| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 103 === Human non-small cell lung cancer (NSCLC) cells harboring epidermal growth factor receptor (EGFR) mutation are sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as Gefitinib (Iressa○R). However, lung cancer patients finally develop acquired resistance after treating with EGFR-TKIs for 9-12 months. EGFR T790M secondary mutation has been reported as the most common resistance mechanism. Recently, WZ8040, a third generation of EGFR-TKI has been introduced for targeting the EGFR T790M mutation. My thesis focused on studying the characteristics of WZ8040 resistance. Six novel acquired WZ-resistant cells, including PC-9/WZ D7, PC-9/WZ F6, PC-9/WZ C3, PC-9/WZ F8, PC-9/WZ C9 and PC-9/WZ C10 were developed by continuous exposure of PC-9/wt NSCLC cells (containing EGFR exon 19 deletion) to escalating concentrations of WZ8040. Using SRB assay, WZ-resistant cells were 60-fold more resistant to WZ8040 than PC-9/wt. Western blot assay showed that WZ-resistant cells had higher protein expression of LC3-II. Using transwell migration assay, PC-9/WZ F6 cell had highest migration ability compared with PC-9/wt and PC-9/WZ D7. Hypoxic treatment enhanced migration in both PC-9/wt and WZ-resistant cells without significant differences. Furthermore, PC-9/WZcells (D7, C3, F8, F6, C10) had lower E-cadherin levels and higher N-cadherin levels. In addition, PC-9/WZ cells (D7, C3, F8) had higher Slug levels and PC-9/WZcells (F6, C10) had higher Snail levels. Moreover, WZ-resistant cells were more resistant to WZ8040-induced inhibition of EGF-induced phosphorylation of AKT and extracellular signal-regulated kinase (ERK). Similarly, WZ8040 induced mild apoptosis in WZ-resistant cells while compared with PC-9/wt cells. To study the potential treatment for WZ-resistance, poly [ADP-ribose] polymerase (PARP) inhibitors (Olaparib, BMN673) were employed. PARP inhibitors alone showed similar cytotoxicity in PC-9/wt and WZ-resistant cells, suggesting that WZ-resistant cells do not resist to PARP inhibitors. Moreover, PARP inhibitors plus WZ8040 had no further effects on PC-9/wt and WZ-resistant cells growth, including cytotoxicity and apoptosis. In addition, cisplatin (a DNA damaging agent) alone was found to induce similar cytotoxic effects in PC-9/wt and WZ resistant cells, indicating that WZ-resistant cells do not resist to DNA damaging agents. Cisplatin plus PARP inhibitors had no further effects on cytotoxicity. To analyze DNA repair capability, PC-9/wt and WZ resistant cells had similar DNA repair capability. These results suggest that DNA repair capability may not play a role in WZ8040 resistance. In vivo model was employed to show that oral administration of gefitinib (50 mg/kg/day) inhibited the tumor growth of PC-9/wt and PC-9/WZ D7; AZD9291 (5 mg/kg/day) inhibited that of PC-9/wt but not PC-9/WZ D7; WZ8040 (50 mg/kg/day) inhibited neither PC-9/wt nor PC-9/WZ D7. Further studies are still required for the potential treatments of WZ-resistance.
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