The Research Of SPARC Affect The Sensitivity Of Hepatocellular Carcinoma Cells To 5-FU By Regulating Glucose Metabolism

[Objectives] To investigate the effect and molecular mechanisms about glycolysis regulation of SPARC in hepatocellular carcinoma, and establish foundation for future therapeutic application of SPARC in resistant hepatocellular carcinoma.[Methods] Hep G2, Hep3 B and Hu H7 human hepatocellular carcinoma cells were stably transfected with vector containing wild type SPARC or si RNA SPARC, Glucose uptake and lactate product, Glut1、Hexokinase and LDHA activity were assayed by colourimetry method. Hep G2 cells were treated with gradually increasing concentrations of 5-FU in regular cell culture conditions for selection of 5-FU resistant cells. Expression of SPARC, AMPK, Thr-172, AMPK in glucose deprivation conditions, and changes of key enzymes of glycolysis in resistant cells or sensitive cells were measured by western blotting. The levels of m RNA of Glut1、HKⅡ and LDHA were measured by RT-PCR. Knockdown of glucose metabolism key enzymes by si RNA, or transfection of SPARC, or co-transfection SPARC and glycolysis enzymes(Glut1, HKⅡ, LDHA), comparing cells sensitivity to 5-FU by trypan blue staining respectively, and detect the effects of SPARC on Hep G2 cell activity under glucose deprivation environment.[Results] The glucose uptake and lactate product were significantly decreased by SPARC in Hep G2, Hep3 B and Huh7 cells, and overexpression of SPARC significantly down regulated the expressions and activity of Glut1, HKII and LDHA. The reverse results were observed when knocked down SPARC by si RNA. Hep G2 cells with overexpression of SPARC showed minor apoptosis under low glucose conditions, and the expression levels of AMPK, Thr-172 AMPK were up regulated.The glucose metabolism key enzymes(Glut1, HKII and LDHA) were up regulated in 5-FU resistant cells. Overexpression of SPARC resulted in an increased sensitivity to 5-FU chemotherapy, and cell viabilities significantly decreased after treated with 5-FU. The re-sensitization obtained by overexpression of SPARC in 5-FU resistant cells were overridden by co-transfection of SPARC and glycolysis enzymes.[Conclusions] SPARC negatively regulates glucose metabolism in hepatocellular carcinoma by down regulating the expression of Glut1, HKII and LDHA in glucose metabolism pathway. Overexpression of SPARC renders Hep G2 cells tolerance to glucose deprivation. 5-FU resistant liver cancer cells exhibit decreased SPARC expression and up regulated glucose metabolism. Overexpression of SPARC re-sensitizes 5-FU resistant Hep G2 cells through down regulation of glycolysis.