Institute of Biotherapy, School of Biotechnology, Southern Medical University, Guangzhou, China.
Am J Chin Med. 2013;41(3):665-82. doi: 10.1142/S0192415X13500468.
Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.
去甲基斑蝥素（NCTD）目前被用于癌症治疗，但确切的作用机制尚不清楚。复制前复合物（pre-RCs）是细胞 DNA 复制所必需的，与恶性增殖高度相关。在这里，我们研究了 NCTD 对 HepG2 细胞中 pre-RC 成分的抑制作用。结果表明，NCTD 以剂量依赖的方式诱导 Cdc6 和 Mcm2 的降解。在 100μM NCTD 浓度下，约 70%的 Cdc6 和 50%的 Mcm2 被降解。此外，NCTD 抑制了 Mcm6 的核转位。进一步针对 G1 同步细胞的研究表明，NCTD 减少了染色质结合的 Cdc6、Mcm2 和 Mcm6。此外，当细胞同步进入细胞周期时，细胞被阻止进入 S 期，并在 G1 期积累。同样，NCTD 抑制了 DNA 复制。最后，与单独使用 NCTD（52%）和 Cdc6 耗竭（39%）相比，NCTD 与 Cdc6 耗竭联合使用导致更严重的细胞毒性（88%）。Cdc6 耗竭和 NCTD 之间观察到协同细胞毒性。总之，我们的结果表明，NCTD 抑制 pre-RC 组装；随后阻止 G1 到 S 期的转变；并抑制 HepG2 细胞中的 DNA 复制。复制前复合物是癌症治疗的一个有趣靶点，值得进一步研究以开发抗癌药物。
