| Summary: | 碩士 === 高雄醫學大學 === 生物化學研究所 === 88 === Non-small cell lung cancers (NSCLC) is a predominant malignancy in Taiwan. The chemotherapy of NSCLC did not improve the mortality of NSCLC patients. CA405, a new alkaloid glycoside, was isolated from a Chinese herb. In this study, MTS and [3H]-thymidine incorporation methods were utilized to determine the cytotoxicity of CA405, Taxol, Cisplatin, Etoposide and Gemcitabine. The results indicated that CA405 possessed a higher cytotoxicity to human lung squamous cell carcinoma (H520) than the other chemotherapeutic agents. The morphological change of H520 induced by CA405 e.g. chromatin condensation and DNA fragmentation was observed in CA405-treated cell, indicating that CA405 induced cell death by apoptosis. The result was varified by Annexin-V flow cytometry analysis. The sub-G1 feature analyzed by a flow cytometer appeared after 3-h of CA405 treatment and the cell population of S phase was increased after treatment. The results implied that CA405 might arrest cell cycle at S phase and drove the cells at G2/M phase to apoptosis. In addition, the death of H520 was utmost in 1 h of treatment, and no increase death of H520 was observed for an extent-incubation of 16 h, suggesting that the effect of CA405 on H520 was irreversible. The synergistic effect of CA405 and cisplatin for H520 cells was also investigated. Combination of CA405 and cisplatin demonstrated an enhancement of cytotoxicity to H520. To define the apoptotic mechanism of CA405 on H520, the change of gene expression after CA405 treatment was studied. A significant up-regulation of TNFR-I and TNFR-II gene expression was obsvered by RT-PCR and specific monoclonal antibodies. In addition, the function of CA405 could be inhibited by TNFR-I and TNFR-II antibodies. Thus, these results indicated that the over expression of TNFR-I and -II might be involved in CA405-induced cell apoptosis of human lung squamous cell carcinoma.
In addition, the TNFR antibodies did not completely inhibit the cytotoxic effect of CA405, revealing that the other mechanisms might also involve in the CA405-induced apoptosis. In fact, TRAIL-R1, TRAIL-R2 and TRAIL-R3 demonstrated a moderated up-regulation after CA405 treatment in H520. The involvement of TRAILR in CA405-induced apoptosis remains to be determined.
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