Department of Urology, Singapore General Hospital, 20 College Road, 169856, Republic of Singapore; Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Republic of Singapore.
Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543, Republic of Singapore.
Life Sci. 2018 Jun 15;203:282-290. doi: 10.1016/j.lfs.2018.04.032. Epub 2018 Apr 22.
To investigate the effect of dioxonaphthoimidazolium analog YM155 on cell cycle progression of the clear-cell variant of renal cell carcinoma (ccRCC).
Cell cycle analysis was performed using bromodeoxyuridine (BrdU) and PI, apoptosis initiation was monitored using Annexin V and proteins expression was determined using western immunoblotting.
Here, we showed that YM155 activated stress-related molecules (histone H2AX, checkpoint kinases Chk1 and Chk2, p53) that mediate DNA damage checkpoint responses. The coordinated activation of these effector molecules disrupts progression of the cell cycle at the S phase as deduced from BrdU pulsing experiments and the ensuing changes in the levels of proteins (cyclins, CDKs, CDK inhibitors, phosphatases) that control cell cycle progression. Notably, we found increases in cyclin E and Cdc2 which regulate transition of cells from G1 to S, even as losses were observed for other CDKs and their cyclin partners. Furthermore, by inducing a loss in total pRb possibly by promoting its degradation, YM155 promoted the E2F transcription of genes that regulate entry into the S phase. After 24 h, cell cycle arrest to repair YM155-inflicted DNA damage was overtaken by p53-mediated apoptosis. YM155 induced increases in pro-apoptotic proteins (Bax and Bad), diminished anti-apoptotic proteins (Mcl-1, Bcl-xl, XIAP, survivin) and initiated cleavage of apoptotic marker proteins caspase 3 and PARP.
Taken together, the added insight provided on the cell cycle perturbative effects of YM155 may assist clinicians in framing rational choices for combining YM155 with other anti-cancer drugs or treatment modalities in ccRCC.
研究二恶并萘并咪唑鎓类似物 YM155 对肾透明细胞癌（ccRCC）透明细胞变异体细胞周期进程的影响。
使用溴脱氧尿苷（BrdU）和 PI 进行细胞周期分析，使用 Annexin V 监测细胞凋亡的起始，使用 Western 免疫印迹法测定蛋白质表达。
在这里，我们表明 YM155 激活了介导 DNA 损伤检查点反应的应激相关分子（组蛋白 H2AX、检查点激酶 Chk1 和 Chk2、p53）。这些效应分子的协调激活会破坏 S 期细胞周期的进展，这可以从 BrdU 脉冲实验推断出来，并且随之而来的是控制细胞周期进程的蛋白质（细胞周期蛋白、CDK、CDK 抑制剂、磷酸酶）水平的变化。值得注意的是，我们发现调节细胞从 G1 到 S 过渡的细胞周期蛋白 E 和 Cdc2 增加，即使其他 CDK 和它们的细胞周期蛋白伴侣丢失。此外，通过诱导总 pRb 的丢失（可能通过促进其降解），YM155 促进了调节进入 S 期的基因的 E2F 转录。24 小时后，细胞周期停滞以修复 YM155 造成的 DNA 损伤被 p53 介导的细胞凋亡所取代。YM155 诱导促凋亡蛋白（Bax 和 Bad）增加，减少抗凋亡蛋白（Mcl-1、Bcl-xl、XIAP、survivin），并启动凋亡标志物蛋白 caspase 3 和 PARP 的切割。
综上所述，YM155 对细胞周期的扰动作用提供了更深入的了解，这可能有助于临床医生在将 YM155 与其他抗癌药物或治疗方式联合用于 ccRCC 时做出合理的选择。
