Camptothecin induces G 2 /M phase arrest through the ATM-Chk2-Cdc25C axis as a result of autophagy-induced cytoprotection: Implications of reactive oxygen species

// Rajapaksha Gedara Prasad Tharanga Jayasooriya 1, 4 , Matharage Gayani Dilshara 1 , Ilandarage Menu Neelaka Molagoda 1 , Cheol Park 2 , Sang Rul Park 1 , Seungheon Lee 1 , Yung Hyun Choi 3 and Gi-Young Kim 1 1 Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea 2 Department of Molecular Biology, College of Natural Sciences and Human Ecology, Dongeui University, Busan 47340, Republic of Korea 3 Department of Biochemistry, College of Oriental Medicine, Dong-Eui University, Busan 47227, Republic of Korea 4 Present address: Department of Bioprocess Technology, Faculty of Technology, University of Rajarata, Mihintale 50300, Sri Lanka Correspondence to: Yung Hyun Choi, email: choiyh@deu.ac.kr Gi-Young Kim, email: immunkim@jejunu.ac.kr Keywords: camptothecin; G 2 /M phase arrest; ROS; ATM; Chk2 Received: May 23, 2016      Accepted: February 28, 2018      Published: April 24, 2018 ABSTRACT In the present study, we report that camptothecin (CPT) caused irreversible cell cycle arrest at the G 2 /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 2 /M phase arrest and moderately induced S phase arrest, indicating that the ROS is required for the regulation of CPT-induced G 2 /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 2 /M phase, indicating that Nrf2 delayed the S phase in response to CPT. We also found that CPT-induced G 2 /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 2 /M phase arrest, suggesting that CPT enhances G 2 /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 2 /M phase arrest, and inhibition of JNK enhanced apoptosis in response to CPT. Finally, we found that CPT-induced G 2 /M phase arrest circumvented apoptosis by activating autophagy through ATM activation. These findings suggest that CPT-induced G 2 /M phase arrest through the ROS-ATM-Chk2-Cdc25C axis is accompanied by the activation of autophagy.