Disintegration of microtubules in Arabidopsis thaliana and bladder cancer cells by isothiocyanates.

Anders Ã˜Verby,Atle M Bones,Ole P Thangstad,Mette S Bã¦Vre

doi:10.3389/fpls.2015.00006

Anders Ã˜Verby, Atle M Bones + Show 2 more

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https://doi.org/10.3389/fpls.2015.00006

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Abstract

Isothiocyanates (ITCs) from biodegradation of glucosinolates comprise a group of electrophiles associated with growth-inhibitory effects in plant- and mammalian cells. The underlying modes of action of this feature are not fully understood. Clarifying this has involved mammalian cancer cells due to ITCs' chemopreventive potential. The binding of ITCs to tubulins has been reported as a mechanism by which ITCs induce cell cycle arrest and apoptosis. In the present study we demonstrate that ITCs disrupt microtubules in Arabidopsis thaliana contributing to the observed inhibited growth phenotype. We also confirmed this in rat bladder cancer cells (AY-27) suggesting that cells from plant and animals share mechanisms by which ITCs affect growth. Exposure of A. thaliana to vapor-phase of allyl ITC (AITC) inhibited growth and induced a concurrent bleaching of leaves in a dose-dependent manner. Transcriptional analysis was used to show an upregulation of heat shock-genes upon AITC-treatment. Transgenic A. thaliana expressing GFP-marked α-tubulin was employed to show a time- and dose-dependent disintegration of microtubules by AITC. Treatment of AY-27 with ITCs resulted in a time- and dose-dependent decrease of cell proliferation and G2/M-arrest. AY-27 transiently transfected to express GFP-tagged α-tubulin were treated with ITCs resulting in a loss of microtubular filaments and the subsequent formation of apoptotic bodies. In conclusion, our data demonstrate an ITC-induced mechanism leading to growth inhibition in A. thaliana and rat bladder cancer cells, and expose clues to the mechanisms underlying the physiological role of glucosinolates in vivo.

Highlights

Occurring isothiocyanates (ITCs) are plant phytochemicals associated with cytotoxicity in several cell types including plant- and mammalian cells
Microtubule filaments were significantly reduced but still detectable when allyl ITC (AITC)-concentration was decreased to 1.5 M, and when 0.5 M AITC was applied the filaments were seemingly unaffected after 2 min of exposure (Figure 3B)
In order to show that the breakdown of microtubules in A. thaliana was not restricted to AITC we repeated the experiments with butyl ITC (BuITC), benzyl ITC (BITC), and phenethyl ITC (PEITC)

Summary

Introduction

Occurring isothiocyanates (ITCs) are plant phytochemicals associated with cytotoxicity in several cell types including plant- and mammalian cells. These electrophilic compounds confer a reactive −N = C = S group linked to an R moiety influencing the potency. The preventive effect has been shown through epidemiological studies in which an inverse association between the intake of ITC-producing cruciferous vegetables and development of cancer in lung, breast, colon, prostate and bladder was observed (London et al, 2000; Seow et al, 2002; Ambrosone et al, 2004; Joseph et al, 2004; Brennan et al, 2005; Tang et al, 2008)

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