Autophagy Inhibition with Monensin Enhances Cell Cycle Arrest and Apoptosis Induced by mTOR or Epidermal Growth Factor Receptor Inhibitors in Lung Cancer Cells

Abstract

BackgroundIn cancer cells, autophagy is generally induced as a pro-survival mechanism in response to treatment-associated genotoxic and metabolic stress. Thus, concurrent autophagy inhibition can be expected to have a synergistic effect with chemotherapy on cancer cell death. Monensin, a polyether antibiotic, is known as an autophagy inhibitor, which interferes with the fusion of autophagosome and lysosome. There have been a few reports of its effect in combination with anticancer drugs. We performed this study to investigate whether erlotinib, an epidermal growth factor receptor inhibitor, or rapamycin, an mammalian target of rapamycin (mTOR) inhibitor, is effective in combination therapy with monensin in non-small cell lung cancer cells.MethodsNCI-H1299 cells were treated with rapamycin or erlotinib, with or without monensin pretreatment, and then subjected to growth inhibition assay, apoptosis analysis by flow cytometry, and cell cycle analysis on the basis of the DNA contents histogram. Finally, a Western blot analysis was done to examine the changes of proteins related to apoptosis and cell cycle control.ResultsMonensin synergistically increases growth inhibition and apoptosis induced by rapamycin or erlotinib. The number of cells in the sub-G1 phase increases noticeably after the combination treatment. Increase of proapoptotic proteins, including bax, cleaved caspase 3, and cleaved poly(ADP-ribose) polymerase, and decrease of anti-apoptotic proteins, bcl-2 and bcl-xL, are augmented by the combination treatment with monensin. The promoters of cell cycle progression, notch3 and skp2, decrease and p21, a cyclin-dependent kinase inhibitor, accumulates within the cell during this process.ConclusionOur findings suggest that concurrent autophagy inhibition could have a role in lung cancer treatment.

A Kinase-Independent Role for EGF Receptor in Autophagy Initiation

“…Thus, though EGFR TKIs block the cellular functions mediated by EGFR kinase signaling, they activate a role for inactive EGFR in autophagy that could potentially provide a survival advantage and TKI resistance in WT EGFR expressing cancers. In fact, there is in vitro and in vivo evidence that co-targeting EGFR and autophagy is a promising strategy to overcome TKI resistance of cancer cells with WT EGFR, even when these cancer cells carry a different oncogenic factor like K-Ras or p53 mutation (Choi et al, 2013; Dragowska et al, 2013; Jutten and Rouschop, 2014; Zou et al, 2013). For example, while erlotinib or hydroxychloroquine (HCQ, a clinically available autophagy inhibitor) alone show no anti-tumor activity in xenografts derived from H460 NSCLC cells expressing WT EGFR but mutant K-Ras, combination of the two drugs resulted in dramatic inhibition of tumor growth (Zou et al, 2013).…”

“…Rapamycin, an autophagy inducer, exhibits cytotoxic effects in cancer cells treated with EGFR tyrosine kinase inhibitors (TKIs), erlotinib or gefitinib (Fung et al, 2012; Gorzalczany et al, 2011). However, autophagy induced by EGFR TKIs or neutralizing antibody shows cytoprotective roles (Choi et al, 2013; Dragowska et al, 2013; Eimer et al, 2011; Han et al, 2011; Li and Fan, 2010; Li et al, 2010b; Sobhakumari et al, 2013; Zou et al, 2013), suggesting that EGFR TKIs (or neutralizing antibody) may induce autophagy by a mechanism distinct from rapamycin. At least some EGFR overexpressing cells and tumors are dependent on autophagy for growth and survival (Jutten et al, 2013; Jutten and Rouschop, 2014).…”

Monensin Inhibits Epidermal Growth Factor Receptor Trafficking and Activation: Synergistic Cytotoxicity in Combination with EGFR Inhibitors

“…It may involve its ability to further inhibit EGFR in an unrelated mechanism to erlotinib enhancing EGFR inhibition and cytotoxicity as suggested in this study. Monensin may also enhance erlotinib activity through its ability to inhibit autophagy, a strategy that has demonstrated the potential to enhance erlotinib cytotoxicity in NSCLC cells (46). Furthermore, lipoprotein lipolysis can lead to the induction of ATF3 resulting in apoptosis (47) and the potential of monensin to target lipid homeostasis suggest this as an alternative mechanism.…”

Pharmacologic agents targeting autophagy

“…Other compounds with CQ-like activities in alkalizing lysosomal compartments have also been identified. Monensin, a polyether antibiotic, is also a lysosomotropic drug that prevents the acidification of lysosomes and interferes with the fusion of autophagosomes and lysosomes (81). In addition, lucanthone, an anti-schistosome agent, inhibits autophagy via a similar mechanism to CQ (82).…”