Effects Of HS100A2 And HS100A6 On Human Colorectal Cancer Cell Line HCT116 And Human Osteosarcoma Cell Line MG63

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 ofβ-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, XhoⅠand 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 40μg/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 ofβ-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 (P<0.05); the inhibition on the cell proliferation of HCT116 in AdS100A6 group began from the 2nd day after treated with AdS100A6(P<0.05). Recombinant hS100A6 protein and AdS100A6 had similar inhibitory effects on HCT116 cell proliferation. The effects of hS100A2 and hS100A6 on MG63 cell proliferation were similar to those on HCT116 cells.These results indicated that both S100A2 and S100A6 had inhibitory effects on cell proliferation of HCT116 cells and MG63 cells.4. The effects of exogenous S100A2 and S100A6 on cell cycle of HCT116 cells and MG63 cells. .HCT116 cell numbers of G0~G1 stage and S stage in experimental group (72h after treated with recombinant hS100A2) were 121% and 58% of those in GST group, respectively (p<0.05). The recombinant hS100A6 protein had similar results for HCT116 cell cycle (p<0.05). HCT116 cell numbers of G0~G1 and S stages in AdS100A6 group were 134%(p<0.05) and 59%(p>0.05) 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 (P<0.05). The apoptosis number of HCT116 cells in AdS100A6 group was 1.13 times as that of AdGFP group (p>0.05) 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<0.01). It showed that S100A2 could obviously inhibit the migration of the two cell lines. The recombinant hS100A6 protein and AdS100A6 showed the similar inhibitory effects to hS100A2 on HCT116 and MG63 cell migration.7. The average fluorescent intensities of MG63 cells in hS100A2 group and hS100A6 group were 1.5 and 1.6 times as that of the control, respectively (p<0.05). Under the LSCM and LM,β-catenin in control group was mainly located in cytoplasm, nuclear membrane and nucleus; In the nucleus, there was 1~2 bright spots of fluorescence, which seemed thatβ-catenin in nucleus was concentrated at nucleolus. The nuclear membrane was intact. After treated with either hS100A2 or hS100A6,β-catenin level increased in both cytoplasm and nucleus, butβ-catenin on nuclear membrane declined, even disappeared. In the nucleus, there were 3~4 bright spots of fluorescence, which also seemed thatβ-catenin in nucleus was concentrated at nucleolus. The nuclear membrane turned discontinuous. The nucleolus increased to 3 or 4 in most cells.CONCLUSIONS:1. Both S100A2 and S100A6 have effects of inhibiting proliferation, blocking cell cycle and promoting apoptosis on human colorectal cancer cell line HCT116 and human osteosarcoma cell line MG63. Blocking cell cycle and promoting apoptosis might contribute to their inhibitory effects on cell proliferation.2. Both S100A2 and S100A6 could significantly inhibit the migration of HCT116 cells and MG63 cells; and S100A2 showed stronger inhibitory effects than S100A6.3. Both S100A2 and S100A6 could increaseβ-catenin level in cytoplasm and nucleus, and change its distribution in MG63 cells:β-catenin decreased in nuclear membrane while the total level ofβ-catenin in cells increased. Furthermore, it seemed thatβ-catenin in nucleus was located at nucleolus.4. Both recombinant hS100A6 proteins and AdS100A6 produced similar biological effects on cells (inhibiting proliferation, blocking cell cycle, promoting apoptosis and changing the expression ofβ-catenin). It indicated that S100A6 was a new member with extracellular roles in S100 family and its extracellular and intracellular effects on the two cell lines were similar.In summary, we found both S100A2 and S100A6 could inhibit cell proliferation, block cell cycle, and promote apoptosis of human colorectal carcimoma cell line HCT116 and human osteosarcoma cell line MG63. Blocking cell cycle and promoting cell apoptosis might contribute to the inhibition of cell proliferation. We also found that both S100A2 and S100A6 could obviously inhibit the migration of the two cell lines. In addition, both S100A2 and S100A6 could increase and redistributeβ-catenin in the two cell lines. Moreover we found that S100A6 had extracellular roles. These results provided some evidences to elucidate the effects and the mechanisms of S100A2 and S100A6.