Ineffective glutathione regeneration on G6PD-knockdown Hep G2 cells

• Main Authors: Yi Ren Huang, 黃奕仁
• Other Authors: P. T. Y. Chiu
• Format: Others
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/48006408331318914410

碩士 === 長庚大學 === 醫學生物技術研究所 === 97 === Glucose-6-phosphate dehydrogenase (G6PD), the first and rate-limiting enzyme of the pentose phosphate pathway, is important to maintain intracellular reduced and oxidized glutathione ( GSH / GSSG ) ratio via NADPH regeneration. G6PD knockdown HepG2 ( Gi ) cells by RNAi technique were used as a model to elucidate the compensatory mechanism for ineffective GSH regeneration in G6PD knockdown cells under oxidative stress. Under basal condition, intracellular levels of NADPH / NADP+ and GSH / GSSG ratio were lower in Gi cells than those in control ( Sc ) cells. When cells were exposed to diamide, Gi cells were more susceptible to diamide-induced cell death compared with Sc cells. GSH regeneration and GSSG clearance were ineffective in Gi cells. NADP+ level was increased and NAD+ level was decreased in both cells. However, the increase of NADP+ and the decrease of NAD+ were larger in Gi cells than those in Sc cells. We found that NAD kinase activity was increased in Gi cells more dramatically than Sc cells when cells were exposed to diamide. Although NADPH level was significantly increased in G6PD knockdown cells, which were with an impaired ability to regenerate glutathione after diamide treatment. Our findings suggest that G6PD confers protection against oxidant-induced cytotoxicity through effective glutathione regeneration.