Department of Urology, Ditmanson Medical Foundation Chiayi Christian Hospital, Chiayi 600, Taiwan.
Department of Health and Nutrition Biotechnology, Asian University, Taichung 41354, Taiwan.
Int J Mol Sci. 2021 Mar 17;22(6):3056. doi: 10.3390/ijms22063056.
Our previous study demonstrated that the glutathione -transferase Mu 5 (GSTM5) gene is highly CpG-methylated in bladder cancer cells and that demethylation by 5-aza-dC activates GSTM5 gene expression. The aim of the present study was to investigate the role of GSTM5 in bladder cancer. The levels of GSTM5 gene expression and DNA methylation were analyzed in patients with bladder cancer, and functional studies of GSTM5 were conducted using GSTM5 overexpression in cultured bladder cancer cells. Clinical analysis revealed that the GSTM5 mRNA expression was lower in bladder cancer tissues than in normal tissues and that the level of GSTM5 DNA methylation was higher in bladder cancer tissues than in normal urine pellets. Overexpression of GSTM5 decreased cell proliferation, migration and colony formation capacity. Glutathione (GSH) assay results indicated that cellular GSH concentration was decreased by GSTM5 expression and that GSH supplementation reversed the decrease in proliferation and migration of cells overexpressing GSTM5. By contrast, a GSH synthesis inhibitor significantly decreased 5637 cell GSH levels, survival and migration. Furthermore, GSTM5 overexpression inhibited the adhesion of cells to the extracellular matrix protein fibronectin. To elucidate the effect of GSTM5 on anticancer drugs used to treat bladder cancer, cellular viability was compared between cells with or without GSTM5 overexpression. GSTM5-overexpressed cells showed no significant change in the cytotoxicity of cisplatin or mitomycin C in 5637, RT4 and BFTC 905 cells. Though a degree of resistance to doxorubicin was noted in 5637 cells overexpressing GSTM5, no such resistance was observed in RT4 and BFTC 905 cells. In summary, GSTM5 plays a tumor suppressor role in bladder cancer cells without significantly affecting chemoresistance to cisplatin and mitomycin C, and the cellular GSH levels highlight a key mechanism underlying the cancer inhibition effect of GSTM5. These findings suggest that low gene expression and high DNA methylation levels of GSTM5 may act as tumor markers for bladder cancer.
我们之前的研究表明，谷胱甘肽 S-转移酶 Mu5（GSTM5）基因在膀胱癌细胞中高度 CpG 甲基化，5-氮杂-2′-脱氧胞苷（5-aza-dC）的去甲基化可激活 GSTM5 基因表达。本研究旨在探讨 GSTM5 在膀胱癌中的作用。分析了膀胱癌患者 GSTM5 基因表达和 DNA 甲基化水平，并通过在培养的膀胱癌细胞中过表达 GSTM5 进行 GSTM5 的功能研究。临床分析显示，GSTM5 mRNA 在膀胱癌组织中的表达低于正常组织，GSTM5 DNA 甲基化水平在膀胱癌组织中高于正常尿沉渣。GSTM5 的过表达降低了细胞的增殖、迁移和集落形成能力。谷胱甘肽（GSH）测定结果表明，细胞内 GSH 浓度因 GSTM5 表达而降低，GSH 补充剂逆转了过表达 GSTM5 的细胞增殖和迁移减少。相比之下，GSH 合成抑制剂显著降低了 5637 细胞的 GSH 水平、存活和迁移。此外，GSTM5 的过表达抑制了细胞对细胞外基质蛋白纤维连接蛋白的黏附。为了阐明 GSTM5 对用于治疗膀胱癌的抗癌药物的影响，比较了有无 GSTM5 过表达的细胞之间的细胞活力。在 5637、RT4 和 BFTC 905 细胞中，GSTM5 过表达的细胞对顺铂或丝裂霉素 C 的细胞毒性没有明显变化。虽然在过表达 GSTM5 的 5637 细胞中观察到对阿霉素的耐药程度有所提高，但在 RT4 和 BFTC 905 细胞中没有观察到这种耐药性。总之，GSTM5 在膀胱癌细胞中发挥肿瘤抑制作用，而对顺铂和丝裂霉素 C 的化疗耐药性没有显著影响，细胞内 GSH 水平突出了 GSTM5 抑制癌症作用的关键机制。这些发现表明，GSTM5 的低基因表达和高 DNA 甲基化水平可能作为膀胱癌的肿瘤标志物。
