Abstract C59: The effects of combined treatment of erlotinib and cisplatin in resistant lung cancer and the role of autophagy in response to chemotherapy

Abstract

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for about 80% of all cases of lung cancer. The treatment of choice for NSCLC is complete surgical resection. However, in advance stages with metastasis and/or aggressive local infiltrate chemotherapy becomes the only option. At high therapeutic doses, chemotherapy can cause significant side effects, and the development of resistance may arise if initial treatment doses of therapy become ineffective. In order to prevent serious side effects and resistance, low-dose combination regimens that offer comparable results to current high-dose regimens are needed. The combination of cisplatin and erlotinib has been shown to inhibit tumor growth more effectively than a single drug in NSCLC cells. However, the mechanism underlying this finding remains unclear. Autophagy has been suggested to play a critical role in cancer and chemotherapy, but there is ongoing controversy among its role (promotion vs. prevention of cancer). In this study, we examined whether the combination of low-dose cisplatin and erlotinib is able to induce higher levels of cancer cell death than each drug alone in both sensitive and erlotinib-resistant lung adenocarcinoma. In addition, we looked into the role of autophagy in cancer cells and its association with chemotherapy. Methods: Alamar blue assay was used to construct dose response curves in which IC 50 values of cisplatin and erlotinib were determined. Erlotinib-resistant PC9 (PC9/ER) cells were developed by culturing PC9 (EGFR overexpression adenocarcinoma) cells in media containing an erlotinib dose 1000 times the IC50 value over a two week period; PC9/ER cells were verified to be 26-fold more resistant than PC9 sensitive cancer cells. Baseline levels of autophagy of normal human bronchial epithelial (NHBE) cells, PC9, and PC9/ER were examined by Western blot with antibodies against LC3I/II. The IC 25 of each drug was chosen as the low-dose treatments (3 uM cisplatin and 10 nM erlotinib). Cells were treated accordingly: control, cisplatin, erlotinib, and combination. Cell death and autophagy levels were measured. Next, cells were pretreated in one day in advance with rapamycin and cell death and changes in autophagy were assessed. Results: At baseline, there were higher levels of LC3II in PC9/ER compared to PC9 and NHBE. Combined treatment of cisplatin and erlotinib was able to induce significantly more cell death than individual drugs at the same dose (p<0.001). In fact, combination treatment provided synergistic killing effects in PC9 and PC9/ER cells. In PC9 cells, erlotinib alone had 79.9% survival, cisplatin 76.3%, and combination 45.0%. In PC9/ER, erlotinib alone had 96.6% survival, cisplatin 89.3%, and combination 61.1%. Levels of autophagy decreased in a similar pattern to killing-effects in both PC9 and PC9/ER cells. In addition, a significant increase in cell survival of about 20% was observed when pretreated with rapamycin (p<0.001). NHBE demonstrated no significant killing and no changes in autophagy levels. Conclusion: Our data is consistent for autophagy's role in promoting cancer cell survival, work as a protective mechanism for cancer-resistant cells, and a critical mechanism in chemotherapy-induced cancer cell death. The combination of erlotinib and cisplatin offers synergistic killing properties in vitro and may be a potential regimen against resistant lung cancer. In addition, the combination treatment does not induce significant cytotoxic effects and death in NHBE cells indicating its safe dosing. We plan to further characterize autophagy with 3-methyladenine pretreatment, changes in apoptosis, and invasiveness of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C59.