| BACKGROUNDS AND OBJECTIVES:The tumorgenesis and progression is a complex biologic process in which various factors and numerous genes are involved, including many signal molecules. The role and the possible mechanisms of these signaling molecules in this process, and the inter-relationship between these signaling molecules have not yet been clarified.The S100 proteins with at least 25 members constitute the largest subfamily of the EF-hand proteins, some members of S100 proteins have been found in tumor cells with abnormal expression and involved in the tumor cell proliferation, apoptosis and migration. S100A6 gene is located at chromosome locus 1q21, which is often abnomal in osteosarcoma patients. At the same time, there is the increased expression of S100A6 in osteosarcoma. It suggests that S100A6 may be involved in the tumorgenesis and progression of osteosarcoma. But the effects of S100A6 and its mechanism on osteosarcoma are not yet clear and should be further studied.Wnt/β-catenin signaling pathway is a classic pathway, which is closely related to tumors. Overexpression ofβ-catenin, which is a central molecule of the Wnt/β-catenin signaling pathway, can cause the activation of this pathway and lead to the occurrence of tumor. Osteosarcoma is no exception.As there are the overexpressions of SA100A6 andβ-catenin in osteoasarcoma at the same time and our previous study found that there were the direct specific interaction between S100A6 and some effector molecules of Wnt signaling pathway (includingβ-catenin), we supposed that the effects of S100A6 on osteosarcoma cell lines may be related to Wnt/β-catenin signaling pathway. Therefore, we will investigate the effects of S100A6 on the expression ofβ-catenin.In order to further investigate the effects of S100A6 on osteosarcoma cells and the possible mechanisms and/ or signal pathways, recombinant protein of hS100A6 and adenoviruses with the genes of hS100A6, SiRNA- hS100A6,β-catenin, SiRNA-β-catenin were used to treat osteosarcoma cells and the effects of S100A6 on the cell proliferation, migration and apoptosis of osteosarcoma cells and the influences of S100A6 onβ-catenin were studied. Then the function ofβ-catenin on these effects were further validated by up-regulation and down-regulation ofβ-catenin.METHODS:1. The preparation and identification of recombinant GST-hS100A6 protein: pGST-Moluc-hS100A6 was transformed in E Coli. (BL21), then induced by IPTG,purified by Glutathione Sepharose 4B beads and identified by SDS-PAGE/ Western Blot. Their concentrations were determined by BCA method.2. The recombinant adenoviruses with hS100A6 gene (AdS100A6), hS100A6-SiRNA gene (AdSiS100A6),β-catenin gene(Adβ-catenin) andβ-catenin-SiRNA gene (AdSiβ-catenin) and control adenoviruses (AdGFP and AdRFP) were amplified in human embryonic kidney cell 293 (HEK293 cell).3. GST-hS100A6(100μg/ml) was added in human osteosarcoma cell lines MG63 and U2OS, MTT and Trypan blue staining were used to detect the cell proliferation; hoechst staining was used to detect the apoptosis; Wound healing assay and Transwell chamber experiments were used to detect the cell migration.4. Adenovirus carrying the human S100A6 and its siRNA gene were used to up-regulate and down-regulate the expression of S100A6, respectively. RT-PCR was used to detectedβ-catenin mRNA; Western blot and immunocytochemitry were used to detect the protein level and distribution ofβ-catenin.5. Adenovirus carrying the humanβ-catenin and its siRNA gene were used to up-regulate and down-regulate the expression ofβ-catenin, respectively. Cell proliferation was measured by MTT method; Cell apoptosis was detected by hoechst staining; Cell migration ability was determined by wound healing assay and transwell chambers.RESULTS:1. After expression of recombinant protein GST-hS100A6 was induced, purificated, and then identificated by SDS-PAGE / Western blot, a specific positive protein band was found in the corresponding position, which showed that the recombinant protein GST-hS100A6 was successful prepared. The concentration of GST-hS100A6 was 5.67 mg/L.2. The green or red fluorescence protein was expressed in 293 cells infected by adenovirus. The infection rate was about 90% after infection for 72 h and the titer of virus solution was about 108-109 pfu / ml.3. The expression of S100A6 was confirmed by SDS-PAGE/Western blot. In MG63 cells infected with AdS100A6 or Adβ-catenin, S100A6 increased 100% than the blank groups andβ-catenin increased 85%. In MG63 cells infected AdSiS100A6 or AdSiβ-catenin, S100A6 reduced 31.34% andβ-catenin reduced 63%. These results showed the genes mediated by adenoviruses could express well in target cells.4. Exogenous S100A6 could inhibit cell proliferation and migration and promote apoptosis in osteosarcoma cells.(1) MTT showed that the OD values of the experimental group were significantly lower than GST group (experimental control) at 48, 72 and 96 h. Proliferation inhibition rates of the experimental group were 25.51%, 16.28% and 11.63% (P <0.05) in MG63 and 29.41%, 15.56% and 11.49% (P <0.05) in U2OS, respectively.The consistent results have been found in Trypan blue staining.(2) Hoechst assay results showed that the apoptosis rate of GST-hS100A6 group increased than GST group at 24, 48 and 72 h in both cells. Compared with GST group, the apoptosis rate of experimental group increased 135.3%, 45.3% and 45.8% (P <0.05) in MG63 cells and 39%, 65.1% and 78.8% in U2OS cells, respectively.(3) Wound healing assay showed that compared with GST group at 24 and 48 h, the healing rate of the experimental group reduced 24.74% and 26.25% (P<0.05), respectively. The results from Transwell experiment were consistent with Wound healing assay and the trans-membrane cell number of experimental group reduced 37.6% than the control group at 48 h (P< 0.05).The healing rates of the blank group were 79.74% in MG63 and 17.74% in U2OS at 24 h; and 100% in MG63 and 29.03% in U2OS at 48 h.These results suggested: 1) that exogenous hS100A6 could inhibit proliferation and promote apoptosis in both MG63 and U2OS cells;2) that exogenous hS100A6 could inhibit MG63 cell migration, but no significant effect on the U2OS cell migration; 3)at the same time, the migration ability of U2OS cells was inferior to that of MG63 cells.5. S100A6 increased the expression ofβ-catenin in osteosarcoma cells MG63 and U2OS(1) With the increased expression of S100A6,β-catenin mRNA levels increased 9% in MG63 and 37% in U2OS, respectively (RT-PCR, P <0.05);β-catenin protein increased 31% in MG63 and 37% in U2OS, respectively (Western blot, P <0.05); Immunocytochemy showed thatβ-catenin protein increased in both cytoplasm and nucleus and the variation was more obvious in nucleus.(2) With the reduced expression of S100A6,β-catenin mRNA levels decreased 22% in MG63 and 21% in U2OS, respectively (P <0.05),β-catenin protein expression decreased 38% in MG63 and 46% in U2OS, respectively (Western blot, P <0.05). Immunocytochemy showedβ-catenin protein decreased in both cytoplasm and nucleus and the variation in nucleus was more obvious, too.These results suggested that S100A6 could up-regulate the mRNA and protein levels ofβ-catenin in human osteosarcoma cell lines U2OS and MG63 and promote transposition ofβ-catenin from cytoplasm to nucleus.6.β-catenin reduced the ability of S100A6 to inhibit proliferation and migration and enhanced the ability of S100A6 to promot apoptosis.After up-regulation ofβ-catenin expression:(1) The ability of S100A6 to inhibit cell proliferation was reduced MTT showed that with up-regulation ofβ-catenin, the OD values of the experimental groups (Adβ-catenin + GST-hS100A6 group) were higher than control group (GST-hS100A6 group). For example, in MG63, compared with the control group at 48, 72 and 96 h, the OD values of experimental group increased 69.86%, 50% and 50.15% (P <0.05), respectively.(2) The ability of S100A6 to promot apoptosis was enhanced Compared with the control group, cell apoptosis rate in experimental group increased. For example, at 24, 48 and 96 h, apoptosis rate in experimental group of MG63 increased 30.25%, 48.83% and 10.81% (P <0.05), respectively.(3) The ability of S100A6 to inhibit migration was weakened Compared with the control group at 24 and 48 h, the healing rate in experimental groups increased 24.16% and 26.25%, respectively (P<0.05); Transwell showed that the trans-membrane cell number of experimental group reduced 35.45% than the control group at 48 h(P< 0.05).After down-regulation ofβ-catenin expression:(1) The ability of S100A6 to inhibit cell proliferation was increased. MTT showed that with down-regulation ofβ-catenin, the OD values of the experimental groups (AdSiβ-catenin + GST-hS100A6 group) were lower than control group (GST-hS100A6 group). For example, compared with the control group of MG63 at 48, 72 and 96 h, the OD values of experimental groups decreased 15.07%, 22.23% and 52.97% (P<0.05), respectively.(2) The ability of S100A6 to promot apoptosis was reducedCell apoptosis rates in experimental groups decreased than the control group. For example, compared with the control group of MG63 at 24, 48 and 96 h, apoptosis rate in experimental group decreased 30.25%, 48.83% and 10.81% (P <0.05), respectively.(3) The ability of S100A6 to inhibit migration was enhanced Compared with the control group at 48 h, the healing rate in experimental decreased 5.88%(P <0.05); The trans-membrane cell number of experimental group reduced 25% than the control group at 48 h(P> 0.05).These results suggested thatβ-catenin could①significantly reduce proliferation inhibition of S100A6 on MG63 and U2OS cells;②enhance the ability of apoptosis promotion of S100A6 on MG63 and U2OS cells;③reduce the ability of migration inhibition of S100A6 on MG63 cells.Conclusions:1. S100A6 could inhibit proliferation and promote apoptosis in both MG63 and U2OS cells.2. S100A6 could inhibit migration of MG63 cell, but exerted no significant effects on the migration of U2OS cells.3. S100A6 could increase mRNA and protein levels ofβ-catenin in human osteosarcoma cell lines MG63 and U2OS and promote transposition ofβ-catenin from cytoplasma to the nucleus. The high expression of S100A6 may be the cause of increased expression ofβ-catenin and one of the deregulation mechanisms of the Wnt /β-catenin signaling pathway in osteosarcoma.4.β-catenin could significantly reduce the ability of S100A6 to inhibit cell proliferation and migration and enhance its ability to pomote apoptosis in osteosarcoma.5. Based on all the results, we supposed that the suppressive effects of S100A6 on osteosarcoma may be mediated by other signaling pathways and were finely adjusted by mutual antagonism between these signaling pathways and Wnt /β-catenin signaling pathway.This study provides an experimental data to illustrate the effects of S100A6 on osteosarcoma cells and the functions ofβ-catenin on these effects.
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