Figure 6
Sensitivity of transformed trastuzumab resistant PTEN− breast cancer to sulforaphane is mediated by inhibition of IL-6/NF-κB signaling loop.
(A) Immunofluorescent staining of NF-κB p65 subunit and DAPI in fixed BT474 PTEN− LTT cells exhibits primarily cytoplasmic staining (left). Translocation of p65 from cytoplasm to nucleus, induced using 50 ng/ml TNF-α, is prevented by pretreatment with SF in a dose dependent manner (right three panels). Red arrows indicate boarder between cytoplasm and nucleus of a representative cell. Original images were obtained using a 20× microscope objective. (B) BT474 PTEN− LTT and BT474 cell lines were transfected with NF-κB luciferase reporter. Luciferase reporter activity was obtained in the absence or presence of 50 ng/ml TNF-α in combination with increasing concentrations of SF. (C) mRNA expression of endogenous inflammatory cytokine IL-6 normalized to GAPDH as identified by real time PCR in the presence of increasing concentration of SF. (D) Quantitative ELISA identifying protein level endogenous NF-κB target protein IL-6 when cell lines are incubated with increasing concentrations of SF for 72 hr. (B–D) N = 3. Data are shown as averages ± SD. *p ≤ 0.05. #p ≤ 0.01.
