Chiao C, Carothers A M, Grunberger D, Solomon G, Preston G A, Barrett J C
Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709, USA.
Cancer Res. 1995 Aug 15;55(16):3576-83.
Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of bee hives, was shown previously to block tumor promoter- and carcinogen-generated oxidative processes in several assays and to engender differential toxicity to some transformed cells. To study the mechanisms of CAPE-induced differential cytotoxicity, nontumorigenic rat embryo fibroblasts (CREF) and adenovirus (type 5)-transformed CREF cells (Wt3A) were used. As shown by nucleosomal-length DNA degradation, morphological alterations by electron microscopy, in situ labeling of 3'-OH ends, and the appearance of a hypodiploid cell population by bivariant flow cytometry, cell death induced by CAPE in the transformed Wt3A cells was apoptosis. Under the same CAPE treatment conditions, CREF cells transiently growth arrested. Both CREF and Wt3A cells were radioresistant, suggesting deficiencies in the proteins controlling the G1 checkpoint. To explore possible mechanisms of CAPE-induced apoptosis, it was determined whether CAPE-induced toxicity was influenced by the redox state of the cells. Depletion of cellular glutathione (GSH) with buthionine sulfoximine before CAPE treatment caused CREF sensitive to CAPE-induced cell death. GSH levels were also determined in CAPE-treated CREF and Wt3A cells. The GSH level in the CREF cells was unaffected by CAPE, whereas the Wt3A cells showed a significant reduction. When the GSH levels were increased in Wt3A cells by treatment with the reducing agent, N-acetyl-cysteine before CAPE treatment, the Wt3A cells were partially rescued. Furthermore, Bcl2, which protects cells from oxidative stress, had a protective effect against CAPE-induced apoptosis in Wt3A cells. Finally, the sensitivity of Wt3A cells to a known oxidant, hydrogen peroxide (H2O2), was examined. Wt3A cells were killed by H2O2-induced apoptosis, whereas CREF cells remained resistant. When Wt3A cells were treated with catalase, a cellular enzyme that inactivates H2O2, CAPE-induced apoptosis in Wt3A cells was reduced, further proving that Wt3A cells were more sensitive than CREF cells to oxidative stress. These results suggest that CAPE can modulate the redox state of cells. Sensitivity of cells to CAPE-induced cell death may be determined by the loss of normal redox state regulation in transformed cells.
咖啡酸苯乙酯（CAPE）源自蜂巢蜂胶，此前在多项试验中显示可阻断肿瘤启动子和致癌物引发的氧化过程，并对某些转化细胞产生不同的毒性。为研究CAPE诱导的差异细胞毒性机制，使用了非致瘤性大鼠胚胎成纤维细胞（CREF）和腺病毒（5型）转化的CREF细胞（Wt3A）。如核小体长度DNA降解、电子显微镜下的形态改变、3'-OH末端的原位标记以及双变量流式细胞术检测到的亚二倍体细胞群的出现所示，CAPE在转化的Wt3A细胞中诱导的细胞死亡为凋亡。在相同的CAPE处理条件下，CREF细胞短暂生长停滞。CREF和Wt3A细胞均具有放射抗性，提示控制G1期检查点的蛋白质存在缺陷。为探究CAPE诱导凋亡的可能机制，确定了CAPE诱导的毒性是否受细胞氧化还原状态的影响。在CAPE处理前用丁硫氨酸亚砜胺耗尽细胞内谷胱甘肽（GSH），使CREF对CAPE诱导的细胞死亡敏感。还测定了CAPE处理的CREF和Wt3A细胞中的GSH水平。CREF细胞中的GSH水平不受CAPE影响，而Wt3A细胞中的GSH水平显著降低。当在CAPE处理前用还原剂N-乙酰半胱氨酸处理使Wt3A细胞中的GSH水平升高时，Wt3A细胞得到部分挽救。此外，保护细胞免受氧化应激的Bcl2对Wt3A细胞中CAPE诱导的凋亡具有保护作用。最后，检测了Wt3A细胞对已知氧化剂过氧化氢（H2O2）的敏感性。H2O2诱导的凋亡可杀死Wt3A细胞，而CREF细胞仍具有抗性。当用使H2O2失活的细胞内酶过氧化氢酶处理Wt3A细胞时，Wt3A细胞中CAPE诱导的凋亡减少，进一步证明Wt3A细胞比CREF细胞对氧化应激更敏感。这些结果表明，CAPE可调节细胞的氧化还原状态。细胞对CAPE诱导的细胞死亡的敏感性可能由转化细胞中正常氧化还原状态调节的丧失所决定。
