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

Figure 5

The expression levels of NOX genes and enzymes in breast cancer and CSCs and the effect of the cholesterol scavenger MβCD on DHTS-induced mammosphere formation inhibition. The expression levels of NOX gene transcripts in MCF-7 and MDA-MB-231 cells were evaluated using specific primers: NOX isoforms, NOX1–5 and DUOX1–2 primers (a). Mammospheres from MCF-7 and MDA-MB-231 cells were treated with 1 μM DHTS, and the expression levels of NOX2 and NOX5 were evaluated by immunoblot analysis (b). Effect of NOX2 and NOX5 protein on mammosphere formation using siRNA knockdown of NOX2 and NOX5. The mammospheres derived from siRNA-treated cells were cultured for 7 days. Representative images of colonies were recorded. The data shown represent of three independent experiments. vs. the DMSO-treated control. NOX2 and NOX5 protein levels were measured by Western blot analyses. All immunoblots are representative of more than 3 independent experiments (c and d). Effect of MβCD on mammosphere formation. Mammospheres from MDA-MB-231 cells were treated with 1 mM and 5 mM MβCD. Mammosphere formation efficiency was assayed using the control as an MβCD-untreated mammosphere (e). Effect of the cholesterol scavenger MβCD on DHTS-induced mammosphere formation inhibition. The dissociated MDA-231-MB cells were seeded in 6-well plates on CSC media and treated with the indicated concentrations of DHTS and DMSO for 7 days. Representative images of colonies were recorded. The data shown represent of three independent experiments. vs. the DMSO-treated control (f). Effect of MβCD on NOX protein depletion in the mammospheres. The mammospheres were treated with 0.5 and 1 mM of MβCD for 1 day. Depletion of NOX2 and NOX5 protein was measured by Western blot analyses. All immunoblots are representative of more than 3 independent experiments. (h) Localization of ROS in the cancer cells with/without DHTA (1 μM). ROS were labeled in MDA-MB-231 treated with DHTS or DMSO for 1 day using fluorogenic probe CellROX Green Reagent (5 μmol/L, final concentration). The images were obtained under a phase-contrast fluorescence microscope (Lionheart FX live cell imager, Biotek, Winooski, VT, USA) at 4x magnification and are representative mammospheres (scale ). Nucleus is blue, and ROS is green. Arrows in lower panels indicated ROS at the plasma membrane (g).

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)