Wang HL, Bai H, Li Y, Sun J, Wang XQ. Rationales for expression and altered expression of apoptotic protease activating factor-1 gene in gastric cancer. World J Gastroenterol 2007; 13(38): 5060-5064 [PMID: 17876870 DOI: 10.3748/wjg.v13.i38.5060]
Corresponding Author of This Article
Yan Li, Department of Gastroenterology, Shengjing Hospital Affiliated to China Medical University, Shenyang110004, Liaoning Province, China. yanli0227@126.com
Article-Type of This Article
Gastric Cancer
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He-Ling Wang, Yan Li, Jun Sun, Xue-Qing Wang, Department of Gastroenterology, Shengjing Hospital Affiliated to China Medical University, Shenyang 110004, Liaoning Province, China
Han Bai, Department of Infectious Diseases, Shengjing Hospital Affiliated to China Medical University, Shengyang 110004, Liaoning Province, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Correspondence to: Yan Li, Department of Gastroenterology, Shengjing Hospital Affiliated to China Medical University, Shenyang110004, Liaoning Province, China. yanli0227@126.com
Telephone: +86-24-83956986 Fax: +86-24-23582697
Received: May 20, 2007 Revised: June 18, 2007 Accepted: June 26, 2007 Published online: October 14, 2007
Abstract
AIM: To elucidate the relationship between apoptotic protease activating factor-1 (Apaf-1) gene and gastric cancer.
METHODS: Thirty-five postoperative cancer and adjacent normal tissue samples were collected in the present study. Expression of the Apaf-1 gene in these samples was analyzed by semi-quantitative RT-PCR. Loss of heterozygosity (LOH) was used to determine whether there was loss of Apaf-1 gene in domain of 12q22-23 in the samples. Promoter methylation of Apaf-1 gene in the samples was analyzed by methylation specific (MSP) PCR.
RESULTS: The expression of Apaf-1 mRNA in gastric cancer tissue samples was 51%. The LOH frequency of D12S346, D12S1706, D12S327, D12S1657 and D12S393 was 33%, 8%, 58%, 12% and 42%, respectively. Fifty percent LOH was found at two sites and 17% LOH at three sites. Apaf-1 mRNA expression decreased significantly in 13 cases (rs = 0.487, P = 0.003). The rate of Apaf-1 promoter methylation was 49% in gastric cancer tissue samples and 23% in para-cancerous tissue samples. Promoter methylation occurred significantly in 16 of 18 gastric cancer tissue samples with decreased expression of Apaf-1 mRNA rs = 0.886, P = 10-6).
CONCLUSION: The expression of Apaf-1 gene is low in gastric cancer tissues. Methylation of Apaf-1 gene promoter and LOH in domain of 12q22-23 are the main reasons for the expression and altered expression of Apaf-1 gene.
Citation: Wang HL, Bai H, Li Y, Sun J, Wang XQ. Rationales for expression and altered expression of apoptotic protease activating factor-1 gene in gastric cancer. World J Gastroenterol 2007; 13(38): 5060-5064
Formation and progression of gastric cancer are a continuous multiple-step process. Cell apoptosis is one of the main mechanisms for tumor genesis[1-3], and Apaf-1 protein is an important apoptosis factor which is expressed abnormally in a series of tumor studies[4-7]. It was recently reported that Apaf-1 gene is closely related to several cancer-inducing genes and tumor suppressor genes, such as p53 and Bcl2[8-10]. However, the expression condition of Apaf-1 gene in gastric cancer and its correlation with the genesis of gastric cancer remain unclear. This study was to study the relation between Apaf-1 gene expression and gastric cancer genesis in promoter domain.
MATERIALS AND METHODS
Materials
Thirty-five postoperative cancer tissue and adjacent normal tissue samples (> 5 cm away from the center of cancer) were collected from gastric cancer patients from January 2005 to September 2005 at No.1 and No.2 Affiliated Hospitals of China Medical University. All these patients were pathologically diagnosed according to the new TNM classification criteria of Union Internationale Contre le Cancer[11]. Of the 35 patients with progressive gastric cancer (25 males and 10 females with a mean age of 58.51 ± 12.24 years), 10 had moderately differentiated gastric cancer and 25 had poorly-differentiated gastric cancer, while 25 had lymphatic metastasis and 10 had no lymphatic metastasis. Tumor and adjacent normal tissues were isolated and maintained at -70°C. Total RNA extracting reagent Trizol (Invitrogen Co.), RT-PCR kit (TaKaRa Co.), methylating reagent (Sigma Co.) and Wizard DNA clean-up (Promega Co.) were used in the study.
Semi-quantitative RT-PCR analysis
Total RNA was extracted with Trizol reagent and cDNA was synthesized with reverse transcriptase and Oliga (dT)20 primer. Apaf-1 primer sequence was designed with primer 3, including functional caspase recruitment domain (CARD), upstream primer (5'-TTGCTGCCCTTCTCC ATG AT-3'), downstream primer (5'-TCCCAACTGAAA CCCAATGC-3') and amplification length (334 bp). The internal reference primer was β-actin, including upstream primer (5'-GTGGGGCGCCCCAGGCACCA-3'), downstream primer (5'-CTCCTTAATGTCACGCAC GATTTC-3') and amplification length (498 bp). Both pairs of primers were added into 25 μL reaction system to react under the proper PCR reaction conditions (denatured at 94°C for 45 s, renatured at 58°C for 45 s and elongated at 72°C for 60 s, for 35 cycles). RT-PCR products were isolated with 2% agarose gel, imaged through Alpha Image 2000 automatic imager and analyzed using Fluorchem V 2.0 Stand Alone software.
LOH analysis
Down-regulation of Apaf-1 gene expression was correlated with allele loss at its 5 sites (D12S327, D12S1657, D12S393, D12S1706 and D12S346). These 5 polymorphic sites were selected for LOH analysis. DNA was extracted from tumor and normal tissues using phenol/chlorine method. Primer sequences derived from genome database (GDB) and amplification conditions are listed in Table 1. Two percent agarose gel electrophoresis revealed target genes but no hybrid band in each specimen. PCR products were added into the denaturing buffer (98% formamide, 10 mmol/L EDTA, 0.025% bromophenol blue and 0.025% xylene cyanol) at a ratio of 1:1, mixed uniformly, denatured at 97°C for 10 min and immediately placed into ice. Fifteen μL of specimens was loaded in each hole, separated through 9% non-denaturing PAGE (in a 100 V low-temperature water bath for 2-3 h) and the electrophoresis results were analyzed through silver staining.
Table 1 Polymorphic sites, primers and amplification conditions (12q22-23).
Site
Repeat
Heterozygosity
Amplification length (kb)
Primer sequence
Reaction conditions
D12S327
Dinucleotide
0.8501
0.1820-0.2010
AAA GTT TCT GGA TGG TAA TAT CG
54°C 40 s
AGA GCA AGA CCT TGT CTC AA
72°C 45 s
D12S1657
Dinucleotide
0.6439
0.1500-0.1600
TCC TAA AGA TGG TGT GCA T
58°C 40 s
AAG TTC CAA TGT TAG TGA ACC
72°C 45 s
D12S393
Tetranucleotide
0.6410
0.2490
ATT ATT GCC AGG ACA TTA AAC G
58°C 30 s
CCT CAC ACA ATG TTG TAA GGG
72°C 40 s
D12S1706
Dinucleotide
0.8916
0.1190-0.1390
CCT ATG ATT TCC CAT CAA GTT T
57°C 40 s
ATT ATT AGG AGA GCC CTG GG
72°C 40 s
D12S346
Dinucleotide
0.8400
0.1660
TGC CC ACCT GCC TGT AAC
58°C 30 s
AAT GGA GGG TAA ATG CCC G
72°C 40 s
Methylation analysis at Apaf-1 promoter domain
DNA extracted from gastric cancer and adjacent normal tissues was treated with Na2SO3, purified and recovered with Wizard DNA clean-up (Promega Co.) for MSP analysis. A 49-378 base zone rich in CpG at Apaf-1 cDNA promoter domain was selected to design MSP primers, including upstream primer (5'-GAGGTGTCGTAG CGGTATTC-3'), downstream primer (5'-CGAAAATTA ACGAAATAAACGTC-3'), PCR reaction conditions (denatured at 94°C for 45 s, renatured at 58°C for 45 s and elongated at 72°C for 60 s, for 38 cycles) and amplification length (221 bp). Methylating primers included upstream primer (5'-ATTTGAGGT GTTGTAGTGGTATTT G-3'), downstream primer (5'-ACCTCCAAAAATTAACAA AATAAACAT-3'), PCR reaction conditions (denatured at 94°C for 45 s, renatured at 56°C for 45 s, elongated at 72°C for 60 s, for 38 cycles) and amplification length (221 bp).
Statistical analysis
Statistical analyses were performed by the SPSS13.0 software package (SPSS, Inc, Chicago, IL). Continuous Data were expressed as mean ± SD. Continuous variables were compared by t-test when appropriate, whereas categorical variables were compared by χ2 test or Fisher’s exact test when appropriate. Spearman rank correlation was analyzed. P < 0.05 was considered statistically significant.
RESULTS
Gene expression
The gene expression was increased 30%-70% in cancer tissue samples with an increased difference between Apaf-1/β-actin cDNA optical density ratios in gastric cancer and adjacent normal tissue samples. In the present study, Apaf-1expression decreased in 48.57% (17/35) of gastric cancer tissue samples (including expression loss in 2 cases), paired t test with SPSS13.0 statistical software showed that the relative Apaf-1 content was 0.96 ± 0.40 in adjacent normal tissue samples and 0.69 ± 0.36 in cancer tissue samples, respectively (t = 3.518, P < 0.01), and Apaf-1expression was significantly higher in adjacent normal tissue samples than in cancer tissue samples. Apaf-1 mRNA expression did not increase in tumor tissue samples (Figures 1 and 2).
Figure 2 Expression of β-actin mRNA.
M: Marker; T: Tumor; N: Normal adjacent tissue; β-actin mRNA: 498 bp; marker: DL2000.
LOH analysis
The data from 2 fragments (i.e. genome DNA was a certain marked heterozygote in normal tissue or adjacent normal tissue samples) were sufficient for LOH analysis, but the data from one fragment were not sufficient for LOH analysis. LOH meant that there was 1 band with less genome DNA PCR amplification product or 1 band with significantly weaker signal of up to 50% in cancer tissue samples than that in adjacent normal tissue samples. As shown by LOH analysis of 5 polymorphic sites for Apaf-1 gene in the present study, the LOH detection rate was 34.29% (12/35), 8.57% (3/35), 57.14% (20/35), 11.43% (4/35) and 42.86% (15/35) at single D12S346, D12S1706, D12S327, D12S1657 and D12S393, respectively. However, the LOH detection rate was 51.43% (18/35) at more than 2 sites (16.67% at 3 sites) (Figure 3), suggesting that the LOH of Apaf-1 gene was correlated with the genesis of gastric cancer. Apaf-1 mRNA expression decreased significantly in 13 cases (rs = 0.487, P = 0.003), indicating that the LOH of Apaf-1 gene was correlated with its decreased expression in gastric cancer (Table 2 and Figure 3).
Table 2 Correlation of Apaf-1, LOH and Apaf-1 mRNA expression in gastric cancer tissue samples.
Figure 3 LOH analysis of polymorphic sites of D12S327 (A), D12S1657 (B), D12S393 (C), D12S1706 (D), and D12S346 (E).
T: Tumor tissue; N: Adjacent normal tissue. Silver staining diagram showing Apaf-1 loss and LOH analysis showing allele loss at 5 polymorphic sites of Apaf-1 gene in gastric cancer tissue.
Promoter methylation
MSP analysis of methylation conditions for Apaf-1 gene promoter showed that the methylation rate of Apaf-1 gene was 48.57% (17/35) in gastric cancer tissue samples and 17.14% (6/35) in adjacent normal tissue samples, respectively (P < 0.05), demonstrating that Apaf-1 methylation was correlated with the genesis of gastric cancer. Significant promoter methylation occurred in 16 of 18 gastric cancer tissue samples while the expression of Apaf-1 mRNA was decreased (rs = 0.886, P = 10-6), displaying that promoter methylation was correlated with decreased expression of Apaf-1 gene (Table 3 and Figure 4). Promoter methylation and LOH of Apaf-1 gene occurred at polymorphic sites in 17 of 18 gastric cancer tissue samples and the expression of Apaf-1 mRNA was decreased.
Table 3 Correlation of Apaf-1 methylation and Apaf-1 mRNA expression in gastric cancer tissue samples.
Figure 4 Methylation of Apaf-1 promoter.
M: 212 bp methylation band; U: 221 bp non-methylation band; T: Gastric cancer tissue.
DISCUSSION
Human Apaf-1 gene is located at chromosome 12q23 to encode cytoplasm protein (130 KD). As an important apoptosis factor, Apaf-1 gene participates in the pathway of mitochondria-mediated apoptosis[12]. However, the condition of Apaf-1 gene expression in gastric cancer and the correlation between expression of Apaf-1 gene and genesis of gastric cancer remain unclear. Although Apaf-1 is an important apoptosis factor and a tumor suppressor gene[12-16], few studies on the relationship between Apaf-1 gene and gastric cancer are available.
In the present study, Apaf-1 gene expression in gastric cancer tissue samples was significantly lower than that in adjacent normal tissue samples, suggesting that Apaf-1 gene plays an important role in genesis and progression of gastric cancer.
According to the epigenetics theory on gene regulation, methylation of gene fragments (especially promoter domain) inhibits gene transcription, and acetylation of gene-related histone up-regulates gene expression. One of the carcinogenic mechanisms is gene silencing caused by hyper-methylation of CpG islet at tumor suppressor gene promoter domain and deacetylation of histone[17-23]. It was reported that Apaf-1 gene is a tumor suppressor gene and seldom mutates, but has functional loss due to LOH and promoter methylation[24]. Apaf-1 gene has different effects and expression patterns in cancer tissue of different sources. The lower (or inactive) expression of Apaf-1 gene is related to methylation silencing in acute leukemia and laryngeal squamous carcinoma[24,25], and LOH in colon cancer, ovarian cancer and malignant melanocarcinoma[26-28].
In the present study, Apaf-1 gene promoter was methylated in 14 gastric cancer tissue samples with a decreased expression of Apaf-1 as detected by the MSP technique. Apaf-1 expression decreased in 13 gastric cancer tissue samples with LOH at more than 2 sites (including promoter methylation in 6 cases) as demonstrated by 9% non-denaturing PAGE. These findings show that decreased expression of Apaf-1 gene in gastric cancer tissue is due to some complex reasons, among which, however, promoter methylation and LOH at polymorphic sites play a major role.
COMMENTS
Background
The expression condition of Apaf-1 gene in gastric cancer and its correlation with the genesis of gastric cancer remain unclear. The purpose of this study was to make it clear.
Research frontiers
The peigenetics theory on gene regulation is the highlight in recent tumor studies. This article describes the role of Apaf-1 gene eigenetics in gastric cancer.
Innovations and breakthroughs
This article focuses on the Apaf-1 expression condition in gastric cancer, and its relation with loss of heterozygosity and methylation in promoter domain.
Applications
Apaf-1 gene is an important apoptosis gene. Its status of methylation and LOH may contribute to the study on the etiology of gastric cancer.
Terminology
Methylation is a term used in chemical sciences to denote the attachment or substitution of a methyl group on various substrates. DNA methylation profiling is gaining momentum as an epigenetic approach to understanding the effects of aberrant methylation (either hyper- or hypomethylation) both in basic research and in clinical applications.
Peer review
Low expression of Apaf-1 gene in gastric cancer tissue was shown in this study, indicating that methylation of Apaf-1 gene promoter and LOH in domain of 12q22-23 are the main reasons for expression and altered expression of Apaf-1 gene in gastric cancer
Sobin LH, Fleming ID. TNM Classification of Malignant Tumors, fifth edition (1997). Union Internationale Contre le Cancer and the American Joint Committee on Cancer.Cancer. 1997;80:1803-1804.
[PubMed] [DOI][Cited in This Article: ]
Deng WG, Kawashima H, Wu G, Jayachandran G, Xu K, Minna JD, Roth JA, Ji L. Synergistic tumor suppression by coexpression of FUS1 and p53 is associated with down-regulation of murine double minute-2 and activation of the apoptotic protease-activating factor 1-dependent apoptotic pathway in human non-small cell lung cancer cells.Cancer Res. 2007;67:709-717.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 35][Cited by in F6Publishing: 42][Article Influence: 2.5][Reference Citation Analysis (0)]
Christoph F, Kempkensteffen C, Weikert S, Köllermann J, Krause H, Miller K, Schostak M, Schrader M. Methylation of tumour suppressor genes APAF-1 and DAPK-1 and in vitro effects of demethylating agents in bladder and kidney cancer.Br J Cancer. 2006;95:1701-1707.
[PubMed] [DOI][Cited in This Article: ][Cited by in Crossref: 62][Cited by in F6Publishing: 70][Article Influence: 3.9][Reference Citation Analysis (0)]
Karpf AR. Epigenomic reactivation screening to identify genes silenced by DNA hypermethylation in human cancer.Curr Opin Mol Ther. 2007;9:231-241.
[PubMed] [DOI][Cited in This Article: ]
Huang DF, Fu WN, Shang C, Xu ZM, Li ZG, Sun KL. Expression and promoter methylation of Apaf-1 gene in laryngeal squamous cell carcinoma.YiChuan XueBao. 2004;31:1327-1331.
[PubMed] [DOI][Cited in This Article: ]
