Curcumin Reverses EGFR-TKIs Resistance In Non-small Cell Lung Cancer By Inducing Autophagy

Background and Objective:Lung cancer remains the leading cause of cancer mortality worldwide.Non-small cell lung cancer（NSCLC）is the most comment histological subtype of lung cancer,accounting for nearly 85%of all cases.The 5-year survival rate of NSCLC patients is approximately 15%owing to insidious onset and asymptomatic at early stage,anddiagnosis at advanced stage.Epidermal growth factor receptor-tyrosine kinase inhibitor（EGFR-TKIs）is the first-line therapeutic regimen for patients with advanced NSCLC with activating EGFR mutation.However,almost all patients inevitably develop drug resistance after 9～13 months of treatment,which seriously weakens the efficacy of EGFR-TKIs for NSCLC.Several studies have shown that autophagy is closely related to EGFR-TKIs resistance,and induction of autophagy can increase sensitivity of NSCLC cell to EGFR-TKIs.Therefore,it is very important to find new effective autophagy inducers for the perspective of overcoming resistance of NSCLC to EGFR-TKI.Curcumin is a hydrophobic polyphenol compound extracted from the rhizome of turmeric.It has a wide range of pharmacological effects,such as anti-inflammatory,antioxidant and anti-tumor effects.Icotinib is the first-generation of EGFR-TKI independently researched and developed in China.Although some studies reported that curcumin can increase sensitivity of NSCLC to EGFR-TKIs by inducing autophagy and autophagy-dependent apoptosis,the underlying molecular mechenism by curcumin reversing EGFR-TKI resistance in NSCLC are not completely elucidated.In the present study,we investigated the effect of curcumin on the reversal of EGFR-TKIs resistance in NSCLC cells as well as their molecular bases by using in vitro experiments.Methods:NSCLC cell lines with different gene background H460（EGFR wild-type）,H1975（EGFR L858R+EGFR T790M mutation）and HCC827（EGFR 19 exon E746-A750 deletion mutation）cell lines were used in the present study.We firstly examined the sensitivity of EGFR-TKI-sensitive and-resistant NSCLC cell lines to icotinib and curcumin by CCK-8 assay,and IC₅₀values of the two drugs were calculated in the three cell lines.Subsequently,the two EGFR-TKI-resistant cell lines were treated with icotinib or curcumin alone or the two combination in the presence or absence of 3-MA which is a autophagy inhibitor,and cell viability,migration ability,expressions of autophagy-relative proteins and autophagosomes were examined by CCK-8,wound healing,western blot and immunofluoscence assays.Results:1.After treatment with different concentrations of icotinib or curcumin for 24 h,cell proliferation was measured by CCK-8 assay in HCC827,H460 and H1975 cells.Results showed that HCC827 cells with activating EGFR mutation displayed more sensitivity to icotinib compared to H460 cells with EGFR wild-type and H1975 cells with EGFR L858R+EGFR T790M mutations（P<0.05）,and icotinib markedly inhibited HCC827 cell proliferation in a dose-dependent manner（P<0.05）.The IC₅₀values of icotinib in H1975 and H460 cells were significantly higher than that of HCC827 cells（P<0.001）.Curcumin suppressed cell proliferation of HCC827,H460and H1975 cells with similar sensitivity in a dose-dependent manner（P<0.05）.There was no significant difference in the IC₅₀values of curcumin in H1975,H460 and HCC827 cells.2.H460 and H1975 cells were treatment with increasing concentrations of icotinib and curcumin combination for 24 h,CCK-8 assay’s results showed that curcumin notably enhanced inhibitory effect of icotinib on the two EGFR-TKI-resistant cell lines in a dose-dependent manner（P<0.05）,the IC₅₀values of icotinib in the two cell lines were significantly reduced by adding curcumin（P<0.05）.The reduced degree of IC₅₀of icotinib in 10μg/ml curcumin group was more significant than that in 5μg/ml curcumin group（P<0.05）.3.The effects of icotinib and curcumin alone or the two combination on the migration ability of H460 and H1975 cells were determined by wound healing assay.The results showed that as compared with the single drug group and the blank control group,the combination treatment of icotinib and curcumin significantly inhibited the migration ability of H460 and H1975 cells in a dose-dependent manner（P<0.05）.4.H460 and H1975 cells were treated with 5μg/ml icotinib,curcumin（5μg/ml,10μg/ml）and the combination of the two drugs（5μg/ml icotinib+5μg/ml curcumin,5μg/ml icotinib+10μg/ml curcumin）,respectively,for 24 h,the expression of autophagy-related proteins were detected by Western Blot assay.The results showed that the two drug combination significantly up-regulated expressions of LC3-II and Beclin-1 proteins,and down-regulated expression of P62/SQSMT1 protein,when compared with the single drug group and the blank control group（P<0.05）.5.The expressions of autophagosomes were detected by indirect immunofluorescence assay in H460 and H1975 cells after treated with 5μg/ml icotinib,10μg/ml curcumin and the two-drug combination for 24 h.The results showed that the two-drug combination treatment resulted in a significant up-regulation of autophagosomes expression as compared with the single-drug group and the blank control group（P<0.05）.6.H460 and H1975 cells were treated with an increasing concentrations of icotinib combined with 10μg/ml curcumin for 24 h in the presence or absence of3-MA（5μg/ml）,cell proliferation rate was determined using CCK-8 assay.Results showed that 3-MA partly and statistically significantly reversed the inhibition of cell’s proliferation rates caused by combination treatment of icotinib and curcumin in H460and H1975 cells（P<0.05）.7.H460 and H1975 cells were treated with 5μg/ml icotinib plus 10μg/ml curcumin in the presence or absence of 3-MA.Western blot assay showed that the addition of 3-MA retarded the two-drug combination-induced the up-regulations of LC3-II and Beclin-1 expressions as well as the down-regulation of P62/SQSMT1expression（P<0.05）.Conclusions:1.Curcumin can inhibit proliferation of HCC827,H460 and H1975 cell lines in a dose-dependent manner.2.Curcumin dose-dependently increased the sensitivity of H460 and H1975cells to icotinib.3.Curcumin synergizes with icotinib to inhibit the migration ability in H460 and H1975 cells.4.The combination of curcumin and icotinib markedly enhanced autophay induction in H460 and H1975 cells,suggesting that curcumin acts synergistically with icotinib to induce autophagic cell death,and thereby reversing resistance of EGFR-TKI-resistant NSCLC cells to icotinib.