BackgroundGlucose-6-phosphate dehydrogenase (G6PD), elevated in tumor cells, catalyzes the first reaction in the pentose-phosphate pathway. The regulation mechanism of G6PD and pathological change in human melanoma growth remains unknown.MethodsHEM (human epidermal melanocyte) cells and human melanoma cells with the wild-type G6PD gene (A375-WT), G6PD deficiency (A375-G6PD∆), G6PD cDNA overexpression (A375-G6PD∆-G6PD-WT), and mutant G6PD cDNA (A375-G6PD∆-G6PD-G487A) were subcutaneously injected into 5 groups of nude mice. Expressions of G6PD, STAT3, STAT5, cell cycle-related proteins, and apoptotic proteins as well as mechanistic exploration of STAT3/STAT5 were determined by quantitative real-time PCR (qRT-PCR), immunohistochemistry and western blot.ResultsDelayed formation and slowed growth were apparent in A375-G6PD∆ cells, compared to A375-WT cells. Significantly decreased G6PD expression and activity were observed in tumor tissues induced by A375-G6PD∆, along with down-regulated cell cycle proteins cyclin D1, cyclin E, p53, and S100A4. Apoptosis-inhibited factors Bcl-2 and Bcl-xl were up-regulated; however, apoptosis factor Fas was down-regulated, compared to A375-WT cells. Moderate protein expressions were observed in A375-G6PD∆-G6PD-WT and A375-G6PD∆-G6PD-G487A cells.ConclusionsG6PD may regulate apoptosis and expression of cell cycle-related proteins through phosphorylation of transcription factors STAT3 and STAT5, thus mediating formation and growth of human melanoma cells. Further study will, however, be required to determine potential clinical applications.
Glucose-6-phosphate dehydrogenase (G6PD) has been implicated in the regulation of cellular antioxidative mechanisms. Tumor cells often lose the balance of oxidation and antioxidation, but the role of G6PD in such an imbalance is still largely unknown. To investigate the related function of G6PD in tumor cells, we established a stable line of A375 human melanoma cells with G6PD gene knockdown by a shRNA lentiviral cloning and expression system. The A375-G6PDDelta cells displayed the stable GFP coexpression after repeated freeze-thaw cycles and multiple passages, accompanied by an 88.83% suppression of the endogenous G6PD expression and a 78.47% decrease in G6PD activity. In comparison with the A375-WT cells, they were characterized by a reduced proliferation with the MTT proliferation assay, a 25% decrease in colony-forming efficiency, and an up to 40% increase of apoptotic rate with flow cytometry analysis. When further challenged by diamide-induced oxidative stress, these cells showed that a median lethal dose (LD(50)) of 1.2 mM decreased from that of the A375-WT cells (1.8 mM), and levels of NADPH and GSH decreased by 2.4-, 8.8-fold, respectively, with a 7.3-fold increase of H(2)O(2), as those of A375-WT cells. These results demonstrated that A375-G6PDDelta is a new, stable G6PD-deficient human tumor cell line, and that silencing G6PD expression decreased tumor-cell proliferation and enhanced apoptosis. In addition, G6PD gene knockdown rendered tumor cells more susceptible to diamide-induced oxidative stress. Together, our data support the important functions of G6PD in the regulation of cell growth and antioxidative capacity of tumor cells.
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