Background: Elevated intracellular Ca2+ ([Ca2+] i ) level could lead to [Ca2+] i overload and promote apoptosis via different pathways. In our previously study, up-regulated expression of transient receptor potential canonical channel (TRPC5) was proven to increase [Ca2+] i level, and resulted in chemoresistance whereas not apoptosis in human colorectal cancer (CRC) cells. The ATP-dependent homeostatic maintenance of resting [Ca2+] i should be important in this process. Increased glycolysis was found to be an important adenosine triphosphate (ATP) source in cancer. This study aimed to explore the potential mechanism of aerobic glycolysis in transient receptor potential channel TRPC5 induced chemoresistance.
Methods: In this study, we examined glucose transporter 1 (GLUT1) expression, glucose consumption and celluar ATP production to determine glycolytic activity. Real-time PCR and western blot were analyzed to determine TRPC5 expression at the mRNA and protein levels in human CRC cells (HCT-8, LoVo), and fluorouracil (5-Fu) resistant CRC cells (HCT-8/5-Fu, LoVo/5-Fu). 3-bromopyruvate (3-BP) and 2-Deoxy-D-glucose (2DG) were used to inhibit glycolysis. Glycolytic activity, intracellular Ca2+ ([Ca2+] i ) and the half maximal inhibitory concentration of 5-Fu (5-Fu IC50) were measured. Western blot was analyzed to determine cleaved Caspase-3 protein level. Flow cytometry was performed to detect the apoptosis rates. Immunohistochemistry staining was performed to determine TRPC5 and GLUT1 expression level in human CRC tissues.
Results: Overproduced of TRPC5 and increased glycolysis were found in HCT-8/5-Fu and LoVo/5-Fu than in HCT-8 and LoVo cells. Compared to HCT-8 cells, the HCT-8/5-Fu cells showed higher [Ca2+] i levels which decreased after treated with TRPC5-specific shRNA. Furthemore, inhibition of glycolysis resulted in decreased ATP production, elevation of [Ca2+] i level and cleaved caspase-3, increased apoptotic cells rate, and a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu cells, while showed no effect in HCT-8 cells. BAPTA-AM, a [Ca2+] i chelator, could reduce the elevation of cleaved caspase-3 and increased apoptotic cells rate due to glycolysis inhibition. Advanced CRC patients with high expression of TRPC5/GLUT1 displayed poorer chemotherapy outcome, and notably, the significant association between high TRPC5 expression and chemoresistance is GLUT1 expression level dependent.
Conclusions: We demonstrated the essential role of glycolysis in TRPC5 induced chemoresistance in human CRC cells via maintaining [Ca2+] i homeostasis.
Keywords: Chemoresistance; Colorectal cancer; Glycolysis; Intracellular Ca2+; Transient receptor potential canonical C5.