Data Availability StatementAll datasets generated because of this study are included in the article/supplementary material. of Cav-1 in the chemoresistance of GC would help to develop novel therapies for a better treatment end result of GC individuals. < 0.05 was considered statistically significant. Results Cav-1 Encourages Resistance of Human being GC Cells to Cisplatin Growing evidences have shown that upregulation of Cav-1 was found in multiple drug-resistance malignancy cells (7C9). To explore the function of Cav-1 in mediating drug resistance of GC cells, AGS cells were transiently transfected with Cav-1 (Cav-1+) or bare vector (EV) for 24, 48, and 72 h, respectively. We found that AGS cells transfected with Cav-1 for 24 h experienced higher levels of Cav-1 mRNA (Number 1A) and protein than those transfected with EV for 24 h (Numbers 1B,C). The endogenous Cav-1 was knocked down by transient transfection of shCav-1 (shCav-1) or bad control shRNA (NC) in MGC803 cells for 24, 48, and 72 h, respectively. Results from real-time PCR showed the mRNA manifestation of Cav-1 was significantly reduced in MGC803 cells after the cells were transfected with shCav-1 for 24 and 48 h (Number 1D), while the protein level of Cav-1 decreased at hours 48 and 72 after transfection (Numbers 1E,F). AGS/Cav-1+ cells and MGC803/shCav-1 cells were then exposed to cisplatin for 24 h. Concentration response curves in CCK-8 assays showed that AGS/Cav-1+ cells were more resistant than EV clones and the IC50 increased significantly from 11.98 to 20.69 (Figure 1G), while MGC803/shCav-1 cells were more sensitive to cisplatin than control cells, and the IC50 dropped from RAC1 8.24 to 4.77 (Figure 1H). Based on the IC50 value of each GC cell line, JMV 390-1 AGS cells were treated with 10 and 20 g/ml cisplatin respectively for 24 h, while MGC803 cells were exposed to 2.5 and 5 g/ml cisplatin, respectively, for 24 h. We found that AGS/Cav-1+ cells showed a significant decrease in cisplatin-induced cell death in compared with the control cells (Figure 1I). However, transient transfection of shCav-1 into MGC803 cells decreased cell survival rate in the present of cisplatin as compared with control cells JMV 390-1 (Figure 1J). Open in a separate window Figure 1 Cav-1 induces the survival of GC cell lines in the presence JMV 390-1 of cisplatin chemotherapy. (A) The mRNA expression level of Cav-1 was significant up-regulated in Cav-1-transfected AGS cells compared with control cells. (B) The protein level was in consistent with the mRNA expression of Cav-1. (C) The relative protein level of Cav-1 in AGS cells was analyzed. (D) The endogenous expression of Cav-1 mRNA in MGC 803 cells was mostly inhibited after the cells were transfected with shCav-1 vector for 24 JMV 390-1 h. (E) The protein level of Cav-1 was decreased after the cells were transfected with shCav-1 for 48 h. (F) The relative protein level of Cav-1 in MGC803 cells was analyzed. (GCJ) Cav-1-overexpression or Crepression GC cells were treated with increasing concentrations of cisplatin for 24 h. Cell viability was assessed by CCK-8 assay. JMV 390-1 AGS/Cav-1+ and MGC803/NC cells were more resistant than AGS/EV (G) and MGC803/shCav-1 cells (H). The survival of AGS/Cav-1+ cells was increased in the presence of cisplatin at the concentration of 10 and 20 g/ml (I). The survival of MGC803/shCav-1 cells was decreased in the presence of cisplatin at the concentration of 2.5 and 5 g/ml (J). The fold change of protein was standardized according to the protein levels in the EV or NC group. Optical density (OD) values are calculated as % survival SEM (= 3) of controls. Data are shown as mean SEM (= 3). *< 0.05, **< 0.01, ***< 0.001 compared with the empty vector or negative control. CDDP, cisplatin. Cav-1 Inhibits the Cisplatin-Induced Apoptosis in GC Cells Inhibition of cell apoptosis is a common mechanism that induces the drug-resistance of cancer cells. Thus, we determined whether Cav-1 could regulate the cisplatin-induced apoptosis in GC cells. Using Annexin V-PE/7-AAD flow cytometry assay, we found the level of apoptosis meaningfully declined with in the presence of Cav-1 after exposed to 20 g/ml cisplatin for 24 h (Figures 2A,B). Whereas, in the absence of Cav-1, the gastric cell apoptosis remarkably increased with 8 g/ml cisplatin for 24 h (Figures 2C,D). The expression of apoptosis-related proteins in both AGS and MGC803 cells were further evaluated. Cisplatin enhanced apoptotic response in the cleavage of caspase-3, caspase-9, and PARP in both AGS and MGC803 cells..