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Anti-Proliferation Activity of Fucoidan in MKN45 Gastric Cancer Cells and Downregulation of Phosphorylated ASK1, a Cell Cycle-Regulated Kinase.

ABSTRACT: BACKGROUND: Fucoidan is a high-molecular polysaccharide whose main constituent is sulfated fucose. We specifically focused on the anti-proliferation activity of fucoidan and examined the underlying mechanism in MKN45 gastric cancer cells. METHODS: MKN45 cell proliferation was analyzed by BrdU assay and fucoidan cytotoxicity was examined by LDH and clonogenic assays. The Agilent Human microarray kit was used to identify upregulated and downregulated genes in response to fucoidan, and western blot analyses evaluated cell cycle proteins. RESULTS: Fucoidan impeded the MKN45 cell cycle by approximately 50%, and inhibited cell proliferation.LDH assays showed no immediate cytotoxic effects of fucoidan at 24 h exposure, however longer time courses revealed cell growth inhibition at 4 days in a dose-dependent manner. Microarray analysis identified MAP3K5, or ASK1 (apoptosis signal-regulating kinase),which was upregulated by 1.38-fold upon fucoidan treatment.Fucoidan increased ASK1 protein levels, while reducing phosphorylated ASK1 levels. Reduction of ASK1 by siRNA decreased proliferation of MKN45 cells. CONCLUSION: Our findings show that fucoidan may suppress cellular proliferation and DNA synthesis in MKN45 cells by suppressing the ASK1-p38 signaling pathway through reduction of phosphorylated ASK1 levels.

SUBMITTER: Yoshimoto M

PROVIDER: S-EPMC4495266 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Anti-Proliferation Activity of Fucoidan in MKN45 Gastric Cancer Cells and Downregulation of Phosphorylated ASK1, a Cell Cycle-Regulated Kinase.

Yoshimoto Miwa M Higaki Katsumi K Nanba Eiji E Ikeguchi Masahide M

Yonago acta medica 20150327 1

<h4>Background</h4>Fucoidan is a high-molecular polysaccharide whose main constituent is sulfated fucose. We specifically focused on the anti-proliferation activity of fucoidan and examined the underlying mechanism in MKN45 gastric cancer cells.<h4>Methods</h4>MKN45 cell proliferation was analyzed by BrdU assay and fucoidan cytotoxicity was examined by LDH and clonogenic assays. The Agilent Human microarray kit was used to identify upregulated and downregulated genes in response to fucoidan, and ...[more]

PMID: 26190891

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Project description:BACKGROUND: Gastric carcinoma is the second leading cause of cancer-associated death worldwide. The high mortality associated with this disease is in part due to limited knowledge about gastric carcinogenesis and a lack of available therapeutic and prevention strategies. MUC1 is a high molecular weight transmembrane mucin protein expressed at the apical surface of most glandular epithelial cells and a major component of the mucus layer above gastric mucosa. Overexpression of MUC1 is found in approximately 95% of human adenocarcinomas, where it is associated with oncogenic activity. The role of MUC1 in gastric cancer progression remains to be clarified. METHODOLOGY: We downregulated MUC1 expression in a gastric carcinoma cell line by RNA interference and studied the effects on cellular proliferation (MTT assay), apoptosis (TUNEL assay), migration (migration assay), invasion (invasion assay) and aggregation (aggregation assay). Global gene expression was evaluated by microarray analysis to identify alterations that are regulated by MUC1 expression. In vivo assays were also performed in mice, in order to study the tumorigenicity of cells with and without MUC1 downregulation in MKN45 gastric carcinoma cell line. RESULTS: Downregulation of MUC1 expression increased proliferation and apoptosis as compared to controls, whereas cell-cell aggregation was decreased. No significant differences were found in terms of migration and invasion between the downregulated clones and the controls. Expression of TCN1, KLK6, ADAM29, LGAL4, TSPAN8 and SHPS-1 was found to be significantly different between MUC1 downregulated clones and the control cells. In vivo assays have shown that mice injected with MUC1 downregulated cells develop smaller tumours when compared to mice injected with the control cells. CONCLUSIONS: These results indicate that MUC1 downregulation alters the phenotype and tumorigenicity of MKN45 gastric carcinoma cells and also the expression of several molecules that can be involved in tumorigenic events. Therefore, MUC1 should be further studied to better clarify its potential as a novel therapeutic target for gastric cancer.

Project description:Aneuploidy and chromosomal instability (CIN) are common features of gastric cancer (GC), but their contribution to carcinogenesis and antitumour therapy response is still poorly understood. Failures in the mitotic checkpoint induced by changes in expression levels of the spindle assembly checkpoint (SAC) proteins cause the missegregation of chromosomes in mitosis as well as aneuploidy. To evaluate the possible contribution of SAC to GC, we analyzed the expression levels of proteins of the mitotic checkpoint complex in a cohort of GC cell lines. We found that the central SAC proteins, Mad2 and BubR1, were the more prominently expressed members in disseminated GC cell lines. Silencing of Mad2 and BubR1 in MKN45 and ST2957 cells decreased their cell proliferation, migration and invasion abilities, indicating that Mad2 and BubR1 could contribute to cellular transformation and tumor progression in GC. We next evaluated whether silencing of SAC proteins could affect the response to microtubule poisons. We discovered that paclitaxel treatment increased cell survival in MKN45 cells interfered for Mad2 or BubR1 expression. However, apoptosis (assessed by caspase-3 activation, PARP proteolysis and levels of antiapoptotic Bcl 2-family members), the DNA damage response (assessed by H2Ax phosphorylation) and exit from mitosis (assessed by Cyclin B degradation and Cdk1 regulation) were activated equally between cells, independently of Mad2 or BubR1-protein levels. In contrast, we observed that the silencing of Mad2 or BubR1 in MKN45 cells showed the induction of a senescence-like phenotype accompanied by cell enlargement, increased senescence-associated ?-galactosidase activity and increased IL-6 and IL-8 expression. In addition, the senescent phenotype is highly increased after treatment with PTX, indicating that senescence could prevent tumorigenesis in GC. In conclusion, the results presented here suggest that Mad2 and BubR1 could be used as prognostic markers of tumor progression and new pharmacological targets in the treatment for GC.

Dataset Information

Anti-Proliferation Activity of Fucoidan in MKN45 Gastric Cancer Cells and Downregulation of Phosphorylated ASK1, a Cell Cycle-Regulated Kinase.

Publications

Anti-Proliferation Activity of Fucoidan in MKN45 Gastric Cancer Cells and Downregulation of Phosphorylated ASK1, a Cell Cycle-Regulated Kinase.

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