Effects Of IGFBP-7 On Expression Of Cx43 In Tumor-stroma Interaction Of Colorectal Carcinoma

Colorectal cancer(CRC) is one of the prevalent malignant tumors that threaten our health.In spite of the development of radiotherapy,chemotherapy and bio-targeted therapy,the mortality declines indistinctively.In China,the incidence grows rapidly, especially in ZheJiang,ShangHai and JiangSu provinces.The low early detection rate is one of the important factors affect the cancer therapy.To improve the early detection rate will promote the cancer therapy.Malignant tumors are composed of tumor cells and stromal cells.The researches mainly focused on the tumor cells.However,the stromal cells receive increasing attention because of their participation at tumor development,progression and metastasis,and of their response to therapy.Fibroblasts are ubiquitous stromal cells which have direct and indirect effects to the tumor cells.Direct interaction includes gap junction,intermediate junction,desmosome and tight junctions,which play vital role in cell growth,differentiation and migration. Connexin 43(Cx43) is a component of gap junction(GJ) channels,related to tumor. development and progression.It also inhibits the development of cancer by inducing the apoptosis of the adjacent cells.As Cx43 are expressed widely in SW480,SW620,RKO and HT-29 CRC lines, we chose SW480 and SW620 as our experiment models because they were derived from the different origin of single patient.We co-culture them with human skin fibroblast(HSF) respectively.By RT-PCR,we found Cx43 expression level got increased after co-culture,and HSF was also activated to be cancer-associated fibroblasts(CAFs) which could prompt tumor cells' growth.For the indirect tumor-stroma interaction,we chose TGF-βsignaling pathway which has been reported to promote cell migration in tumor-stroma interaction.In our pervious study,we found a secreted protein -- IGFBP-7 was expressed intensively in CRC than normal colon mucosa.It had been confirmed that the mechanism for its tumor suppressor role is mainly promoting apoptosis and aging,inhibiting the metastasis of tumor cells,etc.We stimulated SW480 and SW620 with TGF-β1 and found IGFBP-7 expression was increased,implying that TGF-βsignaling played roles in IGFBP-7 expression.Meanwhile,we also found the treatment increased Cx43 expression in SW480 cells.As we have found Cx43 and IGFBP-7 expressed intensively in tumor infiltrating front than in its center by immunohistochemistry,we chosed CW2 and RKO cell lines to see the effects of IGFBP-7 and Cx43.IGFBP-7 was transfected into those cells and the Cx43 mRNA and protein were detected.IGFBP-7 transfection increased Cx43 mRNA and protein expression in CW2 cells,but decreased mRNA expression in RKO cell lines.The expression of P-Smad2 in TGF-β1 signaling was decreased after transfection of IGFBP-7 into CW2 cells and RKO cells,implying that Cx43 expression was increased by inhibiting TGF-β1- Smad2 cell signaling while promoting other TGF-β1 cell signaling.Cx43 locate at the cell membrane of normal cells while they move to cytoplasm in cancer cells.We found Cx43 expressed at cytoplasm in both IGFBP-7 transfected RKO cell and pre- IGFBP-7 transfected RKO cell.We get conclusion from the above results: 1.SW480/SW620-HSF co-culture increased Cx43 expression,prompted growth of SW480 and SW620 and activation of HSF cells.2.TGF-β1 increased expression of IGFBP-7 and Cx43 in SW480 cells mRNA level and the expression of IGFBP-7 in SW620 cells.3.IGFBP-7 increased the expression of Cx43 in CW2 cells but decreased Cx43 mRNA level in RKO cells.