非小細胞肺癌(H460)抗藥性之機轉探討:XIAP蛋白之參與

Abstract

Abstract Non-small cell lung cancer (NSCLC) is the most common and serious cancer worldwide. Cisplatin-based chemotherapy has generally been considered as one of the effective treatments for NSCLC. Cisplatin can prolong survival of NSCLC patients, but the curative effect still has its limit and some NSCLC cells are resistance to cisplatin treatment and result in poor prognosis. Molecular basis underlying the cisplatin-resistance of NSCLC remains unclear and required to be determined. Previously, using three different NSCLC cell lines, CH27 (squamous cell), A549 (adenocarcinoma cell) and H460 (large cell), we had demonstrated that cisplatin-induced cell death of these cells is mitochondria-mediated, H460 cell appears to have higher resistance to cisplatin treatment than the other two cell lines, higher X-linked inhibitor of apoptosis protein (XIAP) level exists in H460 cell, which is not affected by cisplatin. XIAP is known as one of the most potent inhibitors of apoptosis. That led us to further investigate the possible role of XIAP and the other anti-apoptotic protein, survivin, in the development of drug resistance of H460 cells against cisplatin. The possible involvement of NF-κB, a upstream regulator of XIAP, was also examined. Techniques applied in the study included the trypan blue exclusion assay, DAPI staining, annexin V/PI assay, EMSA, western blot analysis, caspase activity assay, siRNA transfection assay, and co-immunoprecipitation. Embelin (XIAP inhibitor), curcumin (inhibitor of survivin and XIAP), wedelolactone (inhibitor of NF-κB) and specific XIAP siRNA were adopted respectively, in the study to assess the importance of XIAP, survivin and NF-κB in development of drug resistance of H460 cell. Results first showed that XIAP played an important role in maintaining the normal growth and development of drug resistance of H460 cells against cisplatin. When XIAP was inhibited by embelin, cisplatin-triggered caspase 3 activity (but not amount of the enzyme) was further increased that subsequently led to increased cell death as well. These results indicated the importance of XIAP in the drug resistance of XIAP. Results from the EMSA assay further demonstrated that XIAP can bind to cisplatin-triggered caspase 3 and restrained its activity in order to protect cells from cisplatin-induced damage. The level of NF-κB was not particularly higher in H460 than the other two cell lines except that NF-κB (both amount and activity) of H460 was not easily affected by cisplatin. XIAP of H460 cells also shared a similar response to cisplatin. When NF-kB was inhibited by wedelolactone, normal growth of three cell lines were all inhibited but only the response of H460 cells to cisplatin was significantly inhibited by wedelolactone. The protein expression levels of XIAP were also suppressed by wedelolactone but not the other around. These findings suggested that NF-κB not only was important for normal growth of NSCLC cells but also played a role in development of drug resistance of H460 against cisplatin. Furthermore, NF-kB-mediated effect may highly act through its regulation of XIAP downstream. Survivin was commonly expressed in all three cell lines and suppressed by cisplatin at 48 h, without cell type specific differences. When survivin was inhibited by curcumin, only the normal growth of these cell lines was inhibited but not the response of these cells to cisplatin. That indicated survivin was important for the normal growth of NSCLC cells but played no part in development of drug resistance of these cells including H460 cells. In addition, the importance of XIAP in development of the drug resistance of H460 was also proved directly by using the XIAP siRNA transfection strategy even though XIAP protein levels remained unstrained by siRNA. In overall, results from the study demonstrated the importance of XIAP and NF-κB in protecting normal growth as well as drug resistance of NSCLC cells (H460). The therapeutic potential of embelin, wedelolactone and XIAP siRNA in future treatment of NSCLCs was also suggested. These results not only strengthened our knowledge about the mechanism underlying the drug resistance of NSCLCs but also suggested a role for XIAP and/or NF-κB in either early diagnosis of NSCLCs or to help in evaluating how effectively a lung cancer patient would respond to chemotherapy. XIAP and NF-κB can also serve as the target proteins used as the basis for future design of new drugs against NSCLCs especially the ones with high drug resistance. Finally, all the information acquired form this thesis can provide valuable inputs to both fundamental research and clinical practice of NSCLCs that can even be helpful for treatment of other types of cancers in the future.