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

¹ Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia and ² Laboratory of Proteomics and Analytical Technologies, Science Applications International Corporation-Frederick, National Cancer Institute-Frederick, Frederick, Maryland

Requests for reprints: Fung-Lung Chung, Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3800 Reservoir Road, LL 128A, Box 571465, Washington, DC 20057. Phone: 202-687-3021; Fax: 202-687-1068; E-mail: flc6{at}georgetown.edu.

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 µmol/L, whereas SFN induced at 40 µmol/L but not at 20 µmol/L. To study binding with cellular proteins and DNA, cells were treated with ¹⁴C-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. [Cancer Res 2007;67(13):6409–16]

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