CIL-102 interacts with microtubule polymerization and causes mitotic arrest following apoptosis in the human prostate cancer PC-3 cell line.
There have been no therapeutic agents that provide a survival advantage in hormone-refractory prostate cancer. Recently, the Food and Drug Administration approved docetaxel combined with prednisone for the treatment of patients with advanced metastatic prostate cancer, and it does show a survival benefit. Hence, anti-microtubule drugs might be of benefit in chemotherapy of hormone-refractory prostate cancer. We used metastatic hormone-refractory prostate cancer PC-3 cells to investigate potential molecular mechanisms for CIL-102, a semisynthetic alkaloid derivative. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide and sulforhodamine B assays indicated that CIL-102 inhibits cell growth dose-dependently. Immunofluorescence microscopy and in vitro tubulin assembly assays indicated that CIL-102 binds to tubulin and disrupts microtubule organization. Flow cytometry showed that CIL-102 causes cells to accumulate in G(2)/M phase and sub-G(0)/G(1) phase. CIL-102-induced apoptosis was also characterized by immunofluorescence microscopy. Western blotting and kinase assays showed that CIL-102 exposure induced up-regulation of cyclin B1 and p34(cdc2) kinase activity and olomoucine, a p34(cdc2) inhibitor, profoundly reduced the number of cells accumulated in mitotic phase. Moreover, Bcl-2 phosphorylation, Cdc25C phosphorylation, and survivin expression were increased. CIL-102-induced apoptosis was associated with activation of caspase-3, but a noncaspase pathway may also be involved, since benzyloxycarbonyl-VAD-fluoromethyl ketone, a pancaspase inhibitor, only partially inhibited the apoptosis, and apoptosis-inducing factor was translocated from mitochondria to cytosol. We conclude that CIL-102 induces mitotic arrest and apoptosis by binding to tubulin and inhibiting tubulin polymerization. CIL-102 causes mitotic arrest, at least partly, by modulating cyclin-dependent kinases and then apoptosis executed by caspase and noncaspase pathways.
Huang
Yao-Ting
YT
Pharmacological Institute and School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan.
Huang
Dong-Ming
DM
Guh
Jih-Hwa
JH
Chen
I-Li
IL
Tzeng
Cherng-Chyi
CC
Teng
Che-Ming
CM
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D004396:Coloring Agents / Q000494:pharmacology; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008297:Male; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects* / Q000378:metabolism; D008938:Mitosis; D009362:Neoplasm Metastasis; D009363:Neoplasm Proteins; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000188:drug therapy*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012235:Rhodamines / Q000494:pharmacology; D000077022:Survivin; D013778:Tetrazolium Salts / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology*; D013997:Time Factors; D015854:Up-Regulation
D000590:Amino Acid Chloromethyl Ketones; D000970:Antineoplastic Agents; C107489:BIRC5 protein, human; C534699:CCNB1 protein, human; D004396:Coloring Agents; D019926:Cyclin B; D056744:Cyclin B1; D004791:Enzyme Inhibitors; D051034:Inhibitor of Apoptosis Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012235:Rhodamines; D000077022:Survivin; D013778:Tetrazolium Salts; D013844:Thiazoles; C484771:benzyloxycarbonyl-valyl-aspartic acid fluoromethyl ketone; C022027:lissamine rhodamine B; D016203:CDC2 Protein Kinase; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; C022616:thiazolyl blue