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Objective: To deeply investigate the effect of S100A6 in the microenvironment on migration of colorectal carcinoma LoVo cells, and to explore the possible molecular mechanism. Methods: The recombinant protein glutathione S-transferase (GST) and the fusion protein GST-human S100A6 (GST-hS1 00A6) were prepared and identifed. After the treatment with GST-hS1 00A6 (30 (μg/mL), the migration of LoVo cells was detected by wound healing assay, the expressions of total protein kinase B (PKB, also known as Akt) and phosphorylation of Akt (p-Akt) in LoVo cells were detected by Western blotting. After the treatment with GST-hS1 00A6 and phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway inhibitor LY294002 alone or in combination, the migration of LoVo cells was detected by wound healing assay. After the macrophages were treated with the conditioned medium of LoVo cells stimulated by GST-hS1 00A6 (named as CM-A6-LoVo), the expression levels of M2 phenotype marker CD206 and M1 phenotype marker inducible nitric oxide synthase (iNOS) in the macrophages were detected by real-time fuorescent quantitative PCR, and the migration ability of LoVo cells co-cultured with the macrophages was detected by wound healing assay. Results: GST-hS100A6 and GST protein (as a control) were successfully prepared. Compared with GST group, the wound healing rate of LoVo cells treated with GST-hS1 00A6 was significantly elevated (P 0.05), and the macrophages co-cultured with LoVo cells could significantly promote the migration of LoVo cells (P < 0.05). Conclusion: S100A6 in microenvironment can directly and indirectly promote the migration of colorectal carcinoma LoVo cells, which maybe related to the regulation of PI3K/Akt signaling pathway and the induction of macrophage polarization to M2 phenotype.
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Objective: To investigate the expressions of S100A6, annexin A2 (AnxA2) and c-myc in patients with multiple myeloma (MM) and their clinical significance. Methods: The expressions of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from 28 cases of MM before and after chemotherapy and 20 controls (whose peripheral blood white cell count and the platelet count were a little lower than the normal values, but the result of bone marrow aspiration was normal) were detected by real-time fluorescent quantitative PCR. The relationships among the expressions of S100A6, AnxA2 and c-myc mRNAs were analyzed. The expressions of S100A6, AnxA2 and c-myc mRNAs and proteins in MM U266 cells after transfection with S100A6 siRNA were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. Results: The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from patients with MM were higher than those from the controls (all P < 0.05). The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from patients with MM before chemotherapy were higher than those after chemotherapy (all P < 0.05). The expression levels of S100A6, AnxA2 and c-myc mRNAs in bone marrow mononuclear cells from MM patients with extramedullary metastasis were higher than those from MM patients not having extramedullary metastasis (all P < 0.05). The expression of S100A6 mRNA was positively correlated with the expressions of AnxA2 and c-myc mRNAs (r = 0.585, P = 0.001; r = 0.540, P =0.004). The expression levels of S100A6, AnxA2 and c-myc mRNAs and proteins in MM U266 cells after transfection with S100A6 siRNA were down-regulated (all P < 0.05). Conclusion: The expression level of S100A6 in patients with MM is higher, and it is positively associated with AnxA2 and c-myc. S100A6 may be involved in the development, progression and extramedullary metastasis of MM.
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Objective To evaluate the potential of S100 calcium binding proteins S100A2 and S100A6 levels in serum as diagnostic markers for non-small cell lung cancer (NSCLC). Methods Enzyme-linked immunosorbent assay (ELISA) was performed to detect the levels of S100A2 and S100A6 in 141 NSCLC patients and 150 healthy subjects. Results The average level of serum S100A2 in NSCLC group was (15.02±0.79) ng/ml, that in healthy control group was (11.18±0.64) ng/ml, and the difference was statistically significant (P=0.002). The average level of serum S100A6 in NSCLC group was (12 760±651.8) pg/ml, that in healthy control group was (8 434±408.2) pg/ml, and the difference was statistically significant (P<0.001). The serum levels of S100A2 and S100A6 in stageⅠ/ⅡNSCLC patients were higher than those in healthy controls (P<0.05), respectively. Receiver operating characteristic (ROC) curve analysis showed that S100A2 and S100A6 could distinguish NSCLC patients from healthy controls [area under the curve (AUC) were 0.646 and 0.668, respectively]. Meanwhile, these two proteins showed notable capabilities for distinguishing stage Ⅰ/ⅡNSCLC from healthy controls (AUC were 0.708 and 0.702, respectively). Conclusion Serum levels of S100A2 and S100A6 are significantly elevated in the early stage for NSCLC patients, which can be the potential biomarkers for the diagnosis of NSCLC.
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Objective To investigate the influence of S100A6 gene RNA interference on the biological behaviors of A549 lung adenocarcinoma cells.Methods The S100A6 gene RNA interference vector was transfected in A549 lung adenocarcinoma by lentivirus.The experiment was divided into three groups:pLenR-GPH group (the vector without S100A6 RNAi gene was transfected),negative control group (no vectors was transfected),and RNAi group (the vector with S100A6 RNAi gene was transfected).S100A6 mRNA and protein were detected using real-time PCR and Western blotting.The biological behavior including cell proliferation,invasion,cell cycle and cell apoptosis were detected by 3-(4,5-dimethyl-2-thiazoly)-2,5-diphenyl-2H-tetrazolium bromide,transwell,and flow cytometer,respectively.Results The expression of S100A6 mRNA of A549 lung adenocarcinoma cell line in RNAi group (0.009 ± 0.001) was significantly decreased than those in negative control group (0.049 ± 0.005) and pLenR-GPH group (0.030 ± 0.006),with statistically significant differences (t =57.56,P =0.000;t =48.21,P =0.000).The expression of S100A6 protein of A549 lung adenocarcinoma cell line in RNAi group (0.107 ± 0.002) was significantly decreased than those in negative control group (0.341 ± 0.005) and pLenR-GPH group (0.311 ± 0.006),with statistically significant differences (t =37.34,P =0.000;t =27.51,P =0.001).The ability of cell proliferation at 48 hours in RNAi group (0.230 ± 0.008) was significantly declined than those in negative control group (0.292 ± 0.038) and pLenR-GPH group (0.307 ± 0.013),with statistically significant differences (t =25.31,P =0.003;t =29.42,P =0.001).The number of transmembrane cells in RNAi group (11.40 ± 1.36) was significantly declined than those in negative control group (26.80 ± 1.83) and pLenR-GPH group (25.80 ± 1.93),with statistically significant differences (t =29.44,P =0.001;t =23.17,P =0.005).The cell proportion of S phase in RNAi group (28.26％ ± 0.38％) was significantly lower than those in pLenR-GPH group (44.73％±0.66％) and negative control group (45.15％ ± 1.69％),with statistically significant (t =63.69,P=0.000;t =71.55,P =0.000).Cell propotion of G2-M phase in RNAi group (26.99％ ± 0.29％) was signi-ficantly higher than those in negative control group (13.26％ ±0.49％) and pLenR-GPH group (12.41％ ± 0.46％),with statistically significant (t =56.31,P =0.000;t =51.39,P =0.000).The cell apoptosis proportion in RNAi group (8.90％ ±0.48％) was significantly higher than those in negative control group (5.84％ ±0.21％) and pLenR-GPH group (5.99％ ±0.37％),with statistically significant (t=51.34,P =0.000;t =47.27,P =0.000).Conclusion S100A6 gene involves the proliferation,invasion,cell cycle and apoptosis of tumor cells,which has close correlation with occurrence,development and metastasis of lung adenocarcinoma.S100A6 gene is hopeful to become a molecular target for the diagnosis and treatment of lung adenocarcinoma.
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S100A6 is the member of the S100 family of EF-hand calcium-binding proteins,which participates in inflammatory reaction and regulates in cell growth,differentiation,growth inhibition and apoptosis.Recently it has been reported that S100A6 plays a key role in neoplastic transformation,proliferation and invasion of tumor,it may become a potential target in the diagnosis and treatment of tumor.Here is to review the recent advances in the research on this molecule,including its structure,function mechanism,and its relationship with the gastrointestinal cancer and so on.
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Objective: To investigate the effects of S100A6 gene silencing on the proliferation and migration of Eca109 human esophagus cancer cells. Methods: The shRNA expression vectors were constructed using the shRNA sequences designed based on human S100A6's coding sequence, and were transfected into Eca109 cells via cationic liposome. The changes of S100A6 mRNA and protein in Eca109 cells transfected with the recombinant vectors were detected using real-time PCR and Western blotting analysis 48 hours after transfection, respectively; the proliferative curves of transfected cells were plotted using MTT assay; furthermore, the change in cellular migration ability was determined using wound healing assay. Results: The eukaryotic expression vector of shRNA targeting S100A6 was successfully constructed. Real-time PCR and Western blotting analysis results showed that S100A6 was effectively silenced by liposome-mediated transfection of the recombinant shRNA vectors in Eca109 cells. Compared with the untransfected cells, S100A6 mRNA and protein in transfected Eca109 cells were significantly decreased (P<0.05, P<0.01). Meanwhile, the proliferative activity of Eca109 cells was significantly inhibited by S100A6 silencing (P<0.01). It was found that the cellular migration was also suppressed by S100A6 gene interference. Conclusion: S100A6 gene can be effectively silenced by shRNA expression vectors, and the silence may lead to inhibition of the proliferation and migration of Eca109 cells.
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Obejective To investigate the effect of exogenous microRNA 136(mir136) on contents of S100 calcium binding protein A6 (S100A6) and proliferation in human colon cancer cell line HCT116 through constructing a eukaryotic expression vector of mir136 (pcDNA-mir136) and cationic liposome-mediated transfection.Methods Human genomic DNA was extracted from HEK293 cells and the DNA fragment containing mir136 precursor sequence was amplified by PCR and cloned into pcDNA to construct pcDNA-mir136. The plasmid was transfected into HCT116 cells via cationic liposome. The content of mir136 was determined by Real-time PCR, S100A6 protein was examined by Western blotting analysis, and apoptosis was detected by Flow Cytometry at 48 h after transfection. The proliferation ability of the tumor cells was examined by CCK-8 at 24 and 48 hours after transfection. Results The eukaryotic expression vector of mir136 was successfully constructed and transfected into HCT116 cells. Expression of mir136 was significantly higher in the transfected group than that in the untransfected group (P<0.01) and the content of S100A6 protein was significantly lower than that of the untransfected group (P<0.05) 48 h after transfection. Meanwhile, cell proliferation activity of the transfected group was significantly lower than that of the untransfected group (P<0.05) and cell apoptosis was significantly increased compared with the untransfected group (P<0.01).Conclusion Mir136 may inhibit proliferation and induce apoptosis of HCT116 cells by inhibiting the expression of S100A6 gene.
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Objective To investigate the effects of S100A6 gene on invasion of human pancreatic cancer cell and possible mechanism. Methods Human pancreatic cancer BxPC3 cell line was transfected with small interfering RNA (siRNA) targeting S1006 gene, the mRNA and protein levels of S100A6 were determined by real time RT-PCR and Western blotting respectively. The invasion ability was evaluated by Transwell chamber. The matrix metalloproteinase-2 (MMP-9) activity of cancer cells was examined by gelatin zymography. Results The levels of mRNA and protein of S100A6 were greatly reduced in a dose and time dependent manner, the number of penetrating cells was greatly reduced in a dose dependent manner. The expression of S100A6 mRNA in 12.5 nmol/L of S100A6 siRNA transfected group decreased from ( 100 ±0.3)％ in control group to (15.3 ±0.2)％ ; while the expression of S100A6 protein decreased from (83.2 ±0. 18 ) ％ to ( 13.5 ± 0. 12) ％ ; the number of penetrating cells decreased from 44.5 ± 2.2 to 7.6 + 1.5 ( P ＜0. 01 ). The MMP-9 activity of siRNA group reduced significantly. Conclusions S100A6 siRNA can inhibit the invasion of pancreatic cancer cells through down-regulation of MMP-9.
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Objective To investigate the effects of human S100A6 on β-catenin in human osteosarcoma cell lines MG63 and U2OS. Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene, AdS100A6 and AdSiS100A6 respectively, to up-regulate and down-regulate the ex-pression of S100A6. Then RT-PCR, Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of β-catenin. Results In both cell lines, with up-regulated S100A6, expression of β-catenin mRNA and protein increased(P <0. 05) and β-catenin protein increase was more obvious in nuclear than in cytoplasma; while down-regulating S100A6, both the mRNA and protein level of β-catenin decreased (P<0. 05) ; β-catenin protein decrease was more obvious in nuclear than in cytoplasma, too. Conclusion In-creasing Wnt/β-catenin signaling activity may be a mechanism that S100A6 involves in tumor development.
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Objective: we investigated the biological effects of exogenous S100A6 on osteosarcoma cell line U2OS and explored its possible mechanism. Methods:MTT assay was used to determine S100A6 influence on U2OS cells proliferation and their conce ntration-dependent relationship. The trypan blue exclusion assay was used to observe S100A6 inhibitory effect on U2OS cells proliferation in a time-dependent manner;Hoechst staining was used to determine cell apoptosis;Western blot and immunocytochemical methods to determine expressions of cyto-?-catenin. Results:We found that ,on the first three days,OD values in cells subjected to the recombinant protein S100A6(GST-S100A6)with the concentrations of 30,100,300 and 1 000?g/ml respectively were obviously lower than those in the GST group,by 20.5%,23.7%,32.2% and 36.8% accordingly(P
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Objective To investigate the effects of human S100A6 on ?-catenin in human osteosarcoma cell lines MG63 and U2OS.Methods Cell lines MG63 and U2OS were infected by recombinant adenoviruses carrying human S100A6 and its siRNA gene,AdS100A6 and AdSiS100A6 respectively,to up-regulate and down-regulate the expression of S100A6. Then RT-PCR,Western blot and immunocytochemistry were used to detect mRNA and protein (level and/or distribution) of ?-catenin.Results In both cell lines,with up-regulated S100A6,expression of ?-catenin mRNA and protein increased(P
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BACKGROUND: A human orthologue of mouse S100A6-binding protein (CacyBP), Siah- 1-interacting protein (SIP) had been shown to be a component of novel ubiquitinylation pathway regulating beta-catenin degradation. The role of the protein seems to be important in cell proliferation and cancer evolution but the expression pattern of SIP in actively dividing cancer tissues has not been known. For the elucidation of the role of SIP protein in carcinogenesis, it is essential to produce monoclonal antibodies specific to the protein. METHODS: cDNA sequence coding for ORF region of human SIP gene was amplified and cloned into an expression vector to produce His-tag fusion protein. Recombinant SIP protein and monoclonal antibody to the protein were produced. The N-terminal specificity of anti-SIP monoclonal antibody was conformed by immunoblot analysis and enzyme linked immunosorbent assay (ELISA). To study the relation between SIP and colon carcinogenesis, the presence of SIP protein in colon carcinoma tissues was visualized by immunostaining using the monoclonal antibody produced in this study. RESULTS: His-tag-SIP (NSIP) recombinant protein was produced and purified. A monoclonal antibody (Korea patent pending; #2003-45296) to the protein was produced and employed to analyze the expression pattern of SIP in colon carcinoma tissues. CONCLUSION: The data suggested that anti-SIP monoclonal antibody produced here was valuable for the diagnosis of colon carcinoma and elucidation of the mechanism of colon carcinogenesis.
Subject(s)
Animals , Humans , Mice , Antibodies, Monoclonal , beta Catenin , Carcinogenesis , Cell Proliferation , Clinical Coding , Clone Cells , Colon , Colorectal Neoplasms , Diagnosis , DNA, Complementary , Ecthyma, Contagious , Enzyme-Linked Immunosorbent Assay , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.
Subject(s)
Animals , Humans , Amino Acid Sequence , Antibodies, Monoclonal , Cell Division , Cell Line , Cell Line, Tumor , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Melanoma , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
BACKGROUND: S100A6 is a calcium-binding protein overexpressed in several tumor cell lines including melanoma with high metastatic activity and involved in various cellular processes such as cell division and differentiation. To detect S100A6 protein in patient' samples (ex, blood or tissue), it is essential to produce a monoclonal antibody specific to the protein. METHODS: First, cDNA coding for ORF region of human S100A6 gene was amplified and cloned into the expression vector for GST fusion protein. We have produced recombinant S100A6 protein and subsequently, monoclonal antibodies to the protein. The specificity of anti-S100A6 monoclonal antibody was confirmed using recombinant S100A recombinant proteins of other S100A family (GST-S100A1, GST-S100A2 and GST-S100A4) and the cell lysates of several human cell lines. Also, to identify the specific recognition site of the monoclonal antibody, we have performed the immunoblot analysis with serially deleted S100A6 recombinant proteins. RESULTS: GST-S100A6 recombinant protein was induced and purified. And then S100A6 protein excluding GST protein was obtained and monoclonal antibody to the protein was produced. Monoclonal antibody (K02C12-1; patent number, 330311) has no cross-reaction to several other S100 family proteins. It appears that anti- S100A6 monoclonal antibody reacts with the region containing the amino acid sequence from 46 to 61 of S100A6 protein. CONCLUSION: These data suggest that anti-S100A6 monoclonal antibody produced can be very useful in development of diagnostic system for S100A6 protein.
Subject(s)
Animals , Humans , Amino Acid Sequence , Antibodies, Monoclonal , Cell Division , Cell Line , Cell Line, Tumor , Clinical Coding , Clone Cells , DNA, Complementary , Ecthyma, Contagious , Melanoma , Recombinant Proteins , Sensitivity and SpecificityABSTRACT
S100A6 (calcyclin) is a member of the S100 family and has been originally isolated from the cDNA library of Syrian baby hamster kidney cells. The S100A6 gene expression is reported to remain high throughout the cell cycle following induction by serum or growth factors, suggesting that the gene may be required for cell cycle progression. Nevertheless, the role that S100A6 may play in tumor progression remains unknown. In this study, we have explored the expression patterns of S100A6 gene in human thyroid tissues by northern blot analysis. Using the S100A6 monoclonal antibody, we carried out the immunohistochemical staining to determine the distribution/localization of S100A6 protein within tumor or non-tumorous cells of the thyroid. To modulate the regulation of endogenously expressed S100A6 protein in the intracellular level, overexpressed or anti-sense treated transfectant was constructed by using the eukaryotic expression vector. As a result, immunohistochemistry for S100A6 showed a strong positivity in the malignant tumors of thyroid and a high expression level of S100A6 protein affected cell proliferation in the overexpressed transfectant. These findings suggest that S100A6 may be involved in the tumor pathogenesis and provides another parameter for the differentiation of malignant and benign lesions. A well defined monoclonal antibody against S100A6 protein is now available for the immunohistochemical studies of the various thyroid tissues.