Caveolin-1 Promotes Chemoresistance of Gastric Cancer Cells to Cisplatin by Activating WNT/β-Catenin Pathway

Abstract

Drug resistance is a major challenge for chemotherapy in treating human gastric cancer (GC), as the underlying molecular mechanism of chemoresistance in GC remains unknown. Caveolin-1 (Cav-1) is a scaffold protein of plasma membrane caveolae that acts as a tumor modulator by interacting with several cell signals. In this research, we showed that the survival rate of GC cells to cisplatin (CDDP) increased in the presence of Cav-1. Moreover, Cav-1 overexpression inhibited cisplatin-induced apoptosis and improved the survival rate of GC cells. Cav-1 overexpression and knock-down experiments indicated that Cav-1 expression stimulated wingless-type MMTV integration site (WNTs) pathway through the phosphorylation of LRP6 and dephosphorylation of β-catenin. Cav-1 was positively associated with the increase of WNT downstream target gene Met, which led to the activation of HER2 signaling. Moreover, our results demonstrated that the expression of Cav-1 and Met were positively associated with the resistance of GC cells to cisplatin. Collectively, Cav-1 enhances the cisplatin-resistance of GC cells by activating the WNT signaling pathway and Met-HER2 crosstalk. Understanding the role of Cav-1 in the chemoresistance of GC would help to develop novel therapies for a better treatment outcome of GC patients.

Keywords: Caveolin-1; WNT/β-catenin pathway; chemoresistance; cisplatin; gastric cancer.

Figure 1

Cav-1 induces the survival of GC cell lines in the presence 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 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. (G–J) Cav-1-overexpression or –repression GC cells were treated with increasing concentrations of cisplatin for 24 h. Cell viability was assessed by CCK-8 assay. 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 μg/ml (I). The survival of MGC803/shCav-1 cells was decreased in the presence of cisplatin at the concentration of 2.5 and 5 μg/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 (n = 3) of controls. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the empty vector or negative control. CDDP, cisplatin.

Figure 2

Cav-1 attenuates apoptotic response induced by cisplatin in GC cells. (A) Overexpression of Cav-1 inhibited the apoptosis of AGS cells. (B) Statistical analysis of the apoptotic rate of AGS cell population after CDDP treatment. (C) Knock-down of Cav-1 enhanced the apoptosis of MGC803 cells. (D) Statistical analysis of the apoptotic rate of MGC803 cell population after CDDP treatment. (E) The expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP were notably down-regulated in AGS/Cav-1+ cells compared with AGS/EV cells. (F) The relative levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP and Cav-1 in AGS cells were analyzed. (G) Knock-down of Cav-1 substantially increased the levels of cleaved caspase-3, cleaved caspase-9, and cleaved PARP in MGC803/shCav-1 cells compared with MGC803/NC cells. (H) The relative levels of cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Cav-1 in MGC803 cells were analyzed. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P <0.001 compared with AGS/EV cells or MGC803/NC cells. ^#P <0.05 compared with CDDP for 24 h after transfected with the empty or negative control vector. CDDP, cisplatin.

Figure 3

Cav-1 regulates the activation of key molecules involved in canonical WNT signaling pathway. (A) Overexpression of Cav-1 increased the phosphorylation of LRP6 but not the expression of Axin1, Dvl2, Dvl3, and total LRP6 in AGS/Cav-1+ cells compared with AGS/EV cells. (B) The relative levels of Axin1, Dvl2, Dvl3, p-LRP6, total LRP6, and Cav-1 in AGS cells were analyzed. (C) The fold change of p-LRP6 relative to total LPR6 in AGS cells was analyzed. (D) Knock-down of Cav-1 obviously inhibited the phosphorylation of LRP6, as well as expression of Axin1, Dvl2, Dvl3, and total LRP6 in MGC803/shCav-1 cells compared with MGC803/NC cells. (E) The relative levels of Axin1, Dvl2, Dvl3, p-LRP6, total LRP6, and Cav-1 in MGC803 cells were analyzed. (F) The fold change of p-LRP6 relative to total LPR6 in MGC803 cells was analyzed. (G) Following the activation in the upstream molecules of β-catenin, the phosphorylation of β-catenin at Ser552 site was notably reduced, while total β-catenin was increased in AGS/Cav-1+ cells compared with AGS/EV cells. (H) The relative levels of p-β-catenin at Ser33/37, Tyr41/Ser45, Ser552, and Ser675 sites, total β-catenin and Cav-1 in AGS cells were analyzed. (I) Knock-down of Cav-1 obviously increased phosphorylation of β-catenin at Thr41/Ser45 sites but decreased total β-catenin in MGC803 cells, although there was a little effect on the dephosphorylation of β-catenin at Ser33/37, Ser552, and Ser675. (J) The relative levels of p-β-catenin at Ser33/37, Tyr41/Ser45, Ser552, and Ser675 sites, total β-catenin and Cav-1 in MGC803 cells were analyzed. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P <0.001 compared with the EV or NC group.

Figure 4

Cav-1 induces the expression of target genes by activating the β-catenin pathway. (A) The immunofluorescence images showed that more β-catenin was located in the nucleus after transfected with Cav-1 for 72 h (as shown in red arrow). (B) TOP/FOP flash luciferase analysis showed that the level of β-catenin-LEF/TCF transcription was remarkably elevated by the overexpression of Cav-1. (C) The expression of Met was significantly increased in AGS cells at 24 h post Cav-1 transfection, while Cav-1 didn't affect the expression of LEF1. (D) The relative levels of LEF1, Met, and Cav-1 in AGS cells were analyzed. (E) The protein levels of LEF1 and Met were both reduced in Cav-1-repressed MGC803 cells at 72 h post Cav-1 transfection. (F) The relative levels of LEF1, Met, and Cav-1 in MGC803 cells were analyzed. Data are shown as mean ± SEM (n = 3). **P < 0.01, ***P <0.001 compared with the EV or NC group.

Figure 5

Cav-1 induces the activation of HER2 in GC cells. (A) Cav-1 stimulated the phosphorylation of HER2 at Tyr1221/1222 sites, but had no effect on the Tyr1248 phosphorylation in AGS/Cav-1+ cells compared with AGS/EV cells. (B) The relative levels of p-HER2 at Tyr1221/1222 and Tyr1248 sites, total HER2 and Cav-1 in AGS cells were analyed. (C) The fold change of p-HER2 relative to total HER2 in AGS cells was analyzed. (D) Both total and phosphorylated HER2 expression levels were attenuated in MGC803/shCav-1 cells compared with MGC803/NC cells. (E) The relative levels of p-HER2 at Tyr1221/1222 and Tyr1248 sites, total HER2 and Cav-1 in MGC803 cells were analyzed. (F) The fold change of p-HER2 relative to total HER2 in MGC803 cells was analyed. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P <0.001 compared with the EV or NC group.

Figure 6

Knock-down of Met prevents the Cav-1-induced cisplatin resistance of AGS cells by targeting the Met and HER2 pathway. (A) The viability of AGS/Cav-1+ cells was elevated compared with AGS cells, while AGS/shMet cells were more sensitive to cisplatin than AGS/Cav-1+ cells. (B) Cav-1 stimulated the activation of Met, p-HER2 at Tyr1221/1222 site and total HER2 in AGS cells. Met silencing inhibited the increase of p-HER2 at Tyr1221/1222 site and total HER2 induced by Cav-1. (C) The relative levels of p-HER2 at Tyr1221/1222, total HER2 and Cav-1 in AGS cells were analyzed. (D) The fold change of p-HER2 relative to total HER2 in AGS cells was analyzed. *P < 0.05, **P < 0.01, ***P < 0.001 compared with control group. ^###P <0.001 compared with Cav-1 overexpression group. CDDP, cisplatin.

Figure 7

Cav-1-dependent increase of Met leads to the cisplatin-resistance of gastric cancer cells. (A) The cell survival rate of AGS/CDDP cells was elevated compared with AGS cells. AGS/CDDP cells showed stronger anti-cisplatin capability than their parental cells. (B) AGS/CDDP cells showed a higher level of Cav-1 mRNA compared with control cells. (C) Met mRNA expression was markedly increased in AGS/CDDP cells. (D) The protein levels of Cav-1 and Met were in consistent with the mRNA expression in AGS cells. (E) The relative levels of Met and Cav-1 in AGS and AGS/CDDP cells were analyzed. Data are shown as mean ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P <0.001 compared with AGS cells. CDDP, cisplatin.