Pro-life (Fas/FasL) and Pro-death (HGF/c-Met) receptor/ligand system interactions regulates invasion in vitro.

Abstract

2077

Introduction: Tumor metastasis is the limiting factor in successful treatment for a wide range of malignancies. Selective targeting of individual molecular pathways provides partial treatment benefit.

The Fas/ FasL system, widely investigated for its pro-apoptotic effects, has been indicated to correlate with tumor progression. Hepatocyte growth factor (HGF) through its receptor c-Met has been shown to enhance the invasive capacity of cancer cells via tyrosine kinase-dependent intracellular signalling pathways. The authors hypothesize that cross-talk between these two receptor/ligand systems and their signalling pathways can regulate proteases essential for tumor invasion and augment metastatic potential.

Methods: A human pancreatic ( PSN-1) and colon cancer (SW1222/ SW620) cancer cell lines were used. Quantitative analysis for presence of Fas/FasL and c-Met by Flow cytometry and qualitative images by fluorescence cytology were obtained. Assays were repeated in response to HGF, TNF-α, IL-6 and IL-8. Inhibition of signaling pathways was investigated by incorporating the potent tyrosine kinase inhibitors Wortmannin and Ly 294002. Fas and c-Met expression in response to Fas ligation was also determined by flow cytometry. HGF production in response to Fas ligation and the pro-inflamatory cytokines was determined by ELISA. Cell supernatants from control and CH-11/HGF treated cells were subjected to substrate zymography for MMP-2/MMP-9 expression. Densitometric analysis was done by Scion image software. The ability of CH-11 treated cells to invade through matrigel treated 8.0μm Transwell filters was analyzed by colorimetric MTS assay and by direct staining of the filters. Transcription factor Nf-kappaB and AP-1,activation was detected by EMSA. ANOVA analysis of variance was the statistical method employed.

Results: Fas and c-Met were constitutively expressed in all cell lines as detected by FACS and confirmed by Fluorescence microscopy. Whilst c-Met was upregulated Fas expression was downregulated in response to cytokines. HGF induced an autocrine upregulation of c-Met. Tyrosine kinase blockade attenuated that response. Protease production in response to HGF and Fas-L was enhanced. The stimulation of Fas with agonist resulted in: Significant dose dependent upregulation (p<0.05) of MMP2/MMP-9; Activation of transcription factors Nf-kappaB and Ap-1; and increased invasiveness.

Conclusions: Our results provide evidence that the invasive capacity of the HGF/c-Met system is strongly influenced by Fas/Fas-L. We have shown a clear interaction between the two receptor/ ligand systems

that may be critical in determining the metastatic potential of a range of gastrointestinal carcinomas. Further research in this direction may identify appropriate therapeutic targets.

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