The Role of Protein Binding in Induction of Apoptosis by Phenethyl Isothiocyanate and Sulforaphane in Human Non–Small Lung Cancer Cells

Lixin Mi,Radoslav Goldman,Thomas P Conrads,Xiantao Wang,Daniel T Saha,Fung-Lung Chung,Sudha Govind,Timothy D Veenstra,Brian L Hood

doi:10.1158/0008-5472.can-07-0340

Abstract

Induction of apoptosis underlies a mechanism for inhibiting tumorigenesis by phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). However, the upstream events by which isothiocyanates (ITC) induce apoptosis have not been fully investigated. As electrophiles, ITCs could trigger apoptosis by binding to DNA or proteins or by inducing oxidative stress. To better understand the molecular mechanisms of apoptosis by ITCs, we examined, as a first step, the role of these events in human non-small lung cancer A549 cells. PEITC was a more potent inducer than SFN; it induced apoptosis at 20 micromol/L, whereas SFN induced at 40 micromol/L but not at 20 micromol/L. To study binding with cellular proteins and DNA, cells were treated with (14)C-ITCs; the initial protein binding by PEITC was almost 3-fold than that of SFN. The binding by PEITC increased with time, whereas binding by SFN remained low. Therefore, 4 h after incubation proteins became the predominant targets for PEITC with a 6-fold binding than that of SFN. To characterize the chemical nature of binding by the ITCs, we used bovine serum albumin (BSA) as a surrogate protein. PEITC also modified BSA covalently to a greater extent than SFN occurring exclusively at cysteine residues. Surprisingly, neither PEITC nor SFN bound to DNA or RNA at detectable levels or caused significant DNA strand breakage. The levels of oxidative damage in cells, measured as reactive oxygen species, 8-oxo-deoxyguanosine, and protein carbonyls formation, were greater in cells treated with SFN than PEITC. Because PEITC is a stronger inducer of apoptosis than SFN, these results indicate that direct covalent binding to cellular proteins is an important early event in the induction of apoptosis by the ITCs.

Highlights

Cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are versatile cancer chemopreventive compounds showing activity to inhibit tumor growth during initiation, promotion, and progression phases [1]
The results indicate that covalent binding to cellular proteins, not DNA or oxidative damage, is likely an important early chemical event leading to the induction of apoptosis
Whereas PEITC and SFN induce apoptosis in cultured cancer cells [2,3,4,5,6,7,8,9,10] and, in a few cases, in tissues obtained from animals where tumor inhibition was observed [5, 7], little is known about the early chemical events that initiate apoptotic responses in cells by these compounds

Summary

Introduction

Cruciferous vegetable-derived phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are versatile cancer chemopreventive compounds showing activity to inhibit tumor growth during initiation, promotion, and progression phases [1]. Evidence obtained from both in vitro cell culture and in vivo animal tissues. PEITC and SFN may covalently bind to cellular proteins, DNA, and RNA These bindings are likely to underlie their biological activities, only limited studies exist on the relationship between the interactions with the macromolecules in cells and their biological functions. Studies showed that SFN induces phase II enzymes through its binding to the cysteine residues of the Kelch-like ECH-associated protein 1 Studies showed that SFN induces phase II enzymes through its binding to the cysteine residues of the Kelch-like ECH-associated protein 1 (Keap; refs. 15–17)

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Conclusion