Abstract
Benzyl isothiocyanate (BITC) is a naturally-occurring isothiocyanate derived from cruciferous vegetables. BITC has been reported to inhibit the proliferation of various cancer cells, which is believed to be important for the inhibition of tumorigenesis. However, the detailed mechanisms of action remain unclear. In this study, we employed a budding yeast Saccharomyces cerevisiae as a model organism for screening. Twelve genes including MTW1 were identified as the overexpression suppressors for the antiproliferative effect of BITC using the genome-wide multi-copy plasmid collection for S. cerevisiae. Overexpression of the kinetochore protein Mtw1 counteracts the antiproliferative effect of BITC in yeast. The inhibitory effect of BITC on the proliferation of human colon cancer HCT-116 cells was consistently suppressed by the overexpression of Mis12, a human orthologue of Mtw1, and enhanced by the knockdown of Mis12. We also found that BITC increased the phosphorylated and ubiquitinated Mis12 level with consequent reduction of Mis12, suggesting that BITC degrades Mis12 through an ubiquitin-proteasome system. Furthermore, cell cycle analysis showed that the change in the Mis12 level affected the cell cycle distribution and the sensitivity to the BITC-induced apoptosis. These results provide evidence that BITC suppresses cell proliferation through the post-transcriptional regulation of the kinetochore protein Mis12.
Highlights
Naturally-occurring isothiocyanates (ITCs), such as benzyl ITC (BITC), phenethyl ITC (PEITC) and sulforaphane (SFN) derived from cruciferous vegetables, have been demonstrated to block the tumor formation initiated by chemicals in experimental animals, and the dietary consumption of ITCs has been shown to strongly correlate with the reduced risk of various cancers in humans[1,2,3,4]
To determine the concentration of Benzyl isothiocyanate (BITC) for the yeast screening, we examined the effect of BITC on the yeast cell growth by calculating the maximum growth rate in the yeast BY4741 strain
We originally developed the yeast screening system as a reliable and convenient tool to identify overexpression suppressors for the antiproliferative effect of BITC
Summary
Naturally-occurring isothiocyanates (ITCs), such as benzyl ITC (BITC), phenethyl ITC (PEITC) and sulforaphane (SFN) derived from cruciferous vegetables, have been demonstrated to block the tumor formation initiated by chemicals in experimental animals, and the dietary consumption of ITCs has been shown to strongly correlate with the reduced risk of various cancers in humans[1,2,3,4]. The yeast screening system would be especially useful for the identification of target molecules contributing to the antiproliferation by ITCs, because ITCs exert an antiproliferative effect in yeast as well as in human cancer cells[18], and antiproliferative agents often target the components of cell division and DNA repair machineries which are highly conserved between humans and yeast. One of the approaches to identify small-molecule targets is a multi-copy suppression screening for genes that provide resistance to a drug on overexpression. This screening is based on the principle that cells overexpressing a small-molecule target should tolerate the higher levels of the drug[19]. Our data indicated that the proteasome-dependent decrease in Mis[12] induces G2/M delay and enhances the BITC-induced apoptosis, which contributes to the suppression of cancer cell proliferation by BITC
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