Abstract
Breast cancer can be divided into a number of subtypes based upon receptor expression. Of these triple-negative breast cancer (TNBC) is the most aggressive subtype and is highly challenging to treat, as it has no targeted therapy and a high recurrence rate after conventional chemotherapy. TNBC cells lack the expression of three key receptors: progesterone, estrogen and HER2, hence cannot be treated using currently approved hormonal or antibody-based therapies. Therefore, TNBC patients have an extremely poor prognosis compared to other subtypes of breast cancer. Hence there is an urgent requirement for new targeted treatment for TNBC. Previous studies have shown that selenium derivatives can treat a range of disorders, particularly diphenyl diselenide (DPDS) has been reported to be cytotoxic against other cancer cell lines at concentrations greater than 30 μM. However, the cytotoxic effect of DPDS on TNBC has not yet been studied. Here we have investigated the effect of two synthetic organoselenium derivatives, DPDS and diphenyl selenide (DPS), with a structural the difference in the number of selenium atoms between the phenyl groups. They were screened against the following cell lines: TNBC: MDA-MB-231, MDA-MB-468 and Hs 578T, estrogen receptor-positive breast cancer MCF-7, the human epidermal growth factor receptor 2 positive breast cancer SKBR3, non-cancerous human breast MCF-10A and normal human dermal fibroblast NHDF.
Firstly, the cytotoxicity induced by DPDS and DPS was determined by MTT and Hoechst/PI double labeling assays. DPS showed no cytotoxic effect against any of the breast cancer and non-cancerous cell lines; however, DPDS showed potent cytotoxicity with IC50s in the range 7-18 μM against TNBC cells, and 27 μM against MCF-7s. However, DPDS did not display cytotoxicity towards Her2 positive SKBR3 breast cancer cells. Additionally, DPDS did not show any cytotoxic effect against any of the non-cancerous cell lines, suggesting selectivity anti-cancer action against subtypes of breast cancers. The data also indicated that DPDS displayed higher cytotoxicity towards MDA-MB-231 cells compared to other breast cancer cell lines. YO-PRO-1 dye, a selective indicator of apoptosis was used to identify if the cell death induced by DPDS was through apoptosis in the TNBC cells and ER+ cells used in this study. The analysis indicated that DPDS induced cell death via apoptosis in the range of 5-40 % after 24 h treatment. A time-dependent study also demonstrated that the cytotoxicity and apoptosis were initiated after 8-10 h DPDS treatment in MDA-MB-231 cells. Mechanistically MDA-MB-231 cells showed a 2-fold increase in the activity of caspase-3, detected following DPDS using Ac-DEVD-AMC caspase-3 fluorogenic substrate assay. These results were further supported by a 2-fold increase in cleaved caspase-3 expression in DPDS-treated MDA-MB-231 cells. Western blot analysis also showed that p53 expression was increased 1.5-fold in MDA-MB-231 cells, and 2-fold in MCF-7 cells after 24 h DPDS treatment.
To determine whether the DPDS induced the intrinsic or extrinsic pathway of apoptosis, the release of cytochrome c from mitochondria in TNBC cells and ER+ cells was determined by immunocytochemistry using confocal microscopy. Only MDA-MB-231 cells released cytochrome c, indicating that these cells underwent the intrinsic-pathway of apoptosis with DPDS treatment. Hence, time-dependent release study of cytochrome c release was conducted. The results identified a correlation between the time of p53 activation and the activation of the mitochondrial pathway of apoptosis, as after 2 h treatment with DPDS, 20-30 % cytochrome c was released and a 2.5-fold increase in p53 expression was detected. Further, the pan-caspase inhibitor Q-VD-OPh was used to determine whether DPDS induce caspase-dependent or caspase-independent pathways of cytotoxicity. Q-VD-OPh reversed the formation of cleaved caspase-3, and the cytotoxicity induced by DPDS in MDA-MB-231 cells, confirming the involvement of caspase-mediated cell death. In summary, the Thesis demonstrates that DPDS induced apoptosis in MDA-MB-231 cells through activation of p53 and the intrinsic caspase-dependent pathway of apoptosis. These results suggest that DPDS is a potential anti-cancer therapeutic agent for TNBC and should be progressed to in vivo preclinical trials.