| Summary: | 博士 === 國立臺灣大學 === 藥理學研究所 === 90 === Prostate cancer is one of the toughest healthy problems in men in the Western as well as in Asia. The thorniest problem in prostate cancer therapy is that no effective treatment regimen is available once the disease has progressed to hormone refractory prostate cancer (HRPC). In the meanwhile, the drug resistance has imposed a heavy burden on the development of effective treatment regimens. The goal of this thesis is to develop new anticancer agents for prostate cancer therapy and to explore the action of mechanism of anticancer drugs resistance on prostate cancer.
Firstly, in a large scale screening test we found an effective compound, TW-01, which caused a time- and concentration-dependent apoptosis of human prostate carcinoma cells (PC-3 cells) using DNA fragmentation assay and TUNEL-reaction technique. By MTT reduction assay, TW-01 showed a potent cytotoxic activity in PC-3 cells under 24 h treatment with an IC50 value of 0.3 mM; whereas it exhibited much less effect on human umbilical veins endothelial cells (HUVECs). Taxol (10 mM) only elicited a modest cancericidal activity toward PC-3 cells; in comparison, TW-01 induced a similar effect at a very low concentration of 30 nM. These results demonstrated that TW-01 is a very potent and selective cancericidal agent in PC-3 cells. TW-01 induced the arrest of cell cycle profound accumulation of cells at G2/M phase before resulting in apoptosis. Using in situ immunofluorescent detection of mitotic spindles and in vitro microtubule dynamics assay, our data showed that TW-01 inhibited microtubule polymerization and mitotic spindles formation, suggesting that TW-01 induces the G2/M arrest and apoptosis in PC-3 cells via causing microtubule disarray. Furthermore, TW-01 significantly induced the activation of olomoucine-inhibitable p34Cdc2 kinase. Furthermore, olomoucine also profoundly inhibited the cell accumulation at G2/M phase and their subsequent apoptosis to TW-01 action. Moreover, TW-01 also showed a promising anticancer activity in SCID mice model. We suggest that TW-01 is a potent and selective anticancer agent against human prostate carcinoma. The cell cycle arrests at G2/M phase via inhibition of microtubule polymerization and p34Cdc2 kinase activation, and the accompanying apoptosis may explain the mechanism of TW-01-evoked cancericidal activity.
Secondly, we found that cardiac glycosides (e.g. ouabain, and digitoxin) induced in vitro resistance of PC-3 cells to tubulin-binding anticancer drugs, such as paclitaxel, colchicine, vincristine, and vinblastine. Cardiac glycosides reversed the G2/M arrest of the cell cycle and cell apoptosis induced by tubulin-binding agents. However, neither ionomycin (a Ca2+ ionophore) nor veratridine (a Na+ ionophore) mimicked the preventive action of cardiac glycosides, indicating that elevation of the intracellular Ca2+ concentration and Na+ accumulation were not involved in this cardiac glycoside action. Furthermore, cardiac glycosides showed little influence on the cytotoxicity effects induced by actinomycin D, anisomycin, and doxorubicin suggesting the selectivity on microtubule-targeted anticancer drugs. Using in situ immunofluorescent detection of mitotic spindles, our data showed that cardiac glycosides diminished paclitaxel-induced accumulation of microtubule spindles; however, in a non-cell assay system, cardiac glycosides had little direct influence on colchicine- and paclitaxel-induced microtubule dynamics. Using an isotope-labeled assay method, we found that ouabain modestly but significantly inhibited the transport of [14C]paclitaxel from the cytosol into the nucleus. It is suggested that cardiac glycosides inhibit the G2/M arrest induced by tubulin-binding anticancer drugs via an indirect blockade of microtubule function. The decline in transport of these drugs into the nucleus may partly explain the cardiac glycoside action. However, the exact action mechanism needs further investigation
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