J Korean Med Sci.  2011 Nov;26(11):1474-1482. 10.3346/jkms.2011.26.11.1474.

Sulforaphane Induces Antioxidative and Antiproliferative Responses by Generating Reactive Oxygen Species in Human Bronchial Epithelial BEAS-2B Cells

• Lee YJ
• Lee SH

• KMID: 1123448
• DOI: http://doi.org/10.3346/jkms.2011.26.11.1474

Abstract

Sulforaphane (SFN) is a naturally occurring compound which is known to induce the phase II antioxidant genes via Nrf2 activation, although the underlying mechanism has not been fully elucidated. In this study, we investigated Nrf2 induction in response to SFN in human bronchial epithelial BEAS-2B cells and determined the signaling pathways involved in this process. SFN treatment reduced cell viability. Prior to cell death, intracellular reactive oxygen species (ROS) were generated at a high rate within a minute of commencing SFN treatment. Pretreatment with antioxidant N-acetylcysteine (NAC) blocked SFN-induced decrease in cell growth. Erk1/2 was activated within 30 min of SFN addition, whereas Akt phosphorylation did not significantly change until the first 8 hr after SFN treatment but then became substantially low until 48 hr. Inhibition of Erk1/2 phosphorylation attenuated SFN-induced loss of cell viability. Nrf2 protein levels in both nuclear and whole cell lysates were increased by SFN treatment, which was dependent on ROS production. Knockdown of Nrf2 with siRNA attenuated SFN-induced heme oxygenase-1 (HO-1) up-regulation. Induction of the Nrf2/HO-1 after SFN treatment was potently suppressed by pretreatment with NAC. Overall, our results indicate that SFN mediates antioxidative and antiproliferative responses by generating ROS in BEAS-2B cells.

Keyword

Sulforaphane; Nrf2; Heme Oxygenase-1; Reactive Oxygen Species; BEAS-2B Cells; Oxidative Stress

MeSH Terms

Acetylcysteine/pharmacology
Anticarcinogenic Agents/pharmacology
Antioxidants/*pharmacology
Bronchi/cytology/*drug effects/metabolism
Cell Line
Cell Proliferation/*drug effects
Epithelial Cells/drug effects/metabolism
Extracellular Signal-Regulated MAP Kinases/metabolism
Free Radical Scavengers/pharmacology
Heme Oxygenase-1/biosynthesis
Humans
NF-E2-Related Factor 2/biosynthesis/genetics
Oxidative Stress/drug effects
Proto-Oncogene Proteins c-akt/metabolism
RNA Interference
RNA, Small Interfering
Reactive Oxygen Species/*metabolism
Respiratory Mucosa/cytology/*drug effects/metabolism
Signal Transduction/drug effects
Thiocyanates/*pharmacology

Figure

• 

  Fig. 1 Effects of SFN on cell proliferation and intracellular ROS levels. (A) BEAS-2B cells were treated with various concentrations (0-20 µM) of SFN for 24, 48, and 72 hr. After XTT assay, absorbance values were measured spectrophotometrically at 450 nm. (B) Cells were treated with SFN (10 µM) for the indicated time. The level of ROS generated was then measured by using the redox-sensitive dye DCF-DA. (C) Cells were treated with NAC (5 mM) for 1 hr prior to incubation with various concentrations (0-20 µM) of SFN for 72 hr. The percentage of viable cells was then determined by XTT assay. Error bars represent the mean ± SEM for three independent experiments. In panel C, values with different letters were significantly different from each other (P < 0.05).*P < 0.05 compared to control.

• 

  Fig. 2 SFN-induced phosphorylation of Akt and Erk1/2. (A) BEAS-2B cells were treated with SFN (10 µM) for the indicated time. (B) Cells were treated with various concentrations (0-20 µM) of SFN for 2 hr or 24 hr. (C) Cells were treated with or without NAC (5 mM) for 1 hr prior to incubation with the indicated concentrations of SFN for 2 hr or 24 hr. Cell lysates were analyzed by Western blot analysis with anti-p-Akt and anti-p-Erk antibodies. The blots were then stripped and re-probed with anti-Akt and anti-Erk antibodies as loading controls. (D) Cells were treated with or without Ly294002 (20 µM) and PD98059 (50 µM) for 1 hr prior to incubation with the indicated concentrations of SFN for 72 hr. The percentage of viable cells was then determined by XTT assay. In the box, values with different letters were significantly different from each other (P < 0.05). *P < 0.05 compared to control.

• 

  Fig. 3 Effects of various chemical inhibitors on SFN-induced Nrf2 activation. (A) BEAS-2B cells were treated with NAC (5 mM) and cycloheximide (CHX, 200 nM) for 1 hr prior to incubation with SFN (10 µM) for 2 hr. Nuclear and cytoplasmic extracts were then analyzed by Western blot analysis. (B) Cells were treated with Ly294002 (20 µM), PD98059 (50 µM), NAC (5 mM) for 1 hr prior to incubation with the indicated concentrations of SFN for 2 hr. Whole cell extracts were then analyzed by Western blot analysis. The blot was then stripped and re-probed with an anti-β-actin antibody as a loading control. The normalized intensity of Nrf2 versus β-actin is presented. Error bars represent the mean ± SEM for three independent experiments. *P < 0.05 compared to control.

• 

  Fig. 4 Nrf2-dependent HO-1 induction. (A) BEAS-2B cells were treated with SFN (10 µM) for the indicated time. (B) Cells were transfected with 10 nM Nrf2-targeting siRNA, or Stealth RNAi control for 24 and 48 hr. (C) Cells were transfected with 10 nM Nrf2-targeting siRNA or Stealth RNAi control for 24 hr prior to incubation with SFN (10 µM) for 24 hr. Cell lysates were analyzed by Western blot analysis with ant-Nrf2 and anti-HO-1 antibodies. The blots were then stripped and re-probed with anti-β-actin as a loading control.

• 

  Fig. 5 Effects of various chemical inhibitors on SFN-induced up-regulation of HO-1 expression. BEAS-2B cells were treated with NAC (5 mM), Ly294002 (20 µM), and PD98059 (50 µM) for 1 hr prior to incubation with the indicated concentrations of SFN for 24 hr. Cell lysates were analyzed by Western blot analysis with anti-Nrf2 and anti-HO-1 antibodies. The blots were then stripped and re-probed with anti-β-actin antibody as a loading control. The normalized intensity of Nrf2 or HO-1 vs. β-actin is presented. Error bars represent the mean ± SEM for three independent experiments. *P < 0.05 compared to control.

• 

  Fig. 6 Comparison of the relative changes in intracellular levels of ROS, Nrf2, HO-1, p-Akt, and p-Erk1/2 after SFN (10 µM) treatment. For each sample, the expression of Nrf2, HO-1, p-Akt, and p-Erk1/2 were quantified by densitometry, and the ratios Nrf2:β-actin, HO-1:β-actin, p-Akt:Akt, and p-Erk1/2:Erk1/2 were calculated. Induction levels are depicted as absolute fold change compared to untreated controls (Y-axis). *P < 0.05 compared to control.

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