Expression Of Inflammatory Molecule S100A9 In Colorectal Cancer And Its Relationship With Disease Progression And Its Effect On Proliferation And Migration Of Colorectal Cancer Cells And Its Molecular Mechanism

Backgrounds and objectivesA key contribution of inflammation to tumor progression is that inflammatory molecules in tumor inflammatory microenvironment exert their promotive role in cancer cell gowth, migration, invasion and the resistance to chemotherapy. The study from epidemiology and molecular biology has been demonstrated that inflammation is closely associated with tumor, also points out that colorectal cancer is an inflammation-related cancer. The association of inflammation with colorectal cancer (CRC) is clear, while the clear-cut mechanism of chronic inflammation contributing to cancer progression is very complex and the specific details in this procress is still not clear, including identification of molecules that have tumor-promoting roles in inflammatory microenvironment and study of their potential molecular mechanisms. Hope to provide useful evidence for prevention and therapy of CRC.S100A9, a member of the S100 family of calcium-binding proteins, has been reported to be associated with inflammation and also recognized an inflammatory molecule. Previous study only demonstrated that S100A9 had a proinflammatory role by stimulating infiltration and adhesion of inflammatory cells to inflammatory lesions and regulating neutrophil cell survival. Recent study showed that there is an elevated S100A9 expression in tumor cells and infiltrated inflammatory cells in surrounding tumor tissues, which suggests that S100A9 might take part in CRC development. Here we investigated the S100A9 expression, biological role, and its potential molecular mechanisms in CRC deveplopment. Hope to provide useful evidence to clarify the effects and molecular mechanism of inflammation on CRC development and to diagnose early and treat efficiently CRC.Methods1 Expression of S100A9 in CRC tissues and cell lines1) RT-PCR, Immunohistochemistry (IHC) and Western bolt were used to detect expression and/or distribution of S100A9 gene and protein in all samples (n=42).2) Western bolt were used to detect the expression of S100A9 protein in CRC cell lines (HCT116, SW480 and LoVo) and human normal colon mucosal epithelial cell line (NCM460).2 Preparation of cell intervention tools1) Amplification and identification of recombinant adenoviruses AdS100A9:The recombinant adenoviruses carrying human S100A9 gene (AdS100A9) and its control adenoviruses (AdGFP) were amplified by infecting human HEK293 cell line; The effectiveness of AdS100A9 was identified by Western blot analysis of S100A9 expression in HCT116 and SW480 cells infected with AdS100A9.2) Preparation of GST-S100A9:The plasmids pGST-Moluc-S100A9 and pGST-Moluc were transformed into E. coli (BL21) following the instructions of calcium chloride transformation. Isopropylthio-b-D-galactoside was used to induce the expression of GST-S100A9 and GST. After the bacteria were sonicated, the supernatants were collected, spun and incubated with glutathione-Sepharose 4B beads, and then GST-S100A9 and GST on the beads were eluted by elution buffer with reduced glutathione. Finally, the proteins were filtered and stored at -80℃.3 The effect of S100A9 on cell proliferation and migration of CRC cellsAfter CRC cells were treated with AdS100A9 or GST-S100A9 protein for indicated time, MTT assay were used to evaluate proliferation and transwell migration assay was used to detect cell migration.4 Molecular mechanisms of S100A9-induced proliferation and migration of CRC cells1) Relationship between S100A9 and RAGE:Immunohistochemistry (IHC), Western bolt and immunofluorescence were used to detect expression and distribution of RAGE in CRC tissues and cells; GST-pull down, co-immunoprecipitation and immunofluorescent double staining assays were used to analyze the interaction of S100A9 with RAGE.2) Relationship between S100A9 and β-catenin:Western blot was used to detect β-catenin and p-GSK3β expression; immunocytochemistry (ICC) and immunofluorescence were used to analyze the P-catenin level and distribution; RT-PCR was used to detect the transcriptional levels of P-catenin, c-myc, MMP7 and Cyclin D.3) Relationship among S100A9, RAGE and p-catenin:Western blot was used to detect expression of β-catenin, p38/p-p38 MAPK and p-AKT/AKT.Results1 The expression of S100A9 in CRC tissues and cells1) The mRNA and protein expression of S100A9 was markedly increased in CRC tissues compared to normal tissues; S100A9 expression was also markedly increased in CRC cell lines HCT116, SW480 and LoVo compared to a normal colon mucosal epithelial cell line NCM460. The positive rate of S100A9 in tumor cells was 69.1%(29/42) in the CRC tissues, compared to 16.7%(7/42) in the normal tissues respectively (P<0.001).2) S100A9 expression in tumor cells was association with histological grade (P<0.05), Dukes stage (P<0.01) and lymph node metastasis (P<0.01), but not with gender, age, tumor location and tumor size. These results suggest that S100A9 might be involved in CRC progression and be a tumor marker for evaluating staging, lymphatic metastasis and prognosis of CRC.2 The effect of S100A9 on proliferation and migration of CRC cells1) Treatment with AdS100A9 and AdGFP in CRC cells for indicated time:there was no significant difference on cell proliferation or migration between AdS100A9-treated and AdGFP-treated HCT116 cells or SW480 cells.2) Treatment with recombinant S100A9 protein in CRC cell lines HCT116 and SW480 for incidated time:Treatment with GST-S100A9 protein at 10, and 20 μg/ml promoted cell proliferation (P<0.05 for each), and the effect at 10 μg/ml was more significant. The concentration of GST-S100A9 for all remaining experiments was 10μg/ml. In addition, GST-S100A9 also promoted clone formation (P<0.05 for each) and cell migration (P<0.05 for each) of HCT116 and SW480 cells.All the results suggest that S100A9 might exert its effects on cell proliferation and migration through being secreted to extracellular space from CRC cells and binding with a possible receptor.3 Molecular mechanisms for S100A9-induced proliferation and migration of CRC1) Relationship between S100A9 and RAGE:RAGE expression is elevated in CRC tissues and cell lines (HCT116 and SW480) compared to normal tissues and a normal colon mucosal epithelial cell line NCM460, respectively. We found that there were a co-localization of S100A9 with RAGE in human CRC intratumoral tissues and the interaction between them. Treating HCT116 cells with RAGE blocking antibody partially blocked the S100A9-induced increase in cell proliferation (P<0.05) and migration (P<0.05). These results suggest that S100A9 can bind to RAGE and then stimulate cell proliferation and migration.2) Relationship between S100A9 and β-catenin:The change of Wnt/β-catenin signaling pathway resulted by GST-S100A9 was as follows: (1) the increased levels of total β-catenin and nuclear β-catenin; (2) no change in the transcriptional level of β-catenin gene, but increased transcriptional levels of the pathway target genes c-myc, MMP7 and Cyclin D (P<0.05 for each); (3) the increased level of p-GSK3β; (4) attenuating the activation of Wnt/p-catenin pathway through knock-down of β-catenin by Adsiβ-catenin partially abolished S100A9-induced cell proliferation (P<0.05) and migration (P<0.05). These results suggest that S100A9-induced proliferation and migration of CRC cells could be partially mediated by upregulating Wnt/β-catenin pathway.3) Relationship among S100A9, RAGE and β-catenin:(1) Treatment with RAGE blocking antibody blocked the increase of β-catenin and p-GSK3β levels caused by S100A9 treatment, suggesting that RAGE is involved in hyperactivation of Wnt/β-catenin signaling. (2) Treatment with RAGE blocking antibody blocked the increase of p38 MAPK and p-AKT levels caused by S100A9 treatment, suggesting that p38 MAPK and AKT is downstream of RAGE. (3) Inhibition of AKT activity by LY294002, but not p38 MPAK activity by SB203580, blocked S100A9-induced β-catenin increase, suggesting that AKT activation is involved in hyperactivation of Wnt/β-catenin signaling.ConclusionS100A9 expression is elevated in the tumor cells of CRC and associated with differentiation, Dukes stage and lymph node metastasis of CRC. Extracellular, but not intracellular, S100A9 could promote cell proliferation and migraton of CRC cells, and its mechanism was partly involved in the activation of RAGE/AKT/β-catenin signaling pathway.Backgrounds and objectivesThe S100A6 protein, a member of the S100 protein family, is overexpressed in many tumors including colorectal carcinoma (CRC). Although recent studies showed that the elevated expression of S100A6 was associated with the stage and lymphatic permeation of CRC, little is known about whether and how S100A6 contributes to CRC development. Here we investigated the S100A6 expression in CRC tissues and cell lines, and explored the molecular mechanisms underlying S100A6’s role in CRC. Hope to provide useful evidence, prevention and therapy for CRC progression.Methods1 Expression of S100A6 in CRC tissues and cell lines1) The expression and distribution of S100A6 in human CRC tissues were detected by Western bolt and immunofluorescence assays. And the difference of S100A6 expression between CRC tissues and normal tissues was analyzed by statistical analysis.2 Preparation of cell intervention tools1) Amplification and identification of recombinant adenoviruses AdS100A6 and AdsiS100A6:The recombinant adeno viruses carrying human S100A6 gene (AdS100A6) and S100A6-siRNA gene (AdsiS100A6) was amplified by infecting human HEK293 cell line; Their intervention effectiveness was identified by Western blot analysis of S100A6 expression in the infected target cells.2) Preparation of S100A6 proteinThe plasmids pGST-Moluc-HRV3C-S100A6 and pGST-Moluc-HRV3C were transformed into E. coli (BL21) per the instructions of calcium chloride transformation. Isopropylthio-β-D-galactoside was used to induce the expression of GST-HRV3C-hS100A6 and pGST-Moluc-HRV3C proteins. Then the bacteria were collected and sonicated on ice, and spun at 4 ℃. The supernatant was incubated with glutathione-Sepharose 4B beads, and GST-HRV3C-hS100A6 and GST-HRV3C on the beads were eluted by elution buffer with reduced glutathione on ice. GST-HRV3C-S100A6 was digested by GST-HRV3C overnight in 4℃, and then the GST tag and GST-HRV3C were removed by glutathione-Sepharose 4B beads. Finally the S100A6 protein was filtered and stored at -80℃.3 The effect of S100A6 on proliferation and migration of CRC cellsAfter CRC cells were treated with AdS100A6, AdsiS100A6 or S100A6 protein for indicated time. MTT assay was used to evaluate cell proliferation and transwell assay was used to evaluate cell migration.4 Molecular mechanisms of S100A6-induced proliferation and migration of CRC cells1) Effect of S100A6 on activation of MAPK signaling:Western blot analysis of p38/p-p38, ERK1/2/p-ERK1/2 and JNK /p-JNK levels.2) Effect of inhibitors of p38 and ERK1/2 on S100A6-induced cell proliferation and migration effects.Results1 The expression of S100A6 in CRC tissues and cell linesS100A6 expression was markedly increased in CRC tissues and CRC cell lines (HCT116, SW480 and LoVo) compared to normal tissues and a normal intestinal epithelial cell line NCM460.2 The effect of S100A6 on proliferation and migration of CRC cellsAfter overexpression of S100A6 in HCT116 cells (a CRC cell line with relative low S100A6 expression) by infection with AdS100A6 and knockdown of S100A6 in LoVo cells (a CRC cell line with relative high S100A6 expression) by infection with AdsiS100A6 for indicated time, cell proliferation and migration abilities were analyzed. Compared with AdGFP group, the increased cell proliferation and migration were found in AdS100A6 group of HCT116 cells (P<0.05 for each); Compared with AdRFP group, the decreased cell proliferation and migration in AdsiS100A6 group of LoVo were found (P<0.05 for each). In addition, treatment with S100A6 protein at 5,10 and 20 μg/ml promoted cell proliferation and migration, and the effect at 20 μg/ml was the most significant. The concentration of S100A6 for all remaining experiments was 20 μg/ml. These results suggest that S1000A6 could promote cell proliferation and migration in CRC.3 Potential molecular mechanisms for S100A6-induced proliferation and migration of CRC cells1) The effect of S100A6 on MAPK:AdS100A6 treatment enhanced the phosphorylation of p38 and ERK1/2 MAPK(P<0.01 for each), and had no effect on p-JNK in HCT116 cells; AdsiS100A6 treatment decreased the phosphorylation of p38 and ERK1/2 MAPK (P<0.01 for each), and had no effect on p-JNK in LoVo cells.2) The effects of p38 and ERK MAPKs activation on S100A6-induced cell proliferation and migration:After this signaling pathway was inhibited by SB203580 (the inhibitor of p38) or PD98059 (the inhibitor of ERK1/2), the AdS100A6- or S100A6 protein-induced cell proliferation of HCT116 cells was partially reversed by the inhibitor of ERK1/2 (P<0.05), the increased cell migration effect was partially reversed by the inhibitor of p38 (P<0.05). All these results suggested that activation of ERK 1/2 and p38 MAPKs signaling pathway was involved in S100A6-induced cell proliferation and migration of CRC cells, respectivelyConclusionS100A6 expression is abnormally elevated in CRC. S100A6 could promote cell proliferation and migration in CRC cells, and its mechanism was partly involved in the activation of ERK1/2 and p38 MAPK signaling pathway.