DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

Abstract

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

Keywords: Nrf2 pathway; Wasabi; gene profile analysis; isothiocyanates; sulforaphane.

Figure 1

Chemical structures of Wasabi-derived ITCs used in the study: (A) 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC, usually called sulforaphane, SFN), (B) 6-(methylsufinyl)hexyl isothiocyanate (6-MSITC), and (C) 6-(methylthio)hexyl isothiocyanate (6-MTITC).

Figure 2

Comparative canonical pathway analyses of differentially expressed genes in IMR-32 neuron cells stimulated with Wasabi ITCs. Differentially upregulated and downregulated genes were evaluated for canonical pathway analyses using IPA software as elaborated in the “Materials and Methods” section. Only five of the top most significant pathways with respect to Wasabi ITCs are shown here. Note: List of corresponding significant pathways is indicated below and their respective level of significance (P < 0.05) denoted by the length of the bars.

Figure 3

Validation of differentially expressed genes in Wasabi ITC-treated IMR-32 cells from DNA microarray analysis by real-time PCR. DNA microarray results were compared to real-time PCR results for selected genes. Real-time PCR was performed using DyNAmo™ SYBR^® Green 2-Step qRT-PCR Kit as described in the “Materials and Methods” section. Fold changes represented the ratio between the treated samples values to that of the untreated samples. Expression changes are depicted as fold change (y-axis). Gene symbols are shown below.

Figure 4

Effect of 6-MSITC on Nrf2 level and Nrf2-mediated induction of typical proteins. (A) IMR-32 cells were treated with 0−20 μM of 6-MSITC for 12 hours. (B) IMR-32 cells were treated with 10 μM of 6-MSITC for 0−24 hours. Nrf2, Keap1, NQO1, TXNRD1, AKR1C1, AKR1C3, and GAPDH were detected using Western blot analysis with their respective antibodies. The induction fold of the protein was calculated as the intensity of the treatment relative to that of control normalized to GAPDH by densitometry. The blots shown are the examples of three separate experiments.

Figure 5

Effect of 6-MSITC on the stability of Nrf2. IMR-32 cells were pretreated with or without 10 μM of 6-MSITC for 3 hours, followed by exposure with 5 mg/mL of CHX for 0–1 hours. Nrf2, Keap1 and GAPDH were detected by Western blot analysis with their respective antibodies. Histograms show the densitometric analysis of Nrf2 compared with the control.

Figure 6

Proposed mechanisms for the neuroprotective effects by Wasabi-derived 6-MSITC in IMR-32 cells. Wasabi-derived ITCs activate Nrf2-mediated oxidative stress pathway and subsequently induce the expression of antioxidant proteins/enzymes to exert the neuroprotective effects. The mRNA and protein of marker in solid box were confirmed by RT-PCR and Western blotting.