Oh-Hashi Kentaro, Irie Nao, Sakai Takayuki, Okuda Kensuke, Nagasawa Hideko, Hirata Yoko, Kiuchi Kazutoshi
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan.
Mol Cell Biochem. 2016 Aug;419(1-2):29-40. doi: 10.1007/s11010-016-2747-5. Epub 2016 Jul 8.
Recently, we developed a variety of phenformin derivatives as selective antitumor agents. Based on previous findings, this study evaluated a promising compound, 2-(2-chlorophenyl)ethylbiguanide (2-Cl-Phen), on the basis of stress responses in the human colon cancer cell line HT-29 under a serum- and glucose-deprived condition. 2-Cl-Phen triggered morphological changes such as shrinkage and plasma membrane disintegration, as well as a decrease in mitochondrial activity and an increase in LDH leakage. To understand intracellular issues relating to 2-Cl-Phen, this study focused on the expression levels of ER stress-inducible genes and several oncogenic genes. Serum and glucose deprivation significantly induced a variety of ER stress-inducible genes, but a 12-h treatment of 2-Cl-Phen down-regulated expression of several ER stress-related genes, with the exception of GADD153. Interestingly, the expression levels of ATF6α, GRP78, MANF, and CRELD2 mRNA were almost completely decreased by 2-Cl-Phen. This study also observed that a 24-h treatment of 2-Cl-Phen attenuated the expression levels of GRP78, GADD153, and c-Myc protein. The decrease in c-Myc protein occurred before the fluctuation of GRP78 protein, while the expression of c-Myc mRNA showed little change with cotreatment of serum and glucose deprivation with 2-Cl-Phen. To further understand the 2-Cl-Phen-induced down-regulation of ATF6-related genes, this study investigated the stability of ATF6α and GRP78 proteins using NanoLuc-tagged constructs. The expression levels of NanoLuc-tagged ATF6α and GRP78 were significantly down-regulated by 2-Cl-Phen in the presence or absence of the translation inhibitor cycloheximide. Taken together, our novel phenformin derivative 2-Cl-Phen has the unique characteristic of diminishing tumor adaptive responses, especially the expression of ATF6-related genes, as well as that of c-Myc protein, in a transcriptional and posttranscriptional manner under a serum- and glucose-deprived condition. Further characterization of cytotoxic mechanisms related to phenformin derivatives may give new insights into developing additional promising anticancer agents.
最近，我们开发了多种苯乙双胍衍生物作为选择性抗肿瘤剂。基于先前的研究结果，本研究在血清和葡萄糖缺乏条件下，根据人结肠癌细胞系HT-29中的应激反应，评估了一种有前景的化合物2-(2-氯苯基)乙基双胍(2-Cl-Phen)。2-Cl-Phen引发了形态学变化，如细胞收缩和质膜解体，以及线粒体活性降低和乳酸脱氢酶泄漏增加。为了了解与2-Cl-Phen相关的细胞内问题，本研究重点关注内质网应激诱导基因和几种致癌基因的表达水平。血清和葡萄糖剥夺显著诱导了多种内质网应激诱导基因，但2-Cl-Phen处理12小时下调了几种内质网应激相关基因的表达，GADD153除外。有趣的是，2-Cl-Phen几乎完全降低了ATF6α、GRP78、MANF和CRELD2 mRNA的表达水平。本研究还观察到，2-Cl-Phen处理24小时可减弱GRP78、GADD153和c-Myc蛋白的表达水平。c-Myc蛋白的减少发生在GRP78蛋白波动之前，而c-Myc mRNA的表达在血清和葡萄糖剥夺与2-Cl-Phen联合处理时变化不大。为了进一步了解2-Cl-Phen诱导的ATF6相关基因下调，本研究使用纳米荧光素酶标记构建体研究了ATF6α和GRP78蛋白的稳定性。在存在或不存在翻译抑制剂环己酰亚胺的情况下，2-Cl-Phen显著下调了纳米荧光素酶标记的ATF6α和GRP78的表达水平。综上所述，我们新型苯乙双胍衍生物2-Cl-Phen具有独特特性，即在血清和葡萄糖缺乏条件下，以转录和转录后方式减少肿瘤适应性反应，特别是ATF6相关基因以及c-Myc蛋白的表达。对苯乙双胍衍生物相关细胞毒性机制的进一步表征可能为开发更多有前景的抗癌药物提供新见解。
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