林慧萱林佳良Chia-Liang Lin醫學檢驗暨生物技術學系碩士班2026-10-159999-12-312013https://handle.ncl.edu.tw/11296/kvjm6ehttps://scholars.csmu.edu.tw/handle/123456789/22067多酚(polyphenols)在先前的研究已被證實能預防癌症，並且能抑制癌細胞的生長與轉移。過去研究也指出洛神葉(Hibiscus sabdariffa leaf)具有豐富的類黃酮(flavonoid)等多酚物質，顯示洛神葉具有抗癌之潛力。因此，本研究擬探討神葉多酚萃取物(H. sabdariffa leaf polyphenolic extracts, HLP)對於抑制前列腺癌細胞的生長作用以及研究相關訊息傳遞路徑。首先以HLP處理人類正常細胞(WS-1)以及三株前列腺癌細胞(LNCaP、PC-3以及DU-145)，發現HLP對於前列腺癌LNCaP細胞具有較強細胞毒性。接著利用HLP和其主要成份ECG (epicatechin gallate)以及植物固醇β-sitosterol處理LNCaP細胞進行trypan blue assay偵測細胞毒性以及BrdU增生試驗分析，結果顯示HLP、ECG和β-sitosterol皆可抑制前列腺癌細胞生長。利用流式細胞儀及DAPI染色分析在相同處理條件下，HLP會促使細胞凋亡(apoptosis)現象發生。另外AVO染色試驗則證實HLP及β-sitosterol會誘導產生細胞自噬(autophagy)現象。上述結果皆顯示出HLP相較於ECG和β-sitosterol具有明顯促使細胞死亡的能力。進一步，利用西方點墨法(Western blotting)探討HLP、ECG和β-sitosterol對於LNCaP細胞凋亡和自噬的調控機轉，結果發現HLP會誘導Fas/FasL、Bax和p53蛋白表現以及caspase-3蛋白活化，此機制可能與促使細胞凋亡有關。而在細胞自噬相關蛋白分析方面，結果得知HLP以及β-sitosterol會促進細胞自噬相關蛋白的表現。並藉由FasL專一性抑制劑NOK-1或細胞自噬抑制劑3-MA分別確認HLP誘導LNCaP細胞兩種死亡機制：細胞凋亡主要是透過Fas/FasL訊息路徑所調控；此外，HLP部分藉由調控PI3K class III/Beclin-1/LC-3路徑，以促進自噬作用。總結，洛神葉多酚萃取物可誘導LNCaP細胞死亡，主要是透過細胞凋亡及自噬作用。本研究結果說明洛神葉具有發展為前列腺癌治療試劑的潛在價值，值得進一步探討。Many studies have shown that polyphenols prevent and inhibit the growth and metastasis of cancer cells. In previous study, Hibiscus sabdariffa leaf was demonstrated be rich in flavonoid, and possess anti-cancer potential. Recently, the data of our laboratory presented the evidence of H. sabdariffa leaf extract (HLE) as an apoptosis inducer in human prostate cancer LNCaP cells. Therefore, the object of the study was to examine the anticancer properties and molecular mechanisma of H. sabdariffa leaf polyphenolic extract (HLP) on LNCaP cells. Trypan blue assay and BrdU assay showed HLP, its main composition epicatechin gallate (ECG) and β-sitosterol, a plant sterol, inhibit the LNCaP cell growth. Our results revealed that the cells presented DAPI-positive morphology, and had an increase in the distribution of hypodiploid phase after a 48-h treatment with HLP. This effect of HLP in LNCaP cells might be mediated via the death receptor (FasL-mediated caspase-8) and/or partially mitochondria (Bax-mediated caspase-9) pathways. In addition, HLP and β-sitosterol could induce mainly cellular autophagy via PI3K class III/Beclin-1/LC3 signaling. Molecular data showed these influences of HLP might be mediated via apoptosis and autophagy pathways, as demonstrated by the treatment of FasL antagonist (NOK-1) or autophagic inhibitor (3-MA), respectively.Based on the above, HLP induced LNCaP cell apoptosis and autophagy. These results suggested that HLP potentially could be developed as an anti-cancer agent, and may open interesting perspectives to the strategy in human prostate cancer treatment.thesis