Fas/FasL Gene Expression In Small Cell Lung Cancer And Its Correlation With Bcl-2 And Apoptosis

IntroductionLung cancer was one of the most dangerous dieases to minkide and both the morbidity and morality of which increasingly rose in the word.In the developed countries and mid large city of China,its mortality was on the top of list of tumor.Althouth interventions such as operation,radiotherapy,chemiotherapy and biological therapy were used,there was no obvious improvement in prognosis change.So,the further researchs of molecular biological feature were impotant to dignosis,therapy and prognosis of lung cancer.Tumor was a kind of diseases mainly resulted from cells mutation.Uncontrolled proliferation was one of the impotant characters of tumors.The activation of oncogene and inactivation of antioncogene had been proved to be important to the development of tumors in recent researches.Further researches showed that some kinds of these genes caused the normal death of the cells.There was a close relationship between tumor agenes and the control of abnomal cell death.So,apoptosis and tumors became a new focus in molecular biological reseaches.Apoptosis was important to the stability of internal environment of the body.The changes of apoptosis contributed to cell proliferation and the development of tumors,which was one of impotant mechanisms in the froming of the tumor.It is now clearly accepted that clonal expansion and tumor growth result not only from the acceleration of intrinsic proliferation,but also from escape from apoptotic cell death.The Fas receptor(APO-1 or CD95)and its ligand(FasL)play a key role in the intiation of one apoptotic pathway.Alterations in this pathway within tumor cells can result in escape from apoptosis and immune surveillance.In malignant cells,their reduced capability to undergo apoptosisin response to some physiological stimuli results in a significant survival advantage and may contribute to tumorigenesis.A previce study reported that loss of Fas expression is an integral part of the process of lung tumorigenesis,by allowing lung tumor cells to escape attack by activated cytotoxic T cell which are frequently present in non small cell lung carcinomas.Fas ligand(FasL)expression in lung carcinomas has also been proposed to play a role in allowing escape from immune surveillance via a mechanism of peripheral delection of tumor-reactive T cell clones.Therefore,the Fas and FasL protein expression is essential for the evalution of potential pathologic alterations of apoptosic pathways in human SCLC,and in fact,the Fas pathway alterations have been reported in variety of solid human tumors including lung carcinomas.Initation of apoptosis occurs principallt by singals from two distinct but convergent pathways,the extrinsic or Fas receptor-mediated pathway and the intrinsic,or mitochondrial pathway.Bcl-2 normally resides in mitochondrial membrance and the cytoplasm,whose overexpression is another protential mechanism for apoptosis resistance.However,its role in the apoptotic resitance and the relationship with Fas expression has not yet been studied well.Materials and methodsMaterials46 paraffin-embedded samples of SCLC(confirmed pathologically) during January 1998 to December 2002 were collected in the department of thoracic surgery, First Affiliated Hospital China Medical University.There were 26 male,20 female patients with an average age of 54(from 26 to 72) years,classified as 14 cases stageâ… , 11 casesâ…¡and 21 casesâ…¢according to 1997 UICC staging Criteria.20 paraffin samples of normal lung tissues were obtained.Then 16 fresh samples of SCLC during January 2004to june 2006 were collected.There were 9 male,7 female patients with age from 33 to 64 years.No cases were received rediotheropy or chemiotheropy before operation.The samples were transferred to liquid nitrogen and then reserved -70℃.Methods1.RT-PCR for Fas mRNA and FasL mRNAThe paraffin samples were treated and deparaffinaged.Total RNA of each sample was isolated by using Trizol Solution,then reverse transcription and PCR were progressed.Reaction system:MgCl₂ 2μl,10X RT Buffer 1μl,RNAse-free water 3.75μl, dNTP Mixture(10mM respectively) 1μl,RNase inhibitor 0.25μl,AMV Reverse Transcriptase 0.5μl,Random 9mers/Oligo dT-Adaptor Primer/Specific Downsteam Primer 0.5μl,RT was performed by the condition:42℃10min,99℃5min and 5℃5min.PCR amplification was performed by the condition:94℃2min,94℃40s,57℃40s,72℃1 min,one cyclye,total 40cycles and 72℃10min extension.The Fas-specific primers:F5'- CCAAATGCAGAAGATGATTGTGTG-3',R5'-TGCCACTGTTTCA GGATTTAAGGTTG3-3'.The FasL-specific primers:F5'-CTGGGGATGTTTC AGCTCTTC-3',R5'-CTTCACTCCAGAAAGCAGGAC-3'.Theβ-actin specific primers:F5'-ATCATGTTTGAGACCTTCAACA-3',R5'-CATCTCTTGCTCGAAG TCCA-3'.PCR products were resolved on a 2%polyacrylamide gels electrophoresis and the bands were visualized and then ascertained with the comparison to Markers (TaKaRa Co).2.Immunohistochemisty of SP method for Fas,FasL and Bcl-2The samples were deparaffinaged and dehydrated,3%H₂O₂ deactivation,antigen repair,preoxydase block,RM incubation,PBS washing.Then the animal-serum was dropwised,and antibody was tapped by biotin,incubated 37℃.The streptomycin-preoxydase was dropwised,RM incubated overnight.Then the samples were washed by PBS,DAB colorated,hematoxylin afterstained,HCL-alcohol differentiated,ladder alcohol dehydrated,xylene tranparentaed,and neutral resin blocked.The posisive were defined as brown to yellow staining in cytoplasm and nucleus.The positive cells were classified according to the number and color intensity of the cells(Monica semi-quantitation method).Statistics were performed with Fisher, Spearman,Kaplan-Meier,Log rank and Breslow by SPSS13.0.3.Flow cytometry to detect apoptosis of SCLC cellsThe tisues were resected and the liquid containing single cell was regulated by PBS into 1×10⁶cell/ml.FITC-Annexin V 5μl and PI liquid 5ml was added to one, 30min at RM in dark,and 400μl 1×Buffer was added,then FCM was performed immediately.Macintosh 9.5 Computer was used for data statistics.Before detection,the CV was regulated fewer than 3.0%.On the FCM picture;normal cells were distributed in R₁,lower-stained by FITC and PI(FITCPI),apoptotic cells in R₂ and FITC⁺PI^-,and dead cells were in R3 and FITC⁺ PI⁺(Figure 3-2).T-test,was used for data analysis by SPSS13.0.Results1.The positive rate of Fas mRNA expression in SCLC was 47.8%,significantly lower than that in normal lung tissue(80%),P＜0.05(χ²=5.907;P=0.015).2.The positive rate of FasL mRNA expression in SCLC was 58.7%;significantly lower than that in normal lung tissue(35%),P＜0.05(χ²=4.591;P=0.032).3.There was no significant correlation between positive Fas mRNA expression and sex,age,pathological subtype,lymph node metastasis.4.There was no significant correlation between positive Fas mRNA expression and sex,age,but there was significant correlation between positive Fas mRNA expression and lymph node metastasis and the TNM staging.5.Spearman correlation analysis showed that the expression of Fas mRNA and FasL mRNA in SCLC was negative correlated with that in normal lung tissue,and the correlation coefficient was r=-0.302,P=0.042.6.There was significant difference in Kaplan-Meiser survival curve of FasL mRNA positive group and FasL mRNA negative group of SCLC patients by Log-rank and Breslow test(χ²=7.596,P=0.006 in log-rank test;χ²=8.286,P=0.01 in Breslow test).7.The Fas,FasL and Bcl-2 protein were found in cytoplasm,partly on membrane as dipersed or adqulis yellow or brown granules.8.The positive expression of Fas protein in SCLC was 39.1%,significant lower than that in normal lung tissue(70%),P＜0.05;the positive expression of FasL protein in SCLC was 54.3%,significant higher than that in normal lung tissue(25%),P＜0.05; the positive expression of Bcl-2 protein in SCLC was 34.8%,significant higher than that in normal lung tissue(10%),P＜0.05.9.There was no significant correlation among the protein expression of Fas,FasL, Bcl-2 and sex,age,PTNM staging,lymph node metastasis or cigarette smoking.10.There was no significant correlation among the protein expression of Fas,FasL, Bcl-2 in SCLC by Spearsman correlation analysis.11.There was significant difference in Kaplan-Meiser survival curve of FasL protein positive group and negative group of SCLC patients by Log-rank and Breslow test(χ²=7.196,P=0,.007 in log-rank test;χ²=5.333,P=0.021 in Breslow test).12.There was no significant difference in Kaplan-Meiser survival curve of Bcl-2 and Fas protein positive group and negative group of SCLC patients by Log-rank and BreSlow test.13.The average ratio of early apoptosis in Fas mRNA positive cases of SCLC was (3.037±0.216)%,and the average ratio of early apoptosis in Fas mRNA negative cases of SCLC was(2.544±0.341)%with significant difference(t=3.321,P＜0.01).14.The average ratio of early apoptosis in FasL mRNA positive cases of SCLC was(2.382±0.356)%,and the average ratio Of early apoptosis in FasL mRNA negative cases of SCLC was(2.818±0.256)%with significant difference(t=2.607, P＜0.05).Conclusion1.The increased expression of Fas mRNA in normal lung tissue and decreased expression in SCLC.2.The decreased expression of FasL mRNA in normal lung tissue and increased expression in SCLC.3.The expression of FasL mRNA may correlate with the lymph node metastasis and survival time of SCLC.Lymph node metastasis is more frequent in the cases with positive expression of FasL mRNA and survival time is also significantly shortened.4.The decreased expression of Bcl-2 protein in in normal lung tissue and increased expression in SCLC.5.The negative expression of Fas mRNA and the positive expression of FasL mRNA have relationship with the apoptosis inhibition in SCLC.