Mechanistic Investigation of the Pharmacological Synergy Between Nigella sativa Thymoquinone and Cisplatin in Triple-Negative Breast Cancer: In Vitro, In Vivo and Proteomic Evidence

Triple-negative breast cancer lacks estrogen receptor, progesterone receptor, and HER2 expression, rendering it refractory to targeted hormonal and biological therapies and dependent on cytotoxic chemotherapy. Cisplatin is active in TNBC but produces significant nephrotoxicity and neurotoxicity at therapeutic doses. Thymoquinone, the principal bioactive constituent of Nigella sativa (black seed), which is extensively cultivated and consumed in North West and North East Nigeria, has demonstrated anticancer and chemoprotective activity in preliminary studies. This study investigated the mechanistic pharmacological synergy between thymoquinone and cisplatin in TNBC using in vitro, in vivo, and proteomic approaches. Synergy was evaluated in MDA-MB-231 and MDA-MB-468 TNBC cell lines using the combination index method. Mechanistic studies examined reactive oxygen species generation, mitochondrial membrane potential, caspase cascade activation, and cell cycle redistribution by flow cytometry. In vivo efficacy was assessed in an orthotopic xenograft mouse model. Quantitative proteomics by SWATH-MS characterised protein expression changes induced by the combination versus single agents. Thymoquinone and cisplatin produced strong synergism with combination index values below 0.6 in both TNBC lines. The combination uniquely activated both intrinsic and extrinsic apoptotic pathways simultaneously. In vivo tumour growth inhibition by the combination was 74% versus 46% for cisplatin alone, and cisplatin-associated kidney histopathological damage was significantly attenuated by thymoquinone co-administration. SWATH proteomics identified suppression of DNA repair proteins RAD51 and BRCA1 as mechanistically explanatory of cisplatin sensitisation. These original mechanistic findings support clinical translation of thymoquinone-cisplatin combination therapy for TNBC. Keywords: thymoquinone, cisplatin, triple-negative breast cancer, synergy, proteomics.