| Overexpression of cyclooxygenase-2 (COX-2) and elevation of its product prostaglandin E2 (PGE2) are implicated in the pathogenesis of human esophageal squamous cell carcinoma. COX-inhibitors also have been demonstrated to overcome multidrug resistance (MDR) in some cancer cells. Therefore, our studies are designed to investigate the role of COX-2 and PGE2 in cell proliferation and drug resistance on human esophageal squamous cell carcinoma cells.PGE2 stimulated cell proliferation of HKESC-1, a human esophageal squamous cell carcinoma cell line. PGE2 exerts its effects through four subtypes of G-protein-coupled receptors, namely EP1 to EP4. In this regard, we showed that all four EP receptor subtypes were expressed in a panel of human esophageal squamous cell carcinoma cell lines (HKESC-1, HKESC-2, HKESC-3, KYSE150, and EC109). Further characterization by pharmacological and RNA interference approaches revealed that EP2 receptor mediated the mitogenic effect of PGE 2 in HKESC-1 cells. EP2 receptor agonist butaprost mimicked the mitogenic effect of PGE2, whereas knockdown of the EP2 receptor attenuated the PGE2-induced proliferation. In relation to the signaling mechanism, PGE2 and butaprost induced phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), whose down-regulation by RNA interference significantly attenuated PGE2-induced cell proliferation. Moreover, ERK1/2 activation by PGE2 was completely abolished by protein kinase C (PKC) inhibitor, Ro-31-8425. In addition, PGE2 and butaprost increased c-Fos expression and activator protein-1 (AP-1) transcriptional activity, which were abolished by the ERK1/2 kinase inhibitor, U0126. AP-1-binding inhibitor, curcumin, also partially reversed the mitogenic effect of PGE2. Apart from c-Fos, PGE2 also increased c-Myc expression and its association with the binding partner Max. Knockdown of c-Myc by RNA interference attenuated PGE 2-induced cell proliferation. Further mechanistic study revealed that PGE2 increased the protein stability and nuclear accumulation of c-Myc via phosphorylation on serine 62 in an ERK1/2-dependent manner. Moreover, the effect of PGE2 on c-Myc expression was mimicked by butaprost. These findings suggest that PGE2 promotes cell proliferation via EP2/PKC/ERK-dependent induction of c-Fos and c-Myc expression in human esophageal squamous cell carcinoma cells.In the event to assess the involvement of COX in cancer cell drug resistance, different COX-inhibitors (indomethacin, SC236, SC560, nimesulide and NS398) were employed. They all substantially suppressed PGE2 production to a similar extent. However, only the non-selective COX inhibitor indomethacin and the COX-2 selective inhibitor SC236 enhanced cytotoxic effects of doxorubicin on HKESC-1 and HKESC-2 cells, and these effects could not be reversed by the addition of PGE2. Knockdown of COX-2 also failed to mimic the enhancing effect of indomethacin or SC236 on cytotoxicity, implicating that their action is COX- and PGE2-independent. To this end, we observed that indomethacin and SC236 directly functioned as non-competitive inhibitors of P-glycoprotein (P-gp), which were manifested as reduction of P-gp ATPase activity. Collectively, these findings suggest that the direct inhibitory effect of indomethacin and SC236 may contribute to their ability to increase the intracellular retention of doxorubicin and thus enhance its cytotoxicity. (Abstract shortened by UMI.)... |
