These data provide evi dence that Cox 2 expression induced by the EGFR path way is associated with invasiveness of MCF 7 DOX cells. PGE2, new post the major end product of Cox 2 activation, is also known to activate EGFR through various pathways. Therefore to clarify whether PGE2 signaling through EPs promotes the PI3K Akt or MAPK pathway mediated invasion primarily, we evaluated the effect of EP1 or EP3 specific agonists or EP inhibitor on the PI3K Akt or MAPK pathways, but we found that PGE2 signaling through EPs didnt affect PI3K Akt and MAPK pathway in the MCF 7 DOX cells. Recent studies have shown that Cox 2 mRNA and protein expression in several cancer cell lines are regu lated by the insulin like growth factor 1R PI3K and nuclear factor kappa B nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor pathways.

In addition to the PI3K Akt pathway, the Ras Raf MAPK pathway is also a downstream transducer of IGF 1R signaling. The IGF 1R signaling pathway plays a major role in cell proliferation, apoptosis, invasion, and angiogenesis. Moreover, IGF 1R has been shown to upregulate Cox 2 mRNA expression and PGE2 synthesis in cancer cells. Although we found that IGF 1R expression was neither increased nor constitutively acti vated in MCF 7 DOX cells, activation of the IGF 1R pathway may still contribute to Cox 2 expression and our efforts are ongoing to determine any further possibility. Treating cells with EGF also increased pAkt and pERK1 2 expression in MCF 7 DOX cells.

To investi gate the role of the PI3K Akt pathway in Cox 2 expres sion, we studied the effect of the PI3K Akt inhibitor LY294002 on EGF induced pAkt and Cox 2 expression. Western blot analysis showed that LY294002 dramati cally suppressed pAkt activation and Cox 2 expression induced by EGF in MCF 7 DOX cells. Because Cox 2 exerts its effects by producing PGE2, which binds to specific EP receptors, we investi gated the role of specific EP receptors in Cox 2 mediated invasion of MCF 7 DOX cells. PGE2 treatment induced expression of the EP1 and EP3 receptors, sug gesting that these two receptors are likely involved in the invasiveness by MCF 7 DOX cells. Both EP1 and EP3 receptors played an important role in Cox 2 induced invasion of MCF 7 DOX cells. We showed that selective inhibition of EP1 and EP3 using siRNAs inhib ited PGE2 induced invasion of MCF 7 DOX cells, as well as expression of MMP 2 and MMP 9.

A previous study showed increased Cox 2 expression in patients with poorly differentiated breast cancer and poor clinical outcomes for patients treated with doxorubicin. However, the expression pattern of EP receptors has never been studied in breast GSK-3 cancer. Therefore, our find ings are the first to suggest a pivotal role for the EP1 and EP3 receptors in doxorubicin resistant breast cancer cells.