雌性素與生長因子已經被證實與乳癌的發生有相當密切之關聯性，然而兩者之間對乳癌細胞的增生作用其詳細機制仍尚未釐清。本實驗中我們針對二十種結構相關性的類黃酮素化合物，探討對雌性素(E2) 及類胰島素生長因子(IGF-I)促進人類乳癌細胞MCF-7大量增生的抑制作用。實驗結果發現，單獨以E2 (10 nM)處理MCF-7細胞時可透過ER的活化來誘導細胞增生，而在共同加入IGF-I (10 ng/ml)處理之下，更能大量的增加E2所誘導的細胞增生並促進細胞群落形成(colony formation)。此外，E2/IGF-I共同處理能大量誘導IRS-1、ERKs和JNKs (而非p38 MAPK)的磷酸化以及c-Jun蛋白質表現，使用ERKs抑制劑PD98059與JNKs抑制劑SP600125則能有效抑制E2/IGF所誘導MAPKs活化、c-Jun蛋白質表現及細胞的增生。除此之外也發現E2/IGF-I能誘導細胞產生微量的活性氧自由基，並藉由活化MAPKs cascade來促進乳癌細胞的大量增生。實驗結果證實當乳癌細胞在E2的存在下同時受到IGF-I的刺激，藉由兩者之間的交互與協同作用會更加促進癌細胞的大量增生與惡性轉型。另外，從二十種結構相似的類黃酮素化合物中，探討不同結構之間對乳癌細胞增生的抑制情形，實驗結果發現3-OH flavone、kaempferol、luteolin、baicalein以及quercetin能有效阻斷E2/IGF-I所誘導ER和c-Jun蛋白質表現並抑制細胞增生和惡性轉型，並證實OH取代基及非醣基部分為主要影響類黃酮素活性之重要因子，因此可做為將來預防腫瘤過度增生的藥物開發的重要依據。

Estrogen (E2) and insulin-like growth factor-I (IGF-I) have been shown to induce cellular proliferation. However, the cross-talk between E2 and IGF-I in the proliferation of breast carcinoma cells is still undefined. In the present study, E2 addition significantly induced the proliferation of human breast cancer cells MCF-7, which was prevented by ER antagonoists Tamoxifen and ICI182,780. Interestingly, addition of IGF-I potentiates the proliferation of cells in the presence of E2. Activation of extracellular signal-related kinase (ERKs) and c-Jun N-terminal kinase (JNKs), but not p38 MAPK, via inducing protein phosphorylation, and subsequently with an increase in c-Jun protein expression was identified in E2/IGF-I-treated cells. An inhibitor of ERKs, PD98059, and an inhibitor of JNKs, SP600125, significantly blocked E2/IGF-I–induced proliferation in according with reducing c-Jun protein expression and ERKs and JNKs protein phosphorylation. Additionally, ROS scavengers, N-acetyl-cysteine (NAC) and Tiron (TIR) significantly prevented E2/IGF-I–induced activation of MAPKs, c-Jun and cell proliferation with a reduction in intracellular peroxide level by flow cytometry analysis. Furthermore, the natural products, 3-OH flavone, kaempferol, luteolin, baicalein and quercetin showed the most-potent inhibitory activities on E2/IGF-I induced events among 20 structure-related flavonoids, and suggest that hydroxyl group and aglycon are importance in the biological activities of flavonoids. These results provide evidence that IGF-I possess ability to enhance E2-induced proliferation and transformation in MCF-7 cells; generation of ROS production followed by activation of MAPKs cascade, c-Jun protein expression are involved. A paracrine effect of IGF-I in breast cancer formation in the presence of E2 was proposed.

目錄………………………………………………………………….. i
圖目錄………………………………………………………………. iv
縮寫表……………………………………………………………….. vii
中文摘要…………………………………………………………….. viii
英文摘要…………………………………………………………….. ix

Ⅰ、前言 ( Introduction )…………………………………………... 1
Ⅱ、實驗材料 ( Materials )………………………………………… 9
Ⅱ-1 材料……………………………………………………. 10
Ⅱ-2 細胞株………………………………………………..... 16
Ⅱ-3 細胞培養液……………………………………………. 16
Ⅱ-4 一般化學試藥及試劑…………………………………. 17
Ⅱ-5 免疫試劑……………………………………………….. 18
Ⅱ-6 儀器…………………………………………………….. 19
Ⅲ、實驗方法 ( Methods )
Ⅲ-1 藥品試液之配製………………………………………. 21
Ⅲ-2 一般溶液之配製………………………………………. 21
Ⅲ-3 MCF-7細胞培養方法………………………………… 24
Ⅲ-4 MCF-7細胞生長曲線………………………………… 26
Ⅲ-5 細胞毒性試驗( MTT assay )………………………….. 26
Ⅲ-6 E2誘導細胞增生之劑量依循性……………………... 27
Ⅲ-7 [3H]-Thymidine incorporation assay
(DNA synthesis assay)………………………………… 27
Ⅲ-8 細胞蛋白質之抽取…………………………………… 27
Ⅲ-9 西方點墨法( Western blot )………………………… 28
Ⅲ-10 自由基群組( Reactive oxygen species )含量分析…... 28
Ⅲ-11 軟瓊脂測試( Soft agar assay )/群落形成測試
(Colony formation assay)……………………………… 29
Ⅲ-12 瓊膠電泳分析法( DNA fragmentation assay )……….. 29
Ⅲ-13 Flavonoids抗自由基能力之探討( DPPH assay ) ……. 29
Ⅲ-14 統計資料( Statistics )………………………………….. . 30
Ⅳ、實驗結果(Results)……………………………………………… 31
Ⅳ-1 MCF-7細胞生長曲線………………………………… 32
Ⅳ-2 E2誘導MCF-7 cells細胞增生之劑量依循性……….. 32
Ⅳ-3 ER拮抗劑Tamoxifen及ICI 182,780對E2誘導細胞
增生的影響…………………………………………….. 32
Ⅳ-4 IGF-I增加E2誘導MCF-7細胞增生………………… 33
Ⅳ-5 IGF-I促進E2對MCF-7細胞群落形成之分析……… 33
Ⅳ-6 IGF-I透過活化IRS-1、ERKs及JNKs促進E2誘導
MCF-7細胞增生……………...………………………. . 34
Ⅳ-7 E2/IGF-I促進MCF-7細胞增生中MAPKs的角色…. 35
Ⅳ-8 MAPKs的活化對細胞增生之影響…………………… 35
Ⅳ-9 ROS在E2/IGF-I誘導MCF-7細胞增生中的角色….. 36
Ⅳ-10 結構相關flavones類化合物對MCF-7細胞增生之探討……………………………………………………….. 36
Ⅳ-11 Flavones類化合物對ER與c-Jun的抑制形……….. . 37
Ⅳ-12 結構相關之flavanones類化合物對MCF-7細胞增生
的探討………………………………………………… . 38
Ⅳ-13 Flavones類化合物抑制E2/IGF-I誘導細胞群落形成之
探討……………………………………………………. . 38
Ⅳ-14 Flavonoids類化合物清除DPPH自由基能力之探討.... 39
Ⅴ、討論……………………………………………………………… 41
Ⅵ、參考文獻………………………………………………………… 49

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