Aquaporin 3 facilitates chemoresistance in gastric cancer cells to cisplatin via autophagy

Abstract

Cisplatin (cDDP) remains one of the first-line chemotherapeutic agents for gastric cancer (GC) treatment, and resistance to cDDP is the major limitation in its clinical application. Mechanisms of cDDP resistance have been shown to be varied and complicated. Aquaporin 3 (AQP3) has been demonstrated to be overexpressed in GC tissues and is thought to be involved in GC carcinogenesis and progression. However, the role of AQP3 in chemosensitivity of GC to cytotoxic agents remains unknown. In this study, we show that AQP3 overexpression induced resistance to cDDP in AGS cells (P<0.05), and AQP3 knockdown increased the chemosensitivity in MGC803 and SGC7901 cells (P<0.05). Moreover, cDDP treatment enhanced AQP3 expression in MGC803, SGC7901 and AGS cells. AQP3 overexpression promoted the conversion of LC3-I to LC3-II in AGS cells, whereas AQP3 knockdown inhibited this conversion in MGC803 and SGC7901 cells. AQP3 upregulation increased Atg5 and Beclin-1 expression, and inhibited P62 expression in AGS cells, whereas AQP3 knockdown showed the opposite results in MGC803 and SGC7901 cells. Chloroquine (CQ), an autophagy inhibitor, enhanced the cytotoxicity of cDDP in GC cells, and CQ reversed the chemoresistance to cDDP caused by AQP3 overexpression in GC cells. Together, our data demonstrate that AQP3 facilitates cisplatin resistance in gastric cancer cells via autophagy, and suggest that the development of AQP3-based tumor therapeutics could play a key role in future GC treatment strategies.

Figure 1

Aquaporin 3 (AQP3) mediates chemoresistance in gastric cancer cells to cisplatin (cDDP). (a) cDDP exerted cytotoxicity in AGS, SGC7901 and MGC803 cells in a dose-dependent manner; *P< 0.05 compared with untreated group. (b) AQP3 expression was upregulated in AGS cells and downregulated in MGC803 and SGC7901 cells after lentiviral transduction. (c) AQP3 overexpression induced resistance to cDDP compared with the null control in AGS cells, and AQP3 knockdown increased chemosensitivity in MGC803 and SGC7901 cells; *P<0.05. (d) AQP3 expression in MGC803, SGC7901 and AGS cells was enhanced after cDDP treatment. UNTR, untreated group.

Figure 2

Aquaporin 3 (AQP3) enhances autophagy in gastric cancer cells. (a) AQP3 overexpression induced the conversion of LC3-I to LC3-II in AGS cells, and AQP3 knockdown inhibited this conversion in MGC803 and SGC7901 cells (b, *P<0.05). AQP3 upregulation increased the expression of Atg5 and Beclin-1, and inhibited P62 expression in AGS cells, whereas AQP3 knockdown produced the opposite results in MGC803 and SGC7901 cells. (c) LC3 immunostaining showed that a larger number of small clusters and intensely stained granules were distributed in the cytoplasm near the nuclei of AGS-AQP3^high cells compared with AGS-NC cells. However, a certain number of granules were detected in MGC803-NC and SGC7901-NC cells, whereas MGC803-AQP3^low and SGC7901-AQP3^low cells showed fewer granules (d, *P<0.05). Original magnification ×200.

Figure 3

Chloroquine (CQ) increases the sensitivity of gastric cancer cells to cisplatin (cDDP) and reverses the chemoresistance induced by aquaporin 3 (AQP3). (a) CQ had little effect on the viability of MGC803, SGC7901 and AGS cells at doses under 50 μM. (b) CQ enhanced the effects of cDDP on the proliferation of GC cells in MGC803, SGC7901 and AGS cells; *P<0.05. (c) The combination of cDDP and CQ showed more severe cytotoxicity in MGC803-AQP3^low and SGC7901-AQP3^low cells than in null control (NC) cells or cDDP treatment alone in MGC803-AQP3^low and SGC7901-AQP3^low cells. The combination therapy significantly reduced the viability of AGS-AQP3^high cells compared with cDDP treatment alone. However, the combination therapy exerted less cytotoxicity in AGS-AQP3^high cells than in AGS-NC cells, and the effect of combination therapy on AGS-AQP3^high cells was similar to cDDP alone on AGS-AQP3^high cells; *P<0.05. UNTR, untreated group.