N-Acetylcysteine Conjugate of Phenethyl Isothiocyanate Enhances Apoptosis in Growth-Stimulated Human Lung Cells

Abstract

We previously showed that dietary treatment with the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) inhibited benzo(a)pyrene-induced lung tumorigenesis in A/J mice, and that tumor inhibition was associated with induction of activator protein-1 (AP-1) activity and stimulation of apoptosis in the lungs of mice. In the present study, we show that PEITC-NAC also induces apoptosis and AP-1 activity in human lung adenocarcinoma A549 cells, and that activation of AP-1 is important in PEITC-NAC induced apoptosis in these cells. PEITC-NAC induced AP-1 binding activity in A549 cells in a dose-and time-dependent manner; peak activity appeared at 10 Mmol/L after 24 hours. At that time, flow cytometric analysis showed a sub-G 1 peak, indicating that f4.5% of the cells had undergone apoptosis. When wild-type c-jun cDNA was transfected into A549 cells, PEITC-NAC-mediated apoptosis was greatly increased in the c-jun-transfected cells compared with the control vector-transfected cells, based on cell morphology and analysis of DNA fragmentation. Furthermore, cells that were pretreated with 100 nmol/L 12-O-tetradecanoyl phorbol-13-acetate, and then treated with 25 Mmol/L PEITC-NAC, underwent enhanced apoptosis compared with cells that were treated with PEITC-NAC alone; cells treated with 12-Otetradecanoyl phorbol-13-acetate alone showed active cell growth without apoptosis. Bivariate flow cytometric analysis of DNA strand breaks versus DNA content showed that apoptosis induced by PEITC-NAC occurred predominantly in the G 2 -M phase. These findings suggest that growth-stimulated cells with an elevated basal AP-1 activity, i.e., A549 cells transfected with wild-type c-jun or treated with a tumor promoter, were more sensitive to PEITC-NAC-mediated apoptosis. The observation that PEITC-NAC induces apoptosis predominantly in growth-promoted cells, such as neoplastic cells, suggests a selective mechanism by which PEITC-NAC inhibits lung carcinogenesis. (Cancer Res 2005; 65(18): 8538-47)

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

“…The lungs of ITC-treated mice show increased apoptotic cells and mitogen-activated protein kinase (MAPK) and activator protein 1 activities. The study of human non-small lung cancer A549 cells reinforced the in vivo observations that the apoptosis was induced by the PEITC conjugate, and the induction was significantly enhanced in the fast growing A549 cells transfected with c-jun or pretreated with 12-O-tetradecanoylphorbol-13-acetate (18).…”

Binding to Protein by Isothiocyanates: A Potential Mechanism for Apoptosis Induction in Human Nonsmall Lung Cancer Cells

“…The study of human nonsmall lung cancer A549 cells further reinforced the observations that apoptosis was induced by PEITC treatment, and the induction was significantly enhanced in the fast growing cells transfected with c-jun or in cells pretreated with 12-O-tetradecanoyl-phorbol-13-acetate (17). It has been reported that multiple and complex signaling pathways are associated with the apoptosis induced by ITCs (1,5,7,8,(18)(19)(20).…”

Covalent Binding to Tubulin by Isothiocyanates

“…ITCs are less potent disrupters of microtubule structure than clinical therapeutic drugs such as vinblastine and colchicine; however, this less potent effect may be desirable for agents designed for use in prevention or treatment of the earlier stages of cancer. Because tubulin is an essential protein for cell division, it is reasonable to speculate that ITCs may induce more cell growth arrest and apoptosis in fast-growing, initiated, pre-neoplastic, or cancer cells than in normal cells, as has been reported (32,33). However, additional studies are needed.…”