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.

Aim: 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". Methods: 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. Results: 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.

Comparative differential cytotoxicity of clinically used SERMs in human cancer lines of different origin and its predictive molecular docking studies of key target genes involved in cancer progression and treatment responses.

SERMS like Tamoxifene, 5-hydroxy tamoxifene, raloxifene and endoxifene has been used for the treatment of hormonal imbalances and dependent cancers owing to their action via Estrogen receptors as in the treatment of estrogen sensitive breast cancers. Due to the adverse side effects, modifications and development of the existing or newer SERMS has always been of immense interest. Ormeloxifene, a SERM molecule manufactured by HLL Lifecare Ltd, India as birth control under the trade names Saheli, Novex, and Novex-DS which is also investigated against mastalgia, fibro-adenoma and abnormal uterine bleeding. Anti-cancer effects have been reported in estrogen dependent and independent cancers which shows its wide scope to be implemented in cancer therapy. Current investigation is a comprehensive effort to find the cytotoxic potential of Ormeloxifene in comparison with clinically used four SERMS in twenty six cancer cell lines of different origin using Adriamycin as positive control. Also the computational studies pertaining to selected target/ligand with respect to tumor progression, development, treatment responses and apoptosis. The studies proved effective cytotoxicity of Ormeloxifene on cancer cell lines with lower TGI, GI50 and LC50 values which are significantly comparable. Also the in silico studies proved that the docking score of the compound suggests the interaction of the compound which could tightly regulate key target genes controlling cancer like ER, EGFR kinase, EGFR-cSRC, HDAC-2, PARP-1 and BRAF. This study brings out the superior efficacy of Ormeloxifene compared to other SERMS with proven safety profile to be repositioned as an anti-cancer drug to treat diverse cancer types.