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

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 Mmol/L, whereas SFN induced at 40 Mmol/L but not at 20 Mmol/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. [Cancer Res 2007;67(13):6409-16]

Covalent Binding to Tubulin by Isothiocyanates

“…Previously we showed that proteins are the major target of PEITC and SFN in cells and concluded that direct binding of ITCs to intracellular proteins may trigger apoptosis (6). In this study, we identified tubulin as one of these in vivo targets for ITC binding, demonstrated covalent binding of BITC, PEITC, and SFN to tubulin, and showed that this binding correlated well with their ability to induce mitosis arrest and apoptosis.…”

“…In fact, the facile reaction between ITCs and cellular thiols is a driving force for enriching intracellular ITC levels up to millimolar levels (4). Recently, we have shown, using 14 C-PEITC and 14 C-SFN, that the initial conjugation predominantly occurs with cellular GSH, but with increasing time, protein binding gradually becomes the major reaction, at least in part because of dissociation of ITC from ITC-GSH conjugates (6). Eventually, proteins are the major binding sites of ITCs inside cells; for example, PEITC-protein conjugates account for 87% of total cellular uptake after 4 h of treatment.…”

Effective elimination of fludarabine-resistant CLL cells by PEITC through a redox-mediated mechanism

“…Recent studies suggest that protein modification and activation of ERK and JNK pathways may contribute to PEITC cytotoxicity. 35,36 Glutathione is a key regulator of cell death and survival of cancer cells, including leukemia cells. 37,38 PEITC is known to conjugate with GSH, leading to its exportation and depletion of cellular glutathione.…”

Are isothiocyanates potential anti-cancer drugs?

“…Recently, they have been implicated in mediating antitumor activities. These compounds have inhibitory effects on the growth of several types of cultured cancer cells, including leukemia [43,44] , prostate cancer [45] , breast cancer [46] , lung cancer [47,48] , cervical cancer [49] , and colorectal cancer [50] .…”