Inhibition of Metastasis and Induction of Apoptosis in Breast Cancer Cells by AC-0

• Main Authors: Pei-Chun Shen, 沈佩君
• Other Authors: 楊新玲
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/44978227354957266641

碩士 === 中國醫藥大學 === 營養學系碩士班 === 100 === 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.