Evaluation of a strategy based on multi-drug targeting of cancer proteins in breast cancer cell lines

Abstract

Therapeutic inefficacy of conventional cancer treatment is a particular dilemma associated with metastatic triple negative breast cancer (TNBC), with patients still facing poor prognosis. The design and development of novel anticancer agents specifically targeted to cancer-associated pathways is of therapeutic interest. The rationale is twofold: firstly, targeted therapy overcomes widespread toxicity and adverse effects of conventional chemotherapy due to the selectivity of the treatment modality. Secondly, synergistic combinations of different classes of highly targeted therapies could hold therapeutic promise to overcome resistance by simultaneously circumventing multiple cancer hallmarks. This study evaluates the in vitro antiproliferative activity of six compounds using breast cancer cell lines as experimental model. Five of these compounds are novel, agents designed in silico to selectively target cancer hallmarks via inhibition of specific cancer-associated proteins. The compounds include an antimitotic (STX1972), three variants of bromodomain 4 (BRD4) inhibitors (Bzt-W41, Bzt-W49 and Bzt-W52), an inhibitor of both sirtuin (SIRT) 1 and 2 (W137) and an inhibitor of janus kinases 1 and 2 (Ruxolitinib). The synergism between paired combinations was also explored. Two breast cancer cell lines, MDA-MB-231 and MCF-7 were used as experimental models. The MDA-MB-231 cell line is oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) negative and is therefore commonly used to model triple negative breast cancer with invasive and metastatic properties. MCF-7 cells are ER and PR positive and represent the hormone-dependent breast cancer model. The endothelial EA.hy926 cell line was used to represent non-cancerous cells. A crystal violet assay was used to determine the half maximal inhibitory concentration (IC50) of the six compounds on the tested cell lines after 48 h exposure. Drug combination studies based on the Chou-Talalay method of paired drug combinations were performed. Effects of treatment on cell morphology was assessed by means of confocal-microscopy. Flow cytometry was used to study the effects on cell cycle progression, apoptosis, autophagy/lysosomal activity, reactive oxygen species (ROS) production, changes in mitochondrial membrane potential (ΔΨm) and the serine 70 phosphorylation status of Bcl-2. Real-time quantitative PCR was used to analyse the effects of the compounds on the mRNA expression levels of p53, c-myc and bcl-2. Quantitative protein expression of c-MYC was analysed by means of enzyme-linked immunosorbent assay. In vitro screening for antiproliferative activity revealed that the compounds showed cancer-selective cytotoxic effects when compared to the EA.hy926 control cell line. The initial screening identified three compounds for further investigative inclusion, namely the antimitotic (STX1972), the BRD4i (Bzt-W41) and the SIRTi (W137). STX1972 was found to inhibit cell growth in the nanomolar concentration range, whilst the rest of the compounds showed growth inhibition in micromolar concentration ranges. Bzt-W41 showed significant preferential selectivity for the TNBC MDA-MB-231 cell line versus the hormone-dependent MCF-7 cell line, while STX1972 and W137 exhibited only slight differential selectivity. Two combinations (STX + Bzt-W41 and Bzt-W41 + W137) exhibited synergism, whilst the STX + W137 combination exhibited antagonistic interaction. Cell cycle and apoptosis analysis revealed that STX1972 and Bzt-W41, alone and in combination, selectively induced cell cycle arrest and apoptosis in cancer cells. However, the W137 +Bzt-W137 combination did not show preferential targeting of breast cancer cell lines, with apoptosis induced equally or even more so in the control EA.hy926 cell line. STX1972 and Bzt-W41, as well as their paired combination, was further probed in aim of deciphering their individual and combined mode of action. STX1972, Bzt-W41 as well as the paired combination proved to selectively inhibit cancer targets resulting in several molecular changes, leading to downstream pathway activation which culminates in both apoptotic and autophagy-related cellular demise. The study contributed towards deducing possible hypotheses regarding the mechanistic behaviours of the individual compounds and elucidated their combined effect during dual treatment. Results warrant future studies to further probe the intricate interaction of pathways involved in the synergistic combination of antimitotics and epigenetic regulators as a novel anticancer therapeutic modality.