Role of TNF Receptor Superfamily in the Mechanism of CA405-induced Apoptosis on Human Lung Adenocarcinoma Cells

• Main Authors: Wei-Ling Lin, 林韋伶
• Other Authors: Kou-Wha Kuo
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/72255000346897856615

碩士 === 高雄醫學大學 === 生物化學研究所 === 88 === Apoptosis is a physiological death manner. When cells are affected by either outside or inside factors, the cell-death program is started to induce the process of suicide. Apoptosis is one of the most important topics in current cancer research. Many reports have demonstrated that the death of cancer cells induced by chemotherapeutic drugs and irradiation are through the process of apoptosis. Therefore, developing a new treatment for cancer via apoptosis is the way that researchers have spent much effort to achieve their goal. Lung cancer is the primary cancer causing death in Taiwan. It can be divided into two major groups, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC). Moreover, drug-resistance to anticancer drugs is the common clinical problem in the treatment of NSCLC. CA405, a new steroidal glycoalkaloid, was isolated from a Chinese herb. The cytotoxicity of CA405 was determined by MTS method. The results indicated that comparing to other four anticancer drugs (Taxol, Etoposide, Cisplatin and Gemcitabine), CA405 possessed a superior cytotoxicity to human adenocarcinoma cells (H441) (IC50 = 3 uM）. In addition, CA405 is a steroid compound, which can diffuse speedly into cells. After CA405 treatment, H441 cells were died within 2 hours. Although the maximum death of H441 was 2 hours, no further death was detected after extented incubation with constant amount of CA405, suggesting that the action of CA405 was irreversible. In order to define the action mechanism of CA405 on H441. The morphological changes, DNA content, and gene expression of H441 cells after CA405 treatment were analyzed. The appearances of chromatin condensation and apoptotic bodies in cells were observed after treatment with relatively high dose of CA405 (24 uM) for one hour. However, the cell membrane started to break after 3 hours. These results indicate that the apoptosis and necrosis may occur simultaneously depend on the dose of CA405. The sub-G1 peak of CA405—treated H441 cells was observed as revealed by flow cytometry. The outside membrane of cellular phosphatidylserine (PS) was increased and the cell shrinked after CA405 treatment were analyzed by Annexin-V staining. These results indicate that CA405 induced cell death by apoptosis. The cellular RNA was extracted and apoptosis-relative factors e.g. TNF receptors, Fas and TRAIL receptors were analyzed by RT-PCR. The results indicated that a parallel up-regulation of Fas, TNFR-I and TRAIL-R gene expression was triggered after CA405 treatment, and the CA405-mediated cytotoxicity could be neutralized with Fas and TNF receptor specific antibodies. The caspase-8 and -3 were also activated after CA405 treatment. To evaluate the synergy of drugs, combined therapy of cisplatin and CA405 on H441 cells has evidenced that the combination increases the cytotoxicity to the cells. The combination inhibits more than 70% of H441 which is originally drug-resistant to cisplatin. In conclusion, CA405 induced cell death of human lung adenocarcinoma cell by apoptosis. The TNF receptors and Fas may be involved in the mechanism of CA405-induced apoptosis. This study demonstrates the action mechanism of CA405, and may shed light on the investigation of Chinese herbs as new chemotherapeutic agents for cancer therapy.