探討棉黃素促進前列腺癌細胞自噬死亡之作用

The study of effect of gossypetin on autophagic cell death in human prostate cancer cells

徐英華(Ying-Hua HSU)

指導教授 ： 林慧萱

中山醫學大學/醫學科技學院/醫學檢驗暨生物技術學系碩士班/碩士(2014年)

摘要

棉黃素(Gossypetin)源自於洛神和棉屬植株分離出的一種類黃酮(flavonoid)物質，過去研究已指出Gossypetin具有抗氧化、抗菌、以及抗動脈粥狀硬化的能力，然而其抗癌活性仍尚未釐清。因此本文以體內與體外試驗探討Gossypetin抗前列腺癌之作用及其分子機制。首先以Gossypetin處理三株前列腺癌細胞(LNCaP、PC3、DU145)進行細胞毒性測試，Trypan blue assay發現Gossypetin對於三株前列腺癌細胞都具有抑制細胞生長之作用。接著利用流式細胞儀與DAPI染色分析觀察到Gossypetin誘導三株前列腺癌細胞凋亡(apoptosis)反應。另外，在AVO染色結果發現LNCaP與PC3也呈現AVO-positive的細胞，其中以LNCaP細胞自噬(autophagy)的情況最為明顯。進一步以西方墨點法探討Gossypetin作用於三株前列腺癌細胞之死亡分子機制，Gossypetin可誘導LNCaP和PC3細胞藉由活化class III PI3K/Beclin 1且增加LC3-II/I比值和Atg5/12 conjugate，促使細胞自噬死亡(autophagic cell death)；但DU145細胞並無自噬路徑的活化，而其caspase-3和PARP-1蛋白的活化表現顯示DU145是走向細胞凋亡途徑。最後，在體內試驗利用前列腺腫瘤移植老鼠模式發現，給予Gossypetin後造成腫瘤生長的抑制，並在分析組織蛋白的表現發現LC3-II和Atg5/12的表現量上升，class III PI3K/Beclin 1路徑的活化，以及促進凋亡蛋白caspase-3和Bax的表現，顯示Gossypetin確實能夠同時誘導LNCaP細胞自噬和細胞凋亡，並且抑制癌細胞之生長。由此推論Gossypetin應用於人類前列腺癌治療的可能性。

關鍵字

棉黃素 ； 洛神植株 ； 棉屬植株 ； 前列腺癌細胞 ； 細胞凋亡 ； 細胞自噬

並列摘要

Gossypetin, a flavone originally isolated from Hibiscus and Gossypium species, has been shown to possess antioxidant, antimicrobial, and anti-atherosclerotic activities. However, the molecular mechanisms involved in chemopreventive activity of gossypetin are poorly understood. In this study we were conducted to examine the mechanism of the anti-cancer potential of gossypetin. We utilized the well-established in vitro and in vivo methods, trypan blue assay, flow cytometric analysis, 4',6-diamidino-2-phenylindole (DAPI) assay, acidic vesicular organelles (AVO) stain and a mouse model, to analyze the effect of gossypetin on cell viability, cell cycle, and programmed cell death of human prostate cancer (CaP) cells, incluging LNCaP, PC3, and DU145 cells. To highlight the anti-cancer mechanisms of gossypetin, the expressions of molecular proteins were measured by Western blotting. Gossypetin could inhibit LNCaP, PC3, and DU145 cell growth in a dose-dependent manner. Gossypetin also was evaluated for apoptotic activities in CaP cells. Our results revealed that the three kinds of cells CaP cells showed the different morphology. LNCaP and PC3 cells presented DAPI-positive morphology, and had an increase in autophagosomes with double-membrane structure after a 24-h treatment with gossypetin. The growth inhibitory effect of gossypetin on DU145 cells only was mediated via apoptotic mechanism. Furthermore, this apoptotic effect of gossypetin in LNCaP cells might be mediated via the regulation of Bcl-2 family and inhibition of class I PI3K/Akt/mTOR pathway. In addition, gossypetin mainly could induce cellular autophagy via class III PI3K/Beclin 1/Atg-5/12/LC3 signaling. Finally, gossypetin was evidenced by its inhibition on the growth of LNCaP cells in xenograft tumor studies. As a result, our data presented the first evidence of gossypetin as an inducer of progressed cell death in LNCaP cells via dual apoptotic and autophagic pathways. These findings may open interesting perspectives to the strategy in human CaP treatment.

並列關鍵字

Gossypetin ； Hibiscus ； Gossypium ； Prostate cancer cells ； Apoptosis ； Autophagy

參考文獻

1. Gronberg H. Prostate cancer epidemiology. Lancet. 361: 859–864,

2003.

2. Kristal AR, Cohen JH, Qu P, and Stanford JL. Associations of energy,

fat, calcium, and vitamin D with prostate cancer risk. Cancer Epidemiol Biomarkers Prev. 11:719–725, 2002.

3. Van der Cruijsen-Koeter IW, Vis AN, Roobol MJ, Wildhagen MF, de Koning HJ, van der Kwast TH, Schroder FH. Comparison of screen detect and clinically diagnosed prostate cancer in the European randomized study of screening for prostate cancer, section Rotterdam. J Urol. 174:121–125, 2005.