蟾蜍靈及華蟾精抑制人類前列腺癌細胞生長之作用機轉
王錫崗
台灣調適科學會
前列腺癌為美國男性最普遍的癌症之一。在台灣，男性罹患前列腺癌的發生率及死亡率逐年增高。許多科學家嘗試不同的方式研究癌症的治療及預防，其中中藥的開發十分受到注目。近年來發現類毛地黃強心藥物，如毛地黃和蟾酥可導致不同腫瘤細胞停止生長或凋亡，但此類藥物對於前列腺癌細胞之影響其作用機制尚不十分清楚。因此，本研究計畫旨在探討蟾酥抑制雄性素依賴性 (LNCaP)、及非雄性素依賴性(DU145和PC3)人類前列腺癌細胞株增殖之效應和其作用機轉。本論文選用之蟾酥類強心藥物中為蟾蜍靈 (bufalin)及華蟾精 (cinobufagin)。首先，將蟾酥類藥物處理過之細胞在顯微鏡下照相，觀察細胞外觀受藥物影響之狀態。其次，以MTT法檢測細胞之增殖速率，再分別計算藥物對於細胞的IC50和倍增時間的影響。另以呈色法觀察藥物處理後細胞內caspase 3, 7, 8, 及9四種酵素活性的變化；再者，將經藥物處理之細胞以流式細胞儀觀察細胞凋亡之情形，並利用西方墨點法、免疫螢光染色法、siRNA..等技術，研究細胞凋亡調控蛋白質的表現，如Fas, cytochrome c, Bcl-2 family、caspases…等。所有數據先以變方分析處理，若呈顯著，再進行Duncans多變域分析。藉著瞭解蟾酥類藥物對於抑制人類前列腺癌細胞生長之作用機轉，以期為臨床提供另一種治療前列腺癌的方式。
關鍵字：蟾蜍二烯羥酸內酯，前列腺癌，細胞凋亡
Inhibitrory Mechanisms of Bufalin and Cinobufagin on the Growth of Human Prostate Cancer Cell Lines
Paulus S. Wang
Society of Adaptive Science in Taiwan
Prostate carcinoma is one of the most common malignant tumors and the seconding case of cancer related death among men in United States. In Taiwan it becomes a more common cancer in men during past 10 years. Numerous chemicals or drugs for therapy of prostate cancer have been developed. Chan su extracts have been known to induce cytostatic or oncolytic effects in a variety of cancers, but few studies about Chan su on prostate cancer cells have been performed. This aim of the present study is to evaluate the anti-proliferation effects and the underlying mechanisms of bufalin and cinobufagin, bufadienolides extracts from Chan su, on the hormone-dependent and -independent prostate cancer cell lines. Cell proliferation of three prostate cancer cell lines, LNCaP, DU145 and PC3, is measured by MTT assay, and calculated to 50% inhibition concentration (IC50) and to doubling time (tD). Western blot and a colorimetric assay are used to analyze the protein expressions and activities of caspases. Other protein expressions of apoptosis modulators, such as mitochondrial Bax, cytosolic cytochrome c, and PARP will be also analyzed by western blot. To investigate whether Fas is involved in the mechanisms that resulted from bufalin and cinobufagin, the techniques of immunocytochemistry and siRNA will be employed. The data will be analyzed by ANOVA and Duncans multiple range tests. We expect that the results will provide a new strategy for therapy of prostate carcinoma in human beings.
關鍵字：bufadienolides, prostate cancer, apoptosis, Fas
