魚藤酮引起MCF-7乳癌細胞凋亡及兒茶素抑制CL1-5肺癌細胞轉移之機制探討

Mechanisms of Rotenone-induced Apoptosis in MCF-7 Human Breast Cancer Cells and Inhibition Effects of EGCG on Metastasis in CL1-5 Human Lung Cancer Cells

鄧雅姿(Yea-Tzy Deng)

指導教授 ： 林仁混

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.00094

摘要

中文摘要
癌症目前對全世界仍是一大威脅，更是台灣持續蟬聯十大死因的頭號殺手，因此，探討癌症的預防及治療方法仍是科學家孜孜不倦的課題。癌細胞由於具有持續增生及切割細胞外基質進而通過血管進入血液而轉移至其它組織的特性，因此，如何使細胞不再不斷增生及如何抑制細胞轉移至其他組織乃為癌症研究的首要目標。目前已有許多研究報告指出，植物中具有許多可以抑制癌細胞生長及轉移的天然物質，且由於癌症治療須耗費大量金錢，也因此，研究如何利用這些大自然天然的寶藏對抗人類的癌症疾病實為當務之急。魚藤酮存在於魚藤植物的根部，其會抑制粒線體內膜上電子傳遞鏈複合體 Ι (electron transfer chain complex Ι)造成細胞內活性氧 (reactive oxygen species; ROS) 的產生。魚藤酮最早於1930年代開始用來毒魚，於約150年前開始用於當作殺蟲劑。然而近年來的文獻指出，魚藤酮會引起黑色素瘤、白血病細胞凋亡。由於引起細胞凋亡乃為對抗癌細胞之重要步驟，又乳癌為威脅女性之惡性腫瘤，因此，我們極欲探討魚藤酮是否可引起乳癌細胞之細胞凋亡。在本論文中，我們利用methylthiazolyldiphenyl-tetrazolium bromide (MTT)試驗、trypan blue exclusion assay、alamar blue試驗、流式細胞儀、螢光顯微鏡觀察細胞核法，發現魚藤酮可引起人類乳癌細胞株MCF-7發生細胞凋亡的現象，此種結果隨著魚藤酮劑量和時間的增加而更明顯。進一步用西方點墨法分析和引起細胞凋亡現象有關的蛋白發現，魚藤酮可促使poly (ADP-ribose) polymerase (PARP) 活化、抗細胞凋亡現象的蛋白Bcl-2 表現下降、及促進細胞凋亡現象發生的蛋白Bax表現上升，並由觀察魚藤酮對粒線體膜電位的影響發現魚藤酮可改變粒線體膜電位差，更證實了魚藤酮確實會引起MCF-7細胞發生細胞凋亡。由於魚藤酮會抑制電子傳遞鏈複合體 Ι 而導致活性氧產生，我們發現隨著處以MCF-7細胞的魚藤酮濃度增高，確實會增加活性氧的產生。進一步利用西方點墨法研究魚藤酮於MCF-7細胞引起細胞凋亡所經過的訊息傳遞途徑，發現魚藤酮引起的活性氧增加會活化c-jun N-terminal kinase (JNK) 及p38 mitogen-activated protein kinase (p38 MAPK) 訊息傳遞，並降低extracellular signal-regulated protein kinase 1/2 (ERK 1/2) 訊息傳遞，進而引起細胞凋亡現象的發生。
另一方面，由於癌細胞轉移會導致癌症治療無法順利進行，因此研究如何抑制癌細胞轉移乃為癌症研究之重要課題。轉移是惡性程度高且預後差的癌症之特性，轉移的一個重要步驟是細胞分泌蛋白酶以切割細胞外的基質，使細胞能通過細胞外基質進入血液中，進而隨著血液轉移至其它組織。因此，促使過量細胞外基質的切割是癌細胞轉移的重要特徵，而扮演切割細胞外基質重要角色的是基質蛋白酶 (matrix metalloproteinases; MMPs)。由於肺癌持續為全世界致死率最高的癌症，且肺癌的高致死率是因其極具轉移的特性，因此我們選用具高轉移能力的肺癌細胞株CL1-5作為研究對象。又茶具有抗癌活性，因此，我們想探究綠茶中主要的茶多酚epigallocatechin-3-gallate (EGCG)是否可抑制高轉移性肺癌細胞株CL1-5轉移的能力。首先利用MTT試驗觀察EGCG對CL1-5細胞生長的影響，發現EGCG濃度高於20 μM時會抑制細胞生長，EGCG濃度於20 μM以下時細胞生長則無明顯影響。進一步探討EGCG於20 μM以上細胞生長的減緩是由於引起細胞凋亡或導致細胞週期停止，以流式細胞儀觀察的結果，EGCG於20 μM以上會導致細胞週期停止於G2時期，且並不會引起細胞凋亡現象的發生。接下來欲探討EGCG對CL1-5細胞轉移能力影響，利用轉移試驗發現EGCG 可抑制CL1-5細胞的轉移能力。由於MMP-2為降解細胞外基質的重要酵素，因此，若MMP-2的表現被抑制，則可抑制細胞降解細胞外基質的能力，進而抑制細胞轉移至其他組織。利用gelatin zymography、西方點墨法、聚合酶連鎖反應，發現EGCG會抑制CL1-5細胞內MMP-2 mRNA及蛋白的表現。進一步研究其中參與的訊息傳遞途徑，發現EGCG會抑制CL1-5細胞內JNK的訊息傳遞，導致MMP-2的轉錄因子NF-κB及Sp1無法由細胞質進入細胞核內進行MMP-2 mRNA的轉錄，而抑制MMP-2 mRNA的形成。觀察EGCG對MMP-2 promoter活性的影響，更證實了EGCG可抑制CL1-5細胞MMP-2 promoter的活性。又因臨床用藥docetaxel具有強副作用如白血球過低、貧血、腹瀉、掉髮，疲倦、虛弱等，因此，我們想探知並用臨床用藥及EGCG是否可減少臨床用藥劑量以期減少副作用的發生，結果指出並用docetaxel與EGCG可在較低劑量的docetaxel即可抑制MMP-2的表現。綜合我們的研究指出，茶中的EGCG及魚藤植物的魚藤酮對癌症預防扮演重要角色！

關鍵字

魚藤酮 ； 細胞凋亡 ； 活性氧 ； EGCG ； 細胞轉移 ； 細胞週期

並列摘要

Cancer is still a serious problem in the world. Therefore, it is important to find ways to prevent or cure cancer. Because cancer cells possess abilities to prolong cell growth and invade to other tissues, it is still thorny to control cancer disease. Therefore, how to suppress cell growth in cancer cells and prevent cancer cells invade to other tissues is of great urgency. Phytochemicals are a group of compounds from natural plants. Recent reports show that they are bioactive components in natural plant to execute chemoprevention or other beneficial effects such as prevent diabetes, protect neuron cells and prevent obesity. Rotenone is a phytochemical from the roots of the Derris and Lonchorcarpus species. Rotenone is an inhibitor of electron transfer chain complex Ι (NADH dehydrogenase complex) and it can promote production of reactive oxygen species (ROS). It has been used as a botanical insecticide for at least 150 years to control crop pests and used even longer as a fish poison by native tribes in South America and East Africa. Recent reports show that rotenone can induce apoptosis in human melanoma cells and leukemia cells. Because anti-apoptosis plays an important role in anti-cancer, we want to determine whether rotenone can induce apoptosis in MCF-7 breast cancer cells which threaten women worldwide. In this study, we found that rotenone can induce apoptosis in MCF-7 human breast cancer cells by using MTT assay, trypan blue exclusion assay, alamar blue assay, flow cytometry and Hoechst 33258 staining and they are dose- and time-dependent effects. To further confirm these results, we used western blot analysis to investigate apoptosis-related proteins, PARP, Bcl-2 and Bax. We showed that rotenone can promote PARP cleavage, downregulate anti-apoptotic Bcl-2 and upregulate apoptotic Bax. In addition, rotenone can reduce mitochondria membrane potential. Because rotenone is attributed to irreversible binding and inactivation of mitochondrial electron transport chain complex Ι, we investigated whether rotenone indeed promote production of ROS in MCF-7 cells. Rotenone can result in an increase of the reactive oxygen species (ROS). We treated MCF-7 cells with rotenone and indeed found that ROS was increased. Then, we want to determine the signaling pathways under treatment of EGCG in MCF-7 cells. Using western blotting analysis, we found that rotenone induced JNK and p38 activation, whereas it attenuated ERK1/2 activation. Then, apoptosis was induced.
In addition, metastasis is a fundamental property of high malignant cancer cells with poor clinical outcome. Therefore, to investigate how to suppress metastasis is an important urgency. Cleavage of extracellular matrix to allow cells invade into blood and transfer to other tissues is an important step in metastasis and MMPs play important roles in this step. Because lung cancer has the highest rate of cancer mortality worldwide and the highest rate of lung cancer mortality is due to their high potential of metastasis, we used highly invasive CL1-5 human lung cancer cells to investigate possible mechanisms of suppression tumor invasion in cancer cells. Green tea contains anti-cancer effects, so we want to determine whether EGCG, the major bioactive compound in green tea, can attenuate metastasis in highly invasive CL1-5 human lung cancer cells. First, we found that EGCG suppress cell growth at concentration higher than 20 μM by using MTT assay. Further, we want to investigate whether EGCG induce apoptosis or cell cycle arrest at concentrations higher than 20 μM. We used flow cytometry analysis and found that EGCG induced G2/M arrest in CL1-5 cells at concentrations of 30, 40 and 50 μM. Next, we want to investigate whether EGCG can attenuate invasion ability of CL1-5 cells. Using transwell invasion assay, EGCG could suppress invasion of CL1-5 cells. Because MMP-2 plays important roles to degrade extracellular matrix and is closely related to invasion in patients of lung cancer, we want to investigate whether EGCG can affect expression of MMP-2 in CL1-5 cells. Using gelatin zymography, western blot analysis and PCR, we found that EGCG can repress MMP-2 expression at the transcriptional level. Furthermore, by western blot analysis we showed that EGCG could suppress activation of JNK and attenuate translocation of MMP-2 transcription factor, NF-κB and Sp1, from the cytosol into the nucleus. In addition, docetaxel, a clinical drug used in the treatment of lung cancer, performs serious side effect, we want to clarify synergistic effects of EGCG and docetaxel. Combine EGCG and docetaxel can repress MMP-2 expression at low dosages of docetaxel.
Taken together, rotenone and EGCG may be potential phytochemicals to chemoprevent breast cancer and lung cancer respectively and they may play synergistic effects with clinical drug.

並列關鍵字

rotenone ； apoptosis ； ROS ； EGCG ； metastasis ； cell cycle

參考文獻

1. Birt DF, Hendrich S, Wang W. Dietary agents in cancer prevention: flavonoids and isoflavonoids. Pharmacol Ther 2001, 90, 157-177.

Neurochem Int 2006, 49, 379-386.

4. Lindahl PE, Oberg KE. The effect of rotenone on respiration and its point of attack. Exp Cell Res 1961, 23, 228-237.

6. Wolvetang EJ, Johnson KL, Krauer K, Ralph SJ, Linnane AW. Mitochondrial

respiratory chain inhibitors induce apoptosis. FEBS Lett 1994, 339, 40-44.