Salinomycin Induces Endoplasmic Reticulum Stress And Autophagy In Gastric Cancer Cells

Objective: To observe the effect of salinomycin on the proliferation of gastric cancer cells,and to determine the changes of autophagy flow and ER stress in gastric cancer cells induced by salinomycin,so as to explore the role of endoplasmic reticulum stress and autophagy in the anti-tumor process of salinomycin.Methods: 1.EdU proliferation and cell cycle methods were used to detect the proliferation and cell cycle progression changes of gastric cancer cell lines SGC-7901 and MGC-803 after treatment with different concentrations of salinomycin for24 hours,and the effect of salinomycin on the proliferation of SGC-7901 and MGC-803 cells was investigated;2.Mitochondrial membrane potential detection(JC-1)method was used to detect the changes of mitochondrial membrane potential in gastric cancer cells SGC-7901 and MGC-803 after treatment with different concentrations of salinomycin for 24 hours to study the antitumor effect of salinomycin on gastric cancer cells;3.The number of GFP-LC3 fluorescent spots in gastric cancer cells SGC-7901 and MGC-803 after treatment with different concentrations of salinomycin for 24 hours was detected,and the focusing of LC3 autophagosomes was detected by cellular immunofluorescence assay to explore the effect of salinomycin on autophagy flux of gastric cancer cells;4.The concentration of endoplasmic reticulum stress standard PDI was detected by immunofluorescence assay;The expression of ER stress signaling pathway proteins BIP and CHOP,and receptor proteins IRE1α and eIF2a were determined by Western-blot after treatment with salinomycin at different concentrations for 24 hours.Real-time quantitative PCR was used to detect the changes of ER stress pathway genes at the mRNA level to explore the effect of salinomycin on ER stress in gastric cancer cells.Results: 1.Compared with the negative control group,G2/M cycle arrest occurred in SGC-7901 and MGC-803 with the increase of salinomycin concentration(0.5μmol/L,1.0μmol/L,2.0μmol/L)in a certain concentration range,which inhibited the cell transformation to S phase,decreased the penetration rate of EdU dye into cells,and inhibited cell proliferation;2.Compared with the negative control group,the JC-1method showed that the red fluorescence decreased gradually,the green fluorescence increased gradually,and the apoptosis of SGC-7901 and MGC-803 gastric cancer cells were treated with salinomycin at different concentrations(0μM,0.5μM,1.0μM,2.0μM)for 24 hours;3.Compared with the negative control group,the number of fluorescence spots in GFP-LC3 fluorescence spot test increased with the increase of drug concentration(0.5μm,1.0μm,2.0μm);Immunofluorescence showed that compared with the negative control group,the LC3 fluorescence expression in SGC-7901 cells was increased and the fluorescence aggregation distribution was obvious after salinomycin treatment,suggesting that salinomycin treatment was positively correlated with the number of autophagosome formation,and salinomycin could induce autophagy in gastric cancer cells;4.Compared with the negative control group,salinomycin treated gastric cancer cell line MGC-803 showed fluorescence aggregation of PDI in the cytoplasm.With the increase of salinomycin concentration(0 μm,0.5 μm,1.0 μm),the ER stress pathway proteins BiP,CHOP and receptor proteins IRE1α were increased in SGC-7901 and MGC-803 cell lines,and the mRNA expressions of ER stress genes ATF6 、 IRE1α and PERK also increased.Conclusion: 1.Salinomycin can inhibit the proliferation of gastric cancer cells in vitro.2.Salinomycin can induce autophagy in gastric cancer cells.3.Salinomycin can trigger endoplasmic reticulum stress in gastric cancer cells.