| Backgrounds and objectives:Breast cancer is the most common malignant tumor in female with an incidence rate of 7%-10% .The etiological factors are multiple which include age, environment, living habit, heredity and so on. As statistic data showed that there are 1.2 million women attacked by this disease and 500 thousands of them die of it each year. The average concealed stage of breast cancer is 12 years. Tumor can rapidly spread once it occurred. Patients are about to suffer the pain of chemotherapy and mastectomy. Better understanding of the molecular mechanisms of breast cancer is important not only for searching the early diagnosis and prognosis biomarkers but also for the finding the new targets of anticancer drugs.S100 protein family is a battery of calcium binding protein with low molecular weight, which play a important role in cell proliferation and differentiation, movement and cell cycle regulation.As yet, 25 kinds of S100 proteins was found, of which 21 kinds located in chromosome 1q21. This chromosome segment is less stable but more rearrangeable and closely related with cancer. S100A6 is a member of S100 protein family and also located in 1q21. S100A6 was highly expressed in various tumors,suggesting that it could be involved in the tumorigenesis and development. But the relationship between S100A6 and tumor is indefinite.Wnt/β-catenin signaling pathway is closely related to the tissue development and tumorigenesis,in whichβ-catenin is a pivotal protein. It displayed that the level ofβ-catenin was relevant to the tumorigenesis and development of breast carcinoma by immunohistochemisty in cancer tissue. Cyclooxygenase (COX) is key enzyme in prostaglandin synthesis.COX-2 is a member of COX, which can be induced to express by cytokines, interleukin, endotoxin, tumor oncogenes and other mitogenic agents. It plays a very important role in the pathogenesis of malignant tumors such as prostate cancer, colorectal cancer and breast cancer.This study will begin with detecting the expression of S100A6 in breast cancer tissues and cell lines; and then investigate the effects of human S100A6 on breast cancer cell MCF-7, including cell proliferation, apoptosis, migration and invasion; and investigate the effects of human S100A6 onβ-catenin and COX-2 expression in MCF-7. All we do will provide an experimental basis to clarify the roles and possible mechanisms of S100A6 in breast cancer. Methods1. Immunohistochemical analysis was used to detect the expression of S100A6 in breast cancer tissues and adjacent tissues.2. RT-PCR and immunocytochemistry analysis of highly metastatic breast cancer cell line (MDA-MB-231), breast cancer cell line (MCF-7) and transformed cell lines of breast (HBL-100) in the S100A6 mRNA and protein levels, respectively.3. Preparation of GST-hS100A6: comparison plasmid pGST-moluc and expression plasmid pGST-moluc-hS100A6 was transformed into E. coli BL21 with calcium chloride(CaCl2) method, then GST and GST-hS100A6 proteins were induced in BL21 by isopropyl-β-thiogalactoside (IPTG), ultrasound was used to prepare bacteria lysate and the proteins were purified by Glutathione-Sepharose 4B beads. Western blot, SDS-PAGE and Quantity One software were used to identify the purified GST and GST-hS100A6, BCA assay was used for protein quantitation, and the protein solution was filtered via 0.22μm membrane and then stored at -80℃for use.4. MTT assay was used to study the effects of hS100A6 on the proliferation of breast cancer line MCF-7 with different concentrations5. According to the results above, a suitable concentration (100μg/ml) of protein was chose. Colony-forming assay, Hoechst staining, Wound healing assay and Transwell chamber experiment were used to detect the abilities or activities of cell cloning formation, cell apoptosis, cell migration and invasion6. Western blot and immunocytochemistry were used to detect the expression and distribution changes ofβ-catenin and COX-2 in MCF-7 treated with hS100A6Results1. Immunohistochemical staining showed that there were S100A6 expression in both the breast cancer tissues and adjacent tissues, but the S100A6 expression in breast cancer tissues is 4.1 times of that in the adjacent tissues (P<0.05); simultaneously, S100A6 mainly located on the membrane and in the cytoplasm of adjacent tissues while mainly located in nucleus in the breast cancer tissues.2. RT-PCR showed that the S100A6 mRNA in MCF-7 is 1.9 times of that in HBL-100(P<0.05), S100A6 mRNA in MDA-MB-231 is 1.7 times of that in MCF-7(P<0.05) and 3.4 times of that in HBL-100(P<0.05). Immunocytochemistry showed that there were S100A6 in all the three cell lines, but the amount in MCF-7 is 2.3 times of HBL-100(P<0.05), the amount in MDA-MB-231 is 2.1 times of that in MCF-7 (P<0.05) and 5.3 times of that in HBL-100(P<0.05). All indicated that the level of S100A6 was positively correlated with the cell malignant degree.3. The recombinant protein we got was 36kDa, 94% purity detected by SDS-PAGE and recognized by S100A6 antibody by Western blot, BCA assay showed that 16.7 mg GST-hS100A6 was harvested from 1L bacteria. 4. MTT showed that after treatment for 48h, the A492nm value of 100μg/ml and 300μg/ml of GST-hS100A6 groups increased by 29.1%and 84.6%compared with GST group, respectively (P<0.05), which indicated that S100A6 can promote the cell proliferation of MCF-7, 100μg/ml concentration was selected for the following tests.5. Colony-forming assay showed that the cloning formation rate of GST-hS100A6 group increased by 38.7% compared with that of GST group (P<0.05), which indicated that S100A6 promote the cloning formation of MCF-7. The results of Hoechst staining showed that cell apoptosis rate of GST-hS100A6 group decreased by 67.8% at 24h(P<0.05) and by 58.4% at 48h compared with those of GST group. It indicated that S100A6 inhibit cell apoptosis. Wound healing assay showed that the healing rate in GST-hS100A6 group was 2.2 times of that in GST group (P<0.05) at 24h. It indicated that S100A6 promote cell migration. Transwell showed that the trans-membrane cell number of GST-hS100A6 group increased by 88.1% (P<0.05) compared with that of GST group at 24h, which indicated S100A6 promote cell invasion.6. Immunocytochemical staining showed that the S100A6 in GST-hS100A6 group was 1.5 times of GST group when adding GST-hS100A6 into MCF-7 for 24h, which indicated that GST-hS100A6 could enter into MCF-7 cells. The amount ofβ-catenin in GST-hS100A6 group was 1.8 fold of that in GST group(P<0.05) and theβ-catenin in nucleus increased; the COX-2 expression was 1.7 times of that in GST group (P<0.05).Western blot showed that intracellularβ-catenin levels increased by 50% compared with that of GST group(P<0.05), COX-2 level increased by 147% compared with that of GST group(P < 0.05). Results of immunocytochemistry and Western blot consistently indicated that S100A6 could increase the levels ofβ-catenin and COX-2.Conclusions1. S100A6 expression was found in normal breast tissues, mainly on the membrane and in the cytoplasm, but S100A6 expression in breast cancer tissues, in which most S100A6 was located in nucleus, was significantly higher than in normal breast tissue.2. There are S100A6 expression both in breast transformed cell lines and cancer cells, and the level of S100A6 was positively correlated with the cell malignant degree.3. The soluble recombinant protein GST-hS100A6 was effectively induced to express in E. coli BL21; the purity reached 94% after the isolation and purification by Glutathione-Sepharose 4B beads. And about 16.7 mg GST-hS100A6 protein was harvested in 1L bacterium solution.4. S100A6 can promote the cell proliferation, colony formation, migration, invasion, and inhibit cell apoptosis on human breast cancer cell line MCF-7, which suggested that S100A6 could have the promoting effects on human breast cancer.5. Exogenous S100A6 could upregulate the levels ofβ-catenin and COX-2 in MCF-7 cells, which indicated the promotional effects of S100A6 on breast cancer cells may be mediated at least in part via upregulatingβ-catenin and COX-2.
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