Studies On The Proliferation And Migration Effects And Mechanisms Of M3 MAchR In NSCLC Cells

Lung cancer,with fast increasing morbidity and mortality,is one of the malignant tumors which threaten human health and life.The lung cancer can be classified into non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).More than 80% of lung cancer is non-small cell lung cancer.Non-small cell lung cancer can be divided into squamous cell carcinoma(25-30%),adenocarcinoma(35-40%)and large cell carcinoma(10-15%.Early NSCLC is mainly subjected to surgical resection and receives the chemotherapy treatment with platinum drugs and antimetabolites,antimicrotubuless or topoisomerase drugs.Owing to the adverse reaction and poor prognosis,they are unable to raise the 5-year survival rates effectively.In recent years,targeted therapy and immunostimulation monoclonal antibody make great progress in specific people,which get extensive attention of the pathogenesis and the therapeutic targets for the cancer.Acetylcholine is a classical neurotransmitter synthesized by nerve cells,and it plays its role by activating mACh R and nAChR.More and more findings show that a wide variety of tumor cells including lung cancer could synthesize and secrete acetylcholine,which acts as a growth factor and promotes the development of cancer.Malignant tumors could express many kinds of mACh R subtypes,and M3 mAChR expression is up-regulated significantly in tumor compared with normal tissue..Studies show that M3 R selective antagonists inhibited cell growth in vitro and caused a decrease in basal levels of MAPK phosphorylation in tumors in nude mice in vivo.These researches provide us a new therapeutic tactic for NSCLC treatment and drug development,meanwhile open a new window for investigating the development mechanism for tumor.In the current researches,we have great interest in the role of M3 mAChR in the proliferation,migration and invasion of NSCLC,and we’d like to research the mechanisms of M3 mAChR in these development progresses,we also hope to validate that M3 AChR could become an anti-tumor target and establish the cholinergic receptor system as a regulation factor for the tumor.To verify our opinion,we performed the experiments as following:(1)CCK-8 assay was used to determine the effect of M3 R antagonists,agonists and PKC inhibitor on NSCLC cell proliferation.(2)The fluo-4,AM was used as Ca2+ fluorescence probe to monitor the dynamic change of intracellular calcium by laser confocal microscopy.(3)The wound-healing assay was used to assess the effect of M3 R antagonists on the migration ability of NSCLC cells.(4)The transwell invasion assay was used to assess the effect of M3 R antagonists on the invasion ability of NSCLC cells.(5)Small interfering RNA(siRNA)transfection was used to knock down M3 R and then detect its effect on NSCLC cell proliferation(6)the western blotting was used to detected the expression of related signal molecular in the cell division cycle.The results of the experiments are listed as following:(1)We first studied the effect of M3 mAChR antagonists on NSCLC cell proliferation,and we found M3 mAChR antagonists R2-8018 and darifenacin both significantly inhibited NSCLC H1299 and H460 cell proliferation in vitro.For H1299 cells,IC50 was 10.6 μmol/L(n=4)after treated by R2-8018 for 48 h.(2)Exogenous cholinergic agonist acetylcholine chloride and carbachol had no effect on NSCLC cell proliferation.(3)M3 protein expression was strongly decreased after 72 h transfection with M3 siRNA compared to the negative control siRNA(p<0.01);CCK-8 assay tested the growth inhibition caused by silencing M3 after 48 h and 72 h,there was a time-dependent reduction of cell growth(48 h:p<0.5,72 h:p<0.01).(4).30 μM carbachol caused an increase of Ca2+ in H1299 cells,M3 R antagonist darifenacin strongly blocked the response of H1299 cells to carbachol.(5).PKC inhibitor staurosporine significantly inhibited H1299 cell proliferation in vitro,and R2-8018 could cause a concentration-dependent down-regulation of PKC-α protein expression(10 μM:p<0.01,20 μM:p<0.01).(6)R2-8018 inhibited the migration and invasion of H1299 cells in a dose-dependent manner after 24 h treatment.(7)Darifenacin down-regulated the protein expression of Thr308p-Akt,Ser9p-GSK3β and cyclinD1 in H1299.(8)R2-8018 up-regulated the level of p21.Conclusion: M3 R antagonists and M3 siRNA both inhibited the cell proliferation of NSCLC cells.M3 R,as a GPCR,inhibiting its function could decrease the intracellular Ca2+ and PKC activity,indicating that antagonizing M3 R may inhibit cell growth through its classical G protein coupled signal;M3R antagonists could inhibit the Akt phosphorylation,relieving GSK3β phosphorylation,then promote cyclin D1 degradation,up-regulate p21,and arrest the transition from G1 to S phase of cell cycle,eventually led to the inhibition of cell proliferation.M3 R antagonists inhibited the migration and invasion of H1299 cell and down-regulated MMP-2 expression in a dose-dependent manner.