Regulation Of Keratinocyte Growth Factor In The Activation Of Aryl Hydrocarbon Receptor In Colorectal Cancer Cells

Background:Colorectal cancer has become a significant global health concern,the third most common cancer and the fourth most common cause of death by cancer worldwide.Keratinocyte growth factor(KGF)which is a member of the fibroblast growth factor group have an important role in epithelial cell growth in a paracrine manner.Recent studies have shown that a high percentage of colorectal cancers overexpress KGF and its receptor.Moreover,overexpression of the mitogenic growth factors may contribute to the cancer cell proliferation in an autocrine or paracrine manner.More interestingly,aryl hydrocarbon receptor(AhR)has been reported to be activated by exogenous ligands could influence the KGF-induced regenerative growth in a zebrafish model.This indicated that an endogenous of AhR might participate in the signaling meditating by FGF.Furthermore,the AhR expression could be regulated by fibroblast growth factor(FGF)in Murine 3T3 fibroblasts.According to this,we speculated that the cancer cell proliferation would be enhanced by KGF stimulation,and KGF administration could up-regulate and activate AhR in cancer cell lines.Furthermore,to investigate the mechanism that KGF promotes colon cancer cell proliferation meditated by AhR.Methods:Firstly,Colon tumor and normal tissue samples from a series of 12 patients were acquired following the routine pathological examine.Real-time PCR analysis was used to detect the expression of KGF,AhR and CYP1A1.Immunohistochemistry was used to observe the localization of AhR.Secondly,Colon cancer cell lines(LoVo and T84 cells)were treated with(100 ng/ml)for 24 h.MTT assay and flow cytometric analyses were performed to measure cell proliferation,cell cycle.Colon cancer cell lines(LoVo and T84 cells)were treated with KGF(100 ng/ml)for 24 h.Immunofluorescence staining,western-blot and real-time PCR analysis were used to detect the expression of AhR,CYP1A1.Thirdly,to inhibit the KGF signaling pathway,an in vitro transfection was performed to silence AhR expression.MTT assay and flow cytometric analyses were performed to measure cell proliferation,cell cycle.Immunofluorescence staining,western-blot or real-time PCR analysis were used to detect the expression of cyclin D1,p Rb and CYP1A1.Results:1.The specimens from 11 of the 12 cancer cases,the KGF m RNA was higher.KGF treatment significantly increased the cell numbers and the percentage of S phase.2.The specimens from 11 of the 12 cancer cases,the AhR m RNA was higher,and from 8 of 12 cancer cases,the CYP1A1 m RNA was higher.In the specimens examined,high immunohistostaining was primarily located in the cytosol of human colorectal epithelial cancer.3.In colon cancer cells culture model,cells were stimulated with KGF for 24 h and observed an elevated(~1.6,~1.8)expression of AhR,CYP1A1 at protein level in LoVo cells and an elevated(~1.5,~1.6)expression of AhR,CYP1A1 at protein level in T84 cells.4.KGF also stimulated an approximately 1.67-fold increase in cell number,which was reduced to 1.10-fold by AhR knockdown in LoVo cells.And an approximately 1.44-fold increase in cell number,which was reduced to 1.14-fold by AhR knockdown in T84 cells.5.A significantly increased cyclin D1 expression in LoVo and T84 cells stimulated by KGF and apparently decreased cyclin D1 expression in the KGF+si AhR group compared with the KGF group were found.6.The LoVo and T84 cells were treated with KGF,there was a significant decrease(~ 27.81% and ~19.72%)in the percentage of cells in the G0/G1 phase compared with control cells at 24 h.However,there was a significant increase(~19.62% and ~17.19%)in the percentage of cells in the G0/G1 phase in KGF+ si AhR group compared with KGF group at 24 hConclusion:KGF administration could promote colon cancer cell proliferation,and AhR plays an important role in the modulation of KGF-induced colon cancer cell proliferation.AhR takes part in the modulation of KGF-induced cell proliferation through KGF/AhR/CyclinD1 signaling pathway.These findings are the first report of mechanism that KGF/AhR/CyclinD1 pathway involved in the regulation of colon cancer cell proliferation by KGF,which indicate that AhR may have a significant effect in the regulation of cancer cell proliferation.