Effects of hS100A2 and hS100A6 on Human Colorectal Cancer Cell ...

[Abstract]

BACKGROUNDS AND OBJECTIVES:The pathogenesis and development of tumor is a complex biologic process that involves various factors and numerous genes, including many signal molecules. Therefore, it is very important to study the effects and the mechanisms of tumor-related signal molecules, and the reciprocal relationships among these signal molecules for elucidating the mechanisms of the pathogenesis and development of tumor.The S100 proteins are small acidic signaling proteins (9?13 kDa) that are found exclusively in vertebrates.With at least 25 members found to date in humans, the S100 proteins constitute the largest subfamily of the EF-hand proteins. S100 proteins are proposed to have intracellular and extracellular roles in the regulation of many diverse processes such as protein phosphorylation, cell growth and motility, cell-cycle regulation, transcription, differentiation and cell survival. 21 members (S100A1?S100A18, trichohylin, filaggrin and repetin) of these proteins have genes clustered at chromosome locus 1q21, which has low stability. A potential role for S100 proteins in neoplasia stems from these activities and from the observation that several S100 proteins (including S100A2 and S100A6) have altered levels of expression in different stages and types of cancers. But the precise roles and the importance of S100 proteins in the development and promotion of cancer are poorly understood.The S100A2 is often downregulated in cancers, including melanoma, breast cancer, prostate cancer, lung tumors, so on. Furthermore, it has been indicated as a potential tumor suppressor by differential hybridization. But there are still opposite reports.The S100A6 protein was first purified from Ehrlich ascites tumor by Kuznicki and Filipek. The overexpression of S100A6 has been reported in several types of cancers, such as epithelial carcinomas (colon cancer, hepatocellular cancer, cholangiocarcinoma, pancreatic cancer and malignant melanoma), osteosarcoma, ovarian cancer, ras tansformed NIH3T3 cells and SV40 transformed mouse fibroblast. But the exact effects of S100A6 in cancers remain unknown.In order to elucidate the effects and the mechanisms of S100A2 and S100A6 on human tumors and to find some new markers for tumor diagnosis and prognosis, and some new targets for tumor therapy, the effects of S100A2 and S100A6 on cell proliferation, cell cycle, apoptosis and migration of human colorectal cancer cell line HCT116 and human osteosarcoma cell line MG63 were investigated; Furthermore, the effects of S100A2 and S100A6 on the level and distribution of?-catenin in MG63 cells were studied.METHODS1. The preparation and identification of recombinant hS100A2 and hS100A6 protein: pGST-Moluc-hS100A2 and pGST-Moluc-hS100A6 were digested by restriction endonucleases, Xho?and EcoR, and then analyzed by agarose gel electrophoresis. E Coli. (BL21) was transformed with the two plasmids respectively and the expressions of GST-hS100A2, and GST-hS100A6 were induced by IPTG. Recombinant hS100A2 and hS100A6 proteins were purified by Glutathione Sepharose 4B beads and identified by SDS-PAGE/ Western Blot. Their concentrations were determined by Bradford.2. The recombinant adenoviruse of S100A6 (AdS100A6) was amplified in human embryonic kidney cell 293(HEK293 cell); hS100A6 protein in the cell lysate of HCT116 infected by AdS100A6 was detected by SDS-PAGE/ Western Blot.3. HCT116 cells and MG63 cells were treated with GST, GST-hS100A2, GST-hS100A6, AdGFP, and AdS100A6, respectively. The final concentration of all proteins was 40?g/ml. Cell proliferation was measured by MTT method and trypan blue stain/cell counting; And cell cycle(PI stain) and cell apoptosis(annexin V-PE and 7AAD stain) were detected by flow cytometry; the cell migration ability was determined by transwell chambers; the changes of?-catenin expression were observed under laser scanning confocal microscope (LSCM) after immunofluorescence stain.RESULTS1. The two plasmids were digested into two fragments, about 300bp and 9kb, respectively. The results were consistent with their maps of restriction endonucleases. hS100A2 and hS100A6 were found in the two purified recombinant proteins ( SDS-PAGE/ Western blot), respectively. The yields of recombinant hS100A2 and hS100A6 were 5mg/L and 3mg/L, respectively (Bradford).2. hS100A6 protein was found in the cell lysate of HCT116 infected by AdS100A6( SDS-PAGE/ Western blot).3. The effects of exogenous S100A2 and S100A6 on cell proliferation of HCT116 cells and MG63 cells.The proliferation of HCT116 cell was inhibited from the 4th day and 5th day after respectively treated with recombinant hS100A2 and hS100A6 proteins (P0.05) of those in AdGFP group, respectively. The effects of hS100A2 and hS100A6 on MG63 cell cycle were similar to theirs on HCT116 cell cycle.These results indicated that both S100A2 and S100A6 might block cell cycles of HCT116 cells and MG63 cells by increasing G0~G1 stage cells and decreasing S stage cells. Recombinant hS100A6 protein and AdS100A6 had similar activities on cell cycles of the two cell lines.5 The effects of exogenous S100A2 and S100A6 on cell apoptosis of HCT116 cells and MG63 cellsAfter HCT116 cells were cultured with recombinant hS100A2 and hS100A6 protein for 48 hours, the apoptosis cell numbers were 2.3 times and 2.0 times as those of GST control groups, respectively (P0.05) and showed a tendency of increase.These results indicated that both S100A2 and S100A6 could promote the apoptosis of HCT116 cells and MG63 cells.6. The effects of exogenous S100A2 and S100A6 on cell migration of HCT 116 cells and MG63 cellsCompared with their own controls, the cell numbers of trans-membrane of HCT116 cells and MG63 cells after treated with recombinant hS100A2 protein for 60 hours decreased by 75 % and 83%, respectively(P

Title: Effects of hS100A2 and hS100A6 on Human Colorectal Cancer Cell Line HCT116 and Human Osteosarcoma Cell Line MG63

Category: Pituitary Tumor

Filename: Effects of hS100A2 and hS100A6 on Human Colorectal Cancer Cell Line HCT116 and Human Osteosarcoma Cell Line MG63.pdf

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