CN108653297B - Application of ganodermanondiol with cell nucleus cathepsin L as target in pharmacy - Google Patents

Abstract

The invention discloses an application of ganodermanondiol with cell nucleus endoplasmin L as a target spot in pharmacy. The experiments of proliferation and apoptosis of in vitro anticancer cells and cell cycle of ganodermanone diol show that ganodermanone diol reduces truncated CUX1 factor (activated form) by inhibiting the activity of CL in colon cancer cell nucleus, and then down regulates the expression of Cyclin E2, finally makes colon cancer cells stay in the S phase of the cell cycle, thereby realizing the effect of inhibiting the growth of cancer cells. Therefore, the ganoderic ketone diol can be used for preventing or treating diseases related to colorectal cancer, including the rectal cancer, the colon cancer, and the diseases related to constipation, hematochezia, abdominal pain, abdominal spasm, intestinal cancer cell infiltration and metastasis and the like caused by the rectal cancer or the colon cancer, and is suitable for popularization and application.

Description

Application of ganodermanondiol with cell nucleus cathepsin L as target in pharmacy

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of ganoderic ketodiol in resisting colorectal cancer.

Background

Large bowel cancers include colon and rectal cancers, and refer to malignancies occurring in the cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum, one of the most common digestive tract malignancies. Recent data of the world health organization show that the number of colorectal cancer deaths is the third of cancer deaths, more than 120 million patients are diagnosed with colorectal cancer every year in the world, and more than 70 million patients die of colorectal cancer directly or indirectly. The incidence rate of the colon cancer is remarkably different in all regions, and generally, the incidence rate of the colon cancer is higher in men than in women. In addition, the incidence of large bowel cancer increases with age. The colorectal cancer is one of the most common malignant tumors in China, and among 136 ten thousand worldwide colorectal cancers diagnosed in 2012, 25 ten thousand new cases in China account for 18.6% of the new cases of the colorectal cancer worldwide, and the colorectal cancer is the country with the most new cases of the colorectal cancer worldwide every year.

Colorectal cancer originates in the mucosal epithelium or submucosal mesenchymal tissue of the large intestine, and the molecular pathogenesis of the cancer is genetic mutation caused by genetic or environmental factors. Epidemiological research shows that: social development, lifestyle and dietary structure are closely related to colorectal cancer. At present, the treatment of colorectal cancer is mainly surgical resection and is combined with radiotherapy, chemotherapy or targeted therapy for comprehensive treatment. The postoperative survival rate of 5 years is closely related to pathological stages of colorectal cancer, the postoperative survival rate of 5 years of early colorectal cancer can reach more than 80 percent, and the survival rate of middle and late stages is only 40 to 10 percent. Chemotherapy is mainly aimed at preventing and reducing recurrence and metastasis, thereby improving the long-term efficacy of surgical treatment, which can be classified into preoperative, intraoperative, and postoperative chemotherapy. The main chemotherapy drugs at present are 5-fluorouracil (5-Fu) as the main drug, and also include calcium folinate (LV), capecitabine, oxaliplatin, irinotecan, bevacizumab which is a biological target therapy drug, and the like. The effective rate of 5-Fu to colorectal cancer is only about 20%, and the combined medication scheme can improve certain effective rate. The current chemotherapy drugs for treating colorectal cancer have the defects of low efficiency, low selectivity and great toxic and side effects. For example, a chemotherapy scheme for treating advanced colorectal cancer by combining 5-FU and LV causes toxic and side effects such as gastrointestinal reaction, bone marrow suppression and the like, oxaliplatin causes toxicity in a nervous system, and a standard scheme for treating advanced colorectal cancer by using irinotecan in combination with 5-FU and LV causes cholinergic syndrome, delayed diarrhea and the like. Monoclonal antibody drugs targeting the blocking of the biological activity of Vascular Endothelial Growth Factor (VEGF) or Epidermal Growth Factor (EGF) pose a threat of failure due to mutations in downstream signaling pathway molecules, in addition to toxicity problems. The search for new drug targets and the development of anti-colorectal cancer drugs with high efficiency and low toxic and side effects are still very urgent needs in the field.

Cathepsin L (Cathepsin L, CL) is one of the members of the cysteine protease family, is 333 amino acids in size, widely exists in various normal tissues and tumor cells of the human body, and is a main secreted protein of lysosomes. Its main functions are to degrade proteins, hydrolyze some precursor proteins (zymogens and prohormones) to their active forms, or activate other proteolytic enzyme systems, thereby participating in various physiological activities of the body. Such as: antigen presentation, spermatogenesis and ovum genesis, bone matrix degradation, central nervous system maturation and development, etc. Under pathological conditions, due to abnormal transcription, translation, modification, localization, maturation, pH change and interaction with inhibitors, the compounds are widely involved in the development of various diseases, such as tumor infiltration and metastasis, arthritis, osteoporosis, Alzheimer's disease, multiple sclerosis and other chronic inflammatory diseases. In recent years, many scholars at home and abroad have studied CL, and found that CL is highly expressed in various malignant tumors such as prostate cancer, melanoma, gastric cancer, colorectal cancer, pancreatic cancer and the like.

CL has been found to exist in a number of different forms within cells, and has been found to enter the nucleus to enhance the DNA binding capacity of CUX1 by cleaving the nuclear transcription factor CUX1 precursor, thereby promoting cell cycle progression in cells. It has also been found that the cell cycle progression of colorectal cancer cells depends on the activity of the enzyme CL in the nucleus. In a study involving tissue sections from 186 colorectal cancer patients, it was found that the level of CL in the nucleus of colorectal cancer cells was positively correlated with the progression and recovery of colorectal cancer. Although these phenomena suggest that nuclear CL is closely related to colorectal cancer, there is no report on anti-colorectal cancer drugs targeting nuclear CL. Therefore, the discovery of the CL inhibitor in the colon nucleus has significant significance for the development of a new drug for highly selective treatment of the colon cancer.

Ganoderma lucidum is a fungus of Ganoderma of Polyporaceae of Basidiomycetes, is a precious product of traditional Chinese medicine, and has a long medicinal history in China. Ganodermanondiol (GMD) is a triterpene compound isolated from Ganoderma fruiting body, and has no reported activity against carcinoma of large intestine. The research on the anti-colorectal cancer activity of the ganoderine diol on SW480 and SW1116 cells of gene microsatellite stabilized (MSS) colon cancer cells by using an MTT method has been carried out for years, but no obvious activity of the ganoderine diol is found. In our research, we use a more sensitive and stable CCK-8 cytotoxicity and proliferation detection method to carry out anti-colorectal cancer activity research and action target analysis on a gene microsatellite instability (MSI) type colorectal cancer cell HCT116, and the evidence shows that the ganodermanondiol has a very significant inhibitory effect on the colorectal cancer cell, and the mechanism of the action is that the cell cycle of the cancer cell is inhibited to achieve an anti-cancer effect by specifically targeting cathepsin CL in the nucleus of the colorectal cancer cell. The discovery has important significance for further developing a novel anti-colorectal cancer drug which has the potential of overcoming the defects of low specificity and large toxic and side effects of the existing drugs.

Disclosure of Invention

The invention provides an application of ganoderic ketone diol in preparing a medicament for preventing or treating colorectal cancer-related diseases, preferably rectal cancer, colon cancer, constipation, hematochezia, abdominal pain, abdominal spasm, intestinal cancer cell infiltration and metastasis and other related diseases caused by the rectal cancer or the colon cancer.

Further, the present invention also provides a pharmaceutical composition for preventing or treating a disease associated with colorectal cancer, comprising ganoderine diol or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier and/or diluent, wherein ganoderine diol or a pharmaceutically acceptable salt thereof inhibits colorectal cancer cells with an IC50 of less than 10 μ M, preferably less than 2.5 μ M.

The pharmaceutical compositions of the present invention may be in the form of preparations for oral or parenteral administration (e.g., intravenous, subcutaneous or intramuscular administration). The preparation form of the oral administration is tablets, sustained release tablets, controlled release tablets, troches, hard or soft capsules, aqueous or oil suspensions, emulsions, dispersible powders or granules, syrups or elixirs, dropping pills, pellets or oral solutions, and the preparation form of the parenteral administration is sterilized aqueous or oil solutions, sterile powders, liposomes, emulsions, microemulsions, nano-emulsions or microcapsules.

The pharmaceutical compositions of the present invention may be obtained by conventional methods using conventional pharmaceutical excipients well known in the art. Thus, compositions for oral use may contain, for example, one or more coloring, sweetening, flavoring and/or preservative agents.

Suitable pharmaceutically acceptable excipients for tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate; granulating and disintegrating agents such as corn starch and alginic acid; binders such as starch; lubricants such as magnesium stearate, stearic acid or talc; preservatives, for example ethyl or propyl p-hydroxybenzoate, and antioxidants, for example ascorbic acid. Tablet formulations may be uncoated or may be coated to modify their disintegration and subsequent absorption of the active ingredient in the gastrointestinal tract or to improve their stability and/or appearance, in any event using conventional coating agents and procedures well known in the art.

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, or soft gelatin capsules wherein the active ingredient is mixed with water or an oil, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions generally contain the active ingredient in micronized form and one or more suspending agents, for example, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, for example, condensation products of lecithin or of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example heptadecaoxyethylene cetyl alcohol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives (e.g., ethyl or propyl paraben), antioxidants (e.g., ascorbic acid), coloring, flavoring, and/or sweetening agents (e.g., sucrose, saccharin and aspartame).

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may also contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient in association with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are as described above. Additional excipients, for example sweetening, flavoring and coloring agents, are also present.

The pharmaceutical compositions of the present invention may also take the form of an oil-in-water emulsion. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be, for example, natural gums such as gum acacia or gum tragacanth, natural phosphatides such as soya bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate), and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavoring and preservative agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, a preservative, a flavouring and/or a colouring agent.

The pharmaceutical compositions may also be in the form of a sterile aqueous or oily suspension for injection which may be formulated according to known methods using one or more suitable dispersing or wetting agents and suspending agents, such as those described above. The sterile injectable preparation may also be a sterile injectable aqueous or oleaginous suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form may vary depending upon the host treated and the particular route of administration. For example, formulations intended for oral administration to humans typically contain, for example, 0.5mg to 2g of the active agent, together with appropriate and conventional amounts of excipients (about 5 to 98% by weight of the total composition). Unit preparations typically contain about 1mg to about 500mg of the active ingredient.

When the ganodermanondiol of the present invention is used in preparing medicine for treating or preventing carcinoma of large intestine, the effective dosage of ganodermanondiol on cancer cell line is 0.01-10 micron, preferably 0.8-5 micron.

The ganodermanone diols of the present invention may be used in monotherapy or in combination with one or more other substances and/or indications. Such combination therapy may be achieved by the simultaneous, sequential or separate administration of the individual therapeutic components. Concurrent therapy may be in the form of a single tablet or in the form of separate tablets. For example, in the treatment of large bowel cancer, the following main categories of treatment may also be included: antineoplastic agents, immunopotentiators.

Drawings

FIG. 1 shows the structure of ganodermanondiol;

FIG. 2 evaluation of cancer cell growth inhibition in vitro by Ganodermanondiol (GMD);

FIG. 3 Ganodermanonediol (GMD) arrests the cell cycle of colorectal cancer in S phase;

FIG. 4 is a diagram of gene chip analysis of gene expression changes in colorectal cancer cells after ganoderic acid diol (GMD) treatment;

FIG. 5 Effect of ganodermanone diol (GMD) on the inhibition of cathepsin L (cathepsin L);

FIG. 6 different nuclear cathepsin L (cathepsin L) expression levels;

FIG. 7 Ganodermanone diol (GMD) reduces the level of expression of a truncated CUX1 transcription factor in the nucleus of a large intestine cell.

Detailed Description

Example 1: anti-colorectal cancer activity experiment and cytotoxicity experiment based on cell growth detection

1.1 subjects: colorectal cancer (HCT116 cell line, Caco2 cell line), breast cancer (MCF-7 cell line), lung cancer (HLC-1 cell line), liver cancer (HepG2 cell line), stomach cancer (HGC-27 cell line), prostate cancer (LNCaP cell line), uterine cancer (Hela cell line) and leukemia (HL60 cell line), 4 kinds of human normal cells (normal skin fibroblast NHDF, normal umbilical cord cell HUC-F2, lung normal fibroblast TIG-1, lung fibroblast HF 19).

1.2 Experimental drugs: ganodermanondiol (obtained from ChemFaces, Inc., Wuhan, Hubei, HPLC. gtoreq.98%).

1.3 Experimental reagents and equipment: CCK-8 kit (available from Dojindo, Japan), 5-fluorouracil (5-Fu, available from InvivoGen, USA), super plate reader (MD, Flexstation 3).

1.4 Experimental methods: each one ofThe seed cell is 1 × 10⁴The density of each hole is inoculated in a 96-hole cell culture plate, after overnight culture cells grow into a monolayer, ganoderic diketone alcohol solution diluted by cell culture medium gradient is added, and meanwhile, a positive drug (5-FU) control group, a normal cell control group and a sample toxicity control group are set in an experiment. After the culture plate was cultured at 37 ℃ for 3 days, the proliferation and toxicity of the cells were measured by using CCK8 cell proliferation and toxicity measuring method using a super microplate reader (Flexstation 3) manufactured by MD. The half inhibitory concentration was then calculated using the Probit Regression function of the software SPSS (IC 50). The Selection Index (SI) of the sample was finally determined by the formula IC50 (cancer cells)/IC 50 (normal cells).

The results show that the IC50 of ganoderic ketone diol on colorectal cancer cells HCT116 and Caco2 is 0.78 +/-0.5 mu M and 2.25 +/-2.0 mu M respectively, and the activity of ganoderic ketone diol on colorectal cancer cells is remarkably and selectively inhibited, wherein the IC50 of ganoderic ketone diol on colorectal cancer cells HCT116 and Caco2 is far lower than that on breast cancer cells 72.08 mu M and other cancer cells and normal cells is more than 200 mu M (figure 2).

Example 2: experiment on apoptosis and cell cycle influence of colorectal cancer cells:

2.1 subjects: colorectal cancer HCT116 cell line.

2.2 Experimental drugs: ganodermanone diol (from ChemFaces, Inc., Wuhan, Hubei, 98% or more by HPLC), Ganoderma manone (from ChemFaces, Inc., Wuhan, Hubei, 98% or more by HPLC), and thymidine (from Beiga Biotech, Inc., Nanjing).

2.3 Experimental reagents and equipment: the fluorochrome PI (propidium iodide, available from Shenzhen Leber Biotech, Inc.), the apoptosis annexin V-FITC kit (available from Abcam, Inc., UK), and the flow cytometer SH800 (Sony, Japan).

2.4 Experimental methods colorectal cancer cells HCT116 3 × 10⁵The density of each well was inoculated in a 6-well plate, and after overnight culture to grow a monolayer of cells, the culture medium containing 10% fetal bovine serum was changed to that containing 1% fetal bovine serum. After 16h of culture in the low serum culture medium, the cells were added to a 100. mu.M sample solution, and the triterpene compound, namely, polypyristin (GM) which has a similar structure but no significant inhibitory activity on the proliferation of colorectal cancer cells was obtainedT) and thymidine (thymidine), a cell S-cycle blocker, served as negative and positive controls, respectively. Continuously culturing at 37 ℃, collecting cells after 24h, 48h and 72h respectively, fixing the cells by ethanol, performing PI staining, and finally performing cell cycle detection by a flow cytometer. For the apoptosis effect test, annexin V-FITC reagent is used for detection.

The results showed that GMD had no significant effect on apoptosis of colon cancer cells, but rather arrested the colon cancer cells in the S phase at the cell cycle, thereby inhibiting their growth (fig. 3).

Example 3: gene chip analysis experiment:

3.1 subjects: colorectal cancer HCT116 cell line.

3.2 Experimental drugs: ganodermanone diol (obtained from ChemFaces GmbH, Wuhan, Hubei province, HPLC ≥ 98%), and Ganoderma manone (obtained from ChemFaces GmbH, Wuhan, Hubei province, HPLC ≥ 98%).

3.3 Experimental reagents and equipment: agilent Human expression profiling Gene chip Sureprint G3Human GEMicroarray 8x60Kv2.0 (available from Agilent, USA), RNA extraction kit (available from InvivoGen, USA).

3.4 Experimental methods: HCT116 cells were first seeded into 10cm diameter dishes and cultured until the cell monolayer covered 90% of the surface, then 100. mu.M ganodermanone diol (GMD) was added, along with a solvent DMSO solvent control and a Ganoderma lucidum ketone (GMT) negative control. After culturing at 37 ℃ for 48 hours, the cells were collected and RNA was extracted, followed by human genome expression profiling using an Agilent human expression profiling gene chip (Cell Innovator, Japan).

The results show that GMD down-regulates the expression of Cyclin E2 in colorectal cancer cells, further confirming that GMD inhibits the proliferation of colorectal cancer cells by regulating the cell cycle (fig. 4).

Example 4: experiments for inhibiting the activity of cell cathepsin L (cathepsin L, CL):

4.1 Experimental materials ganodermanondiol and other triterpenoids (from ChemFaces, Inc., Wuhan, Hubei, HPLC. gtoreq. 98%), cathepsin L inhibitor screening kit (from Biovision, USA), and enzyme-linked immunosorbent assay (MD, Flexstation 3).

4.2 Experimental methods: cell cathepsin L enzyme activity inhibition experiments were performed on ganodermanondiol using a commercial kit. 49. mu.L of an assay buffer (kit-of-parts), 1. mu.L (0.2 mU/. mu.L) of cell cathepsin L and 1. mu.L of LDTT were sequentially added to each well of a 96-well cell culture plate, and mixed well to prepare an enzyme reaction solution. Then, each sample to be analyzed was diluted with an analysis buffer and added to the enzyme reaction solution in an amount of 10. mu.L per well, mixed well and reacted at room temperature for 15 minutes. Positive and negative controls were set up for the experiment, 6 replicates per concentration. After completion of the reaction at room temperature, 40. mu.L of the diluted substrate solution was added to each well and mixed well. Finally, dynamic fluorescence signal detection was performed at 37 ℃ for 30 minutes on a microplate reader using a wavelength of Ex/Em of 400/500 nm.

The results show that the ganodermanondiol can obviously inhibit the activity of cell cathepsin L and IC₅₀The value was 15.56. mu.M, which was more effective than other triterpene compounds (FIG. 5).

Example 5: experiment for confirming expression of Cathepsin L (CL) in nucleus of colorectal cancer cell

5.1 subjects: colon cancer cells HCT116, Caco2, breast cancer cells (MCF-7), cervical cancer cells (Hela), normal human fibroblast HFLI and normal large intestine cells CCD 841.

5.2 Experimental reagents: rabbit anti-Cathepsin L monoclonal antibody (ab58991, available from abcam, uk), FITC-labelled goat anti-rabbit IgG H & L monoclonal antibody (ab6717, available from abcam, uk), nuclear dye Hoechst33528 (available from MCE, usa), confocal laser microscopy (Leica TCS SP8STED, germany).

5.3 Experimental methods cells were cultured at 0.5 × 10⁴The density of each well was inoculated into a 96-well cell culture plate and cultured in a cell culture medium containing 1% fetal bovine serum, and after 24 hours, the cell culture medium was replaced with a cell culture medium containing 10% fetal bovine serum and the culture was continued for 24 hours. The culture was then removed and the cells were washed once with PBS and fixed with pre-cooled methanol (100. mu.L/well) for 5 minutes at room temperature. After fixation, the methanol was removed and the cells were washed three times with PBS for 5 minutes each. Then, the user can use the device to perform the operation,mu.L of cell-permeabilizing solution containing 0.25% Trito X-100 was added to each well and treated for 10 minutes at room temperature. After three PBS washes, blocking was performed with PBS solution containing 10% goat serum and 0.1% tween 20 for 30 minutes at room temperature. After blocking, a rabbit anti-Cathepsin L monoclonal antibody (1/100 dilution) was added and incubated overnight at 4 ℃. PBST three times after washing with secondary antibody HRP labeled goat anti rabbit IgG H&The L monoclonal antibody (1/250 diluted) was incubated for 1 hour at room temperature in the dark. After PBST was washed three times, Hoechst nuclear staining (0.2. mu.g/mL) was performed for 10 minutes at room temperature. Finally, the staining solution was removed, washed with PBS, observed and photographed by a confocal laser fluorescence microscope (FITC: Ex/Em ═ 495/528nm, Hoechst: Ex/Em ═ 352/461nm), and the pictures were processed and analyzed by software (CellProfiler) to calculate the ratio of the intracellular and extranuclear CL expression levels of the respective cells.

The results showed that the expression level of CL in nuclei was higher in both the colon cancer cells and normal colon cells than in other cells (fig. 6).

Example 6: experiments to reduce the level of a truncated CUX1 transcription factor downstream of CL in the nucleus

Cathepsin CL in the nucleus can produce an activated truncated CUX1 by cleavage of CUX1, and then modulate Cyclin E2 to promote cell cycle progression. To further confirm that intracellular CL is the target of action of ganodermanondiol, it was tested.

6.1 subjects: large intestine cancer cell HCT116 and normal large intestine cell CCD 841.

6.2 Experimental drugs: ganodermanondiol (obtained from ChemFaces, Inc., Wuhan, Hubei, HPLC. gtoreq.98%).

6.3 Experimental reagents: rabbit anti-CUX 1 monoclonal antibody (PA5-25788, available from Invitrogen, Inc., USA), rabbit anti-HDAC 1 monoclonal antibody (ab32103, available from Abcam, Inc., UK), HRP-labeled goat anti-rabbit IgG H & L monoclonal antibody (ab6721, available from Abcam, Inc., UK), subcellular proteome extraction kit (available from EMBBIOSciences, USA), ECL chemiluminescent substrate (available from Thermo, USA).

6.4 Experimental methods: culturing cells in 10cm diameter culture dish, adding ganodermanone diol (GMD) with different concentrations after growth into monolayer, and adding drug solvent (DMSO) as control group. After culturing for another 48 hours, the cells were harvested and the subcellular proteome was separated using the EMD kit to obtain nuclear and cytoplasmic proteome samples. The subformonal sample was subjected to SDS-PAGE followed by western blotting analysis, followed by blocking with 5% milk solution after membrane transfer, and then incubation with anti-CUX 1 antibody or anti-HDAC 1 antibody, respectively. Finally, the secondary antibody is washed and detected by using an ECL substrate.

The results show that 50 μ M GMD treatment significantly reduced the level of truncated CUX1 in colon cancer or normal cells of the large intestine, indirectly confirming the fact that cathepsin CL in the nucleus is the GMD target molecule (fig. 7).

From the results of in vitro anticancer cell proliferation experiments and apoptosis and cell cycle experiments, ganoderic acid diol can specifically inhibit the proliferation of colorectal cancer cells and can arrest the cell cycle of colorectal cancer cells in S phase. The analysis of action mechanism shows that the Cathepsin L (CL) in nucleus is the action target point, GMD reduces the truncated CUX1 factor (activated form) by inhibiting the activity of CL in the nucleus of the colorectal cancer cell, then down regulates the expression of Cyclin E2, finally makes the colorectal cancer cell stay in the S phase of the cell cycle, thereby realizing the effect of inhibiting the growth of the cancer cell.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims (2)

1. Use of ganodermanondiol in the preparation of a reagent for inhibiting HCT116 and Caco2 cells, wherein the effective dose of ganodermanondiol is 0.01-10 μ M.

2. Use according to claim 1, characterized in that: the effective dose of ganoderic ketone diol is 0.8-5 μ M.

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