Novel indole Schiff base β-diiminato compound as an anti-cancer agent against triple-negative breast cancer: In vitro anticancer activity evaluation and in vivo acute toxicity study

Breast cancer is the most prevalent cancer among women globally, with triple-negative breast cancer (TNBC) associated with poor prognosis and low five-year survival rates. Schiff base compounds, known for their extensive pharmacological activities, have garnered significant attention in cancer drug research. This study aimed to evaluate the anticancer potential of a novel β-diiminato compound and elucidate its mechanism of action. The compound's effect on cell viability was assessed using MTT assays in breast cancer cell lines including MCF-7 and MDA-MB-231. Cytotoxic effects were further analyzed using trypan blue exclusion and lactate dehydrogenase (LDH) release assays. In order to assess the mechanism of inhibitory activity and mode of cell death induced by this compound, flow cytometry of cell cycle distribution and apoptosis analysis were carried out. Apoptosis incidence was initially assessed through cell and nuclear morphological changes (Hoechst 33342/Propidium iodide (PI) staining) and further confirmed by Annexin V/PI staining and flow cytometry analysis. In addition, the effect of this compound on the disruption of mitochondrial membrane potential (MMP) and generation of the reactive oxygen species (ROS) was determined using the JC-1 indicator and DCFDA dye, respectively. The results demonstrated that the 24 h treatment with β-diiminato compound significantly suppressed the viability of MDA-MB-231 and MCF-7 cancer cells in a dose-dependent manner with the IC₅₀ value of 2.41 ± 0.29 and 3.51 ± 0.14, respectively. The cytotoxic effect of the compound was further confirmed with a dose-dependent increase in the number of dead cells and enhanced LDH level in the culture medium. This compound exerted its anti-proliferative effect by G2/M phase cell growth arrest in MDA-MB-231 breast cancer cells and induced apoptosis-mediated cell death, which involved characteristic changes in cell and nuclear morphology, phosphatidylserine externalization, mitochondrial membrane depolarization, and increased ROS level. Neither hepatotoxicity nor nephrotoxicity was detected in the biochemical and histopathological analysis confirming the safety characterization of this compound usage. Therefore, the results significantly confirmed the potential anticancer activity of a novel β-diiminato compound, as evidenced by the induction of cell cycle arrest and apoptosis, which might be driven by the ROS‑mediated mitochondrial death pathway. This compound can be a promising candidate for future anticancer drug design and TNBC treatment, and further preclinical and clinical studies are warranted.