Blocking PI3K/AKT Pathway Inhibits Cisplatin Induce Gastric Cancer Cells Migration And Invasion

Background and ObjectiveChemotherapy is currently one of the most commonly used methods for the treatment of tumors in clinical practice.However,most patients with tumors will still relapse and metastasize after chemotherapy,which is also the main cause of cancer patients’ clinical death.In recent years;research on the correlation between chemotherapeutic drugs and biological characteristics of tumor cells has found that the chemotherapeutic drug cisplatin can induce tumor cells to undergo epithelial-mesenchymal transition(EMT)after the action of cisplatin on colon cancer and other tumor cells.Migration and invasion.The results of previous studies indicate that epirubicin,a chemotherapeutic drug,can activate the PI3K/AKT signaling pathway to induce EMT and promote the migration and invasion of gastric cancer cells.However,it is unclear whether the chemotherapeutic drug cisplatin can promote the migration and invasion of gastric cancer cells through the PI3K/AKT signaling pathway.Therefore,this study used the chemotherapeutic drug cisplatin to treat gastric cancer cells to investigate the effect of cisplatin on the migration and invasion of gastric cancer and Its possible mechanism.Materials and methods1.MTT assay to detect the toxic effect of cisplatin on gastric cancer cellsTake the gastric cancer cell BGC 823 in logarithmic growth phase and spread it into a 96-well plate with 1 × 105/ml cells.When the cell monolayer coating rate reaches about 50%,use 0 μg/ml、0.5 μg/ml、1 μg/ml2.5 μg/ml、and 5 μg/mlof cisplatin treated with Gastric cancer cells for 36 h,and the toxic effect of the chemotherapeutic drug cisplatin on gastric cancer cells was detected.2.Detect the effect of cisplatin on epithelial-mesenchymal transition in gastric cancer cells(1)Observation of cell morphology:Take the gastric cancer cell BGC 823 in logarithmic growth phase and digest it,and spread it into a 12-well plate.When the cell monolayer spreading rate reaches about 50%,the control group changes the fresh medium,and the experimental group Treated with 1 μg/ml cisplatin,and observed with an inverted microscope and photographed 36 hours after dosing,and compared the morphological changes of the two groups of cells.(2)Western blot:Digest logarithmic gastric cancer cells BGC 823,digest them,and spread them in a 10 cm diameter dish.When the cell monolayer spreading rate reaches about 50%,the control group changes the fresh medium,and the experimental group has gastric cancer.Cells were treated with 1μg/ml cisplatin,and the total protein of gastric cancer cells was extracted at 0 h、12 h、24 h and 36 h,and the expression levels of the marker proteins E-cadherin and N-cadherin of epithelial-mesenchymal transition of tumor cells were detected.3.Detect the effects of cisplatin on the migration and invasion of gastric cancer cells(1)Scratch experiment:Use a marker to mark the bottom of a 24-well plate in advance every 0.5 cm,and take a log phase digestion plate of gastric cancer cells.When the cell fusion reaches 100%,use 10 μl autoclave The tip of the pipette is scratched perpendicular to the bottom line with a sterilized ruler.Aspirate the original culture solution,wash it gently with PBS twice,add the serum-free medium in the control group,and add 1 μg/ml cisplatin in the experimental group.Serum-free medium was photographed under an inverted microscope to record the cell migration status at 0 h and 36 h after scratching,and the degree of scratch healing was calculated to determine the effect of cisplatin on the migration capacity of gastric cancer cells.(2)Transwell experiment:Take the gastric cancer cell plate and cell culture bottle in the logarithmic growth phase,and when the cell monolayer spreading rate reaches about 50%,the control group changes the fresh medium,and the experimental group contains 1 μg/ml cis The platinum medium was digested at 36 hours after dosing and spread into the Transwell chamber.The culture was continued for 24 hours in a constant temperature incubator,fixed,stained,dried,and observed under an inverted microscope.Three high-power fields were used to record the invading cells.The amount of cisplatin to detect the invasion ability of gastric cancer cells.(3)Western blot:Gastric cancer cells BGC 823 in logarithmic growth phase were evenly spread into 10 cm diameter dishes,and the total protein of cells treated with cisplatin for 0 h、12 h、24 h and 36 h was extracted to detect the correlation between cisplatin and gastric cancer cell metastasis.The effect of the protein expression of the proteins MMP-2,MMP-9.4.Test whether cisplatin can activate the PI3K/AKT signaling pathway and its role in epithelial-mesenchymal transition,gastric cancer cell migration and invasion ability(1)Western blot to detect whether cisplatin can activate the PI3K/AKT signaling pathway:extract the total protein of gastric cancer cells after adding 1μg/ml cisplatin for 0 h、12 h、24 h 36 h,and detect the protein of AKT and p-AKT after cisplatin action the amount of expression changes.(2)Scratch test:When the degree of cell fusion reaches 100%,the control group was replaced with serum-free medium,the cisplatin group was added with a serum-free medium containing 1 μg/ml of cisplatin,and the inhibitor group was added with 1 μg/ml.In the serum-free medium of cisplatin+inhibitor,at 0 h and 36 h of scratch,observe and photograph under the inverted microscope,calculate the scratch healing degree of each group,and detect the cisplatin pair after blocking the PI3K/AKT signaling pathway.Impact of Gastric Cancer Cell Migration Ability.(3)Transwell experiment:The gastric cancer cells BGC 823 in logarithmic growth phase were spread into culture flasks,the control group was replaced with fresh medium,the cisplatin group was added with a medium containing 1 μg/ml cisplatin,and the inhibitor group was added with 1 μg/ml cisplatin+inhibitor medium was treated for 36 hours,digested and spread into the Transwell chamber,and then cultured in a constant temperature incubator for 24 hours.After fixing,staining,and drying,observe and record the number of invaded cells in each group,and detect blocking effect of cisplatin on the invasion ability of gastric cancer cells after PI3K/AKT signaling pathway.(4)Western blot to detect the effects of blocked PI3K/AKT signaling pathway on cisplatin-induced gastric cancer cell epithelial-mesenchymal transition,migration and invasion ability:uniformly spread gastric cancer cells BGC 823 in logarithmic growth phase into 10 cm diameter when the cell monolayer spreading rate reached about 50%,the control group was replaced with fresh medium,the cisplatin group was added with 1 μg/ml cisplatin-containing medium,and the inhibitor group was added with 1μg/ml cisplatin+inhibitor.The medium was extracted from gastric cancer cells for 36 hours,and the effects of cisplatin on epithelial-mesenchymal transition and metastasis-related proteins in gastric cancer cells after blocking the PI3K/AKT signaling pathway were detected.Results1.MTT experiments show that cisplatin has a killing effect on gastric cancer cells BGC 823,and the mortality of gastric cancer cells is positively correlated with drug concentration.2.Cell morphology observation showed that compared with the control group,some gastric cancer cells changed into fusiform interstitial-like cells with enlarged spaces after cisplatin treatment,suggesting that gastric cancer cells may have undergone epithelial-interstitial transformation;Western blot The results showed that compared with 0 h,the expression level of E-cadherin protein in gastric cancer epithelial cells decreased,and the expression level of N-cadherin protein in mesenchymal cells increased at 12 h,24 h and 36 h.It suggests that cisplatin can induce epithelial-mesenchymal transition in gastric cancer cells.3.The results of the scratch test showed that compared with the control group,the healing of scratches increased when cisplatin was treated for 36 h,suggesting that cisplatin can enhance the migration potential of gastric cancer cells;Transwell test results showed that,compared with the control group,the cisplatin effect at 36 h,the number of cells that invaded the lower chamber increased,suggesting that cisplatin can enhance the invasion potential of gastric cancer cells;Western blot results show that compared with 0 h,when cisplatin acts at 12 h,24 h,and 36 h,gastric cancer cell metastasis-related protein MMP-2,The expression level of MMP-9 increased significantly,suggesting that cisplatin can promote the migration and invasion of gastric cancer cells.4.Western blot results showed that compared with 0 h,AKT phosphorylation level in gastric cancer cells was significantly increased after cisplatin treatment for different time,while AKT expression level was basically unchanged,suggesting that cisplatin can activate the PI3K/AKT signaling pathway.5.The scratch test showed that compared with the control group,the 36 hour scratch healing degree of the cisplatin group was increased,and the inhibitor group was reduced by 36 hours compared with the cisplatin-treated group,suggesting that cisplatin may pass the PI3K/AKT signaling Pathway enhances the migration capacity of gastric cancer cells;Transwell results show that compared with the control group,the number of cells invading into the lower chamber was significantly increased,and that of the inhibitor group was invading into the lower chamber compared with the cisplatin-treated group the number is significantly reduced,and cisplatin may enhance the invasion ability of gastric cancer cells through the PI3K/AKT signaling pathway;Western blot results showed that compared with the control group,the expression level of E-cadherin in the cisplatin group was reduced and the expression level of N-cadherin protein was increased.Compared with the cisplatin-treated group,the expression level of E-cadherin in the inhibitor group Increased and decreased expression of N-cadherin protein,suggesting that the blocked PI3K/AKT signaling pathway can partially reverse cisplatin-induced epithelial-mesenchymal transition;compared with the control group,the AKT phosphorylation level in the cisplatin group was increased,compared with cisplatin.Compared with the treatment group,the level of AKT phosphorylation in the inhibitor group was reduced,suggesting that the inhibitor Wortmannin can effectively inhibit the PI3K/AKT signaling pathway;compared with the control group,the protein expression levels of transfer-related proteins MMP-2 and MMP-9 were increased in the cisplatin group,and Compared with the cisplatin-treated group,the protein expression levels of the transfer-related proteins MMP-2 and MMP-9 in the inhibitor group were reduced,suggesting that the blocked PI3K/AKT signaling pathway can partially reverse the promotion effect of cisplatin on the migration and invasion of gastric cancer cells.conclusionThe chemotherapeutic drug cisplatin can induce epithelial mesenchymal transformation(EMT)in gastric cancer cells through the PI3K/AKT signaling pathway,thus promoting the migration and invasion of gastric cancer cells.