Cox 2 expression was markedly find more information suppressed when both MCF 7 DOX and MDA MB 231 cells were transfected with EGFR specific siRNAs. In addition, Western blot analyses of MCF 7 DOX cells revealed that Cox 2 expression was induced within 2 h of EGF treatment. We further confirmed that blocking the EGFR pathway suppressed Cox 2 expression using a selective EGFR tyrosine kinase inhibi tor, gefitinib. Treating cells for 24 h with gefitinib effec tively suppressed EGF induced Cox 2 expression in MCF 7 DOX cells. We then tested the effects of the EGFR inhibitor on invasion of MCF 7 DOX cells. Gefitinib decreased the invasive potential of MCF 7 DOX cells, indicating that EGFR contributes to invasiveness of MCF 7 DOX cells by upregulating Cox 2.
Effect of EGFR on PI3K Akt and MAPK mediated Cox 2 expression in MCF 7 DOX cells Because EGFR controls several other pathways, such as the PI3K Akt and MAPK pathways, we next investigated which downstream pathway was involved in EGFR mediated Cox 2 expression. As expected, treating MCF 7 DOX cells with EGF for 8 h to induce Cox 2 expression activated both the PI3K Akt and MAPK pathways. To confirm the role of the PI3K Akt and MAPK pathways in Cox 2 expression, we stu died the effect of the PI3K Akt inhibitor LY294002 and the MAPK inhibitor U0126 on EGF induced expression of pAkt and Cox 2 in MCF 7 DOX cells. Western blot analysis showed that LY294002 and U0126 dramatically suppressed activation of pAkt and pERK1 2, respec tively, and downregulated EGF induced Cox 2 expres sion.
To investigate the role of the PI3K Akt pathway in invasiveness of MCF 7 DOX cells, we deter mined whether blocking the PI3K Akt pathway would inhibit invasion of MCF 7 DOX cells in an in vitro inva sion assay. Blocking the PI3K Akt pathway with LY294002 or the MAPK pathway with U0126 dramati cally inhibited invasion of MCF 7 DOX cells, but did not affect proliferation of the cells. Because we wondered whether PI3K Akt or MAPK sig naling alone not through Cox 2 PGE2 can increase expression of MMP 2, MMP 9, and uPA, we tested MMP 2, MMP 9, and uPA mRNA expression in MCF 7 DOX cells transfected with siRNA specific for Cox 2. Transfection with siRNAs targeting Cox 2 specifically inhibited MMP 2, MMP 9, or uPA RNA expression in MCF 7 DOX cells, whereas control scrambled siRNA demonstrated no effect.
Expression of MMP 2, MMP 9 and uPA mRNA was suppressed by the PI3K Akt inhibitor LY294002 and the MAPK inhibitor U0126 in MCF 7 DOX cells transfected with control siRNA. however, their expressions were less affected by either LY294002 or U0126 in MCF 7 DOX cells transfected with Cox 2 siRNA. Role of EP1 and EP3 receptors in Cox 2 mediated invasion of MCF Anacetrapib 7 DOX cells PGE2, which is produced by Cox 2, exerts its effect by binding to specific prostanoid receptors, which are a family of G protein coupled receptors.