title: Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma.
abstract: OBJECTIVE
  To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis.
  
  
  METHODS
  Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2'-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.
  
  
  RESULTS
  SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2'-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro (47.17% ± 15.61% vs 17% ± 3.6%, P = 0.031) and tumor growth in nude mice (917.86 ± 249.35 mm(3)vs 337.23 ± 124.43 mm(3), P < 0.05). Using flow cytometry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells (P = 0.025). The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts.
  
  
  CONCLUSIONS
  Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.
authors.affiliation: Department of Gastroenterology, Beijing Friendship Hospital, Capital Medical University, 10050 Beijing, China.
authors.forename: Xiao-Wen
authors.initials: XW
authors.lastname: Hao
authors.forename: Sheng-Tao
authors.initials: ST
authors.lastname: Zhu
authors.forename: Yuan-Long
authors.initials: YL
authors.lastname: He
authors.forename: Peng
authors.initials: P
authors.lastname: Li
authors.forename: Yong-Jun
authors.initials: YJ
authors.lastname: Wang
authors.forename: Shu-Tian
authors.initials: ST
authors.lastname: Zhang
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D020868:Gene Silencing; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008819:Mice, Nude; D011401:Promoter Regions, Genetic; D000094742:Secreted Frizzled-Related Proteins
chemical_list: D008565:Membrane Proteins; C109618:SFRP2 protein, human; D000094742:Secreted Frizzled-Related Proteins