CN114159427A

CN114159427A - Application of composition containing android alcohol in preparation of medicine for inhibiting growth of liver cancer cells or liver cancer stem cells

Info

Publication number: CN114159427A
Application number: CN202010953233.9A
Authority: CN
Inventors: 曾耀铭; 杨震; 叶淇台; 吴骏翃; 龚建贤; 常青; 曾德毓
Original assignee: Gansu Anzhuoxing Pharmaceutical Co ltd; Singapore Green Chemical Engineering Co ltd; Yuwei Biotechnology Co ltd
Current assignee: Gansu Anzhuoxing Pharmaceutical Co ltd; Singapore Green Chemical Engineering Co ltd; Yuwei Biotechnology Co ltd
Priority date: 2020-09-11
Filing date: 2020-09-11
Publication date: 2022-03-11

Abstract

The invention provides application of a composition containing antrocin alcohol (Antrocinol; (3aS,4R,6aS,10aR) -4- (hydroxymethyl) -7, 7-dimethyldecahydro-1H-naphthol [1,8a-c ] furan-1-one) in preparing a medicine for inhibiting growth of liver cancer cells or liver cancer stem cells, wherein the composition contains effective dose of antrocin alcohol.

Description

Application of composition containing android alcohol in preparation of medicine for inhibiting growth of liver cancer cells or liver cancer stem cells

Technical Field

The invention relates to application of a composition in preparing a medicine for inhibiting growth of liver cancer cells or liver cancer stem cells, in particular to application of a composition containing antrocin (Antrocinol) in preparing a medicine for inhibiting growth of liver cancer cells or liver cancer stem cells.

Background

Liver cancer refers to malignant tumor that occurs in or begins from the liver, and is the fifth most common cancer and the second most fatal cancer worldwide, and the incidence rate is high in asian countries, and the pathogenic cause of liver cancer may be caused by hepatitis B, hepatitis C or alcoholic cirrhosis, or by diseases including aflatoxin or non-alcoholic fatty liver disease. The FDA in the united states approved, in 2005, one of the Multiple Kinase Inhibitors (MKI) Sorafenib (Sorafenib) developed by bayer as first-line molecular targeted drugs in patients with advanced HCC; this sorafenib was the first anti-liver cancer drug approved by the FDA in the united states since last hundred years established in 6 months 1906. After the treatment of sorafenib, the survival time of the patients with advanced liver cancer (HCC) is prolonged by 2.8 months. Two MKI drugs, Regorafenib and Lenvatinib, developed by Bayer, were also used as second-line and first-line treatments for advanced HCC patients under FDA approval, and despite the approval of the two MKI drugs by FDA, the overall survival rate of HCC patients still showed non-inferiority to sorafenib (non-inhibitory response), and the overall results of the method of treatment with MKIs were disappointing due to Objective Remission Rates (ORR) of less than 10% and the emergence of drug-resistant phenotypes.

Antrodia cinnamomea (scientific name: Antrodia cinnamomea) has the functions of resisting inflammation, resisting oxidation and resisting vascular proliferation, is widely used for health-care food for preventing cancer and protecting liver at present, and past researches indicate that Antrodia cinnamomea is an effective antagonist for a plurality of cancer cells (such as liver cancer, lung cancer, breast cancer and the like) and can affect non-small lung cancer cells (non-small lung cancer) by inhibiting JAK/STAT3 information transmission channels.

Disclosure of Invention

The invention synthesizes a new small molecular compound, namely, the Antrocin (Antrocinol; (3aS,4R,6aS,10aR) -4- (hydroxymethyl) -7, 7-dimethyldecahydro-1H-naphthol [1,8a-c ] furan-1-ketone), which is obtained by adding hydroxyl to the No. 12 carbon atom position of the Antrocin (Antrocin).

The terms "a" or "an" in this specification are used to describe elements and components of the invention, and they are used merely for convenience in description and to give a basic idea of the invention, and further, this description should be construed to include one or at least one and also to include the plural unless it is explicitly stated otherwise. The terms "a" or "an," when used in conjunction with the term "comprising" in the claims, may mean one or more than one.

The term "or" in this specification means "and/or".

The invention relates to an application of a composition in preparing a medicine for inhibiting growth of liver cancer cells or liver cancer stem cells, wherein the composition comprises an effective amount of a compound (3aS,4R,6aS,10aR) -4- (hydroxymethyl) -7, 7-dimethyldecahydro-1H-naphthol [1,8a-c ] furan-1-one) shown in a formula (I) (Chinese name: antrocin; English name: Antrocinol; chemical formula: (3aS,4R,6aS,10aR), or a tautomeric form, a stereoisomer, a racemate, a metabolite, a polymorph, a salt or a solvate thereof.

The structural formula of the compound of formula (I) is as follows:

in one embodiment, wherein the tautomeric form, the stereoisomer, the racemate, the metabolite, the polymorph, the salt, or the solvate of the compound of formula (I) has the same effect on inhibiting growth of hepatoma cells or hepatoma stem cells as the published anti-cancer mechanism of action of the compound of formula (I).

In one embodiment, wherein the composition further comprises a pharmaceutically acceptable salt or carrier.

In one embodiment, the medicament is a medicament for treating or preventing metastasis or recurrence of liver cancer cells or liver cancer stem cells.

In one embodiment, the effective amount of the compound of formula (I) is 0.01 μ M to 1000 μ M.

In a preferred embodiment, the effective amount of the compound of formula (I) is 0.5 μ M to 1000 μ M.

In one embodiment, the effective amount is 0.08mg/kg to 0.8mg/kg (0.08mg/kgBW to 0.8mg/kgBW) of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate thereof, administered to a human per kg of body weight.

In a preferred embodiment, the effective amount is 0.24mg/kgBW to 0.64mg/kgBW of the compound of formula (I) or its tautomeric forms, its stereoisomers, its racemates, its metabolites, its polymorphs, its salts, or its solvates administered to a human.

In a more preferred embodiment, the effective amount is 0.4mg/kg BW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate thereof, administered to a human.

In a more preferred embodiment, the medicament is administered orally, by inhalation, or by injection.

The composition of the present invention may be mixed with carbolic acid, thymol, eucalyptol, benzalkonium chloride, cetylpyridinium chloride, methylparaben, hydrogen peroxide, domiphen bromide, fluoride, bio-enzyme, calcium, water, sweeteners (such as sorbitol, sucrose, sucralose, sodium saccharin and xylitol), etc. to make liquid or paste preparations, such as mouthwash, toothpaste or oral topical preparations.

The composition of the present invention may be used in the form of a spray in combination with a humectant (e.g., propylene glycol), an emulsifier (e.g., polysorbate, lanolin), etc., and may further form an antibacterial film on the medical device.

The compositions of the present invention can be in the form of solids, solutions, emulsions, dispersions, micelles, liposomes, and other products such as compositions containing one or more of the ingredients of the present invention as active ingredients, or mixed with organic or inorganic carriers or excipients suitable for enteral or parenteral administration. The active ingredients may be mixed for use, for example, in pharmaceutically acceptable, generally non-toxic vehicles such as tablets, pills, capsules, suppositories, solutions, emulsions, suspensions and any other suitable form. Carriers which may be used include glucose, lactose, gum arabic, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silicon dioxide, potato starch, urea, medium-chain triglycerides, dextran, and other carriers suitable for use in the preparation of formulations, solid, semi-solid, or liquid forms, and additionally, stabilizers, thickeners, and coloring agents and flavors may be used as an adjunct.

The compositions of the present invention may be in the form of tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs for oral administration, for example. Compositions for oral use may be prepared according to any of the known methods for preparing pharmaceutical compositions and such compositions may contain one or more sweetening agents such as sucrose, lactose or saccharin, flavoring agents such as peppermint, oil of wintergreen or cherry, coloring agents and preservatives to provide a pharmaceutically acceptable aesthetic and taste profile. Tablets incorporating the active ingredient in admixture with pharmaceutically acceptable non-toxic excipients may also be manufactured by known methods. Excipients which may be used are for example: (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch, potato starch or alginic acid; (3) binding agents, such as tragacanth, corn starch, gelatin or acacia, and (4) lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed as described in, for example, U.S. Pat. Nos. 4256108; 4160452, respectively; and 4,265,874 to produce osmotic therapeutic tablets with controlled release of the drug effect.

In some cases, the compositions for oral use may be in the form of hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.

Compositions embodiments of the present invention may also be in the form of sterile injectable suspensions. This suspension can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils such as sesame oil, coconut oil, peanut oil, cottonseed oil and the like, or synthetic fatty acid carriers such as ethyl oleate or the like. Buffers, preservatives, antioxidants and the like may be incorporated as necessary.

The composition of the present invention may also be mixed with a moisturizer (e.g., urea; PCA-Na) and a base and applied to the skin in the form of an ointment.

Drawings

FIG. 1 shows the chemical structure of antrocin ((3aS,4R,6aS,10aR) -4- (hydroxymethyl) -7, 7-dimethyldecahydro-1H-naphthol [1,8a-c ] furan-1-one).

FIG. 2 shows the results of inhibition of hepatoma cell proliferation by Antrocinol; figure 2A shows toxicity of antrocin on HCC cells; figure 2B shows that, in Huh7 cells, the effect of antrocin (Antrocinol) as an antiproliferative agent is superior to Sorafenib (Sorafenib) and cancer regel (Stivarga); figure 2C shows that the time and dose dependent properties of android alcohol inhibit Huh7 cell proliferation; figure 2D shows dose-dependent downregulation of cell proliferation and cell cycle-associated biomarkers of android cortisol; fig. 2E and 2F show that antrocin inhibited the cell number and cell population formation of Huh7 cells; all experimental results were replicated in triplicate and expressed as mean ± standard deviation.

FIG. 3 is a graph showing that to further understand the enhanced signaling pathway of Antrocinol in sensitive cells, referring to FIG. 3A, the present invention compares gene expression profiles between the most sensitive cells (Huh7) and the less sensitive cells (SNU387), both of which are derived from cBioportal website, and analyzed using the Gene set enrichment analysis GSEA4.0.3 software (Broad Institute), as shown in FIG. 3B, with the KRAS/MAPK signaling pathway being significantly up-regulated in Huh7 cells, RAS binding to and activating a protein kinase receptor upstream of the MAPK signaling pathway; FIGS. 3C and 3D show that android-well alcohol significantly inhibited the KRAS/MAPK signaling pathway in Huh7 cells. Notably, antrocin significantly reduced protein expression and activation of KRAS, ERK1/2, and AKT in a dose-dependent manner (fig. 3E). These findings indicate that antrocin provides a new option for HCC treatment by inhibiting KRAS and MAPK anti-cancer cell proliferation.

Figure 4 shows that antrocin induces apoptosis of hepatoma cells; figures 4A and 4B show that Annexin a5(Annexin V) analysis results demonstrate that android-well alcohol promotes apoptosis in Huh7 cells in a dose-dependent manner; FIG. 4C shows that as the dose of android alcohol was increased, the biomarkers Caspase-3, Caspase-7, Bak and Bax of apoptosis increased; and decreased expression of the anti-apoptotic biomarkers Bcl2 and Bcl-xL; FIG. 4D shows that the Bsx/Bcl2 ratio reflects apoptosis; the results of the experiments were all triplicated and expressed as mean ± standard deviation, ± representing p <0.05, ± representing p <0.01, ± representing p < 0.001; NS means no significant difference.

FIG. 5 shows the dryness of cancer cells in which android alcohol inhibited hepatoma cells; figure 5A shows that antrocin can reduce the size of Huh7 cell spherical tumors (Tumor sphere); the quantified values for the spherical tumors of figure 5B show that antrocin significantly inhibited the size of the tumors in a dose-dependent manner; figure 5C shows that inhibition of altrenol is associated with a decrease in protein labeling of cancer stem cells; the results of the experiments were all triplicated and expressed as mean ± standard deviation.

Figure 6 shows that antrocin inhibits hepatoma tumorigenesis in mice. Figure 6A shows tumor burden (tumor burden) versus time for Huh7 with a spherical tumor for different treatments, with the antrocin treatment significantly inhibiting tumor growth compared to the control and Sorafenib (Sorafenib) groups and the antrocin + Sorafenib combined treatment group was most effective with the lowest tumor burden among all groups; figure 6B shows the mean body weight versus time curves and the results show that all mice appear normal with no sudden decrease or increase in body weight, indicating that all treatments do not cause cytotoxicity; figure 6C is an immunohistological stain showing that decreased ERK and AKT expression, but increased BAX expression was observed in both the antrocin group and the combination treatment group compared to the control group; figure 6D is a comparison of the capacity of spherical tumor cells to form, and the number of tumor cells cultured in all groups under serum-free conditions, showing that the combined treatment group showed the lowest capacity of spherical tumor formation, followed by antrocin group, representing p <0.05, representing p <0.01, representing p < 0.001; NS means no significant difference.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.

Experimental example 1 preparation of Antrocinol

The novel small molecule android alcohol (Antrocinol; (3aS,4R,6aS,10aR) -4- (hydroxymethyl) -7, 7-dimethyldecahydro-1H-naphthol [1,8a-c ] furan-1-one) is synthesized according to the synthesis method disclosed in the prior literature (chem. Commun.,2016,52:12426-12429) for one of the intermediates of the asymmetric synthesis series of compounds with a given stereoconfiguration, namely, the android alcohol, by adding a hydroxyl group to the 12 th carbon atom of Antrocin.

The antrocin alcohol has the following chemical structural formula:

experimental example 2 analysis of biological Activity of Antrocinol

1. Activation of cryopreserved cells

The principle of activation of cryopreserved cells is rapid thawing to avoid the damage to the cells caused by the recrystallization of ice crystals, which leads to the death of the cells, and after the cells are activated, it takes several days or passes from generation to generation, and the cells are restored to normal (e.g., producing single antibodies or other proteins).

The method for quickly thawing the cryopreserved cells comprises the following steps: taking out the freezing tube from a liquid nitrogen or dry ice container, immediately placing the freezing tube into a 37 ℃ water bath for quick thawing, slightly shaking the freezing tube to completely thaw the freezing tube within 3 minutes, wiping the outside of the preserving tube with 70% alcohol, transferring the freezing tube into a sterile cell operating platform, taking out a thawed cell suspension, slowly adding the cell suspension into a culture container containing a culture medium (the dilution ratio is 1: 10-1: 15), uniformly mixing, and then placing CO into the culture container₂The culture medium is replaced after every other day of culture in the incubator.

2. Culture of human cancer cells

Human HCC cell lines SNU387, Mahlavu, Hep3B, Huh7, J5, and the like were obtained from the American Type Culture Collection (ATCC; Manassas, USA).

Cells were cultured in RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (Invitrogen brand by Life Technologies, Carlsbad, CA, usa) and in a carbon dioxide incubator at 37 ℃ and 5% humidity, passaging was performed when the cells grew to 95% confluence, or medium was changed every 72 hours.

To perform the cytotoxicity assay of the drug, cells were treated with varying concentrations of antrocin for different durations.

3. Android alcohol cytotoxicity test (cytoxicity)

3.5X 10 inoculations per well in 96-well plates³Cells of SNU387, Mahlava, Hep3B, Huh7 and J5 cell lines were cultured for 24 hours, the cells were treated with various concentrations of ambroxol, and 24 or 48 hours after the treatment, the treated cells were washed with PBS, further fixed with 10% Trichloroacetic acid (TCA) for 1 hour, and washed with distilled water, and the live cells were incubated for 1 hour at room temperature in 1% acetic acid containing 0.4% SRB (w/v), washed three times with 1% acetic acid to remove unbound dye and air-dried in 96-well plates, and then the attached dye was dissolved with 10mM Trizma base (Trizma base), and absorbance was read at a wavelength of 570nm in a 96-well plate spectrometer.

4. Western blot analysis of Antrocinol

Mu.g of protein samples were electrophoresed in 10% SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes using the Bio-Rad Mini-protein system (Bio-Rad Laboratories, Inc., Hercules, CA.A.), non-specific binding was blocked using 5% skim milk membranes configured with Tris-buffered saline (Tris-buffered saline; TBST) containing Polysorbate twenty (Tween 20) for 1 hour, and GSK-3 (1: 1000, Cell Signaling Technology, TCF 1/Protean, TCF 6754/TCF 671, Tech 1000, 361000, Tech-Dairy technologies; Tech-Dairy, Inc., Tech-Dairy, Inc., Teiry-Dairy, Inc., Cell-Dairy, Teiry, Inc., 5% skim milk membranes configured with Tris-buffered saline (Tris-buffered saline; TBST), santa Cruz corporation), Stat3 (1: 1000, Santa Cruz corporation), KLF4 (1: 1000, Santa Cruz Corp.), c Myc (1: 1000, Santa Cruz corporation), Nanog, CD133, KLF4 (1: 1000, Cell Signaling Technology), Slug (1: 1000, Cell Signaling Technology corporation) and CD44, SOX2, GAPDH (1: 500, Santa Cruz Co.) and the like at 4 ℃ overnight.

After overnight detection with primary antibody, the membrane was incubated with horseradish peroxidase (HRP) conjugated secondary antibody for 1 hour, then washed three times with PBS; protein band information was detected and visualized using an Enhanced Chemiluminescence (ECL) detection system (Thermo Fisher Scientific, llc, Waltham, usa MA) and protein bands were quantified using ImageJ software.

Example 3 results of animal test assays for Antrochol

1. Laboratory animal

The immunodeficient mice used in the present invention were purchased from leszidae biotech corp (NOD/SCID female mice about 4-6 weeks old), were bred in standard laboratory animals without specific pathogens, and after one week of acclimation, the experiments were started.

2. Animal experiments

Treating Huh7 cells with 0.05% trypsin-ethylene diamine tetraacetic acid (trypsin-EDTA) for 3-5 minutes to make the cells in suspension, adding a serum-containing culture medium to neutralize the action of trypsin, centrifuging at 1000rpm and 20 ℃ for 5 minutes, removing supernatant, gently scattering precipitated cells, dissolving the cells back in a culture solution with a proper volume, uniformly mixing, taking a little cell sap, counting the cells by a hemocytometer, diluting the cells to 10 per milliliter⁷About 0.15 ml of each cell was taken and dispensed into a 1.5 ml small centrifuge tube.

6 to 8 week old female NOD/SCID mice (n-20) from Lescow Biotechnology GmbH were bred in standard experimental animal pathogen free conditions. NOD/SCID mice (5/treatment 4 groups) 0.5X 10 in 0.5ml PBS⁶When the average size of the tumor reaches more than or equal to 150mm, the Huh7 liver cancer cells³The treatment was started between

days

7 and 10. Treatment group 1 included intraperitoneal (i.p) injections of 5mg/kg of android chonol in 0.5ml of PBS three times a week for up to 4 weeks; treatment group 2 included three intraperitoneal (i.p) injections of 10mg/kg Sorafenib (Sorafenib) in 0.5ml PBS for 4 weeks; treatment group 3 consisted of intraperitoneal (i.p) injections of 5mg/kg of ambroxol combined with 10mg/kg of Soraf (Soraf) three times a weekenib) up to 4 weeks long; while control treatment group 4 included intraperitoneal (i.p) injections of PBS three times per week.

The tumor size was measured once a week, the longest and shortest diameters of the tumor were measured using a light gauge, and the tumor size was measured by the same person during the experiment to ensure the accuracy of the measurement, and finally, the mice were sacrificed, tumor tissues were taken, photographed and stored, and fixed with formalin.

Tumor volume calculation formula:

the longest diameter is a, and the shortest diameter is b; tumor size ═ a × b²)/2. The change in tumor size is graphed by fold calculation.

Tumor size change fold (fold change in tumor volume) is tumor size (N)/tumor size (N-1).

N is the number of weeks.

Tumor growth was measured twice weekly and tumor volume (v) was calculated using the formula: when v is 2 × length/2, animals are followed for another 3 weeks after the last antrocin treatment (i.e. 7 weeks after tumor inoculation), tumor-small mice are allowed to follow for another 8 to 12 weeks, and then are sacrificed humanely under the treatment and control groups with extremely large tumors.

Assessment of tumor growth was performed for each experiment and statistical analysis was determined using Sigma plot 13 edition (Stystat Software, Calif., USA) with student's t assay, P <0.05 was considered statistically significant, all experimental animal procedures were approved and performed in accordance with institutional laboratory animal care and use Committee/group (IACUC/P) approval protocol LAC-2015-0386.

Results

The chemical structure of the novel small molecule, antrocin alcohol (antrocin) of the invention is shown in figure 1.

The invention discovers the anti-cancer capacity of the android alcohol in liver cancer (HCC) for the first time, and in order to research the influence of the android alcohol on the proliferation of liver cancer cells, the invention carries out the analysis of the survival rate of the sulfadopa B (SRB) on several liver cancer cell lines such as SNU387, Mahlavu, Hep3B, Huh7, J5 and the like, as shown in figure 2A, the corresponding maximum half-maximum of the android alcohol in the cell linesNumber inhibitory dose (IC)₅₀) SNU387 (5. mu.M), Mahlavu (4.9. mu.M), Hep3B (4.6. mu.M) and J5 (4.4. mu.M), respectively; notably, the Huh7 cell line is the most sensitive cell to android alcohol, with its IC₅₀It was 3.8. mu.M.

Furthermore, as shown in figure 2B, the present inventors found that the effect of android-well-alcohol as an antiproliferative agent for hepatoma cells or hepatoma stem cells was unexpectedly better than "android-well; antrocin (IC)₅₀9 μ M), "cancer regig; stivarga' (IC)₅₀11 μ M) and sorafenib; sorafenib (IC)₅₀12.5 μ M), and the results of fig. 2C also demonstrate that the time and dose dependent properties of antrocin inhibit Huh7 cell proliferation.

Given the results of fig. 2B, it was demonstrated that the android alcohol significantly inhibited survival (viability) and proliferation (proliferation) of HCC cells more effectively than the clinically used multiple kinase inhibitors canerger (Stivarga) and Sorafenib (Sorafenib). The present invention further assessed relevant biomarkers of the cell cycle by western blotting to confirm the antiproliferative effect of altrenol, and referring to fig. 2D, in Huh7 cell line, the antiproliferative effect of altrenol was dose-positively correlated with the decrease in biomarkers CDK2, CDK4, Ki67 and Cyclin D; in functional studies, as shown in fig. 2E and 2F, antrocin reduced cell number and cell colony formation dose-dependently.

To further understand the enhanced signaling pathway of anzhuyol in sensitive cells, referring to fig. 3A, the present invention compares gene expression between the most sensitive cells (Huh7) and the less sensitive cells (SNU387), both from the cbiotort website, and analyzed using the gene set enrichment analysis GSEA4.0.3 software (Broad Institute), which showed that KRAS/MAPK signaling pathway was significantly up-regulated in Huh7 cells (fig. 3B), RAS binding to and activating protein kinase receptors upstream of the MAPK signaling pathway. The antrocin can obviously inhibit KRAS/MAPK information transmission paths in Huh7 cells (FIG. 3C and FIG. 3D).

Recent studies found that down-regulation of MAPK signaling pathway could inhibit proliferation and growth of HCC cells, and therefore the present inventors believe that alterosol may be able to exert anti-proliferative capacity through inhibition of MAPK signaling pathway.

To provide evidence for the inhibitory effect of antrocin on the MAPK signaling pathway, we evaluated ERK1/2 and AKT downstream of the MAPK signaling pathway, noting that antrocin significantly reduced protein expression and activation of ERK1/2 and AKT in a dose-dependent manner (fig. 3E). These findings indicate that antrocin provides a new option for HCC treatment by inhibiting MAPK anti-cancer cell proliferation.

To investigate whether treatment with antrocin induces apoptosis in HCC cells, in one embodiment, Huh7 cells were treated with different doses of antrocin (0 μ M, 5 μ M, 10 μ M, 20 μ M and 40 μ M), respectively, and the apoptosis rate was assessed using apoptosis analysis and the associated protein marker changes in apoptosis were analyzed by Western blot (Western blot).

Referring to figures 4A and 4B, groups treated with android alcohol had more apoptotic cells than the control group, increasing the android alcohol dose from 5 μ M to 40 μ M significantly increased early and late apoptosis; results of Western blot analysis show that altrenol activated caspase-3 and caspase-7 in a dose-dependent manner to promote apoptosis in Huh7 cells, while anti-apoptotic proteins Bcl2 and Bcl-xL expression both decreased significantly as the altrenol dose increased from 5 μ M to 40 μ M, treatment with altrenol significantly increased expression of pro-apoptotic proteins Bax and Bak compared to control (fig. 4C), and a significant increase in Bax/Bcl2 ratio after increasing the altrenol dose (fig. 4D), which indicates that altrenol may induce apoptosis by inhibiting the MAPK signaling pathway.

Liver Cancer Stem Cells (LCSCs) are a group of cells with self-renewal (self-renewal) and differentiation capacity (differentiation) characterized by a high level of EpCAM, CD44, CD133, CD90 and CD13, and in HCC, the activation of MAPK promotes the manifestation of cancer sternness.

To provide evidence that alterosol inhibits liver cancer stem cells by down-regulating MAPK, we treated Huh7 cells with different doses of alterosol and performed cancer-like stem cell cultures and evaluated protein-tagged performance of LCSCs.

In one embodiment, as shown in fig. 5A and 5B, we found that all doses of altrenol treatment (5 μ M, 10 μ M, 20 μ M, and 40 μ M) significantly inhibited the size of Huh7 spherical tumors, which finding provided first evidence that altrenol reduced the LCSCs phenotype; in Huh7 cells, referring to fig. 5C, compared to the control group, the presence of proteins like Nanog, CD133, KLF4, CD44, OCT4, SOX2 and cMyc was inhibited by ancanol in a dose-dependent manner, and this data suggests that ancanol might provide a new alternative for targeted treatment of LCSCs against HCC cells.

Figure 6A shows the results of different treatments of Huh7 with spherical tumors, the alterosol treated group significantly inhibited tumor growth compared to the control and Sorafenib (Sorafenib) groups, although the Sorafenib (Sorafenib) treatment also showed some degree of inhibition, which was inferior to the alterosol, whereas the best tumor growth-delaying effect was observed in the combined alterosol + Sorafenib (Sorafenib) treated group, notably, all treatment regimens used in the present invention did not result in significant weight loss in the mice (figure 6B), indicating that all treatments were not cytotoxic to the animals throughout the experiment.

Immunohistological staining (IHC) of the tumor samples was consistent with in vitro observations, as shown in fig. 6C, treatment with antrocin reduced AKT and ERK protein expression and increased BAX, whereas the combined treatment group of antrocin + Sorafenib (Sorafenib) had a more pronounced effect on the tumor samples, and more importantly, significantly reduced the number of spherical tumor formations in mice of the antrocin treatment and the combined treatment group of antrocin + Sorafenib (Sorafenib) compared to the control and Sorafenib (Sorafenib) groups.

The results presented by the foregoing embodiments, that altrenol is more effective than canceriga (Stivarga) and even Sorafenib (Sorafenib) in inhibiting Huh7 cell growth, and the ERK/AKT protein signaling pathway in Huh7 cells is the pathway of greatest change in gene expression following treatment with altrenol, demonstrating that altrenol significantly inhibits the expression of AKT, p-AKT, ERK1/2, and p-ERK 1/2; results from global tumor analysis showed that treatment with antrocin significantly inhibited global tumor formation in Huh7 cells and expression of tumor stem cell biomarkers (including CD133, KLF4, CD44, OCT4, SOX2, and c-MYC) in Huh7 cells.

In addition, the sole use of the anxelol can obviously inhibit the tumor growth of mice implanted with Huh7 type cancer stem cells (cancer stem-like cells), and the combined use of the anxelol and the Sorafenib (Sorafenib) shows the synergistic effect of inhibiting the tumor, so that the anxelol can enhance the effect of the Sorafenib (Sorafenib), and the results of the immune tissue staining analysis of tumor samples show that the groups using the combination of the anxelol and the Sorafenib (Sorafenib) have reduced expression of ERK and AKT and increased expression of BAX; in the analysis of spherical neoplasia, the combination of antrocin and Sorafenib (Sorafenib) was the most effective in inhibiting spherical neoplasia ability.

In summary, the present invention provides the first invention of the anticancer ability of the android alcohol against hepatocellular carcinoma (HCC), and proves that the android alcohol is more effective in inhibiting the growth of hepatoma cells or hepatoma stem cells than the Sorafenib (Sorafenib), and the android alcohol may become a new drug substitute for hepatoma patients with poor therapeutic effect or drug resistance (resistance).

Claims

1. Use of a composition for the manufacture of a medicament for inhibiting the growth of a hepatoma cell or a hepatoma stem cell, said composition comprising an effective amount of a compound of formula (I):

or a tautomeric form thereof, a stereoisomer thereof, a racemate thereof, a metabolite thereof, a polymorph thereof, a salt thereof, or a solvate thereof.

2. The use of claim 1, wherein said composition further comprises a pharmaceutically acceptable salt or carrier.

3. The use of claim 1, wherein the medicament is a medicament for treating or preventing metastasis or recurrence of liver cancer cells.

4. The use of claim 1, wherein the effective amount is 0.08mg/kgBW to 0.8mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate thereof, administered to a human.

5. The use of claim 1, wherein the effective amount is 0.24mg/kgBW to 0.64mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate thereof, administered to a human.

6. The use of claim 1, wherein the effective amount is 0.4mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate thereof.

7. The use of claim 1, wherein the medicament is administered orally, by inhalation, or by injection.