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 IC 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.

中文翻译：

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新型吲哚希夫碱β-二亚胺化合物作为三阴性乳腺癌抗癌剂：体外抗癌活性评价和体内急性毒性研究


乳腺癌是全球女性中最常见的癌症，三阴性乳腺癌 (TNBC) 与预后不良和五年生存率低相关。希夫碱化合物以其广泛的药理活性而闻名，在癌症药物研究中引起了极大的关注。本研究旨在评估新型β-二亚胺化合物的抗癌潜力并阐明其作用机制。在乳腺癌细胞系（包括 MCF-7 和 MDA-MB-231）中使用 MTT 测定评估了该化合物对细胞活力的影响。使用台盼蓝排除和乳酸脱氢酶（LDH）释放测定进一步分析细胞毒性作用。为了评估该化合物的抑制活性机制和诱导细胞死亡的方式，进行了细胞周期分布的流式细胞术和细胞凋亡分析。细胞凋亡发生率最初通过细胞和核形态变化（Hoechst 33342/碘化丙啶 (PI) 染色）进行评估，并通过膜联蛋白 V/PI 染色和流式细胞术分析进一步证实。此外，分别使用 JC-1 指示剂和 DCFDA 染料测定了该化合物对线粒体膜电位 (MMP) 破坏和活性氧 (ROS) 生成的影响。结果表明，β-二亚胺化合物处理 24 h 可显着抑制 MDA-MB-231 和 MCF-7 癌细胞的活力，且呈剂量依赖性，IC 值分别为 2.41 ± 0.29 和 3.51 ± 0.14。该化合物的细胞毒性作用通过死亡细胞数量的剂量依赖性增加和培养基中 LDH 水平的增强得到进一步证实。 该化合物通过 MDA-MB-231 乳腺癌细胞中 G2/M 期细胞生长停滞发挥抗增殖作用，并诱导细胞凋亡介导的细胞死亡，其中涉及细胞和核形态的特征性变化、磷脂酰丝氨酸外化、线粒体膜去极化、并增加ROS水平。在生化和组织病理学分析中未检测到肝毒性或肾毒性，证实了该化合物使用的安全性。因此，结果显着证实了一种新型 β-二亚胺化合物的潜在抗癌活性，诱导细胞周期停滞和细胞凋亡就证明了这一点，这可能是由 ROS 介导的线粒体死亡途径驱动的。该化合物可能成为未来抗癌药物设计和 TNBC 治疗的有希望的候选者，并且需要进一步的临床前和临床研究。