| Summary: | 碩士 === 國立臺灣大學 === 藥理學研究所 === 90 === Cytotoxic chemotherapy has been used to treat patients with metastatic colorectal cancer with limited success. Therefore novel chemotherapeutic agents are needed. In the present study, we demonstrated that TW-01, a novel synthetic chemical compound, triggered the arrest of cell cycle in G2/M phase and induced apoptosis in human colorectal carcinoma cell line HT-29 as measured by TUNEL staining and FACS analysis. This increase in apoptosis is concentration- and time-dependent and associated with G2/M arrest. Since microtubule plays a pivotal role in the regulation of G2/M arrest, we explored the effects of TW-01 on the microtubule formation. In contrast with the stablization of microtubule polymerization by taxol, TW-01 significantly induced the microtubule depolymerization in HT-29 cells. In addition to the inhibition on microtubule polymerization, olomoucine, a p34cdc2 kinase inhibitor, partially protected against G2/M arrest and apoptosis mediated by TW-01 suggesting the involvement of p34cdc2 kinase. Furthermore, we examined whether caspase-3 involved in TW-01-induced apoptotic mechanism. Our studies revealed that TW-01-induced apoptosis was independent of caspase-3 activation. We also examined the expression of Bcl-2 family proteins, such as Bcl-2 and Bcl-XL in this study. However, there was little alteration of these protein expressions denying their participation in TW-01-induced effect. On the other hand, the increased expression of proapoptotic protein, Bax, was detected as early as 4 h after TW-01 treatment. Interestingly, preincubation of cells with pyrolidine dithiocarbamate (PDTC) statistically significantly protected against apoptosis mediated by TW-01 evoking the consideration that if reactive oxygen species (ROS) or the transcription factor NF-kB played a role on TW-01-induced effect. In a parallel experiment, the time-dependent increase of ROS formation after TW-01 treatment was detected; PDTC and trolox, an antioxidant, profoundly inhibited the ROS formation to TW-01 action. However, trolox could not mimic PDTC-mediated inhibition on TW-01-induced apoptosis by functional MTT assay method suggesting that PDTC did not behave as an antioxidant against the TW-01 action. Moreover, TW-01 was not able to significantly induce NF-kB activation revealing that the effect induced by PDTC was not throught the inhibition on NF-kB activation. We conclude that TW-01 induced the apoptosis in HT-29 cells via the inhibition of microtubule polymerization and the G2/M arrest of the cell cycle. The p34cdc2 kinase activation and Bax upregulation might play a role on TW-01-mediated apoptotic mechanism.
|
|---|
