荷葉萃取物抑制乳腺癌生長、血管新生及轉移作用之研究

乳腺癌是世界上女性普遍常見的惡性腫瘤，癌細胞的轉移與血管新生則是癌症擴展的主要步驟。結締組織生長因子(Connective tissue growth factor，CTGF)則在血管新生與轉移扮演了重要的調控角色。荷葉是睡蓮科多年生草本植物，其化學成分含有多種荷葉鹼(pronuciferine)、抗氧化物質類黃酮等，先前的研究已證實荷葉具有抗氧化及降低乳腺癌生長的能力。在本篇研究中我們想要探討荷葉萃取物(Nelumbo nucifera leaves extracts，NLE)是否可透過CTGF進而抑制乳腺癌細胞的血管新生與轉移。我們首先利用wound-healing與Boyden chamber進行分析，發現NLE隨著劑量的增加，抑制MDA-MB-231乳腺癌細胞的移動(migration)與侵襲(invasion)能力也隨之遞增。此外，NLE在雞胚胎尿囊絨膜試驗(chicken chorioallantoic membrane, CAM)中亦可有效降低微血管的形成。進一步我們將NLE餵食裸鼠，進行Matrigel plugs與CD31免疫染色分析，結果證實NLE確實可有效降低裸鼠之血管生成。最後，我們利用西方墨點法分析NLE抑制乳腺癌細胞血管新生與轉移的機制，結果指出CTGF調控了NLE 抑制血管新生的過程，且是透過降低Ras/MEK/ERK及PI3K/Akt/NF-kB訊息路徑。此外，我們利用NMU (N-methyl-N-nitrosourea)誘導乳腺癌的產生，並處理不同劑量NLE與臨床藥物 Herceptin及Tamoxifen進行比較。結果顯示無論腫瘤發生率、數量及腫瘤大小，在NLE的處理之下皆顯著下降，NLE 2%組別效果更優於Tamoxifen之組別。我們亦以人類乳腺癌細胞株BT-474為實驗模式，探討NLE對乳腺癌生成的影響。將細胞注射於裸鼠，並使用不同劑量NLE與Tamoxifen餵食裸鼠，結果證實NLE確實可有效降低裸鼠之腫瘤生成，NLE 2%組別抑制效果更優於Tamoxifen之組別。接著利用免疫染色分析與西方墨點法分析腫瘤蛋白之表現，發現隨著NLE 劑量之提升，FASN及phospho–ERα的表現量隨著NLE及NLPE 的劑量增加而逐漸減少，顯示NLE 確實會造成ERα 的磷酸化表現下降。所有NLE組別，Tamoxifen組跟對照組相比較下，結果顯示HER2蛋白並無顯著變化；phospho–HER2的減少則以NLPE較為顯著。總結這些結果也正可以說明NLE降低乳腺癌的發生是透過減少FASN，ERα 及HER2的活化，因此荷葉萃取物也許可考慮作為治療乳腺癌生長、轉移與血管新生的輔助策略。

Nelumbo nucifera Gaertn (Nymphaeaceae) has been recognized as a medicinal plant, which was distributed throughout the Asia. The aqueous extract of Nelumbo nucifera leaves extract (NLE) has various biologically active components such as polyphenols, flavonoids, and oligomeric procyanidines. However, the role of NLE in breast cancer therapy is poorly understood. The purpose of this study was to identify the hypothesis that NLE can suppress tumor angiogenesis and metastasis through CTGF (connective tissue growth factor), which has been implicated in tumor angiogenesis and progression in breast cancer MDA-MB-231 cells. We examined the effects of NLE on angiogenesis in the chicken chorioallantoic membrane (CAM) model. The data showed that NLE could reduce the chorionic plexus at day 17 in CAM and the duration of this inhibition was dose-dependent. In Xenograft model, NLE treatment significantly reduced tumor weight and CD31 (capillary density) over control, respectively. We examined the role of angiogenesis involved restructuring of endothelium using human umbilical vein endothelial cell (HUVEC) in Matrigel angiogenesis model. The results indicated that vascular-like structure formation was further blocked by NLE treatment. Moreover, knockdown of CTGF expression markedly reduced the expression of MMP2 as well as VEGF, and attenuated PI3K-AkT-ERK activation, indication that these signaling pathways are crucial in mediating CTGF function. Additionally, we found that NLE extracted from nuciferine leaf suppressed the chemical induced incidence, multiplicity and tumor volume of mammary tumors in NMU induce rats。In murine xenograft model also confirmed that NLE inhibited tumor growth. Immunohistochemical and Western blot analysis of FASN (fatty acid synthase) and phospho–ERα show dose dependent decreased protein level. Protein expression has no significant change in HER2. However, phospho-HER2 in NLPE has been suppressed significantly; the other group and control group had no significant differences. The result revealed that NLE reduce the incidence and growth of breast cancer is by reducing the activation of FASN， ERα and HER2. The present results suggest that NLE might be useful for treatment in therapy-resistance breast cancer progression.