NF ��B activation was found to be significantly and positively co

NF ��B activation was found to be significantly and positively correlated with STAT3 activation and MMP9 expression. Similarly, STAT3 activation was also correlated with MMP9 expression. I��BM overexpression reduces STAT3 expression and activation Since the relationship between NF ��B and STAT3 has been dependent on the cellular context and cell type, we performed in vitro experiments. To investi gate whether STAT3 is regulated by NF ��B, we produced stable cell lines from SNU 638 and MKN1 cells overex pressing I��BM. Immunoblotting analysis was performed to determine the protein expression of NF ��B p65 subunit phosphorylated at serine 536 in addition to the protein expression of total NF ��B p65, because an important site of phosphorylation of NF ��B p65 subunit is at serine 536, and this phosphoryl ation is involved in regulation of transcriptional activity, nuclear localization, and protein stability.

Our results showed that NF ��B activation was down regulated, whereas total RelA protein expression was not modulated. Consistently, luciferase reporter assay also showed that NF ��B transcriptional activity markedly decreased in I��BM overexpressing cells. Then, we assessed whether NF ��B reg ulates the STAT3 activation by immunoblotting and found that I��BM overexpression decreased the STAT3 expression and activation. STAT luciferase reporter assay also showed that STAT transcriptional activity was decreased in I��BM overexpressing cells. In addition, double immunofluorescence staining showed that pRelA and STAT3 were colocalized in the nucleus of the same gastric cancer cells, which was reduced in I��BM overexpressing cells.

Next, to investigate whether there is a crosstalk between NF ��B and STAT3, STAT3 was silenced by transfection of STAT3 siRNA. Immunoblotting showed that STAT3 silencing decreased STAT3 expression and activation, but neither total RelA nor pRelA expression was changed in STAT3 silenced cells. In addition, luciferase reporter assay confirmed that STAT3 silencing did not modulate NF ��B transcriptional activity. Taken together, these findings suggest that STAT3 acts as a downstream molecule of NF ��B in NF ��B pathway. NF ��B suppression decreases the migration and invasion through the regulation of EMT markers In the initial steps of metastasis of carcinoma cells, epi thelial cancer cells change their phenotype to mesenchy mal phenotype and become motile and invasive by a process called epithelial mesenchymal transition.

This process includes down regulation of epithelial markers and up regulation of mesenchymal markers. To confirm the effect of NF ��B Anacetrapib activation on gastric can cer cell motility, we used a stable SNU 638 and MKN1 cells overexpressing I��BM. Wound healing assay showed that I��BM overexpression significantly decreased migra tion of gastric cancer cells compared with control cells infected with an empty vector.

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