ONCOLOGY REPORTS 28: 1785-1791, 2012 Frequent silencing of protocadherin 8 by promoter methylation, a candidate tumor suppressor for human gastric cancer DANJIE ZHANG, WEI ZHAO, XINHUA LIAO, TIEQIANG BI, HAIJUN LI and XIANGMING CHE Department of General Surgery, First Affiliated Hospital of Medical College of Xi'an JiaoTong University, Xi'an 710061, P.R. China Received May 10, 2012; Accepted July 30, 2012 DOI: 10.3892/or.2012.1997 Abstract. The cadherins are a family of cell surface glycopro- These abnormalities can also define biological characteristics of teins responsible for cell adhesion which play an important role gastric cancer, which can play a role in therapy (3,4). Although in cell morphology, contact inhibition and signal transduction genetic abnormalities which include gene mutation and dele- during tumorigenesis. Protocadherin 8 (PCDH8), a member tion are remarkable in causing oncogene activation and tumor of the cadherin family, has been reported to act as a tumor suppressor gene inactivation, epigenetic silence of tumor suppressor involved in oncogenesis in breast cancer. In this study, suppressor genes through aberrant promoter hypermethylation we aimed to investigate the epigenetic inactivation of PCDH8 have also been confirmed to be frequent in gastric carcinoma and its tumor suppressor function in gastric cancer. The expres- (5,6). Gene silencing by promoter hypermethylation has been sion of PCDH8 was markedly reduced or silenced in gastric affirmed in several genes in gastric cancer, including CDH1, cancer cell lines compared with normal gastric cells or tissues. which is involved in cell adhesion, and hMLH1, which is associ- Methylation of the PCDH8 gene promoter was observed in 100% ated with DNA mismatch repair and the cell cycle regulator p16. (4/4) of cell lines and 55.38% (36/65) of the primary gastric In addition, promoter hypermethylation of MAL has been shown cancer by methylation-specific PCR, but not in normal gastric to be an independent prognostic marker for gastric cancer (7-9). mucosa (0/10). Methylated PCDH8 was significantly associated DNA high methylation of tumor suppressor genes frequently with lymph node metastasis in a logistic regression analysis. The occurs in the early stage of human carcinogenesis, so investi- demethylation reagent 5-aza-2'-deoxycytidine was able to restore gating the methylation of these gene promoters may contribute to or upregulate PCDH8 expression in gastric cancer cell lines. the diagnosis, prognosis and target therapy in gastric carcinoma. Ectopic expression of PCDH8 in silenced gastric cancer cells Cadherin molecules are pivotal in producing and preserving significantly inhibited cell migration and induced apoptosis. For tissue structure in tumorigenesis (10,11). Such as E-cadherin, the first time, our study demonstrates the epigenetic inactivation it is a classical tumor suppressor which is mutated in gastric of PCDH8 by promoter methylation and its tumor suppressor carcinoma and lobular breast carcinoma (12). Accumulating function in human gastric cancer. Thus, PCDH8 could be identi- data have suggested that protocadherins can function as tumor fied as a candidate tumor suppressor in human gastric cancer. suppressors. Protocadherins 10 and 20 are frequently silenced in carcinomas of the nasopharynx and lung due to promoter Introduction methylation and inhibit cell migration and proliferation (13,14). Protocadherin 17 in esophageal squamous cell carcinoma is Gastric cancer is the second leading cause of cancer death in the also frequently silenced because of promoter methylation (15). world behind lung cancer (1,2). Gastric carcinogenesis is a multi- The protocadherin 8 (PCDH8) gene localizes on the human step process including many genetic and epigenetic alterations, chromosome 13q14.3, is an adhesion protein with six cadherin such as abnormalities in DNA mismatch repair genes, growth repeats that organizes the formation and polarity of developing factors/receptors, angiogenic factors or cell cycle regulators. cellular structures in embryos (16). It has been shown that PCDH8 is epigenetically silenced caused by promoter hypermethylation in most of breast tumors and it can suppress breast epithelial migration and proliferation (17). To the best of our knowledge, the expres- Correspondence to: Professor Xiangming Che, Department of General Surgery, First Affiliated Hospital of Medical College of sion of PCDH8 and its promoter hypermethylation has not Xi'an JiaoTong University, Yanta West Road 277, Xi'an 710061, yet been shown in gastric cancers. Therefore, in this study, P. R. Ch i na we first confirmed the expression of PCDH8 and the methyla- E-mail: [email protected] tion of its gene promoter in human gastric cancer cell lines and determined the role of 5-aza-2'-deoxycytidine (5-AZA, Key words: protocadherin 8, gene methylation, 5-aza-2'-deoxycytidine, a drug that inhibits the DNA methyltransferase (DNMT)- gastric cancer mediated hypermethylation of promoter region CpG islands) in regulation of PCDH8 expression in gastric cancer cells. We also detected the methylation of PCDH8 gene promoter 1786 ZHANG et al: CANDIDATE TUMOR SUPPRESSOR PCDH8 IN GASTRIC CANCER in tissue specimens and found the association between CTC GTC G 3'. Each MSP reaction incorporated ~100 ng of PCDH8 gene promoter methylation and clinicopathological bisulfite-treated DNA, 25 pmol of each primer, 100 pmol dNTPs, characteristics of gastric cancer. 2.5 µl 10X PCR buffer, and 1 U of JumpStart RedTaq Polymerase (Sigma) in a final reaction volume of 25 µl. The PCR amplifica- Materials and methods tion conditions were an initial 95˚C for 5 min and then 35 cycles of 95˚C for 30 sec, 60˚C for 30 sec, and 72˚C for 30 sec and a Human gastric samples. A total of 65 primary gastric adeno- final extension at 72˚C for 5 min and then stored at 4˚C. The MSP carcinomas and their adjacent non-cancer specimens, and products were separated on 2% agarose gel electrophoresis and 10 normal gastric specimens from patients with normal endos- visualized under the ultraviolet (UV) light. copy results were obtained from the Department of General Surgery, First Affiliated Hospital of the Medical College of RNA isolation and semi-quantitative reverse transcription Xi'an Jiaotong University, Xi'an, China between January 2009 PCR. Total cellular RNA from the cell lines was isolated and January 2010. All specimens were immediately frozen in using the TRIzol reagent (Invitrogen) according to the WJG liquid nitrogen and stored at -80˚C until use. The gastric cancer manufacturer's instructions. RNA quality and quantity were cases were clinically and pathologically verified. Standard assessed using agarose gel electrophoresis (1%) and spectro- protocols established by the Hospital's Protection of Human photometric analysis of 260/280 ratios. The RNA was stored Subjects Committee were followed in this study. at -70˚C prior to use. The first strand cDNA was synthesized with oligo-(dT) primer using a reverse transcriptase kit from Cell lines and culture. A total of four gastric cancer cell lines Invitrogen. RNA (2 µg) was subjected to the first strand cDNA (SGC7901, MKN45, MKN28, and BCG823) and the immortal- synthesis, and 1 µl cDNA from RT reaction was subjected to ized gastric mucosal epithelial cell line (GES-1) were purchased PCR amplification of gene expression in a total 25-µl reaction from the Laboratory Animal Research Centre of the Fourth volume. The PCR amplification was carried out using primer Military Medical University at Xi'an, China. All cell lines were sets derived from the published PCDH8 gene sequences: cultured in RPMI-1640 medium (Gibco BRL, Gaithersburg, PCDH8 primers: 5'-TGGCGGTGTGGAAAGGACA-3' and MD, USA) supplemented with 10% fetal bovine serum in a 5'-CGGAGTGACCTGTATATGTG-3' (17). This primer set, humidified incubator with 5% CO2 and 95% air at 37˚C. designed to cross the intronic sequences, can prevent from These cells were passaged at a ratio of 1:3 with trypsin once amplification of genomic DNA for control of genomic DNA they reached confluence (~106 cells) into 752 cm culture flasks contamination during RNA isolation. A total of 32 cycles (Sarstedt, Newton, NC). For treatment with 5-aza-2'-deoxy- of PCR amplification were performed based on our pre- cytidine, these cell lines were split and cultured at a low density experiment for semi-quantitative measurement of PCDH8 gene (30% confluence) overnight and then treated with 5-aza-2'-deoxy- expression levels. Glyceraldehyde-3-phosphate dehydrogenase cytidine (Sigma, St. Louis, MO) at a concentration of 1 µmol/l for (GAPDH) was amplified for 25 cycles as an internal control of up to 72 h. The growth medium was refreshed every 24 h, and at equal loading and cDNA quality and quantity. The sequence of the end of the treatment, DNA and RNA from these cells were GAPDH primers: 5'-GACCACAGTCCATGCCATCAC-3' and isolated as described below. 5'-GTCCACCACCCTGTTGCTGTA-3'. The PCR products (PCDH8, 251 bp; GAPDH, 150 bp) were then electrophoresed Bisulphite treatment of DNA, methylation-specific polymerase in 1.5% agarose gels containing ethidium bromide and reviewed chain reaction (MSP). Genomic DNA from these cell lines and under the UV light. Primers were designed according to tissue specimens were extracted by a DNA mini kit (Qiagen, GenBank, NCBI. For the validation, each experiment was done Valencia, CA, USA). Methylation status of PCDH8 in gastric in triplicate. tissues and cell lines were determined by MSP. Briefly, 2 mg of genomic DNA was bisulphite-treated with Zymo DNA Protein extraction and western blotting. The cells were grown Modification kit (Zymo Research, Orange, CA, USA). Bisulphite- and treated with or without 1 µM 5-aza-2'-deoxycytidine for treated DNA was used as a template for MSP and quantitative 72 h and total cellular protein was then extracted from these PCR by ABI 2700 thermocycler (Applied Biosystems, CA, cells in 200 µl ice-cold mild lysis buffer containing 10 µl USA) and LightCycler (Roche Diagnostic), respectively.