Application of mediodarin in preparation of gastric cancer medicine for inhibiting proliferation of human gastric cancer cell HGC-27

Abstract

The invention relates to an application of mediodine in preparation of a gastric cancer drug for inhibiting proliferation of human gastric cancer cells HGC-27. The pterocarpin induces the occurrence of apoptosis of gastric cancer cells by up-regulating the expression of a pro-apoptotic protein (CARP). The invention relates to a method for inducing apoptosis of gastric cancer cells by down-regulating expression of anti-apoptosis protein Mcl-1, and inducing apoptosis of gastric cancer cells by down-regulating expression of anti-apoptosis protein Mcl-1, or inducing apoptosis of gastric cancer cells by down-regulating expression of p-Ak protein by down-regulating expression of p-mTOR protein by down-regulating expression of p-mTOR protein by inhibiting activation of mTOR signal channel. Or inhibiting activation of a Stat3 signal channel and down-regulating expression of p-Stat3 protein to induce apoptosis of the gastric cancer cells. Compared with the prior art, the application has the advantages that the mendinosin has obvious effects of inhibiting proliferation and migration and promoting apoptosis on human gastric cancer cells HGC-27, can be used as a main active component to develop anti-gastric cancer drugs and prepare corresponding pharmaceutical preparations, aims to realize growth inhibition and apoptosis induction of the gastric cancer cells by inhibiting Akt / mTOR / Stat3 signal channels, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to use of medipterostilbene in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. Background Art

[0002] Gastric cancer (GC) is one of the most common malignant tumors worldwide. At present, the main treatments for gastric cancer include endoscopic submucosal dissection (ESD), radical surgery, chemotherapy, radiotherapy, immunotherapy and molecular targeted therapy. For early gastric cancer, endoscopic treatment can effectively remove the diseased tissue, but it is only suitable for patients with a very low possibility of lymph node metastasis. For advanced and late gastric cancer, the therapeutic effect of surgical resection is limited, the incidence of postoperative recurrence and distant metastasis is high, and the long-term survival rate is low. In addition, although chemotherapy can improve the survival prognosis of patients to a certain extent, its application is limited by drug resistance and toxic side effects. Molecular targeted therapy shows good efficacy and safety in some patients, but the applicable population is limited and there is a problem of drug resistance.

[0003] Therefore, in view of the shortcomings of the prior art, it is necessary to provide a use of medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27 to solve the shortcomings of the prior art. Summary of the invention

[0004] The purpose of the present invention is to avoid the shortcomings of the prior art and provide a use of medipterocarpin in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. In this use, medipterocarpin can significantly inhibit the proliferation, migration and apoptosis of gastric cancer cells.

[0005] The above-mentioned purpose of the present invention is achieved by the following technical measures: Provided is the use of medipterocarpin in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27.

[0006] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene induces the occurrence of apoptosis of gastric cancer cells by upregulating the expression of the pro-apoptotic protein Cleaved PARP.

[0007] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene induces the occurrence of apoptosis of gastric cancer cells by downregulating the expression of the anti-apoptotic protein Mcl-1.

[0008] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene inhibits the activation of the Akt signaling pathway, downregulates the expression of p-Ak protein, and induces the occurrence of apoptosis of gastric cancer cells.

[0009] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene inhibits the activation of the mTOR signaling pathway, downregulates the expression of the p-mTOR protein, and induces the occurrence of apoptosis of gastric cancer cells.

[0010] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene inhibits the activation of the Stat3 signaling pathway, downregulates the expression of the p-Stat3 protein, and induces the occurrence of apoptosis of gastric cancer cells.

[0011] The invention discloses a use of the medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene induces the occurrence of gastric cancer cell apoptosis by destroying the cell microtubule network structure and actin fiber structure.

[0012] The present invention discloses a use of medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

[0013] The invention discloses a use of medipterostilbene in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The medipterostilbene is dissolved in dimethyl sulfoxide, and the concentration of the medipterostilbene is 100 μmol / L to 200 μmol / L.

[0014] The use of the medipterostilbene in the present invention in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27 can enhance the cell proliferation inhibition rate and inhibit the cloning ability, scratch healing ability and migration ability of gastric cancer cells by increasing at least one of the concentration of medipterostilbene or prolonging the action time of medipterostilbene.

[0015] The invention discloses a use of medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27. The invention verifies the anti-gastric cancer effect of medipterostilbene through in vitro experiments, and the research results show that medipterostilbene can significantly inhibit the proliferation, migration and apoptosis of gastric cancer cells, and has good drug development potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention is further described with reference to the accompanying drawings, but the contents in the accompanying drawings do not constitute any limitation to the present invention.

[0017] Figure 1The effect of medipterostilbene on the cell proliferation inhibition rate of human gastric cancer cell HGC-27 after treatment with 12h, 24h and 48h at different concentrations.

[0018] Figure 2 The effect of medipterostilbene on the colony-forming ability of human gastric cancer cell HGC-27 after 48h of treatment with different concentrations of medipterostilbene. Figure 2 A is a picture of cell colony formation visualized by crystal violet staining; Figure 2 B is a quantitative graph of colony formation in each treatment group.

[0019] Figure 3 The effect of medipterostilbene on scratch healing of human gastric cancer cells HGC-27 after 24h of treatment with medipterostilbene.

[0020] Figure 4 The effect of medipterostilbene on the migration ability of human gastric cancer cell HGC-27 after 48h treatment with different concentrations of medipterostilbene. Figure 4 A is a picture of the migration of HGC-27 cells observed by crystal violet staining after treatment with different concentrations of medipterostilbene for 48 h; Figure 4 B is a statistical analysis of the number of migrating cells after treatment with different concentrations of medipterone, and the data are expressed as mean ± standard deviation (Mean ± SD).

[0021] Figure 5 This figure shows the effect of medipterostilbene on the microtubule network structure of human gastric cancer cell HGC-27 after 48h of treatment with medipterostilbene at different concentrations.

[0022] Figure 6 This figure shows the effect of medipterostilbene on the actin fiber structure of human gastric cancer cell HGC-27 after 48h of treatment with medipterostilbene at different concentrations.

[0023] Figure 7 This is the effect of 0 μmol / L medipterostilbene on the apoptosis rate of human gastric cancer cells HGC-27 after treatment for 48 hours.

[0024] Figure 8 This is the effect of 100 μmol / L medipterostilbene on the apoptosis rate of human gastric cancer cells HGC-27 after treatment for 48 hours.

[0025] Fig. 9 This is the effect of 150 μmol / L medipterostilbene on the apoptosis rate of human gastric cancer cells HGC-27 after treatment for 48 hours.

[0026] Fig.10 This is the effect of 200 μmol / L medipterostilbene on the apoptosis rate of human gastric cancer cells HGC-27 after 48 hours of treatment.

[0027] Fig.11 The expression levels of apoptosis-related proteins and cell proliferation-related proteins in human gastric cancer cell HGC-27 after treatment with different concentrations of medipterostilbene for 48 h. Fig.11 A is the expression level diagram of key proteins (PARP, p-Akt, p-mTOR, p-Stat3 and Mcl-1) analyzed by Western Blot; Fig.11 B is the quantitative result of gray value analysis based on Image-J software, showing the fold change of each protein expression relative to GAPDH.

[0028] Fig.12 The effects of medipterostilbene alone and in combination with Akt agonist IGF-1 on the proliferation and apoptosis-related protein expressions of human gastric cancer cell HGC-27. Fig.12 A is the expression level diagram of key proteins (p-Akt, p-mTOR, p-Stat3, Mcl-1 and PARP) analyzed by Western Blot; Fig.12 B is the quantitative result of grayscale analysis based on Image-J software, showing the fold change of each protein expression relative to the internal reference GAPDH. DETAILED DESCRIPTION

[0029] The technical scheme of the present invention is further described in conjunction with the following examples. The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagent materials, etc. used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical companies unless otherwise specified.

[0030] The cells used in the present invention are human gastric cancer undifferentiated cells HGC-27, and human gastric cancer undifferentiated cells HGC-27 were purchased from the Chinese Academy of Sciences.

[0031] RPMI Medium 1640 basic was purchased from Gibco, USA, with the catalog number C11875500BT.

[0032] Phosphate buffered saline (PBS buffer) was purchased from Solarbio, China, with the catalog number P1020.

[0033] Fetal bovine serum (France Origin) was purchased from ZETA, USA, with the product number Z7186FBS-500.

[0034] Trypsin-EDTA (0.25% Trypsin-EDTA, containing phenol red) was purchased from Gibco, USA, with the catalog number 25200-072.

[0035] CCK-8 Cell Counting Kit was purchased from China Novozymes Co., Ltd. with the catalog number A311-01.

[0036] BCA Protein Assay Kit was purchased from China Kangrun Company with the catalog number E162-01.

[0037] The cell apoptosis detection kit (Annexin V PE Apoptosis Detection Kit, 100 Test) was purchased from BD Company, USA, with the catalog number 559763.

[0038] The substrate color development reagent (Immobilon Forte Western HRP substrate) was purchased from Millipore, USA, with the catalog number WBLUF0500.

[0039] Anti-PARP antibody (Anti-PARP (46D11) antibody (9532S)), anti-Phospho-Akt antibody (Anti-phospho-Akt antibody (4060S)), anti-Phospho-mTOR antibody (Anti-phospho-mTORantibody (5536P)), anti-Phospho-Stat3 antibody (Anti-phospho-Stat3 antibody (9145S)), anti-Mcl-1 antibody (Anti-Mcl-1 antibody (5453S)), and anti-GAPDH antibody (Anti-GAPDH antibody(2118S)) were all purchased from CST, USA.

[0040] Example 1 A use of medipterostilbene in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, wherein medipterostilbene can upregulate the expression of the pro-apoptotic protein Cleaved PARP and downregulate the expression of the anti-apoptotic protein Mcl-1, thereby inducing the occurrence of apoptosis in gastric cancer cells.

[0041] The medipterostilbene of the present invention can induce apoptosis of gastric cancer cells by inhibiting the activation of Akt signaling pathway and down-regulating the expression of p-Ak protein. medipterostilbene can also induce apoptosis of gastric cancer cells by inhibiting the activation of mTOR signaling pathway and down-regulating the expression of p-mTOR protein. medipterostilbene can also induce apoptosis of gastric cancer cells by inhibiting the activation of Stat3 signaling pathway and down-regulating the expression of p-Stat3 protein.

[0042] Specifically, the Akt agonist insulin-like growth factor 1 (IGF-1) can significantly reverse the down-regulation of intracellular p-Akt, p-mTOR, p-Stat3 and Mcl-1 protein expression levels induced by medipterostilbene, and effectively inhibit the apoptosis process of gastric cancer cells.

[0043] Moreover, the medipterostilbene of the present invention can also induce apoptosis of gastric cancer cells by destroying the cell microtubule network structure and actin fiber structure.

[0044] The applicant further discovered that the target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

[0045] The present application also treats gastric cancer cells with different concentrations of medipterostilbene to observe the inhibitory effect of medipterostilbene on the proliferation of gastric cancer cells. When medipterostilbene is dissolved in dimethyl sulfoxide and the concentration of medipterostilbene is in the range of 100 μmol / L to 200 μmol / L, it can significantly destroy the microtubule network and F-actin structure of HGC-27 cells, leading to microtubule depolymerization, cytoskeleton collapse, F-actin fiber reduction and decreased adhesion ability, and presents a concentration-dependent effect. When medipterostilbene increases the action time at the same concentration, the inhibitory effect on HGC-27 cell proliferation shows a dependent trend. Therefore, the present invention can enhance the cell proliferation inhibition rate and inhibit the cloning ability, scratch healing ability and migration ability of gastric cancer cells by increasing at least one of the concentration of medipterostilbene or extending the action time of medipterostilbene.

[0046] In summary, medipterostilbene has a significant pro-apoptotic effect on gastric cancer cells and can be used as a drug for the treatment of gastric cancer.

[0047] Example 2 A use of medipterostilbene in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, wherein the gastric cancer is caused by the proliferation of human gastric cancer cells, and the human gastric cancer cells are undifferentiated cells HGC-27.

[0048] Medipterocarpin can induce apoptosis of gastric cancer cells by upregulating the expression of the pro-apoptotic protein Cleaved PARP and downregulating the expression of the anti-apoptotic protein Mcl-1.

[0049] Medipterocarpin inhibits the activation of Akt signaling pathway, downregulates the expression of p-Ak protein, and induces apoptosis of gastric cancer cells.

[0050] Medipterocarpin can also inhibit the activation of the mTOR signaling pathway, downregulate the expression of p-mTOR protein, and induce apoptosis of gastric cancer cells. Medipterocarpin can also inhibit the activation of the Stat3 signaling pathway, downregulate the expression of p-Stat3 protein, and induce apoptosis of gastric cancer cells.

[0051] Moreover, the medipterostilbene of the present invention can also induce apoptosis of gastric cancer cells by destroying the cell microtubule network structure and actin fiber structure.

[0052] The applicant further discovered that the target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

[0053] The present application also treated gastric cancer cells with different concentrations of medipterostilbene to observe the inhibitory effect of medipterostilbene on the proliferation of gastric cancer cells. The experiment found that when medipterostilbene was dissolved in dimethyl sulfoxide and the concentration of medipterostilbene was 100 μmol / L, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

[0054] Compared with Example 1, the medipterostilbene in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

[0055] Example 3 A use of medipterostilbene in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, wherein the gastric cancer is caused by the proliferation of human gastric cancer cells, and the human gastric cancer cells are undifferentiated cells HGC-27.

[0056] Medipterocarpin can induce apoptosis of gastric cancer cells by upregulating the expression of the pro-apoptotic protein Cleaved PARP and downregulating the expression of the anti-apoptotic protein Mcl-1.

[0057] Medipterocarpin inhibits the activation of Akt signaling pathway, downregulates the expression of p-Ak protein, and induces apoptosis of gastric cancer cells.

[0058] Medipterocarpin can also inhibit the activation of the mTOR signaling pathway, downregulate the expression of p-mTOR protein, and induce apoptosis of gastric cancer cells. Medipterocarpin can also inhibit the activation of the Stat3 signaling pathway, downregulate the expression of p-Stat3 protein, and induce apoptosis of gastric cancer cells.

[0059] Moreover, the medipterostilbene of the present invention can also induce apoptosis of gastric cancer cells by destroying the cell microtubule network structure and actin fiber structure.

[0060] The applicant further discovered that the target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

[0061] The present application also treated gastric cancer cells with different concentrations of medipterostilbene to observe the inhibitory effect of medipterostilbene on the proliferation of gastric cancer cells. The experiment found that when medipterostilbene was dissolved in dimethyl sulfoxide and the concentration of medipterostilbene was 150 μmol / L, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

[0062] Compared with Example 1, the medipterostilbene in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

[0063] Example 4 A use of medipterostilbene in preparing a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, wherein the gastric cancer is caused by the proliferation of human gastric cancer cells, and the human gastric cancer cells are undifferentiated cells HGC-27.

[0064] Medipterocarpin can induce apoptosis of gastric cancer cells by upregulating the expression of the pro-apoptotic protein Cleaved PARP and downregulating the expression of the anti-apoptotic protein Mcl-1.

[0065] Medipterocarpin inhibits the activation of Akt signaling pathway, downregulates the expression of p-Ak protein, and induces apoptosis of gastric cancer cells.

[0066] Medipterocarpin can also inhibit the activation of the mTOR signaling pathway, downregulate the expression of p-mTOR protein, and induce apoptosis of gastric cancer cells. Medipterocarpin can also inhibit the activation of the Stat3 signaling pathway, downregulate the expression of p-Stat3 protein, and induce apoptosis of gastric cancer cells.

[0067] Moreover, the medipterostilbene of the present invention can also induce apoptosis of gastric cancer cells by destroying the cell microtubule network structure and actin fiber structure.

[0068] The applicant further discovered that the target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

[0069] The present application also treated gastric cancer cells with different concentrations of medipterostilbene to observe the inhibitory effect of medipterostilbene on the proliferation of gastric cancer cells. The experiment found that when medipterostilbene was dissolved in dimethyl sulfoxide and the concentration of medipterostilbene was 200 μmol / L, the inhibitory effect on the proliferation of gastric cancer cells was obvious.

[0070] Compared with Example 1, the medipterostilbene in this example has a better effect of promoting apoptosis of gastric cancer cells and can be used to prepare drugs for treating gastric cancer.

[0071] Effect example 1. Culture of human gastric cancer cells HGC-27 1.1. Cell passaging: When the human gastric cancer cell HGC-27 grows to a density of 70% to 80%, the culture medium is discarded, and after washing twice with phosphate buffered saline (PBS), trypsin-EDTA (0.25%) solution is added for digestion for about 1 min. After the cells shrink and become round, RPMI Medium 1640 culture medium containing 10% fetal bovine serum is added to terminate the digestion, and a single cell suspension is formed by gentle pipetting. Centrifuge at 1000 rpm for 3 min, discard the supernatant, resuspend the cells and pass them at a ratio of 1:2 or 1:3, and culture them in an incubator at 37°C and 5% CO2.

[0072] 1.2. Cell counting: After cell digestion and resuspending, use a cell counting plate to count cells. Follow the principle of counting the top but not the bottom, and counting the left but not the right. Repeat the counting twice to calculate the concentration of the cell suspension.

[0073] 1.3. Cell plating: According to experimental requirements, a certain number of HGC-27 cells are inoculated into culture dishes or well plates for subsequent experiments.

[0074] 2. Inhibitory effect of medipterostilbene on proliferation of human gastric cancer cell HGC-27 2.1. CCK-8 method to evaluate the time- and concentration-dependent inhibitory effects of medipterostilbene on HGC-27 cell proliferation HGC-27 cells in the logarithmic growth phase were trypsinized and cultured at 8×10 3 Cell suspension was prepared at a density of 10 cells / 100 μL and inoculated into a 96-well plate (100 μL per well). After culturing in a cell culture incubator at 37°C and 5% CO2 for 12 h, the cells were treated with 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) of medipholtziacin, respectively, with 5 replicate wells set for each concentration group. After 12 h, 24 h and 48 h of treatment, the cell viability was detected using the CCK-8 kit, and the cell proliferation inhibition rate was calculated, as shown in Figure 2. Figure 1 shown.

[0075] from Figure 1 The results showed that medipterostilbene could significantly inhibit the proliferation of HGC-27 cells in a time- and concentration-dependent manner; moreover, with the increase of medipterostilbene concentration, the cell proliferation inhibition rate gradually increased, and the inhibitory effect was further enhanced with the extension of the action time.

[0076] Specifically, at a concentration of 100 µmol / L, the inhibition rates of HGC-27 cells were 42.86% (12h), 50.54% (24h) and 51.25% (48h), respectively; at a concentration of 150 µmol / L, the inhibition rates were 65.47% (12h), 72.33% (24h) and 78.50% (48h), respectively; at a concentration of 200 µmol / L, the inhibition rates were further increased, reaching 85.53% (12h), 90.57% (24h) and 95.35% (48h), respectively.

[0077] In summary, medipterostilbene can significantly inhibit the proliferation of HGC-27 cells, and its inhibitory effect shows a dose-dependent and time-dependent trend, suggesting that it has potential anti-gastric cancer activity. Therefore, the cell proliferation inhibition rate can be enhanced by at least increasing the concentration of medipterostilbene or prolonging the action time of medipterostilbene.

[0078] 2.2 Colony formation assay to evaluate the inhibitory effect of medipterostilbene on HGC-27 cell colony formation Human gastric cancer cells HGC-27 in the logarithmic growth phase were prepared into a cell suspension of 500 cells / 2 mL and inoculated into a 6-well plate with 2 mL per well. The cells were cultured in an incubator at 37°C and 5% CO2 for 7 days, and the solution was changed every 2 days. The experimental concentrations of 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) were set for the treatment groups of medipterostilbene. After 48 hours of action, they were fixed with 1 mL / well of methanol for 20 min, rinsed with PBS, and stained with 1 mL / well of crystal violet for 25 min, then rinsed with PBS again and dried, and the images were recorded, as shown in Figure 2 shown.

[0079] like Figure 1 and Figure 2 As shown, medipterostilbene significantly inhibited the colony-forming ability of HGC-27 cells at different concentrations, and showed a concentration-dependent trend.

[0080] When the concentration of medipterostilbene was 0 µmol / L, more cell colonies were formed, and when the concentration of medipterostilbene was 100 µmol / L, the number of colonies was significantly reduced, and when the concentration of medipterostilbene was 150 µmol / L, the number further decreased, and when the concentration of medipterostilbene was 200 µmol / L, almost no colonies were formed.

[0081] It can be seen that medipterostilbene can significantly inhibit the colony-forming ability of HGC-27 cells, and the inhibitory effect increases with increasing concentration.

[0082] And in Figure 1The results showed that when the concentration of medipterostilbene was 200 μmol / L for 48 hours, cell proliferation was inhibited by more than 90%, indicating that this concentration basically achieved the maximum inhibitory effect. Therefore, further increasing the concentration may not bring additional significant effects, but may introduce nonspecific cytotoxicity or other interfering factors.

[0083] 3. Inhibitory effect of medipterostilbene on migration of human gastric cancer cell HGC-27 3.1. Cell scratch assay to evaluate cell migration ability After marking the positioning line on the back of the 6-well plate, the digested HGC-27 cells were inoculated into the well plate and cultured until the cell monolayer was confluent. Subsequently, a 200 μL pipette tip was used to scratch in the vertical direction, the culture medium was removed, and the cells were rinsed three times with PBS to remove the detached cells. The experimental groups were treated with 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) of medipholtzia in the concentrations, and the scratch healing was recorded at 0 h and 24 h, as shown in Figure 2. Figure 3 shown.

[0084] The results showed that medipterostilbene significantly inhibited the migration ability of HGC-27 cells in a concentration-dependent manner. As the concentration of medipterostilbene increased, the scratch closure rate gradually decreased, and the scratch closure rate in the 200 μmol / L group was the lowest, with almost no obvious migration.

[0085] 3.2 Transwell assay to evaluate cell migration ability Take HGC-27 cells in the logarithmic growth phase and prepare a single cell suspension of 5×104 cells / mL. 200 μL of serum-free cell suspension treated with medipholtzia in concentrations of 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) was inoculated into the upper chamber of the Transwel chamber, and 600 μL of culture medium containing 10% FBS was added to the lower chamber to provide a chemotactic signal. After 48 hours of culture, the chamber was removed and rinsed in deionized water to remove residual culture medium and non-adherent cells. Subsequently, 500 μL of methanol was used to fix for 20 minutes, and the non-migrated cells in the upper chamber were gently wiped with a cotton swab. After rinsing with deionized water, 400 μL of crystal violet solution was added and stained at 37°C in the dark for 25 minutes. After staining, rinse thoroughly and image after drying, as shown Figure 4 shown.

[0086] The experimental results showed that medipterostilbene can significantly inhibit the migration of HGC-27 cells in a concentration-dependent manner. As the concentration of medipterostilbene increases, the number of cells migrating to the lower chamber gradually decreases, and the migration inhibition effect of the medipterostilbene group with a concentration of 200 μmol / L is the most significant. It can be seen that medipterostilbene can exert its anti-tumor effect by inhibiting the migration ability of gastric cancer cells. Therefore, it can inhibit the cloning ability, scratch healing ability and migration ability of gastric cancer cells by increasing the concentration of medipterostilbene or prolonging the action time of medipterostilbene.

[0087] 4. Immunofluorescence staining to evaluate the effect of medipterostilbene on the skeleton structure of human gastric cancer cell HGC-27 4.1 Effects of Medipterocarpin on the Microtubule Network HGC-27 cells were evenly seeded on pre-sterilized coverslips (12-well plates, 2 × 10 4 / well). After 48 hours of treatment with 0μmol / L, 100μmol / L (Example 2), 150μmol / L (Example 3) and 200μmol / L (Example 4) of medipholin, α-tubulin (α-Tubulin) immunofluorescence staining was used to detect changes in the cell microtubule network. After the cells were fixed, they were permeabilized with 0.5% Triton X-100 to block nonspecific binding sites, and then the α-tubulin primary antibody was added and incubated overnight at 4°C in the dark. The secondary antibody was added the next day and incubated at room temperature in the dark, and DAPI was used to stain the cell nucleus. After sealing, an Olympus laser confocal microscope was used to observe changes in the microtubule structure and record images, as shown Figure 5 shown.

[0088] exist Figure 5 In the experiment, the results of α-tubulin immunofluorescence staining showed that the microtubule network of HGC-27 cells in the untreated group (0 μmol / L) was intact, the microtubules were evenly distributed in a reticular pattern, and the cell morphology was normal. With the increase of the concentration of medipterostilbene, the cell microtubules gradually disassembled, the cytoskeleton showed a trend of collapse, the microtubules broke significantly, and the cell morphology tended to shrink. After high-concentration treatment (150 μmol / L and 200 μmol / L), the microtubules almost completely disappeared, leaving only a small amount of α-tubulin fluorescence signal, and the number of cells decreased significantly. The above results show that medipterostilbene can significantly destroy the microtubule structure of HGC-27 cells and show a concentration-dependent effect.

[0089] 4.2 Effects of Medipterocarpin on Actin Fibers HGC-27 cells were evenly seeded on sterilized coverslips and plated in 12-well plates (2 × 10 4 / well). After 48h of treatment with 0μmol / L, 100μmol / L (Example 2), 150μmol / L (Example 3) and 200μmol / L (Example 4) of medipholtzia, phalloidin immunofluorescence staining was used to detect changes in the cell actin (F-actin) network. After the cells were fixed, they were permeabilized with 0.5% Triton X-100, and then phalloidin was added for F-actin staining. The cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) and then sealed. Laser confocal microscopy was used for observation and imaging, as shown in Figure 6 shown.

[0090] pass Figure 6 Phalloidin immunofluorescence staining results showed that the F-actin fibers of HGC-27 cells in the untreated group (0 μmol / L) were evenly distributed, the cells were in an extended state, and the adhesion ability was good. With the increase of the concentration of phalloidin, the F-actin fibers gradually decreased, the cell morphology tended to be round, the extension ability decreased, and the cell adhesion ability was significantly weakened. After treatment with high concentrations (150 μmol / L and 200 μmol / L), the F-actin structure was almost completely disaggregated, only a small amount of fluorescent signal remained, and the number of cells was significantly reduced. The above results show that phalloidin can significantly affect the F-actin structure of HGC-27 cells, weaken their adhesion and migration abilities, and lead to cytoskeleton instability.

[0091] 5. Induction of apoptosis of human gastric cancer cell HGC-27 by Medipterostilbene 5.1. Detection of cell apoptosis rate by flow cytometry After being treated with 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) mediptertin for 48 h, the cells were collected, washed twice with PBS buffer, resuspended with 100 μL Binding Buffer buffer, transferred to a flow tube, added with 2.5 μL 7-aminoactinomycin D (7-AAD) staining solution and 2.5 μL phycoerythrin (PE) staining solution, incubated at room temperature in the dark for 15 min, then added with 400 μL Binding Buffer buffer and mixed, and detected by flow cytometry, as shown in FIG. Figure 7-10 shown.

[0092] exist Figure 7-10The results showed that with the increase of the concentration of medipterostilbene, the total apoptosis rate ratio (Q2+Q3) showed a dose-dependent increase trend. The total apoptosis rate of the control group (0μmol / L) was 5.5±0.6%, while that of the drug-treated group reached 27.5±1.5% (100μmol / L), 46.6±2.5% (150μmol / L) and 61.9±1.4% (200μmol / L), respectively. It suggests that medipterostilbene can significantly induce apoptosis of human gastric cancer cells HGC-27, and mainly promote the occurrence of late apoptosis.

[0093] 5.2. Western Blot detection of cell proliferation and apoptosis-related proteins After 48 h of treatment with 0 μmol / L, 100 μmol / L (Example 2), 150 μmol / L (Example 3) and 200 μmol / L (Example 4) of medipholtziacin, cells were collected and proteins were extracted, subjected to SDS-PAGE gel electrophoresis, and transferred to a PVDF membrane. Subsequently, the cells were blocked with 5% skim milk powder for 1 h, incubated with primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 1 h at room temperature. Finally, the Azure Biosystems C500 near-infrared imaging system was used for image acquisition, as shown in Figure 2. Fig.11 shown.

[0094] exist Fig.11 Western Blot results showed that with the increase of medipterostilbene concentration, the expression of pro-apoptotic protein Cleaved PARP was significantly upregulated, while the intact PARP protein gradually decreased, indicating that medipterostilbene can induce apoptosis of HGC-27 cells in a concentration-dependent manner. In addition, the expression levels of p-Akt, p-mTOR, p-Stat3 and Mcl-1 proteins gradually decreased with the increase of medipterostilbene concentration, suggesting that medipterostilbene may induce apoptosis by inhibiting the Akt / mTOR / Stat3 signaling pathway and downregulating Mcl-1.

[0095] 5.3. Medipterocarpin induces apoptosis of human gastric cancer cell HGC-27 via Akt / mTOR / Stat3 signaling pathway In order to further explore the role of Akt / mTOR / Stat3 signaling pathway in medipharmacon-induced HGC-27 cell apoptosis, the present invention provides the following experimental implementation scheme: (1) control group (untreated); (2) IGF-1 treatment group (300 ng / mL); (3) Medicarpin treatment group (150 µmol / L); (4) Medicarpin+IGF-1 co-treatment group (150 µmol / LMedicarpin+300 ng / mL IGF-1). After 48 h of cell culture, total protein was extracted and Western blot was performed, as shown in Figure 2. Fig.12 shown.

[0096] exist Fig.12 Western blot results showed that medipterostilbene treatment could significantly inhibit the expression of p-Akt, p-mTOR, p-Stat3 and Mcl-1, and up-regulated Cleaved PARP, suggesting that medipterostilbene could induce apoptosis of HGC-27 cells by inhibiting the Akt / mTOR / Stat3 signaling pathway and down-regulating Mcl-1. IGF-1 treatment could partially restore the expression of p-Akt, p-mTOR, p-Stat3 and Mcl-1, and reduce the level of Cleaved PARP, indicating that the activation of the Akt / mTOR / Stat3 axis could antagonize the pro-apoptotic effect induced by medipterostilbene to a certain extent.

[0097] The present invention reveals the molecular mechanism by which medipterostilbene induces apoptosis of HGC-27 cells by inhibiting the Akt / mTOR / Stat3 signaling pathway and downregulating Mcl-1. Further studies have shown that IGF-1, as an agonist of the Akt signaling pathway, can partially reverse the pro-apoptotic effect of medipterostilbene, further confirming the key regulatory role of the Akt / mTOR / Stat3 signaling pathway in the survival and apoptosis of gastric cancer cells. The results of the present invention indicate that medipterostilbene may be a potential candidate drug for the treatment of gastric cancer and provide an experimental basis for targeted intervention of the Akt / mTOR / Stat3 axis.

[0098] In summary, Medipterocarbone inhibits the activation of the Akt / mTOR / Stat3 signaling pathway, downregulates the phosphorylation levels of p-Akt, p-mTOR and p-Stat3, thereby inhibiting the expression of the anti-apoptotic protein Mcl-1, and ultimately promoting the expression of Cleaved PARP and inducing cell apoptosis. Moreover, Medipterocarbone can also regulate the cytoskeleton structure, further enhancing its pro-apoptotic effect. The present invention discovers the anti-gastric cancer mechanism of Medipterocarbone for the first time, providing a new scientific basis and potential target for the research and development of gastric cancer therapeutic drugs. Medipterocarbone can be used as the main active ingredient to develop anti-gastric cancer drugs and prepare corresponding pharmaceutical preparations, aiming to achieve growth inhibition and apoptosis induction of gastric cancer cells by inhibiting the Akt / mTOR / Stat3 signaling pathway, and has good clinical application prospects.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of medipterostilbene in the preparation of gastric cancer drugs for inhibiting the proliferation of human gastric cancer cells HGC-27.

2. The use of medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin induces apoptosis of gastric cancer cells by upregulating the expression of the pro-apoptotic protein Cleaved PARP.

3. Use of the medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin induces apoptosis of gastric cancer cells by downregulating the expression of anti-apoptotic protein Mcl-1.

4. The use of medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin inhibits the activation of Akt signaling pathway, downregulates the expression of p-Ak protein, and induces apoptosis of gastric cancer cells.

5. Use of the medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin inhibits the activation of the mTOR signaling pathway, downregulates the expression of p-mTOR protein, and induces apoptosis of gastric cancer cells.

6. Use of the medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin inhibits the activation of Stat3 signaling pathway, downregulates the expression of p-Stat3 protein, and induces apoptosis of gastric cancer cells.

7. Use of medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin induces apoptosis of gastric cancer cells by destroying the cell microtubule network structure and actin fiber structure.

8. Use of the medipterocarpin according to claim 1 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: The target of medipterostilbene is at least one of the Akt signaling pathway, the mTOR signaling pathway or the Stat3 signaling pathway.

9. Use of the medipterocarpin according to any one of claims 1 to 8 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: Medipterocarpin is dissolved in dimethyl sulfoxide, and the concentration of Medipterocarpin is 100 μmol / L to 200 μmol / L.

10. Use of medipterocarpin according to claim 9 in the preparation of a gastric cancer drug for inhibiting the proliferation of human gastric cancer cells HGC-27, characterized in that: By increasing at least one of the concentration of medipterostilbene or prolonging the action time of medipterostilbene, the cell proliferation inhibition rate is enhanced, and the cloning ability, scratch healing ability and migration ability of gastric cancer cells are inhibited.

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