| 摘要: | 攝護腺癌目前的治療著重在抑制雄性素在體內濃度，然而往往會復發成非雄性素依賴性，所以發展新藥對抗攝護腺腫瘤是迫切需的。我們從中藥南柴胡中篩選到的全新純化物新穎 r 丁內酯化合物 (4-benzo[1,3]dioxol-5-ylmethyl-3-(3,4,5-trimethoxylbenzylidene)dihydro-furan-2-one) 分子量為 398.41 kDa，簡稱 K8，其擁有一個對掌中心並可分離出兩個立體異構物 (E-K8和Z-K8)。E-form與Z-form的K8皆可抑制多種癌細胞的增生，包含攝護腺癌、肺癌、肝癌、大腸直腸癌、惡性腦膠質瘤等。其中，Z -form K8已證實能降低雄性素受體的表現、並調控EGR-1 與 NAG-1 的表現。由於雄性素受體 (AR) 是臨床上攝護腺癌主要治療的標的。因此深入去探討 Z-K8 對 LNCaP 細胞內的 AR 基因的影響。實驗結果顯示 Z-K8 能顯著的抑制LNCaP細胞株的增生，並透過提高 caspase-3 的活性誘導細胞凋亡。AR mRNA和蛋白質也會隨著處理Z-K8的時間增加而抑制更多。此外，也發現 Z-K8 能增加 JNK 的磷酸化，而前處理 JNK 的抑制劑 (SP600125) 能部分的降低受到 Z-K8 誘導的細胞死亡；且抑制 JNK 路徑後Z-K8抑制AR蛋白的能力顯著降低。 我們另轉染帶有突變Sp1 site 的AR 啟動子，與控制組相比，Z-K8 的抑制能力也是降低的。本實驗室並建立攝護腺癌的裸鼠異位移植腫瘤動物模式，並以此動物模式作為抑制攝護腺癌增生的藥物測試平台，探討 Z-K8抑制攝護腺癌的效果。實驗發現以15 mg/kg 較低劑量 Z-K8 可以抑制 60 % 的裸鼠腫瘤生長。在本篇論文中，我們另外針對Z-K8對攝護腺癌細胞內miRNA的表現影響和差異，並且深入探討miR-301a在此條件下所扮演的角色。我們發現Z-K8處理後的攝護腺癌細胞的miR-301a表現量比處理前有明顯的上升，轉染miR-301a入細胞時 AR也隨著下降，所以，我們證實 miR-301a 會調控 AR 的表現。試驗另加入miR-301a抑制劑於攝護腺癌細胞中，攝護腺癌細胞經抑制miR-301a後，細胞能增加其耐受性。綜合以上結果指出 Z-K8 不只能誘導攝護腺癌細胞凋亡並能抑制 AR 的表現，因此期望未來Z-K8能開發為治療攝護腺癌的新藥。
On the average, the prostate cancer (PCa) therapy is emphasized on the suppression of androgen, because PCa cells rely on androgens for proliferation and survival. However, the effectiveness of androgen deprivation therapy is temporary, and tumors in the majority of patients eventually relapse and evolve into castration resistant prostate cancer (CRPC). We previously demonstrated that the crude acetone extract of Bupleurum scorzonerifolium (BS-AE) and its active anti-proliferative component, (4-benzo[1,3]dioxol-5-ylmethyl-3-(3,4,5-trimethoxylbenzyli- dene)dihydro-furan-2-one). The molecular weight is 398.41 kDa, abbreviated as K8, which has a chiral centerring and two stereoisomeric racemic forms (E-K8 and Z-K8). In one aspect, the above two kinds of compounds can inhibit the hyperplasia of the cancer cells, include PCa, lung cancer, hepatocarcinoma, colon cancer, glioblastoma multiformis. Among them, Z-Form K8 has already verified that can reduce the androgen receptor (AR) expression, and up-regulation the EGR-1 as NAG-1 expression. Because AR is a main therapeutic target for treatment of PCa, so we chose the androgen-dependent human PCa cell line LNCaP and the androgen-independent cell lines DU145 and PC-3 to study the anti-tumor potency and AR regulation mediated by Z-K8. In LNCaP cells, Z-K8 significantly repressed cell proliferation, induced apoptosis, repressed AR mRNA and protein expression in a time-dependent manner. We then confirm Z-K8 can induce phosphorylation of c-Jun-N-terminal kinase (JNK), and JNK inhibitor partially reduced Z-K8 induced cell death. Furthermore, treatment with a JNK inhibitor significantly abolished Z-K8-induced AR downregulation. Z-K8 did not significantly inhibit reporter gene expression of constructs containing the AR promoter when it contained a mutated Sp1 binding site. We establish the LNCaP nude mice xenograft tumor model and use this animal model to be a drug-test platform. In our preliminary data, to inject 15 mg/kg in nude mice xenograft tumor model can inhibit tumour about 60 percentage. In this study, we also examine the change of miRNAs profile and the role of miR-301a in human PCa cell lines following the treatment without or with Z-K8. We here found the increased level of miR-301a expressions in LNCaP cells after Z-K8 treatment. Therefore, we examined the role of miR-301a in the mechanism of Z-K8-induced PCa cell death. To elucidate the role of miR-301a, downregulation of AR was tested in LNCaP cells. Here, we identified miR-301a as a regulator of expression of AR. Our data showed that suppression of miR-301a significantly increased the survival rate of LNCaP cells after exposure to Z-K8. Z-K8 not only induced LNCaP apoptosis but also reduced AR expression. These results indicate that Z-K8 may be a potential anti-tumor drug for PCa therapy. |
