抗癌藥物 Moscatilin, Denbinobin 與 CHM-1對於血管新生抑制作用機轉之探討

Abstract

血管新生 (angiogenesis)，是指由原來已存在的血管分化形成新血管之一個多重 且複雜的過程。一些疾病，如腫瘤、慢性發炎 (類風濕性關節炎)、糖尿病引起之視 網膜病變等與血管過度增生有關。因此，抑制血管新生可應用於各種因血管增生所 衍生的疾病。在本論文中，將探索具有研發潛力之抗血管新生藥物，並對其作用機 轉進行研究。 本論文的第一個部分主要在探討黃草石斛 (Dendrobrium loddigesii) 莖部萃取 物 moscatilin 對 VEGF 及 bFGF 所引起人類臍靜脈內皮細胞 (Human umbilical vein endothelial cells；HUVECs) 的生長抑制作用及其可能作用的分子機轉。實驗發 現 moscatilin 能使人類臍靜脈內皮細胞生長產生抑制作用，且其抑制效果和 moscatilin 的濃度呈正相關性。Moscatilin 抑制血管新生是透過阻斷 VEGF 及 bFGF 所引起的 ERK1/2、Akt 和 eNOS 之活化。我們也進行了 in vivo matrigel plug assay 和腫瘤的異體移植實驗，確認 moscatilin 在體內的抗血管新生效果。綜合以 上實驗證明 moscatilin 是個值得進一步研發的抗血管新生先導藥物。 本論文的第二個部分在探討大爪石斛 (Ephemerantha lonchophylla) 莖部萃取 物 denbinobin 對於第一型類胰島素生長因子 (insulin-like growth factor-1；IGF-1) 所引發血管新生的抑制作用。從體內動物實驗證實 denbinobin 能有效抑制腫瘤細胞 的生長並阻斷 IGF-1 誘發之血管新生作用。此外，denbinobin 也能抑制體外人類臍 靜脈內皮細胞的增生、移行及管柱形成作用。進一步實驗證實 denbinobin 能顯著地 抑制人類臍靜脈內皮細胞中，由 IGF-1 所引起的 IGF-1/IGF-1R 下游相關訊息傳遞 分子 ERK1/2、Akt、mTOR、p70S6K 和 cyclin D1 之活化。因此我們認為 denbinobin 是相當值得未來開發及應用於血管新生相關疾病，例如癌症，的治療上。 本論文的第三個部分在探討 2-phenyl-4 quinolone 類化合物 CHM-1 在體內及 體外模式抑制血管新生的作用機轉。在動物實驗中，CHM-1 可以有效抑制腫瘤的 生長與血管生成。體外試驗也發現 CHM-1 能抑制人類臍靜脈內皮細胞的生長、遷 2 移及管腔形成。進一步實驗證實 CHM-1 能抑制微管蛋白的聚合作用，並透過使細 胞內 p53 蛋白增加，提高 DR5 蛋白質的表現量，而誘發細胞外生性凋亡路徑 (extrinsic apoptotic pathway)，最後造成人類臍靜脈內皮細胞的凋亡，達到破壞腫瘤 血管的效果。以上實驗證明 CHM-1 是個具有潛力的抗血管新生先導藥物。 綜合以上所述，本論文主要以抑制血管新生為標的，陸續證實石斛成分 moscatilin 和 denbinobin，以及化學小分子 CHM-1 能顯著地阻斷體外與體內之血 管新生作用，進而治療與血管新生相關的疾病，並發展為具療效之先導藥物。

Angiogenesis is a multiple and complex process involving the growth of new blood vessels from pre-existing vessels. Various pathological conditions, including cancer, chronic inflammation (e.g. rheumatoid arthritis) and diabetic retinopathy are related to angiogenesis, thus inhibition of angiogenesis might have implication in various angiogenesis-mediated disorders. In this thesis, we focused on the discovery of potential novel antiangiogenic agents, and further investigated the mechanisms of these agents. The first part is to investigate the anti-proliferation effect and molecular mechanisms of moscatilin, a bibenzyl derivative from the stem of an India orchid Dendrobrium loddigesii, on human umbilical vein endothelial cells (HUVECs). We found moscatilin significantly inhibited HUVECs growth in a concentration-dependent manner. The antiangiogenic effects of moscatilin are proposed to inhibition of ERK1/2, Akt, and eNOS signaling pathways. In vivo Matrigel plug assay and tumor xenograft were also performed to support the potential of moscatilin as an angiognenesis inhibitor for cancer therapy. Moscatilin is a promising lead compound worthy of further development into a drug candidate for anti-angiogenesis. The second part is aimed to evaluate the roles of denbinobin, a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla, in suppressing the IGF-1-induced angiogenesis and elucidating the underlying molecular mechanisms. Denbinobin inhibited tumor growth and IGF-1-induced angiogenesis in vivo. Additionally, denbinobin also suppressed IGF-1-induced HUVECs proliferation, migration and tube formation in vitro. We further found that denbinobin caused more efficient inhibition of IGF-1-induced activation of IGF-1R and its downstream signaling targets, including ERK, Akt, mTOR, p70S6K, 4EBP, and cyclin D1. Our findings suggest that denbinobin could be a potential therapeutic agent against angiogenesisrelated diseases, such as cancer. The third part is to study the antiangiogenic activity and mechanism of CHM-1, a 2-phenyl-4 quinolone derivative, in vitro and in vivo. Results of animal models indicated 4 that CHM-1 inhibited tumor growth and induced vascular shutdown within tumor. CHM-1 also suppressed the proliferation, migration and tube formation of HUVECs in vitro. Besides, CHM-1 inhibited microtubule assembly as well as upregulated the expression of p53, DR5. Then, apoptosis executed by an extrinsic apoptotic pathway. Taken together, we suggest CHM-1 has potential to be an antivascular and antitumor therapeutic lead compound. In conclusion, the aim of this study is to examine the anti-angiogenetic effects of moscatilin, denbinobin and CHM-1. We found that these agents can inhibit angiogenesis in vitro and in vivo. We suggest that moscatilin, denbinobin and CHM-1 have potential to be a lead compound for treating angiogenesis-dependent diseases such as cancer.