The study of cytotoxic mechanisms of paclitaxel and GL331 in human nasopharyngeal carcinoma cells

• Main Authors: Chih-Hung Shu, 許志宏
• Other Authors: Wen K. Yang
• Format: Others
• Language: zh-TW
• Published: 1998
• Online Access: http://ndltd.ncl.edu.tw/handle/88433503316887251158

博士 === 國立陽明大學 === 臨床醫學研究所 === 87 === Nasopharyngeal carcinoma (NPC) is one of the ten major cancers in Taiwan. The main treatment of NPC, radiotherapy, can effectively control the locoregional disease of NPC. However, distant metastasis attributes to most treatment failure of NPC. The current trend is to explore chemotherapy to improve the prognosis of NPC. Investigations of new chemotherapeutic agents in vitro will help their clinical application in the future. Paclitaxel, derived from the bark of Taxus brevifolia, is effective against ovarian, breast, lung and head and neck cancers. The action of paclitaxel was preliminary considered to enhance polymerization or suppress dynamic instability of microtubule. Cells treated with paclitaxel is blocked in G2/M. Apoptosis, observed in paclitaxel-treated cancer cells, is an important form of cell death. Many gene products have been identified as apoptosis regulation proteins, such as Bax and Bcl-2, which act as apoptosis inducer and inhibitor, respectively. Abnormal activation of cyclin B1/CDC 2 kinase induces apoptosis. In this study, two nasopharyngeal carcinoma (NPC) cell lines were treated with a range of doses of paclitaxel or GL331 to investigate their cytotoxic mechanisms. The LD50 values of paclitaxel were 0.7 to 0.9 nM. NPC cells treated with paclitaxel exhibited convoluted nuclei, condensed chromatin and decreased cellular and nuclear volume, and also showed genomic DNA degradation into multiple oligonucleosomal fragments, suggesting that paclitaxel induced apoptosis in NPC cells. Most NPC cells were arrested at the G2/M phase after treatment with 0.1 or 1 mM of paclitaxel for 6 hours and progressed to an enlarged sub-G2 peak in flow cytometric histographs. NPC cells treated with 5 nM paclitaxel showed a progressively enlarged sub-G1 peak. Either sub-G2 peak or sub-G1 peak indicated apoptosis proceeding in the NPC cells. The time-course flow-cytometric analysis of 5 nM paclitaxel at 1-h interval showed that transient G2/M blockage was a prerequisite step for paclitaxel-elicited sub-G1 apoptosis. The levels of Bcl-2 or Bax proteins remained unchanged in NPC cells treated with paclitaxel or GL331. There were no significant changes in the levels of cell cycle proteins including cyclins A, B1, D1, E, CDC 2, CDK 2, and PCNA after treatment with paclitaxel. Furthermore, cyclin A, D1 and E-associated histone H1 kinase activities remained unchanged after paclitaxel treatment, but histone H1 kinase associated with cyclin B1/CDC 2 was dramatically elevated. The degree of elevation was progressive with increase of paclitaxel concentration from 1 nM to 1 mM. These findings suggest that CDC 2 plays an important role in paclitaxel-induced apoptosis. CDK 7 kinase and CDC 25A phosphatase were two known regulators of CDC 2 kinase activity. The levels of CDK 7 and CDC 25A were not affected by paclitaxel treatment. However, CDK 7 immunoprecipitate from paclitaxel treated cells was able to increase cyclin B1-associated CDC 2 kinase activity, indicating that stimulating effect of paclitaxel treatment on cyclin B1-associated CDC 2 kinase was mediated through the activation of CDK 7 kinase. GL331 is a new semisynthetic podophyllotoxin compound developed to cope with the multiple drug resistance property evolved in cancer cells against its congener etoposide. GL331 also causes abnormal CDC 2 kinase activation and subsequent apoptosis in human NPC cell lines. GL331 treatment elevated the CDC 25A phosphatase activity and caused the dephosphorylation of CDC 2 proteins on Tyrosine-15 and Threonine-14 residues, suggesting that paclitaxel and GL331 elicited distinct mechanisms leading to apoptosis. We further determined the cytotoxic effect by combining paclitaxel with GL331. Our results reveal that the treatment with paclitaxel plus GL331 was less cytotoxic than the treatment with paclitaxel or GL331 alone. Both the activation of CDC 2 kinase and the induction of apoptosis were dramatically inhibited in NPC cells treated with 0.1 mM of paclitaxel and 1 mM of GL331 simultaneously. In conclusion, both paclitaxel and GL331 were cytotoxic to NPC cells and induced apoptotic reaction in NPC cells. Cyclin B1-associated CDC 2 kinase was activated in NPC cells treated with either paclitaxel or GL331. Paclitaxel and GL331 activated different upstream regulators of cyclin B1/CDC 2 kinase. Paclitaxel activated CDK 7 kinase while GL331 activated CDC 25A phosphatase. The different mechanisms of cytotoxic effect of paclitaxel and GL331 didn''t contribute to the cytotoxicity of combination of paclitaxel with GL331. Contrarily, combination of paclitaxel with GL331 revealed decreased cytotoxicity. The information is valuable for clinical application of paclitaxel and GL331 in the future.