Effects Of HS100A6 On Tumor Cells

Backgrounds and objectives:S100 protein family is a kind of calcium-binding protein with EF-hand, can be combined with calcium and play a variety of physiological functions involved in cell proliferation and differentiation, calcium homeostasis, protein phosphorylation,enzyme activity and transcription factor regulation . So far, at least 25 kinds of protein have been identified as S100 protein, of which 21 kinds located in chromosome 1q21. The chromosome segment is less stable and more rearrangements, and closely related with cancer. S100A6 is an S100 protein and its gene is in chromosome 1q21. There are S100A6 high expression in a variety of tumors, suggesting that it could be involved in the tumorigenesis and development.The deregulation of Wnt /β-catenin signaling pathway is closely related to the tumorigenesis and development. The high expression ofβ-catenin, the increased activity of Wnt/β-catenin signaling pathway and the high expression of S100A6 often present in tumor cells at the same time, but the exact relationship between them is not yet clear.In order to clarify the role of S100A6 in tumor development and to get the experimental evidence of S100A6 effect on Wnt /β-catenin signaling pathway, this study will explore hS100A6 effects on cell proliferation, apoptosis and Wnt /β-catenin signal pathway in seven tumor cell lines. They are murine melanoma cell line (B16), human ovarian cancer cell lines (low transfer line HO8910 and highly metastatic strain HO8910 PM), human colon cancer cell lines (HCT116 and SW480) and human osteosarcoma cells lines (MG63 and U2OS) . Methods:1. Preparation and identification of hS100A6 recombinant protein:1) Transform pGST-moluc and pGST-moluc-S100A6 into E Coli. (BL21) by CaCl2 method; 2) induce the expression of GST or GST-S100A6 by IPTG; 3) prepare the bacteria lysate by ultrasound on ice; 4) isolate and purify GST and GST-S100A6 from the lysate with the Glutathione Sepharose 4B beads. And 5) identify and quantify the recombinant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Bradford, respectively. Store in aliquots at -80℃.2. Seven kinds of tumor cell lines were treated with different concentrations (30, 100, 300μg/ml and 1000μg/ml) of GST-hS100A6, their proliferation was detected with MTT method.3. Based on the test results, an appropriate GST-hS100A6 concentration was selected to treat those tumor cell lines, the cell proliferation was detected with trypan blue exclusion staining method at different points(1,2,3,4 d and 5 d).4. Western blot, immunocytochemistry and immunofluorescence methods were used to detect the expression and distribution ofβ-catenin in those tumor cell lines treated with hS100A6.5. Tumor cell lines were transfected with pTOP-LUC and then were treated with GST-hS100A6. After 36 h, the cell lysate was collected and subjected to luciferase activity assays (pFOP-LUC as a negative control ).6. Immunocytochemical method was used to detect c-myc expression in B16, HCT116 and MG63 cell lines treated with GST-hS100A6.Results:1.Target fragment from digestion of pGST-moluc-hS100A6 by XhoⅠand EcoRⅠwas about 300bp, which was consistent with restriction enzyme map of pGST-moluc-hS100A6. The purity of recombinant protein reached 92% and specific positive reaction bands of anti-S100A6 appeared in Western blot analysis after induced expression, isolation and purification. It indicated that expression plasmid was correctly constructed and could be induced to express GST-hS100A6. The concentration of GST-hS100A6 was 5.67mg/L bacterium solution detected by bicinchoninic acid (BCA) protein assay. And GST is the control protein.2. Exogenous hS100A6 could inhibit the proliferation of seven tumor cell lines in a dose-dependent manner. For example, after treatment of GST-hS100A6 for 72 h, the OD value of 30, 100, 300μg/ml and 1000μg/ml group of B16 decreased 15%, 25%, 31% and 33% than GST group, respectively (P<0.05) while the differences between 3μg/ml and 10μg/ml groups were not significant (P>0.05).The effects of GST-hS100A6 of different concentrations on other six cell lines were consistent with B16.3. Exogenous hS100A6 could inhibit the proliferation of tumor cell lines in a time-dependent manner. For example, the viable counts of HO8910 PM group were not significant differences compared with GST group in the previous two days, and they were 71.4%, 68.0% and 63.7% of GST group in 3, 4 and 5 days(P<0.05).The effects of GST-hS100A6 of different time on other six cell lines were consistent with B16 HO8910.4. Exogenous hS100A6 incresed the apoptosis rate of seven tumor cell lines 1.43 to 4.3 times, which was significantly different compared with control groups(P<0.01). For example, the apoptosis rate of HO8910 cells after hS100A6 treatment increased 3.33 times compared with GST group(P<0.01). 5.β-catenin level of seven tumor cell lines analyzed by Western Blot increased 36.7%-52.1% after infected by AdS100A6 for 72h(P<0.01). For example, the gray value ofβ-catenin in B16 cells after infected by AdS100A6 for 72h increased 43.9% compared with that of AdGFP group, which was significant(P<0.01) while there were no obvious differences between AdGFP group and blank control (P>0.05).6. After treated by hS100A6,β-catenin expression levels of seven tumor cell lines increased 23.8%-52.7% respectively and the expression location was from cytoplasma to nuclei(by ICC). For example, in blank control of B16 cells,β-catenin mainly located in cytoplasma and nuclear membrane, less in nuclei. In GST-hS100A6 group after treatment for 72h, the staining was much stronger ( the IOD value increased 23.7% ) than GST group (P<0.01) and theβ-catenin increase was more obvious in nuclei than cytoplasma. It indicated that hS100A6 could generally increaseβ-catenin expression in cells and even promoteβ-catenin transport to nuclei.7. Analyzed by immunofluorescence assay, the total amount ofβ-catenin in B16, U2OS and MG63 after treated by GST-hS100A6 increased, which was consistent with results of ICC. For example,β-catenin of blank control in U2OS cells mainly located in cytoplasma and nuclear membrane, less in nuclei. The fluorescence intensity of cytoplasma and nuclear both enhanced after treated by GST-hS100A6.8. The transcription activities ofβ-catenin/TCF4 in GST-hS100A6 groups of seven tumor cell lines increased 6.4-14.3 times. For example, differences of the transcription activities ofβ-catenin/TCF4 in SW480 between GST-hS100A6 group and GST group after transfected pFOP-LUC were not significant (P > 0.05), while the transcription activities of experimental group were 6.5 times of corresponding GST group after transfected by pTOP-LUC (P<0.01). It indicated that S100A6 could up-regulate the transcription activities ofβ-catenin/TCF4 in these cell lines.9.The expression of c-myc analyzed by ICC in HO8910, MG63 and HCT116 after treated for 72h and increased 105.6%, 75.7% and 92.6%, respectively (P<0.01).Conclusions:1. pGST-Moluc-hS100A6 could express the soluble recombinant protein GST-hS100A6 in E. coli BL21 effectively, the purity could reach 92% after the isolation and purification by Glutathione - Sepharose 4B beads. And about 5.67 mg GST-hS100A6 protein was harvested in 1L bacterium solution.2. Exogenous S100A6 could inhibit the proliferation of seven tumor cell lines in a dose-dependent and time-dependent manner and could promote the apoptosis of tumor cell lines.3. S100A6 could lead toβ-catenin accumulation in the cytoplasm and shift into the nucleus, thereby enhanced the activity of intracellular Wnt /β-catenin signaling pathway, and turned on the expression of the downstream target gene c-myc. So, S100A6 could be a potential factor which lead to deregulation of Wnt /β-catenin signaling pathway in tumor cells.4. S100A6 has complicated biologic effects on tumor cells, just like to inhibit the proliferation of tumor cells as while as increase the activity of Wnt/β-catenin pathway. Every relative effects are synergetic or antagonistic to realize the impalpable modulation of S100A6 on osteosarcoma cells and to determine the final effects in cell level.