Amphiregulin (AR)是上皮生長因子(epidermal growth factor)的一員，不僅會刺激角化細胞(kerati-nocyte)的增生，而且也會促進口腔癌細胞株的生長移動及侵犯能力的增加。表皮生長因子接受器1(epidermal growth factor receptor 1)，AR的主要受體之一，在許多癌細胞的表現量比正常高，同時受體活化常造成mitogen-activated protein kinases (MAPK)的磷酸化，並進一步誘使cyclooxy- genase-2(COX-2)產生。另一方面， 在許多種惡性前期與惡性組織中，COX-2 常常是過度表現的。然而，AR在調控COX-2表現的作用機轉，現在仍不清楚。目前我們發現在口腔癌細胞中，AR誘導COX-2蛋白質表現，是藉由影響COX-2的轉錄，但不會影響COX-2蛋白質新合成及其mRNA的穩定性。這個結果與我們另一個發現一致，AR確實會調控COX-2啟動子的轉錄活性，並且調控情形會隨著劑量的增加而加強。因此，我們將利用連續性分段COX-2啟動子之構築基因與合併使用不同激酶抑制劑，進一步探討AR對COX-2 啟動子的影響與其中涉及的訊息傳遞路徑。EGFR1含有數個酪胺酸殘基，當被配體活化後會導致EGFR1的酪胺酸殘基磷酸化。因此，我們以西方點墨法分析AR會誘導EGFR1上哪些酪胺酸殘基(Y-845，992， 1045，1068和1173)的磷酸化，並分析有哪些蛋白質激酶參與AR誘導COX-2表現。我們發現AR會誘導酪胺酸殘基(Y845,1068)的磷酸化。而Y845與Y-1068的下游包含PKC，c-Src， PI3K及MAPK傳遞路徑，因此，這些激酶有可能參與AR誘導COX-2的表現。在我們抑制PKC，c-Src，PI3K 或 MAPKs的活化後，發現AR誘導COX-2的表現會部分經由c-Src 與 MAPK 但不會透過 PKC 或 PI3K。 另外，我們確實發現AR會促使口腔癌細胞(KB或 SCC-25)型態明顯的改變，即所謂上皮細胞向間質細胞轉換(epithelial mesenchymal transition, EMT)。綜合以上發現，我們想進一步研究AR誘導COX-2表現更詳細的機轉，並探討AR調節口腔癌細胞產生EMT現象中是否COX-2的表現在此現象中也扮演重要的角色。

Amphiregulin (AR), a member of epidermal growth factor (EGF)-like family, not only stimulates keratinocyte proliferation, but also promotes the proliferation, migration and invasion abilities of oral cancer cells. Activating EGFR1, a primary receptor of AR, often leads to MAPK activation and subsequent induction of COX-2. Moreover, overexpressions of EGFR or COX2 are frequently observed in various cancerous tissues. However, the role of AR in the regulation of COX-2 expression remains elusive. We demonstrated that AR-induced expression of COX-2 protein primarily mediated the synthesis but not stability of COX-2 mRNA and no de novo protein synthesis was required for the induction. Consistent with this finding, AR dose-dependently induced the expression of COX-2 promoter-driven reporter activity. Serial truncated COX-2-promoter reporter constructs together with pharma-cological inhibition were used to address the signaling pathways involved in the COX-2 promoter mediation. Following activation, several tyrosine residues on the C-terminal of EGFR1 are phosphorylated. We next examined by Western blotting analysis if AR induced any phosphorylation of EGFR1 on the tyrosine (Y) residues 845, 992, 1045, 1068 and 1173, and if any protein kinases were involved in the AR-induced COX-2 expression. AR-induced tyrosine phosphorylation was preferentially at Y845 and Y1068, suggesting the involvement of c-Src, PI3K and MAPK. Pharmacological inhibition of PKC, c-Src, PI3K or MAPK indicated that AR-induced COX-2 was in part through activation of c-Src and MAPK but neither PKC nor PI3K. In addition, AR was able to induce oral squamous cells, KB or SCC-25, to undergo a morphological change, reminiscence of epithelial mesenchymal transi- tion (EMT). More studies are warranted to address the detailed mechanism whereby AR induced the expression of COX-2 and whether the induced COX-2 plays any role in the AR-mediated EMT.

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