Oridonin confers protection against arsenic-induced toxicity through activation of the Nrf2-mediated defensive response

Abstract

Background: Groundwater contaminated with arsenic imposes a big challenge to human health worldwide. Using natural compounds to subvert the detrimental effects of arsenic represents an attractive strategy. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a critical regulator of the cellular antioxidant response and xenobiotic metabolism. Recently, activation of the Nrf2 signaling pathway has been reported to confer protection against arsenic-induced toxicity in a cell culture model.

Objectives: The goal of the present work was to identify a potent Nrf2 activator from plants as a chemopreventive compound and to demonstrate the efficacy of the compound in battling arsenic-induced toxicity.

Results: Oridonin activated the Nrf2 signaling pathway at a low subtoxic dose and was able to stabilize Nrf2 by blocking Nrf2 ubiquitination and degradation, leading to accumulation of the Nrf2 protein and activation of the Nrf2-dependent cytoprotective response. Pretreatment of UROtsa cells with 1.4 muM oridonin significantly enhanced the cellular redox capacity, reduced formation of reactive oxygen species (ROS), and improved cell survival after arsenic challenge.

Conclusions: We identified oridonin as representing a novel class of Nrf2 activators and illustrated the mechanism by which the Nrf2 pathway is activated. Furthermore, we demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to protect humans from various environmental insults that may occur daily.

Keywords: ARE; Keap1; Nrf2; antioxidant responsive element; antitumor; arsenic; chemoprevention; diterpenoid; oridonin; oxidative stress; rubescensin.

Figure 1

(A) Structure of the diterpenoid oridonin. (B, C) Luciferase reporter gene assays in MDA-MB-231 cells expressing ARE-luciferase. (B) Luciferase activity showing oridonin as an Nrf2 activator using a high-throughput screening system. The stable MDA-MB-231 cells expressing ARE-luciferase were seeded in 96-well plates; cells were grown to 90% confluence and treated with oridonin for 24 hr before analysis of luciferase activity. (C) Luciferase activity in MDA-MB-231 cells cotransfected with a plasmid containing an ARE-luciferase reporter gene and a plasmid encoding renilla luciferase driven by the herpes simplex virus thymidine kinase promoter. The transfected cells were treated with oridonin for 24 hr prior to measurement of firefly and renilla luciferase activities in cell lysates. All luciferase reporter gene assays were run in triplicate and expressed as mean ± SD.

Figure 2

Effects of oridonin on MDA-MB-231 cells. (A) An aliquot of cell lysates from the dual luciferase reporter gene assay was subjected to immunoblot analysis with anti-Nrf2, anti-Keap1, and anti-β-actin. (B) Total cell lysates from MDA-MB-231 cells treated with oridonin for 24 hr were subjected to immunoblot analysis with anti-Nrf2, anti-Keap1, and anti-β-actin antibodies. (C) mRNA from similarly treated cells was extracted and reverse transcribed into cDNA prior to real-time PCR analysis for detection of mRNA for Nrf2 (top), NQO1 (center), and HO-1 (bottom).

Figure 3

Effects of oridonin on Nrf2 and Keap1 ubiquitination and protein half-life in MDA-MB-231 cells or UROtsa cells. Abbreviations: IB, immunoblot; IP, immunoprecipitation; t_1/2, half-life; Ub, ubiquitin. (A) MDA-MB-231 cells were cotransfected with expression vectors for HA-ubiquitin, a Gal4–Neh2 fusion protein, and Keap1; the transfected cells were left untreated or treated with 8.4 μM oridonin or 100 μM tBHQ for 4 hr, along with 10 μM MG132. Cells were lysed in 2% SDS and immediately heated. Anti-Gal4 (left) and anti-Keap1 (center) immunoprecipitates analyzed by immunoblot with anti-HA antibodies for detection of the ubiquitin-conjugated Neh2 or Keap1. (Right) Ubiquitination of endogenous Nrf2 assessed in UROtsa cells treated with DMSO, 8.4 μM oridonin, or 100 μM tBHQ for 4 hr, along with 10 μM MG132; Nrf2 was immunoprecipitated with an anti-Nrf2 antibody, and ubiquitinated Nrf2 was detected with an anti-ubiquitin antibody. (B) Protein half-life in MDA-MB-231 cells (left) and UROtsa cells (right) left untreated or treated with 8.4 μM oridonin for 4 hr. Cycloheximide (50 μM) was added to block protein synthesis. Cells were lysed at the indicated time points, and lysates were subjected for immunoblot analysis with anti-Nrf2 and anti-β-actin antibodies (top). Intensity of the bands was quantified using Quantity One software (bottom). In B, the bottom left and right graphs represent the bands in the top left and right, respectively.

Figure 4

Effect of oridonin on UROtsa cells. Abbreviations: AnnV, Annexin V; DCF, 2′,7′-dichlorodihydrofluorescein; PI, propidium iodide. (A) Immunoblot showing Nrf2, Keap1, and β-actin in UROtsa cells treated with oridonin for 24 hr; cell lysates were collected and subjected to immunoblot analysis with anti-Nrf2, anti-Keap1, and anti-β-actin antibodies. (B) Intracellular glutathione concentrations in UROtsa cells either untreated (control) or treated with 1.4 μM oridonin. Concentrations were measured using the QuantiChrom glutathione assay kit; values shown are the mean ± SD of experiments run in triplicate. (C) ROS analysis in UROtsa cells untreated or pretreated with oridonin for 24 hr and then further treated with As(III) or As(III) plus oridonin, respectively, for another 24 hr. ROS were measured by dichlorofluorescein/flow cytometry; values shown are the mean ± SD of experiments run in triplicate. (D) Cell survival in UROtsa cells untreated or pretreated with 1.4 μM oridonin for 24 hr and then treated with As(III) in the absence or presence of 1.4 μM oridonin for another 48 hr; values shown are the mean ± SD of experiments run in triplicate. (D, top) Cell survival measured by the MTT assay. (D, center, at right) Immunoblot analysis showing Nrf2 protein levels in UROtsa cells transfected with control siRNA or Nrf2-siRNA for 48 hr; Nrf2 protein levels were assessed by immunoblot analysis with an anti-Nrf2 antibody to con-firm knockdown of Nrf2 expression. (D, center) Cell survival in Nrf2-siRNA transfected cells at 48 hr posttransfection measured by the MTT assay; 200 cells in 35-mm plates were pretreated and cotreated in the same manner as in the MTT assay. (D, bottom) Cell survival measured by the colony formation assay. Values shown are the mean ± SD of experiments run in triplicate. (E) Apoptotic cell death in UROtsa cells untreated or pretreated with 1.4 μM oridonin for 24 hr and then treated with As(III) in the absence or presence of 1.4 μM oridonin for another 48 hr. Apoptotic cell death was detected using Annexin V-FITC and flow cytometry; the mean ± SD was calculated from experiments run in triplicate (center). (E, bottom) Apoptois in UROtsa cells grown on cover slides were pretreated and cotreated in the same way. Apoptotic cells were visualized by condensed nuclei using Hoechst staining; bars = 25 μm. The experiment was repeated, and similar results were obtained. *p < 0.05. **p < 0.01.