The antitumor and drug resistant mechanisms of chemotherapy in non-small cell lung cancer cells

• Main Authors: Kun-Chieh Chen, 陳焜結
• Other Authors: 陳健尉
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/87015347589704602123

博士 === 國立中興大學 === 生物醫學研究所 === 103 === Lung cancer is one of leading causes of cancer death in Taiwan. Non-small cell lung cancer (NSCLC) compromises more than 85-90% of all lung cancers. Systemic therapy with chemotherapy is an important part of therapy in NSCLC patients. Chemotherapy provides survival benefits and relive disease-related symptoms. However, the overall response rate to chemotherapy is less than 50%. Therefore, understanding the anti-tumor and drug resistance mechanisms wound help us to better select patients more likely to benefit from chemotherapy. Herein, our study was to investigate the role of the activated Akt in pemetrexed-induced apoptosis and the association between tumor suppressor gene, HLJ1, and sensitivity of chemotherapy. Pemetrexed is approved for treatment of patients with advanced nonsquamous, non-small-cell lung cancer (NSCLC). The protein kinase Akt/protein kinase B is a well-known regulator of cell survival which is activated by pemetrexed, but its role in pemetrexed-mediated cell death and its molecular mechanisms are unclear. This study showed that stimulation with pemetrexed induced S-phase arrest and cell apoptosis and parallel increase in sustained AKT phosphorylation and nuclear accumulation in the NSCLC A549 cell line. Inhibition of Akt expression by Akt specific siRNA blocked S-phase arrest and protected cells from apoptosis, indicating an unexpected proapoptotic role of Akt in the pemetrexed-mediated toxicity. Treatment of A549 cells with pharmacological inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and Ly294002, similarly inhibited pemetrexed-induced S phase arrest and apoptosis and Akt phosphorylation, indicating that PI3K is an upstream mediator of Akt and is involved in pemetrexed-mediated cell death. Previously, we identified cyclin A-associated cyclin-dependent kinase 2 (Cdk2) as the principal kinase that was required for pemetrexed-induced S phase arrest and apoptosis. The current study showed that inhibition of Akt function and expression by pharmacological inhibitors as well as Akt siRNA drastically inhibited cyclin A/Cdk2 activation. These pemetrexed-mediated biological and molecular events were also observed in a H1299 cell line. Overall, our results indicate that , in contrast to its normal prosurvival role, the activated Akt plays a proapoptotic role in pemetrexed-mediated S phase arrest and cell death through a mechanism that involves cdk2/cyclin A activation. HLJ1, a tumor suppressor gene, may inhibit lung cancer cell proliferation, tumorigenesis, cell mobility, invasion and slow cell cycle progression and its expression is associated with low recurrence rate and survival of lung cancer patients. However, the effect of HLJ1 expression on drug susceptibility of chemotherapeutic agents is not clear. To investigate the effect of HLJ1 expression on chemosensitivity, lung cancer cells CL1-0 and its HLJ1-knockdown subclones were treated with various chemotherapeutic agents, which used commonly for treatment of NSCLC patients. The results showed that HLJ1-knockdown cells expressed more Cyclin D1 that that of parental CL1-0 cells. Additionally, HLJ1-knockdown cells were highly sensitive to paclitaxel, but not to cisplatin, gemcitabine or genfitinib. Downregulation of HLJ1 enhanced paclitaxel-induced apoptosis and decreased XIAP expression in lung cancer cell lines. Because XIAP is an endogenous inhibitor of caspases and associated with inhibition of apoptosis and chemo-resistant. Data from immunoprecipitation assay showed that HLJ1 and XIAP proteins were directly binding with each other. Therefore, we hypothesize that decrease of XIAP may be one of the mechanisms of paclitaxel sensitivity in HLJ1 knockdown cells. Our results showed that the activated Akt played the important role in pemetrexed-induced apoptosis and downregulation of HLJ1 enhanced paclitaxel-induced apoptosis. Further clinical studies are needed to verify these association. In the future, we may use these biomakers as predictive factors of chemotherapy, so that patients can select appropriate and effective chemotherapy.