Yeh Chi-Tai, Yen Gow-Chin
Department of Food Science and Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan.
Carcinogenesis. 2005 Dec;26(12):2138-48. doi: 10.1093/carcin/bgi185. Epub 2005 Jul 20.
The molecular mechanism of sulforaphane on the induction of metallothionein (MT) genes in HepG2 cells and the antiproliferative effects of sulforaphane were investigated in this study. Treatment of the cells with sulforaphane at non-toxicity concentration (0-20 microM) resulted in coordinate increases in the induction of MT-I and MT-II mRNA, followed by corresponding increases in MT protein expression. Western blot analysis revealed the increased level of the transcription factor, Nrf2 in a time-dependent manner from sulforaphane-treated cells. Furthermore, sulforaphane activated the extracellular signal-regulated protein kinase (ERK), p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways. SB203580, a specific inhibitor of p38 and PD98059, a specific inhibitor of ERK, abolished sulforaphane-induced MT protein expression, whereas SP600125, a specific inhibitor of JNK, had no significant effect. At relatively high concentration (30-100 microM), sulforaphane is a cell growth modulator, as it induced apoptotic cell death characterized by internucleosomal DNA fragmentation and caused a rapid induction of caspase 3 activity, according to the appearance of the caspase 3 fragments and stimulated proteolytic cleavage of poly (ADP-ribose) polymerase in a time-dependent manner. Moreover, sulforaphane-induced apoptotic cell death was accompanied by upregulation of Bax and downregulation of Bcl-2 and Bcl-X(l) protein. Sulforaphane-induced DNA fragmentation was blocked by the N-acetyl-L-cysteine and catalase, suggesting that the death signaling was triggered by oxidative stress. Taken together these results strongly suggest that at low concentrations of sulforaphane, activation of MAPKs, such as ERK and p38 pathway, lead to Nrf2-mediated MT gene expression. Whereas at a higher concentration, sulforaphane is an effective apoptosis inducer in HepG(2) cells through regulation of Bcl-2 family molecular and activation of ICE/Ced-3 protease (caspase 3) cascade. The results from this study may provide more evidence for its chemopreventive function.
本研究探讨了萝卜硫素诱导HepG2细胞中金属硫蛋白（MT）基因的分子机制以及萝卜硫素的抗增殖作用。用无毒浓度（0 - 20微摩尔）的萝卜硫素处理细胞，导致MT - I和MT - II mRNA的诱导协同增加，随后MT蛋白表达相应增加。蛋白质印迹分析显示，经萝卜硫素处理的细胞中，转录因子Nrf2的水平呈时间依赖性增加。此外，萝卜硫素激活了细胞外信号调节蛋白激酶（ERK）、p38和c - Jun氨基末端激酶（JNK）丝裂原活化蛋白激酶（MAPK）途径。p38的特异性抑制剂SB203580和ERK的特异性抑制剂PD98059消除了萝卜硫素诱导的MT蛋白表达，而JNK的特异性抑制剂SP600125则无显著影响。在相对较高浓度（30 - 100微摩尔）下，萝卜硫素是一种细胞生长调节剂，因为它诱导了以核小体间DNA片段化为特征的凋亡细胞死亡，并导致caspase 3活性迅速诱导，这可根据caspase 3片段的出现以及聚（ADP - 核糖）聚合酶的蛋白水解裂解呈时间依赖性刺激来判断。此外，萝卜硫素诱导的凋亡细胞死亡伴随着Bax的上调以及Bcl - 2和Bcl - X(l)蛋白的下调。萝卜硫素诱导的DNA片段化被N - 乙酰 - L - 半胱氨酸和过氧化氢酶阻断，表明死亡信号是由氧化应激触发的。综上所述，这些结果强烈表明，在低浓度的萝卜硫素下，ERK和p38等MAPK的激活导致Nrf2介导的MT基因表达。而在较高浓度下，萝卜硫素通过调节Bcl - 2家族分子和激活ICE/Ced - 3蛋白酶（caspase 3）级联反应，是HepG(2)细胞中一种有效的凋亡诱导剂。本研究结果可能为其化学预防功能提供更多证据。
