Repurposing loperamide to overcome gefitinib resistance by triggering apoptosis independent of autophagy induction in KRAS mutant NSCLC cells


Session type:


Christy Wing Sum Tong1,Mia Mingxia Wu1,Wei Yan1,Kenneth Kin Wah To1
1The Chinese University of Hong Kong



The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib, provides significant clinical benefit for non-small cell lung cancer (NSCLC) patients with activating EGFR mutations. However, primary resistance remains a major obstacle to effective gefitinib therapy in patients with activating KRAS mutations. Autophagy induction was reported to promote autophagic cell death in RAS-driven cancer cells. Therefore, we investigated the combination of gefitinib and autophagy inducers to circumvent drug resistance in KRAS-mutated NSCLC cells.


NSCLC cell lines harboring KRAS mutation (A549 and H460) and EGFR mutation (H1975 and H1650) were employed. Quantitative real-time PCR and Western blot analysis were used to examine the expression of autophagy-related genes and proteins, respectively. Sulforhodamine B assay was employed to evaluate the cytotoxicity of drug combinations. Annexin-V/propidium iodide apoptosis assay was used to investigate the apoptotic effect after the treatment.


Loperamide, commonly used to relieve the dose-limiting adverse effect of diarrhea from gefitinib, was found to induce autophagy by increasing autophagy-related genes, ATG2B and RGS1, and an autophagy-related protein, LC3-II. Combination treatment of gefitinib and loperamide produced synergistic cytotoxic effect only in KRAS-mutated cell lines (A549 and H460) with combination indexes of 0.37 ± 0.04 and 0.68 ± 0.10, respectively. Knocking down Atg5 did not reverse the synergistic cytotoxic effect, suggesting that autophagy induction did not directly cause cancer cell death. However, this drug combination produced a remarkable synergistic apoptotic effect. Inhibition of autophagy induction either chemically (by 3-methyladenine) or genetically (by Atg5 silencing) did not reverse the synergistic apoptotic effect, indicating that the apoptotic cell death was not mediated by autophagy induction.


Our results indicated that loperamide could be repurposed to combine with gefitinib to produce synergistic anti-cancer effect in KRAS mutant NSCLC cell lines by further inducing synergistic apoptosis, which is independent of autophagy induction.