Goniothalamin與 tubocapsenolide A引發人類乳癌細胞MDA-MB-231凋亡作用機轉之探討

Studies on the apoptotic effects and mechanisms of action of goniothalamin and tubocapsenolide A in human breast cancer MDA-MB-231 cells

陳文穎(Wen-Ying Chen)

指導教授 ： 吳永昌 ； 吳志中

高雄醫學大學/藥學院/天然藥物研究所碩士班/博士(2008年)

摘要

由於腫瘤的形成及化學治療的抗藥性與腫瘤細胞中凋亡訊號的不活化息息相關，因此促進細胞凋亡 (apoptosis) 是最有效的癌症治療策略之一。本研究即以人類乳癌細胞株MDA-MB-231細胞為材料，深入探討goniothalamin (GT) 及tubocapsenolide A (TA) 在癌細胞中誘發凋亡的作用機轉，以期在新藥開發的領域中能有些許的貢獻。
Goniothalamin是一個新發現的styryl-lactone類天然成分，具有潛在抗癌活性，但其作用機轉未曾被深入探討。本實驗室發現goniothalamin會明顯地誘導MDA-MB-231細胞週期停滯在G2/M期以及造成細胞凋亡。若利用細胞週期抑制劑使細胞停留在特定週期下，以處於G2/M期的細胞對goniothalamin的毒性最敏感。此外，當細胞以goniothalamin處理後，細胞內reactive oxygen species (ROS) 含量增加而glutathione (GSH) 含量減少。這種細胞內氧化還原狀態失衡的現象伴隨Cdc 25C的分解。而細胞以抗氧化劑N-acetylcysteine (NAC) 前處理，會完全防止goniothalamin引起的細胞週期停滯及凋亡；相反的，若以GSH合成抑制劑DL-buthionine-(S, R)-sulfoximine (BSO) 前處理，則會協同性增加goniothalamin的作用。這些結果證明出goniothalamin在MDA-MB-231細胞造成的細胞毒性是因為使細胞產生氧化壓力誘發Cdc 25C分解，導致細胞停滯在G2/M期而對凋亡訊息敏感致死。
本研究探討的另一個成分tubocapsenolide A，則是一個withanolide 結構的類固醇化合物，先前的研究顯示tubocapsenolide A對於許多人類癌症細胞株皆有很強的細胞毒殺效果，但其作用機轉並不清楚。本實驗室發現以tubocapsenolide A處理MDA-MB-231細胞，會導致細胞週期停滯在G1期以及引起細胞凋亡。這些作用與抑制熱休克蛋白90 (heat shock protein； Hsp90)的功能，進而引發Cdk4、 cyclin D1、Raf-1、Akt 和 mutant p53等Hsp90所保護的蛋白質 (client proteins) 產生proteasome依賴性的分解有關。Tubocapsenolide A藉短暫增加細胞內ROS形成及減少GSH 含量來干擾細胞的氧化還原狀態。當以nonreducing SDS-polyacrylamide gels (SDS-PAGE)來分析，發現不論是在完整的細胞或純化的重組蛋白，tubocapsenolide A均快速及選擇性地誘導Hsp90和Hsp70氧化並使之凝集。此外，螢光酵素再摺疊分析 (luciferase refolding assay) 的結果也顯示，tubocapsenolide A會明顯抑制Hsp90和Hsp70複合物的chaperone 活性。為進一步探討氧化還原狀態在tubocapsenolide A的作用中參與的程度，發現以thiol抗氧化劑NAC處理，可以防止tubocapsenolide A引起的所有反應，包括熱休克蛋白的氧化，Hsp90 client proteins的分解以及細胞凋亡。然而非thiol抗氧化劑 trolox 和 vitamin C 卻不能防止Hsp90 被抑制後所造成的反應及細胞死亡。這些結果首次證明tubocapsenolide A在MDA-MB-231細胞引起毒殺作用的機轉至少部分是經由直接氧化細胞內Hsp90和Hsp70 分子上的thiol基團，因而抑制含Hsp90和Hsp70複合物的活性，隨後導致Hsp90的 client proteins不穩定而被proteasome分解，以致引起MDA-MB-231細胞週期停滯及細胞死亡。由於tubocapsenolide A藉氧化作用抑制熱休克蛋白而多重影響癌細胞內複雜的細胞生長及凋亡訊息傳遞路徑，因此，tubocapsenolide A不但可被視為是一個新型的Hsp90和Hsp70 複合物抑制劑，亦被認為在癌症治療上非常具有發展成臨床抗癌新藥的潛力。

關鍵字

恆春哥納香 ； 龍珠 ； 乳癌細胞 ； 氧化還原狀態 ； 熱休克蛋白

並列摘要

Apoptosis is presently one of the most obvious targets for cancer treatment as its relatively common inactivation in tumors, contributes to carcinogenesis, as well as resistance to chemotherapy. In the present study, the anticancer mechanisms of goniothalamin and tubocapsenolide A were investigated at molecular levels in human breast cancer MDA-MB-231 cells, respectively.
Goniothalamin is a styryl-lactone with putative anticancer activities, but its mechanism of action has never been explored before. Goniothalamin treatment of cells significantly induced cell cycle arrest at G2/M phase and apoptosis. By means of cell-cycle synchronization, the G2/M phase cells proved to be the most sensitive fraction to goniothalamin-induced apoptosis. Cells treated with goniothalamin revealed an increase in intracellular reactive oxygen species (ROS) and a decrease in intracellular glutathione (GSH) contents. The disruption of intracellular redox balance caused by goniothalamin was associated an enhancement of Cdc 25C degradation. Furthermore, the antioxidant N-acetylcysteine (NAC) and the GSH synthesis inhibitor DL-buthionine-(S, R)-sulfoximine (BSO), inhibited and enhanced, respectively, the effects of goniothalamin on cell cycle arrest and apoptosis. Taken together, our result demonstrates for the first time that goniothalamin disrupts intracellular redox balance and induces Cdc 25C degradation, which in turn causes cell cycle arrest and cell death maximally at G2/M phase in MDA-MB-231 cells.
Tubocapsenolide A, a novel withanolide-type steroid, has been reported to exhibit potent cytotoxicity against several human cancer cell lines. Treatment of MDA-MB-231 cells with tubocapsenolide A led to cell cycle arrest at G1 phase and apoptosis. The actions of tubocapsenolide A were correlated with proteasome-dependent degradation of Cdk4, cyclin D1, Raf-1, Akt and mutant p53, which are heat shock protein 90 (Hsp90) client proteins. The redox states of cells treated with tubocapsenolide A were interfered by transiently increasing the ROS formation and decreasing the intracellular GSH contents. Nonreducing SDS-PAGE revealed that tubocapsenolide A rapidly and selectively induced thiol oxidation and aggregation of Hsp90 and Hsp70, both in intact cells and in cell-free systems using purified recombinant proteins. Furthermore, tubocapsenolide A inhibited the chaperon activity of Hsp90-Hsp70 complex in the luciferase refolding assay. NAC, a thiol antioxidant, prevented all of tubocapsenolide A-induced effects, including oxidation of heat shock proteins, degradation of Hsp90 client proteins as well as apoptosis. In contrast, non-thiol antioxidants (trolox and vitamin C) were ineffective to prevent Hsp90 inhibition and cell death. Taken together, our results demonstrate that the tubocapsenolide A inhibits the activity of Hsp90-Hsp70 chaperone complex, at least in part, by a direct thiol oxidation, which in turn leads to the destabilization and depletion of Hsp90 client proteins, and thus causes cell cycle arrest and apoptosis in MDA-MB-231 cells. Therefore, tubocapsenolide A can be considered as a new type of inhibitor of Hsp90-Hsp70 chaperone complex, which has the potential to be developed as a novel strategy for cancer treatment.

並列關鍵字

goniothalamin ； tubocapsenolide A ； breast cancer cells ； redox state ； heat shock proteins

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