Antiapoptosis Gene, Livin Expression In Small Cell Lung Cancer And Its Correlation With Caspase-3, P53, Proliferation And Apoptosis

IntroductionSince 70's in 20 centuries,cancer morbidity and mortality are increasing significantly,and now have become the leading cause of death in both town and countryside in China.The morbidity and mortality of lung cancer is particularly going up.Despite of many progressions in treating the deadly disease,which including multimodality and individual therapy,the fatality rate of lung cancer ranks as the No.1 in malignancies of human beings.So,the further researchs of molecular biological feature were important to therapy and prognosis of lung cancer.Inhibitor of apoptosis protein(IAP) family is first found by Losis Miller.etc,who found it in baculovirus group(CpGC) and Orgyia pseudotsugata multinucleusd virus (OPMNV).It is a new kind of anti-apoptotic protein which is independent of Bcl-2. Eight human inhibitors of apoptosis protein have been identified in recent years.Each contains one or more baculovirus IAP repeat domains,which are necessary to bind specifically to a terminal effector cell-death protease(for instance,caspases-3 and -7). Binding results substantially reduced caspase activity and reduced cell death in response to diverse apoptotic stimuli.Livin found recently was a novel inhibitor of apoptosis family members.The distribution of Livin had a high selectivity, tissue-specific expression in human fetal tissues and nearly all solid malignancies. There were little expression of Livin in normal adult tissues,such as spleen,thymus, prostate,testis,small lintestin,colon,ovary,liver,lung,brain,skeletal muscle,lymph cell and peripheral blood.The similar extends of amino acids sequence among Livin and the other members of IAPs family separately are:NAIP is 25.5%,cIAP-1 is 24.1%, cIAP-2 is 26.1%,XIAP is 34.7%,Survivin is 26.3%.Its senior structure is much more similar with Survivin,including BIR domain with 4αhelix and 1βantiparallel fold(including one Ser/Thr phosphatizing site,RING structure domain in C terminal, but no helix-helix structure domain of Survivin.There are some data displayed that,the anti-apoptotic activity of Livin is much stronger than Survivin.The effect of Livin inhibiting cell apoptosis is mainly taken on by the combination of its BIR domain and Caspase to inhibit the activity of Caspase,especially inhibit the activity of Caspase 3/7 and Caspase 9.It has been suggested that suppression of apoptosis may contribute to the development and progression of cancer.Anti-apoptotic Livin gene,like suvivin,is highly expressed in cancer cells and transformed cells,but shows little or no expression in normal differentiated tissues.However,there are no available data concerning Livin expression in small cell lung cancer(SCLC).Therefore,the expression of LivinαmRNA and LivinβmRNA in 30 SCLC tissues and 16 para-cancerous lung tissues was measured by reverse transcription polymerase chain reaction(RT-PCR) technique, Western blot method was used to studay Livin protein in these tissues. Immunohistochemical SP method was used to detect the expression of Livin, Caspase-3 and P53 proteins in these tissues.We used flow cytometry to detect the level of SCLC cell proliferation and apoptosis and tried to draw a conclusion that the expression of Livin correlated with proliferation and apoptosis.Materials and methodsMaterialsAll samples during January 2003 to October 2006 were collected in the department of thoracic surgery,First Affiliated Hospital China Medical University and Dalian Medical University.30 SCLC tissues and 16 para-cancerous lung tissues were obtained.There were 22 male,8 female patients with an average age of 54.53(from 34 to 71)years.According to 1997 UICC staging criteria,30 cases were classified as stageⅠ(12cases),stageⅡ(10cases) and stageⅢ(8cases).There were 17 cases of lymph node metastasis in 30 SCLC cases.No cases were received rediotheropy or chemiotheropy before operation.Methods1.RT-PCR for Livin mRNATotal RNA form each sample(contain 100mg tissue) were isolated by using Trizol Solution,then the concentration and purity of RNA PCR Buffer 1μl,dNTP 1μl,MgCl2 2μl,Random 9 mers 0.5μl,RNA 1μl,RNase inhibitor 0.25μl,Reverse Transcriptase 0.5μl,ddH20 3.75μl.Followed by the condition:10min at 30℃,30min at 42℃,5min at 99℃and 5min at 5℃.The gene-specific primers:FR5'-CATGGGTCTCCGT CCCTCG-3' and RV5'- CAG GGAGCCCACTCTGCA-3' PCR amplification was performed on a PCR thermal cycler.2.5μlof cDNA mixture was added to 10μl amplification solution containing the following:ddH20 7.16μl,20pmol/L each of 5' and 3' primers 0.15μl,Taq Hs 0.04μl and 5×Buffer 2.5μl,PCR conditions included initial denaturation for 2min at 95℃,followed by 30 cycles of denaturation of 95℃for 1 min and annealing at 72℃for 1min,then extention at 72℃for 7min.The efficiency were detected byβ-actin.PCR products were resolved on a 2%polyacrylamide gels electrophoresis and the bands were visualized,then ascertained with the comparison to Makers(TaKaRa Co).2.Western-blot analysisTissue samples were lysed in 200μl RIPA buffer.The protein concentration was measured according to the Lowry method;polyacrylamide gel eletrophoresis;After 2h of transferring to PVDF membrane,the blot was probed with rabbit multiclonal anti-Livin(1:3000,IMGENEX,USA) and murine monoclonal anti-β-actin antibody (1:400,SIGMA,USA),incubated 2h in room temperature.The secondary antibody was goat anti-rabbit and horse anti-murine monoclonal antibody,challenged with the appropriate second antibody conjugated with alkaline phosphatase for 2h at room temperature,stained with alkaline phosphatase 15min.The positive blot were scanned and then measured for intensity by using the UPV gel imaging-analyzing system (Chemi Imager 5500,USA).3.Immunohistochemisty for Livin,Caspase-3 and P53 proteinsImmunohistochemical tests were performed by S-P method.The S-P Kit was produced by FuZhou Maixin Biological Corp.China.The primary antibodies were multiclonal rabbit antibody against Livin(1:2000),multiclonal rabbit antibody against Caspase-3(1:200) and multiclonal rabbit antibody against P53(1:100).All the procedures were followed by the instruction.The negative control was prepared in the PBS,the positive control for staining was obtained from the sections of gastric cancer. The positive cells were defined as brown to yellow staining in cytoplasm,nuclear membrane or nucleus.The positive cells were classified into three degrees according to the number and color intensity of the cells.The three degrees were weak positive(+), positive(++) and intense positive(+++),respectively.4.Flow cytometry to detect proliferation and apoptosisThe tissues were resected in deep-low temperature.The liquid containing single cell was regulated by PBS into 1×106cell/ml.PI liquid(PI 5mg.Rnase 2mg. TritonX-100 1ml,normal saline 65ml,sodium citrate 100mg,add distilled water to100ml) 1ml was added to one,30min at 4℃in dark,and FITC-AnnexinⅤand PI each 5μl were added to the other,15min at room temperature in dark.Stained cells were analyzed by means of a FACSCalibu(B.D,USA) and relative modifat software in Macintosh 9.5 computer.10000 cells were counted in every specimen.Before detection,the CV was regulated under 3.0%.PI couldreflect activity of proliferation.The early apoptosis cells were counted on two-dimensional lattice picture.Statistical analysisChi-squared test was used in sample rate contrastion of Livin expression. Chi-squared test of two-way orderly tabulate data was used in the correlation of Caspase-3,P53 and Livin.T-test was used in data of proliferation and apoptosis.The data was analyzed by the SPSS 13.0 software,P＜0.05 has importance in statistics.Results1.The expression of Livin geneThe positive rate of Livin gene was 63.33%(19/30) in 30 SCLC tissues,but Livin gene expressed low levels in 16 para-cancerous lung tissues,its positive rate was 12.5%(2/16)(P＜0.05),the expression of both variants was simultaneous basically.The RT-PCR and Western-blot methods have identical results.2.Correlation between expression of Livin gene and clinicopatho- logical factorsThere was no significant correlation between positive Livin gene expression and age,sex,TNM staging,lymph node metastasis and tumor diameter(P＞0.05).3.The expression of Livin protein in SCLC and its correlation with Caspase-3, P53 proteins The positive rate of Livin protein was 56.67%in 30 SCLC tissues,but Livin protein expressed low levels in 16 para-cancerous tissues,its positive rate was 12.50% (P＜0.05).The ration of positive expression of Caspase-3 was 11.77%(2/17) among the Livin protein positive expression and the ration of positive expression of Caspase-3 was 46.15%(6/13 )among the Livin protein negative expression(r=-0.359,χ2 = 4.455, P = 0.035 ).The ration of positive expression of P53 was 70.59%(12/17) among the Livin protein positive expression and the ration of positive expression of P53 was 15.39%(2/13) among the Livin protein negative expression(r = 0.481,χ2 = 9.020, P = 0.003).4.Correlation between the expression of Livin gene and tumor cell proliferation in SCLC tissuesThe average proliferation ratio in 30 SCLC tissues was(30.89±7.17)%. According to RT-PCR and Western-blot results,the average PI of 19 cases with positive Livin expression and 11 cases with negative expression were respectively (35.50±4.08%) and(22.92±3.01)%,with significant deference(t=8.891,p＜0.01).5.Correlation between the expression of Livin gene and cell apoptosis in SCLC tissuesThe average ratio of early apoptosis in 30 SCLC tissues was(3.24±0.74)%. According to RT-PCR and Western-blot results,the average apoptosis ratio of 19 cases with positive Livin expression and 11 cases with negvative expression were respectively(2.77±0.34)%and(4.04±0.51)%,with significant deference(t=8.194, p＜0.01).Conclusion1.Livin gene is highly expressed in SCLC tissues,but shows little expression in para-cancerous lung tissues.2.There was no significant correlation between positive Livin gene expression and age,sex,TNM staging,lymph node metastasis and tumor diameter.3.The expression of Livin protein was negatively related to the expression of Caspase-3 protein,but the expression of Livin protein was positively related to the expression of P53 protein. 4.The expression of Livin gene in SCLC tissues had correlation with tumor cell proliferation.5.The expression of Livin gene in SCLC tissues had correlation with tumor cell early apoptosis.