Lai Jin-Ping, Chien Jeremy R, Moser David R, Staub Julie K, Aderca Ileana, Montoya Damian P, Matthews Tori A, Nagorney David M, Cunningham Julie M, Smith David I, Greene Eddie L, Shridhar Viji, Roberts Lewis R
Division of Gastroenterology, Mayo Clinic College of Medicine, Rochester, MN 55905, USA.
Gastroenterology. 2004 Jan;126(1):231-48. doi: 10.1053/j.gastro.2003.09.043.
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.
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.
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.
Down-regulation of hSulf1 contributes to hepatocarcinogenesis by enhancing heparin-binding growth factor signaling and resistance to apoptosis.
肝素结合生长因子成纤维细胞生长因子（FGF）和肝细胞生长因子（HGF）是肝细胞癌（HCC）的强效促有丝分裂原。肝素结合生长因子信号传导受细胞表面硫酸乙酰肝素蛋白聚糖（HSPG）硫酸化作用的调节。我们推测，最近发现的硫酸酯酶hSulf1可调节HCC中的生长信号传导。
通过实时PCR测定hSulf1在人HCC肿瘤中的表达。通过分析hSulf1基因座的杂合性缺失（LOH）以及DNA甲基化抑制剂5-氮杂脱氧胞苷对hSulf1表达的影响，研究hSulf1表达的下调情况。通过免疫细胞化学评估hSulf1的亚细胞定位和细胞表面HSPG的硫酸化状态。通过磷酸特异性免疫印迹分析检测FGF和HGF信号传导。通过台盼蓝排斥法测量细胞生长，并通过荧光显微镜对MTT法和细胞凋亡进行定量分析。
29%的HCC和82%的HCC细胞系中hSulf1表达降低。42%的HCC中hSulf1基因座存在LOH。用5-氮杂脱氧胞苷处理可使hSulf1阴性细胞系中的hSulf1表达重新激活。低hSulf1表达细胞显示细胞表面HSPG硫酸化增加、FGF和HGF介导的信号增强以及HCC细胞生长增加。相反，hSulf1的强制表达降低了细胞表面HSPG的硫酸化并消除了生长信号。高hSulf1表达的HCC细胞对星形孢菌素或顺铂诱导的细胞凋亡敏感，而低表达细胞则具有抗性。将hSulf1转染到hSulf1阴性细胞中可恢复对星形孢菌素和顺铂的敏感性。
hSulf1的下调通过增强肝素结合生长因子信号传导和抗凋亡能力促进肝癌发生。
