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Title: FXR activation induces mitochondrial mediated apoptosis in breast cancer and synergizes with tamoxifen
Author: Mohan, Rati
ISNI:       0000 0004 5918 7922
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 2016
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Breast Cancer is one of the major causes of mortality among women in the world. During normal tissue development, cell growth is controlled by a mechanism of cell death called apoptosis. However, during cancer, the balance between cell division & apoptosis is altered, leading to cell survival, cell proliferation and tumour formation. The nuclear receptor Farnesoid X Receptor (FXR) is expressed in human breast cancer tissue and the breast cancer cell lines MCF-7 and MDA-MB-468. In these cells, activators of FXR caused apoptosis, but the exact mechanism of how FXR regulates apoptosis in breast cancer is unknown. The aim of this research is to elucidate the mechanism of FXR-mediated apoptosis using human breast cancer cell lines MCF-7 and MDA-MB-231. Cell viability after treatment with FXR agonists chenodeoxycholic acid (CDCA) & GW4064 was measured by MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. The effect of staurosporine was also examined as a positive control for induction of apoptosis. All three chemicals significantly decreased the viability of MCF-7 and MDA-MB-231 cells in a concentration dependent manner, with IC50 values determined as 35µM, 245µM and 6µM for GW4064, CDCA and staurosporine, respectively for MCF-7 cells and 22µM, 440µM and 4µM for GW4064, CDCA and staurosporine, respectively for MDA-MB-231 cells. The difference in efficacy of the two FXR agonists corresponds to differing potency as GW4064 is a high affinity synthetic ligand for FXR, whereas CDCA is an endogenous bile acid that has much lower affinity for FXR. FXR expression was confirmed in both MCF-7 and MDA-MB-231 cell lines by Western blotting. Poly-ADP ribose polymerase (PARP) cleavage is the classical marker for apoptosis. Cleaved PARP, indicative of active caspase-3, was observed in MCF-7 and MDA-MB-231 cells exposed to staurosporine (positive control for apoptosis) or the FXR agonists GW4064 and CDCA. Luminescent analysis for caspases: 3/7, 9 and 8 were measured for MCF-7 and MDA-MB-231 cells treated with staurosporine (positive control for apoptosis), FXR agonists GW4064 and CDCA. Further research focused more specifically on the mechanism of apoptosis. Proteins involved in apoptotic pathway such as BAX, Bcl-2, and cytochrome c were studied through Western blotting performed on MCF-7 and MDA-MB-231 cell lines treated with staurosporine and FXR agonists. Reactive oxygen species were studied on MCF-7 and MDA-MB-231 cell lines treated with FXR agonists, measured using ROS luminescence assay. The results showed that FXR agonists GW4064 and CDCA induced intrinsic apoptotic pathway via ROS activation in both MCF-7 and MDA-MB-231 cell lines. Chemotherapy has been extensively used to treat patients with breast cancer. Tamoxifen (Tam) has been in use for over 30 years to treat patients. However, due to its toxicity and resistance, there is constant need for new therapeutic alternatives. In this study, the interaction between tam and FXR agonists was studied using MTT and clonogenic assay. The results showed a synergistic interaction of GW4064 with tam in both MCF-7 and MDA-MB-231 cell line. However, the mechanism needs further investigation. In summary, FXR agonists induce mitochondrial dependent apoptosis in breast cancer cells. Also GW4064 synergises with tam and reduces cell proliferation in MCF-7 and MDA-MB-231 irrespective of their estrogen status.
Supervisor: Plant, Nick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available