Synergistic Effects Of Resveratrol And Gefitinib To Overcome Drug Resistance In Human Non-small Lung Cancer Cell Line PC9/G

The incidence of lung cancer, one of the highest morbidity and mortality of cancers worldwide, shows an increasing trend in China. Non-small cell lung cancer (NSCLC) accounts for approximately80%of lung cancers. Molecular targeted therapy gives a hope for the treatment of lung cancer due to limited effects for the traditional chemotherapy drugs. Gefitinib is a targeted drug in the treatment of NSCLC, with characteristics of high specificity, good tolerance, and less side effects. However, the acquired drug-resistance is the main causes of gefitinib treatment failure in clinically. Resveratrol, as one of natural polyphenol compounds, has been shown to have the anti-tumor effects in three stages of carcinogenesis, promotion, and development by enhancing the activity of anticancer drugs. The present study aimed to examine the effect and its mechanism of resveratrol on resistant NSCLC, and thus provide new ideas for solving the problems of gefitinib drug resistant in treatment of NSCLC and experimental evidence for resveratrol drug development and its clinical application. The study contained three parts:Part I The antiproliferative effects of combined resveratrol with gefitinib on PC9/G cell lineTo establish gefitinib-resistant NSCLC PC9/G cell line; to evaluate the anti-proliferation effects of gefitinib, resveratrol treatment alone or different combination treatments on PC9/G cell and optimize the most potent mode of combination treatments. Methods:PC9cells were treated with increasing concentrations of gefitinib (0.01-1μM) in a stepwise manner for3months until they eventually acquired resistance to gefitinib. Then the most resistant cell line (PC9/G) was selected through single-cell cloning. The difference of morphology and proliferation characteristics between PC9and PC9/G cell were compared. The antiproliferation effects of different treatments on PC9/G cell were assayed by MTT. Combination Index (CI) value were used to evaluate the combination effect of different combination treatments. Results:Compared with PC9cell, the morphology and proliferation characteristic of PC9/G cell were significantly changed and the resistant index of PC9/G to gefitinib was about310; the growth rate quotient was increased (2.0-fold, P<0.05) and double time was decreased (0.7-fold, P<0.05). The IC50value of gefitinib and Resveratrol in PC9/G cell were6.54μM and33.9μM, respectively. Co-treatment with resveratrol and gefitinib possesses a strong synergistic anti-proliferation effect (CI<0.9) in PC9/G cell. Conclusion:Gefitinib-resistant NSCLC cell line (PC9/G cell line) was established. Resveratrol can significantly enhance the anti-proliferation effect of gefitinib on PC9/G cell. The method of simultaneous of co-treatment with resveratrol and gefitinib showed a strong synergistic effect and was the most potent mode of combination treatments in PC9/G cell.Part II The effects of resveratrol on cellular pharmacokinetics and the EGFR autophosphorylation of gefitinib in PC9/G cell lineObjective:To investigate the effects of the alternation of intracellular gefitinib concentration on the EGFR autophosphorylation in PC9and PC9/G cells; to illuminate the effect and the mechanism of resveratrol on the change of intracellular pharmacokinetics and the EGFR autophosphorylation of gefitinib in PC9/G; Methods: A sensitive method of high performance liquid chromatography-mass spectrometry (HPLC-MS/MS) to detect intracellular concentration of gefitinib was established. Western blot assay was applied to detect the expressing level of phosphorylated protein of EGFR, and the expression protein of ABCG2, ABCB5, CYP1A1and CYP2D6. The linear relationship between inhibition rate of phosphorylated EGFR and intracellular gefitinib was analysised using regression algorithms in PC9and PC9/G cells. Results:①A method of HPLC-MS/MS to quantify of gefitinib in intracellular was developed and validated (P>0.99);②when the extracellular concentration of gefitinib was1μM to20μM, the intracellular content of gefitinb and the inhibition of EGFR phosphorylation in PC9/G was less than in PC9cell. The inhibition rate of EGFR phosphorylation in PC9and PC9/G cells was correlated with intracellular concentration of gefitinib (R2>0.89).③Resveratrol significantly increased the intracellular concentration of gefitinib in the PC9/G cells, increasing the AUC and Cmax of intracellular gefitinib with the increasing of intracellular concentration of gefitinib, and reduced clearance of gefitinib.④Compared with PC9cells, the expression of ABCG2and ABCB5protein in PC9/G cells was significantly increased, which was1.6fold,1.3fold and3.7fold (P<0.05, P<0.01).No significant differences were found in the protein expression of CYP2D6(P>0.05). Compared with the control of PC9/G cells, the expression level of ABCG2and CYP1A1protein was significantly lower in the group of combination of resveratrol and gefitinib, which was respectively33%and17%(P<0.01) of the control, while no significant effect on the expression of ABCB5and CYP2D6was found (P>0.05).Conclusions:The inhibition rate of EGFR phosphorylation was correlated with intracellular concentration of gefitinib in PC9and PC9/G cells. ABCG2, ABCB5and CYP1A1might contribute to drug resistance of PC9/G cells. Resveratrol could significantly influence the pharmacokinetics of gefitinib by reducing the transport and metabolism rate. It indicated that the combination of resveratrol and gefitinib could improve the inhibition rate of EGFR phosphorylation in PC9/G cell by increasing intracellular concentration of gefitinib. This process was realized through reducing metabolism and excretion of gefitinib in PC9/G cell.Part III The effects of combined resveratrol with gefitinib on apoptosis, autophagy and senescence in PC9/G cell lineObjective:To assess whether multiple modes of cell death (apoptosis, autophagy and senescence) could mediate the effect of combined treatment of resveratrol with gefitinib in PC9/G cells; to clarify the exact mechanism of apoptosis, autophagy and senescence in the antiproliferation effect of combined treatment of resveratrol with gefitinib on PC9/G cell, and the relationship among apoptosis, autophagy and senescence.Method:DAPI and monodansylcadaverine (MDC) were used to stain cell and autophagosome. The apoptosis rate, autophagy rate and cell cycle distribution were measured by flow cytometry; senescence cells were labeled by senescence-associated β-Galactosidase (SA-β-Gal) staining kit. Results:①Compared with control group, the apoptosis rate, autophagy rate and SA-β-Gal positive rate of combination treatment was significantly increased(P<0.01). Moreover, there was a significantly difference in the apoptosis rate, autophagy rate and SA-P-Gal positive rate of between combination group with gefitinib treatment alone. Cell cycle analysis showed that cell distribution in G2/M phase of combination treatment group was significantly higher compared to gefitinib treatment alone (P<0.01).②Compared to gefitinib treatment group, combination treatment group significantly up regulated the protein expression of cleaved caspase-3, LC3BII, p53and p21by7.2-fold (P<0.01),7.3-fold (P<0.01),3.6-fold and3.5-fold(P<0.01), while there is no change in Beclin lexpression (P>0.05).③In comparison to combination group, DEVD combination group did not have antiproliferation, effect (P>0.05).3-MA combination group had a stronger antiproliferation effect (P<0.05). Besides, compared with combination group, DEVD combination group had accelerated apoptosis rate (P<0.05), had no effect of autophagy rate and SA-β-Gal positive rate (P>0.05);3-MA combination group had decreased autophagy rate (P<0.01), increased apoptosis rate (P<0.01) and decelerated senescence (P<0.01). Conclusion:The protein expression of p53and p21were up regulated, autophagy and senescence were accelerated, cell-cycle arrest was blocked and proliferation was inhibited in PC9/G cells exposed to combination treatment of resveratrol with gefitinib. The combination of the two drugs also induced apoptosis through caspase-3pathway. This indicated that resveratrol synergismly overcome gefitinib resistant in PC9/G cell line through inducing autophagy and senescence.