Serine phosphorylation of glutathione S-transferase P1 (GSTP1) by PKCα enhances GSTP1-dependent cisplatin metabolism and resistance in human glioma cells.
Recently, we reported that the human GSTP1 is phosphorylated and functionally activated by the PKC class of serine/threonine kinases. In this study, we investigated the contribution of this post-translational modification of GSTP1 to tumor cisplatin resistance. Using two malignant glioma cell lines, MGR1 and MGR3, the ability of PKCα-phosphorylated GSTP1 to catalyze the conjugation of cisplatin to glutathione was assessed and correlated with cisplatin sensitivity and cisplatin-induced DNA interstrand cross-links and apoptosis of the cells. The results showed PKCα activation and associated phosphorylation of GSTP1 to correlate significantly with increased glutathionylplatinum formation, decreased DNA interstrand cross-link formation and increased cisplatin resistance. Following PKC activation, the IC(50) of cisplatin increased from 13.63μM to 36.49μM in MGR1 and from 20.75μM to 38.45μM in MGR3. In both cell lines, siRNA-mediated GSTP1 or PKCα transcriptional suppression similarly decreased cisplatin IC(50) and was associated with decreased intracellular levels of glutathionylplatinum metabolite. Combined inhibition/transcriptional suppression of both PKCα and GSTP1 was synergistic in enhancing cisplatin sensitivity. Although, cisplatin-induced apoptosis was associated with the translocation of Bax to mitochondria, release of cytochrome c and caspase-3/7 activation, the levels of relocalized Bax and cytochrome c were significantly greater following GSTP1 knockdown. These results support a mechanism of cisplatin resistance mediated by the PKCα-dependent serine phosphorylation of GSTP1 and its associated increased cisplatin metabolism, and suggest the potential of simultaneous targeting of GSTP1 and PKCα to improve the efficacy of cisplatin therapy.
Singh
Simendra
S
Department of Surgery, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA. simendra.singh@duke.edu
Okamura
Tatsunori
T
Ali-Osman
Francis
F
D000970:Antineoplastic Agents / Q000378:metabolism*; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000378:metabolism* / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D005910:Glioma / Q000188:drug therapy* / Q000473:pathology; D051549:Glutathione S-Transferase pi / Q000037:antagonists & inhibitors / Q000502:physiology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D010766:Phosphorylation; D051571:Protein Kinase C-alpha / Q000502:physiology*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000970:Antineoplastic Agents; C494826:BAX protein, human; D007211:Indoles; D008301:Maleimides; D034741:RNA, Small Interfering; D051028:bcl-2-Associated X Protein; D012694:Serine; C496556:GSTP1 protein, human; D051549:Glutathione S-Transferase pi; C496628:PRKCA protein, human; D051571:Protein Kinase C-alpha; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002945:Cisplatin