Institute of Biomedical Sciences, National Chung Hsing University, Taichung, 40227, Taiwan.
Department of Surgery, Feng-Yuan Hospital, Ministry of Health and Welfare, Taichung, 42055, Taiwan.
J Exp Clin Cancer Res. 2018 Jul 20;37(1):161. doi: 10.1186/s13046-018-0837-9.
Oxaliplatin belongs to the platinum-based drug family and has shown promise in treating cancer by binding to DNA to induce cytotoxicity. However, individual patients show diverse therapeutic responses toward oxaliplatin due to yet-unknown underlying mechanisms. We recently established that oxaliplatin also exert its anti-cancer activity in gastric cancer cell lines by targeting tumor-associated NADH oxidase (tNOX), attenuate NAD generation and reduce NAD-dependent sirtuin 1 (SIRT1) deacetylase activity, which in turn enhances p53 acetylation and apoptosis.
In this study, differential cellular outcomes in response to oxaliplatin exposure of p53-wild-type versus p53-null HCT116 human colon cancer cells were examined. Cell growth profile was determined by cell impedance measurements and apoptosis was analyzed by flow cytometry. The engagement between oxaliplatin and tNOX protein was studied by cellular thermal shift assay. Furthermore, western blot analysis revealed that p53 was important in regulating tNOX expression in these cell lines.
In p53-wild-type cells, we found that oxaliplatin inhibited cell growth by inducing apoptosis and concurrently down-regulating tNOX at both the transcriptional and translational levels. In p53-null cells, in contrast, oxaliplatin moderately up-regulated tNOX expression and yielded no apoptosis and much less cytotoxicity. Further experiments revealed that in p53-wild-type cells, oxaliplatin enhanced ROS generation and p53 transcriptional activation, leading to down-regulation of the transcriptional factor, POU3F2, which enhances the expression of tNOX. Moreover, the addition of a ROS scavenger reversed the p53 activation, POU3F2 down-regulation, and apoptosis induced by oxaliplatin in p53-wild-type cells. In the p53-null line, on the other hand, oxaliplatin treatment triggered less ROS generation and no p53 protein, such that POU3F2 and tNOX were not down-regulated and oxaliplatin-mediated cytotoxicity was attenuated.
Our results show that oxaliplatin mediates differential cellular responses in colon cancer cells depending on their p53 status, and demonstrate that the ROS-p53 axis is important for regulating POU3F2 and its downstream target, tNOX. Notably, the depletion of tNOX sensitizes p53-null cells to both spontaneous and oxaliplatin-induced apoptosis. Our work thus clearly shows a scenario in which targeting of tNOX may be a potential strategy for cancer therapy in a p53-inactivated system.
奥沙利铂属于铂类药物家族，通过与 DNA 结合诱导细胞毒性，显示出治疗癌症的潜力。然而，由于未知的潜在机制，个体患者对奥沙利铂的治疗反应存在差异。我们最近发现，奥沙利铂还通过靶向肿瘤相关的 NADH 氧化酶（tNOX）在胃癌细胞系中发挥其抗癌活性，减弱 NAD 的产生并降低 NAD 依赖性 SIRT1 去乙酰化酶活性，进而增强 p53 乙酰化和细胞凋亡。
本研究检测了 p53 野生型与 p53 缺失的 HCT116 人结肠癌细胞对奥沙利铂暴露的不同细胞反应。通过细胞阻抗测量法测定细胞生长曲线，通过流式细胞术分析细胞凋亡。通过细胞热转移分析研究奥沙利铂与 tNOX 蛋白的结合。此外，Western blot 分析表明，p53 在这些细胞系中对 tNOX 表达的调节很重要。
在 p53 野生型细胞中，我们发现奥沙利铂通过诱导细胞凋亡，同时在转录和翻译水平下调 tNOX，从而抑制细胞生长。相比之下，在 p53 缺失细胞中，奥沙利铂适度上调 tNOX 表达，不产生细胞凋亡，细胞毒性也大大降低。进一步的实验表明，在 p53 野生型细胞中，奥沙利铂增强 ROS 的生成和 p53 的转录激活，导致转录因子 POU3F2 的下调，从而增强 tNOX 的表达。此外，添加 ROS 清除剂可逆转 p53 激活、POU3F2 下调和奥沙利铂在 p53 野生型细胞中诱导的细胞凋亡。另一方面，在 p53 缺失系中，奥沙利铂处理触发的 ROS 生成较少，p53 蛋白也较少，因此 POU3F2 和 tNOX 未下调，奥沙利铂介导的细胞毒性减弱。
我们的结果表明，奥沙利铂通过其对 p53 状态的不同作用，在结肠癌细胞中介导不同的细胞反应，并表明 ROS-p53 轴对于调节 POU3F2 及其下游靶标 tNOX 很重要。值得注意的是，tNOX 的耗竭使 p53 缺失细胞对自发性和奥沙利铂诱导的细胞凋亡敏感。我们的工作清楚地表明，针对 tNOX 可能是一种在 p53 失活系统中用于癌症治疗的潜在策略。
