黃花蜜菜抽出物治療前列腺癌功能與機轉之研究

Abstract

對賀爾蒙療法有抗性的癌症與轉移的癌細胞是前列腺癌治療上的一大挑戰，發展前列腺癌動物模式來研究癌症的惡化過程和有效的治療方式是重要且急迫的任務。在這個研究中，我們利用前列腺癌細胞株(22Rv1) 建立原位腫瘤小鼠模式，以初代培養的方式獲得高轉移性的腫瘤細胞。我們的研究發現，隨著前列腺癌症的惡化過程中癌細胞表現的間質蛋白酶(matriptase)會活化而促進癌細胞轉移。因為，matriptase的抑制蛋白，第二型肝細胞生長因子活化抑制因子(HAI-2)，表現量會隨著癌惡化呈下降的趨勢。此外，在草藥治療前列腺癌的研究中，我們經由PSA啟動子活性的測定，鑑定出黃花蜜菜酒精抽出物中主要的活性成分（ Wedelolectone ，Luteolin，Apigenin），研究發現這些活性成分在抑制前列腺癌細胞生長有協同作用。然而，傳統草藥缺乏詳細、正確的有效成分組成的資料，使其在臨床使用上受到限制。因此，我們使用製備級的液相層析儀截取出富含有效成分的區段，並以LC/MS/MS定性定量分析主要的成分。在比較不同批次之後，通過此定義的草藥精華(WCE)，皆含有一定比例的活性成分且能有效的抑制腫瘤生長。在活性成分間對抑制前列腺癌具有協同作用，此外，我們以藥物動力學分析證明了WCE草藥的組成可以增加活性成分的吸收並減緩活性成分的清除，因而增加在體內的作用時間，進一步增加療效。抗癌機制分析上，我們發現WCE有效地抑制雄性激素受體 (androgen receptor)、 HER2/3和AKT信號路徑與回饋機制，因此增強了荷爾蒙療法的效果，防止初期療效反應之後經常發生的抗藥性。儘管HER2/3和AKT的抑制未能引起對賀爾蒙治療有抗性的前列腺癌細胞(PC-3)體外的凋亡， 然而在動物實驗中，WCE卻可以抑制癌細胞引起的骨髓細胞(MDSCs)的趨化作用，進而抑制前列腺腫瘤的生長和轉移。因此，WCE可以結合賀爾蒙療法去提升治療成效，也可以調節癌症免疫而抑制前列腺癌細胞(PC-3)的轉移。

Recurrences of hormone-refractory and metastatic prostate cancer (PCa) both highly affect the treatment outcome. Developing the orthotopic PCa animal model to study the cancer progression and effective therapy is the critical mission. Here, we established the orthotopic xenograft mouse model of PCa cell line, 22Rv1, cultured the different stages of tumor cells, and repeated the same procedure to get highly metastatic cells. In this model, we found that HAI-2, a matriptase inhibitor, is decreased during PCa progression, which enhanced the invasive growth and metastasis capacity of PCa cells. This effect was reversed by either ectopic expression of HAI-2 or matriptase knockdown, indicating that derepression of matriptase from HAI-2 contributed to the cancer metastasis. Besides, we used Prostatic Specific Antigen (PSA) promoter activity assay to identify the principal active compounds (wedelolectone, luteolin, and apigenin) in the herbal ethanol extract of Wedelia chinensis and demonstrated the active compounds synergistically inhibited AR-positive PCa cell growth in vitro and in vivo. To promote the potential clinical use as a botanical drug, we developed a standardized preparation of a W. chinensis extract (WCE) by enrichment and quantification of the principal active compounds and analyses of the in vivo biological activities. Pharmacokinetic analyses of active compounds upon oral administration with WCE indicated that presence of copurifiied other compounds in WCE prolonged the systemic exposure to active compounds over a formula of the pure active compounds. In agreement, the resulting anti-tumor efficacy of WCE was higher than the formula of pure active compounds. Furthermore, WCE effectively disrupted the androgen receptor, HER2/3, and AKT signaling network and therefore enhanced therapeutic efficacy of androgen ablation in PCa. While HER2/3 and AKT inhibition by WCE failed to induce apoptosis in hormone-refractory PCa cells, WCE curbed PCa-induced chemotaxis of myeloid cells and the tumor-microenvironment interactions, thus impairing PCa growth and metastasis. In conclusion, the imbalance between HAI-2 and matriptase expression led to matriptase activation, thereby increasing PCa metastasis. In herbal medicine study, we demonstrated that standardized, characterized preparations of WCE improved the outcome of PCa therapy either as an add-on to hormonal therapy for androgen-dependent disease or as a monotherapy for hormone-refractory disease.