Investigation of anti-tumor mechanisms of K2154: characterization of tubulin isotypes, mitotic arrest and apoptotic machinery.
Microtubules are crucial targets for cancer chemotherapeutic drugs, and new microtubule-directed agents are of continued interest in drug development. A novel microtubule-directed agent, ethyl-2-[N-rho-chlorobenzyl-(2'-methoxy)]-anilino-4-oxo -4, 5-dihydro-furan-3-carboxylate, was identified. The compound, designated K2154, inhibited cell proliferation, with IC(50) values of 10.3, 15.3, 9.6, 11.2, 12.8 and 12.1 muM in prostate cancer PC-3, hepatocellular carcinoma Hep3B, non-small cell lung cancer A549, colorectal cancer HT29 and HCT116, and P-glycoprotein-rich breast cancer NCI/ADR-RES cells, respectively. Because NCI/ADR-RES cells were susceptible to inhibition by K2154, it indicated that this compound is a poor substrate for P-glycoprotein. In this study, PC-3 cells were used to identify the anticancer mechanisms of K2154. K2154 induced an arrest of the cell cycle at G2/M phase and a subsequent increase of hypodiploid phase in PC-3 cells, whereas it only induced a moderate level of G2/M arrest with little increase of hypodiploid phase in normal prostate cells. K2154 inhibited microtubule assembly in both in vitro turbidity assay and in vivo microtubule spin-down experiment. Immunochemical examination showed that K2154 caused formation of abnormal mitotic characteristics with bipolar spindles, particularly, in beta(II)- and beta(III)-tubulin staining. It also induced several pathways, including cyclin B1 up-regulation, dephosphorylation on Tyr(15) and phosphorylation on Thr(161) of Cdk1 and Cdc25C phosphorylation, and roscovitine (a Cdk1 inhibitor) significantly inhibited K2154-induced apoptosis, suggesting a pro-apoptotic role of Cdk1. Phosphorylation of Bcl-2 and Bcl-xL and cleavage of Mcl-1, together with activation of caspase-9 and -3, indicated that mitochondrial pathway played a central role in K2154-mediated apoptotic cell death. Additionally, AIF contributed to a late phase of K2154-induced apoptotic pathway. In conclusion, it is suggested that K2154 displays an anticancer activity through a target on microtubules and a subsequent signaling cascade on cell cycle regulation and apoptotic machinery.
Lu
Pin-Hsuan
PH
School of Pharmacy, College of Medicine, National Taiwan University, Taipei, Taiwan.
Kung
Fan-Lu
FL
Kuo
Sheng-Chu
SC
Chueh
Shih-Chieh
SC
Guh
Jih-Hwa
JH
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000814:Aniline Compounds / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D051033:Apoptosis Inducing Factor / Q000378:metabolism; D020169:Caspases / Q000187:drug effects / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D019926:Cyclin B / Q000187:drug effects / Q000378:metabolism; D056744:Cyclin B1; D045304:Cytochromes c / Q000378:metabolism; D016503:Drug Delivery Systems; D005663:Furans / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008870:Microtubules / Q000187:drug effects; D008928:Mitochondria / Q000378:metabolism; D008938:Mitosis / Q000187:drug effects; D010766:Phosphorylation; D014404:Tubulin / Q000187:drug effects* / Q000378:metabolism; D015854:Up-Regulation
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000814:Aniline Compounds; D000970:Antineoplastic Agents; D051033:Apoptosis Inducing Factor; C534699:CCNB1 protein, human; D019926:Cyclin B; D056744:Cyclin B1; D005663:Furans; D014404:Tubulin; C521415:ethyl 2-(N-p-chlorobenzyl-(2'-methoxy))anilino-4-oxo-4,5-dihydrofuran-3-carboxylate; D045304:Cytochromes c; D020169:Caspases