Antiapoptosis Gene, Livin Expression In Esophegeal Carcinoma And Its Correlation With P₅₃ And Bcl-2 Expression, Cell Proliferation And Apoptosis

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: c-IAP 1, c-IAP2, XIAP, NAIP, ILP-2, Bruce, survivin, and Livin. 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 Livih 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 4 a helix 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 esophageal carcinoma. Therefore, the expression of LivinαmRNA and LivinβmRNA in 36 esophageal carcinoma tissues and 18 para-cancerous tissues was measured by reverse transcription polymerase chain reaction (RT-PCR) combined with silver staining technique, Western blot method was used to studay Livin protein in these tissues. Immunohistochemical SP method was used to detect the expression of Livin, P₅₃ and Bcl-2 proteins in these tissues. We used flow cytometry to detect the level of esophageal carcinoma 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 April 2005 to October 2005 were collected in the department of thoracic surgery, First Affiliated Hospital China Medical University. 36 esophageal carcinoma tissues and 18 para-cancerous tissues were obtained. There were 33 male, 3 female patients with an average age of 56.9(from 41 to 81)years. According to the differentiation degree, 36 cases were classified as highly differentiated (15cases), moderately, differentiated(18cases) and lowly diferentiated (3cases). According to pathological subtype, there were 35 cases of squamous cell carcinoma and 1 case of adenocarcinoma. There were 18 cases of lymph node metastasis in 36 cases of esophageal carcinoma. No cases were received rediotheropy or chemiotheropy before operation.Methods1. RT-PCR for Livin mRNA Total 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,MgCl₂2μl,Random 9 mers 0.5μl, RNA 1μl,RNase inhibitor 0.25μl,Reverse Transcriptase 0.5μl, ddH₂O 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: ddH₂O 7.16μl, 20pmol/L each of 5' and 3' primers 0.15μl, Taq Hs 0.04βl 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 1 min, then extention at 72℃for 7min. The efficiency were detected byβ-actin. PCR products were loaded onto a 8% polyacrylamide gels electrophoresis and the bands were visualized by silver staining, 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 muiticlonal anti-Livin (1: 400, BIOCHEMICALS, 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; P₅₃ and Bcl-2 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: 200), mouse monoclonal antiboldy against P₅₃ and mouse anti-human Bcl-2, and 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 control for staining was obtained from the sections of gastric cancer. The posisive 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 ceils. 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 5rag. Rnase 2mg. TritonX-100 1ml, normal saline 65ml, sodium citrate 100rag, add distilled water to100ml) 1ml was added to one, 30min at 4℃in dark, and FITC-Annexin V and PIeach 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 data of quantity. T-test was used in data of quality. The data was analYzed by the SPSS 13.0 software.Results1. The expression of Livin geneThe positive rate of Livin gene was 50.0 % in 36 esophageal carcinoma tissues, but Livin gene expressed low levels in 18 para-cancerous tissues, its positive rate was 5.6%(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 clinicopathological factorsThere was no significant correlation between positive Livin gene expression and age, sex, histological subtype (P＞0.05). The expression of Livin gene was closely related to infiltration degree of carcinoma tissues, lymph node metastasis and TNM staging (P＜0.05).3. The expression of Livin protein and the relationship of Livin, P₅₃ and Bcl-2 proteins in esophageal carcinomaThe positive rate of Livin protein was 47.2 %in 36 esophageal carcinoma tissues, but Livin protein expressed low levels in 18 para-cancerous tissues, its positive rate was 5.6 % (P＜0.05).The ration of positive expression of Livin was 52.94% (9/17) among the p₅₃ protein positive expression and the ration of positive expression of Livin was 42,11% (8/19) among the P₅₃ protein negative expression (P＞0.05). The ration of positive expression of Livin was 64.00% (16/25) among the Bcl-2 protein positive expression and the ration of positive expression of Livin was 9.09% (1/11) among the Bcl-2 protein negative expression (P＜0.01, r=0.452)4. Correlation between the expression of Livin gene and tumor cell proliferation in esophageal carcinoma tissuesThe average proliferation ratio in 36 esophageal carcinoma tissues was (29.27±5.72) %. According to RT-PCR and Western-blot results, the average PI of 18 cases with positive Livin expression and 18 cases with negative expression were respectively (31.55±6.19% and (27.00±4.26)%, with significant deference (t=2.566, P＜0.05). According to Immunohistochemisty result, the average PI in 17 cases with positive Livin protein expression and 19 cases with negative expression were respectively (32.22±5.66)% and (26.64±4.43)%, with significant deference (t=3.310, P＜0.01).5. Correlation between the expression of Livin gene and cell apoptosis in esophageal carcinoma tissues The average ratio of early apoptosis in 36 esophageal carcinoma tissues was (2.43±0.91)%. According to RT-PCR and Western-blot results, the average apoptosis ratio of 18 cases with positive Livin expression and 18 cases with negvative expression were respectively (2.08±0.95)% and (2.78±0.73)% , with significant deference (t=2.472, P＜0.05). According to Immunohistochemisty result, the average apoptosis ratio of 17 caseswith positive Livin protein expression and 19 cases with negative expression were respectively (1.91±0.63)% and (2.89±0.87)%, with significant deference (t=3.837, p＜0.01).Conclusion1. Livin gene is highly expressed in esophageal carcinoma tissues, but shows little expression in para-cancerous tissues.2. There was no significant correlation between Livin gene expression and age, sex, histological subtype, but the expression of Livin gene was closely related to infiltration degree of carcinoma tissues, lymph node metastasis and TNM staging.3. The expression of Livin protein was not related to the expression of P53 protein, but the expression of Livin protein was positively related to the expression of Bcl-2 protein.4. The expression of Livin gene in esophageal carcinoma tissues had correlation with cell apoptosis.5. The expression of Livin gene in esophageal carcinoma tissues was correlated with tumor cell proliferation.