Transcriptional silencing of Dickkopf gene family by CpG island hypermethylation in human gastrointestinal cancer

AIM： To clarify alterations of Dickkopfs （Dkks） and Kremen2 （Krm2） in gastrointestinal cancer. METHODS： We investigated the expression profiles and epigenetic alterations of Dkks and Krm2 genes in gastrointestinal cancer using RT-PCR, tissue microarray analysis, and methylation specific PCR （MSP）. Cancer cells were treated with the demethylating agent and/or histone deacetylase inhibitor. WST-8 assays and/n y/tro invasion assays after treatment with specific siRNA for those genes were performed. RESULTS： Dkks and Krm2 expression levels were reduced in a certain subset of the gastrointestinal cancer cell lines and cancer tissues. This was correlated with promoter hypermethylation. There were significant correlations between Dkks over-expression levels and beta-catenin over-expression in colorectal cancer. In colorectal cancers with beta-catenin over-expression, Dkk-1 expression levels were significantly lower in those with lymph node metastases than in those without. Down-regulation of Dkks expression by siRNA resulted in a significant increase in cancer cell growth and invasiveness in vitro.CONCLUSION： Down-regulation of the Dkks associated to promoter hypermethylation appears to be frequently involved in gastrointestinal tumorigenesis.

Association of glypican-3 expression with growth signaling molecules in hepatocellular carcinoma

AIM:To clarify the association of glypican-3(GPC3)expression with Wnt and other growth signaling molecules in hepatocellular carcinoma(HCC). METHODS:Expression of GPC3,Wnt,matrix metalloproteinases(MMPs),sulfatase(SULF)1,SULF2,and other growth signaling molecules was analyzed in HCC cell lines and tissue samples by real-time reverse transcriptionpolymerase chain reaction,immunoblotting,and/or immunostaining.Expression of various genes in GPC3 siRNA-transfected HCC cells was analyzed. RESULTS:GPC3 was overexpressed in most HCCs at mRNA and protein levels and its serum levels weresignificantly higher in patients with HCC than in non- HCC subjects(P<0.05).Altered expressions of various MMPs and growth signaling molecules,some of which were correlated with GPC3 expression,were observed in HCCs.Down-regulation of GPC3 expression by siRNA in GPC3-overexpressing HCC cell lines resulted in a significant decrease in expressions of MMP2,MMP14,fibroblast growth factor receptor 1,insulin-like growth factor 1 receptor.GPC3 expression was significantly correlated with nuclear/cytoplasmic localization ofβ-catenin. CONCLUSION:These results suggest that GPC3,in conjunction with MMPs and growth signaling molecules, might play an important role in the progression of HCC.