Methylation Of ASC Promoter In NSCLC And Demethylation Agent 5-aza-CdR Inhibited The Proliferation Of Lung Cancer Cells In Vitro

IntroductionLung cancer is the primary cause of death in patients that suffered from cancer worldwide. Though the detection and therapeutic strategies of lung cancer have been greatly improved in the past 20 years, the total five - year survival rates were still less than 15% . What's more, there was almost no significant improvements in the survival rates of lung cancer in the past 20 years. Non - small cell lung carcinoma (NSCLC) is the most common type which accounts for 85% of the lung cancer. Due to its convenience to obtain patho - specimen, NSCLC was the type that has been in - depthly studied. To profoundly investigate NSCLC is essential for the improvement of long term survival rate in lung cancer patients, and is the focus of researches in recent years.It has been generally accepted that the alteration of oncogenes and tumor suppressor gene is the underlaying mechanism for tumorigenesis. Previous researches were mainly focused on gene structure and quantity, and it was presumed that chromosomal changes such as the mutation or depletion of certain gene might play a key role in the development of tumor. In Recent years, with the advent of postgenome epoch, epigenetics has attracted more and more attention. DNA methylation is the most widely investigated mechanism in epigenetics. Recent studies indicate that methylation of tumor related genes not only plays a prominent role in the formation of tumor, but also has the potential to become the molecular biological parameter for the early diagnosis of tumor and the target for tumor gene therapy.Methylation of tumor suppressor gene was the focus of tumor DNA methyla-tion researches. Methylation of tumor suppressor gene leads to the inactivity of tumor suppressor gene, which occurred in the promoter domain that abundantly contains CpG dinucleotide and links to the 5'- terminal of gene, and this domain was named CpG Island. Methylated DNA recruits methylium binding protein, attracts a chromatosome remodeling complex which is connected with proteins, then modifies histones by deacetylation, thus turns off gene transcription.To date, there is extensive investigation focus on the role of tumor suppress genes (TSGs ) methylation in the pathogenesis of lung cancer. TSGs methylation plays an important role in the pathogenesis of lung cancer, and might be an early event in the formation of lung cancer;what's more, TSGs methylation might be a biological marker with high degree of specificity and sensitivity for the early diagnosis of lung cancer and monitoring of therapeutic effects and prognosis;demethylation drugs made TSGs re - expression, and have the potential to be a new approach for the treatment of lung cancer.Recently, Masumoto and Conway isolated and identified a new gene ASC (apoptosis - associated speck - like protein containing a CARD) , also namely TMS1 (Target of methylation - inducing silencing) respectively, with different methods. ASC gene was located in 16pll. 2 - 12. 1, encoding a 22 kDa protein containing CARD °C aspase recruitment domain) with anti - apoptosis effects. It has been shown that there was gene silencing resulted from ASC gene methylation in tissue and cell lines from melanoma, colon cancer and ovarian cancer. ASC gene methylation was associated with the formation and development of the above tumors. There was little information about the exact role of ASC gene in lung cancer. There was only one paper concerning the role of ASC gene methylation in lung cancer. It was reported by Arvind in 2003 that ASC gene abnormal methylation can be observed in 48% NSCLC cell line and 40% NSCLC tumor tissue, and they thought ASC gene methylation might be related to the formation of NSCLC.Based on these reports, the present study was designed to investigate ASC gene abnormal methylation in NSCLC tissue, normal lung tissue besides tumor and NSCLC cell line and analyze the relationship between ASC gene abnormal methylation and NSCLC cell apoptosis;seek the role of ASC gene abnormalmethylation in the development of NSCLC, furthermore to provide a theoretical basement for taking ASC as a gene therapy target in demethylation drugs treatment of lung cancer.Materials and methods1. Material48 NSCLC frozen tumor tissues and corresponding normal tissue specimen were obtained NSCLC patients when they accepted chest surgery in our hospital during Oct 2004 to Mar 2005. After excision, the tumor was instantly preserved in hquid nitrogen and stored in - 80t refrigerator. We avoid collecting the liquefaction and necrosis portion of tumor tissue, and corresponding normal tissue was 5 cm faraway from tumor tissue. Among the 48 patients, 33 are male and 15 are female, aged 34 ~73 years old, with the average age 52.3 years old. Patho-histology showed 25 are squamous carcinoma, 20 are adenocarcinoma and 3 are alveolar cell carcinoma.70 paraffin wax specimen of tumor tissue used in the present study were from NSCLC patients when they accepted radical excision in our hospital during Jan 1999 to Aus 2000. Among the 70 patients, 53 are male and 17 are female, aged 33 ~71 years old. Pathohistology showed 39 are squamous carcinoma, 26 are adenocarcinoma and 5 are large cell carcinoma. All patients accepted radical excision including mediastinal lymph node clearance, and 59 accepted pulmonary lobectomy (including sleeve excision) , 11 accepted complete pneumonoresec-tion. All patients were followed over 5 years.Lung carcinoma cell line A2>calu3 and A549 were obtained from Academia Sinica Shanghai cell bank.2. AgentsXylene, saturate phenol, chloroform, protease K, absolute alcohol, sodium acetate, hydroquinone were purchased from Shanghai reagent factory. Ammonia ethanedioic acid, sodium hydrate, natrium bisulfurosum and agarose were from Sigma;TaqDNA pclymerase was from Takara;5 - aza - CdR and DMSO were from sigma;fetal bovine serum was from huamei;RPMI1640 and DMEMwere from Gibco. Primers were synthesized by Shanghai Bioengineering Corporation;Wizard DNA clean - up system was obtained from Promega. Etoposide (Vp-16, Lianyungang pharmaceutical factory);Cisplatin (DDP, Shangdong Dezhou pharmaceutical factory);adriamycin ( ADM, Shenzhen Main Luck Pharmaceuticals Incorporation).3. Experimental methods(l)cell cultureLung carcinoma cell line A2%calu3 and A549 were obtained from Academia Sinica Shanghai cell bank. A2 and A549 were grown in RPMI1640 medium supplemented with 10% (v/v) fetal bovine serum and incubated at 37T! in a humidified atmosphere of 5% CO2;calu3 was grown in DMEM medium under the above - mentioned conditions. Cells in logarithmic growth were used in the stud-y-(2) Specific PCR detection of methylationDNA extractionDNA was extracted by protease digestion and phenol/chloroform extract according to the routine methods described in Molecular Cloning (Second Edition ). The obtained DNA was quantified spectrophotometrically to guarantee the DNA purity between 1.7 and 1.8, and qualified DNA was stored in -20t refrigerator.MSP detectionMSP was used to detect the methylation state of ASC gene promoter. Extracted DNA genome wasvmodified by hydrosulfite. Primers for methylated ASC gene: forward 5 ' - CGATITrGGCGTnTrCGACGGTT - 3 ' , reverse 5 ' -CCGCTCACCCCG CTACAACCGC - 3 ';Primers for non - methylated ASC gene: forward 5' - TTGTT GGAGGGTAATGGATT - 3' , reverse: 5' - ATAC-CCACATAAAACACCC - 3 '. Reaction system was as follows: 20 yj reaction mixture containing 10 x PCR buffer 2 |xl, dNTP 2 yj, DMSO 2 pi, DNA 4 jxl ? Taq - E 0.2 |ji%dd H2O 9. 8 |xlxforward and reverse primer 2 jxl respectively. PCR was performed as follows: denaturation step at 95 °C for 2 min, followed by 38 cycles of 94*C for 40 s, annealing at 62*0 for non - methylated ASC gene and 65 *￡ for methylated ASC gene, extension at 70°C for 5 min. The PCR prod-uct was separated on a 2 % agarose gel and scanned with UVP system.(1)RT - PCR detectionRT - PCR was applied to detect ASC gene expression in lung carcinoma cell lines before and after demethylation reagents treatment.Total RNA was extracted from three lung carcinoma cell lines according to the routine methods described in Molecular Cloning ( Second Edition) before and after demethylation reagents treatment. After quantified spectrophotomet-rically for 260/280 value, RNA was stored in -lOX.RTTwo g total RNA was reverse - transcribed in a total volume of 20 1 reaction mixture containing 5 Reverse Transcriptase Buffer 4 1, dNTP Mixture (2. 5mM each) 8 1 x RNase Inhibitor (40u/u.l) 0. 5jxl* oligo ( dT) 18 Primer (50pmol/ jml) ljxlNAMV Reverse Transiptase (5u/(xl) 2jxl>DEPC - treated H2O 3. 5ui at room temperature for 10 min followed by 42 °C for 1 h and cooling in cold water for 2 min.PCRASC specific primers were forward 5' - TGGGCCTGCAGGAGATG -3'and reverse 5' - ATTTGGTGGGATTGCCAG - 3'. Two jud RT products were PCR amplified in reaction mixture containing TakaRa Ex Taq(5u/jxl) 0.1/xlx10 x Ex Taq Buffer 2jud,dNTP Mixture.( each 2.5mM) 16uJU3 ~ actin forward primer 0. 4jxUp - actin reverse primer 0. 4 ul> forward primer for ASC 0. 8 }jdN reverse primer for ASC 0. 8 jxlx sterile purified water 11. 9jju1o PCR was performed as follows;an initial denaturation step at 94 °C for 5 min, followed by 35 cycles of 941 for 30 s, 60T: for 30 s, 72X for 90 s, and a final extension at 12X for 7 min.Electrophoresis and analysisThe PCR product (10|xl) was separated on a 2 % agarose gel electrophoresis in 0.5 xTBE buffer, 100 V for 60 min. After staining with EBf the products were scanned with UVP system.(3)Flow cytometry detection of cell apoptosisCells in logarithmic growth were synchronized by culture in 1640 medium without supplement of serum for 24 h. 5 - aza - CdR with different final concen-trations were added to 1640 medium supplemented with FCS to treat the cells for 72 h as an intervention. Control group did not accept any treatment. After incubation , cells were washed twice with precooling PBS, and harvested 106cells/ml cell suspension. Binding buffer (200 jxl) were added, then Annexin - V (10ui) %PI (5 jxl) were added, and kept away from light at room temperature for 15 min. After reaction, 300 |xl binding buffer were added and FCM detection was performed within an hour.?MIT assay for cell inhibition rateCells in logarithmic growth were seeded onto a 96 well plate. After 18 h, media were replaced with fresh RPMI1640 containing different final concentrations of 5 - aza - CdR as an intervention. Control group did not accept 5 - aza -CdR treatment. Cells were further cultured for certain periods. After these treatment, 20 jxl MTT were added to each well and incubation for 4 h. Then the media was discarded carefully, and 150ui DMSO was added and shakes for 10 min. Absorbance at 490 nm were measured on the appearance within 1 h. Each experiment was performed triplicate and for 3 independent times. (§) Fluorescence microscope observation of cell apoptosis Clean coverslips were soaked in 70% ethanol for 5 min or longer. Cells were washed three times with PBS, and then washed with culture media once. Coverclips were placed in 6 - well plate, and cells were seeded and cultured o-vernight to reach 50% ~ 80% confluence. Cells were divided as control and intervention group. After cultured for 48 h, cells were washed with precooling PBS twice, and fixed with95% ethanol at 4t for 10 min, and then stained with PBS that contains 0.01% acridine orange and 100 mg/L PI for 30 min. Liquid that against fluorescence quench was applied to glass slide, which were then covered by coverslips with cells. 340 nm ultraviolet light were used to observe cells and then photographed.(^Transmission electron microscope observation of cell infrastructure Cells in logarithmic growth were suspensed, washed with PBS, centrifuged, fixed with glutaraldehyde, dehydrated by acetone and embedded. Semithin section with ljxm thickness was made with ultramicrotome;ultrathin section were stained with uranium and lead, and then for observation.Results1 Relationship between ASC gene promoter methylation and clinical features of NSCLCStudy on ASC gene methylation in 48 NSCLC frozen tumor tissues and corresponding normal tissue specimen indicate that the incidence of ASC gene promoter methylation were 47.9% (23/48) and 8. 3% (4/48) in NSCLC frozen tumor tissues and corresponding normal tissue specimen, respectively ( P < 0. 001). Normal tissue specimen that has ASC gene methylation was all from squa-mous carcinoma patients with a severe smoking history;and their corresponding tumor tissue has ASC gene methylation. The incidence of ASC gene methylation was significantly higher in tumor tissues of patients with a severe smoking history (p = 0. 0089, x2 = 6. 8363 ). The size of NSCLC is also associated with ASC gene methylation: ASC gene methylation was significantly higher in smaller tissues than in larer tumor tissues ( p = 0. 335 ,x2 =4. 5223 ). There was no relationship between ASC gene methylation with patients' age, sex, and pathological type of tumor or metastasis of lymph node.2. Relationship between ASC gene promoter methylation and prognosis of NSCLCWe performed MSP detection of 70 paraffin wax specimen from NSCLC patients with completed follow - up data and analyzed the relationship between ASC gene promoter methylation and prognosis of NSCLC,. It demonstrated that pathological stages, tumor size, metastasis of lymph node and ASC gene promoter methylation all have significant relationship with 5 year survival possibility. 5 year survival rate was significantly lower in patients with ASC gene promoter methylation (P =0.007, x2 =7.338). We further set a Cox ratio risk model to perform regression analysis: besides pathological stages and metastasis of lymph node, ASC gene promoter methylation was an independent prognostic factor(P = 0.009, regression coefficient - 1. 035, risk degree 0. 355,95% confidence interval 0.164 - 0.769).3. ASC gene promoter methylation in lung carcinoma cell lineMSP showed that only A2 cell line has significant ASC gene promoter methylation. There was no ASC gene promoter methylation in A549 and calu -3.4. Reverse of methylated ASC gene expression and induction apoptosis by 5- aza - CdR5 - aza - CdR was used to induce A2 cell line that has ASC gene promoter abnormal methylation. After 5 - aza - CdR treatment, RNA expression of ASC has reversed from nonexpression to re - expression. With the increase of 5 - aza- CdR concentration, the ASG gene expression had a tendency to enhance. FCM showed that 5 - aza - CdR induced marked apoptosis in A649%A2xcalu -3, no matter there is a ASC gene promoter abnormal methylation or not. There was no significant difference in the apoptosis rate between the 3 cell lines, no matter there is a ASC gene promoter abnormal methylation or not.5. Inhibitory effects of 5 - aza - CdR on cell proliferationMTT showed 5 - aza - CdR inhibited the proliferation of lung cancer cell in a time and dose dependent manner. When its concentration is 1. Oul, there was an inhibitory effect on cell growth and IC50 in 48 h was 4.20 0.12umol/L.6 Synergism of 5 - aza - CdR with chemotherapeuticsLD50 of Vp - 16, ADM and DDP on A2 cell were 0.25umol/Lx5. 0ug/mls 0.25ug/ml, respectively. Induction with lower concentration of 5 - aza - CdR for 24 h significantly reduced the survival rate of Vp - 16, ADM - treated cells by 25.3 ±2.6%and 27.1 ±3.1% Respectively;While did not significantly reduced the survival rate of DDP - treated cells.Conclusion1. There are frequent methylations of ASC gene promoter in the tumor tissue of NSCLC.2. The methylation of ASC gene promoter may be associated with the patho-genesis of smoke — induced lung squamous carcinoma.3. The methylation of ASC gene promoter might be an early event in the pathogenesis of NSCLC.4. The methylation of ASC gene promoter is an independent prognostic fac-tor for NSCLC patients.5. There was depletion of ASC gene expression resulted from methylation in lung cancer cell line.6. Mathylated ASC gene can be reversed to re - expess by 5 - aza - CdR induction.1.5- aza. - CdR can induce apoptosis in lung cancer cell lines.8. 5 - aza - CdR induced apoptosis was not only due to ASC gene demethy-lation.9. 5 - aza - CdR has significant inhibitory effects on the proliferation of A2 cell line.10. 5 - aza - CdR and Vp - 16, ADM have synergism in inhibition the proliferation of A2 cell line...