Abstract

BackgroundHeparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2). Human endosulfatase 1 (HSULF-1) is an enzyme that selectively removes 6-O sulfate groups from HS side chains and alter their level and pattern of sulfation and thus biological activity. It is known that HSULF-1 is expressed at low levels in some cancer cell lines and its enhanced expression can inhibit cancer cell growth or induce apoptosis, but the mechanism(s) involved has not been identified.MethodsHSULF-1 mRNA expression was assessed in five normal cells (primary human lung alveolar type 2 (hAT2) cells, adult lung fibroblasts (16Lu), fetal lung fibroblasts (HFL), human bronchial epithelial cells (HBE), and primary human lung fibroblasts (HLF)) and five lung cancer cell lines (A549, H292, H1975, H661, and H1703) using quantitative real time polymerase chain reaction (qRT-PCR). H292 and hAT2 cells over-expressing HSULF-1 were analyzed for cell viability, apoptosis, and ERK/Akt signaling, by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and Western Blot, respectively. Apoptosis pathway activation was confirmed by PCR array in hAT2, H292, and A549 cells.ResultsHSULF-1 was expressed at a significantly lower level in epithelial cancer cell lines compared to normal cells. Infection with recombinant adenovirus for HSULF-1 over-expression resulted in decreased cell viability in H292 cells, but not in normal hAT2 cells. HSULF-1 over-expression induced apoptosis in H292 cells, but not in hAT2 cells. In addition, apoptosis pathways were activated in both H292 and A549 cells, but not in hAT2 cells. HSULF-1 over-expression reduced ERK and Akt signaling activation in H292 cells, which further demonstrated its inhibitory effects on signaling related to proliferation.ConclusionsThese results indicate that HSULF-1 is expressed at lower levels in H292 lung cancer cells than in normal human alveolar cells and that its over-expression reduced cell viability in H292 cells by inducing apoptotic pathways, at least in part by inhibiting ERK/Akt signaling. We hypothesize that HSULF-1 plays important roles in cancer cells and functions to modify cell signaling, inhibit cancer proliferation, and promote cancer cell death.

Highlights

  • Heparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2)

  • Human endosulfatase 1 (HSULF-1) basal expression is lower in lung cancer cells than in normal lung cells To evaluate the expression of HSULF-1 in cells of pulmonary origin, five normal lung cells (fibroblasts (16Lu), fetal lung fibroblasts (HFL), primary lung fibroblasts (HLF), primary alveolar type 2 cells, and bronchial epithelial cells (HBE)) and five lung epithelial cancer cell lines (A549, H292, H1975, H661, and H1703) were cultured and mRNAs were analyzed

  • Over-expression of HSULF-1 decreased cell density in H292 cancer cells but not in human primary hAT2 cells H292 and hAT2 cells were infected with adenovirus at various Multiplicities of infection (MOI) for lac operon Z (lacZ) or HSULF-1 over-expression

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Summary

Introduction

Heparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2). Heparan sulfate (HS) proteoglycans are major components of extracellular matrix (ECM) and cell surfaces. They function as dynamic interfaces between cells and their external environment. They help cells affix to and maintain the extracellular scaffolding of the ECM as well as directly internalize lipid factors. Their shed ectodomain fragments can even neutralize injurious agents [1,2,3,4]. 6-O-desulfated heparin, which binds to FGF-2 ligands but fails to bind to the FGFR, can decrease the FGF-2-induced proliferation of CHO677 cells [15]

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