Gastroenterology

Gastroenterology

Volume 126, Issue 1, January 2004, Pages 231-248
Gastroenterology

Basic-liver, pancreas, and biliary tract
hSulf1 sulfatase promotes apoptosis of hepatocellular cancer cells by decreasing heparin-binding growth factor signaling

https://doi.org/10.1053/j.gastro.2003.09.043Get rights and content

Abstract

Background & aims: The heparin-binding growth factors fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) are potent mitogens for hepatocellular carcinomas (HCCs). Heparin-binding growth factor signaling is regulated by sulfation of cell-surface heparan sulfate proteoglycans (HSPGs). We hypothesized that hSulf1, a recently described sulfatase, regulates growth signaling in HCCs. Methods:Expression of hSulf1 in human HCC tumors was determined by real-time PCR. Down-regulation of hSulf1 expression was investigated by analyzing loss of heterozygosity (LOH) at the hSulf1 locus and the effect of the DNA methylation inhibitor 5-aza-deoxycytidine on hSulf1 expression. The subcellular location of hSulf1 and sulfation state of cell-surface HSPGs were assessed by immunocytochemistry. FGF and HGF signaling was examined by phospho-specific immunoblot analysis. Cell growth was measured by trypan blue exclusion, and the MTT assay and apoptosis were quantitated by fluorescence microscopy. Results:hSulf1 expression was decreased in 29% of HCCs and 82% of HCC cell lines. There was LOH at the hSulf1 locus in 42% of HCCs. Treatment with 5-aza-deoxycytidine reactivated hSulf1 expression in hSulf1-negative cell lines. Low hSulf1-expressing cells showed increased sulfation of cell-surface HSPGs, enhanced FGF and HGF-mediated signaling, and increased HCC cell growth. Conversely, forced expression of hSulf1 decreased sulfation of cell-surface HSPGs and abrogated growth signaling. HCC cells with high-level hSulf1 expression were sensitive to staurosporine- or cisplatin-induced apoptosis, whereas low expressing cells were resistant. Transfection of hSulf1 into hSulf1-negative cells restored staurosporine and cisplatin sensitivity. Conclusions:Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.

Section snippets

Tumor samples

Thirty-one HCC tumors were used for the real-time PCR experiments and 94 HCCs for the loss of heterozygosity (LOH) experiments. For each case, tumor samples with matched adjacent benign tissue were collected during surgical resections at the Mayo Clinic between 1991 and 2001, frozen in liquid nitrogen, and stored at −80°C. Sections from each specimen were examined by a pathologist and graded histologically. The study was approved by the Mayo Clinic Institutional Review Board.

HCC cell lines

The following HCC

Expression of the hSulf1 sulfatase mRNA is decreased in a proportion of primary HCC and HCC cell lines

Our previous results demonstrated down-regulation of hSulf1 in 77% (23/30) of ovarian carcinomas and in the majority of cancer cell lines of ovarian, breast, pancreas, kidney, and liver origin.11, 12 To determine whether hSulf1 was also down-regulated in primary HCCs, we evaluated hSulf1 expression in 31 primary HCCs by quantitative real-time PCR. Twenty-two of the HCCs were randomly selected, and 9 additional tumors were selected based on known LOH at the hSulf1 locus. Of the 31 total HCCs

Discussion

In the present study, we have demonstrated that the newly identified sulfatase gene hSulf1 is down-regulated in approximately 30% of primary HCC tumors and 80% of HCC cell lines and has a growth-inhibitory, proapoptotic function in HCC cell lines. We showed that hSulf1 is localized to the cell surface and has a measurable sulfatase activity in hSulf1-expressing HCC cell lines. We also demonstrated that the substrates of hSulf1 include sulfated heparan sulfate proteoglycans (HSPGs) at the cell

Acknowledgements

The authors thank Kristi Simmons and Eric Bungum for secretarial assistance; Steve Bronk and Drs. Ali Canbay, Hong Cao, Jing Chen, Haidong Dong, Eugenia Guicciardi, and Hajime Higuchi for technical assistance; and Drs. Gregory Gores and Scott Kaufmann for critical review of the manuscript.

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    Supported by the National Institutes of Health (CA82862 and CA100882 to L.R.R.), an Industry Research Scholar Award from the Foundation for Digestive Health and Nutrition (to L.R.R.), a Minority Medical Faculty Development Awards from The Robert Wood Johnson Foundation (to L.R.R. and E.L.G.), DOD grant DAMD17-99-1-9504 (to V.S. and D.I.S.), a John W. Anderson Foundation grant (to V.S.), and the Mayo Foundation.

    1

    V.S. and L.R.R. contributed equally to this work.

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