WO2022052055A1 - Use of composition comprising antrocinol in preparation of medications for inhibiting growth of liver cancer cells or liver cancer stem cells - Google Patents

Abstract

The present invention provides a use of composition comprising antrocinol ((3aS, 4R, 6aS, 10aR)-4-(hydroxymethyl)-7,7-dimethyl decahydro-1H-naphthol[1, 8a-c]furan-1-one) in preparation of medications for inhibiting growth of liver cancer cells or liver cancer stem cells. The composition comprises an effective dose of antrocinol.

Description

Use of a composition containing android for the preparation of a drug for inhibiting the growth of liver cancer cells or liver cancer stem cells technical field

The invention relates to the use of a composition for preparing a drug for inhibiting the growth of liver cancer cells or liver cancer stem cells, in particular to the use of a composition comprising Antrocinol for preparing a drug for inhibiting the growth of liver cancer cells or liver cancer stem cells .

Background technique

Liver cancer is a malignant tumor that occurs in the liver or starts from the liver. It is also the fifth most common cancer in the world and the second most deadly cancer. Its incidence is relatively high in Asian countries. The cause of liver cancer may be caused by B. Hepatitis C, hepatitis C, or alcoholic cirrhosis, or diseases including aflatoxins or non-alcoholic fatty liver disease. In 2005, the US FDA approved Sorafenib, a multi-kinase inhibitor (MKI) developed by Bayer, as the first-line molecular target drug for patients with advanced HCC; Established in June 1906, it is the first anti-cancer drug approved for nearly 100 years. After treatment with sorafenib, the survival of patients with advanced liver cancer (Hepatocellular carcinoma; HCC) was prolonged by 2.8 months. Subsequently, the two MKI drugs Regorafenib and Lenvatinib developed by Bayer were also approved by the FDA as second-line and first-line treatments for advanced HCC patients. The non-inferiority response to sorafenib, and the overall outcome of the approach to MKIs, is in line with the objective response rate (ORR) of less than 10% and the emergence of resistant phenotypes. people disappointed.

Antrodia cinnamomea (scientific name: Antrodia cinnamomea) has anti-inflammatory, antioxidant and anti-angiogenesis functions, and has been widely used in anti-cancer and liver-protecting health food. It is an effective antagonist of lung cancer and breast cancer, etc., and can affect non-small lung cancer cells by inhibiting the JAK/STAT3 signaling pathway.

SUMMARY OF THE INVENTION

The present invention has synthesized a new small molecule compound Antrocinol (Antrocinol; (3aS, 4R, 6aS, 10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphthol [ 1,8a-c]furan-1-one), which is a hydroxyl group added to the No. 12 carbon atom of Antrocin. The present invention is the first to discover the anticancer ability of Antrocin in liver cancer.

The term "a" or "an" in this specification is used to describe the components and components of the present invention. This term is only for convenience of description and to give the basic concept of the present invention. Further, the description should be understood as including one or a At least one, and unless expressly stated otherwise, the singular also includes the plural. When used with the word "comprising" in the scope of the claims, the term "a" can mean one or more than one.

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

The present invention is the use of a composition for preparing a drug for inhibiting the growth of liver cancer cells or liver cancer stem cells, wherein the composition comprises an effective amount of a compound of formula (I) (Chinese name: Antrocinol; English name: Antrocinol; Chemical formula: (3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphthol[1,8a-c]furan-1-one) or its mutual Variant forms, stereoisomers thereof, racemates thereof, metabolites thereof, polymorphs thereof, salts thereof, or solvates 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) is The action mechanism for inhibiting the growth of liver cancer cells or liver cancer stem cells has the same effect as the disclosed anti-cancer action mechanism of the compound of formula (I).

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

In one embodiment, the drug is a drug 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 μM to 1000 μM.

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

In one embodiment, the effective amount is 0.08 mg/kg to 0.8 mg/kg (0.08 mg/kg BW to 0.8 mg/kg BW) of the compound of formula (I) or a tautomeric form thereof per kilogram of body weight administered to a human , its stereoisomers, its racemates, its metabolites, its polymorphs, its salts, or its solvates.

In a preferred embodiment, the effective amount is 0.24mg/kgBW～0.64mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, Its metabolites, its polymorphs, its salts, or its solvates.

In a more preferred embodiment, the effective amount is 0.4 mg/kg BW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemate, its metabolite, its Polymorphs, their salts, or their solvates.

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

The composition of the present invention can be mixed with carbolic acid, thymol, cineole, benzalkonium chloride, cetylpyridinium chloride, methylparaben, hydrogen peroxide, dumefene, fluoride, biological enzymes, calcium, water, Sweeteners (such as sorbitol, sucrose, sucralose, sodium saccharin and xylitol) and the like are mixed to prepare liquid or paste preparations, such as mouthwash, toothpaste or oral topical preparations.

The composition of the present invention can be mixed with a moisturizing agent (such as propylene glycol, propylene glycol), an emulsifier (such as: polysorbate, lanolin), etc. to be used in the form of a spray, and can further form a layer on the medical device Antibacterial film.

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 with organic or Inorganic carriers or excipients are mixed to be suitable for enteral or parenteral administration. The active ingredient may be mixed, for example, with pharmaceutically acceptable generally nontoxic vehicles such as tablets, pills, capsules, suppositories, solutions, emulsions, suspensions and any other suitable form for use. Carriers that can be used include dextrose, lactose, acacia, 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, in addition, stabilizers, thickening agents and coloring and flavoring agents may also be used as auxiliaries.

The compositions of the present invention may be in oral form, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions for oral use may be prepared according to various known methods of preparing pharmaceutical compositions, and the compositions may contain one or more sweetening agents such as sucrose, lactose or saccharin, such as peppermint, oil of wintergreen or cherry, etc. Flavoring agents, coloring agents and preservatives to provide pharmaceutical aesthetics and taste. Tablets incorporating the active ingredient with non-toxic pharmaceutically acceptable excipients can also be manufactured by known methods. Excipients that can be used are: (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) ) binders such as tragacanth, cornstarch, gelatin or acacia, and (4) lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated, or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a longer duration of action. For example, time delay materials such as glyceryl monostearate or glyceryl distearate can be used, and can also be coated by techniques such as those described in US Pat. Osmotic therapeutic tablet.

In certain instances, compositions for oral use may be in the form of hard gelatin capsules in which the active ingredient is admixed with an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules in which the active ingredient is mixed with an aqueous or oily medium, such as peanut oil, liquid paraffin or olive oil.

Composition embodiments of the present invention may also be in the form of sterile injectable suspensions. This suspension may 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 nontoxic 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, etc., or synthetic fatty acid carriers Such as ethyl oleate or the like. Buffers, preservatives, antioxidants, etc. can be combined as desired.

The composition embodiments of the present invention can also be mixed with a moisturizing agent (eg: urea; PCA-Na) and a base, and applied to the skin in the form of an ointment.

Description of drawings

Figure 1 is android ((3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphthol[1,8a-c]furan-1- ketone) chemical structure diagram.

Figure 2 shows the results of Antrocinol inhibiting the proliferation of liver cancer cells; Figure 2A shows the toxicity of Antrocinol to HCC cells; Figure 2B shows that in Huh7 cells, the effect of Antrocinol as an antiproliferative agent is better than that of Antrocinol Sorafenib and Stivarga; Figure 2C shows that androxenol inhibits Huh7 cell proliferation in a time- and dose-dependent manner; Figure 2D shows that androxenol dose-dependently down-regulates cell proliferation and cell cycle Relevant biomarkers; Figure 2E and Figure 2F show that android inhibits cell number and cell population formation of Huh7 cells; all experimental results are repeated in triplicate and are expressed as mean ± standard deviation.

Fig. 3 is to further understand the enhanced information transmission pathway of Andrewsinol in sensitive cells, referring to Fig. 3A, the present invention compares the relationship between the most sensitive cells (Huh7) and the less sensitive cells (SNU387). Gene expression. The gene expression maps of the two cells were obtained from the cBioportal website, and were analyzed using the Gene Set Enrichment Analysis GSEA 4.0.3 software (Broad Institute). The results are shown in Figure 3B. The KRAS/MAPK information transmission pathway is in Huh7 Cells were significantly up-regulated, and RAS would bind to and activate the protein kinase receptor upstream of the MAPK information transmission pathway; Figure 3C and Figure 3D show that android can significantly inhibit the KRAS/MAPK information transmission pathway in Huh7 cells. Notably, androxanthin significantly decreased protein expression and activation of KRAS, ERK1/2 and AKT in a dose-dependent manner (Fig. 3E). These findings suggest that Andrewsinol inhibits the proliferation of cancer cells by inhibiting KRAS and MAPK, providing a new option for HCC treatment.

Figure 4 shows that Andrews can induce apoptosis of liver cancer cells; Figure 4A and Figure 4B show that Annexin A5 (Annexin V) analysis results prove that Andrews can promote the apoptosis of Huh7 cells in a dose-dependent manner; Figure 4C shows that The apoptosis biomarkers Caspase-3, Caspase-7, Bak and Bax increased with increasing doses of androxanthin; while the expression of anti-apoptotic biomarkers Bcl2 and Bcl-xL decreased; Figure 4D shows the Bsx/Bcl2 ratio It reflects the situation of cell apoptosis; the experimental results were repeated in triplicate and expressed as mean ± standard deviation, * means p<0.05, ** means p<0.01, *** means p<0.001; NS means no significant difference.

Fig. 5 shows that Andrewsinol inhibits cancer cell stemness of hepatoma cells; Fig. 5A shows that androidxantol can reduce the size of Huh7 cell Tumor spheres; Fig. 5B shows that androidxantol can reduce the size of Tumor spheres in a dose-dependent manner. Figure 5C shows that the inhibitory effect of androxenol is related to the reduction of protein markers of cancer stem cells; the experimental results were repeated in triplicate and expressed as mean ± standard deviation.

Figure 6 shows that android inhibits the tumorigenesis of hepatocellular carcinoma in mice. Figure 6A shows tumor burden versus time for different treatments for Huh7 mice with spherical tumors, compared with control and Sorafenib groups, androxanthinol Treatment significantly inhibited tumor growth, while the combination treatment group of android and Sorafenib had the most significant effect, with the lowest tumor burden among all groups; Figure 6B shows the relationship between average body weight and time, the results show that all groups The weight of the mice all appeared to be normal, with no sudden loss or increase, indicating that none of the treatments caused cytotoxicity; Figure 6C is an immunohistochemical staining showing that, compared with the control group, both the android and combination treatment groups observed The expression of ERK and AKT was reduced, while the expression of BAX was increased; Figure 6D is a comparison of the formation ability of spherical tumor cells. Under serum-free conditions, the number of tumor cells cultured in all groups was compared, and the results showed that the combination treatment group showed the lowest Spherical tumor formation ability, followed by android group, * means p<0.05, ** means p<0.01, *** means p<0.001; NS means no significant difference.

detailed description

The technical solutions of the present invention are further described below through specific embodiments, which do not represent limitations on the protection scope of the present invention. Some non-essential modifications and adjustments made by others according to the concept of the present invention still belong to the protection scope of the present invention.

Experimental Example 1 Preparation of Androsol

The novel small molecule Antrocinol of the present invention (Antrocinol; (3aS, 4R, 6aS, 10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphthol [1,8a- c] Furan-1-one) is based on the synthesis of one of the intermediate products of the asymmetric synthesis series of compounds with a given stereo configuration disclosed in the previous literature (Chem. Commun., 2016, 52: 12426-12429) "Androsinol" The method was synthesized by adding a hydroxyl group on the 12th carbon atom of Antrocin.

The android has the following chemical structural formula:

(3aS,4R,6aS,10aR)-4-(hydroxymethyl)-7,7-dimethyldecahydro-1H-naphthol[1,8a-c]furan-1-one_

Formula (I)

Experimental Example 2 Analysis of the biological activity of Andrews

1. Activation of cryopreserved cells

The activation principle of cryopreserved cells is to thaw quickly to avoid ice crystals recrystallizing and causing damage to cells, leading to cell death. Normal (eg production of monoclonal antibodies or other proteins).

The method for quick thawing of cryopreserved cells is as follows: take the cryovial out of the liquid nitrogen or dry ice container, immediately place it in a 37°C water bath for rapid thawing, and gently shake the cryovial to thaw it all within 3 minutes. Wipe the outside of the preservation tube with % alcohol, move it into a sterile cell operating table, take out the thawed cell suspension, slowly add it to the culture container containing the medium (dilution ratio is 1:10~1:15), mix well, Then put it into a CO ₂ incubator and change the medium every other day.

2. Culture of human cancer cells

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

Cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Invitrogen brand from Life Technologies, Carlsbad, CA) at 37°C and 5% humidity In a carbon dioxide incubator, cells were passaged when they had grown to 95% confluence, or the medium was changed every 72 hours.

For drug cytotoxicity assays, cells were treated with different concentrations of androxenol for different durations.

3. Cytotoxicity test of android

Inoculate 3.5×10 ³ cells of SNU387, Mahlavu, Hep3B, Huh7 and J5 cell lines in each well of 96-well plate. After culturing for 24 hours, the cells were treated with different concentrations of android and 24 or 48 hours after treatment. The filtered cells were washed with PBS, fixed with 10% Trichloroacetic acid (TCA) for 1 hour, washed with distilled water, and the viable cells were incubated at room temperature for 1 hour in 1 hour with 0.4% SRB (w/v). % acetic acid, washed three times with 1% acetic acid to remove unbound dye and air-dried the 96-well plate, then dissolved the attached dye with 10 mM Trizma base and read at 570 nm in a 96-well plate spectrometer absorbance value.

4. Western blot analysis of Andrews

10 μg protein samples were electrophoresed in a 10% SDS-PAGE gel using the Bio-Rad Mini-Protean system (Bio-Rad Laboratories, Inc., Hercules, CA) and blotted into polyvinylidene fluoride (PVDF). ) membrane, incubate the membrane with 5% skim milk in Tris-buffered saline (Tris-buffered saline; TBST) containing Tween 20 for 1 hour to block Nonspecific binding was followed by targeting all β-catenin (1:1000, Cell Signaling Technology), CDK2 (1:1000, Cell Signaling Technology), CDK4 (1:1000, Cell Signaling Technology), GSK-3 ( 1:1000, Cell Signaling Technology Company), TCF1/TCF7 (1:1000, Cell Signaling Technology Company), LEF1 (1:1000, Cell Signaling Technology Company), p-Stat3 (1:1000, Santa Cruz Company), Stat3 (1:1000, Santa Cruz Company), KLF4 (1:1000, Santa Cruz Company), c Myc (1:1000, Santa Cruz Company), Nanog, CD133, KLF4 (1:1000, Cell Signaling Technology Company), Slug (1:1000, Cell Signaling Technology Co., Ltd.) and primary antibodies of CD44, SOX2, GAPDH (1:500, Santa Cruz Co., Ltd.) were reacted overnight at 4°C.

After overnight probing with primary antibodies, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour, and then washed three times with PBS; an enhanced chemiluminescence (ECL) detection system was used ( Thermo Fisher Scientific Inc., Waltham, MA) to detect and visualize protein band information, and quantify protein bands using ImageJ software.

The result of the animal test determination of embodiment 3 androxin alcohol

1. Experimental animals

The immunodeficient mice used in the present invention were purchased from Lesco Biotechnology Co., Ltd. (NOD/SCID female mice about 4-6 weeks old), and were raised under standard experimental animals without specific pathogens, and were domesticated for one week. After that, start the test.

2. Animal experiments

The Huh7 cells were treated with 0.05% trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA) for 3 to 5 minutes to make the cells in a suspended state, and serum-containing medium was added to neutralize the effect of trypsin, and the cells were heated at 1000rpm and 20°C. , Centrifuge for 5 minutes, then remove the supernatant, gently disperse the precipitated cells, then dissolve the cells back into an appropriate volume of culture medium and mix evenly, take a little cell fluid, count the cells with a hemocytometer, and dilute the cells into At a concentration of 10 ⁷ cells per milliliter, approximately 0.15 milliliters were aliquoted into small 1.5 milliliter centrifuge tubes.

6- to 8-week-old female NOD/SCID mice (n=20) from Lesco Biotechnology Co., Ltd. were housed under standard laboratory animal specific pathogen-free conditions. NOD/SCID mice (5/4 treatment groups) with 0.5 x 106 Huh7 hepatoma cells in 0.5 ml PBS, were treated between days ⁷ and 10 when tumors reached an average size of ≥150 ^mm3 . Treatment group 1 consisted of 5 mg/kg androxenol in 0.5 ml PBS injected three times per week for up to 4 weeks; treatment group 2 consisted of 10 mg intraperitoneal (ip) injected in 0.5 ml PBS three times per week /kg Sorafenib for up to 4 weeks; Treatment Group 3 consisted of three weekly intraperitoneal (ip) injections of 5 mg/kg androxitol combined with 10 mg/kg Sorafenib for up to 4 weeks; and Control group Treatment group 4 consisted of three weekly intraperitoneal (ip) injections of PBS.

The tumor size was measured once a week, and the longest and shortest diameters of the tumor were measured with a cursor ruler. To ensure the accuracy of the measurement, the same person measured the tumor size during the experiment. The tumor tissue was photographed and archived, and then fixed with formalin.

Tumor volume calculation formula:

The longest diameter is a, the shortest diameter is b; tumor size=(a×b ² )/2. Changes in tumor size are graphed as fold calculations.

The fold change in tumor volume = tumor size (N)/tumor size (N-1).

N is the number of weeks.

Tumor growth was measured twice a week and tumor volume (v) was calculated using the formula: v = (width) 2 x length/2, and animals were followed for 3 weeks after the last android treatment (ie 7 weeks after tumor inoculation). ), mice with small tumors were allowed to continue tracking for an additional 8 to 12 weeks before being humanely sacrificed in treated and control groups with extremely large tumors.

Tumor growth was assessed for each experiment and statistical analysis was determined with Student's t-test using Sigma plot version 13 (Stystat Software, CA, USA), p < 0.05 was considered statistically significant, and all experimental animal procedures were approved and All were performed in accordance with the Institutional Animal Care and Use Committee/Panel (IACUC/P) approved protocol LAC-2015-0386.

result

The chemical structure of the novel small molecule Antrocinol of the present invention is shown in FIG. 1 .

The present invention discovers for the first time the anti-cancer ability of androxenol in liver cancer (HCC). Dopaphenol B (SRB) cell viability analysis, as shown in Figure 2A, the maximal half-inhibitory dose (IC ₅₀ ) of Andrews in these cell lines were SNU387 (5μM), Mahlavu (4.9μM), Hep3B (4.6 μM) and J5 (4.4 μM); it is worth noting that the Huh7 cell line was the most sensitive to androxenol with an _IC50 of 3.8 μM.

In addition, as shown in Figure 2B, the present invention found that the effect of Antrocin as an anti-proliferative agent for liver cancer cells or liver cancer stem cells was unexpectedly stronger than that of "Antrocin" (IC ₅₀ : 9 μM), "Antrocin;Stivarga""(IC ₅₀ : 11 μM) and "Sorafenib;Sorafenib" (IC ₅₀ : 12.5 μM) were both good, and the results in Figure 2C also proved that androxenol inhibited Huh7 cells in a time- and dose-dependent manner. proliferation.

In view of the results in Figure 2B, it was confirmed that androxitol was significantly more effective than the clinically used multiple kinase inhibitors Stivarga and Sorafenib in inhibiting the viability and proliferation of HCC cells ( proliferation). The present invention further evaluates cell cycle-related biomarkers by Western blotting to confirm the anti-proliferative effect of androxenol. Referring to Figure 2D, in the Huh7 cell line, the anti-proliferative effect of androxenol is related to that of CDK2, CDK4, Ki67 and Cyclin. The reduction in biomarkers such as D was dose-related; in functional studies, as shown in Figures 2E and 2F, androxenol dose-dependently reduced cell number and colony formation.

In order to further understand the enhanced information transmission pathway of androxenol in sensitive cells, referring to Figure 3A, the present invention compares the gene expression between the most sensitive cells (Huh7) and the less sensitive cells (SNU387), The gene expression profiles of both cells were obtained from the cBioportal website and analyzed using the Gene Set Enrichment Analysis GSEA4.0.3 software (Broad Institute), and the results showed that the KRAS/MAPK signaling pathway was significantly upregulated in Huh7 cells (Fig. 3B). ), RAS binds to and activates protein kinase receptors upstream of the MAPK signaling pathway. Androsinol could significantly inhibit the KRAS/MAPK signaling pathway in Huh7 cells (Figure 3C and Figure 3D).

Recent studies have found that the down-regulation of the MAPK information transmission pathway can inhibit the proliferation and growth of HCC cells. Therefore, the present inventors believe that android may have anti-proliferative ability by inhibiting the MAPK information transmission pathway.

To provide evidence for the inhibitory effect of androxenol on the MAPK messaging pathway, we evaluated ERK1/2 and AKT downstream of the MAPK messaging pathway. It is worth noting that androxenol significantly reduced ERK1/2/AKT in a dose-dependent manner. 2 and AKT protein expression and activation (Figure 3E). These findings suggest that androxenol inhibits the proliferation of cancer cells by inhibiting MAPK, providing a new option for HCC treatment.

The MAPK messaging pathway is an evolutionarily conserved kinase that regulates basic biological processes, including cell survival and apoptosis. To investigate whether androxanthin treatment can induce apoptosis in HCC cells, in one embodiment, The present invention treats Huh7 cells with different doses of Andrews (0 μM, 5 μM, 10 μM, 20 μM and 40 μM) respectively, and uses apoptosis analysis to evaluate the apoptosis rate and Western blotting (Western blot) to analyze the apoptosis rate. Associated protein marker changes.

Compared with the control group, referring to Fig. 4A and Fig. 4B, there were more apoptotic cells in the group treated with androxenol, and increasing the dose of androxenol from 5 μM to 40 μM significantly increased early and late apoptosis; protein The results of Western blot analysis showed that androxenol activated caspase-3 and caspase-7 in a dose-dependent manner to promote the apoptosis of Huh7 cells, while with the dose of androxenol increasing from 5 μM to 40 μM, anti-apoptotic effect was observed. The expression of proteins Bcl2 and Bcl-xL were significantly decreased. Compared with the control group, treatment with android significantly increased the expression of the pro-apoptotic proteins Bax and Bak (Fig. 4C). The ratio of Bax/Bcl2 after increasing the dose of android significantly increased (Fig. 4D), these findings suggest that android may induce apoptosis by inhibiting the MAPK signaling pathway.

Liver cancer stem cells (LCSCs) are a group of cells with self-renewal and differentiation ability, which are characterized by the high expression of EpCAM, CD44, CD133, CD90 and CD13. In HCC, MAPK Activation of cancer promotes the manifestation of cancer stemness.

To provide evidence that androxenol inhibits HCC stem cells by downregulating MAPK, we treated Huh7 cells with various doses of androxenol, performed carcinoid stem cell culture and assessed the protein labelling performance of LCSCs.

In one embodiment, as shown in Figures 5A and 5B, we found that all doses of androxenol treatment (5 μM, 10 μM, 20 μM, and 40 μM) significantly inhibited the size of Huh7 spherical tumors, a finding that provides androxenol First evidence to reduce the phenotype of LCSCs; in Huh7 cells, referring to Figure 5C, androxenol inhibited proteins such as Nanog, CD133, KLF4, CD44, OCT4, SOX2 and cMyc in a dose-dependent manner compared to controls , against HCC cells, the data suggest that androxenol may provide a new option for targeted therapy of LCSCs.

Figure 6A shows the results of different treatments for Huh7 mice with spherical tumors. Compared with the control and Sorafenib groups, the androxantol treatment group significantly inhibited tumor growth, although the The treatment of rafenib (Sorafenib) also showed a certain degree of inhibition, but the inhibitory effect was worse than that of the androxenol group, and the best effect could be observed in the combination treatment group of androxenol + sorafenib (Sorafenib). Delayed tumor growth effect, it is worth noting that none of the treatment regimens used in the present invention resulted in significant weight loss in mice (Figure 6B), indicating that all treatments were not cytotoxic to animals throughout the experiment.

The immunohistochemical staining (IHC) of the tumor samples was consistent with the in vitro observations. As shown in Figure 6C, treatment with androxenol decreased the expression of AKT and ERK proteins, and increased BAX, while androxenol + sorafenib ( The effect of the combination treatment group with Sorafenib on tumor samples was more pronounced, and more importantly, compared with the control group and the Sorafenib (Sorafenib) group, the combination treatment with androidxitol and androidxitol + sorafenib (Sorafenib) ) in the combination treatment group showed a significant reduction in the number of spherical tumor formations.

According to the results presented in the previous embodiments, Andrews can more effectively inhibit the growth of Huh7 cells than Stivarga or even Sorafenib, and the ERK/AKT protein information transmission pathway in Huh7 cells is Andrews. The path with the greatest change in gene expression after glucosinolate treatment, confirming that droitol can significantly inhibit the expression of AKT, p-AKT, ERK1/2 and p-ERK1/2; the results of spherical tumor analysis showed that drogenol treatment significantly inhibited the expression of AKT, p-AKT, ERK1/2 and p-ERK1/2. Spherical tumor formation of Huh7 cells and markedly inhibited expression of tumor stem cell biomarkers including CD133, KLF4, CD44, OCT4, SOX2 and c-MYC in Huh7 cells.

In addition, the use of androxenol alone significantly inhibited tumor growth in mice implanted with Huh7 cancer stem-like cells, and the combination of androxenol and sorafenib showed synergistic tumor inhibition. The results of the immunohistochemical staining analysis of tumor samples showed that in the group of combined use of android and sorafenib, its ERK The expression level of AKT and AKT was decreased and the expression level of BAX was increased; in the spherical tumor formation assay, the combination of android and Sorafenib was the most effective in inhibiting the spherical tumor formation ability.

Based on the above results, the present invention is the first invention to propose the anticancer ability of androxenol on hepatocellular carcinoma (HCC), and proves that androxenol is more effective in inhibiting liver cancer cells or liver cancer stem cells than sorafenib. For patients with liver cancer who are ineffective or resistant to sorafenib treatment, androxenol may become a new alternative drug.

Claims (7)

1. The application of a composition in the preparation of a medicine for inhibiting the growth of liver cancer cells or liver cancer stem cells, characterized in that the composition comprises an effective amount of the compound of formula (I):

   

   or its tautomeric form, its stereoisomer, its racemate, its metabolite, its polymorph, its salt, or its solvate.
2. The use according to claim 1, wherein the composition further comprises a pharmaceutically acceptable salt or carrier.
3. The application according to claim 1, wherein the medicine is a medicine for treating or preventing the metastasis or recurrence of liver cancer cells.
4. The use according to claim 1, wherein the effective amount is 0.08mg/kgBW～0.8mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomeric form administered to the human body compounds, their racemates, their metabolites, their polymorphs, their salts, or their solvates.
5. The use according to claim 1, wherein the effective amount is 0.24mg/kgBW～0.64mg/kgBW of the compound of formula (I) or its tautomeric form, its stereoisomeric form administered to the human body compounds, their racemates, their metabolites, their polymorphs, their salts, or their solvates.
6. The use according to claim 1, wherein the effective amount is 0.4 mg/kg BW of the compound of formula (I) or its tautomeric form, its stereoisomer, its racemic compounds, their metabolites, their polymorphs, their salts, or their solvates.
7. The use according to claim 1, wherein the medicine is administered orally, inhaled or by injection.

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