Possible Contribution of Zerumbone-Induced Proteo-Stress to Its Anti-Inflammatory Functions via the Activation of Heat Shock Factor 1.

Yoko Igarashi,Akira Murakami,Kazuhiro Irie,Kohta Ohnishi

doi:10.1371/journal.pone.0161282

Abstract

Zerumbone is a sesquiterpene present in Zinger zerumbet. Many studies have demonstrated its marked anti-inflammatory and anti-carcinogenesis activities. Recently, we showed that zerumbone binds to numerous proteins with scant selectivity and induces the expression of heat shock proteins (HSPs) in hepatocytes. To dampen proteo-toxic stress, organisms have a stress-responsive molecular machinery, known as heat shock response. Heat shock factor 1 (HSF1) plays a key role in this protein quality control system by promoting activation of HSPs. In this study, we investigated whether zerumbone-induced HSF1 activation contributes to its anti-inflammatory functions in stimulated macrophages. Our findings showed that zerumbone increased cellular protein aggregates and promoted nuclear translocation of HSF1 for HSP expression. Interestingly, HSF1 down-regulation attenuated the suppressive effects of zerumbone on mRNA and protein expressions of pro-inflammatory genes, including inducible nitric oxide synthase and interlukin-1β. These results suggest that proteo-stress induced by zerumbone activates HSF1 for exhibiting its anti-inflammatory functions.

Highlights

Zerumbone is a sesquiterpenoid present in the subtropical ginger Zingiber zerumbet Smith, a plant that has been traditionally utilized in Southeast Asia for various purposes, including antiinflammatory medicine
Zerumbone in 5% ethyl acetate fraction was recrystallized from methanol Abs were obtained from the following sources: mouse anti-ubiquitin, rabbit anti-Heat shock factor 1 (HSF1), and anti-rabbit horseradish peroxidase (HRP)-linked IgG Abs were from Cell Signaling Technology (Beverly, MA); mouse anti-HSP70 Ab was from Enzo Biochem Inc. (New York, NY); mouse anti-α-tubulin Ab was from EMD Bioscience (La Jolla, CA); goat anti-COX2, rabbit anti-inducible nitric oxide synthase (iNOS), and goat anti-lamin B Abs were from Santa Cruz Biotechnology (Santa Cruz, CA); and HRP-conjugated anti-mouse and HRP-conjugated anti-goat Abs were from DAKO (Tokyo, Japan). siRNAs and LipofectamineTM RNAiMAX were purchased from Invitrogen (Carlsbad, CA)
To confirm its reactivity to cellular proteins in RAW264.7 mouse macrophages, we treated those cells with the vehicle alone or zerumbone for 6 hours, cell lysates were subjected to western blotting with an Ab against zerumbone-adducts

Summary

Introduction

Zerumbone is a sesquiterpenoid present in the subtropical ginger Zingiber zerumbet Smith, a plant that has been traditionally utilized in Southeast Asia for various purposes, including antiinflammatory medicine. Recent in vitro and in vivo studies have revealed several biological properties of this phytochemical, e.g., inhibitory effects toward free radical generation, expression of pro-inflammatory genes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and carcinogenesis [1,2,3,4,5,6,7]. The underlying molecular mechanisms of its cancer preventive activity have not been fully elucidated, we previously reported that zerumbone activated nuclear factor erythroid 2-related factor-2 (Nrf2) presumably by binding to PLOS ONE | DOI:10.1371/journal.pone.0161282. The underlying molecular mechanisms of its cancer preventive activity have not been fully elucidated, we previously reported that zerumbone activated nuclear factor erythroid 2-related factor-2 (Nrf2) presumably by binding to PLOS ONE | DOI:10.1371/journal.pone.0161282 August 18, 2016

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