探討去甲斑蝥素誘發人類前列腺癌細胞凋亡之分子機制

前列腺癌近年來已成為男性國人惡性腫瘤第六位，但往往延誤治療或對荷爾蒙療法產生抗藥性，造成腫瘤復發及轉移。因此尋找並研究天然化合物作為癌症預防或是治療藥物是目前重要課題之一。去甲斑蝥素(Norcantharidin, NCTD) 從斑蝥分離出的斑蝥素(cantharidin)的結構上去甲基所得的衍生物，過去研究指出具有抗癌、凋亡，抑制侵襲及血管新生等多重功用。本研究目的為探討去甲斑蝥素誘導人類前列腺癌細胞凋亡的分子機制。本研究利用MTT證實NCTD抑制四株前列腺癌細胞(PC3、DU145、LNCaP和22Rv1) 生長。利用DAPI染色、Annexin V/PI、JC-1及西方墨點實驗證實NCTD誘導細胞核濃染，粒線體膜電位改變、抑制Bcl-2和Mcl-1表現，增加caspase-3，-6，-7，-9和PARP活化造成細胞凋亡。並進一步證實NCTD透過兩種方式來誘導細胞凋亡：(1) NCTD抑制AKT活化，造成FOXO4轉位到細胞核並結合Mcl-1啟動子來促進細胞凋亡途徑，最後以裸鼠模式證實NCTD抑制腫瘤形成。(2) NCTD誘導miR-320d表現增加並抑制Mcl-1表現造成細胞凋亡。我們證明NCTD透過AKT/ FOXO4/Mcl-1及miR-320d調控Mcl-1誘導前列腺癌細胞凋亡，綜合以上研究證實NCTD在前列腺癌中做為抗癌藥物的發展上深具潛力。

Prostate cancer (PCa) is one of the most lethal malignancies, and it is the second leading cause of cancer death in American men. However, after about few years of androgen and chemo-therapy, almost all tumors will eventually develop into drug-resistant PCa with the distant metastases and poor prognosis. Therefore, development of novel therapy from nature compound is an important topic. Norcantharidin (NCTD), it has been reported to have the anti-proliferation, apoptosis, anti-metastasis and angiogenesis properties in some human cancer cell lines. This study investigates the molecular mechanism of the antitumor effects of NCTD on human prostate cancer cells. In this study, we found that NCTD inhibited the proliferation of four human prostate cancer cell line (PC3, DU145, 22Rv1 and LNcap). DAPI stain, Annexin V/PI stain, JC-1 assay and Western blot indicated that NCTD significantly induced cell apoptosis via increasing of the chromatin condensation, mitochondria dysfunction, suppressing the Bcl-2 and Mcl-1 protein expression, and activating of cleavages of caspases-3, -6, -7, -9, and -PARP. Mechanistic investigations suggested two signaling pathway involved in NCTD induced mitochondria dependent apoptosis. (1) NCTD induced FOXO4 increasment of nuclear translocation and interaction with the Mcl-1 promoter through AKT-dependent pathway, resulting in an apoptotic effect. In vivo animal model also revealed that NCTD significantly reduced tumor growth in mice with PC3 tumor xenografts. (2) Induction of miR-320d by NCTD promoted mitochondria-dependent apoptosis by targeting Mcl-1. Thus, our results provide new insights into the critical role of NCTD in PCa, which is a promising candidate of further anticancer agents for the treatment of Pca.