臺灣博碩士論文加值系統

第一部份:
類黃酮是由多種植物所產生的一群多樣的植物化合物。在類黃酮化合物中，apigenin (4'',5,7-trihydroxyflavone)是一種低毒性及無誘導突變性的植物多酚類(phytopolyphenol)及蛋白質磷酸酶抑制劑(protein kinase inhibitor)。同時它也具有抑制人類乳癌細胞生長的弁遄C在我們最近的實驗中發現，在不同HER2/neu致癌基因表現量的乳癌細胞中，apigenin對過度表現HER2/neu乳癌細胞抑制生長效果比基礎量HER2/neu的乳癌細胞來的好。Apigenin會誘發過度表現HER2/neu的乳癌細胞細胞凋亡，然而其分子機制仍有待探討。在過度表現HER2/neu的乳癌細胞中，一個包含phosphatidylinositol 3’-kinase(PI3K)及AKT的訊息傳遞途徑，已知對抑制細胞死亡扮演一個重要的角色。因此促使我們去探討此訊息傳遞途徑是否對apigenin誘發過度表現HER2/neu的乳癌細胞細胞凋亡扮演一個重要的角色。研究結果發現apigenin抑制癌症細胞中AKT的活性是一種複合式的方式:首先，apigenin直接抑制PI3K的活性然而並非直接抑制AKT的活性；第二，藉由減少HER2/neu蛋白質的量而使HER2/neu蛋白質磷酸化及磷酸化其他受質的能力下降；此外，apigenin藉由抑制PI3K結合到HER2/HER3 二聚物來達到抑制AKT的活性。由此我們推測apigenin所擁有的細胞活性主要是藉由減少HER2/neu及HER3蛋白表現量來達到抑制跟細胞存活及抑制細胞凋亡有關的PI3K及AKT訊息傳遞途徑。這表示抑制HER2/HER3二聚物的弁鄍i以對於過度表現HER2/neu的乳癌細胞提供一個專一有效的治療策略。研究結果同時證實apigenin可以將HER2/neu與一個跟HER2/neu穩定有關的蛋白質GRP94分離。Apigenin減少成熟HER2/neu蛋白質量的過程，主要包含了將HER2/neu polyubiquitination及接下來藉由proteasome將HER2/neu水解。這些發現證實對於過度表現HER2/neu的乳癌細胞而言，apigenin具有有效的治療或預防的效果。接著，為了探討此訊息傳遞途徑對抗癌效果的重要性，將過度表現HER2/neu的乳癌細胞處理apigenin，發現它會藉由cytochrome c 的釋放、快速活化caspase-3的活性及刺激DFF-45的分解來引起細胞凋亡。此外，apigenin 會降低cyclin D1 及 cyclin D3的表現及增加 p27 蛋白質的表現。軟膠的群落實驗是細胞轉形的一種重要特徵，apigenin 對HER2/neu過度表現的乳癌細胞有明顯抑制的效果。 最後在結構活性的研究中發現：(1) B 環的位置及(2) 第2位置上芳香環上的3'', 4''-hydroxyl 是類黃酮降低HER2/neu 蛋白表現的重要結構。本研究結果提供了關於類黃酮類化合物結構活性關係上的一個重要的資訊。

第二部份:
Aromatase主要的作用是將雄烯二酮轉換為雌性激素，進而達到調節雌激素促進赫爾蒙需要型乳癌細胞生長的能力。在本篇研究當中，調查了紅茶茶多酚對aromatase的抑制效果，發現紅茶茶多酚TF1， TF2 及 TF3可以顯著的抑制老鼠及人類胎盤的aromatse酵素活性。此外，利用一個細胞體內的模式發現紅茶亦可以抑制100 nM DHEA所引起的MCF-7細胞增生。轉殖HER2/neu 基因至MCF-7細胞中會使得它對赫爾蒙治療產生抗藥性。有趣的是不似選擇性雌激素接受子調節者(SERM) tamoxifen ，紅茶茶多酚對於這種具有抗藥性的乳癌細胞具有抑制細胞增殖的效果。這種抑制抗藥性乳癌細胞增殖的弁鄍D要是由於紅茶茶多酚可以抑制HER2/neu 接受子的酪氨酸的磷酸化。這些發現證實使用紅茶茶多酚可以對赫爾蒙依賴型的乳癌細胞作為一個有效的化學預防效果，對於赫爾蒙不依賴型的乳癌細胞可以克服赫爾蒙療法所產生的抗藥性問題。

Part I:
Flavonoids are a diverse group of phytochemicals that are produced by various plants. Among the flavonoids, apigenin (4'',5,7-trihydroxyflavone) is one of the most effective in inducing cell growth inhibition and is relatively nontoxic and nonmutagenic. Here we examined several human breast cancer cell lines having different levels of HER2/neu expression, and found that apigenin exhibited potent growth-inhibitory activity in HER2/neu-overexpressing breast cancer cells, but was much less effective for those cells expressing basal levels of HER2/neu. Induction of apoptosis was also observed in HER2/neu-overexpressing breast cancer cells in a dose- and time-dependent manner. However, the molecular mechanism(s) of apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells remained to be elucidated. A cell survival pathway involving phosphatidylinositol-3�S-kinase (PI3K) and Akt is known to play an important role in inhibiting apoptosis in response to HER2/neu-overexpressing breast cancer cells, which prompted us to investigate whether this pathway plays a role in apigenin-induced apoptosis in HER2/neu-overexpressing breast cancer cells. Our results showed that apigenin inhibits Akt function in tumor cells in a complex manner. First, apigenin directly inhibited the PI3K activity while indirectly inhibited the Akt kinase activity. Second, inhibition of HER2/neu autophosphorylation and transphosphorylation resulting from depleting HER2/neu protein in vivo was also observed. In addition, apigenin inhibited Akt kinase activity by preventing the docking of PI3K to HER2/HER3 heterodimers. Therefore, we proposed that apigenin-induced cellular effects result from loss of HER2/neu and HER3 expression with subsequent inactivation of PI3K and AKT in cells that are dependent on this pathway for cell proliferation and inhibition of apoptosis. This implies that the inhibition of the HER2/HER3 heterodimer function provided an especially effective strategy for blocking the HER2/neu-mediated transformation of breast cancer cells. Our results also demonstrated that apigenin dissociated the complex of HER2/neu and GRP94 that preceded the depletion of HER2/neu. Apigenin-induced degradation of mature HER2/neu involves polyubiquitination of HER2/neu and subsequent hydrolysis by the proteasome. These findings suggest that apigenin may be an effective chemotherapeutic and preventive agent against HER2/neu-overexpressing breast cancer cells. Next, to examine whether inhibition of this pathway play a role in the antitumor effect, we demonstrated that treatment with apigenin induced apoptosis through cytochrome c release and caused a rapid induction of caspase-3 activity and stimulated proteolytic cleavage of DFF-45. Furthermore, apigenin down-regulated cyclin D1 and cyclin D3, and increased p27 protein levels. Colony formation in the soft agar assay, a hallmark of the transformation phenotype, was preferentially suppressed in HER2/neu-overexpressing breast cancer cells by apigenin. A structure-activity study indicated that (1) the position of B ring; and (2) the existence of the 3'', 4''-hydroxyl group on the 2-phenyl group are important factors affecting flavonoids depleted HER2/neu protein. This provides new information on the structure-activity relationship of flavonoids.

Part II:
The aromatase enzyme, which converts androstenedione to estrone, regulates the availability of estrogen to support the growth of hormone-dependent breast tumors. In this study, we investigated the inhibitory effects on aromatase activities by the black tea polyphenols. We found that black tea polyphenols, TF1, TF2 and TF3 significantly inhibited rat ovarian and human placental aromatase activities. In addition, using an in vivo model, these black tea polyphenols also inhibited the proliferation induced by 100 nM DHEA in MCF-7 cells. Transfection of HER2/neu in MCF-7 breast cancer cells appeared to be associated with an increased resistance of the cells to hormonal therapy. Interestingly, unlike the selective estrogen receptor modulator (SERM) tamoxifen, black tea polyphenols expressed antiproliferation effects in breast cancer cells with hormonal resistance. The inhibitory effect of black tea polyphenols on hormone-resistant breast cancer cells suppressed the basal receptor tyrosine phosphorylation in HER2/neu-overexpressing MCF-7 cells. These findings suggest the use of black tea polyphenols may be beneficial in chemoprevention of hormone-dependent breast tumors and represent a possible remedy to overcome hormonal resistance of hormone-independent breast tumors.

Table of Contents
Abbreviations 1

Part I: Mechanism studies by which apigenin inhibits HER2/neu-overexpressing breast cancer cells growth 3
摘要 4
Abstract 6
List of Figures 8
Chapter1. Overview and Rationale 9
Chapter2.
Introduction 14
Materials and Methods 19
Results 25
Discussion 35
References 43
Figures and Figure legends 51

Part II: Mechanism studies by which black tea polyphenol theaflavins inhibit aromatase activity and attenuate tamoxifen resistance in HER2/neu-transfected breast cancer cells 74
摘要 75
Abstract 76
List of Figures 77
Chapter 1. Overview and Rationale 78
Chapter 2.
Introduction 82
Materials and Methods 85
Results 89
Discussion 93
References 98
Figures and Figure legends 100

Vita 110

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