AC-0對人類乳癌細胞之抗腫瘤機制探討

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題名:	AC-0對人類乳癌細胞之抗腫瘤機制探討 Inhibition of Metastasis and Induction of Apoptosis in Breast Cancer Cells by AC-0
作者:	沈佩君;Shen, Pei-Chun
貢獻者:	營養學系碩士班
關鍵詞:	AC-0;乳癌;轉移;PI3K/Akt/NF-κB;上皮-間質細胞型態轉換;細胞週期;細胞凋亡;AC-0;Breast cancer;Metastasis;PI3K/Akt;NF-κB;EMT;Cell-cycle;Apoptosis
日期:	2012-07-18
上傳時間:	2012-08-31 16:34:15 (UTC+8)
出版者:	中國醫藥大學
摘要:	台灣行政院衛生署統計資料顯示，民國100年國人的十大死因之首依然為惡性腫瘤，又稱癌症。而其中乳癌佔居十大癌症死因之第四位，並於女性癌症中佔居第一名，對女性健康是一重大威脅。造成乳癌患者的死亡原因大都是癌細胞生長及癌細胞轉移至其他組織和器官所引起。人類乳癌細胞MDA-MB-231因缺乏estrogen receptor (ER)、progesterone receptor (PR) 、Her2/neu 等接受器，具有高度轉移及對於預後較差的特性。故本研究主要探討乳癌細胞MDA-MB-231給予AC-0在抑制轉移及誘發細胞凋亡之相關機制。研究結果顯示AC-0給予MDA-MB-231人類乳癌細胞，其細胞存活率隨著AC-0濃度的增加而下降(IC50=10.48)。因此本實驗分為兩部分，首先是MDA-MB-231乳癌細胞作用於低濃度的AC-0 (0-2μM，>IC80)，其細胞遷移(migration assay)及侵襲(invasion assay)能力隨著給予AC-0濃度的增加而抑制；另外，由colony formation assay 可發現，隨著給予AC-0濃度的增加，其癌細胞分裂增生的現象減少；以西方墨點法及zymography實驗顯示，MMP-2、MMP-9、uPA和VEGF等轉移相關的蛋白表現量及MMP-2、MMP-9和uPA等活性皆有隨著AC-0濃度的增加而降低，而且TIMP-2和TIMP-1則隨著給予AC-0的濃度增加而增加；進一步探討其訊息路徑發現， AC-0可經由抑制PI3K/Akt路徑進而向下調控核內NFκB表現以及可經由增加IκB蛋白表現量而穩定細胞質中的NFκB (p65)進而抑制NFκB入核，另外，分別給予PI3K抑制劑LY294002及NFκB抑制劑Celastrol可以抑制MMP-9表現，若再加上AC-0則更能顯著抑制MMP-9表現；而由Reporter assay中亦可發現，AC-0具有抑制NFκB轉錄活性。在侵入及轉移能力強的癌間質型態細胞中，可發現低量的E-cadherin表現，但當這些高轉移性的癌細胞重新高度表現E-cadherin後，則可轉變為較不具轉移性之癌上皮型態細胞，其現象稱為上皮-間質細胞型態轉換 (EMT﹐Epitheilial-Mesenchymal Transition)。當AC-0給予MDA-MB-231細胞可增加E-cadherin和Occludin等上皮細胞指標蛋白表現，亦可減少Vimentin、β-catenin、Twist等間質細胞指標蛋白表現，並觀察其細胞型態由本來的紡錘狀(梭狀)轉變成鵝卵石狀。藉由Reporter assay實驗發現，給予AC-0及β-catenin、NFκB抑制劑後，比較單獨給予AC-0組發現，可更加促進E-cadherin轉錄活性；另外， E-cadherin siRNA 處理後之 Control 組相較於E-cadherin siRNA 處理後給予AC-0 組，可回復E-cadherin 之上皮細胞指標蛋白表現。以上結果顯示，低濃度AC-0可經由PI3K/Akt/NFκB及E-cadherin/NFκB和E-cadherin/β-catenin訊息傳遞路徑，進而達到抑制乳癌細胞之轉移作用。第二部分實驗中，人類乳癌細胞MDA-MB-231給予高濃度AC-0 (0-15μM)時，可經由誘發ROS的產生進而活化p53及p21之蛋白表現，使細胞週期停滯於G2/M期，並經由TUNEL assay、Annexin V等實驗發現，乳癌細胞隨著給予AC-0的濃度增加其凋亡程度也隨之增加；經由西方墨點法顯示，給予高濃度AC-0會伴隨著凋亡相關蛋白cytochrom c釋放、caspase-9/-3裂解活化、PARP裂解及Bax/Bcl-2比例增加，此外，AC-0加入抗氧化劑NAC也可發現，乳癌細胞的凋亡須透過誘發ROS進而活化p53，增加Bax/Bcl-2比例，而誘發細胞凋亡。裸鼠異位移植腫瘤模式實驗得知，AC-0的確可以抑制MDA-MB-231乳癌細胞植入所誘發之腫瘤大小、體積、及重量，並經由腫瘤蛋白及免疫組織染色分析結果顯示，其相關轉移、訊息傳遞、細胞週期、細胞凋亡蛋白之表現均可受到影響。總結，當MDA-MB-231乳癌細胞給予低濃度AC-0時，可有效抑制細胞遷移/侵襲及轉移；當給予高濃度AC-0時，則可有效促進細胞凋亡，故AC-0可能具有抗乳癌的功效，也許可做為日後輔助治療乳癌細胞的方法之一。 Statistical report from the Department of health of Taiwan showed that the top ten causes of death of people in Republic of China for the last 100 years are being the malignant tumors, also known as cancer. Female breast cancer occupies the fourth place of the top ten leading causes of cancer-related death in human and a major threat to women's health. The major cause of death by breast cancer is metastasis of cancerous cells to other tissues and organs. Currently, many studies pointed out that due to the lack of estrogen receptor (ER) or progesterone receptor (PR) or the over-expression of Her2/neu and other receptors triggered highly metastatic features and poor prognosis in human breast cancer cells. However, the inhibition of breast cancer metastasis and their mechanism are remains unclear. Therefore, the present study was aimed to explore the anti-tumor effect of AC-0 against human breast cancer cell lines in vitro and in vivo. The first set of experiment, we observed that AC-0 treatment significantly decreased cell viability in MDA-MB-231 human breast cancer cells, then, the experiment was divided into two parts. First, we observed AC-0 (0-2 μM) treatment significantly decreased cell invasion and cell migration in human breast cancer cells MDA-MB-231 using invasion and wound healing assays, respectively. In addition, colony formation assay showed that AC-0 treatment also significantly decreased cell growth and and proliferation of MDA-MB-231 cells. Next, we subjected Western blot and zymography analyses to observe the protein expression levels of metastasis and angiogenic regulatory proteins. Results indicate that treatment with AC-0 significantly down-regulated MMP-2, MMP-9, uPA, VEGF and other metastasis-related protein expression and activity. Interestingly, AC-0 treatment caused a remarkable increase inTIMP-2, TIMP-1, PAI-1 protein expression, which are the endogenous inhibitors of MMPs and uPA. To further explore the signaling pathways, we monitored the activation of NF-κB and their up-stream cascades. Results showed that AC-0 significantly inhibited the nuclear translocation of NF-κB through the inhibition of I-κB proteasomal degradation via down-regulation of PI3K/Akt cascades. Luciferase reporter assay also confirmed that AC-0 significantly inhibited the NF-κB transcriptional activity in MDA-MB-231 cells. In addition, inhibition of MMP-9 expression was observed when cells were treated with PI3K inhibitor LY294002 and NF-κB inhibitor Celastrol. Previous studies indicate that lower amount of E-cadherin expression involved in high invasion and metastasis of cancer cells. When this type of cancer cells re-express E-cadherin that could be transformed into non-metastatic type of cancer cell morphology, the process called EMT (Epitheilial-Mesenchymal Transition). We also observed that AC-0 treatment significantly increased epithelial cell protein expression, including E-cadherin and Occludin in human breast cancer cells MDA-MB-231, and reduced mesenchymal cell proteins including Vimentin, β-catenin and Twist. Next, we observed that AC-0 treatment significantly suppressed the cell morphology change from spindle-shaped (fusiform) into cobblestone-like shaped. In addition, results of luciferase reporter assay confirmed that inhibition of β-catenin and NF-κB activity significantly increased E-caherin expression in MDA-MB-231 cells. These results show that low concentrations of AC-0 could down-regulate PI3K/Akt/NFκB, and E-cadherin/β-catenin pathway to inhibit the metastasis of breast cancer cells. In the second part, we observed that treatment of AC-0 (0-15 μM) significantly arrest G2 to M-phase transition followed by the suppression of Cyclin A, Cyclin B, CDK1 expression, and induce ROS to increased in p21, p53 levels. In addition, TUNEL and Annexin V assays revealed that AC-0 treatment induced apoptosis, followed by the induce ROS, activation of p53, dis-regulation of Bax/Bcl-2 ratio, cytochrome c release to cytosol and the down-regulation of pro-caspase- 9, pro-caspase-3 and pro-PARP. Further in vivo studies showed that AC-0 significantly decreased the growth of MDA-MB-231-derived tumors development in the xenograft athymic nude mice. The decrease of tumor volume is associated with a down-regulation of PI3K/Akt/NFκB, and EMT target genes. AC-0 treatment also caused apoptosis and cell-cycle arrest in the MDA-MB-231 xenografted nude mice. In conclusion, we demonstrated that when MDA-MB-231 breast cancer cells were treated with non-cytotoxic concentrations of AC-0 effectively inhibits breast cancer cell migration/invasion and metastasis. In parallel, the higher cytotoxic concentrations of AC-0 induce cell-cycle arrest and apoptosis in human breast cancer cells. Therefore, AC-0 might be a potential chemo-preventive agent for breast cancer treatment in the future.
顯示於類別:	[營養學系暨碩士班 ] 博碩士論文

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