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Dual Inhibition of Cdc7 and Cdk9 by PHA-767491 Suppresses Hepatocarcinoma Synergistically with 5-Fluorouracil

Significance Statement

Upon DNA damage, checkpoint kinase 1 (Chk1) is activated through ATM/ATR-mediated phosphorylation to facilitate cell division, a mechanism essential in the chemoresistance of highly malignant tumors to 5-fluorouracil (5-FU) and other antimetabolite family of drugs. At the replication checkpoint, Cell division cycle kinase 7 (Cdc7)  interacts with and subsequently phosphorylates Claspin, which binds to Chk1 to result in its activation. Inhibition of the Cdc7/Chk1 pathway has become a tentative therapeutic strategy to treat chemoresistant tumors. The PHA-767491 compound is a dual inhibitor of both Cdc7 kinase and cyclin-dependent kinase 9 (Cdk9), a member of the Cdk family of kinases which bind to cyclins and phosphorylate their substrates to regulate transcription as well as cell differentiation. Ours is the first study to examine whether PHA-767491could sensitize hepatocarcinoma (HCC) to 5-FU treatment. Our data demonstrated that PHA-76749 and 5-FU has synergistic antitumor effect to suppress human HCC cells both in vitro and in vivo. PHA-767491 in combination with 5-FU exhibited strong cytotoxicity and induced significant apoptosis manifested by remarkably increased caspase 3 activation and poly(ADP-Ribose) polymerase fragmentation in HCC cells. PHA-767491 directly counteracted the 5-FU-induced phosphorylation of Chk1, a substrate of Cdc7; and decreased the expression of the anti-apoptotic protein myeloid leukemia cell 1 (Mcl1), which is downstream of Cdk9. Tumor tissues sectioned from nude mice HCC xenografts also showed decreased Chk1 phosphorylation and increased in situ cell apoptosis. Our study suggests that PHA-767491 enhances the efficacy of 5-FU by inhibiting Chk1 phosphorylation and down-regulating Mcl1 expression through inhibition of Cdc7 and Cdk9, thus combinational administration of PHA-767491 with 5-FU could be potentially beneficial to HCC patients. There is evidence that PHA-767491 could be well tolerated by animals due to the relatively slow division rate of normal cells, but systematic evaluation of the in vivo toxicity of the compound is still lacking. Our findings support the feasibility of further clinical evaluation of co-administration of antimetabolites together with PHA-767491, a small molecule that inhibits cell division checkpoint kinases. The combinational protocol may provide an effective and tolerable therapeutic modality for advanced and resistant HCC.

Figure Legend: Theme of the downstream signaling events of PHA-767491 and 5-FU in regulation of cell fate.

 

Dual Inhibition of Cdc7 and Cdk9 by PHA-767491 Suppresses Hepatocarcinoma Synergistically with 5-Fluorouracil. Global Medical Discovery

 

 

 

 

 

 

 

 

Journal Reference

Wei LI, Zhao XL, Shang SQ, Shen HQ, Chen XI. Curr Cancer Drug Targets. 2015 Feb 11.

No. 57 Zhu Gan Xiang, Hangzhou, Zhejiang 310003, China. [email protected]

Abstract

Activation of checkpoint kinase 1 (Chk1) is essential in chemoresistance of hepatocarcinoma (HCC) to 5-fluorouracil (5-FU) and other antimetabolite family of drugs. In this study, we demonstrated that PHA-767491, a dual  inhibitor of two cell cycle checkpoint kinases, cell division cycle kinase 7 (Cdc7) and cyclin-dependent kinase 9 (Cdk9), has synergistic antitumor effect with 5-FU to suppress human HCC cells both in vitro and in vivo. Compared with the sole use of each agent, PHA-767491 in combination with 5-FU exhibited much stronger cytotoxicity and induced significant apoptosis manifested by remarkably increased caspase 3 activation and poly(ADP-Ribose) polymerase fragmentation in HCC cells. PHA-767491directly counteracted the 5-FU-induced phosphorylation of Chk1, a substrate of Cdc7; and decreased the expression of the anti-apoptotic protein myeloid leukemia cell 1, a downstream target of Cdk9. In tumor tissues sectioned from nude mice HCC xenografts, administration of PHA-767491also decreased Chk1 phosphorylation and increased in situ cell apoptosis. Our study suggests that PHA-767491 could enhance the efficacy of 5-FU by inhibiting Chk1 phosphorylation and down-regulating Mcl1 expression through inhibition of Cdc7 and Cdk9, thus combinational administration of PHA-767491 with 5-FU could be potentially beneficial to patients with advanced and resistant HCC.

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