The antiproliferative and antimigratory properties of the 3-biotin-B-norcholesteryl benzimidazole derivative in MCF-7 cells are mediated through G1 phase arrest and apoptosis.

Our research group has previously constructed a series of 3-biotin-B-norcholesteryl benzimidazole derivatives; nevertheless, their underlying biological mechanisms against breast cancer remain poorly defined. In the present work, three representative analogs were assessed in MCF-7 human breast cancer cells to explore whether the optimal lead candidate restrains tumor cell proliferation and migration via modulating apoptosis and cell-cycle progression. MTT and colony formation assays identified compound 1 as the most potent antiproliferative agent, with an IC50 of 6.53 μM in MCF-7 cells, which outperformed tamoxifen under identical experimental conditions. By contrast, compound 1 exhibited a markedly elevated IC50 of 15.19 μM in non-tumorigenic MCF10A human mammary epithelial cells, yielding a selectivity index of 2.33 toward MCF-7 cells. Furthermore, compound 1 suppressed colony formation and scratch wound closure in a concentration-dependent fashion. Morphological examination combined with Hoechst 33258 staining revealed hallmark apoptotic phenotypes, including cellular shrinkage, apoptotic body generation, as well as nuclear condensation and fragmentation. Annexin V-FITC/PI double-staining flow cytometry validated concentration-dependent apoptosis predominantly featured by early apoptotic populations, while PI cell cycle staining demonstrated prominent G1-phase cell cycle arrest. RT-qPCR quantification revealed that compound 1 treatment markedly upregulated the transcript levels of p53 and c-Myc yet downregulated MDM2, whereas the expression of ERα remained statistically unaltered. Collectively, these data demonstrate that compound 1 elicits antiproliferative and antimigratory activities in MCF-7 cells by triggering robust early apoptosis and G1-phase arrest, an effect mediated at least partially through regulation of the MDM2-p53 signaling axis. Taken together, these observations establish compound 1 as a promising antitumor lead scaffold for subsequent structural modification. Further investigations regarding its selectivity profile, systemic toxicity, pharmacokinetic properties, and in vivo therapeutic efficacy are therefore warranted.