Abstract
Tyrosine kinase inhibitors such as erlotinib are commonly used as a therapeutic agent against cancer due to its relatively low side-effect profile and, at times, greater efficacy. However, erlotinib resistance (ER) in non-small cell lung cancer is being recognized as a major problem. Therefore, understanding the mechanism behind ER and developing effective regimens are needed. Autophagy’s role in cancer has been controversial and remains unclear. In this study, we examined the effectiveness of low dose erlotinib-cisplatin combination in erlotinib resistant lung adenocarcinoma (ERPC9) cells and the role of autophagy in ER. ERPC9 cells were established from erlotinib sensitive PC9 cells. Appropriate treatments were done over two days and cell survival was quantified with Alamar Blue assay. LC3II and regulatory proteins of autophagy were measured by western blot. Small interfering RNA (siRNA) was utilized to inhibit translation of the protein of interest. In ERPC9 cells, combination treatment induced synergistic cell death and a significant decrease in autophagy. At baseline, ERPC9 cells had a significantly higher LC3II and lower p-mTOR levels compared to PC9 cells. The addition of rapamycin increased resistance and 3-methyladenine sensitized ERPC9 cells, indicating autophagy may be acting as a protective mechanism. Further examination revealed that ERPC9 cells harbored high baseline Atg3 levels. The high basal Atg3 was targeted and significantly lowered with combination treatment. siRNA transfection of Atg3 resulted in the reversal of ER; 42.0% more cells died in erlotinib-alone treatment with transfection compared to non-transfected ERPC9 cells. We reveal a novel role for Atg3 in the promotion of ER as the inhibition of Atg3 translation was able to result in the re-sensitization of ERPC9 cells to erlotinib-alone treatment. Also, we demonstrate that combination erlotinib-cisplatin is an effective treatment against erlotinib resistant cancer by targeting (down-regulating) Atg3 mediated autophagy and induction of apoptotic cell death.
Highlights
Lung cancer remains the leading cause of cancer-related deaths and has one of the lowest survival rates among all cancers, with a reported five-year survival of 13% [1]
When cells were treated with erlotinib-cisplatin combination, 61.1% of erlotinib resistant PC9 (ERPC9) cells survived; this was significantly lower compared to cells treated with erlotinib-alone (96.6%) and cisplatin-alone (89.3%)
We demonstrated that low dose erlotinib-cisplatin combination is able to overcome erlotinib resistance (ER) and induce synergistic cell death in both ERPC9 and the parental PC9 cell line while remaining relatively non-toxic to Normal human bronchial epithelial (NHBE) cells
Summary
Lung cancer remains the leading cause of cancer-related deaths and has one of the lowest survival rates among all cancers, with a reported five-year survival of 13% [1]. Lung cancer can be broadly categorized into two main groups for prognostic and treatment purposes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). 85% are NSCLC [2] and is further subdivide into three groups based on their histological characteristics: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma [2,3,4]. NSCLC tends to be less sensitive to chemotherapy than SCLC, and even with surgical resection of early stage primary tumors, up to 50% of patients show recurrence of their primary cancers [4,5]. Platinum based chemotherapeutic agents such as Cis-diamminedichloroplatinum (II) (cisplatin) have been traditionally the main agents used to treat NSCLC and have been shown to improve patient survival [6]. Cisplatin carries significant toxic side-effects, especially at high doses, such as: nausea, emesis, renal failure, ototoxicity and neurotoxicity due to its non-specific cytotoxic effects on both cancer and normal cells [7,8]
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