronger cytotoxic activity than its parent compound in various cell lines, with EC50 values ranging from 6.2 to 63.8 ⁇ after 24h exposure.
  • phloridzin fatty acid esters function as a broad spectrum antiproliferative agent in human cancer cells and are more effective than their natural occurring parent compounds from which they were derived.
  • quercetin-3-O-glucoside (Q3G) acylated enzymatically were used for determining their antiproliferative action in comparison to precursor compounds (quercetin, Q3G and six fatty acids namely, stearic acid, oliec acid, linolec acid, alpha-linolenic acid, eicosapentaenoic and docosahexanoic acids) using HepG2 cells in vitro.
  • precursor compounds quercetin, Q3G and six fatty acids namely, stearic acid, oliec acid, linolec acid, alpha-linolenic acid, eicosapentaenoic and docosahexanoic acids
  • Long chain fatty acid esters of Q3G showed significant inhibition of cell proliferation (approximately 85% to 90%) in HepG2 cells compared to the precursor compounds and two prescribed drugs (sorafenib and cisplatin) for liver cancer chemotherapy (P-value ⁇ 0.05) after 6 hrs and 24 hrs of treatment.
  • the cell death due to these novel compounds was associated with cell cycle arrest and apoptosis based on DNA fragmentation, cell cycle analysis by flow cytometer, fluorescent microscopy and elevated caspase-3 activity.
  • the long chain fatty acid esters of Q3G exhibited strong DNA topoisomerase II inhibition.
  • oleic acid ester displayed the greatest antiproliferation action and a high potential as a cancer therapeutic.
  • Cell Titer 96TM Aqueous One solution cell proliferation (MTS) assay and CytoTox 96® nonradioactive cytotoxicity (LDH) Assay kits were purchased from Promega (Madison, WI). ApoTargetTM Quick apoptotic DNA ladder detection kit from Invitrogen (Burlington, ON). Caspase 3 colorimetric assay kit was purchased from
  • Enzymatic reactions were initiated by the addition of lipase (Novozyme 435 ); with an activity of 10,000 propyl laurate units).
  • the mixture was stirred and heated at 45 °C for 12- 24 h. and was monitored by thin layer chromatography (TLC), followed by staining with anisaldehyde spray reagent and then heating at 110 °C. After completion of reaction, it was filtered, evaporated and column chromatography (acetone/toluene; 35:75 to 50:50) was performed to get the pure fatty acid esters of Q3G. The pure compounds were then analyzed
  • HepG2 cell culture system HepG2 cells were obtained from American Type Culture Collection (ATCC, 8065) and maintained according to ATCC's instructions. Briefly, the cells were cultured in Eagle's Minimum Essential Growth Medium (EMEM) with 2 mM L-glutamine and 10% Fetal Bovine Serum (FBS) at 37°C and 5% C02. T-75 tissue culture flasks with 12-15 ml of media were used for regular culturing. Sub-culturing was performed in 1 :4 or 1 :5 ratio every 3 to 4 days when cells reached a confluency of 70-80%. Cells were counted under Nikon Eclipse TS 100 phase contrast microscope using haemocytometer and then transferred to fresh flasks.
  • EMEM Eagle's Minimum Essential Growth Medium
  • FBS Fetal Bovine Serum
  • HepG2 cells in the exponential growth phase were collected and seeded in 96-well microplate in density of 2 x
  • concentration for 100 ⁇ of test compounds in all assays was less than 1%.
  • the plates were then incubated for different time intervals (6 and 24 hrs) in culture incubator (37° C, 5% C02, 90% humidity). According to manual instructions, 20 ⁇ of MTS was added to each well (5 g/L in PBS) and again incubated for 1-4 hrs. Absorbance was recorded directly at 490 nm using Fluostar Optima micro plate reader (BMG Labtech, Ortenberg, Germany).
  • the microplates were placed in culture incubator in standard conditions (37° C with 5% C0 2 ) and cultured for 24 hrs. After incubation, 100 ⁇ of the long chain fatty acid esters of Q3G and control samples in fresh media were added to each well in triplicates. The plates were incubated for different time intervals (mainly, 6 and 24 hrs) in culture incubator (37° C, 5% C0 2 , 90% humidity). After treatment, the 96-well microplate was centrifuged and supernatant was transferred to a fresh 96-well microplate and subjected to LDH assay. Absorbance was taken at 490-492 nm using Fluostar Optima micro plate reader (BMG Labtech, Ortenberg, Germany).
  • LDH has a half-life of 8-9 hrs (Lisa et al., 2004 and Riss et al., 2004) and because the compounds cause a significant amount (over 85%) of cell death within 6 hours, by 24 hrs LDH gets degraded in the medium and hence 24 hours incubation readings were seen comparatively lesser than 6 hours readings. Nevertheless, the LDH release which signifies the cytotoxic action of the long chain fatty acid esters of Q3G was significantly greater than the precursor compounds and control cancer drug, Cisplatin (P ⁇ 0.05) .
  • Oleic acid ester of Q3G emerged as the most effective compound showing the greatest antiproliferative action (over 95%), while all other long chain fatty acid esters of Q3G except steric acid ester showed relatively lesser antiproliferative action ( ⁇ over 85%).
  • DNA fragmentation This assay was performed by utilizing commercially available ApoTargetTM Quick Apoptotic DNA Ladder Detection Kit. The manufacturer' s instructions were followed for the assay. Briefly, HepG2 cells (5 x 10 5 cells/well) were grown in 12 well culture plate (75-80%) confluency and then treated with 100 ⁇ of test compounds for 24 and 48 hrs. Cells were collected and total DNA was isolated from each sample. Extracted DNA pellet was dissolved in 30 ⁇ of DNA suspension buffer (provided
  • the long chain fatty acid esters of Q3G treated cells showed consistent DNA damage and fragmentation within 24 hours as seen on the gel image.
  • the intensity of the damage and fragmentation increased at 48 hours incubation.
  • Caspase Assay The caspases activation was quantified by utilizing Caspase- 3/CPP32 Colorimetric Assay Kit. The assay was performed according to the manufacturer's
  • HepG2 cells (1 x 10 cells/well) were plated in six-well tissue culture plate. After treatment with the test compounds for 24 hrs, the cells were lysed with lysis buffer provided by the manufacturer and centrifuged at 13000 rpm. After collecting the supernatant, the protein was quantified using BCA protein quantification kit and 250 ⁇ g of protein per treatment sample was used for the assay. Reaction buffer (50 ⁇ ) was added to each treatment well of microplate reader followed by addition of 5 ⁇ DEVD-pNA (Asp-Glu- Val-Asp p-nitroanilide) caspase substrate. The microplate was incubated at 37° C for 1-2 hour.
  • DEVD-pNA Asp-Glu- Val-Asp p-nitroanilide
  • the absorbance of the samples was read at 405 nm in Fluostar Optima micro plate reader (BMG Labtech, Ortenberg, Germany). After subtracting background readings from all the samples (induced and uninduced), fold-increase in CPP32 activity was determined by comparing these results with the level of the uninduced control.
  • Topoisomerase Assay Commercially available topoisomerase II drug screening kit (TopoGEN, Inc., Columbus, Ohio, USA) was utilized and assay was performed as explained by Patra et al., (2011). Briefly, substrate supercoiled pHotl DNA (0.25 ⁇ g) was incubated with four units (2 ⁇ ) of human DNA topoisomerase II, test compounds (2 ⁇ ) and assay buffer (4 ⁇ ) in 37° C for 30 minutes. The reaction was terminated by the addition of 10% sodium dodecyl sulphate (2 ⁇ ) followed by digestion with proteinase K (50 ⁇ g/ ⁇ l) at 37° C for 15 minutes. After incubation DNA was run on 1% agarose gel in BioRad gel TM
  • the assay was performed to test whether long chain fatty acid esters of Q3G act as a poison and increase the DNA cleavage via topoisomearse II.
  • the esters did not stabilize topoisomerase II cleavage complexes and failed to exhibit the formation of single linear DNA, and increased the supercoiled DNA intensity, whereas, positive control drug VP 16 increased the formation of linear DNA.
  • This result shows that long chain fatty acid esters of Q3G do not act as human topoisomearse II poison but as a catalytic inhibitor by inhibiting the DNA relaxation activity.
  • the intensity of the supercoiled bands in comparison to the negative control (DMSO) is very high clearly suggesting that due to DNA topoisomearse II inhibition, the supercoiled DNA did not get relaxed
  • apoptotic cells Briefly, 2 x 10 HepG2 cells were seeded on two- well chambered cover slides (Sigma- Aldrich) and grown to about 75% confluency; this was followed by treatment with 100 ⁇ long chain fatty acid esters of Q3G for 24 hours. Adherent cells were stained according to the manufacturer' s instructions by dual detection reagent (containing annexin V coupled with PI). The dual-labeled cells were visualized by fluorescence microscopy with a Leica DMBL (20x/.040) fluorescent microscope (Houston, TX) incorporated with Nikon Cool Pix 4500 Digital camera (Mississauga, Ontario). Cells with bound annexin-V show green staining in the plasma membrane.
  • Cell cycle analysis HepG2 cells were plated in six well culture plate (1 x 10 cells/well). After 24 hours of incubation at 37° C, 5% C02, the cells were treated with 100 ⁇ long chain fatty acid esters of Q3G for another 24 hours. Briefly, cells were trypsinized and centrifuged at 1200 rpm at 4 ° C for 10 minutes followed by another PBS wash. The pellet was resuspended in 0.3 ml of PBS. The cells were then fixed by adding 0.7 ml of ice cold ethanol for 2 hours.
  • the cells were centrifuged again at 1200 rpm at 4 ° C for 10 minutes and cell pellet was re-suspended in 0.25 ml of PBS with the addition of 5 ⁇ of 10 mg/ml Rnase A (the final concentration being 0.2-0.5 mg/ml) and incubation at 37°C for 1 hour. After incubation 10 ⁇ of 1 mg/ml PI solution (the final concentration being 10 ⁇ g/ml) was added to the cell suspension and kept in the dark at 4°C until analysis. The cells were then analyzed for cell cycle using flowcytometer FACS Caliber (BD Biosciences, San Jose, California) with an excitation wavelength at 488 nm and emission at 670 nm. DNA content was determined by MotFit LTTM software, version 4.0 (Topsham, ME), which provided histograms to evaluate cell cycle distribution.
  • long chain fatty acid esters of Q3G increased the population in the S phase with a corresponding decrease of cells in the Gl phase after 24 hours of treatment, implying that the DNA synthesis was retarded.
  • long chain fatty acid esters of Q3G also appeared to increase the cell population in the G2-M phase, implying the cell mitosis stage was inhibited for the cells that managed to move from S phase to G2-M phase.
  • the novel synthesized long chain fatty acid esters of Q3G can inhibit liver cancer cell proliferation (HepG2) through induction of apoptosis by the activation of caspase-3 family followed by necrosis, through cell cycle changes, and possibly through inhibition of DNA topoisomerase II activity.
  • HepG2 liver cancer cell proliferation
  • long chain fatty acid esters of Q3G exhibited much stronger anti-proliferative property than precursor compounds
  • the test compounds caused cytotoxicty to the HepG2 cells resulting in the cell membrane shrinkage and eventually breakage. This result was further assessed by the membrane integrity test via LDH release assay which showed that there was clear membrane breakage when compared with untreated control cells. Interestingly, the strong inhibition of cell proliferation by the fatty acid esters of Q3G when compared to the precursor compounds alone and the chemotherapy drugs is noteworthy.
  • the long chain fatty acid esters of Q3G also showed significantly lower cytotoxic effect to normal hepatocytes as compared to the transformed HepG2 suggesting their specific action on HepG2 cells. Additionally, fluorescence microscopy showed cell membrane breakage suggesting symptoms of late apoptosis and necrosis. To distinguish between apoptosis and necrosis, cells were analyzed after staining with Annexin V and PI through fluorescence microscopy. After 24 h of treatment of HepG2 cells with long chain fatty acid esters of Q3G showed that, some treated cells were positive for PI and some for both
  • OA-Q3G oleic acid ester of Q3G
  • results from the cancer drug target RT-PCR array suggested that antiproliferative effects of OA-Q3G in vitro could be related to enhanced gene expression of pro-apoptotic genes BCL2 (2-fold), RHOB (3.3fold), IRF5 (6.3-fold); down-regulation of cell cycle genes CDK1 (2-fold), CDK2 (2fold), CDK8 (12.2-fold), growth factor-receptor EGFR (12.2-fold), protein kinases AURKB (2- fold), PRKCA (2-fold) and DNA topoisomerase II (2.7fold).
  • OA-Q3G treatment considerably down-regulated gene expression of certain genes which are found over- expressed in cancer cells such as HIF1A (2.4-fold), NFKB1 (2.2-fold), HDAC6 (25.9-fold) and BIRC5 (11.2-fold).
  • results from ELISA and ELISA array revealed a significant increase in the protein expression of pro-apoptotic genes phospho-p53, phospho- Bad, cleaved-caspase-3, cleaved-parp, ERK1/2, and decrease in protein expression of anti- apoptotic and cell survival genes AKT, PRAS40 and p70S6kinase in HepG2 cells upon treatment with OA-Q3G.
  • this study provides possible molecular targets of OA- Q3G in HepG2 cells through the attenuated regulation of genes involved in cell cycle, survival and apoptosis.
  • HepG2 cell culture system HepG2 cells were obtained from American Type Culture Collection (ATCC, 8065) and maintained according to ATCC's instructions. Briefly, the cells were cultured in Eagle's Minimum Essential Growth Medium (EMEM) with 2 mM L-glutamine and 10% Fetal Bovine Serum (FBS) at 37°C and 5% C0 2 . T-75 tissue culture flasks with 12-15 ml of the media were used for regular culturing. Cells were counted under Nikon Eclipse TS 100 phase contrast microscope (Mississauga, ON) using haemocytometer and then transferred to fresh flasks.
  • EMEM Eagle's Minimum Essential Growth Medium
  • FBS Fetal Bovine Serum
  • RNA-related genes expression profiling To identify possible molecular targets for antiproliferation action of OA-Q3G, Human Cancer Drug Target RT2 ProfilerTM PCR Array (SABioscience, Frederick, MD) was used. Assay was performed according to manufacturer's instructions. Briefly, HepG2 cells were treated with OA-Q3G for 24 h. Total RNA was isolated using AurumTM Total RNA Mini Kit (Bio-Rad, Mississauga, ON) according to the manufacturer' s protocol. RNA concentration was measured with NanoDrop 1000 Spectrophotometer (Thermo Scientific, Ottawa, ON), RNA quality was evaluated by electrophoresis.
  • cDNA was synthesized from 500 ⁇ g total RNA using RT2 First Strand cDNA Synthesis kit (Qiagen, Toronto, ON). Real-time PCR was performed using CFX96TM Real-Time PCR detection System (Bio-Rad, Mississauga, ON) according to manufacturer's instructions. Obtained data were analyzed with Excel-based PCR Array Data Analysis Software (SABioscience, Frederick, MD). This system profiles the expression of 84 actively sought targets for anticancer therapeutics and drug development which includes genes dysregulated during carcinogenesis, including those involved in key cellular growth pathways such as apoptosis, DNA damage repair, epigenetics, and growth factor and other signaling pathways.
  • Cell cycle and survival down-regulation of cell cycle proteins, CDK1 (-2-fold), CDK2 (-2-fold), CDK (-12.2-fold), CDC25A (-2-fold), topoisomerase II TOP2A (-2.7-fold), growth factors and receptors, EGFR (-12.2-fold), GRB2 (-2-fold) receptor tyrosine kinase AKT2 (-2.1 -fold), protein kinases, AURKB (-2-fold), PRKCA (-2-fold) and histone deacetylases HDAC2 (-2-fold), HDAC6 (-25.9-fold).
  • Multi-Target Sandwich ELISA analysis To investigate the effect of OA-Q3G treatment on intracellular apoptotic markers, PathScan® Apoptosis Multi-target Sandwich ELISA kit (Cell Signaling Technology, Danvers, MA) was used.
  • the assay is a solid phase is a solid phase sandwich enzymelinked immunosorbent assay (ELISA) that combines the reagents necessary to detect endogenous levels of p53 protein, phospho-p53 protein (Serl5), Bad, phospho-Bad (Serl l2), Cleaved Caspase-3 (Aspl75) and Cleaved PARP (Asp214), which are key signaling proteins of cell survival and apoptosis pathways.
  • ELISA solid phase sandwich enzymelinked immunosorbent assay
  • the assay was performed according to manufacturer's instructions. Briefly, HepG2 cells were treated with OA-Q3G for 0, 6 and 24 h in fresh media. Cells were harvested under non-denaturing conditions by adding IX cell lysis buffer (provided with the kit) supplemented with IX protease inhibitor (Sigma-Aldrich, Mississauga, ON) and kept on ice for 10 min. Lysates were then microcentrifuged for 10 min at 4 °C and supernatents were transferred in fresh tubes and stored at -80 °C in single-use aliquotes.
  • the assay was performed using 500 ⁇ g/ml protein quantified by BCA Protein Assay (Thermo Scientific, Ottawa, ON) according to the manufacturer's instructions.
  • BCA Protein Assay Thermo Scientific, Ottawa, ON
  • 100 ⁇ of each cell lysate was added and incubated the plate overnight at 4 °C. After incubation, wells were washed four times with IX wash buffer (provided with kit). After washing, 100 ⁇ of detection antibody (provided with the kit) for each protein in respective wells and incubated the plate for 1 h at 37 °C followed by washing with wash buffer. After washing, 100 ⁇ of HRP-linked secondary antibody was added to corresponding wells and incubated the plate for 45 min at 37 °C followed by another wash procedure.
  • TMB substrate was added to each well and plate was incubated for 30 min at 37 °C followed by addition of stop solution. Protein activity was measured by reading absorbance at 450 nm in Fluostar Optima micro plate reader (BMG Labtech, Ortenberg, Germany).
  • Results from sandwich ELISA showed that total p-53 and phosphorylated p-53 amounts significantly increased at 6 h of incubation and comparatively decreased after 24 h of incubation. Similar results were seen by densitometry analysis from ELISA array which showed a significant 2-fold increase in levels of phospho p-53 within 6 h of treatment with OA-Q3G.
  • Multi-target ELISA Array analysis To indentify intracellular protein level targets of OA-Q3G in HepG2 cells, PathScan® Intracellular Signaling Array kit (Cell Signaling Technology, Danvers, MA) was used. It is a slide-based antibody array founded upon the sandwich immunoassay principle. The array kit allows for the simultaneous detection of 18 important and well-characterized signaling molecules when phosphorylated or cleaved. The glass slides are nitrocellulose-coated spotted with target- specific capture antibodies. The assay was performed according to the manufacturer's instructions. Briefly, cell lysate were prepared and quantified similar to sandwich ELISA explained above.
  • Protein 500 ⁇ g/ml were incubated on the slide overnight at 4 °C followed by addition of a biotinylated detection antibody cocktail (provided with kit). Streptavidin-conjugated HRP and LumiGLO® Reagent (provided with the kit) was then used to visualise the bound detection antibody by chemiluminesence. The bands were developed using Carestream® Kodak film (Sigma- Aldrich, Mississauga, ON) and were quantified using Image J 1.47 software according to NIH guidelines.
  • Results showed an up-regulation in pro-apoptotic proteins ERK1/2, Bad, p-53, SAPK/JNK, PARP and caspase-3 active products expression and down-regulation of cell survival and growth proteins AKT, p70 S6 kinase, GSK 3 ⁇ , Stat3 and PRAS40 within 6 h of treatment with OA-Q3G.
  • results from 24 h treatment showed a decrease in the expression of ERK1/2, Statl, HSP27 and p53 active products and an increase in the expression of p38, SAPK/JNK, PARP and caspase-3 active products.
  • Results from this analysis suggest a strong role of OA-Q3G in active regulation of cell survival and cell death factors.
  • results from the RT Profiler PCR array analysis showed a 2-fold downregulation in the expression of CDK1 and an up- regulation in the expression of pro-apoptotic Bcl-2 family as compared to untreated control cells within 24 h of treatment with OA-Q3G. Additionally, results from sandwich ELISA showed a significant increase in the expression of pro-apoptotic Bad after 6 and 24 h of treatment with OA-Q3G.
  • phloridzin fatty acid esters were investigated in comparison with the parent compounds, phloridzin, aglycone phloretin, the six free fatty acids, and a standard hepatocellular carcinoma drug, sorafenib (Nexavar®) using human hepatocellular carcinoma cells, HepG2, and normal human liver cells.
  • sorafenib sorafenib
  • the antiproliferative potency of phloridzin fatty acid esters was comparable or greater than the potency of sorafenib.
  • the activities of these new compounds on DNA topoisomerases ⁇ activity, cell cycle and apoptosis were determined. Phloridzin fatty acid esters inhibited DNA topoisomerases ⁇ activity that might induce G0/G1 phase arrest, and apoptosis via activation of caspase-3 in HepG2 cells.
  • Phloridzin fatty acid esters have the anticancer potential against liver cancer cells in vitro and deserve further investigation.
  • Test Compounds and chemicals Fatty acid esters of phloridzin (Pz) viz. stearic acid ester of Pz (Pz-stearic acid), oleic acid ester of Pz (Pz-oleic acid), linoleic acid ester of Pz (Pz-linoleic acid), a-linolenic acid ester of Pz (Pz-a-linolenic acid), DHA ester of Pz (Pz-DHA) and eicosapentaenoic acid ester (EPA) of Pz (Pz-EPA) were synthesised as described in Example 1 above.
  • Pz Fatty acid esters of phloridzin
  • Phloridzin, phloretin, caspase 3 colorimetric assay kit, propidium iodide, fatty acids namely oleic, stearic, linoleic, a-linolenic, eicosapentaenoic and docosahexanoic acids were purchased from Sigma-Aldrich (Mississauga, ON, Canada).
  • MTS One solution cell proliferation
  • LDH non-radioactive cytotoxicity
  • DMSO Sterile dimethyl sulfoxide
  • GFP- certifiedTM apoptosis/necrosis detection kit for microscopy from Enzo Lifesciences
  • HepG2 Human hepatocellular carcinoma cell line
  • ATCC American Type Culture Collection
  • FBS FBS
  • ATCC Rockville, MD
  • penicillin-streptomycin ATCC, Rockville, MD, USA
  • THP-1 cells were cultured in RPMI-1640 media supplemented 0.05 mM 2-mercaptoethanol and 10% fetal bovine serum to a final concentration of 10%.
  • MDA-MB-231 breast cancer cells (ATCC HTB-26TM) were obtained from Cedarlane, Berlington, ON, Canada) and were maintained in DMEM medium (Sigma- Aldrich Canada) supplemented with 100 u/mL penicillin, 100 ⁇ g/mL streptomycin, 2 mM L-glutamine, 5 mM HEPES (pH 7.4) and 10% heat-inactivated fetal bovine serum (Invitrogen, Burlington, ON, Canada).
  • Cryopreserved normal human hepatocytes HP-F
  • hepatocyte plating medium hepatocyte maintenance medium
  • Normal human hepatocytes plated on 96 well collagen 1 coated cell culture plates (Life Technologies) and maintained in hepatocyte maintenance medium for 24 h to allow for cell recovery and attachment.
  • Rat hepatocytes (RTCP10), thawing media and incubation media were purchased from Life Technologies.
  • Rat hepatocytes were plated in collagen 1 coated 96 well plates (Life Technologies, Burlington, ON, Canada) using thawing media and maintained in incubation medium. All cell types were maintained at 37°C in an incubator under 5% C0 2 /95% air atmosphere at constant humidity.
  • Cell Proliferation assay Cell viability was determined by using the MTS assay. In brief, HepG2 cells (5 x 10 3 cells/100 pIJwell), MDA-MB-231 (5 x 10 3 cells/100 MlJwell), THP-1 (25 x 10 3 /100 ⁇ ), normal human and rat hepatocytes (1 x 10 4 cells/ 100 pIJwell) were plated in triplicate, in a 96 well sterile flat bottom tissue culture plates.
  • phloridzin fatty esters, phloridzin, phloretin, free fatty acids of respective esters or sorafenib were prepared in media and 100 ⁇ L ⁇ of each treatment was added to each well, each treatment in three replications. Thereby, cells were exposed to various concentrations (0.1, 1, 10, 25, 50, 75, 100 ⁇ ) of each treatment. Controls consist of cells with media containing DMSO ( ⁇ 0.5%), test blank wells contained the test compound in media with no cells and blank wells contained media with no cells.
  • phenazine methosulfate were added to the wells and cells were incubated in a humidified incubator for 3 h.
  • Absorbance at 490 nm was measured by using a Flurostar Optima microplate reader (BMG Labtech, Cary, NC, USA) to obtain the number of viable cells relative to the control population. Percentage of viability of the test compound treated cells are expressed as percentage compared to control ( ⁇ 0.5% DMSO).
  • EC5 0 values concentration required to reduce cells viability by 50% as compared to control cells
  • Phloretin showed inhibition at 200 ⁇ from 6 h where as its lower concentration showed no effect.
  • the parent compounds of phloridzin fatty esters viz, phloridzin and free fatty acids showed no inhibition on HepG2 cell proliferation.
  • LDH Lactate dehydrogenase
  • LDH actVity was measured using CytoTox 96 Non-Radio ac tive Cytotoxicity A ssay (Promega, Madison, WI), in which LDH released in culture supernatants is measured with a coupled enzymatic assay, resulting in conversion of a tetrazolium salt into a red formazan product.
  • HepG2 cells (5000/100 ⁇ /well) were seeded and treated with 100 ⁇ of phloridzin fatty acid esters, phloridzin, phloretin, free fatty acids of respective esters or sorafenib prepared in serum free media and incubated (37°C 1 5% C0 2 ) for 6 h.
  • HepG2 cells were equally seeded in 24-well flat bottom tissue culture treated plates (BD Biosciences ), and then treated with 100 ⁇ of phloridzin fatty acid esters, phloridzin, phloretin, sorafenib and DMSO ( ⁇ 0.5%) control. After 24 h of treatment, the morphology of HepG2 cells was observed under an inverted phase contrast microscope (Nikon Eclipse E 100, Nikon, ON) and images were captured at 400X magnification using Infinity digital microscopy camera (Lumenera corporation, Ott a wa, ON, C a nada).
  • the attached cells were then treated either with 100 ⁇ phloridzin fatty acid esters, phloridzin, phloretin, sorafenib or DMSO vehicle (as control) for 24 h.
  • the slides were washed with PBS. After removing the chamber, each slide was added with 50 ⁇ of Dual Detection Reagent containing apoptosis detection reagent (Annexin V-EnzoGold) and necrosis detection reagent (7-AAD) in IX binding buffer.
  • the samples were incubated at room temperature for 15 min in the dark. After staining, the cells were washed with binding buffer and covered with a glass coverslip.
  • the stained cells were observed under a fluorescence Zeiss Axiovert 200 m inverted microscope ( Carl Zeiss, ON, Canada) at magnification of x40 with a filter set for Annexin V-EnzoGold (Ex/Em: 550/570 nm) and 7-AAD (Ex/Em: 546/647 nm).
  • Fluorescence imaging was conducted to visually differentiate between apoptosis induced and necrotic cell death. After incubation of HepG2 cells with 100 ⁇ of phloridzin fatty acid esters for 24 h, the number of cells remaining as an adherent monolayer was greatly decreased compared to the control cells. In addition, floating cells showed morphological changes, with characteristics similar to apoptosis or necrosis. The cells were co-incubated with AnnexinV Enzogold (enhanced cyanine), an early marker of phosphatidylserine extemalization at the cell membrane and red emitting dye 7-AAD, marker of late apoptosis or necrosis.
  • AnnexinV Enzogold enhanced cyanine
  • ApoTargetTM Quick Apoptotic DNA Ladder Detection Kit (Invitrogen, Burlington, ON) according to the manufacturer's protocol. The principle involves detecting the internucleosomal DNA fragments formed during apoptosis. Briefly, floating dead cells and trypsinized adherent cells were collected and centrifuged at 1 ,000 rpm for 10 min. After washing with PBS, the cells were lysed with 35 ⁇ TE lysis buffer (a kit component). To the lysate, 5 ⁇ of Enzyme A (a kit component) was added and incubated at 37°C for 10 min.
  • Enzyme B (a kit component) was added, gently mixed and incubated at 50°C for 30 min.
  • the DNA was precipitated with the ammonium acetate and absolute ethanol at -20°C. After centrifugation (10 minutes at 12,000 rpm) and air drying, the DNA pellet was dissolved in 30 ⁇ of DNA suspension buffer.
  • the extracted DNA samples were run on a 1.2% agarose gel containing 0.5 ⁇ g/ml gel red in Tris-Borate-EDTA (TBE) buffer. After electrophoresis, the gel image was captured using Gel Doc 100 system (Bio-Rad, Mississauga, ON, Canada).
  • caspase-3 Activity The activity of caspase 3 enzyme was measured using caspase 3 colorimetric assay kit purchased from Sigma- Aldrich (Mississauga, ON, Canada). HepG2 cells (2 x 10 6 cells/well), grown in 6-well plates, were treated either with 100 ⁇ phloridzin fatty acid esters, phloridzin, phloretin, sorafenib or DMSO vehicle (as control). Cells were lysed and the protein content of cell lysate was quantified by the BCA protein assay (Thermo Fisher Scientific Inc., Ottawa, ON, Canada).
  • Caspase-3 activity was measured in the 200 ⁇ g of cell lysate using the caspase-specific peptide substrate, DEVD (Asp-Glu-Val-Asp), conjugated to reporter p-nitroanaline (p-NA) molecules. Cleavage of this peptide by caspase releases the chromophore which is measured colorimetrically at a wavelength of 405 nm as described in the supplier' s protocol.
  • the fixed cells were washed with PBS and centrifuged at 2000 x g for 10 min. After suspending cells in 0.3 ml PBS, 8 ⁇ of DNAase free RNAse (10 mg/ml) was added and incubated for 1 h. After adding, 15 ⁇ of propidium iodide (0.5 mg/ml), cells were incubated in 4°C for 30 minutes. The cells were analyzed for cell cycle using flow cytometer FACS calibur (Beckman Coulter, Fullerton, CA) with an excitation wavelength of 488 nm and emission at 670 nm. DNA content was determined by ModFit software (Verity Software House, Topsham, ME), which provided histograms to evaluate cell cycle distribution.
  • ATP level assay Cellular ATP levels were measured with CellTiter-Glo® luminescent assay kit obtained from Promega according to the manufacturer's instructions. HepG2 cells plated on a black walled clear bottom 96- well plate were incubated with 100 ⁇ fatty acid esters of phloridzin, phloridzin, phloretin, sorafenib, free fatty acids or DMSO ( ⁇ 0.5%) control in media. After 24 h, CellTiter-Glo®Reagent equal to the volume of cell culture medium present in each well and mixed contents for 2 min on an orbital shaker to induce cell lysis. Luminescence was recorded on Flurostar Optima microplate reader (BMG Lab tech) after incubation at room temperature for 10 min to stabilize luminescent signal. The level of ATP in a sample was presented as percentage compared to untreated control.
  • Mitochondrial membrane potential HepG2 cells were seeded in a black walled clear bottom 96-well sterile flat bottom tissue culture plates (BD Biosciences, USA) at a density of 5 x 10 4 cells/well (100 ⁇ ) and incubated in a C0 2 incubator for 24 h at 37 °C. Cells were treated with 100 ⁇ fatty acid esters of phloridzin, phloridzin, phloretin, sorafenib, free fatty acids or DMSO ( ⁇ 0.5%) control prepared in media and incubated for 24 h. The staining solution JC-1 was prepared with PBS and 5 ⁇ was added to each well.
  • the cells were further incubated in a C0 2 incubator at 37 °C for 1 h. After washing the plate with PBS twice, the fluorescence was measured using a Fluostar Optima microplate reader (BMG Lab tech) at 535 nm for JC-1 monomers and at 590 nm for JC-1 aggregates
  • topoisomerase Hot (topo Hot) Catalytic Activity The topo ⁇ catalytic activity was monitored via electrophoresis using topoisomerase II drug screening kit (TopoGEN, Inc., Columbus, Oh, USA). Briefly, 20 ⁇ of reaction mixtures contained 0.5 M Tris-HCl, pH 8.0, 1.50 M NaCl, 100 mM MgCl 2 , 20 mM ATP, 300 ⁇ g BSA/ml and 5 mM dithiothreitol. Supercoiled DNA (pHOTl DNA), supercoiled provided in the kit was determined to be ideal for this assay because it is small and easy to handle and has a large number of topo ⁇ recognition elements.
  • topoisomerase II activity was defined as the minimum amount of enzyme required to achieve complete relaxation of 0.5 mg superhelical pHOTl DNA in 30 min at 37° C. Inhibition of topoisomerase II relaxation activity was investigated by the same procedure using four units of enzyme and 100 ⁇ test compounds. The percent of inhibition was calculated by the following formula:
  • S contro i is the percent of supercoiled DNA in the control lane (without enzyme and test compounds)
  • So is the percent of supercoiled DNA in the lane without test compounds
  • S is the percent of supercoiled DNA in the lane with test compounds and enzyme.
  • RNA extraction was performed using Arum Total RNA minikit (Bio-Rad, Hercules, CA, USA). RNA concentration and purity was determined by measuring the absorbance using a NanoDrop (NanoDrop Technologies, Wilmington, DE, USA). RNA integrity was assessed by formaldehyde agarose gel electrophoresis. RNA (400 ng) was used to synthesize cDNA using RT 2 First Strand kit (SABiosciences, Frederick, MD, USA).
  • RT 2 RNA QC PCR arrays (SABiosciences, Frederick, MD, USA) was used to assess the quality of cDNA samples before characterization with the human cancer drug targets RT 2 profilerTM PCR array (SABiosciences, Frederick, MD, USA).
  • Gene expression profiles of 84 genes were investigated using the human cancer drug targets RT 2 profilerTM PCR array (PAHS-507ZD) on a Bio-Rad CFX Connect (Bio-Rad, Hercules, CA, USA) using RT 2 real-time SYBR green PCR master mix (SABiosciences, Frederick, MD, USA).
  • the array also has five reference genes (beta-2-microglobulin (B2M), hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L13a (RPL13A), glyceraldehyde-3 -phosphate dehydrogenase (GAPDH), and actin beta (ACTB), three reverse transcription controls (RTCs), three positive PCR controls (PPCs), and one genomic DNA control (GDC), making up to 96 assays.
  • B2M beta-2-microglobulin
  • HPRT1 hypoxanthine phosphoribosyltransferase 1
  • RPL13A ribosomal protein L13a
  • GPDH glyceraldehyde-3 -phosphate dehydrogenase
  • ACTB actin beta
  • RTCs reverse transcription controls
  • PPCs positive PCR controls
  • GDC genomic DNA control
  • RNA QC PCR data showed no genomic DNA contamination (Ct ⁇ 35 will indicate least GDC) or presence of impurities in RNA samples based on the Ct value of PPC (Ct should be 20 + 2 on each array) and showed no inhibition of reverse transcription based on the Ct values of RTC and PPC. Reproducibility was maintained by using three biological replicates from three individual experiments.
  • Phloridzin-DHA ester has the greatest potential and efficacy as a chemopreventive agent. These esters showed very low cytotoxicity to normal cells revealing their specific action against the cancer cells.
  • the HepG2 cell growth inhibition mechanism of the fatty acid esters of phloridzin is related to inhibition of apoptosis and cell cycle arrest. Novel compounds inhibited topo ⁇ and triggered DNA damage.
  • DHA ester of phloridzin has the greatest potential and efficacy as a chemopreventive agent. The anti-proliferative properties of the ester seems to be through a mechanism that down regulate key signalling pathways including PI3K/AKT/mTOR.

Abstract

La présente invention concerne un dérivé de flavonoïde acylé dérivé de flavonoïdes et d'acides gras naturels et synthétiques. Les dérivés de flavonoïdes acylés peuvent faire partie d'une composition pharmaceutique. Un procédé de diminution de la prolifération et de la viabilité de cellules cancéreuses chez un sujet comprend l'administration de la composition pharmaceutique décrite.
PCT/IB2014/002520 2013-08-08 2014-08-08 Dérivés acylés de phloridzine et d'isoquercétrine en tant qu'agents thérapeutiques anticancéreux et leurs procédés d'utilisation WO2015019193A2 (fr)

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CN109852645A (zh) * 2019-02-26 2019-06-07 山东省千佛山医院 一种槲皮素酯及其复合物
CN110279869A (zh) * 2019-07-01 2019-09-27 大连民族大学 一种葡聚糖-槲皮素前药聚合物的制备方法
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CN110540566A (zh) * 2019-08-08 2019-12-06 云南农业大学 紫云英苷衍生物及其应用
WO2019236765A1 (fr) * 2018-06-05 2019-12-12 Flagship Pioneering Innovations V, Inc. Polyphénols de catéchine acylés et leurs procédés d'utilisation pour le traitement du cancer
US10953027B2 (en) 2018-06-05 2021-03-23 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease
WO2021073132A1 (fr) * 2019-10-17 2021-04-22 南京康齐生物科技有限公司 Application d'acide nervonique de haute pureté dans le blanchiment et procédé de préparation d'acide nervonique de haute pureté
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US10953027B2 (en) 2018-06-05 2021-03-23 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease
US11813272B2 (en) 2018-06-05 2023-11-14 Flagship Pioneering Innovations V, Inc. Active agents and methods of their use for the treatment of metabolic disorders and nonalcoholic fatty liver disease
WO2019236765A1 (fr) * 2018-06-05 2019-12-12 Flagship Pioneering Innovations V, Inc. Polyphénols de catéchine acylés et leurs procédés d'utilisation pour le traitement du cancer
CN109852645A (zh) * 2019-02-26 2019-06-07 山东省千佛山医院 一种槲皮素酯及其复合物
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CN110302391B (zh) * 2019-07-01 2022-11-08 大连民族大学 一种葡聚糖-槲皮素聚合物载药胶束制剂及其制备方法
CN110279869A (zh) * 2019-07-01 2019-09-27 大连民族大学 一种葡聚糖-槲皮素前药聚合物的制备方法
CN110540566A (zh) * 2019-08-08 2019-12-06 云南农业大学 紫云英苷衍生物及其应用
WO2021073132A1 (fr) * 2019-10-17 2021-04-22 南京康齐生物科技有限公司 Application d'acide nervonique de haute pureté dans le blanchiment et procédé de préparation d'acide nervonique de haute pureté
RU2800457C1 (ru) * 2022-06-03 2023-07-21 федеральное государственное бюджетное образовательное учреждение высшего образования "Волгоградский государственный медицинский университет" Министерства здравоохранения Российской Федерации Синтез сложных эфиров флавоноидов нарингенина, кверцетина, гесперетина
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