希仙草萃取物及其有效成分對子宮內膜癌細胞生長與轉移之抑制作用及其機轉探討

Inhibitory effects and mechanisms of Siegesbeckia orientalis extract and its bioactive ingredients on proliferation and metastasis of endometrial cancer cells

張基昌(Chi-Chang Chang)

指導教授 ： 洪哲穎

義守大學/理工學院/化學工程學系暨生物技術與化學工程研究所/博士(2015年)

https://doi.org/10.6343/ISU.2015.00221

摘要

子宮內膜癌為歐美常見的惡性子宮體癌，台灣地區子宮內膜癌的發生率僅次於子宮頸癌，近年來其病患數有逐漸升高的趨勢。子宮內膜癌的治療方式通常是手術，以及術後合併以放射線治療、化學治療以及賀爾蒙進行輔助治療。然而，此些術後輔助治療方式的效果仍有限，因此有效輔助治療藥物的開發可謂當務之急。希仙草屬雙子葉菊科草本植物，學名為Siegesbeckia orientalis linne，具有治療四肢麻痺、風濕關節痛、筋骨疼痛、腰膝無力、高血壓、疔瘡腫毒、外傷出血等功效。儘管有相關文獻指出希仙草之效用，但科學驗證之報導仍然極少。本研究主要在探討希仙草乙醇萃取物 (S. orientailis ethanol extract, SOE) 對子宮內膜癌細胞株之毒殺效果，並針對其抑制癌細胞生長及誘發凋亡之作用進行深入之探討，後續再針對SOE及其主要成分caryophyllene oxide (CPO) 抑制子宮內膜癌轉移之效果及其機制做進一步探討。首先，實驗中可發現SOE對RL95-2子宮內膜癌細胞之毒殺作用會隨劑量增加及投藥時間延長而愈趨明顯。以顯微鏡與微影像電子目鏡觀察細胞型態之變化，SOE會誘導RL95-2細胞皺縮、空泡化、細胞膜脹破，並造成細胞懸浮，顯示為細胞走向計畫性凋亡之特徵。由流式細胞儀分析細胞週期及凋亡程度結果顯示，SOE能使RL95-2細胞停滯於G2/M生長期，並隨投藥時間增加，最終誘導細胞走向凋亡。其次，在成份分析實驗中發現CPO可能為SOE誘導RL95-2細胞凋亡之主要活性成份。
Transforming growth factor β (TGFβ) 是具多功能的細胞激素，可參與人類腫瘤的生成過程，包括癌細胞的增生、生存、轉移、侵襲及血管新生等。於本研究細胞型態觀察實驗發現，SOE可逆轉由TGFβ1引起之上皮細胞間質轉化作用的發生。SOE及CPO也可抑制由TGFβ1誘導之子宮內膜癌RL95-2 及 HEC-1A細胞株的癒合、移行與侵襲作用，且隨SOE及CPO濃度增加有呈劑量效應關係。此外，本研究亦觀察到在子宮內膜癌細胞株中，SOE及CPO對於TGFβ1所刺激之MAPK pathway、p-Akt、uPA、MMP-2、MMP-9等訊息傳遞分子的活化具有顯著的抑制效果，可進一步阻止癌細胞降解細胞外間質，最終達到抗轉移之效果。

關鍵字

希仙草 ； 丁香烯氧化物 ； 子宮內膜癌 ； 生長 ； 移行 ； 侵襲 ； 轉移

並列摘要

Endometrial cancer is one of the most common uterine cancers in the United States and Europe. In Taiwan, the incidence of endometrial cancer is second only to cervical cancer and its cases are increasing gradually. Treatment for endometrial cancer usually includes surgery and the postoperative combination therapy with radiation, chemotherapy and hormonal treatment for advanced endometrial cancer. However, the efficiency of these therapeutic strategies remains limited. Therefore, developing an efficacious adjuvant drug for endometrial cancer might be most pressing matter of the moment. Siegesbeckia orientalis linne is an herbaceous and dicotyledonous plant, which belongs to Asteraceae. It is a traditional Chinese medicinal herb endowing with the medicinal facts to treat limb paralysis, rheumatism, joint pain, aching pain, lassitude, weakness, hypertension, detumescence, detoxification, trauma, bleeding. However, these bioactivities have not yet been well examined scientifically. In this study, we investigated whether S. orientailis crude extract (SOE) and its potential active agents induce endometrial cancer cell apoptosis and whether they can inhibit metastasis of endometrial cancer cells. The results revealed that SOE had a significant cytotoxic effect on endometrial carcinoma RL95-2 cells and arrested cells in the G2/M phase of cell cycle. From the result of GC-MS analysis, caryophyllene oxide (CPO) was the major bioactive ingredients of SOE.
Transforming growth factor β (TGFβ) is a multifunctional cytokine involved in tumorigenesis in human cancer including proliferation, cell survival, angiogenesis, migration and invasion during tumor progression. This study shows that SOE and CPO can inhibit TGFβ1-induced cell wound healing, cell invasion and cell migration in a dose-dependent manner in two endometrial cancer cell lines, RL95-2 and HEC-1A cells. The TGFβ1-induced epithelial-mesenchymal transition, including the loss of cell-cell junction and the lamellipodia-like structures, could be reversed by SOE and CPO. Moreover, SOE and CPO inhibited significantly the expression of MAPK pathway, p-Akt, MMP-9 and MMP-2 in RL95-2 cells dose-dependently. The inhibition of activation of pro-MMPs to MMPs by suppressing the uPA activity may be involved in the possible molecular mechanism.

並列關鍵字

Siegesbeckia orientalis linne ； caryophyllene oxide ； endometrial cancer cells ； proliferation ； migration ； invasion ； metastasis

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