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Caspase-Dependent Apoptosis Induction by Phenethyl Isothiocyanate, a Cruciferous Vegetable-Derived Cancer Chemopreventive Agent, Is Mediated by Bak and Bax

Authors' Affiliations: Departments of ¹ Pharmacology, and ² Urology, and University of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania

Requests for reprints: Shivendra V. Singh, Hillman Cancer Center Research Pavilion, Suite 2.32A, University of Pittsburgh Cancer Institute, 5117 Centre Avenue, Pittsburgh, PA 15213. Phone: 412-623-3263; Fax: 412-623-7828; E-mail: singhs{at}upmc.edu.

Purpose: The present study was undertaken to gain insights into the molecular mechanism of apoptosis induction by phenethyl isothiocyanate (PEITC) using prostate cancer cell lines derived from transgenic adenocarcinoma mouse prostate (TRAMP) mice (TRAMP-C1 and TRAMP-C2).

Experimental Design and Results: The viability of TRAMP-C1 and TRAMP-C2 cells was reduced significantly in the presence of PEITC in a concentration-dependent manner as determined by sulforhodamine B and trypan blue dye exclusion assays. Treatment of TRAMP-derived cells with PEITC revealed features characteristic of apoptosis induction, including appearance of subdiploid cells (determined by flow cytometry), cytoplasmic histone-associated DNA fragmentation (determined by an ELISA assay), and cleavage of caspase-3 (determined by immunoblotting). The PEITC-induced apoptosis in TRAMP-derived cells was associated with a marked increase in the level of proapoptotic protein Bak and/or a decrease in the levels of antiapoptotic protein Mcl-1 or Bcl-x_L and disruption of mitochondrial membrane potential. The SV40 immortalized mouse embryonic fibroblasts derived from Bak and Bax double knockout mice were significantly more resistant to PEITC-induced DNA fragmentation compared with wild-type or Bak^–/– mouse embryonic fibroblasts. The PEITC-induced apoptosis in both cell lines was significantly attenuated in the presence of caspase inhibitors zVAD-fmk, zLEHD-fmk, and zIETD-fmk. Oral administration of PEITC (9 or 12 µmol PEITC/d, Monday-Friday) significantly retarded growth of TRAMP-C1 xenografts in nude mice without causing weight loss or any other side effects.

Conclusion: The results of the present study indicate that caspase-dependent apoptosis by PEITC is mediated by Bak and Bax proteins.

Key Words: Isothiocyanates • Prostate Cancer • Bcl-2 • Apoptosis • Chemoprevention

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