Dihydrotanshinone-Induced NOX5 Activation Inhibits Breast Cancer Stem Cell through the ROS/Stat3 Signaling Pathway

Figure 7

Effects of DHTS on the Stat3 pathway activation in the mammospheres and CSC loads in breast cancer. The activation of Stat3 and NF-κB was determined in the mammospheres with antibodies to pStat3, Stat3, p65, and lamin b. DHTS decreased the nuclear pStat3 level in the mammospheres (a). EMSA assay using the mammosphere lysates treated with DHTS. Nuclear lysates were incubated with a Stat3 probe and separated by 6% PAGE. Lane 1: probe only; lane 2: nuclear extracts with probe; lane 3: DHTS-treated nuclear extracts with probe; lane 4: self-competition; lane 5: nuclear extracts incubated with mutated Stat3 probe (b). Immunoblot of extracellular fluids from mammosphere cultured solution with an anti-IL-6 and the number of cancer cell/well as internal control (c). The effects of DHTS and NAC on pStat3 phosphorylation. DHTS-induced dephosphorylation of pStat3 was ameliorated by NAC (d). Transcriptional levels of the CSC markers Nanog, Sox2, Oct4, c-Myc, and CD44 were determined in DHTS-treated mammospheres using gene-specific primers and real-time RT-PCR. β-Actin acts as an internal control (e). Effect of DHTS on mammosphere growth. DHTS prevented mammosphere growth. The DHTS-treated mammospheres for 2 days were dissociated into single cells and plated in 6 cm dishes with equal numbers of cells. Twenty-four hours after plating, the cells were counted. At 2 and 3 days after, the cells were counted in triplicate and plotted as the mean value. The data shown represent of three independent experiments. vs. the control (f). The proposed model for CSC death by DHTS. DHTS induced calcium release and activated NOX5, and then NOX5 produced ROS. ROS induced dephosphorylation of Stat3 and reduced secreted IL-6. The secreted IL-6 can convert NSCCs to CSCs and regulate dynamic equilibrium from NSCCs to CSCs. DHTS deregulates equilibrium from NSCCs to CSCs through dephosphorylation of Stat3 and deregulation of IL-6 and kills CSCs (g).

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