CPT promotes G2/M phase arrest along with Chk2 and Cdc25C, resulting from coordinative DNA damage-mediated ATP and ROS generation. The JNK and ERK signal pathway also involved CPT-induced G2/M phase arrest accompanied by autophagy. These converging views may offer great opportunities for pharmacological intervention of CPT in cell cycle regulation and apoptosis.
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Chapter 3
Camptothecin induces mitotic arrest in LNCaP cells, resulting from Mad2-mediated cyclin B1 and Cdk1
expression: Implication of tubulin polymerization
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Abstract
Camptothecin (CPT) was first discovered as a topoisomerase I inhibitor, thus expanding to use clinical trial; however, its use has been limited because of adverse effect. Nevertheless, CPT is a prominent therapeutic model because CPT accurately targets topoisomerase I which is consistently active in cancer cells. Therefore, we investigated how CPT regulates cell cycle progression in human prostate cancer LNCaP cells. Our finding showed that treatment with CPT induced microtubule polymerization, not actin polymerization, resulting in remarkable upregulation of histone H3 phosphorylation which is a mitotic-specific marker and complex between Mad2 and Cdc20 known as mitotic checkpoint proteins, and thereby may increase mitotic prometaphase arrest. CPT also enhanced expression and activity of cyclin B1 and cyclin-dependent kinase 1 (Cdk1); thus, depletion of Mad2 completely restored cell cycle progression from CPT-induced mitotic arrest, accompanied by loss of cyclin B1 and Cdk1, suggesting that Mad-mediated cyclin B1 and Cdk1 complex is an important axis in CPT-induced mitotic arrest. Moreover, we found that c-Jun N-terminal kinase (JNK) is an upstream molecule for transcription factor Sp1 in response to CPT, which regulates p21-mediated mitotic arrest; however, knockdown of p21 slightly restored cell cycle progression from CPT-induced mitotic arrest. Nevertheless, inhibition of Cdks which are downstream targets of p21 completely restored from CPT-induced mitotic arrest. During mitotic arrest in response to CPT, we made a hypothesis that some cell survival signals block apoptosis, leading to enhance mitotic arrest. As presumed, a caspase-9 inhibitor, z-LEHD-FMK, and an autophagy inhibitor, 3-methyladenine (3MA), significantly diminished CPT-induced mitotic arrest with no apoptosis, suggesting that capase-9 and autophagy are associated to with CPT-induced mitotic arrest. On the other hand, in depletion of Mad2, z-LEHD-FMK and 3MA remarkably increased apoptosis, accompanied by restoration from CPT-mediated cell cycle
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progression. Taken together, these results indicate that CPT may be promising for decoding the molecular modes of topoisomerase I-mediated tubulin targeting drugs.
3.1 Introduction
The spindle assembly checkpoints (SACs) ensure cell cycle to delay until all duplicated chromosomes align and attach to the spindle, which guarantee genomic stability during mitosis (M) [Lara-Gonzalez et al., 2012]. Once mitotic spindles tightly bind to kinetochores through tubulin polymerization from prometaphase to metaphase, the spindle assembly checkpoints are inactivated, which allows tubulin depolymerization at anaphase [Wang et al., 2014]; when microtubule polymerization and depolymerization are incorrectly regulated, kinetochores trigger the spindle assembly checkpoint signal pathway, which halt cell cycle distribution in M phase. Therefore, microtubule-targeting strategy which disrupts or hyper-stabilizes spindle microtubules, has been anti-mitotic therapeutics to treat various cancers. In particular, microtubule polymerization inhibitors such as colchicines and vinblastine, and microtubule depolymerization inhibitor such as paclitaxel are intensively used for binding to tubulins, which disrupt microtubule dynamics and induce M phase arrest [Jordan et al., 2004]. Additionally, recent studies showed that microtubule-targeting drugs has been successfully used in clinical trial with much interest because their action is totally different from other DNA-targeting drugs [Tsimberidou et al., 2011; Field et al., 2014]. In fact, SACs arrest cell cycle distribution in M phase in response to signal from kinetochores as a results of impaired binding to spindle microtubules due to lack of microtubule-kinetochore attachment or improper tension imposed on sister kinetochores [Musacchio et al., 2015]. In particular, improper tension of microtubules on kinetochores renders catalytic site for Mad2, which inhibits anaphase onset by suppressing the anaphase promoting complex APC with Cdc20
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[Zhou et al., 2002]. Interaction among mitotic checkpoint complex such as Mad2 and Cdc20 inhibits the APC-mediated ubiquitination of securin and cyclin B1 [Tian et al., 2012].
Cyclin-dependent kinases (Cdks) are associated with different stages where cyclins regulate cell cycle distribution by inducing nuclear localization, phosphorylation, and dephosphorylation of the Cdks and degradation of the regulatory cyclin subunits [Dash et al., 2005]. Cyclins and Cdks bind p21 through the CRRL consensus site at N-terminal end required for inhibition of cell cycle progression [Rousseau et al., 1999]. In particular, p21 -/-mice have markedly decreased the number of mitotic embryonic fibroblasts cells with cyclin B1, suggesting which value of p21 promotes late G2/M phase arrest by regulating cyclin B1-Cdk1 activity [Dash et al., 2005]. Induction of chromosomal instability by depletion of Mad2 reduces the time available to proper oriented chromosome in metaphase leading to activate JNK signaling to tolerate chromosomal instability induced by spindle checkpoint defects [Wong et al., 2014]. This activation and phosphorylation of JNK regulates of the stability of transcription factor Sp1 during mitosis [Chuang et al., 2008]. Phosphorylated p21 binds to cyclin B1 and Cdc2 phosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21/cyclin B1 complex activated as a kinase which required Sp1 transcription [Dash et al., 2005; Kim et al., 2014]. Additionally, transient knockdown of cyclin B or Cdk1 inhibit or decreased the cells in M phase. Similarly, treatment with tubulin polymerization inhibitors nocodazole or tubulin depolymerization inhibitor paclitaxel also regulate the G2/M phase with marked induction of cyclin B1 and Cdk1 [Dash et al., 2005; Chadebech et al., 2000]. Choi et al. reported that selective knockdown of Mad2 resulted in degradation of cyclin B in nocodazole-treated G2/M stage; whereas the accumulation of cyclin B1 and Cdc2 is trigger the development of chromosomal condensation and segregation in prometaphase arrested cells [Choi et al., 2011].
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Xiao et al., reported that camptothecin (CPT)-induced S phase arrest which activate Chk1 and cause the rapid proteolysis of Cdc25A, whereas elimination of Chk1 expression through knockdown abrogate S phase and protect Cdc25A degradation [Choi et al., 2011].
CPT also induced apoptosis in cancer cells by miR-125b mediated mitochondrial pathways via targeting to the 3’UTR regions of p53, Bak1, and Mcl1 [Zeng et al., 2012]. Nevertheless, a little has been known on the molecular action of CPT in G2/M phase. In the present study, we investigated the molecular mechanisms underline activities of CPT in M phase of LNCaP cells. Additionally, we found that CPT-induced M phase arrest is regulated by Mad2-Cdc20 complex through JNK-dependent Sp1 pathway.