Dehydrocostuslactone、MPT0G013與MPT0B271對於血管新生抑制作用及抗癌機轉之探討

Investigation of anti-angiogenic and anti-cancer mechanisms of dehydrocostuslactone, MPT0G013 and MPT0B271 in vitro and in vivo

王之雅(Chih-Ya Wang)

指導教授 ： 鄧哲明 ； 潘秀玲

國立臺灣大學/醫學院/藥理學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.01029

摘要

血管新生 (angiogenesis) 是從已存在的血管分化形成新血管網路的過程，對於生理器官發育、組織修復及生殖占有重要調控的角色。在病理中，腫瘤的生長、進展都必須仰賴血管新生，來得到足夠的氧氣和養分供應，以及排除細胞的代謝廢棄物。此外，自體免疫疾病、退化性關節炎及老年性黃斑部病變也與血管過度新生相關，因此，抑制血管新生可應用於血管生成相關的疾病。在本論文中，將探討具有發展潛力的血管新生抑制劑之機轉。
本論文的第一個部分主要在探討來自中草藥廣木香 (Saussurea costus (Falc.) Lipschitz) 的萃取物dehydrocostuslactone (DHC) 對血管新生促進因子 (endothelial growth factors；EGM-2) 引起血管新生的抑制作用與分子機轉。實驗發現，DHC能夠抑制人類臍靜脈內皮細胞的增生及類血管管腔的形成，但是不會影響細胞的移行，在in vivo matrigel plug assay中，DHC能抑制體內的血管新生。DHC能夠透過抑制Akt/GSK-3b、mTOR的磷酸化，明顯減少cyclin D1的蛋白表現，當細胞過度表現持續性活化的 myristoylated (myr)-Akt，DHC抑制細胞增生、管腔形成以及減少cyclin D1都會被逆轉而回復。若是將內皮細胞共同處理LiCl及DHC，則能夠明顯地逆轉DHC抑制細胞生長的作用。總結以上結果，本研究證實Akt/GSK-3b/cyclin D1 以及 mTOR訊息路徑是DHC重要的標靶，因此能夠應用於如癌症及血管新生相關的疾病。
本論文的第二個部分在探討arylsulfonamide 衍生物MPT0G013在體內及體外具有抗血管新生之活性。MPT0G013能明顯抑制內皮細胞增生、移行和類血管管腔形成，經由基因晶片檢測分析，MPT0G013能明顯誘導tissue inhibitors of metalloproteinases 3 (TIMP3) 的表現，將細胞轉染TIMP3 siRNA，能夠逆轉MPT0G013抑制血管新生的作用及回復Akt、ERK蛋白的磷酸化；除此之外，利用in vivo matrigel plug assay和腫瘤的異體移植實驗，證實MPT0G013在體內具有明顯抑制血管生成的作用。因此，我們推論MPT0G013是一個值得進一步研發的抗血管新生藥物。
目前被用來治療癌症的taxanes及vinca生物鹼等微管結合藥物，缺點為抗藥性的產生及必須透過靜脈注射投與，因此迫切需要研發新的藥物。MPT0B271是一個口服生體可用的微管標靶全合成藥物，在體內及體外具有強力的抗癌效果，MPT0B271除了導致微管的去聚合，且能抑制許多癌細胞株生長及降地存活率，包括multidrug-resistant癌細胞株NCI/ADR-RES。利用流式細胞儀分析rhodamine-123 (Rh-123) 的輸出及利用calcein acetoxymethyl ester (calcein AM) assay，發現MPT0B271不是p-glycoprotein (p-gp) 的受質；此外，MPT0B271 也會誘導細胞G2/M期停滯以及細胞凋亡。我們也發現在體內及體外，與單獨給藥相較之下，MPT0B271合併erlotinib (Tarceva) 能明顯抑制人類非小細胞肺癌A549細胞的生長，這些結果證實MPT0B271是一個具有潛力治療各種癌症的新穎的微管結合藥物。
綜合以上所述，本論文主要以抗血管新生及抗癌為研究對象，證實DHC和MPT0G013能顯著抑制體內及體外之血管新生作用，MPT0B271是新穎的抗癌藥物，能夠發展成為治療癌症的先導藥物。

關鍵字

血管新生 ； 癌症

並列摘要

Angiogenesis, which is the process of formation of new blood vessels from pre-existing ones, takes place throughout physiological development, tissue repair, and reproduction. In pathological conditions, angiogenesis is also essential for tumor growth and progression to ensure that more oxygen and nutrients are delivered from the host’s vascular system. Therefore, angiogenesis is a promising target for anticancer treatments. In this thesis, we focused on the discovery of potential novel antiangiogenic agents, and further investigated the mechanism of these agents.
In the first part, we investigated the traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, which has been shown to have anti-cancer activity. DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on HUVEC proliferation and capillary-like tube formation in vitro. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, we demonstrated that DHC significantly inhibited Akt expression, resulting in the suppression of GSK-3b phosphorylation and mTOR expression. Furthermore, the degradation of cyclin D1 and the abrogation of tube formation induced by DHC were significantly reversed by constitutively active myristoylated (myr)-Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC.
Tissue inhibitors of metalloproteinases 3 (TIMP3) were originally characterized as inhibitors of matrix metalloproteinases (MMPs), acting as potent antiangiogenic proteins. In the second part, we demonstrated that the arylsulfonamide derivative MPT0G013 has potent antiangiogenic activities in vitro and in vivo via inducing TIMP3 expression. Treatments with MPT0G013 significantly inhibited endothelial cell functions, such as cell proliferation, migration, and tube formation. Subsequent microarray analysis showed significant induction of TIMP3 gene expression by MPT0G013, and siRNA-mediated blockage of TIMP3 up-regulation abrogated the antiangiogenic activities of MPT0G013 and prevented inhibition of p-AKT and p-ERK proteins. Importantly, MPT0G013 exhibited antiangiogenic activities in in vivo Matrigel plug assays, inhibited tumor growth and up-regulated TIMP3 and p21 proteins in HCT116 mouse xenograft models.
MPT0B271, an orally active microtubule-targeting agent, is a completely synthetic compound that possesses potent anticancer effects in vitro and in vivo. MPT0B271 caused depolymerization of tubulin at both molecular and cellular levels and reduced cell growth and viability at nanomolar concentrations in numerous cancer cell lines, including a multidrug-resistant cancer cell line NCI/ADR-RES. Further studies indicated that MPT0B271 is not a substrate of p-gp, as determined by flow cytometric analysis of Rh-123 dye efflux and the calcein AM assay. MPT0B271 also caused G2/M cell-cycle arrest, accompanied by induction of cell apoptosis. We demonstrated that MPT0B271 in combination with erlotinib significantly inhibits the growth of the NSCLC A549 cells as compared with erlotinib treatment alone, both in vitro and in vivo. These findings identify MPT0B271 as a promising new tubulin-binding compound for the treatment of various cancers.
Taken together, the present studies have highlighted the potential application of DHC and MPT0G013 in angiogenesis-related diseases, such as cancer. And the anti-tumor agent MPT0B271 may foster novel therapeutic strategies for various cancer cell lines.

並列關鍵字

angiogenesis ； cancer

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