Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer

Abstract

The role of secreted frizzled-related protein (SFRP) genes in gastric cancer remains largely unknown. We determined the frequency and functional significance of SFRPs hypermethylation in human gastric cancer. The expression and methylation status of four SFRP members (SFRP1, 2, 4, and 5) in primary gastric cancer samples was screened. The biological effects of SFRP were analysed by flow cytometry, cell viability assay and in vivo tumour growth in nude mice. Among the four SFRPs, only SFRP2 was significantly downregulated in gastric cancer as compared to adjacent non-cancer samples (P<0.01). Promoter hypermethylation of SFRP2 was detected in 73.3% primary gastric cancer tissues, 37.5% of samples showing intestinal metaplasia and 20% adjacent normal gastric tissues. Bisulphite DNA sequencing confirmed the densely methylated SFRP2 promoter region. Demethylation treatment restored the expression of SFRP2 in gastric cancer cell lines. Forced expression of SFRP2 induced cell apoptosis, inhibited proliferation of gastric cancer cells and suppressed tumour growth in vivo. Moreover, methylated SFRP2 was detected in 66.7% of serum samples from cancer patients but not in normal controls. In conclusion, epigenetic inactivation of SFRP2 is a common and early event contributing to gastric carcinogenesis and may be a potential biomarker for gastric cancer.

Figure 1

mRNA expression levels of secreted frizzled-related proteins (SFRPs) in primary gastric cancer (Tumour) and their adjacent non-cancer tissues (Normal) as determined by quantitative real-time PCR. The results were expressed as the ratio of copies of target gene relevant to GAPDH form three independent experiments (n=14). Data are expressed as mean±s.d., ^*P<0.01.

Figure 2

(A) The mRNA expression of secreted frizzled-related protein 2 (SFRP2) in gastric cancer cell lines (MKN28, MKN45, AGS, Kato III, NCI87, SNU1, and SNU16) treated with or without demethylation agent 5′Aza-DC as determined by RT–PCR. Pharmacologic treatment with 5′Aza-DC (5′Aza) restored the expression of SFRPs in tumour cell lines. (B) The methylation status of SFRP2 in gastric cancer cell lines treated with or without 5′-Aza-DC as determined by methylation specific PCR. M: methylated primers; U: unmethylated primers.

Figure 3

A representative picture of bisulphite sequencing in secreted frizzled-related protein 2 (SFRP2) gene. The CpG island, MSP region (−219 to −81) and bisulphite sequence region (−362 to +9) were shown in the upper panel. A 371-bp region with 34 CpG site was analysed. Each row of CpG sites represented an individual allele of the SFRP2 promoter analysed. Percentage methylation was determined as percentage of methylated cytosine from 8 to 10 randomly sequenced colonies.

Figure 4

Transient transfection of secreted frizzled-related protein 2 (SFRP2) gene in MKN45 cell lines. (A) Strong SFRP2 mRNA expression was observed in MKN45 cells transfected with pcDNA3.1(+)SFRP2, but not in cells transfected with the empty vector. (B) Strong SFRP2 protein expression was observed in MKN45 cells transfected with pcDNA3.1(+)SFRP2, but not in cells with empty vector of in parental MKN45. (C) SFRP2 significantly suppressed cell viability at 48 h. Values are expressed as the mean±s.d. from three independent experiments (^*P<0.001).

Figure 5

Effect of SFRP2 on apoptosis of human gastric cancer cells. SFRP2 increased the rate of apoptosis in MKN45 cells transfected with pcDNA3.1(+)SFRP2, as determined by the number of cells with sub-G₁ DNA content by flow cytometry. Values are expressed as the mean±s.d. of three replicate experiments. ^*P<0.001.

Figure 6

Secreted frizzled-related protein 2 (SFRP2) inhibits growth of tumours derived from MKN45 in vivo. (A) A representative picture of nude mice: at week 3 nude mice injected with MKN45/3.1 and MKN45/SFRP2. (B) The tumour volume of vector (pcDNA3.1) transfected MKN45 cell in nude mice (n=15) was indicated as mean tumour volume±s.d. (mm³). Tumour mean volume of MKN45/SFRP2 mice was significantly smaller than the MKN45/3.1 nude mice group (^*P<0.001).