Erlotinib Induces The Human NCLC Cells Apoptosis Via Activating ROS-dependent JNK Pathways And Clinical Study

Lung cancer,as one of the most malignant cancer endangering human health and lives,is the main reason resulting in relevant death caused by cancer.The combined chemotherapy mainly of platinum is an important corner stone and main means of traditional treatment for advanced non-small cell lung cancer(III B phase and IV phase).Wi th gradually research into the pathogenesis of lung cancer,the target treatment of lung cancer and immunotherapy has become the research hot spot for advanced NSCLC.The molecule target medicine clinical application research represented by epidermal growth factor receptor,tyrosine kinase inhibitor(EGFR-TKI)found that it could obviously improve the survival phase of later NSCLC and life quality.Moreover,adverse effect relevant to such medicines such as cardiotoxicity,harms on myelosupression,and fun ction of liver and kidney obviously reduced.As a result,it has become the hotspot of research on later NSCLC.The first generation of EGFR-TKI is the reversible EGFR tyrosine kinase inhibitor which is with the widest application.But the treatment averagely lasts for about 9 month.As long as with EGFR-TKI treatment,evidence of drug resistance would occur and this is when the disease develops with fast speed.As a result,how to solve the drug resistance of EGFR-TKI is the very key and difficult problem for clinical treatment.Erlotinib,one of the EGFR-TKI,can ihibit and slow down the growth of tumor while also has the problem of drug resistance.With research going deeper,the treatment with it and its drug resistance mechanism as well as pathway still remain unclear for many parts.ROS,the reactive oxygen species produced in the process of human cell aerob ic respiration and aerobic metabolism,closely participates in adusting various physiological and pathological processes.The production of large amount of ROS in some environments can trigger the destruction of structures such as intracellular proteins an d organelle.As a result,clinical practice commonly use anti tumor medicines to kill tumor cell and can directly or indirectly function through ROS.Meanwhile,regulatory T cell(Treg)/ Regulatory B cell(Breg),as one kind of cell subsets with unique immune negative regulatory functions is gradually known and used in tumor immune tolerance and treatment processes.A lot of researches showed that immune cells such as Breg and Treg play the role of immune regulation under tumor micro environment,and take part in survival,proliferation and neoplasm metastasis of tumor cells.Given that ROS is the main factor for inducing tumor cell death and immune cells play an important role on tumor immune tolerance and treatment,the concrete function and mechanism and the regulatory mechanism of immune cells such as Treg and Breg under tumor micro environment relevant to ROS inducted by EGRF-TKI is still not completely clear to us.It demands for immediate deep research to illustrate.So we carry out new molecule biotherapy on tumor to improve the progn osis of patients.Objective:In this study,the anti-cancer effect of ERL,against human lung cancer A549 cells,and the underlying mechanisms were investigated.A nd to investigate the phenotype of circulat ing regulatory T / Bcells in advanced NSCLC patients and their potential role in tumorgenesis.Methods:Part one:The anti-cell growth ability of ERL on A549 was assessed by MTT assay.Apoptosis was determined by Hoechst33342 staining and FACS assay.Then,the intracellular reactive oxygen species(ROS)was monitored using DCFH-DA and mitochondrial membrane potential(MMP)was measured with JC-1 staining.Finally,the effect of ERL on phosphorylation state of JNK protein and downstream apoptosis related protein was determined by Western blotting.Results: the data showed that ERL significantly inhibited cell growth of A549 with the concentration rising up in vitro.Hoechst33342 staining confirmed ERL induced human non-small cell lung cancer cells apoptosis with the concentration rising up.Furthermore,exposure of A549 cells to ERL increased the production of ROS,which activated the pro-apoptotic JNK signaling pathway and inhibited the activation of EFGR signaling pathway.At the same time,we found that ERL could induce cell-cycle arrest at G0/G1 period.Phosphorylated JNK decreased mitochondria membrane potential and down-regulated expression of anti-apoptotic protein Bcl-2 concomitant with the up-regulation expression of pro-apoptotic protein Bax.In addition,ERL-induced the phosphorylation of JNK further increase the activation of c-Jun and cleaved-caspase-3.All of these pro-apoptosis effect of ERL was reversed by administration of NAC which performed as a ROS-scavenger.Part two:19 Patients with advanced NSCLC and 15 healthy donors were enrolled in the study.Frequencies of Tregs and Bregs were measured by flow cytometry.1 The pro-apoptosis effect of ERL on A549 cellsA549 lung cancer cells were incubated with various concentrations of ERL(0,5,10,15,20,25,30,35,40,45 μmol/L)for 24 h.As shown in Fig.1B,ERL significantly increased the death of A549 cells.The higher concentration of the ERL,the stronger apoptosis effect emerged on A549 cells.According to the FACS results,we determined t he IC-50 values of the ERL in the A549 cells,which was 23 umol/L.On the basis of IC-50 values,we used the 25 umol/L as ERL standard treatment concentration.2 The effect of ERL on A549 cells by Hoechst33342 stainingTo further investigate the mechanisms about the ERL on A549 cell,we chose ERL at 0,5,15,25,35 and 45μmol/L in our study.As shown in Figure 2,after treatment with ERL,morphological characteristics indicative of apop totic cells were observed in Hoechst 33342-stained A549 cells.In control groups,A549 cells showed regular and round nuclei as observed under a microscope,whereas condensation and fragmentation of nuclei were evident after cells being exposed to ERL for 24 h,which was the characteristic of apoptotic cells.In conclusion,the result suggested that ERL could promote cell apoptosis with concentration rising up.3 Effects of ERL on the levels of ROS in lung cancer A549 cellsTo determine whether treatment of cells with ERL is associated with the generation of ROS,A549 cells were incubated with DCFH-DA.in A549 cells,the levels of ROS increased significantly after treated with various consentrations of ERL for 24 h(p<0.05),suggesting the ERL induced the generation of ROS in A549 cells.The higher concentration of the ERL,t he stronger generation of ROS emerged on A549 cells.These data conclusively suggested that ERL-induced A549 cells apoptosis tightly related to the level changes of ROS in human lung cancer A549 cells.4 Effects of ERL on the MMP in A549 cellsIt has been demonstrated that decreased MMP is one of main characteristic of early apoptosis.Therefore,to investigate whether mitochondria were involved in ERL-induced apoptosis,we measured MMP in A549 cells by flow cytometry using JC-1 staining.A549 cells treated with ERL showed a significant loss of MMP.The higher concentration of the ERL,the stronger decreasing of MMP emerged on A549 cells.As expected,the positive control treatment CCCP(10 μM)resulted in a significant decrease in MMP.5 Underlying mechanism of ERL induced cell apoptosis and cell-cycle arrestIn order to further confirm the effect of ROS in cell apoptosis,we put the ROS scavenger(NAC)on our study.As we expected,the pro-apoptosis effect of ERL was obviously reversed by Results: the administration of NAC,This result confirmed that ERL-induced the generation of ROS played a critical role in the antitumor effect of ERL in A549 cells.Next,we analyzed the effect of ERL on the cell-cycle progression by FACS.Treatment of ERL induced a marked G0/G1 arrest in A549 cells(p<0.05).Similarly,treatment with NAC could reverse the ERL’s cell-cycle arrest effect.Combined with previous results,we confirmed that the generation of ROS was a key factor in the p ro-apoptosis effect of ERL.6 Effects of ERL on the activation of p-JNK in A549 cellsThe activation of the redox sensitive JNK signaling pathway is followed by an increase in ROS production.To investigate whether ERL-induced ROS leads to the activation of JNK in A549 cells,we determined the phosphorylation state of JNK in A549 cells treated with ERL.The results were shown: ERL treatment for 24 h significantly increased the phosphorylation of JNK in A549 cells compared with the control group(p<0.05).Be sides,the administration of NAC significantly inhibited the over-expression of p-JNK induced by ERL,These data conclusively suggested that ERL-induced the generation of ROS contributed to the activation of JNK signaling pathway in A549 cells.7 Effects of ERL on inhibition of p-EGFR in A549 cellsAs we all know,ERL is a reversible inhibitor of EGFR,so we confirmed the effect of ERL on the activation of the protein EGFR.As is shown,the results showed that ERL significantly inhibited the expression of p-EGFR compared with the control group(p<0.05).Besides,the administration of NAC obviously reversed this effect of ERL.We may conclude that ERL-induced the generation of ROS also had a tight relationship with the down regulation of EGFR signaling pathway in A549 cells.8 Effects of ERL on the expression of apoptosis related protein.Studies have demonstrated that c-Jun is a nuclear substrate of JNK and the activated JNK,in turn,phosphorylates c-Jun,which further activate those proteins associated with apoptosis such as Bcl-2,Bax and caspase-3.Thereby,it is essential to address the effect of ERL on the phosphorylation of c-Jun,the expression of Bcl-2,Bax and caspase-3.Figures shows the results of western blotting with anti-Bcl-2 and anti-Bax antibodies in A549 cells after ERL treatment for 24 h.Compared with the control group,the protein expression of Bcl-2 significantly decreased and Bax increased in ERL treatment groups(p <0.05).Moreover,the relative ratio of Bcl-2/Bax protein also reduced in ERL treatment groups compared with the control group(p < 0.05).The administration of NAC obviously reversed this effect of ERL on the decreased ratio of Bcl/Bax.In addition,ERL treatment significantly increased the phosphorylation of c-Jun and the expression of cleaved-caspase-3 in A549 cells compared with the control group(p < 0.05).Similarly with other apoptotic proteins,the administration of NAC obviously reversed the activation of pro-apoptosis proteins of ERL.The frequency of CD4+CD25+ Treg and Breg was higher in patients with non-small cell lung cancer than healthy controls.However further analysis showed that only a Treg were highly expressed in the CD4+ of NSCLC patients compared to healthy controls.The frequency of r Treg and non-Treg didn’t show any significant.The frequency of peripheral Bregs in CD19 + cells inlung cancer patients was significantly higher than in the healthy.Conclusions:Our results showed that ERL induced apoptosis concomitant with increased ROS generation in A549 cells,indicating that ERL may exert anticancer activity by increasing the production of ROS.ERL induces A549 cells apoptosis via activating ROS-dependent JNK pathways in human non-small lung cancer cells that provide a new experimental foundation for cancer therapy.The frequency of peripheral a Tregs in CD4 + T cells from advanced NSCLC patients was higher than in the healthy,while the frequency of peripheral Bregs in CD19 + B cells was significantly higher.The immune system plays a significant role in the control of tumor progression,although the regulatory mechanism of interaction between two systems remains unclear.