Effect of 5-aza-2’-deoxycytidine on Cell Proliferation of Non-small Cell Lung Cancer Cell Line A549 Cells and Expression of the TFPI-2 Gene

Objective: The present study employed 5-aza-2’-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene. Methods: Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 μmol/L 5-Aza-CdR, a specific demethylating agent, for 24 ,48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression. Results: MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 μmol/L 5-Aza-CdR). After treatment with 0, 1, 5, 10 μmol/L 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was 69.7±0.99%, 76.1±0.83%, 83.8±0.35%, 95.5±0.55% respectively ( P <0.05), and the proportion in S was 29.8±0.43%, 23.7±0.96%, 15.7±0.75%, 1.73±0.45%, respectively ( P <0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 μmol/L 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 μmol/L 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were 1±0, 1.49±0.14, 1.86±0.09 and 5.80±0.15 ( P <0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were 0.12±0.01, 0.23±0.02, 0.31±0.02, 0.62±0.03 ( P <0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration. Conclusions: TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.


Introduction
Lung cancer remains the leading cause of cancer deaths worldwide, More than 80% of lung cancers are NSCLC (Jemal et al., 2009). Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15% (Sculier et al., 2008;Carvalho et al., 2009). In stage I to III NSCLC, the occurrence of extrathoracic metastasis also leads to a poor 5-year survival rate of 50% (Friedel et al., 2004). Surgery is the best curative therapeutic approach in the early stages. However, even in these patients, the mean 5-year overall survival rate is less than 70%. The scarcity of effective tools for early detection and therapy strategies is the reason of the poor 5-year overall survival rate. Consequently, developing molecular markers for early detection, predicting prognosis, and exploiting new therapy agents of lung cancer are urgently needed.
It has shown that the occurrence of NSCLC is a multigene-involved, multi-stage pathological process under the action of both genetic and epigenetic factors including DNA methylation, and that epigenetic mechanism plays an important role in the occurrence and development of NSCLC (Fischer et al., 2006;Basseres et al., 2012;Dmitriev et al., 2012;Nelson et al., 2012). Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin which activates matrix metalloproteinases (MMPs) involved in degradation of the extracellular matrix. Its secretion in the tumor microenvironment makes TFPI-2 as potential inhibitor of tumor invasion and metastasis (Gaud et al., 2011). The gene that codes for TFPI-2 has been mapped to chromosome 7q22 by fluorescence in situ hybridization (Kempaiah et al., 2007). The expression of TFPI-2 gene in tumors is inversely related to an increasing degree of malignancy, which may suggest a role for TFPI-2 in the maintenance of tumor stability and inhibition of the growth of neoplasms, so, TFPI-2 is considered as a candidate tumor suppressor gene (Sierko et al., 2007). Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers was widely documented to be responsible for diminished expression of mRNA encoding TFPI-2 and decreased or inhibited synthesis of TFPI-2 protein during cancer progression and invasion (Rao et al., 2001;Hube et al., 2003;Jee et al., 2009;Ribarska et al., 2010;Takada et al., 2010;Tang et al., 2011). Abnormal methylation is reversible, demathylating agents could be used to induce re-expression of the inactivated genes due to methylation. 5-Aza-CdR is a specific inhibitor of DNA methyltransferase (DNMTs), which induces the reexpression of tumor suppressor genes by demethylating promoter CpG island (Ding et al., 2012;Jin et al., 2012).
In the present study, the lung cancer cell line A549 were treated in vitro with demethylating agent 5-Aza-CdR so as to investigate the changes in methylation status and expression of TFPI-2 gene after 5-Aza-CdR treatment, the results will provide the experimental evidences for the clinical therapy.

Cell culture
A549 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin and 100 mg/ml streptomycin at 37℃ in a humidified incubator with 5% CO 2 . At the logarithmic phase of cell growth, with a percentage of viable cells over 95%, the cells were remained hungry for 24 h in the RPMI 1640 medium containing 1% fetal bovine serum, followed by the drug treatment. Cells were treated with 0, 1, 5, 10 μmol/L 5-Aza-CdR, a specific demethylating agent for 24 to 72 h. Cells were harvested for further analysis. The cells which were treated with 0 μmol/L 5-Aza-CdR were used as the control group.

MTT Assay
A549 cells were trypsinized and seeded in 96-well plates at a density of 1×10 4 cells/well and cultured for 24 h. And then, 0, 1, 5, 10 μmol/L 5-Aza-CdR was added and the plates were incubated at 37℃, 5% CO 2 for 24, 48, 72h. At the end of incubation, 20 μL of 5 mg/mL MTT was added into each well followed by incubation for another 4 h. The medium was then aspirated and 150 μL dimethylsulfoxide (DMSO) was added into each well. The plates were mixed gently by rocking back and forth until the blue sedimentation was completely dissolved. optical density (OD) was measured at 490 nm using an ELIASA and the growth inhibition rate was calculated using the following formula: Inhibition rate(%)=(Average OD value of experimental group-Average OD value of control group)/Average OD value of control groupx100%. Each experiment was repeated three times.

FIow Cytometer
Cell cycle distribution was analyzed using propidium iodide staining.CelIs were seeded at 1×10 6 cells/well in 6-well plates.and exposed to 0, 1, 5, 10×10 -6 μmol/L 5-Aza-CdR for 72 h. The cells were harvested and washed twice with PBS, then fixed with ice-cold 70% ethanol. The sample was concentrated by removing ethanol and re-suspended in a PBS solution containing propidium iodide (0.05g/L) and RNaseA (100 mg/ml) for 3h at 37℃ in the dark. The samples were then measured by flow cytometry.Cell division activity was measured by proliferation index(PI), PI=(S+G2/M)/(G0/G1+S+G2/M). Each sample was repeated three times.
Methylation-specific polymerase chain reaction( MSPCR). Genomic DNA was obtained from cultured cells using standard phenol-chloroform protocols. The DNAs were treated with sodium bisulfite. Total reaction mixture volume of 25 μL, containing 2 μL DNA, 2.5 μL10 × PCR buffer, 0.5 μL each of the sense and antisense primers, 2 μL dNTPs, 0.2 μL Taq enzyme, and 17.3 μL double-distilled water. Reaction condition: pre-denatured at 94℃ for 4 min, followed by 35 cycles of 94℃ for 30 s, 56℃ for 30 s, and 72℃ for 30 s, and with a final extension at 72℃ for 5 min. The methyltransferase SssItreated and untreated placenta DNA were used as the positive and negative controls respectively, while the double-distilled water as the blank control. The 1000 bp DL2000 was used as the molecular weight markers. The primer set used for methylation analysis of TFPI-2 was 5'-TTTATGTTTTTAAGAGGTGGATTTC-3' (sense) and 5'-TCTATCCTCCAGCAAGCATCGT-3' (antisense).The PCR products were separated by electrophoresis on the 2% agarosegel at 4V for 1min. The gels were photographed using a laser density scanner (Pharmacia LKB Ultroscan) for analysis. Each experiment was repeated three times.
Real-time polymerase chain reaction (real-time PCR). The TFPI-2 mRNA expression was determined with real-time PCR. The total RNA was extracted from A549 cells using Trizol method according to the manufacturer`s instruction. The cDNAs were synthesized from the templates in presence of reverse transcriptase and oligo (dT) 20 primers, As the internal control, β-actin transcripts were amplified from the same cDNA samples. The Primer sequences and condi¬tions for the real-time PCR product were previously described (forward primer: 5'-TTCTCCGTTACTACTACGACAGG-3'; reverse 5-aza-2'-deoxycytidine Effects on Proliferation of NSCLC A549 Cells and TFPI-2 Gene Expression primer: 5'-TCTATCCTCCAGCAAGCATCGT-3'. The housekeeping gene β-actin was used as an internal control for the real-time PCR (forward primer: 5'-GTCCACCGCAAATGCTTCTA-3'; reverse primer: 5'-TGCTGTCACCTTCACCGTTC-3'). 25 µL PCR reaction mixture was pre-denatured at 95℃ for 1 min, and 40 cycles of 94℃ for 15 s, 58℃ for 20 s, and 72℃ for 20s were performed with a final extension at 72℃ for 5 min. SYBR green was used for the and each sample was repeated for 3 times. Comparative delta-delta Ct method was used for final result calculation: rCt=Ct TFPI-2 −Ct β-actin , rrCt=rCt experiment group −rCt control group , the relative mRNA level =2 -rrCt . Each experiment was repeated three times.

Western blotting
100 μL of cell lysis buffer was mixed with 40 μL harvested cells to isolate the total cellular protein, and then quantitated by the Bradford method, 100 μL of the total protein was denaturated for 10 min, and 25 μL was taken for the 10% polyacrylamide gel electrophoresis. The proteins separated by the electrophoresis were then transferred onto the PVDF membrane, and the 10% skim milk used to block the PVDF membrane at room temperature for 1 h. The PVDF membranes were incubated in the 1:200 dilution of the primary antibody at 4℃ overnight, and then in the 1:2000 diluted alkaline phosphatase-labeled secondary antibody solution at 37℃ for 2 h, followed by the DAB coloration. The PVDF membranes were photographed using a ChemiImager 5500 AlPhaInn Ch, and the subsequent data acquisition and mining were conducted with the Fluorchem V2.0 system. Taking the expression level of β-actin protein as the internal control, the ratio of scanning density of TFPI-2 protein to that of β-actin was finally calculated and used as the analyzing index to further investigate the changes in protein expression of TFPI-2 and compare them between different experimental groups. Each experiment was repeated three times.

Statistical analysis
Statistical analyses were performed using the SPSS13.0 software package. The results were expressed as mean ± standard deviation (SD). Continuous variables were compared by the analysis of variance (ANOVA), and multiple comparisons were analyzed by the LSD. A P-value of 0.05 or less was considered significant.

Discussion
Multiple genetic and/or epigenetic changes including DNA methylation, has been suggested to contribute to development and progression of human cancers including lung cancer. Epigenetic alterations which result in aberrant gene expression without any concomitant change in DNA sequence, is a rapidly expanding field that focuses on stable changes in gene expression (Boumber and Issa, 2011). Epigenetic changes have been shown to play important roles in determining when and where as well as whether a gene would express or silence. DNA methylation and the modification of histones including acetylation and deacetylation are important components of epigenetics, however, the most common modification is DNA methylation, which is considered as the best-characterized and most easily quantifiable epigenetic mechanism underlying gene expression or silencing (Li et al., 2012). DNA methylation is a type of covalent modification in which a methyl group is added to a cytosine in the genome via S-adenosylmethionine. This process occurs as an enzymatic reaction after DNA replication (Zuo et al., 2009). In mammalian cells, methylation of DNA is typically restricted to the 5-position of the pyrimidine ring of cytosine residues that are located in CpG dinucleotides (Goel, 2010). DNMTs are important for methylation, When DNMT1 is removed, the level of methylation of the whole genome is reduced by 3%, and when DNMT3 is removed, it is reduced by 4%. When they are both removed, the level is lowered by 98% (Gopalakrishnan et al., 2008).
TFPI-2 is a 32 kDa Kunitz-type serine proteinase inhibitor ,which is a potential inhibitor of the plasmin within the extracellular matrix, was reported to be Asian Pacific Journal of Cancer Prevention, Vol 14, 2013 4425 DOI:http://dx.doi.org/10.7314/APJCP.2013.14.7.4421 5-aza-2'-deoxycytidine Effects on Proliferation of NSCLC A549 Cells and TFPI-2 Gene Expression frequently methylated in various other cancers as well as in lung cancer (Glöckner et al., 2009;Takadaa et al., 2010;Wang et al., 2010). Silencing of the TFPI-2 gene by hypermethylation might contribute to tumor progression in NSCLC (Rollin et al., 2005). Methylation of TFPI-2 gene is an independent factor for an unfavorable prognosis in patients with NSCLC (Wu et al., 2012). Downregulation of TFPI-2 gene because of methylation could promote the migration and invasion abilities in lung cancer cell lines (Lakka et al., 2000). 5-Aza-CdR, a nucleoside analogue, could incorporate into DNA and exert direct cytotoxic and anti-proliferative effects on tumor cells. However, to our knowledge, there are few studies on effect of 5-aza-2'-deoxycytidine on TFPI-2 gene in non-small cell lung cancer A549 cell line.
In our study, hypermethylation in the promoter region of TFPI-2 gene was detected in A549 cells which were not treated with 5-Aza-CdR, and only partial demethylation of the TFPI-2 gene promoter was detected in A549 cells after 1, 5 μmol/L 5-Aza-CdR treatment, and unmethylation of the TFPI-2 gene promoter was detected in A549 cells after 10 μmol/L 5-Aza-CdR treatment. A549 cells treated with 5-Aza-CdR revealed demethylation of the TFPI-2 gene. The expression of TFPI-2 gene mRNA and protein were very weak in A549 cells which were not treated with 5-Aza-CdR, however, it were enhanced considerably after 5-Aza-CdR treatment.This result suggested that the weak expression of TFPI-2 gene in A549 cells may be due to the hypermethylation of CpG islands in the promoter of TFPI-2 gene. It was found the proliferation of A549 cells was inhibited significantly after various 5-Aza-CdR concentration treatment, 5-Aza-CdR significantly increased inhibition rate of proliferation of A549 cells with increasing 5-Aza-CdR concentration and delaying treatment time. The cell cycle was arrested in the G0/G1. The results indicated the anti-tumor role of 5-Aza-CdR. According to the mechanism of 5-Aza-CdR, this drug could induce the re-expression of certain silencing genes by demethylation, therefore, recover the function of some tumor suppressor genes. This suggested that the inhibitory role of 5-Aza-CdR in A549 cells had a close correlation with the re-expression of TFPI-2 gene.
So, aberrant hypermethylation of promoter regions is an alternative mechanism for inactivation of tumor suppressor genes. 5-Aza-CdR could efficiently reverse the expression of tumor suppressor gene silencing by DNA hypermethylation, which provided a new pathway for the early diagnosis and treatment of the lung cancer (Dolle et al., 2006). These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with demethylation agents 5-Aza-CdR. TFPI-2 will be one of the molecular markers for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.