| Backgrounds and objectives:S100A8 is one of S100 protein family. S100 protein family is a class of low molecular weight of calcium-binding protein with EF-hand. So far, there are 25 types of S100 protein, of which 21 kinds located in chromosome 1q21, including S100A8. This chromosome segment is less stable and more rearrangements, and closely related with cancer. The physiological roles of S100 protein family include cell proliferation and apoptosis, enzyme activity and protein phosphorylation and so on. It was found that there are abnormal expressions of some S100 proteins in tumor cells and these S100 proteins are involved in the tumor cell proliferation, apoptosis and migration and invasion.S100A8 is a multifunctional molecule, which usually form a heterodimer with S100A9. It was first discovered as an immunoglobulin. Recent studies showed that: S100A8 alone could also play a biological role. S100A8. S100A8 is also involved in myeloid cell differentiation, embryonic development and immune and inflammatory reactions. There are abnormal expressions of S100A8 in some tumor cells and infiltration of immune cells, suggesting that it could be involved in the tumorigenesis and development of inflammation-related cancer.Cervical cancer is the second most common malignancy of women in the world, and the death rate is only second to breast cancer. According to statistics, there are about 20 million women died from this disease each year and the age of incidence is gradually younger. The processes of cervical cancer which includes normal cervical epithelium, atypical hyperplasia to carcinoma in situ and then invasive carcinoma involves multi-factor and multiple genes. Although research showed that more than 90% of cervical cancer were associated with human papillomavirus (HPV) infection, but the exact pathogenesis of cervical cancer remains unclear.According to the points which just mentioned, we investigate the effects(proliferation, apoptosis, colony formation and migration and invasion) and mechanisms of S100A8 on Hela cell lines, which can provide experimental evidences to clarify the role of S100A8 in the development of cervical cancer and its related mechanism.Methods:1. Preparation and identification of recombinant protein GST-hS100A8: pGST-Moluc-hS100A8 was transformed in E Coli BL21, after induced by IPTG,GST-hS100A8 was purified by Glutathione Sepharose 4B beads and identified by SDS-PAGE and Western Blot. Its concentration was determined by BCA method. Store in aliquots at -80℃.2. Amplification of the recombinant adenovirus(AdS100A8) and determination of its titer : AdS100A8 was amplified in HEK293 cells, then the viral titer was determined.3. Detection the effects of S100A8 on Hela:When the concentration of GST-hS100A8 was 100μg/ml, MTT was used to detect the cell proliferation; Hoechst staining was used to detect the cell apoptosis; Flow cytometry was used to detect the variation of cell cycle; Colony-forming assay was used to detect the ability of colony formation; Wound healing assay and Transwell chamber experiment were used to detect the cell migration and invasion ability.4. Detection ofβ-catenin:Western blot,Immunofluorescence and immunohistochemistry were used to detect the expression and distribution ofβ-catenin in Hela which was treated with exogenous S100A8.5. Detection of MAPKs: Western blot was used to detect the expression of Phosphorylation of P38,ERK and JNK of MAPKs in Hela which was treated with GST-hS100A8(100μg/ml).6. Detection the growth of subcutaneous tumor in nude mice : Subcutaneous tumor model was used to observe the formation, the volume and weight of subcutaneous tumor .Results:1.It was displayed that the purity of recombinant protein (GST-hS100A8) was about 94%by SDS-PAGE and the recombinant protein was specific recognized by antibody of hS100A8 by Western blot. It indicated that the plasmid was successfully induced to express GST-hS100A8. Bradford method showed that 13.5mg GST-S100A8 was obtained from 1L bacteria culture.2. The recombinant adenovirus (AdS100A8) can be amplified in HEK 293 cells and then the titer was 1011 IU/ml.3. GST-hS100A8 inhibited cell proliferation, colony formation, migration, and invasion and induced cell apoptosis of Hela cell line.(1) After treated with GST-hS100A8 for 3d, the A value of 100, 300 and 1000 mg/L group of GST-hS100A8 decreased by 13.64%, 19.29% and 25.06% compared with GST group, respectively (P<0.05) .In the group of 100 mg/L, the A value of GST-hS100A8 decreased in a time-dependent manner(P<0.05).(2) At 72h, cell apoptosis rate in GST-hS100A8 group increased by 5.18 times compared with GST group (P<0.05). Meanwhile, the apoptosis peak value of GST-hS100A8 group was (19.90±0.76) % by FCM, while the groups of GST and blank had no apoptotic peak.(3) The effect of GST-hS100A8 on cell cycle was not significant.(4) The colony formation rate of GST-hS100A8 decreased by 30.2% (P<0.05).(5) The healing rate of GST-hS100A8 group reduced by 30.1% compared with GST group (P<0.05).(6) The transmembrane cell number of GST-hS100A8 group reduced by 48.9 %( P<0.05) compared with GST group.These results indicate that S100A8 could have the inhibitive effects on cervical cancer.4.GST-hS100A8 could increase theβ-catenin level andβ-catenin translocation from cytoplasm into nuclear in Hela cell line.(1) In Western blot, the gray value ofβ-catenin increased by 70.1% and 75.6%(P<0.01)after treated with S100A8 for 24h and 48h ,respectively.(2)Compared with GST group, the fluorescence intensity ofβ-catenin in GST-hS100A8 group was stronger and the fluorescence intensity in nuclear was stronger than that in cytoplasm.(3)Immunohistochemistry results showed the brown particles in AdS100A8 group was more than those of AdGFP group, especially in nuclear; the average optical density ofβ-catenin of AdS100A8 group increased by 2.31 times(P<0.01)compared with AdGFP group.5.In Western blot, the gray value of the phosphorylation of P38, ERK in GST-hS100A8 group increased 43% and 78.1%(P<0.01)respectively compared with GST group, which indicated GST-hS100A8 could increase phosphorylation of P38 and ERK of MAPKs in Hela cell line.6. The results from subcutaneous tumor model showed the rate of tumor formation was 80%. From 21d, the tumor volume in AdhS100A8 group was less than that of AdGFP group(P<0.01). At 40d,the the volume and weight of tumor of AdS100A8 group reduced by 65.3% and 1.7 times (P<0.01)respectively, compared with AdGFP group. There was no statistically significant between AdGFP group and blank group.Conclusions:1. Recombinant protein (GST-hS100A8) was successful preparation, 13.5 mg GST-hS100A8 obtained from 1L bacterial culture and the purity was 94%. It has biological activity identified by Follow-up test.2. The recombinant adenovirus (AdS100A8) was amplified in HEK 293 cells successfully and its titer was 1011 IU/ml.3. Exogenous S100A8 can inhibit cell proliferation, colony formation, migration, and invasion and induce cell apoptosis of Hela cell line in vitro.4. Exogenous S100A8 can inhibit the growth of subcutaneous tumor in nude mice.5. Exogenous S100A8 can increase the expression ofβ-catenin and transferβ-catenin from cytoplasm into nuclear in Hela cell line.6. Exogenous S100A8 can increase the phosphorylation levels of P38 and ERK of MAPKs in Hela cell line.In a summary, S100A8 has the inhibitive effects on cervical cancer Hela cell line and these effects may be mediated by up-regulation of Wnt/β-catenin and partly activating MAPKs signaling pathway.This study provides an experimental data to illustrate the effects and mechanisms of S100A8 on cervical cancer.
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