Mi Lixin, Wang Xiantao, Govind Sudha, Hood Brian L, Veenstra Timothy D, Conrads Thomas P, Saha Daniel T, Goldman Radoslav, Chung Fung-Lung
Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University, Washington, District of Columbia 20057, USA.
Cancer Res. 2007 Jul 1;67(13):6409-16. doi: 10.1158/0008-5472.CAN-07-0340.
Induction of apoptosis underlies a mechanism for inhibiting tumorigenesis by phenethyl isothiocyanate (PEITC) and sulforaphane (SFN). However, the upstream events by which isothiocyanates (ITC) induce apoptosis have not been fully investigated. As electrophiles, ITCs could trigger apoptosis by binding to DNA or proteins or by inducing oxidative stress. To better understand the molecular mechanisms of apoptosis by ITCs, we examined, as a first step, the role of these events in human non-small lung cancer A549 cells. PEITC was a more potent inducer than SFN; it induced apoptosis at 20 micromol/L, whereas SFN induced at 40 micromol/L but not at 20 micromol/L. To study binding with cellular proteins and DNA, cells were treated with (14)C-ITCs; the initial protein binding by PEITC was almost 3-fold than that of SFN. The binding by PEITC increased with time, whereas binding by SFN remained low. Therefore, 4 h after incubation proteins became the predominant targets for PEITC with a 6-fold binding than that of SFN. To characterize the chemical nature of binding by the ITCs, we used bovine serum albumin (BSA) as a surrogate protein. PEITC also modified BSA covalently to a greater extent than SFN occurring exclusively at cysteine residues. Surprisingly, neither PEITC nor SFN bound to DNA or RNA at detectable levels or caused significant DNA strand breakage. The levels of oxidative damage in cells, measured as reactive oxygen species, 8-oxo-deoxyguanosine, and protein carbonyls formation, were greater in cells treated with SFN than PEITC. Because PEITC is a stronger inducer of apoptosis than SFN, these results indicate that direct covalent binding to cellular proteins is an important early event in the induction of apoptosis by the ITCs.
苯乙基异硫氰酸酯（PEITC）和萝卜硫素（SFN）通过诱导细胞凋亡构成一种抑制肿瘤发生的机制。然而，异硫氰酸酯（ITC）诱导细胞凋亡的上游事件尚未得到充分研究。作为亲电试剂，ITC可通过与DNA或蛋白质结合或诱导氧化应激来触发细胞凋亡。为了更好地理解ITC诱导细胞凋亡的分子机制，我们作为第一步，研究了这些事件在人非小细胞肺癌A549细胞中的作用。PEITC比SFN是更强效的诱导剂；它在20微摩尔/升时诱导细胞凋亡，而SFN在40微摩尔/升时诱导细胞凋亡，在20微摩尔/升时则不诱导。为了研究与细胞蛋白质和DNA的结合，用（14）C-ITC处理细胞；PEITC与蛋白质的初始结合几乎是SFN的3倍。PEITC的结合随时间增加，而SFN的结合保持较低水平。因此，孵育4小时后，蛋白质成为PEITC的主要靶标，其结合量是SFN的6倍。为了表征ITC结合的化学性质，我们使用牛血清白蛋白（BSA）作为替代蛋白质。PEITC对BSA的共价修饰程度也比SFN更大，且仅发生在半胱氨酸残基处。令人惊讶的是，PEITC和SFN均未以可检测水平与DNA或RNA结合，也未导致明显的DNA链断裂。以活性氧、8-氧代脱氧鸟苷和蛋白质羰基形成来衡量的细胞氧化损伤水平，在用SFN处理的细胞中比用PEITC处理的细胞中更高。由于PEITC比SFN是更强的细胞凋亡诱导剂，这些结果表明与细胞蛋白质的直接共价结合是ITC诱导细胞凋亡的重要早期事件。
