Bioactive food components prevent carcinogenic stress via Nrf2 activation in BRCA1 deficient breast epithelial cells

Abstract

Although BRCA1 is the most prevalent genetic factor in breast cancer, the pathologic mechanism of tumorigenesis caused by its deficiency has not been elucidated. We have previously demonstrated that BRCA1 can modulate responses to xenobiotic stress by regulating expression of genes involved in metabolic activation, detoxification and antioxidant reactions. In this study, we examined whether BRCA1 deficiency is more vulnerable to xenobiotic stress by employing an in vitro cell model system. Benzo[a]pyrene (B[a]P), used as a xenobiotic insult, increased intracellular reactive oxygen species (ROS) levels in breast epithelial cells. Accumulation of ROS upon B[a]P exposure was significantly augmented by abrogation of BRCA1 compared to the control. Overexpression of Nrf2 in BRCA1 deficient cells reduced elevated ROS to the control levels. Bioactive food components such as sulforaphane (SFN) and resveratrol (RSV) significantly reduced B[a]P-induced ROS accumulation regardless of BRCA1 presence. In addition, these bioactive food components increased Nrf2 levels and Nrf2 transcriptional activity, which led to attenuation of B[a]P-induced DNA damages. Likewise, incubation with bioactive food components reduced B[a]P-mediated DNA damage in BRCA1 deficient cells. In conclusion, we demonstrated that the lack of BRCA1 renders cells more susceptible to ROS-induced DNA damage, which may eventually result in tumorigenesis, and that administration of Nrf2-activating bioactive food components can reduce those risks.

Figure 1

ROS accumulation by B[a]P in BRCA1 defective cells. MCF10A cells pretreated with control or BRCA1 specific siRNA for 72 hrs were further incubated with B[a]P for 24 hrs. Then cellular ROS and NO levels were measured using (A) CM-H₂DCFDA, (B) Lucigenin, and (C) DAF-FM diacetate. * indicates p < 0.05 (control siRNA vs. BRCA1 siRNA treated cells).

Figure 2

Reduction of ROS accumulation after Nrf2 overexpression. MCF10A cells pretreated with control or BRCA1 specific siRNA for 72 hrs were transfected with pCDNA or Flag-Nrf2 vectors for 24 hrs. Then indicated concentrations of B[a]P were further incubated for 24 hrs. The level of cellular ROS and NO were measured using (A) CM-H₂DCFDA, (B) Lucigenin, and (C) DAF-FM diacetate. Open square, control-siRNA + pCDNA3; light gray square, control-siRNA + Flag-Nrf2; dark gray square, BRCA1-siRNA + pCDNA3; and black square, BRCA1-siRNA + Flag-Nrf2. (D) Expression levels of BRCA1 and Flag-Nrf2 after knockdown of BRCA1 and overexpression of Nrf2 were analyzed by Western blotting.

Figure 3

Nrf2 activation by bioactive food components. (A) Control or BRCA1 specific siRNA treated cells were transfected with the NQO1-ARE-Luc vector. Indicated concentrations of bioactive food components or chemopreventive agents were incubated for 24 hrs. Data show fold increases of luciferase activity compare to control. * indicates p < 0.05 (control vs. bioactive food components treated in control-siRNA treated cells), and ^# indicates p < 0.05 (control vs. bioactive food components treated in BRCA1-siRNA treated cells). (B) After treatment of 5 μM of SFN for indicated time, expression of Nrf2 target proteins were measured by Western blotting. (C) ROS levels were measured after knockdown of BRCA1 and overexpression of UGT1A1.

Figure 4

ROS accumulation after preincubation of bioactive food components. MCF10A cells pretreated with control or BRCA1 specific siRNA were incubated with indicated concentrations of SFN (A), RSV (B), OTP (C), and I3C (D) for 6 hrs then further incubated with B[a]P for 24 hrs. ROS accumulation was determined using CM-H₂DCFDA.

Figure 5

Reduction of DNA damage by bioactive food components. Control or BRCA1 specific siRNA treated cells were transfected with Nrf2 expression vector for 24 hrs or preincubated with SFN, RSV, or I3C for 6 hrs, then incubated with B[a]P for 24 hrs. DNA damages by B[a]P were determined by measuring 8-oxo-guanine levels. Protection of DNA damage by (A) Nrf2 overexpression, (B) SFN, (C) RSV, and (D) I3C were compared to each control.