探討Steviol活性衍生物在人類賀爾蒙不依賴型轉移性前列腺癌細胞之抗癌作用機轉

Mechanism Study of Active Steviol Derivative against Human Hormone-Refractory Metastatic Prostate Cancer

林庭卉(Ting-Huei Lin)

指導教授 ： 顧記華

國立臺灣大學/醫學院/藥學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703547

摘要

Steviol glycosides是由甜菊(Stevia rebaudiana)萃取而來的diterpene glycosides，近來有許多Stevioside相關代謝產物在營養以及藥理活性方面的研究，其中Steviol衍生物的討論以及調查相關藥理活性的相關研究更是引起極大的興趣。因此，我們的同仁合成了一系列的Steviol衍生物，本實驗室使用sulforhodamine B assay以PC-3細胞來篩選(一種對賀爾蒙療法具抵抗性並且已轉移的前列腺癌細胞株) ，結果發現STD-30具有最佳抗增生活性，其IC50為1.09 μM。在長時間的細胞群落生長試驗中更可以觀察到STD-30對PC-3細胞株的抑制效果有明顯的提升，其IC50為0.11 μM。藉由流式細胞儀分析經過propidium iodide染色的細胞，可以觀察到經過STD-30藥物處理後，代表細胞凋亡的Sub-G1細胞明顯的提高；另外，分析經過carboxyfluorescein succinimidyl ester (CFSE)染色的細胞也能觀察到STD-30的抗細胞增生活性。使用西方點墨法可以觀察到STD-30會造成Bcl-2家族蛋白中促細胞凋亡的Bid磷酸化現象以及Bak cleavage form的產生，以及JNK與c-Jun的活化。再更進一步使用流式細胞儀分析經由dichlorodihydrofluorescein diacetate (DCFH-DA)染色的細胞，可以觀察到STD-30會使細胞內活性氧化物質(ROS)明顯的上升，並且能被N-acetylcysteine (NAC, 抗氧化物)以及BAPTA (鈣離子螯合劑)抑制。另外，STD-30還會導致細胞外鈣離子進入細胞的量增加，此現象能被NAC抑制。這些研究顯示在STD-30作用下，ROS的生成會和細胞內鈣離子有交互作用。更進一步使用不同抑制劑觀察是否會影響藥物對細胞凋亡的效果以及在Bid、Bak、c-Jun產生的現象，結果顯示NAC、diphenyleneiodonium (NADPH oxidase抑制劑)、trolox (水溶性維他命E類似物), SP600125 (JNK抑制劑)以及Go 6983 (廣範圍protein kinase C抑制劑)都會逆轉細胞凋亡的現象；NAC、SP600125、移除細胞外鈣離子(Hank's Balanced Salt Solution + EGTA)都能夠逆轉Bid、Bak、c-Jun的現象。綜合上述實驗結果，steviol的衍生物STD-30在PC-3細胞中抑制細胞增生及導致細胞凋亡活性是來自ROS以及增加細胞內鈣離子濃度，雖然其上下游還有些尚待釐清的部分，但它展示了能夠被更進一步發展及研究的抗癌潛力。

關鍵字

Steviol ； JNK ； Bid ； 活性氧化物 ； 鈣離子

並列摘要

Steviol glycosides are a group of sweet diterpene glycosides from Stevia rebaudiana. In recent decades, there are increasing studies reporting the nutritional and pharmacological benefits of these secondary metabolites. With many interests, the derivatives of steviol have been produced and the related pharmacological effects have been examined. Our colleagues have synthesized a number of steviol derivatives and, after an anti-proliferative screening test using sulforhodamine B assay in human hormone-refractory metastatic prostate cancer (HRMPC) cell line PC-3, the most effective derivative STD-30 stood out with an IC50 of 1.09 μM. A long-term exposure of cells to STD-30 for ten days resulted in a dramatic inhibition of colony formation with an IC50 of 0.11 μM. The flow cytofluorometric analysis of propidium iodide staining demonstrated that STD-30 caused an increase of sub-G1 population (apoptosis) and the analysis of carboxyfluorescein succinimidyl ester (CFSE) staining substantiated the anti-proliferative activity. Furthermore, the flow cytofluorometric analysis of dichlorodihydrofluorescein diacetate (DCFH-DA) staining demonstrated that STD-30 induced a significant production of reactive oxygen species (ROS), which was inhibitable by N-acetylcysteine (NAC, an antioxidant) and BAPTA (a calcium chelator). Furthermore, STD-30 induced an increase of extracellular Ca2+ influx that was NAC-inhibitable. The data suggested a crosstalk between the ROS production and intracellular Ca2+ mobilization to STD-30 action. Further study demonstrated that the apoptosis induced by STD-30 was significantly rescued by NAC, diphenyleneiodonium (an NADPH oxidase inhibitor), trolox (a water-soluble vitamin E analog), SP600125 (a JNK inhibitor) and Go 6983 (a broad spectrum protein kinase C inhibitor). Taken together, the data suggest that STD-30 induces both anti-proliferative and apoptotic activities in PC-3 cells through a crosstalk interaction between ROS production and Ca2+ influx. Although the functional contribution of NADPH oxidase, JNK and PKC needs further elucidation, the steviol derivative STD-30 displays anticancer potential for further development.

並列關鍵字

Steviol ； JNK ； Bid ； ROS ； Calcium

參考文獻

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