Research On The Relationship Between HSulf-1 And Hedgehog Signaling Pathway In Human Gastric Cancer

Gastric cancer is one of the most common malignant tumors, and its genesis involved in the abnormal activation of some signaling pathways. Recently, the investigations on Hedgehog signaling pathway have revealed that its aberrant activation played a critical role in the gastric cancerogenesis. Since HSulf-1 can regulate the sulfation states of HSPGs, thereby affect the Hedgehog signaling pathway, we researched the relationship between HSulf-1 and Hedgehog signaling pathway in human gastric cancer cells.HSulf-1 gene encoded a secreted protein, which had functions in the neutral condition. In the extracellular matrix (ECM), HSulf-1 can decrease the degree of sulfation of heparan sulfate proteoglycans (HSPGs) by which the signaling molecules bind to their homologous receptors, then modulate the signaling pathway, thereby induce a series of biological responses, such as cell proliferation, angiopoiesis, tumorigenesis and so on. The previous report suggested that Hedgehog signaling pathway not only played a significant role in the embryonic development, but also had a pronounced effect in the cancerogenesis. In our prior observation, the expression of HSulf-1 was down-regulated due to the hypermethylation of its promoter. It was supposed that HSulf-1 could attenuate Hedgehog pathway which were aberrantly activated in the human gastric cancer cells.After obtaining stable transfected MNK28 cell line expressing HSulf-1 by G418 screening and identification using the method of semi-quantitative RT-PCR analysis at the transcriptional level as well as western blotting analysis at the translational level, the remarkable inhibition of the growth of cells (P<0.05) was observed, comparing to the cell transfected with control plasmid as empty control. As the clone mumber was decreased for MNK28 transfected with HSulf-1, comparing to the control with the empty plasmid, the observation was confirmed by the application of clone formation assay. Given that HSulf-1 can regulate the sulfation states of HSPGs, thereby affect the Hedgehog signaling pathway, we measured the expressions of the pathway targeted genes as well as some genes involved in the cell proliferation such as C-MYC、 MYCN、CCND1、BCL2 et al., at the transcriptional level by semi-quantitative RT-PCR analysis, and found that the expressions of almost all genes measured were down-regulated in the stable transfected MNK28 with HSulf-1 compared to the control cells. So, we speculated that the suppression of the growth due to HSulf-1 maybe caused by the down-regulation of Hedgehog pathway induced by HSulf-1.HSulf-1, as a sulfatase functioning in ECM(extracellular matrix), can alter the state of sulfate of HSPGs then affect the combination between signaling molecules and their homologous receptors, thereby control the signaling pathway. It implies that HSulf-1 has a potential utilization in medicine and industry. So, we tried to express the functional fragment of HSulf-1 using pGEX-6p-1 expression vector in the prokaryotic expression system and measured the enzyme activity of the production employing 4-Mus as the substrate. The result showed that the purified recombinant protein could be achieved, however, the protein had no enzyme activity. Possible reasons were explored in the discussion. The further investigation on HSulf-1 would be launched with its functions and roles in the mechanism so as to make it available to utilize in the medicine and industry.