Breast cancer remains the leading cause of cancer death among women worldwide. This is in part due to late diagnosis, high recurrence rate and the development of drug resistance. Indeed, even though oestrogen receptor-positive breast cancers are known to respond to hormonal therapy, drug resistance is a common occurrence. Furthermore, triple negative breast cancers lack a specific therapeutic target, which has led to poor treatment outcomes. Hence, there is a critical need for new therapeutic approaches. Our laboratory previously identified a novel palladium-based compound, AJ-5, that exhibit potent anti-cancer activity in triple negative and oestrogen receptor-positive breast cancer cells. However, AJ-5 is poorly soluble, therefore, a series of water soluble AJ-5-based compounds were synthesized. The aim of this study was to test and characterise the anti-cancer activity of one of these AJ-5 analogues, BTC2, in triple negative (MDA-MB-231) and oestrogen receptor-positive (MCF7) breast cancer cell lines. Cytotoxicity assays were performed and BTC2 was shown to inhibit the proliferative rates of breast cancer cells with calculated IC50 values of 0.49μM in MCF7 cells and 0.58μM in MDA-MB-231 cells. BTC2 did not display considerable selectivity to breast cancer cells as the calculated IC50 value for the normal fibroblast cell line (FG0) was found to be 0.85μM and thus the selectivity index was less than 2 in both cell lines. Clonogenic assays were performed and BTC2 was shown to inhibit the long term (10 to 21 days) survival of MCF7 and MDA-MB-231 cells as it reduced their colony forming ability. Western blot analyses and immunofluorescence with an antibody to ƴH2AX, a robust marker of DNA double strand breaks, indicated that BTC2 acts by inducing DNA damage as the levels of this protein increased in drug treated cells. Light microscopy revealed that BTC2 induced morphological features of apoptosis (membrane blebbing and cell shrinkage) and autophagy (vacuoles reminiscent of autophagosomes). To further characterise the molecular mechanism underpinning the cytotoxic effects of BTC2, western blotting was performed with antibodies against key protein markers of stress signalling, cell cycle, apoptosis and autophagy. The results indicated that BTC2 activated the p38 MAP kinase signalling pathway and the p53 response in MCF7 cells. It is worth noting that MDA-MB-231 cells have a mutant p53 but that the p53 target protein, p21, was upregulated in both MCF7 and MDA-MB-231 cells. This suggests that p21 is regulated by a p53-independent mechanism in the MDA-MB-231 cells. BTC2 was shown to induce apoptosis and autophagy in both breast cancer cell lines as demonstrated by increased levels of cleaved PARP and LC3-II respectively. Apoptosis was confirmed by Annexin V-FITC/ propidium iodide double staining using flow cytometry. Taken together, data from this study suggest that BTC2 represents a promising anti-cancer drug for the treatment of triple negative and oestrogen receptor-positive breast cancer cells.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/24901 |
| Date | January 2017 |
| Creators | Irene, Ikponmwosa |
| Contributors | Prince, Sharon, Kimani, Serah |
| Publisher | University of Cape Town, Faculty of Health Sciences, Department of Human Biology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Master Thesis, Masters, MSc (Med) |
| Format | application/pdf |
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