TRPC Inhibitor SKF96365 Combined With Cisplatin In The Treatment Of Gastric Cancer

Background: Gastric cancer is a common gastrointestinal malignant tumor in China,and its pathogenesis is complex.Abnormal calcium signal is closely related to the occurrence and development of gastric cancer.Calcium ions,as the second intracellular messenger,participate in the regulation of many important biological behaviors of tumor cells such as proliferation,migration,invasion and apoptosis.Therefore,calcium homeostasis in tumor cells is critical for cell function and survival.Transient receptor potential channels（TRPC）are important calcium channels in cell membrane that regulate the changes of intracellular and intracellular calcium concentration.TRPC targeting inhibitors are expected to become new antitumor drugs.SKF96365 is a broad spectrum inhibitor of TRPC.Existing studies have found that SKF96365 can inhibit the proliferation of esophageal cancer,breast cancer,colon cancer and other tumor cells.At present,the antitumor effect of SKF96365 in gastric cancer and its molecular mechanism remain unclear.Objective: To explore the inhibitory effect of SKF96365,a broad spectrum inhibitor of TRPC,combined with cisplatin on human gastric cancer cells and its molecular mechanism.Methods: TRPC gene subtypes in gastric cancer tissues and different gastric cancer cells were identified by immunofluorescence and RT-PCR;CCK-8 method was used to detect the drug inhibition rate of SKF96365 combined with cisplatin on gastric cancer cells;The morphological changes of gastric cancer cells were observed under light microscope;The effects of combined administration on proliferation and migration of human gastric cancer cells were investigated by cell clone formation assay and cell scratch assay;Hoechst staining and flow cytometry were used to detect the apoptosis rate after combined administration;The mitochondrial membrane potential loss was detected by JC-1 staining;Mito ROS mitochondrial reactive oxygen fluorescence probe,Rhod-2/AM fluorescence probe were used to detect the changes of intracellular mitochondrial reactive oxygen concentration and mitochondrial calcium concentration after combined administration;Western blot was used to detect apoptosis,autophagy protein levels and PI3K/AKT/BAD signaling pathway-related protein levels.Results: RT-PCR results showed that TRPC m RNA was expressed in human gastric cancer cell lines MGC-803 and HGC-27;Immunofluorescence showed that the expression of TRPC6 in different stages of gastric cancer was positively correlated with the progression of gastric cancer;CCK-8 results showed that TRPC inhibitor SKF96365 combined with cisplatin could inhibit the activity of human gastric cancer cells;Morphological changes such as unclear cell contour,chromatin shrinkage and nuclear fragmentation were observed under light microscope after combined administration;Clone formation and cell scratch test indicated that SKF96365 combined with cisplatin could significantly inhibit the proliferation and migration of human gastric cancer cell lines in vitro;The results of Hoechst staining and flow cytometry showed that the apoptosis rate of the two human gastric cancer cell lines increased after SKF96365 combined with cisplatin in vitro;JC-1 staining showed that combined administration could significantly reduce the mitochondrial membrane potential of gastric cancer cells,leading to mitochondrial dysfunction;The detection results of Mito ROS mitochondrial reactive oxygen fluorescence probe,Rhod-2/AM fluorescence probe showed that the concentration of mitochondrial reactive oxygen in gastric cancer cells increased and mitochondrial calcium overload after SKF96365 combined with cisplatin;Western Bolt results showed that combined administration increased the levels of apoptosis and autophagy related proteins in gastric cancer cells,and significantly inhibited the phosphorylation level of PI3K/AKT/BAD signaling pathway in gastric cancer cells,suggesting that the inhibition of SKF96365 combined with cisplatin on gastric cancer cells was related to the inhibition of PI3K/AKT/BAD signaling pathway.Conclusion: SKF96365 combined with cisplatin induces gastric cancer cells to initiate mitochondrial apoptosis pathway and protective autophagy by inhibiting the phosphorylation level of PI3K/Akt/Bad pathway.