The Role Of Intracellular Calcium In The ET-1-induced Proliferation Of Human Lung Adenocarcinoma Cells SPC-A1

Non-small cell lung cancer(NSCLC) is the leading cause of cancer death in the world.The platinum-based combination chemotherapy regimens have emerged as the standard approach in advanced NSCLC.Meta-analyses have demonstrated a 2-month increase in median survival and 10%improvement in the 1-year survival rate after platinum-based therapy vs.best supportive care.Despite their contributions,the combination chemotherapy for advanced NSCLC has reached a therapeutic plateau, with response rates seldom exceeding 40%and 1-year survival rates stable between 30%and 40%.It is hoped that the introduction of new anticancer agents with greater specificity and less toxicity will improve the outcome for advanced NSCLC patients. In recent years,target therapy has been used for the treatment of NSCLC,which is focused on epidermal growth factor receptor and vascular endothelial growth factor. Even though they reach a certern clinical response,the problem is that the response rate is modest.Therefore,the finding of new therapeutic target for NSCLC and the combination of these different target therapies are the tendency in the treatment of NSCLC.Endothelin-1(ET-1),the most potent vasoconstrictor,is distributed not only in cardiovascular system,but also in a wide variety of tissues,and appears to be involved in a wide variety of physiological functions.It exerts biological actions through at least two major G protein-coupled receptor subtypes,ETA receptor(ET_AR) and ET_B receptor(ET_BR).It has been reported that ET-1 stimulates mitogenic responses and expression of protooncogenes(c-myc,c-fos)in vascular smooth muscle cells,fibroblasts,and glomerular mesangial cells,suggesting its potential role as a growth factor.Interest in the role of ET-1 in cancer has grown over the last decade.From the evidence to date,it is well established that ET-1 is an important factor involved in several processes affecting tumor progression such as cell proliferation,angiogenesis,apoptosis and bone metastasis.ET-1 messenger RNA (mRNA) has been identified in a variety of human lung cancer tissues and lung cancer cell lines.ETRs and endothelin converting enzyme-1(ECE-1) were also found in several lung cancer cell lines.These findings strongly suggest that ET-1 play a role in the the process of lung cancer.It is well known that the ET-1-induced elevation of intracellular free Ca²⁺([Ca²⁺]_i) plays a great role in vascular contraction and vascular smooth muscle cell proliferation.Previous studies showed that the elevation of[Ca²⁺]_i induced by ET-1 is resulted from both transmembrane Ca²⁺ influx and intracellular Ca²⁺ release from inosital triphosphate receptor(IP₃R) and ryanodine receptor(RyR) gated Ca²⁺ stores. It was found that[Ca²⁺]_i is closely related to the ET-1-induced cell proliferation in a number of cancer including cervix carcinoma and ovarian carcinoma.However,at present,no information is yet available about its biological role in lung cancer cell and its possible cellular mechanism.In the present study,we firstly determine the mitogenic effect of ET-1 and ETRs in the human lung adenocarcinoma cell SPC-A1 and then explore the role of[Ca²⁺]_i in this process. Partâ… The effect of ET-1 on the cell proliferation of human lung adenocarcioma cell SPC-A1To explore the role of ET-1 in proliferation of human lung adenocarcioma cell SPC-A1,we used MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay to mesure tumor cell number.Data are presented as mean±S.E.M. where n refers to the number of experiments.Experiments between two groups were analyzed by t test.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.The results were shown as follows:1.At concentration of 10^-15mol/L～10^-8mol/L,ET-1 induced significant increase of cell number in a dose-dependent manner,with the maximal effect beginning at 10^-10 mol/L.10^-10mol/L～10^-8mol/L produced saturation.2.To define the receptor-mediated action of ET-1 on the proliferation of SPC-A1 cells,highly selective ET_AR antagonist and ET_BR antagonist were used in this study. The cell proliferation induced by ET-1(10^-10M) was completely abolished by BQ₁₂₃ at 10^-7M(P=0.001＜0.05),a highly selective ET_AR antagonist,not by BQ₇₈₈ at 10^-7M(P=0.872＞0.05),a highly selective ET_BR antagonist.Partâ…¡The role of intracellular free calcium in the ET-1-induced proliferation of human lung adenocarcinoma cells SPC-A1In the present study we used Fura-2/AM fluorescent assay to measure intracellular free calcium([Ca²⁺]_i) and to explore its role in the ET-1-induced cell proliferation in human lung adenocarcinoma cells SPC-A1.Data are presented as mean±S.E.M,where n refers to the number of experiments.Experiments between two groups were analyzed by t test.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.The results were shown as follows:1.At concentration of 10^-15～10^-8M,ET-1 induced biphasic increases of[Ca²⁺]_i in a dose-dependent manner.The[Ca²⁺]_i response typically consisted of a spike phase followed by a prolonged plateau phase.The ET-1-induced(10^-10M) increase of[Ca²⁺]_i was blocked by the ET_A-selective receptor antagonist BQ₁₂₃(10^-7M)(P= 0.001＜0.05),not by the ET_B-selective receptor antagonist BQ₇₈₈(P=0.680＞0.05).2.Chelating extracellular Ca²⁺ significantly reduced ET-1-induced increase of [Ca²⁺]_i in SPC-A1 cells(P=0.001＜0.05).Inhibition of L-type Ca²⁺ channels with nifedipine(10^-6mol/L) or with non-specific ion channel inhibitor SKF₉₆₃₆₅ (10^-7mol/L) also reduced the ET-1-induced increase of[Ca²⁺]_i in SPC-A1 cells(P =0.001＜0.05).The effect between nifedipine and 8KF₉₆₃₆₅ has no significant difference(P=0.720＞0.05).3.Ryanodine receptor activator ryanodine(50×10^-6 mol/L) significantly reduced the ET-1-induced increase of[Ca²⁺]_i in SPC-A1 cells(t=11.353,P=0.004＜0.05).The ET-1-induced increase of[Ca²⁺]_i was significantly reduced by U₇₃₁₂₂ at 10^-5M(P=0.015＜0.05),the specific inhibitor of phospholipase C(PLC),but not by U₇₃₄₃₃(P=0.921＞0.05),it's inactive analogue.However,staurosporine(2×10^-9 mol/L),a protein kinase C inhibitor,exerted no significant effect on the ET-1-induced(10^-10 mol/L) increase of[Ca²⁺]_i(t=0.712,P=0.486＞0.05).Partâ…¢The role of intracellular calcium in the ET-1-induced proliferation of human lung adenocarcinoma cells SPC-A1Based on the results of Partâ… and Partâ…¢,we observed the changes in the ET-1-induced cell growth and increase of[Ca²⁺]_i at the same time to probe the role of [Ca²⁺]_i in ET-1-induced proliferation of human lung adenocarcinoma cells SPC-A1. Data are presented as mean±S.E.M,where n refers to the number of experiments. Experiments between two groups were analyzed by t test.Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.The following was the results of this part: 1.Chelating extracellular Ca²⁺,which significantly reduced the ET-1-induced [Ca²⁺]_i increase,significantly blocked the ET-1-induced cell proliferation(P= 0.001＜0.05).Inhibition of L-type Ca²⁺ channels with nifedipine(10^-6mol/L),which significantly reduced the ET-1-induced[Ca²⁺]_i increase,also reduced the ET-1-induced cell proliferation of SPC-A1 cells(P=0.001＜0.05).2.ET-1-induced cell proliferation was significantly reduced by U₇₃₁₂₂ at 10^-5M (P=0.001＜0.05),the specific inhibitor of phospholipase C(PLC),but not by U₇₃₄₃₃ (P=0.986＞0.05),it's inactive analogue.Conclusion:ET-1,acting as an growth factor,enhances the proliferation of SPC-A1 cell line and increase of[Ca²⁺]_i via activation of ET_AR.Both phosphoinositol/Ca²⁺ pathway and Ca²⁺ influx through voltage dependent Ca²⁺ channel contribute to this process.