槲皮素和白藜蘆醇調控Sirt-1來抑制人類乳腺癌細胞(MCF-7)的轉移機制之探討

Investigation of inhibitory effects of quercetin and resveratrol through Sirt-1 regulation in Human breast cancer cell(MCF-7) metastasis

黃昫璁(Hsu-Tsung Huang)

指導教授 ： 陳兆勛

國立臺灣大學/工學院/應用力學研究所/碩士(2016年)

https://doi.org/10.6342/NTU201601955

摘要

大部分多酚化合物都有保護正常細胞和減少腫瘤生長的作用，然而不同類型的多酚化合物的作用機制可能不一樣。而槲皮素(Quercetin，Qu)是類黃酮型的多酚化合物，可從多種天然植物以及食物中獲取。因為目前槲皮素對於調控乳腺癌的侵襲效果尚不明確。所以本論文選用與槲皮素性質最相近的白藜蘆醇(二苯二烯型的多酚化合物)作為對照組，來觀察比較槲皮素對人類乳腺癌細胞株MCF-7轉移能力的影響，並探討槲皮素影響轉移的機制。
　　實驗結果如下：(1)傷口癒合試驗結果顯示：乳腺癌細胞株MCF-7經槲皮素和白藜蘆醇(10,20,40,80,100μM)作用24或48小時後，發現槲皮素和白藜蘆醇皆有抑制傷口癒合的能力，且在統計學上有明顯的差異，並隨著藥物濃度的增加，抑制的能力則越強，此顯示兩種藥物對人類乳腺癌細胞株MCF-7的遷移能力皆有抑制效果。此外，有文獻指出MMP-9參與乳癌的侵襲和轉移，為了進一步探討藥物對乳腺癌細胞株MCF-7的MMP-9蛋白是否有影響，我們以西方墨點法試驗來觀察。(2)西方墨點法試驗結果顯示：藥物濃度為40μM的槲皮素和白藜蘆醇對人類乳腺癌細胞株MCF-7的MMP-9蛋白皆有下調的趨勢。又因為有文獻指出，白藜蘆醇可透過上調Sirt-1蛋白表達量來調控不同的信號通路，以發揮多種生物學功能，進而調控癌症的侵襲及轉移。因此我們也觀察了藥物對人類乳腺癌細胞株MCF-7的Sirt-1蛋白之影響，發現槲皮素和白藜蘆醇都會讓Sirt-1蛋白的表現量增加。(3)細胞遷移試驗和細胞侵襲試驗結果顯示：為了證明槲皮素和白藜蘆醇調控的MMP-9蛋白，即可使人類乳腺癌細胞株MCF-7轉移能力下降，我們採用此兩種體外細胞試驗來輔佐證明。發現槲皮素和白藜蘆醇確實可以抑制人類乳腺癌細胞株MCF-7的遷移和侵襲。另外我們也發現人類乳腺癌細胞株MDA-MB-231經槲皮素和白藜蘆醇(40μM)作用6小時對細胞遷移有顯著的抑制效果。而人類乳腺癌細胞株MDA-MB-231經槲皮素和白藜蘆醇(40μM)作用24小時對細胞侵襲亦有顯著的抑制效果。(4)細胞增殖試驗結果顯示：槲皮素和白藜蘆醇(40μM)皆能抑制人類乳腺癌細胞株MCF-7的細胞增殖。
　　綜合以上結果顯示：槲皮素能透過下調人類乳腺癌細胞株MCF-7的MMP-9蛋白表現量，來抑制人類乳腺癌細胞株MCF-7的遷移和侵襲。另一方面，槲皮素也可以上調人類乳腺癌細胞株MCF-7的Sirt-1蛋白表現量。因此我們可以推斷，槲皮素有可能與白藜蘆醇相似，透過上調Sirt-1蛋白表現量來抑制人類乳腺癌細胞株MCF-7的遷移和侵襲。此外，槲皮素亦能抑制轉移能力較強的人類乳腺癌細胞株MDA-MB-231的遷移和侵襲。

關鍵字

MCF-7 ； 乳癌轉移 ； 白藜蘆醇 ； 槲皮素 ； Sirt-1 ； MMP-9

並列摘要

Most of polyphenol compounds have ability to protect normal cells and reduce tumor growth. Different types of polyphenols might have different mechanisms. Quercetin (Qu), which can be extracted from various natural plants and foods, is known as a flavonoid-type polyphenol. However, the effect of regulation of Quercetin on mammary cancer is still unclear. In this study, the mechanism of Quercetin’s influence on metastasis ability of MCF-7 was investigated. In order to observe and compare the effect on metastasis of MCF-7, Resveratrol, a stilbenes-type polyphenol compound with qualities most similar to Quercetin, was chosen as the control group in comparison.
The results show below: (1) In wound healing assay, both Quercetin and Resveratrol (10, 20, 40, 80 100 μM) have ability of inhibition on wound healing of MCF-7 after reacting 24 or 48 hours with a significant sifference. Along with the increase of the concentration of the agents, the inhibiting ability increases, showing that both Quercetin and Resveratrol have effect on inhibition of metastasis of MCF-7. In addition, since previous studies proposed that MMP-9 involved in invasion and metastasis of mammary cancer, we did a further investigation on whether MMP-9 of MCF-7 was influenced by medicine with West blot assay. (2) The result of Western blot assay shows MMP-9 was down-regulated both in the condition of 40 μM of Quercetin and Resveratrol. Furthermore, studies also proposed that Resveratrol, by up-regulating expressing quantity of Sirt-1, could regulate different signal paths to work biologically on regulating invasion and metastasis of cancers. For this reason, we also observed the effect of the medicine on Sirt-1 of MCF-7, and discovered both Quercetin and Resveratrol have influence on increasing the expressing quantity of Sirt-1. (3) In cell migration assay and cell invasion assay, in order to confirm that Quercetin and Resveratrol can reduce the ability of metastasis by regulating MMP-9, we did examination on two in vitro cell lines. It was realized beyond and doubt that Quercetin and Resveratrol can inhibit the invasion and metastasis of MCF-7. On the other hand, Quercetin and Resveratrol (40 μM) inhibited the metastasis of MDA-MB-231 in 6-hour reaction and the invasion of MDA-MB-231 in 24-hour reaction significantly as well. (4) The result of cell proliferation assay shows both Quercetin and Resveratrol (40 μM) can inhibit cell proliferation of human breast cancer cell line MCF-7.
According to the results of our experiments, on the one hand, Quercetin can inhibit the invasion and metastasis of MCF-7 by down-regulating the expressing quantity of MMP-9. On the other hand, Quercetin can up-regulate the expressing quantity of Sirt-1 of MCF-7. Therefore, we can deduce that, like Resveratrol, Quercetin can inhibit the invasion and metastasis of MCF-7 by up-regulating the expressing quantity of Sirt-1. Besides, Quercetin can also inhibit the invasion and metastasis of MDA-MB-231, which was understood much powerful in metastasis.

並列關鍵字

MCF-7 ； Breast Metastasis ； Resveratrol ； Quercetin ； Sirt-1 ； MMP-9

參考文獻

6. Holliday, D.L. and V. Speirs, Choosing the right cell line for breast cancer research. Breast Cancer Res, 2011. 13(4): p. 215.

7. Goldhirsch, A., et al., Strategies for subtypes—dealing with the diversity of breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2011. Annals of oncology, 2011: p. mdr304.

8. Yanagawa, M., et al., Luminal A and luminal B (HER2 negative) subtypes of breast cancer consist of a mixture of tumors with different genotype. BMC research notes, 2012. 5(1): p. 376.

10. 陳虹瑋 and 張家靖, 癌症標靶材料作用於細胞與分子機制之探討: 1. 多光子趨動標靶化奈米鑽石誘發癌細胞死亡之機制; 2. anti-CEACAM6 與 anti-HER-2 單一抗原結合功能區抗體於乳癌標靶治療之研究. 2010.

11. Han, D., et al., Disulfiram inhibits TGF-β-induced epithelial-mesenchymal transition and stem-like features in breast cancer via ERK/NF-κB/Snail pathway. Oncotarget, 2015. 6(38): p. 40907.