Prasad Tharanga Jayasooriya Rajapaksha Gedara, Dilshara Matharage Gayani, Neelaka Molagoda Ilandarage Menu, Park Cheol, Park Sang Rul, Lee Seungheon, Choi Yung Hyun, Kim Gi-Young
Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea.
Present address: Department of Bioprocess Technology, Faculty of Technology, University of Rajarata, Mihintale 50300, Sri Lanka.
Oncotarget. 2018 Apr 24;9(31):21744-21757. doi: 10.18632/oncotarget.24934.
In the present study, we report that camptothecin (CPT) caused irreversible cell cycle arrest at the G/M phase, and was associated with decreased levels of cell division cycle 25C (Cdc25C) and increased levels of cyclin B1, p21, and phospho-H3. Interestingly, the reactive oxygen species (ROS) inhibitor, glutathione, decreased CPT-induced G/M phase arrest and moderately induced S phase arrest, indicating that the ROS is required for the regulation of CPT-induced G/M phase arrest. Furthermore, transient knockdown of nuclear factor-erythroid 2-related factor 2 (Nrf2), in the presence of CPT, increased the ROS' level and further shifted the cell cycle from early S phase to the G/M phase, indicating that Nrf2 delayed the S phase in response to CPT. We also found that CPT-induced G/M phase arrest increased, along with the ataxia telangiectasia-mutated (ATM)-checkpoint kinase 2 (Chk2)-Cdc25C axis. Additionally, the proteasome inhibitor, MG132, restored the decrease in Cdc25C levels in response to CPT, and significantly downregulated CPT-induced G/M phase arrest, suggesting that CPT enhances G/M phase arrest through proteasome-mediated Cdc25C degradation. Our data also indicated that inhibition of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibited CPT-induced p21 and cyclin B1 levels; however, inhibition of ERK blocked CPT-induced G/M phase arrest, and inhibition of JNK enhanced apoptosis in response to CPT. Finally, we found that CPT-induced G/M phase arrest circumvented apoptosis by activating autophagy through ATM activation. These findings suggest that CPT-induced G/M phase arrest through the ROS-ATM-Chk2-Cdc25C axis is accompanied by the activation of autophagy.
在本研究中，我们报告喜树碱（CPT）导致细胞周期在G/M期发生不可逆阻滞，并与细胞分裂周期25C（Cdc25C）水平降低以及细胞周期蛋白B1、p21和磷酸化组蛋白H3水平升高相关。有趣的是，活性氧（ROS）抑制剂谷胱甘肽减少了CPT诱导的G/M期阻滞，并适度诱导了S期阻滞，表明ROS是CPT诱导的G/M期阻滞调节所必需的。此外，在CPT存在的情况下，瞬时敲低核因子红系2相关因子2（Nrf2）会增加ROS水平，并使细胞周期进一步从S期早期转变为G/M期，表明Nrf2在响应CPT时延迟了S期。我们还发现，CPT诱导的G/M期阻滞随着共济失调毛细血管扩张突变（ATM）-检查点激酶2（Chk2）-Cdc25C轴的增加而增加。此外，蛋白酶体抑制剂MG132恢复了CPT作用下Cdc25C水平的降低，并显著下调了CPT诱导的G/M期阻滞，表明CPT通过蛋白酶体介导的Cdc25C降解增强了G/M期阻滞。我们的数据还表明，抑制细胞外信号调节激酶（ERK）和c-Jun氨基末端激酶（JNK）可抑制CPT诱导的p21和细胞周期蛋白B1水平；然而，抑制ERK可阻断CPT诱导的G/M期阻滞，抑制JNK则增强了CPT诱导的细胞凋亡。最后，我们发现CPT诱导的G/M期阻滞通过激活自噬（通过ATM激活）规避了细胞凋亡。这些发现表明，CPT通过ROS-ATM-Chk2-Cdc25C轴诱导的G/M期阻滞伴随着自噬的激活。
