Camptothecin induces mitotic arrest through Mad2-Cdc20 complex by activating the JNK-mediated Sp1 pathway

Abstract

Camptothecin (CPT) is a popular therapeutic agent that targets topoisomerase I. Our findings demonstrated that CPT-induced microtubule polymerization results in markedly increased histone H3 phosphorylation. CPT also enhanced interactions between the mitotic checkpoint proteins, Mad2 and Cdc20, and thereby increased mitotic arrest. Transient knockdown of Mad2 completely restored cell cycle progression from CPT-induced mitotic arrest, while simultaneously reduced cyclin B1 and Cdk1 expression. Moreover, we found that c-Jun N-terminal kinase (JNK) acts upstream of Sp1, which upregulates p21-mediated mitotic arrest in response to CPT; furthermore, knockdown of p21 restored cell cycle progression, while inhibition of Cdks completely restored cell cycle progression from CPT-induced mitotic arrest. We hypothesized that, during mitotic arrest in response to CPT, cell survival signaling blocks apoptosis, thereby enhancing mitotic arrest. As expected, a caspase-9 inhibitor, z-LEHD-FMK, and an autophagy inhibitor, 3-methyladenine (3 MA), significantly diminished CPT-induced mitotic arrest. On the other hand, when Mad2 was depleted, z-LEHD-FMK and 3 MA markedly increased apoptosis, and restored cell cycle progression. Taken together, these results suggest that CPT decodes the action of topoisomerase I-mediated tubulin targeting drugs, leading to mitotic arrest by upregulating Mad2 through the JNK-mediated Sp1 pathway and autophagy formation from tubulin polymerization.