Mycoplasma hyorhinis infection in gastric carcinoma and its effects on the malignant phenotypes of gastric cancer cells

<p>Abstract</p> <p>Background</p> <p><it>Mycoplasma hyorhinis </it>infection has been postulated to play a role in the development of several types of cancer, but the direct evidence and mechanism remained to be determined.</p> <p>Methods</p> <p>Immunohistochemistry assay and nested polymerase-chain reaction (PCR) were performed to examine the <it>mycoplasma hyorhinis </it>infection in gastric cancer tissues. Statistical analysis was used to check the association between mycoplasma infection and clinicopathologic parameters. Transwell chamber assay and metastasis assay were used to evaluate <it>mycoplasma hyorhinis</it>' effects on metastasis in vitro and in vivo. <it>Mycoplasma hyorhinis</it>-induced extracellular signal-regulated kinase (ERK) and epidermal growth factor receptor (EGFR) activation were investigated by Western blot.</p> <p>Results</p> <p>My<it>coplasma hyorhinis </it>infection in gastric cancer tissues was revealed and statistical analysis indicated a significant association between mycoplasma infections and lymph node metastasis, Lauren's Classification, TNM stage, and age of the patients. <it>Mycoplasma hyorhinis </it>promoted tumor cell migration, invasion and metastasis <it>in vitro </it>and <it>in vivo</it>, which was possibly associated with the enhanced phosphorylation of EGFR and ERK1/2. The antibody against p37 protein of <it>Mycoplasma hyorhinis </it>could inhibit the migration of the infected cells.</p> <p>Conclusions</p> <p>The infection of <it>m</it>y<it>coplasma hyorhinis </it>may contribute to the development of gastric cancer and <it>Mycoplasma hyorhinis</it>-induced malignant phenotypes were possibly mediated by p37.</p

PRL-3 promotes the motility, invasion, and metastasis of LoVo colon cancer cells through PRL-3-integrin β1-ERK1/2 and-MMP2 signaling

<p>Abstract</p> <p>Background</p> <p>Phosphatase of regenerating liver-3 (PRL-3) plays a causative role in tumor metastasis, but the underlying mechanisms are not well understood. In our previous study, we observed that PRL-3 could decrease tyrosine phosphorylation of integrin β1 and enhance activation of ERK1/2 in HEK293 cells. Herein we aim to explore the association of PRL-3 with integrin β1 signaling and its functional implications in motility, invasion, and metastasis of colon cancer cell LoVo.</p> <p>Methods</p> <p>Transwell chamber assay and nude mouse model were used to study motility and invasion, and metastsis of LoVo colon cancer cells, respectively. Knockdown of integrin β1 by siRNA or lentivirus were detected with Western blot and RT-PCR. The effect of PRL-3 on integrin β1, ERK1/2, and MMPs that mediate motility, invasion, and metastasis were measured by Western blot, immunofluorencence, co-immunoprecipitation and zymographic assays.</p> <p>Results</p> <p>We demonstrated that PRL-3 associated with integrin β1 and its expression was positively correlated with ERK1/2 phosphorylation in colon cancer tissues. Depletion of integrin β1 with siRNA, not only abrogated the activation of ERK1/2 stimulated by PRL-3, but also abolished PRL-3-induced motility and invasion of LoVo cells in vitro. Similarly, inhibition of ERK1/2 phosphorylation with U0126 or MMP activity with GM6001 also impaired PRL-3-induced invasion. In addition, PRL-3 promoted gelatinolytic activity of MMP2, and this stimulation correlated with decreased TIMP2 expression. Moreover, PRL-3-stimulated lung metastasis of LoVo cells in a nude mouse model was inhibited when integrin β1 expression was interfered with shRNA.</p> <p>Conclusion</p> <p>Our results suggest that PRL-3's roles in motility, invasion, and metastasis in colon cancer are critically controlled by the integrin β1-ERK1/2-MMP2 signaling.</p

Synuclein gamma predicts poor clinical outcome in colon cancer with normal levels of carcinoembryonic antigen

<p>Abstract</p> <p>Background</p> <p>Synuclein gamma (SNCG), initially identified as a breast cancer specific gene, is aberrantly expressed in many different malignant tumors but rarely expressed in matched nonneoplastic adjacent tissues. In this study, we investigated the prognostic potential of SNCG in colon cancer particularly in the patients with normal carcinoembryonic antigen (CEA) levels.</p> <p>Methods</p> <p>SNCG levels were assessed immunohistochemically in cancer tissues from 229 colon adenocarcinoma patients with a mean follow-up of 44 months. Correlations between SNCG levels and clinicopathologic features, preoperative serum CEA level, and clinical outcome were analyzed statistically using SPSS.</p> <p>Results</p> <p>SNCG levels in colon adenocarcinoma were closely associated with intravascular embolus and tumor recurrence but independent of preoperative serum CEA levels. SNCG expression was an independent prognostic factor of a shorter disease-free survival (DFS) and overall survival (OS) (<it>P </it>< 0.0001). Multivariate analysis revealed that both tissue SNCG and serum CEA were independent prognostic factors of DFS (<it>P </it>= 0.001, <0.0001, respectively) for 170 patients with colon adenocarcinomas. Importantly, SNCG remained a prognostic determinant of DFS and OS (<it>P </it>= 0.001, 0.002) for 97 patients with normal preoperative serum CEA level.</p> <p>Conclusions</p> <p>Our results suggest for the first time that SNCG is a new independent predicator for poor prognosis in patients with colon adenocarcinoma, including those with normal CEA levels. Combination of CEA with SNCG improves prognostic evaluation for patients with colon adenocarcinoma.</p

Mycoplasma hyorhinis and Mycoplasma fermentans induce cell apoptosis and changes in gene expression profiles of 32D cells

Infection of mycoplasmas has been linked to various human diseases including arthritis, pneumonia, infertility and cancer. While Mycoplasma hyorhinis and Mycoplasma fermentans have been detected in gastric adenocarcinomas, the mechanisms underlyine the pathogenesis are unknown. In this study, cell growth kinetics, Hoechst 33258 staining, DNA ladder assays, Western blotting analysis and cDNA microarray assays were performed to investigate the roles of M. hyorhinis and M. fermentans during infection of mammalian cells. Our data demonstrated that these mycoplasmas inhibid the growth of immortalised cell lines (32D and COS-7) ane tumor cell lines (HeLa and AGS). In addition, the infection of the 32D cell line with M. hyorhinis and M. fermentans induced compression of the nucleus, degradation of the cell genome and dysregulation of the expression of genes related to proliferation, apoptosis, tumorigenesis, signaling pathway and metabolism. Apoptosis related proteins Bcl-2, Bid and p53 were down-regulated, Fas was up-regulated and Bax was dysregulated in mycoplasma-infected 32D cells. Together, our data demonstrated that infection of mycoplasmas inhibitd cele growts through modification of gene expression profiles and post-translation modification of proliferation and apoptosis related proteins

N-Terminal Polypeptide of Annexin A2 Decreases Infection of Mycoplasma hyorhinis to Gastric Cancer Cells.

Mycoplasma infection in human and its contamination in cell cultures are worldwide problems. The drugs currently available for preventing or treating mycoplasma infection suffer from low sensitivity, strong resistance and high toxicity. Our previous work showed that Mycoplasma hyorhinis (M. hyorhinis) infection was mediated by the interaction between p37 of M. hyorhinis and Annexin A2 (ANXA2) of host cells, however the translational value of this mechanism was unknown. Herein, we synthesized the N-terminal of ANXA2 polypeptide (A2PP) and found that A2PP could decrease the infection of M. hyorhinis to gastric cancer cells and block M. hyorhinis infection-induced cell migration. Furthermore, we found that A2PP could reduce M. hyorhinis contamination of passage cells. Moreover, compared with the commercial antibiotics commonly used in cell culture to prevent M. hyorhinis infection, A2PP demonstrated a more effectiveness but a low toxicity on cell growth. Thus, our study for the first time revealed A2PP's potential for the treatment and prevention of M. hyorhinis infection

Tumor necrosis factor-a polymorphisms and colorectal cancer risk: a meta-analysis.

BACKGROUND AND OBJECTIVES: Tumor necrosis factor-alpha (TNF-a) was related to inflammation and involved in the development of colorectal cancer. Polymorphisms located in TNF-a promoter region, such as 308G/A and 238G/A, could affect the risk of various types of cancer by regulating TNF-a production. In this study, a meta-analysis was performed to investigate the association between common polymorphisms of TNF-a promoter region and colorectal cancer susceptibility. METHODS: Searching of several databases was performed for all publications on the association between TNF-a polymorphisms and colorectal cancer. Summary odds ratios (ORs) with their 95% confidence intervals (95% CIs) were calculated using random-effects models. Stratified analyses based on ethnicity and control population source were also conducted. RESULTS: Overall, TNF-a 308A polymorphism showed a significant association with increased risk of colorectal cancer in worldwide populations under homozygote comparison [AA vs. GG, OR (95% CI) = 1.46 (1.07-1.97)] other than heterozygote comparison [AG vs. GG, OR (95% CI) = 1.05 (0.93-1.19)]. TNF-a 238A was not associated with colorectal cancer risk under homozygote or heterozygote comparisons. In stratified analysis, significant association was observed only in Western populations [AA vs. GG, OR (95% CI) = 1.39 (1.01-1.91)] other than in Eastern populations under homozygote comparison. No significant difference was observed between population-based subgroup and hospital-based subgroup. CONCLUSIONS: TNF-a 308A was moderately associated with an increased risk of colorectal cancer in Western populations, and TNF-a 238A polymorphism was not significantly associated with colorectal cancer risk

Extracellular gamma-synuclein promotes tumor cell motility by activating β1 integrin-focal adhesion kinase signaling pathway and increasing matrix metalloproteinase-24, -2 protein secretion

Abstract Background Increasing evidence reveals a significant correlation between gamma-synuclein (SNCG) level and tumor invasion and metastasis in various human cancers. Our previous investigation showed that SNCG could secrete into extracellular environment and promoted tumor cell motility, but the mechanism is unknown. Methods The membrane binding ability of SNCG was characterized by immunohistochemical staining, immunofluorescence staining and fractionation of colorectal cancer (CRC) cell membrane. Association between SNCG and β1 integrin was validated by coimmunoprecipitation and far Western blot. After inhibition of β1 integrin and focal adhesion kinase (FAK), effect of SNCG on cell motility was measured by transwell chamber assays and changes of protein levels were detected by Western blot. Association between SNCG and activated β1 integrin levels in human CRC tissues was determined by Spearman’s rank correlation analysis. Secreted proteins in conditioned medium (CM) were screened by antibody array. Results Extracellular SNCG bound β1 integrin on CRC cell membrane and increased levels of activated β1 integrin and FAK. Correspondingly, SNCG-enhanced cell motility was counteracted by knockdown or inhibition of β1 integrin or FAK. Further study revealed that high SNCG level indicated poor outcome and SNCG levels positively correlated with those of activated β1 integrin and phospho-FAK (Tyr397) in human CRC tissues. Additionally, extracellular SNCG promoted secretion of fibronectin (FN), vitronectin (VN), matrix metalloproteinase (MMP)-2, and MMP-24 from HCT116 cells. Protease activity of MMP-2 in the CM of HCT116 cells was increased by treatment with SNCG, which was abolished by inhibiting β1 integrin. Conclusion Our results highlight the potential role of SNCG in remodeling extracellular microenvironment and inducing β1 integrin-FAK signal pathway of CRC cells

A2PP has minimal effects on EGFR-ANXA2 signaling, migration of gastric cancer cells, or the localization of ANXA2.

<p>(A) Western blotting of phospho-EGFR, phospho-ANXA2, EGFR, and ANXA2 from AGS and BGC823 cells treated with A2PP for 24 hr. GAPDH was used as loading control. (B) Representative images of migration of AGS and BGC823 cells treated with A2PP for 24 hr. Scale bars, 200 μm. (C) Statistical summary of migration assay. Mean ± SD from three experiments with triplicate samples. ns, no significance. (D) Immunofluorescence of ANXA2 localization (red) in AGS and BGC823 cells treated with A2PP for 24 hr. Scale bars, 5 μm.</p

A2PP binds to p37 of <i>M</i>. <i>hyorhinis</i> and has minimal effect on the proliferation of gastric cancer cell.

<p>(A) Schematic diagram of amino acid sequence of N-terminal of ANXA2 polypeptide (A2PP). (B) Solid-phase binding assay. OD490 of 15 nM biotin-A2PP to GST was set as 1 and relative bindings were caculated. Mean ± SD of three independent assays with triplicate samples. ***, P < 0.001. (C) Streptavidin pull-down assays identified Biotin-A2PP as a GST-p37 binding polypeptide. (D) Cell morphology of AGS and BGC823 following indicated concentrations of A2PP treatment for 24 hr and 48 hr. (E) Proliferation of gastric cancer cell lines (AGS and BGC823) treated with increasing concentration of A2PP for 72 hr. Mean ± SD from three experiments with triplicate samples.</p

A2PP reduces <i>M</i>. <i>hyorhinis</i> infection.

<p>Cell ELISA analysis of p37 (OD 490 nm) of p37 protein in AGS and BGC823 cells infected with 10⁵ CCU (color changing units)/ml of <i>M</i>. <i>hyorhinis</i> and treated with A2PP or ConP for 24 hr. <i>M</i>. <i>hy</i>, <i>M</i>. <i>hyorhinis</i>. Mean ± SD from 3 experiments with triplicate for each sample. (B) Quantitative PCR (qPCR) analysis of <i>p37</i> in AGS and BGC823 cells infected and treated as in (A). Mean ± SD from 3 experiments with triplicate for each sample. (C) Western blotting of p37, p-EGFR, EGFR, p-ANXA2 and ANXA2 from AGS and BGC823 cells treated as in (A). (D) Quantification of p37 protein levels of in (C). Levels of p37 were normalized to those of GAPDH. Mean ± SD from 3 independent experiments. (E) Quantification of p-EGFR and p-ANXA2 levels in (C). Levels of p-EGFR or p-ANXA2 were normalized to those of EGFR or ANXA2. Mean ± SD from 3 independent experiments. **, P < 0.01; **, P < 0.001; n.s, no significance.</p