Study On Expression And Promoter Methylation Of APAF-1 Gene In Non-Small Cell Lung Cancer And Effects Of 5-Aza-2'-deoxycytidine On Proliferation Of Human Lung Adenocarcinoma Cell Lines

ObjectiveLung caner was one of the most common malignant tumor, in our country the incidence was increasingly raise. Non — small cell lung cancer ( NSCLC ) accounted for approximately 75% ~ 80% of the lung cancer cases and lots of genes assotiated with NSCLC were found such as ras gene, myc gene, erbB gene, P53 , Rb. Apoptosis which closely associated with oncogenesis is the major reason for the resistance to the chemotherapy and side effect caused by chemotherapy or radio therapy. Abnormal expression of apoptosis associated gene ,such as BCL -2,BAX,P53, may play an important role in the progression of NSCLC. APAF -1 (apoptotic protease activating factor - 1) gene locus at 12q23 is a key factor in cytochrome C - dependent apoptotic pathway. APAF - 1 gene mutations were seldom , therefor the mechanism for abnormal gene function includes LOH , Promoter Methylation . "Gene silence"caused by promoter methylation is the major reason. Study on expression and promoter methylation of APAF - 1 Gene in NSCLC and explore the correlation of APAF - 1 Gene with NSCLC.MethodsSamplesAll lung cancer tissue and matched normal tissues confirmed pathologically were obtained from Iiaoning Province Tumor Hospital. 45 NSCLC cases aged from 42 ~68 including male 39 cases,female 6 cases;squamous carcinoma 18 cases , adenocarcinoma 23 cases, adenosquamouscarcinoma 4 cases;TNM staging:I —>-H30 cases, HI—>-!Vl5 cases;lymph node metastasis( + ) 24 cases, lymph node metastasis ( - ) 21 cases. After resected tumor tissue and normal lung tissue were conserved in -70t refigerabo immediately.1. ImmunohistochemistryNormal lung tissues and NSCLC tissues were fixed with 10% formalin embedded in paraffin. A standard avidin - biotin complex method with alkaline phosphatase detection was carried out,then statistical analysis was implemented according to the stain in the cytoplasm.2. Expression of APAF - 1 mRNATotal RNA were refined by TRIZOL,then cDNA was synthesized by Olingo (dT) 20 primer. RT - PCR primer sequence was designed by Primer3. The production included CARD(caspase recruitment domain) was separated by 1.5% agarose, then images were taken with Alphalmage 2000 auto - imaging apparatus and analyzed by Fluorchem v2. 0 Stand Alone software.3. Methylation - specific PCR (MSP) analysis of the APAF - 1 promoterMethylation and unmethylation specific primers were selected based upon the sequences identified by the computer software CpG Island Searcher. Totally 1 g DNA was denatured by NaOH and treated with 3M sodium bisulfite, 10 mM hydroquinbne for 16 hours at 50^1. The modified DNA,which was purified using Wizard DNA purification system ,was used for MSP assays. The expected PCR products were visualized by 2.0% agarose gel with ethidium bromide staining.4. Statistics analysisSPSS10.0 software was used in Statistics analysis, when P <0. 05 there was statistic significance.Results1. ImmunohistochemistryObserved with Light microscope: APAF -1 protein mainly localized in normal lung tissue cell plasm, stain in light yellow totan. In contrast to normal lungtissues the expression oiAPAF - 1 in NSCLC obviously decreased.2. Expression oiAPAF - 1 mRNA in NSCLCSemi - quantitative RT - PCR analysis of APAF — 1 mRNA showed that 60% (27/45 ) of NSCLC were down - regulation including 15 cases which showed no APAF - 1 mRNA. Compared with normal lung tissues, there was statistical significance( P < 0. 05 ).3. The relationship of APAF -1 gene expression and NSCLC clinical pathological characterThere was no relationship between APAF - 1 gene expression and sex, histo-logic subtype. There was obvious association between APAF - 1 gene expression and lymph node metastases, which showed relationship between APAF - 1 gene expression and NSCLC patients prognosis.4. Methylation analysis of APAF - 1 gene promoter;MSP analysis displayed 19 cases of APAF - 1 gene methylation in 27 cases with obvious down - regulation expression of APAF - 1 , though 5 cases methylation were found in 18 .cases in which the expression of APAF - 1 gene had no obvious change. The difference of the two groups had statistical significance ( P < 0.05). No methylation was found in 45 normal lung tissues.Conclusion1. APAF - 1 participated in the pathogenesis of NSCLC gene expression was down -regulated in NSCLC.2. There was relationship between APAF - 1 gene expression and NSCLC patients prognosis.3. Methylation of promoter regions plays an important role in down regulation of APAF - 1 gene in NSCLC.ObjectiveHuman lung adenocarcinoma associated with lots of genes inactivation, especially APAF — 1 ( apoptotic protease activating factor — 1) gene which induced apoptosis as tumor surpress gene. A major mechanism of APAF - 1 gene inactivation was abnormal methylation in gene promoter domain which had closely association with up -regulated methyltransferase. 5 - Aza -2'-deoxycytidine, as a well - established inhibitor of DNA methylation, could decreased the expression of methyltransf erase, recovered "silence gene" transcription activity. So 5 - Aza - 2' - deoxycytidine was used to treat human lung adenocarcinoma cell line SPC — A — 1, afer observing the change of malignant biology behaviour and analysising its mechanism, we discussed the mechanism of lung cancer occur-rency as well as quesed new therapeutic method.Methods1. Main reagentHuman lung adenocarcinoma cell line SPC - A - 1 were purchased from Shanghai Cell laboratory,5 -Aza -2'-deoxycytidine was purchased from Sigma company, TRIZO1 reagent and Taq DNA polymerase, transcription kit were purchased from Promega company. APAF - 1 - IgG was purchased from Santa Cruz Company and other reagents from sigma company.2. Human lung adenocarcinoma cell line culture, treatment with 5 -Aza - 2- deoxycytidineLogarithm growth period cells were applied in this study. All cell cultures were carried out in RPMI1640 medium, supplemented with 10% fetal calf serumand lOOU/ml benzylpenicillin ,100U/ml streptaquaine at 37°C in a 5% CO2 atmosphere. After 48 hours,cells were added 5 - Aza -2'- deoxycytidine to the end concentration 5 x 10~7mol/L . After 5 - Aza - 2'- deoxycytidine effection 24 hours,cells were exposured in ultraviolet rays 3 minutes,then were cultured 36 hours.3. Western - blotting detect APAF -1 protein expression4. Expression of APAF - 1 mRNA, methyltransferase mRNATotal RNA were refined by TRIZOL, then cDNA was synthesized by Olingo (dT) 20 primer. RT - PCR primer sequence was designed by Primer3. The production was separated by 1.5% agarose,then images were taken with Alphalm-age 2000 auto - imaging apparatus and analyzed by Fluorchem v2. 0 Stand Alone software.5. Cell cycle distribution, apoptotic rate were observed under flow cytometerTo calculate the percentage of apoptosis, Flow cyto - meter assay were performed according to the recommend of Anexin V kit. Briefly, SPC - Al cells, which were treated with different concentration 5 - Aza — 2' — deoxycytidine, were diluted to 1 x 10 cells/ml with PBS, then cells were washed with PBS and centrifugated in 1,000rpm at 4T) for 10 min. Subsequence, cells were stained by PI for 15 min and detected by Flow cytometer. The data of apoptotic cells were averaged from three times of experiments use for further analysis.6. Statistics analysisSPSS10. 0 software was used in Statistics analysis, when P <0. 05 there was statistic significance.Results:1. Expression of APAF -1 protein in NSCLC cell line After treatment with 5 - Aza - 2'- deoxycytidineResults of Western - blot after treatment with 5 - Aza -1'- deoxycytidinethe expression of APAF -1 protein was obviously increased in NSCLC cell lines.2. Expression of APAF - 1 mRNA and DNMT mRNA after treatment with 5 - Aza -2'- deoxycytidineAfter treatment with 5 - Aza - 2'- deoxycytidine the expression of DNMT mRNA was obviously decreased,whereas the expression oiAPAF - 1 mRNA was increased.3. After treatment with 5 - Aza -2'- deoxycytidine NSCLC cell line cell cycle ,apoptosis rate changeSPC - A - 1 cells after treatment with 5 - Aza -2'- deoxycytidine ,The expression of APAF - 1 mRNA and protein increased, but the expression of meth-yltransferase mRNA decreased in cell lines. SPC - A - 1 cells displayed a slowed growth. The percentage of cells in G0/G1 increased to (80. 2 ±2. 88) % , the percentage of cells in S reduced to (6.4 ±0. 62) % , the percentage of cells in G2 + M increased to (13.4 ±0. 93 ) % , and apoptotic rate increased to (13.5 ±0.97)%.Conclusion1. Pulmonary carcinoma is associated with alternation of DNA methylation.2. 5 - Aza - 2'- deoxycytidine, as a well - established inhibitor of DNA methylation, may slow the growth of tumor cells by inhibited DNA methyltrans-ferase and reactivating growth - regulatory genes silenced by de novo methylation and genes silenced by promoter hypermathylation ( such as tumor suppressor APAF - 1 gene.) .