Mechanisms of HER-2/neu induced drug sensitivity to methotrexate in bladder cancer cell lines: signal transduction and cell cycle analysis

• Main Author: 許家誠
• Other Authors: 賴明德
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/14589573838431475228

碩士 === 國立成功大學 === 生物化學研究所 === 88 === Chemotherapy plays an important role in the clinical tumor treatment, but the tumor cells may acquire resistance to those drugs. Recent study established that HER-2/neu and p53 are correlated with chemotherapeutic drug resistance. HER-2/neu, a member of erbB family, encodes a 185-KDa transmembrane receptor (p185erbB) which has a tyrosine kinase activity and is essential for cell growth and differentiation in many cell types. Overexpression of HER-2/neu is frequently observed in many tumor types such as non-small-cell-lung-cancer (NSCLC) and breast cancer. To investigate the role of HER-2/neu in bladder cancer, we overexpressed exogenous wild-type HER-2/neu in TCCSUP human bladder transitional carcinoma cell lines：TCCSUP-N5 and TCCSUP-N10 previously. We also created an active version of HER-2/neu (Val664-to-Glu) transfectants：TCCSUP-NA1 and TCCSUP-NA2. Then, we tested many anti-cancer drugs on these transfectants to determine the relationships between drug resistance and HER-2/neu level. The results demonstrated that the chemosensitivity to methotrexate (MTX) in wild-type HER-2/neu overexpressed TCCSUP transfectants is higher than both parental and control cell lines. The cell death of these transfectants during MTX treat is prone to undergo apoptic pathway. Because HER-2/neu is an oncogene, we can’t overexpress this gene in the cancer cells to improve the drug sensivity to methotrexate. We need to understand the mechanisms of HER-2/neu-induced sensitivity to methotrexate, then design apparent method to modify these signal pathways and increase drug sensivity to methotrexate. Therefore, we examined HER-2/neu mediated signal pathways, including ERK MAPK pathway, p38 MAPK pathway, SAPK/JNK pathway and PI3K pathway. I used phospho-specific antibodies to detect the activity of these kinases. The western blotting results indicated that ERK MAPK pathway, p38 MAPK pathway and PI3K pathway didn’t play a determinative role in MTX-induced cell death. In contrast, p46 SAPK/JNK activity increased only in TCCSUP-neu transfectants after treated with MTX. However, the sensitivity to methotrexate did not change when JNK dominant negative plasmids were transfected. SAPK/JNK may not play an essential role in methotrexate-induced cell death. To study which phase of cells in the cell cycle are prone to apoptosis, I stained the cells with propidium iodide and TUNEL, then analyzed by flowcytometer. The results showed that methotrexate treatment caused a G1 phase arrest in both TCCSUP and N5 cells. It was interesting to note that TCC-SUP-N5 entered apoptosis in all phases of the cell cycle, while TCCSUP cells were prone to apoptosis in S or G2/M phase. This difference might explain why neu transfectants had higher durg sensitivity to methotrexate in bladder cancer cell lines.