Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma

Figure 1 Expression and promoter methylation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma cell lines. A: Secreted frizzled-related protein 2 (SFRP2) mRNA expression as determined by reverse-transcription polymerase chain reaction (RT-PCR); B: The methylation status of the SFRP2 promoter as determined by methylation specific PCR; C: mRNA expression of SFRP2 (determined by RT-PCR) restored after treatment with the demethylation agent 5-Aza-dC in three esophageal squamous cell carcinoma cell lines. M: Methylated; U: Unmethylated.

Figure 2 A representative picture of bisulfite genomic sequencing in the secreted frizzled-related protein 2 gene promoter. A-293bp region with 34 CpG site was analyzed. B

Figure 3 Expression of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma and paired non-cancerous tissues. A: Reverse-transcription polymerase chain reaction (RT-PCR) analysis of secreted frizzled-related protein 2 (SFRP2) mRNA expression in 10 primary esophageal squamous cell carcinoma (ESCC) tissues and their adjacent non-cancerous tissues; B: The SFRP2 mRNA expression level normalized according to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. Data are expressed as mean ± SD. ESCC tissues vs adjacent non-tumor tissues (^aP < 0.01).

Figure 4 Effect of secreted frizzled-related protein 2 ectopic expression on cell proliferation. A: Strong secreted frizzled-related protein 2 (SFRP2) mRNA expression in KYSE30 cells transfected with pcDNA3.1/SFRP2, but not in KYSE30 and cells transfected with the empty vector; B: SFRP2-transfected KYSE30 cells growing significantly slower than control and parental cells (^aP < 0.05).

Figure 5 Ectopic expression of SFRP2 induces apoptosis in KYSE30 cells. A: The representative images of flow cytometry analysis; B: The data are the percentage of early apoptotic out of the total cell population of KYSE30 cells transfected with SFRP2 and cells with the empty vector.The early apoptotic cells in SFRP2-transfected cells were significantly higher than that in the control cells (^aP < 0.05).

Figure 6 Colony formation assays. A: The representative dishes of transfection with pcDNA3.1V5HisA/secreted frizzled-related protein 2 (SFRP2) or empty vector (pcDNA3.1 V5HisA). Left: colonies formed by control cells. Right: colonies formed by cell strains with stably transfected SFRP2; B: Quantitative analyses of colony formation efficiency. Values are the mean ± SD of at least 3 independent experiments. The relative colony formation efficiency of SFRP2-transfected esophageal squamous cell carcinoma cells were lower than those of mock-transfected cells (^aP < 0.05).

Figure 7 Secreted frizzled-related protein 2 inhibits growth of tumors derived from KYSE-30 in vivo. A: A representative picture of nude mice: at week 5 nude mice injected with KYSE-30, KYSE-30/vector, KYSE30/SFRP2 and phosphate buffered saline; B: The tumor growth curves of nude mice. The growth of tumors derived from the SFRP2-transfected esophageal squamous cell carcinoma cells cells were significantly slower than those from the mock-transfected cells (^aP < 0.05).