A new G6PD knockdown tumor-cell line with reduced proliferation and increased susceptibility to oxidative stress.
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.
Li
Danyi
D
Department of Biochemistry, Kunming Medical University, 191 West Renmin Road, Kunming, People's Republic of China.
Zhu
Yuechun
Y
Tang
Qiongling
Q
Lu
Huiru
H
Li
Hongjun
H
Yang
Yinfeng
Y
Li
Zhigang
Z
Tong
Shufen
S
D000975:Antioxidants / Q000378:metabolism; D017209:Apoptosis; D001483:Base Sequence; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008545:Melanoma / Q000378:metabolism; D008969:Molecular Sequence Data; D018384:Oxidative Stress; D010957:Plasmids / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D014162:Transfection
D000975:Antioxidants; D034741:RNA, Small Interfering; D013778:Tetrazolium Salts; D013844:Thiazoles; D005954:Glucosephosphate Dehydrogenase; C022616:thiazolyl blue