Niclosamide Treatment Suppressed Metastatic, Apoptotic, and Proliferative Characteristics of MDA-MB-231 Cancer Stem Cells

This study evaluated the efficacy of niclosamide after the invasion of aggressive TNBC breast CSCs into a 3D bone-mimicking model. Initially, the optional dose required for triggering apoptosis was determined for MDA-MB-231 CSCs (CD44+ and CD24−). Our findings revealed that approximately 50% of the cells showed apoptotic properties, as assessed with Annexin V/7AAD assay and WST-1 at IC₅₀ = 100 μM (6 h). Additionally, this treatment suppressed p-STAT3 protein levels and increased Bax levels (p < 0.05), as determined by Western Blot. The expression of genes associated with metastasis and cell migration (CXCR4, MMP2, MMP9), drug resistance (ABCG1, ABCG2), stemness (OCT4, NANOG) and cell cycle and proliferation (CYCLIN D1) was found to be significantly suppressed (p < 0.05). Therefore, after validating the efficacy of the 100 μM dose on CSCs, cell cycle, ELISA, Western Blot, and RT-qPCR analyses were conducted in the 3D model. It was found that the cells were arrested in the G0-G1 phase (p < 0.05). 100 μM Niclosamide suppressed the levels of EMT markers, Vimentin (p > 0.05) and ZEB1 (p < 0.05). Additionally, RT-qPCR results indicated a significant downregulation of CXCR4, ABCG1, ABCG2, MMP2, OCT4, CCND1, AXIN2, and LGR5 gene expressions following niclosamide treatment in both CD133+ and CD133- groups (p < 0.05). The increase in the Bax protein, a key player in apoptosis induction, along with the decrease in the anti-apoptotic protein Bcl-2, suggests the activation of cell death mechanisms. Notably, its targeted impact on the CD44+/CD24- population suggests that niclosamide could enhance the sensitivity of CSCs to treatment, thereby preventing tumor recurrence.