5A). Induction of CpG methylation with SssI methylase decreased the activity to minimal levels. Figure 5 Cellular function of OSMR. Oncostatin M (OSM) is an interleukin-6 (IL-6)-type cytokine, but more active than IL-6 in inhibiting the proliferation of numerous solid tumor cell lines [19], [20]. Recently, a correlation of resistance to growth inhibition by www.selleckchem.com/products/Belinostat.html OSM with specific loss of the OSMR and Stat3 signaling was reported [15]. In order to examine CRC cell resistance to growth inhibition by OSM, we transiently transfected a siRNA pool targeting OSMR and a non-targeting control siRNA into OSMR-expressing HCT116 cells, and performed a standard cell growth assay after treatment of cells with a recombinant human OSM (rhOSM). We observed significant growth inhibition by rhOSM in HCT116 cells, and the inhibition was reversed by knock-down of OSMR (Fig.

5B, left). A Stat3 inhibitor peptide (Stat3 Inh) that abrogated Stat3 activation (Fig. 5D) blocked the rhOSM-induced growth inhibition in HCT116 cells (Fig. 5B, right). Consistently, suppression of cell growth by rhOSM was not observed in SW480 and DLD-1 cells with OSMR promoter methylation (Fig. 5C). Interestingly, the inhibition of cell growth by 5-Aza-dC treatment in SW480 and DLD-1 cells (*, P<0.05) was significantly enhanced by the treatment of rhOSM (#, P<0.05). Finally we observed that rhOSM activated Stat3 phosphorylation in HCT116 cells, regardless OSMR expression levels (Fig. 5D, left). Expression of gp130 and LIFR mRNA was observed in HCT116 cells (Fig.

5E), indicating that the activation of Stat3 in HCT116 cells with very low OSMR expression caused by siRNA transfection may be through gp130/LIFR (type I OSM receptor)-mediated signaling. Erk phosphorylation increased in HCT116 cells transfected with siRNA targeting OSMR, and the basal level of phospho-Erk in SW480 cells with OSMR methylation was higher than in HCT116 cells (Fig. 5D). 5-Aza-dC partially decreased activated Erk in SW480 cells, and rhOSM recovered basal Erk phopshorylation in the presence of 5-Aza-dC. Thus, methylated OSMR markedly decreased tumor-inhibiting signals from rhOSM and key downstream signaling events. Discussion Promoter methylation of key regulatory genes drives the cancer process and in the right context can serve as a diagnostic marker and a therapeutic target. Cancer-specific methylation serves as an important biomarker for the early detection of cancer.

Such markers may supplement the cytopathological assessment of tissue biopsies or potentially stand on their own as markers of disease in various bodily fluids such as stool. To this end, the methylation frequency of GSK-3 genes identified in primary tissues has important clinical implications. A number of genes are commonly hypermethylated in colorectal cancer (CRC) including hMLH1, p16INK4a, p14ARF, RAR-��, APC, MGMT, cyclin A1, CDX1, MYOD1, COX-2 and WT-1 [21], [22], [23].