Application of Clavipine A in preparation of medicine for treating gastric cancer

Abstract

The invention relates to the technical field of medicines, in particular to application of Clavipine A in preparation of a medicine for treating gastric cancer. The Clavipine A is a meroterpenoid compound extracted and separated from the pterocarpus clavatus, and can inhibit the proliferation of gastric cancer cells by regulating and controlling cell cycle-related proteins and activate a mitochondrial apoptosis pathway to induce the apoptosis of the gastric cancer cells. The Clavipine A disclosed by the invention has remarkable proliferation inhibition and apoptosis induction effects on gastric cancer cells, is relatively low in toxicity to normal cells, has relatively high selectivity and relatively low side effects, and provides a theoretical basis and an experimental support for developing a novel anti-gastric cancer drug.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to a new use of the meroterpenoid compound Clavipine A extracted from Clitocybe clavipes in the preparation of drugs for treating gastric cancer, especially for its mechanism of inhibiting the proliferation of gastric cancer cells by regulating the cell cycle and mitochondrial apoptosis pathway. Background Art

[0002] Gastric cancer is one of the main causes of cancer death globally. Traditional chemotherapy drugs (such as cisplatin and fluorouracil) have serious side effects and drug resistance problems. In recent years, natural products have become a research hotspot for anti-tumor drugs due to their low toxicity and multi-target characteristics. Meroterpenoid compounds have anti-tumor activity, but existing research mainly focuses on liver cancer and lung cancer, and there is no report on their anti-gastric cancer activity.

[0003] Clavipine A is a novel meroterpenoid compound isolated from the fungus Clitocybe clavipes. Previous studies have shown that it has significant inhibitory effects on HepG2 and A549 cells, but there is no report on its anti-gastric cancer activity and mechanism of action. The present invention has verified through experiments that Clavipine A can efficiently inhibit gastric cancer cells through cell cycle arrest and mitochondrial apoptosis pathway, filling the gap in this field. Summary of the Invention

[0004] The Clavipine A of the present invention is a meroterpenoid compound extracted and isolated from Clitocybe clavipes, and its structural formula is as shown in the appendix Figure 1 shown.

[0005] The application of the Clavipine A of the present invention in the preparation of drugs for treating gastric cancer is characterized in that: Clavipine A can inhibit the proliferation of gastric cancer cells by regulating cell cycle-related proteins and activate the mitochondrial apoptosis pathway to induce apoptosis of gastric cancer cells.

[0006] Specifically, the inhibitory effect of Clavipine A on the proliferation of gastric cancer cells is mainly reflected in the following aspects:

[0007] 1. The results of MTT colorimetric assay show that Clavipine A has obvious inhibitory effects on the cell viability of human gastric cancer cells HGC-27 and AGS, and shows a certain dose-dependence. The IC 50 values are 6.9 μM and 4.9 μM respectively, and its activity is superior to that of the positive control drug cisplatin (DDP), and its cytotoxicity to primary rat cardiomyocytes and rat vascular smooth muscle cells is relatively low. The IC 50 values are 56.1 μM and 26 μM respectively.

[0008] 2. The results of the colony formation assay showed that the number of cell colonies formed by single-cell proliferation in the Clavipine A treatment group was significantly less than that in the control group, showing a dose-dependent manner, indicating that Clavipine A could significantly inhibit the replication and proliferation ability of single gastric cancer cells.

[0009] 3. The results of flow cytometry experiments showed that Clavipine A could induce dose-dependent cell cycle arrest in gastric cancer cell lines, selectively arresting them in the S phase.

[0010] 4. The results of Western Blot experiments showed that after the treatment with Clavipine A, the protein expression levels of cell cycle S-phase checkpoint proteins p-CHK1, p-CHK2, and p-BRCA1 in the two gastric cancer cells were significantly up-regulated. At the same time, the protein expression levels of cell cycle promoting proteins Cyclin D3 and CDK6 were significantly decreased, while the protein expression level of the cell cycle inhibitor protein p21 was significantly increased.

[0011] The apoptotic effect of Clavipine A on gastric cancer cells is mainly reflected in the following aspects:

[0012] 1) The results of optical microscopy observation showed that obvious morphological changes such as a significant decrease in the number of gastric cancer cells and cell shrinkage and fragmentation occurred after treatment with Clavipine A.

[0013] 2) The results of AO / EB staining showed that after the treatment with Clavipine A, the number of cells with orange-red fluorescence representing apoptosis increased in a dose-dependent manner.

[0014] 3) The results of Hoechst 33258 staining showed that the number of apoptotic nucleosomes emitting bright blue fluorescence in the Clavipine A treatment group increased in a dose-dependent manner.

[0015] 4) The results of fluorescence observation by JC-1 staining and detection by microplate reader showed that Clavipine A could significantly reduce the mitochondrial membrane potential of gastric cancer cells.

[0016] 5) Western Blot results showed that after the treatment with Clavipine A, the protein levels of Pro-caspase-3 and apoptosis inhibitor XIAP in the mitochondrial apoptosis pathway were significantly down-regulated, while the protein levels of Cleaved-caspase-3 and Cleaved-caspase-8 were significantly up-regulated.

[0017] Effects of the Invention

[0018] The present invention has the following beneficial effects:

[0019] 1. Clavipine A has significant inhibitory effects on the proliferation of gastric cancer cells and induces apoptosis, with low toxicity to normal cells, high selectivity, and low side effects.

[0020] 2. The mechanism of action of Clavipine A is clear. It exerts its anti-gastric cancer effect by regulating cell cycle-related proteins and activating the mitochondrial apoptosis pathway, providing a theoretical basis and experimental support for the development of new anti-gastric cancer drugs.

[0021] 3. This invention provides a new drug option for the treatment of gastric cancer, is expected to overcome the deficiencies of existing gastric cancer treatment drugs, and improve the survival rate and quality of life of gastric cancer patients.

[0022] Patent advantages:

[0023] 1. High efficiency and low toxicity: It has strong inhibitory activity against gastric cancer cells, and the toxicity to normal cells is significantly lower than that of traditional chemotherapy drugs.

[0024] 2. Dual mechanisms: Through the synergistic effects of cell cycle arrest and apoptosis induction, the risk of drug resistance is reduced.

[0025] 3. Potential for combination application: It can be used in combination with existing chemotherapy regimens to enhance the therapeutic effect and reduce dose-dependent toxicity. Description of the drawings

[0026] Figure 1 : Chemical structure diagram of Clavipine A.

[0027] Figure 2 : Effects of Clavipine A on the survival rates of gastric cancer cells and normal cells determined by the MTT method.

[0028] Figure 3 : Effects of Clavipine A on the colony formation of HGC-27 and AGS cells detected by the colony formation assay.

[0029] Figure 4 : Effects of Clavipine A on the cell cycle of HGC-27 and AGS cells detected by flow cytometry.

[0030] Figure 5 : Effects of Clavipine A on the expression of cell cycle regulatory proteins in HGC-27 and AGS cells detected by Western-Blot.

[0031] Figure 6 : Effects of Clavipine A on the morphology of HGC-27 and AGS cells observed by optical microscopy.

[0032] Figure 7: Observe the effect of Clavipine A on apoptosis of HGC-27 and AGS cells by AO / EB fluorescence staining and Hoechst 33258 nuclear staining.

[0033] Figure 8 : Observe the effect of Clavipine A on mitochondrial membrane potential of HGC-27 and AGS cells by JC-1 staining.

[0034] Figure 9 : Detect the effect of Clavipine A on the expression of apoptosis pathway proteins in HGC-27 and AGS cells by Western Blot. Specific implementation mode

[0035] The present invention will be further described below through specific examples, but the present invention is not limited to the following examples.

[0036] Example 1: Inhibitory effect of Clavipine A on the proliferation of gastric cancer cells.

[0037] 1. Cell culture: After resuscitating human gastric cancer cell lines HGC-27 and AGS, inoculate them into RPMI1640 medium containing 10% fetal bovine serum, culture in an incubator at 37°C and 5% CO2, passage regularly, and take cells in the logarithmic growth phase for experiments.

[0038] 2. MTT colorimetric method for determining cell viability:

[0039] a) Inoculate HGC-27 and AGS cells into 96-well plates respectively, 100 μL per well, and the cell densities are 6×10 3 cells / well and 8×10 3 cells / well respectively. Set 3 replicates for each group and culture for 12 - 24 h to allow the cells to adhere.

[0040] b) Add Clavipine A solutions at different concentrations. The final concentrations for HGC-27 cells are 0.5625 μM, 1.125 μM, 2.25 μM, 4.5 μM, 9 μM, 18 μM, and 36 μM respectively; the final concentrations for AGS cells are 0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, and 16 μM respectively. At the same time, set a blank control group (add an equal volume of medium).

[0041] c) After continuing to culture for 48 h, add 10 μL of MTT solution to each well and continue to culture for 4 h.

[0042] d) Discard the liquid in the wells, add 150 μL of DMSO to each well, gently shake to fully dissolve the crystals, and measure the absorbance value at 570 nm.

[0043] e) Calculate cell viability: Viability (%) = (OD value of the drug-treated group / OD value of the control group) × 100%. 2. Colony formation assay to detect the proliferative ability of single cells:

[0044] a) Seed HGC-27 and AGS cells into six-well plates, with 300 or 200 cells per well, and culture for 24 h to allow the cells to adhere.

[0045] b) Add Clavipine A solutions at different concentrations. The final concentrations for HGC-27 cells are 0.04 μM, 0.08 μM, 0.16 μM, 0.38 μM, and 0.75 μM respectively; the final concentrations for AGS cells are 0.03 μM, 0.06 μM, 0.13 μM, 0.25 μM, and 0.5 μM respectively. Meanwhile, set up a blank control group.

[0046] c) Continue to culture for 10 - 14 days and observe the cell colony formation.

[0047] d) Fix the cells with 4% paraformaldehyde, add 0.1% crystal violet for staining for 15 min, slowly wash away the staining solution with running water, air dry at room temperature, and then take pictures.

[0048] 3. Flow cytometry to detect cell cycle distribution:

[0049] a) Seed HGC-27 and AGS cells into six-well plates, with 1 mL of cell suspension per well, and the cell concentration is 2.0×10 5 / mL. Culture for 24 h to allow the cells to adhere.

[0050] b) Add Clavipine A solutions at different concentrations. The final concentrations for HGC-27 cells are 3 μM, 9 μM, and 18 μM respectively; the final concentrations for AGS cells are 3 μM, 6 μM, and 12 μM respectively. Meanwhile, set up a blank control group.

[0051] c) After continuing to culture for 48 h, collect the cells, digest the cells with trypsin and centrifuge to collect the cells. Wash the cells with pre-cooled PBS, add 70% ethanol to fix the cells, and store them in a -20°C refrigerator overnight.

[0052] d) Centrifuge to precipitate the cells, add propidium iodide staining solution, incubate at 37°C in the dark for 30 min, filter through a 300-mesh nylon net, and then detect with a flow cytometer.

[0053] e) Analyze the cell cycle distribution using Flowjo software.

[0054] 4. Western Blot to detect the expression of cell cycle regulatory proteins:

[0055] a) Seed HGC-27 and AGS cells in culture dishes respectively. When the cell density reaches about 70%, add Clavipine A solutions at different concentrations. The final concentrations of Clavipine A for HGC-27 cells are 5 μM, 10 μM, and 20 μM respectively; the final concentrations for AGS cells are 4.5 μM, 9 μM, and 18 μM respectively. At the same time, set up a blank control group.

[0056] b) After continuing to culture for 48 h, collect the cells, extract proteins, and measure the protein concentration.

[0057] c) Prepare 10% or 12% SDS-PAGE gels, electrophoretically separate the protein samples, and transfer them to PVDF membranes.

[0058] d) Block with 5% non-fat milk powder at room temperature for 2 h, add primary antibodies (p-CHK1, p-CHK2, p-BRCA1, Cyclin D3, CDK6, p21, etc.), and incubate overnight at 4°C.

[0059] e) The next day, wash the membranes with TBST, add secondary antibodies, incubate at room temperature for 2 h, wash the membranes again, and then perform chemiluminescent imaging.

[0060] Example 2: Effect of Clavipine A on inducing apoptosis of gastric cancer cells

[0061] 1. Observe the morphological changes of cells under an optical microscope:

[0062] a) Seed HGC-27 and AGS cells in six-well plates respectively, with 1 mL of cell suspension in each well, and culture for 24 h to allow the cells to adhere.

[0063] b) Add Clavipine A solutions at different concentrations. The final concentrations of Clavipine A for HGC-27 cells are 3 μM, 9 μM, and 18 μM respectively; the final concentrations for AGS cells are 3 μM, 6 μM, and 12 μM respectively. At the same time, set up a blank control group.

[0064] c) After continuing to culture for 48 h, discard the liquid in the wells, add 4% paraformaldehyde phosphate buffer to fix the cells for 10 - 20 min.

[0065] d) Observe the morphological changes of the cells under an optical microscope.

[0066] 2. AO / EB fluorescence staining experiment:

[0067] a) Prepare stock solutions of AO and EB: Weigh 2 mg of AO, add 2 mL of PBS to make a 1 mg / mL stock solution; take 20 μL of EB and dissolve it in 1980 μL of PBS to make a 100 μg / mL stock solution, and store both at 4°C.

[0068] b) Seed HGC-27 and AGS cells into six-well plates, 1 mL of cell suspension per well, and culture for 24 h to allow the cells to adhere to the wall.

[0069] c) Add Clavipine A solutions at different concentrations. The final concentrations for HGC-27 cells are 3 μM, 9 μM, and 18 μM respectively; the final concentrations for AGS cells are 3 μM, 6 μM, and 12 μM respectively. Meanwhile, set up a blank control group.

[0070] d) After continuing to culture for 48 h, collect the cells, digest the cells with trypsin and centrifuge to collect the cells, add the prepared AO / EB staining solution, and incubate in the dark for 10 - 15 min.

[0071] e) Observe the apoptotic morphology of the cells under a fluorescence microscope.

[0072] 3. Hoechst 33258 nuclear staining experiment:

[0073] a) Prepare the Hoechst 33258 dye stock solution: Mix 25 mg of Hoechst dye with 2.5 mL of pure water to make a 10 mg / mL stock solution. After aliquoting, store it in the dark at 4°C. Dilute it to 5 μg / mL with PBS before staining.

[0074] b) Seed HGC-27 and AGS cells into six-well plates, 1 mL of cell suspension per well, and culture for 24 h to allow the cells to adhere to the wall.

[0075] c) Add Clavipine A solutions at different concentrations. The final concentrations for HGC-27 cells are 3 μM, 9 μM, and 18 μM respectively; the final concentrations for AGS cells are 3 μM, 6 μM, and 12 μM respectively. Meanwhile, set up a blank control group.

[0076] d) After continuing to culture for 48 h, collect the cells, fix the cells with 4% paraformaldehyde for 15 min, aspirate the fixing solution, and wash the cells twice with PBS.

[0077] e) Add the diluted Hoechst 33258 dye, incubate in the dark at room temperature for 30 min, then wash twice with PBS, and observe the apoptotic morphology of the cell nuclei under a fluorescence microscope.

[0078] 4. Detection of mitochondrial membrane potential by JC-1 staining experiment:

[0079] a) Prepare the JC-1 working solution: Dissolve 1 mg of JC-1 in 200 μL of DMSO solution to prepare the JC-1 working solution.

[0080] b) Seed HGC-27 and AGS cells into six-well plates, 1 mL of cell suspension per well, and culture for 24 h to allow the cells to adhere to the wall.

[0081] c) Add Clavipine A solutions at different concentrations, with the final concentrations of HGC-27 cells being 3 μM, 9 μM, and 18 μM respectively; and the final concentrations of AGS cells being 3 μM, 6 μM, and 12 μM respectively. Meanwhile, set up a blank control group.

[0082] d) After continued culture for 48 h, collect the cells, digest them with trypsin and centrifuge to collect the cells. Then add JC-1 staining agent and incubate in the dark for 20 min.

[0083] e) After centrifuging to discard the supernatant, resuspend the cells twice with pre-cooled PBS, and finally resuspend them with 20 μL PBS. Observe the changes in the mitochondrial membrane potential of the cells under a fluorescence microscope.

[0084] f) For quantitative detection, transfer the stained cells into a black 96-well plate, and measure the red fluorescence and green fluorescence signals respectively (set the excitation wavelength at 550 nm and the emission wavelength at 600 nm for red fluorescence; set the excitation wavelength at 485 nm and the emission wavelength at 535 nm for green fluorescence), and calculate the red / green fluorescence ratio.

[0085] 5. Detection of apoptosis pathway protein expression by Western Blot:

[0086] a) Seed HGC-27 and AGS cells in culture dishes respectively. When the cell density reaches about 70%, add Clavipine A solutions at different concentrations, with the final concentrations of HGC-27 cells being 5 μM, 10 μM, and 20 μM respectively; and the final concentrations of AGS cells being 4.5 μM, 9 μM, and 18 μM respectively. Meanwhile, set up a blank control group.

[0087] b) After continued culture for 48 h, collect the cells, extract proteins, and measure the protein concentration.

[0088] c) Prepare 10% or 12% SDS-PAGE gel, electrophoretically separate the protein samples, and transfer them onto a PVDF membrane.

[0089] d) Block the membrane with 5% non-fat milk at room temperature for 2 h, add primary antibodies (XIAP, Cleaved-caspase-8, Pro-caspase-3, Cleaved-caspase-3, etc.), and incubate overnight at 4°C.

[0090] e) The next day, wash the membrane with TBST, add secondary antibody, incubate at room temperature for 2 h, wash the membrane again, and then perform chemiluminescent imaging.

Claims

1. A use of Clavipine A in the preparation of a drug for treating gastric cancer.

2. The use according to claim 1, characterized in that: The Clavipine A is a meropentenoid compound extracted and separated from Clitocybe clavipes, and its structural formula is shown in FIG1 .

3. The use according to claim 1, characterized in that: The drug can inhibit the proliferation of gastric cancer cells by regulating cell cycle-related proteins, and activate the mitochondrial apoptosis pathway to induce apoptosis of gastric cancer cells.

4. The use according to claim 3, characterized in that: The cell cycle-related proteins include p-CHK1, p-CHK2, p-BRCA1, Cyclin D3, CDK6 and p21.

5. The use according to claim 3, characterized in that: The mitochondrial apoptosis pathway-related proteins include XIAP, Pro-caspase3, Cleaved-caspase-8 and Cleaved-caspase-3.

6. The use according to claim 1, characterized in that: The drug can be used alone or in combination with other anti-gastric cancer drugs.

7. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule, injection or oral solution.

Images