Recombinant Human FKN Enhances The Migration And Invasion In H446: A Small Cell Lung Cancer Cell Line And The Potential Factors On CX3CR1Gene Expression Of H446

Objective: Fractalkine，also known as CX3CL1,is the unique member of the fourthclass of chemokines and mediates both chemotaxis and adhesion of inflammatorycells and some tumor cells via its highly selective receptor CX3CR1.However,theexpression of CX3CR1or its effects on small cell lung cancer has not been shown.The goal of this study is to determine the influence of FKN/CX3CR1activation onmigration and invasion of small cell lung cancer cells. We also investigated the role ofTNF-α and TGF-β in the expression of CX3CR1gene in small cell lung cancer cells.Materials and Methods: PT-PCR and Western-blot were used to detect mRNA andprotein expression of CX3CR1in H446.For migration and invasion studies,H446cellssuspended in RPMI1640media with or without neutralizing antibody againstCX3CR1were placed in the upper chambers of transwell plates with FKN(80ng/ml)added to the upper or lower chambers. Cells were cultured with FNK(0ng/ml20ng/ml40ng/ml80ng/ml120ng/ml),the MMP9and MMP2gene expression weredetermined by Western-blot at24h.H446also cultured with FKN((80ng/ml),thephosphorylation status of AKT,ERK,NF-κB and STAT3were measured withWestern-blot at0min,5min,15min,30min and60min.We also analysed the effect ofserial concentrations (0ng/ml,0.01ng/ml,0.1ng/ml,10ng/ml,100ng/ml) of TNF-α and TGF-β on CX3CR1expression by RT-PCR,western-blot and immunofluorescenceassay.Results:.We produce evidence,for the first time,that CX3CR1is expressed by H446:a human small cell lung cancer cell line. The migration and invasion of H446cells isincreased with FKN added either in upper or lower chamber and this phenomenon issignificantly reduced by a neutralizing antibody against CX3CR1.Fractalkine activesthe STAT3pathway in H446cells.There is no significant influence of TNF-α onCX3CR1gene expression.While the expression of CX3CR1mRNA is significantlypromoted by TGF-β at the concentration of1ng/ml,but, the expression is inhibited bythe higher concertration(100ng/ml). CX3CR1protein level is increased at differentconcentration of TGF-β and at1ng/ml the enlargement is the most.Conclusions:1. FKN is associated with a significant augmentation of H446cells chemotaxisvia the expression of CX3CR1on H446.2. FKN/CX3CR1enhances the expression and activity of MMP9via the STAT3pathway in H446.3. CX3CR1protein level is increased at different concentration of TGF-β and theenlargement is the most at1ng/ml.Our findings support a role for FKN/CX3CR1in H446adhesion and migration,and pave the way for a novel therapeutic approach targeting this molecule.