辣椒素（Capsaicin）是存在於辣椒屬中多種不同紅辣椒植物的主要刺激性成分，屬於vanilloid成員之一。研究指出辣椒素對許多癌細胞株（例如肝癌、子宮頸癌及食道癌等）均具有生物活性，能抑制細胞生長。在不表現凋亡蛋白酶3（caspase-3）的人類乳房腫瘤細胞株MCF-7中，辣椒素亦會抑制細胞生長，且隨著辣椒素濃度的增加，其抑制作用愈明顯，並可觀察到細胞凋亡的發生。本研究進一步探討辣椒素在MCF-7細胞中引發細胞凋亡的分子機制，並分析辣椒素與凋亡蛋白酶非依賴型路徑（caspase-independent pathway）的關係。MCF-7細胞經過24小時辣椒素處理後會發生劑量依存型（dose-dependent）細胞凋亡。在辣椒素處理5分鐘後，細胞內活性氧分子（reactive oxygen species; ROS）即有微量下降趨勢；處理2小時後，細胞內鈣離子濃度也有上升現象。MCF-7細胞的粒線體膜電位也會因辣椒素的處理而下降。辣椒素處理24小時後，凋亡誘導因子（apoptosis-inducing factor；AIF）會從粒線體中被釋放並進入細胞核內，這意味著辣椒素可能經由凋亡蛋白酶非依賴型路徑引發MCF-7細胞凋亡。此外，辣椒素可能直接進入細胞或穿透細胞膜誘發細胞死亡，而TRPV1蛋白會增加細胞對辣椒素的敏感性。因此，辣椒素具有成為乳癌治療藥物的潛力。

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a major pungent ingredient in a variety of red peppers of the genus Capsicum, is a type of vanilloids. It has been shown to exert biological activities in many cancer cell lines. It was found that capsaicin induces dose-dependent growth inhibition of MCF-7 cells, which do not express caspase-3. In this study, we investigated the molecular mechanism of capsaicin-induced apoptosis in MCF-7 cells. Treatment with capsaicin for 24 hours resulted in a dose-dependent apoptosis in MCF-7 cells. After addition of capsaicin, levels of reactive oxygen species (ROS) reduced slightly in the earlier time of treatment. Interestingly, an elevation of intracellular calcium ion (Ca2+) concentration was also detected in MCF-7 cells. It is worth noting that apoptosis-inducing factor (AIF) translocated into the cytosol and nucleus from mitochondria, suggesting that capsaicin may induce cellular apoptosis through caspase-independent pathway in MCF-7 cells. Furthermore, capsaicin may directly enter the cell or penetrate cell membrane to induce cell death and the expression of TRPV1 could increase the sensitivity of capsaicin for cells. Hence, capsaicin may be a potent drug in breast cancer therapy.

目錄
頁數
中文摘要 ……………………………1
英文摘要 ……………………………………………..…..…………2
第 一 章 前言 …...……………………………..……..………..… 3
第 二 章 文 獻 探 討 …………………………………………5
第ㄧ節 植物化學物質…………………………………5
第二節 辣椒素 (Capsaicin) ……………….….………6
第三節 乳癌 …………………………………………8
第四節 乳癌的治療 ………………………………… 9
第五節 細胞凋亡(Apoptosis ) .................................. .. 12
第六節 人類乳癌細胞缺乏caspase-3 ……………… 16
第三章 目標 …………………………………………………… 18
第四章 材料和方法 …………………………………………… 19
第ㄧ節 化學藥劑和試劑 …………………………… 19
第二節 人類乳癌細胞植株 ………………………… 19
第三節 細胞存活試驗 ……………………………… 19
第四節 細胞週期的進行和細胞凋亡的分析 ……… 20
第五節 鹼性單一細胞膠體電泳(Comet assay) ….… 21
第六節 活性氧化物的偵測…………………………21
第七節 細胞內鈣離子濃度的偵測…………………22
第八節 粒線體膜電位的偵測………………………23
第九節 西方墨點法 ………………………………. 24
第十節 去氧核醣核酸（DNA）分裂 …………… 25
第十一節 免疫螢光染色和共焦顯微鏡檢 ……… 25
第十二節 統計分析 ……………………………… = 26
第五章 結果
第ㄧ節 辣椒素誘導的細胞死亡不需要caspase-3…27
第二節 透過辣椒素誘導MCF-7細胞DNA受損…28
第三節 透過flow cytometry觀察給予不同濃度的辣椒
素後MCF-7細胞中ROS的產生情形…… 29
第四節 辣椒素對MCF-7細胞中鈣離子生成的影響..29
第五節 辣椒素可以改變粒線體膜電位(MMP)...…. 30
第六節 MCF-7細胞中辣椒素影響p53, Bcl-2和Bax的
表現………………………………………… 30
第七節 辣椒素誘導PARP-1的分裂 ……………… 31
第八節 在辣椒素治療的MCF-7細胞中沒有低分子量的DNA碎片生成 ………………………… 31

第九節 辣椒素誘導AIF轉錄…………………………32

第六章 討論………………………………………………………… 33
第七章 結論………………………………………………………… 37
參考文獻 …………………………………………………………… 70
圖與表 ……………………………………………………………… 83
附錄………………………………………………………………… 104

















Contents

Chapter1.Introduction …..………………………………………… 38
Chapter 2. Literature Review……....................................................40
2.1 Phytochemicals……………...……………………………40
2.2 Capsaicin …………………………………………………41
2.3 Breast cancer………………………………………………43
2.4 Therapies of breast cancers 44
2.5 Apoptosis 46
2.6 Caspase-3 deficiency in human breast cancer cells 51

Chapter 3. Aim …………………………………………………….. 52

Chapter 4. Materials and Methods………………………………..…53
4.1 Chemicals and reagents 53
4.2 Human breast cancer cell lines 53
4.3 Cell viability assay 53
4.4 Cell cycle progression and apoptosis analysis 54
4.5 Alkaline single-cell gel electrophoresis (Comet assay) 54
4.6 Detection of ROS 55
4.7 Detection of intracellular Ca2+ concentration 56
4.8 Detection of mitochondrial membrane potential 56
4.9 Western blot 57
4.10 DNA Fragmentation 58
4.11 Immunofluorescence staining and confocal microscopy 58
4.12 Statistical analysis 59

Chapter 5. Results……………………………………………………60
5.1 Caspase-3 is not required in capsaicin-induced cell death 60
5.2 Induction of DNA damage on MCF-7 cells by capsaicin. 61
5.3 The production of ROS in MCF-7 cells after treated with various concentrations of capsaicin by flow cytometry. 61
5.4 Effects of capsaicin on the production of Ca2+ from MCF7 cells. 62
5.5 Capsaicin could alter mitochondria membrane potential (MMP) 62
5.6 Capsaicin affected the expressions of p53, Bcl-2 and Bax in MCF-7 cells. 63
5.7 Capsaicin induces the cleavage of PARP-1 63
5.8 No low molecular weight DNA fragmentation occurred in capsaicin- treated MCF-7 cells. 64
5.9 Capsaicin-induced AIF translocation 64

Chapter 6. Discussion………………………………………………...65

Chapter 7. Conclusion ……………………………………………… 69

References………….. 70
Contents of Figures and Table 83
Appendix ……………………………………………………………... 104



圖 表 目 錄
Figure 1. The chemical structure of capsaicin. 83
Figure 2. Caspase-dependent and -independent execution of apoptosis. 84
Figure 3. Effects of capsaicin on the cell viability and cell cycle. 85
Figure 4. Morphological changes of human breast cancer MCF-7 cells in response to capsaicin. 86
Figure 5. PI staining analysis for the effects of capsaicin on apoptosis in human breast cancer MCF-7 cells. 87
Figure 6. The expression of TRPV1 in breast cancer cells. 88
Figure 7. DNA damage determination for the effects of capsaicin on MCF-7 cell’s DNA. 89
Figure 8. Flow cytometric analysis of ROS in human breast MCF-7 cells with 150 μM capsaicin for various time periods. 90
Figure 9. Flow cytometric analysis of intracellular Ca2+ concentration in human breast MCF-7 cells with 150 μM capsaicin for various time periods. 92
Figure 10. The alteration of mitochondria membrane potential after capsaicin treatment. 94
Figure 11. Representative results of Western blotting showed changes on the levels of Bcl-2, P53 and Bax in MCF-7 cells after treated with capsaicin. 95
Figure 12. The cleavage of PARP-1 after capsaicin treatment. 96
Figure 13. The effect of capsaicin on the activation of procaspase-7 in breast cancer cells. 97
Figure 14. DNA fragmentation assay of MCF-7 cells after capsaicin treatment for 24 hours. 98
Figure 15. Capsaicin-induced AIF translocation in breast cancer cells. 99
Table 1. Chemicals and Reagents………………………………… 100

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