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 (<i>p</i> < 0.05), as determined by Western Blot. The expression of genes associated with metastasis and cell migration (<i>CXCR4, MMP2, MMP9</i>), drug resistance (<i>ABCG1, ABCG2</i>), stemness (<i>OCT4, NANOG</i>) and cell cycle and proliferation (<i>CYCLIN D1</i>) was found to be significantly suppressed (<i>p</i> < 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 (<i>p</i> < 0.05). 100 μM Niclosamide suppressed the levels of EMT markers, Vimentin (<i>p</i> > 0.05) and ZEB1 (<i>p</i> < 0.05). Additionally, RT-qPCR results indicated a significant downregulation of <i>CXCR4, ABCG1, ABCG2, MMP2, OCT4, CCND1, AXIN2, and LGR5</i> gene expressions following niclosamide treatment in both CD133+ and CD133- groups (<i>p</i> < 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.