Department of Surgery, The Preston Robert Tisch Brain Tumor Center, Durham, NC 27710, USA.
Biochem Pharmacol. 2010 Nov 1;80(9):1343-55. doi: 10.1016/j.bcp.2010.07.019. Epub 2010 Jul 21.
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.
最近，我们报道人类 GSTP1 可被 PKC 类丝氨酸/苏氨酸激酶磷酸化并具有功能活性。在这项研究中，我们研究了 GSTP1 的这种翻译后修饰对肿瘤顺铂耐药性的贡献。使用两种恶性神经胶质瘤细胞系 MGR1 和 MGR3，评估了 PKCα 磷酸化 GSTP1 催化顺铂与谷胱甘肽结合的能力，并将其与细胞对顺铂的敏感性、顺铂诱导的 DNA 链间交联和细胞凋亡相关联。结果表明 PKCα 的激活和 GSTP1 的相关磷酸化与谷胱甘肽结合铂的形成增加、DNA 链间交联形成减少以及顺铂耐药性增加显著相关。在 PKC 激活后，MGR1 中顺铂的 IC50 从 13.63μM 增加到 36.49μM，MGR3 中从 20.75μM 增加到 38.45μM。在这两种细胞系中，siRNA 介导的 GSTP1 或 PKCα 转录抑制同样降低了顺铂的 IC50，并且与细胞内谷胱甘肽结合铂代谢物水平降低相关。同时抑制/转录抑制 PKCα 和 GSTP1 具有协同作用，增强了顺铂的敏感性。尽管顺铂诱导的细胞凋亡与 Bax 向线粒体易位、细胞色素 c 释放和 caspase-3/7 激活有关，但在 GSTP1 敲低后，重新定位的 Bax 和细胞色素 c 水平明显更高。这些结果支持了 GSTP1 的 PKCα 依赖性丝氨酸磷酸化及其相关的增加的顺铂代谢介导的顺铂耐药机制，并表明同时靶向 GSTP1 和 PKCα 以提高顺铂治疗效果的潜力。
