The Antagonistic Effect And Underlying Mechanisms Of Combination Therapy Of Gefitinib With Cisplatin In Non-small Cell Lung Cancer

Background:Lung cancer is the most common tumor of the respiratory system, and has a relatively high morbidity and mortality worldwide. Non-small cell lung cancer(NSCLC) account for the majority of the morbidity of lung cancer, and surgery is still the main method for treatment of NSCLC. However, for surgical patients, the recurrence rate was about 40% within two years. Therefore, for patients with recurrent NSCLC after surgery and advanced NSCLC patients with contraindications for surgery, systemic chemotherapy and targeted therapy are still the main treatment method. Nevertheless, the therapeutic efficacy of current common first-line chemotherapy for NSCLC is still not satisfactory, and the efficacy is only about 30%,hence it is necessary to find a new method and strategy for treatment. Whether the combination of targeted therapy and chemotherapy could improve the therapeutic efficacy of chemotherapy has become an important research topic domestically and abroad.At present, combined chemotherapy with platinum and third generation of new drugs is still the only standard first-line chemotherapy for advanced NSCLC, among which the first-generation platinum drugs, cisplatin, is the cornerstone of NSCLC chemotherapy. However, the individual differences in drug therapeutic efficacy is a common phenomenon, which has attracted widespread clinical attention. The difference in performance is more obvious when tumor patients are receiving chemotherapy, as the same chemotherapy regimen with the same dose may have completely different effects in different patients, which may vary from effective to lethal doses. Many clinical and pathological factors can affect the body’s reaction to drugs, and the drug resistance of platinum drugs seriously influences the therapeutic efficacy of NSCLC chemotherapy.Gefitinib is an epidermal growth factor receptor(EGFR) tyrosine kinase inhibitor, and the enzyme is usually expressed in solid tumors of epithelial origin. As the first generation of small molecule tyrosine kinase inhibitor,current related large-scale phase III multicenter clinical trials have verified that among first-line treatments of advanced NSCLC, gefitinib has significant advantage for progression-free survival(PFS) in Asians, and patients with adenocarcinoma and EGFR mutations compared with chemotherapy. However, in terms of whether it can be combined with chemotherapy, four large-scale multi-center clinical trials have confirmed that epidermal growth factor receptor tyrosine-kinase Inhibitor(TKI), such as gefitinib and erlotinib, provide no significant benefit for overall survival(OS) of advanced NSCLC when combined with chemotherapy drugs. It is suggested that there may be antagonistic mechanism in the combination of gefitinib and chemotherapy drugs, including cisplatin, where the drug resistance of cisplatin is one of the possible reasons for the antagonistic mechanism.This topic can be divided into two parts to verify the mechanism and effect of whether the synergistic action of gefitinib and cisplatin would cause the drug resistance of cisplatin, which in turn would provide new methods and ideas for the clinical treatment of NSCLC.Part1 Autophagy inhibition overcomes the antagonistic effect between gefitinib and cisplatin in EGFR mutant NSCLC cellMethods:(1) The combination of gefitinib and different concentrations of cisplatin were used on human NSCLC cell line PC9, and the MTT method was performed to measure the effect of different concentrations of cisplatin on the proliferation of PC9 cells. The interaction index between gefitinib and different concentrations of cisplatin were also calculated to analyze the effect of gefitinib on different concentrations of cisplatin.Western Blot was used to analyze the changes of autophagy marker proteins, LC3-II and P62, after PC9 cells were treated with the combination of gefitinib and cisplatin,and with cisplatin alone. Electron microscopy was used to understand the changes in the number of autophagosome in the two groups above. The aim was to understand the influence of gefitinib on autophagy level induced by cisplatin.(2) In order to accurately assess the intensity of autophagy, 5μg/ml of lysosomal inhibitor chloroquine(CQ), which is an autophagy inhibitor that inhibits lysosomal degradation, was employed to pretreat the cells for 1 hour. Transmission electron microscopy(TEM) were used to confirm that CQ was sufficient to inhibit the effect of autolysosome degradation. Then, gefitinib and/or cisplatin were added, and the changes in the expression of LC3 were compared with the group without CQ. Thus,we could determine whether that the changes in autophagy level of PC9 cells were induced by gefitinib and/or cisplatin, instead of defective autophagy degradation.(3) To understand the impact of autophagy inhibition on the antagonistic effects of gefitinib and cisplatin, MTT method was applied to test the PC9 cells in the gefitinib and cisplatin group after CQ treatment; the optical density(OD) values obtained were then used to calculate the coefficient of drug interaction(CDI), in order to verify the role of autophagy on the process of gefitinib leading to drug resistance of cisplatin from another perspective.(4) Annexin V-FITC/PI and flow cytometry were performed to detect the apoptosis rate of each group after the above treatments. In addition, the apoptosis proteins,Bcl-2 and Bax, in the respective groups of PC9 cells were extracted, in order to understand the effect of changes in autophagy levels on the apoptosis of PC9 cells.Several studies have reported that gefitinib could cause G1 phase arrest, thereby resulting in the antagonism between gefitinib and chemotherapy drugs. Therefore,flow cytometry(FCM) was utilized for cell cycle analysis, and the comparison of cisplatin, gefitinib, and combination groups with/without CQ was performed to understand the impact of autophagy on the cell cycle of PC9.Results:(1) Combination of gefitinib and cisplatin resulted in antagonistic effects MTT method was performed to detect the inhibitory effects of different concentrations of gefitinib and cisplatin on PC9 cell proliferation. The results indicated that the inhibitory effects of gefitinib and cisplatin on NSCLC cells showed time and concentration dependence. Compared with the cisplatin or gefitinib monotherapy group, MTT of the combination therapy group showed no significant reduction. Coefficient of drug interaction(CDI) was utilized to assess the properties of drug action; CDI value after treatment with different concentrations of gefitinib and cisplatin was 1.19±0.1(1.07-1.35), suggesting the presence of antagonism.(2) Combination of gefitinib and cisplatin induced autophagy Western blot was used to observe the expression of autophagy markers, LC3 and p62, in PC9 cells after treatment with gefitinib and/or cisplatin. The results showed that, compared with the untreated control group, gefitinib and cisplatin could induce and increase the transformation of LC3-II, while the transformation of LC3-II showed no difference between the combination of gefitinib and cisplatin, and gefitinib alone.Gefitinib and/or cisplatin significantly downregulated p62 levels. Compared with monotherapy stimulation, the combination of gefitinib and cisplatin led to significant reduction of p62 protein, which indicated that the combination of gefitinib and cisplatin could induce higher levels of autophagy when compared with monotherapy.Transmission electron microscopy(TEM) was performed to observe the changes of autophagosome in PC9 cells after treatment with gefitinib and/or cisplatin. The results indicated that the number of autophagosomes in the combination of gefitinib and cisplatin was larger than that in the monotherapy treatment groups, which also suggest that the combination of gefitinib and cisplatin could induce a higher level of autophagy when compared with monotherapy. Western blot method was used to observe the expression of autophagy markers, LC3 and p62, in PC9 cells after pretreatment with CQ. The result indicated that the autophagy of PC9 cells was indeed induced by gefitinib and/or cisplatin, instead of defective autophagy degradation.(3) Autophagy inhibition could resist the antagonistic effects between gefitinib and cisplatin TEM was employed to detect the changes of autophagosomes in PC9 cells treated with gefitinib and/or cisplatin after pretreatment with CQ. The results showed that, compared with the control group and CQ only group, the intensity of green fluorescence was significantly enhanced by the addition of gefitinib and/or cisplatin,suggesting a significant increase in autophagy lysosome. TEM results also suggested that CQ could inhibit the degradation of lysosome in the process of autophagy. MTT was utilized to detect the proliferation of PC9 cells treated with gefitinib and/or cisplatin after pretreatment with CQ. The results showed that, compared with the group without CQ treatment, gefitinib and cisplatin group pretreated with CQ significantly reduced cellular activity. In addition, the synergistic effect of gefitinib and cisplatin was relatively increased after CQ pretreatment. Annexin V-FITC/PI apoptosis assay was applied to investigate whether CQ pretreatment could increase the proportion of apoptotic PC9 cells in all groups when compared with that without CQ pretreatment. Both monotherapy and combination therapy of the two drugs could significantly increase the proportion of apoptotic cells with CQ pretreatment,suggesting that autophagy played an important role in the process of apoptosis.(4) Inhibition of autophagy could promote apoptosis Flow cytometry(FCM) was performed for PC9 cell cycle analysis. The results indicated that both gefitinib and cisplatin monotherapy could lead to G1 phase arrest;however, the combination of gefitinib and cisplatin only slightly increased the proportion of PC9 cells in the G1 phase. To further confirm the relationship between autophagy and cell cycle arrest, gefitinib and/or cisplatin were pretreated for 1 hr using CQ(5 μg/ml). The results showed that autophagy inhibition of CQ could not reverse cell cycle arrest induced by gefitinib and/or cisplatin. Although cell cycle arrest plays an important role in the antagonistic effects of gefitinib and cisplatin, the synergistic effect generated by autophagy inhibition was not regulated by cell cycle arrest, hence it is likely that other mechanisms were involved with its occurrence.Western blot was applied to detect whether CQ pretreatment could affect the expression levels of pro-apoptotic proteins and anti-apoptotic proteins in PC9 cells.After CQ treatment, the expression levels of Bcl-2(a classic anti-apoptotic protein)and Bax(pro-apoptotic protein) were significantly decreased and increased,respectively. However, compared with the group without CQ, there was no significant difference in Bax/Bcl-2 ratio for the gefitinib and/or cisplatin groups with CQ pretreatment(p=0.592, 0.09 and 0.06, respectively). These results suggested that autophagy inhibition by CQ could promote apoptosis of PC9 cell by upregulating Bax expression and downregulating Bcl-2 expression.Part2 Exosomes derived from gefitinib treated EGFR mutant lung cancer cells alter cisplatin sensitivity via up-regulating autophagyMethods:(1) Exo Quick precipitation solution was used to isolate PC9 cells in normal control group or the exosomes of group treated with gefitinib(1μM)/cisplatin(7.5μM).Exosomes were extracted from the liquid supernatant of PC9 cells treated with gefitinib or cisplatin, which were divided into Exo-Con(Exosomes derived from negative Control), Exo-GF(Exosomes derived from gefitinib), Exo-DDP(Exosomes derived from gefitinib treated EGFR mutant lung cancer cells). BCA protein detection kit was used for standard quantitation of different extracellular proteins.(2) Transmission electron microscopy(TEM) was used to observe the formation of exosomes, and to determine whether the extraction was successful.(3) CCK-8 method was utilized to detect the cell inhibition rate in each group after pretreatment with exosomes. A certain concentration of exosomes was used for pre-incubation for 24 hours, and 1 μM gefitinib or 7.5 μm cisplatin was added for incubation for 24 hours. CCK-8 method was applied to calculate the cell proliferation activity, and the cell inhibition rate was calculated. Coefficient of Drug Interaction(CDI) was also calculated. Then, exosome inhibitor GW4869 was applied for each drug treatment group to understand the effects of gefitinib or cisplatin on PC9 cells after exosome secretion inhibition.(4) Each group of exosomes and drug were used in combination to incubate the cells.Western Blot was applied to detect changes in autophagy marker proteins, p62 and LC3, in order to understand the correlation between exosomes and autophagy.Annexin V-FITC/PI counter-staining reagents and flow cytometry(FCM) were employed to detect the apoptosis of cells for the groups above. Western blot was utilized to detect apoptotic proteins, Bcl-2 and Bax, and to determine the influence of exosomes on the apoptosis of PC9 cells.Results:(1) Combination of gefitinib and cisplatin resulted in antagonistic effects Use CCK-8 was used to detect the effect of gefitinib and/or cisplatin on the proliferation ability of PC9 cells. The results showed that compared with different concentrations of gefitinib monotherapy(0.4-2μΜ), the anti-proliferative effects of gefitinib showed only a slight increase in the combination therapy with cisplatin. CDI values of the two drugs were also calculated in PC9 and A549 cells. The results suggested that mutual antagonistic effects existed when the combination of gefitinib and cisplatin was applied on EGFR mutant and wild-type NSCLC PC9 cell lines.(2) Exo-GF reduced the anti-tumor activity of cisplatin We investigated whether the antagonistic effects between gefitinib and cisplatin were mediated by Exo-GF or Exo-DDP. Exosomes were collected, and a certain concentration of cisplatin or gefitinib was added to the PC9 cells together. The results of CCK-8 exhibited that Exo-GF showed dose-dependent antagonism against the anti-tumor effects of cisplatin, while Exo-Con had no significant effect on proliferation inhibition induced by cisplatin, and Exo-DDP had almost no effect on gefitinib. CCK-8 method was performed to detect the inhibition rate of gefitinib and/or cisplatin in PC9 cells after pretreatment with exosome inhibitor GW4869. The results showed that after adding GW4869, the effect of gefitinib and cisplatin monotherapy on inhibition rate of PC9 cells only increased slightly. However, the inhibition rate increased significantly with the combination of gefitinib and cisplatin after GW4869 pretreatment, while the CDI values of the two drugs decreased significantly after the addition of GW4869, suggesting that inhibition of Exo-GF secretion could reverse antagonistic effects between gefitinib and cisplatin.(3) Exo-GF upregulated autophagy caused by cisplatin on PC9 cells Western blot was employed to detect the effects of Exo-Con, Exo-GF and Exo-DDP on LC3 and p62 expression in PC9 cells. When compared with the control group of untreated PC9 cells, Exo-Con, Exo-GF and Exo-DDP could all significantly increase autophagy activity. When compared with the cisplatin monotherapy group,the autophagy of cisplatin with Exo-GF pretreatment was significantly enhanced.Semi-quantitative analysis confirmed Exo-GF could significantly promote autophagy induced by cisplatin, but Exo-DDP pretreatment had almost no effect on autophagy induced by gefitinib.(4) Exo-GF downregulated cell apoptosis induced by cisplatin Flow cytometry(FCM) was applied to detect the effect of Exo-GF on apoptosis of PC9 cells induced by cisplatin. The results indicated that compared with the cisplatin group with Exo-Con pretreatment and cisplatin monotherapy group, the number of apoptotic cells was significantly reduced in the combination of Exo-GF pretreatment and cisplatin monotherapy group. Consistent with CCK-8 test results,Exo-DDP had no effect on apoptosis induced by gefitinib.Western blot test was further employed to detect the protein expression levels of Bcl-2 and Bax in PC9 cells in the above treatment groups. In accordance with expectations, when compared with the cisplatin group or Exo-Con pretreatment plus cisplatin group, expressions of Bcl-2and Bax were significantly increased and decreased, respectively, in PC9 cells of the Exo-GF pretreatment plus cisplatin group. When compared with the cisplatin monotherapy group, Bcl-2/Bax ratio was significantly higher in the Exo-GF pretreatment plus cisplatin group. However, when compared with the gefitinib monotherapy group, apoptosis level of PC9 cells showed no significant difference in the Exo-DDP pretreatment plus Iressa group. Therefore, Exo-GF could significantly downregulate cell apoptosis induced by cisplatin.Conclusions:(1) Gefitinib combined with Cisplatin, when acting on EGFR mutational NSCLC cells, can cause antagonistic effect.(2) Gefitinib treatment for exosomes produced by PC9 cells can fight Cisplatin induced cell apoptosis through autophagy up-regulation.(3) Inhibitation of exosome secretion can reverse the antagonistic effect between Gefitinib and Cisplatin via downregulating autophagy.