APOPTOSIS AND ALTERED REDOX STATE INDUCED BY CAFFEIC ACID PHENETHYL ESTER (CAPE) IN TRANSFORMED RAT FIBROBLAST CELLS

Caffeic acid phenethyl ester (CAPE), which is derived from the propoli s of bee hives, was shown previously to block tumor promoter- and carc inogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechani sms of CAPE-induced differential cytotoxicity, nontumorigenic rat embr yo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells ( Wt3A) were used. As shown by nucleosomal-length DNA degradation, morph ological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivarian t flow cytometry, cell death induced by CAFE in the transformed Wt3A c ells was apoptosis. Under the same CAFE treatment condition, CREF cell s transiently growth arrested. Both CREF and Wt3A cells were radioresi stant, suggesting deficiencies in the proteins controlling the G(1) ch eckpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the re dox state of the cells, Depletion of cellular glutathione (GSH) with b uthionine sulfoximine before CAPE treatment caused CREF sensitive to C APE-induced cell death. GSH levels were also determined in CAPE-treate d CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When t he GSH levels were increased in Wt3A cells by treatment with the reduc ing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells wer e partially rescued. Furthermore, Bcl2, which protects cells from oxid ative stress, had a protective effect against CAPE-induced apoptosis i n Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidan t, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H 2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3 A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further provi ng that Wt3A cells were more sensitive than CREF cells to oxidative st ress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determin ed by the loss of normal redox state regulation in transformed cells.