色胺酮在乳癌细胞MCF-7的作用

Abstract

多重抗藥性（mutidrug resistance）是癌症化學治療常見的問題，經常伴隨某些酵素或細胞生物標記表現量或功能上的改變，如P-gp（P-glycoprotein），MRP（mutidrug resistance-releated protein），topoisomerase或glutathione等。我們利用乳癌細胞株MCF-7/WT和具有對adriamycin類抗癌藥有抗藥性的細胞株MCF-7/ADR，研究在此兩種細胞株表現量有差異的酵素或蛋白質，包括ER-α (estrogen receptor α)、ER-β (estrogen receptorβ)、PR (progestrone receptor)、VEGF（vascular endothelial growth factor）、HIF-1α ( hypoxia-inducible factor-1α ) 、FoxO1（forkhead box-containing protein，O subfamily）、C/EBP β（CCAAT/enhancer-binding protein β）、CtBP1(C-terminal binding protein 1)、dicer 1、argonaute 2、PIM-1以及PKc α (protein kinase c α)，藉以深入研究多重抗藥性的機制。 色胺酮(tryptanthrin)是一種吲哚類衍生物，併用tryptanthrin和doxorubicin可使MCF-7/ADR細胞對doxorubicin的敏感性上升、doxorubicin IC50值大幅降低。 我們以tryptanthrin為工具，利用聚合酶連鎖反應法(PCR)、即時定量-聚合酶連鎖反應法 (real-time PCR)以及西方墨點法(Western blot) 觀察MCF-7/WT和MCF-7ADR細胞在加了10-6 M tryptanthrin五天後，對前述目標蛋白質的影響。在篩選的過程中雖然沒有發現新的方向來解釋MCF-7/ADR細胞抗藥性產生的原因，但發現tryptanthrin具有抑制核蛋白受體表現的能力。MCF-7/WT細胞加入tryptanthrin後，ER-α蛋白的表現下降了一半。並且進一步藉由EMSA實驗證實tryptanthrin會影響ER-α啟動子上IEF-1區域與SP1家族蛋白的鍵結，暗示著tryptanthrin抑制ER-α基因的表現可能是經由減少ER-α啟動子與SP1家族蛋白的鍵結。MCF-7/WT細胞的PR基因比ER-α基因更快受到tryptanthrin的抑制，但文獻報導PR基因位於ER-α基因下游，暗示著PR基因受到tryptanthrin的影響可能不是經由ER-α基因的改變。另外還藉由siRNA抑制MCF-/WT細胞的ER-α基因，發現MDR1基因並沒有因此而表現，所以tryptanthrin抑制ER-α基因的表現和MCF-7/ADR細胞表現MDR1基因並沒有直接相關。

Multidrug resistance (MDR), the resistance of tumor cells to anticancer agents, remains a major cause of treatment failure for cancer patients. MDR usually occurs with alteration of function and expression of some proteins and enzymes, for example P-gp (P-glycoprotein), MRP (multidrug resistance-related protein), topoisomerase and glutathione. Recently, the genes which show differential expressions in MCF-7/WT and its doxorubicin-resistant counterpart MCF-7/ADR include ER-α (estrogen receptor α), ER-β (estrogen receptor β), PR (progesterone receptor), VEGF（vascular endothelial growth factor）, HIF-1α ( hypoxia-inducible factor-1α ) , FoxO1（forkhead box-containing protein，O subfamily）, C/EBP β（CCAAT/enhancer-binding protein β）, CtBP1(C-terminal binding protein 1), dicer 1, argonaute 2, PIM-1 and PKc α (protein kinase c α). Our lab previously demonstrated that tryptanthrin could reverse the resistance to doxorubicin in MCF-7/ADR. In order to extensively understand the mechanisms of multidrug resistance, MCF-7/WT and MCF-7/ADR were treated with trptanthrin to examine the changes in expression of the above genes in this study, using RT-PCR, realtime-PCR and Western blot. Results show that tryptanthrin suppresses the expression of nuclear receptor genes in MCF-7/WT. The expression of ER-α is 50％ down. Furthermore, the binding of SP1 family proteins to ER-α promoter site IEF-1 decreases upon tryptanthrin treatment. In mRNA level, tryptanthrin inhibits the expression of PR prior to the expression of ER-α. As PR is downstream protein of ER-α, it seems that tryptanthrin acts on PR and ER-α via different pathways. When ER-α expression was knockdowned by siRNA in MCF-7/WT, the expression of MDR1 gene did not induced, suggesting the decrease in ER-α was not related to MDR1 gene expression in MCF-7/ADR.