Figure 8

(a) Inhibition effect of compound C1 on primary, secondary, and tertiary mammosphere-forming units. In the absence of drug, the second and third passages derived from Complex C1-treated primary mammospheres yielded smaller numbers of spheres in comparison with the controls. The size of the mammospheres was estimated using V = (4/3) πR3. Complex C1 hinders mammosphere formation and prevents self-renewal of MDA-MB-231 primary, secondary, and tertiary mammosphere-forming units. (b and c) Aldefluor assay of MDA-MB-231 cancer stem cells: Single cells obtained from cell cultures were incubated for 50 minutes at 37 °C in Aldefluor assay buffer comprising an ADH substrate, BODIPY-aminoacetaldehyde (1 μmol/L per 1 × 10⁶ cells). (b) Cell population (R2) with high ALDH activity was informed to develop mammary stem/progenitor cells. (c) Quantitative analysis of the inhibitory effects of C1 on ALDH-positive cell populations MDA-MB-231. Cancer stem cells were treated with complex C1 at 1, 2, and 4 μg/mL concentrations for 4 days and were subjected to an Aldefluor assay and flow cytometry analysis. Complex C1 decreased the percentage of ALDH-positive cells. (d) Western blot analysis of the Wnt/β-catenin self-renewal pathway markers: Treatment with 2.5 μg/mL (IC₅₀) of compound C1 after 24, 48, and 72 hours increased protein expression levels of P-β-catenin and decreased β-catenin, cyclin D1, and P-GSK3β. The expression level of GSK3β was almost unchanged. The data represent the means ± standard deviation (SDs) of 3 independent tests. Statistical analysis is defined as significant if *P < 0.05.