Kong, Wei Yang
(2019)
Zebularine and trichostatin a sensitized human breast adenocarcinoma cells towards tumour necrosis-factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis.
MRes thesis, University of Nottingham.
Abstract
Triple-negative breast cancer (TNBC) is characterised by the absence of hormone receptors (HR) and human epidermal growth factor receptor 2 (HER2). It is highly aggressive and has the worst clinical outcome worldwide. Current TNBC treatment is limited to chemotherapy which causes adverse side effects to patients. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent due to its selective killing on cancer cells while sparing the normal cells. Nevertheless, human breast adenocarcinoma cells can develop TRAIL resistance. To overcome this problem, combinational therapy is imperative to reverse TRAIL resistance and augment the apoptosis effects towards human breast adenocarcinoma cells. The administration of epigenetic drugs as sensitizers was reported to enhance the TRAIL-mediated apoptosis in cancerous cells. Therefore, this project aimed to investigate the anti-cancer effects of epigenetic drugs zebularine (Zeb) and trichostatin A (TSA) as sensitizers to human breast adenocarcinoma cells towards TRAIL-induced apoptosis (TZT) and this treatment regimen was compared with the natural anti-cancer compound curcumin (Cur) and standard chemotherapeutic drug doxorubicin (Dox).
In 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT) cell proliferation studies, TZT treatment hampered the cell proliferation of human breast adenocarcinoma cells MDA-MB-231 (TNBC) significantly but not MCF-7 (HR-positive which served as control) as compared to the TRAIL alone treatment. Nevertheless, there was no reduction in the cell proliferation of human breast epithelial cells MCF10A. In addition, Cur and Dox treatments induced a significant reduction in cell proliferation in human breast adenocarcinoma and epithelial cells. Based on flow cytometric cell cycle analysis, there were no changes in cell cycle distribution in both TRAIL and TZT treatments in human breast adenocarcinoma and epithelial cells. Nevertheless, Cur treatment induced G2/M arrest in MDA-MB-231, MCF-7 and S phase arrest in MCF10A. Dox treatment induced G2/M phase in both human breast adenocarcinoma cells and epithelial cells.
From morphological assessment via hematoxylin and eosin staining, the features of apoptosis such as rounded cell shape, nuclear chromatin condensation and membrane blebbing were more prominent in TZT-treated MDA-MB-231 and MCF-7 as compared to MCF10A. Moreover, apoptotic features were observed in MDA-MB-231, MCF-7 and MCF10A treated with Cur and Dox. Quantitative flow cytometric apoptotic assays demonstrated TZT treatment induced significantly higher apoptotic activity in MDA-MB-231 at 24 hours post-treatment as compared to TRAIL treatment. Compared to TZT treatment, Cur and Dox treatments induced less apoptotic activity in MDA-MB-231. Apoptotic activity in MCF10A treated with TRAIL, TZT and Dox were rather subtle but significant apoptotic activity was observed in Cur treatment. Following this, the expression of cleaved poly (ADP-ribose) polymerase (PARP) protein as a hallmark of apoptosis was evaluated using Western blot analysis. In MDA-MB-231, both TRAIL and TZT treatment caused the cleavage of PARP protein while Cur and Dox treatments showed lesser expression of cleaved PARP protein in MDA-MB-231. Besides, no cleaved PARP protein was detected in MCF10A treated with TRAIL, TZT and Dox.
Collectively, these results demonstrated that Zeb and TSA collectively enhanced TRAIL-induced apoptosis in MDA-MB-231 at 24 hours post-treatment. Nevertheless, further molecular analyses are required to validate the enhancement of apoptosis induction. With these, TZT treatment can be an alternative therapeutic strategy in improving clinical outcome of TNBC patients in the near future.
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