Evaluation of molecular effects associated with apoptosis, tumour progression, angiogenesis and metastasis by a novel combination of drugs with ormeloxifene in triple negative breast cancer cells

To investigate the molecular effects of a novel combination [sertraline and plumbagin (comb) with ormeloxifene (Orm)] for anticancer activity in triple negative breast cancer cell line "MDA-MB-231".

Collectively this study reveals the efficacy of Orm + comb as more significant than the clinically used tamoxifen (Tam). The study elucidates the promising novelty of the combination as a potential chemotherapeutic intervention for mitigating the aggressiveness of triple negative breast cancer and it addresses the intrinsic resistance caused by single drug treatments.

The cytotoxic effect of the drugs was analyzed by the MTT assay and nuclear morphological changes by acridine orange/ethidium bromide (AO/EB) staining. Induction of apoptosis by annexin V-FITC staining, active caspase-3 detection and cell cycle analysis were studied in vitro on "MDA-MB-231" cells. The qRT-PCR was done to explore the upregulation and down regulation of targeted genes for angiogenesis, metastasis, tumor suppression and protein folding on the triple negative breast cancer cells. The preliminary anti-angiogenic effect of the drugs was assessed by chorioallantoic membrane (CAM) assay.

Orm showed inhibitory effects in "MDA-MB-231" cells in a dose and time dependent manner whereas; the drugs in combination gave better cytotoxic effects in the screening MTT assay. Orm + comb was more effective than Orm alone in eliciting apoptosis as well as inhibited the single cell to grow into a colony. CAM assay using Orm and Orm + comb suggested the anti-angiogenic potential which was further confirmed by the downregulation of VEGF in "MDA-MB-231" cells by qRT-PCR studies. The combination was found to effectively upregulate the expression of P53 and P21 and downregulate the gene expression of zinc finger E-box binding homeobox 1 (ZEB1) and heat shock protein 70 (HSP70) in "MDA-MB-231" cancer cells. Conclusions: Collectively this study reveals the efficacy of Orm + comb as more significant than the clinically used tamoxifen (Tam). The study elucidates the promising novelty of the combination as a potential chemotherapeutic intervention for mitigating the aggressiveness of triple negative breast cancer and it addresses the intrinsic resistance caused by single drug treatments.