Overcoming the acquired resistance to gefitinib in lung cancer brain metastasis in vitro and in vivo.

Abstract

In our previous work, PC-9-Br, a PC-9 brain seeking line established via a preclinical animal model of lung cancer brain metastasis (LCBM), exhibited not only resistance to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) gefitinib in vitro, but also chemotherapy regimens of cisplatin plus etoposide in vivo. Using this cell line, we investigated novel potential targeted therapeutics for treating LCBM in vitro and in vivo to combat drug resistance. Significant increases in mRNA and protein expression levels of Bcl-2 were found in PC-9-Br compared with parental PC-9 (PC-9-P), but no significant changes of Bcl-XL were observed. A remarkable synergistic effect between EGFR-TKI gefitinib and Bcl-2 inhibitors ABT-263 (0.17 ± 0.010µM at 48h and 0.02 ± 0.004µM at 72h), or ABT-199 (0.22 ± 0.008µM at 48h and 0.02 ± 0.001µM at 72h) to overcome acquired resistance to gefitinib (> 0.5µM at 48h and 0.10 ± 0.007µM at 72h) in PC-9-Br was observed in MTT assays. AZD9291 was also shown to overcome acquired resistance to gefitinib in PC-9-Br in MTT assays (0.23 ± 0.031µM at 48h and 0.03 ± 0.008µM at 72h). Western blot showed significantly decreased phospho-Erk1/2 and increased cleaved-caspase-3 expressions were potential synergistic mechanisms for gefitinib + ABT263/ABT199 in PC-9-Br. Significantly decreased protein expressions of phospho-EGFR, phospho-Akt, p21, and survivin were specific synergistic mechanism for gefitinib + ABT199 in PC-9-Br. In vivo studies demonstrated afatinib (30mg/kg) and AZD9291 (25mg/kg) could significantly reduce the LCBM in vivo and increase survival percentages of treated mice compared with mice treated with vehicle and gefitinib (6.25mg/kg). In conclusion, our study demonstrated gefitinib + ABT263/ABT199, afatinib, and AZD9291 have clinical potential to treat LCBM.