Stockwin Luke H, Bumke Maja A, Yu Sherry X, Webb Simon P, Collins Jack R, Hollingshead Melinda G, Newton Dianne L
Developmental Therapeutics Program, Science Applications International Corporation Frederick, Frederick, Maryland 21702, USA.
Clin Cancer Res. 2007 Jun 15;13(12):3667-81. doi: 10.1158/1078-0432.CCR-07-0025.
Activities distinct from inhibition of Bcr/abl have led to adaphostin (NSC 680410) being described as "a drug in search of a mechanism." In this study, proteomic analysis of adaphostin-treated myeloid leukemia cell lines was used to further elucidate a mechanism of action.
HL60 and K562 cells treated with adaphostin for 6, 12, or 24 h were analyzed using two-dimensional PAGE. Differentially expressed spots were excised, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry. The contribution of the redox-active hydroquinone group in adaphostin was also examined by carrying out proteomic analysis of HL60 cells treated with a simple hydroquinone (1,4-dihydroxybenzene) or H(2)O(2).
Analysis of adaphostin-treated cells identified 49 differentially expressed proteins, the majority being implicated in the response to oxidative stress (e.g., CALM, ERP29, GSTP1, PDIA1) or induction of apoptosis (e.g., LAMA, FLNA, TPR, GDIS). Interestingly, modulation of these proteins was almost fully prevented by inclusion of an antioxidant, N-acetylcysteine. Validation of the proteomic data confirmed GSTP1 as an adaphostin resistance gene. Subsequent analysis of HL60 cells treated with 1,4-dihydroxybenzene or H(2)O(2) showed similar increases in intracellular peroxides and an almost identical proteomic profiles to that of adaphostin treatment. Western blotting of a panel of cell lines identified Cu/Zn superoxide dismutase (SOD) as correlating with adaphostin resistance. The role of SOD as a second adaphostin resistance gene was confirmed by demonstrating that inhibition of SOD using diethyldithiocarbamate increased adaphostin sensitivity, whereas transfection of SOD I attenuated toxicity. Importantly, treatment with 1,4-dihydroxybenzene or H(2)O(2) replicated adaphostin-induced Bcr/abl polypeptide degradation, suggesting that kinase inhibition is a ROS-dependent phenomenon.
Adaphostin should be classified as a redox-active-substituted dihydroquinone.
与抑制Bcr/abl不同的活性导致阿地福司汀（NSC 680410）被描述为“一种寻找作用机制的药物”。在本研究中，对经阿地福司汀处理的髓系白血病细胞系进行蛋白质组学分析，以进一步阐明其作用机制。
用二维聚丙烯酰胺凝胶电泳分析经阿地福司汀处理6、12或24小时的HL60和K562细胞。切除差异表达的斑点，用胰蛋白酶消化，并用液相色谱-串联质谱分析。还通过对用简单对苯二酚（1,4-二羟基苯）或过氧化氢处理的HL60细胞进行蛋白质组学分析，研究了阿地福司汀中具有氧化还原活性的对苯二酚基团的作用。
对经阿地福司汀处理的细胞的分析鉴定出49种差异表达的蛋白质，大多数与氧化应激反应（如钙调蛋白、内质网蛋白29、谷胱甘肽S-转移酶P1、蛋白二硫异构酶1）或凋亡诱导（如层粘连蛋白、细丝蛋白A、转甲状腺素蛋白、生长分化抑制因子）有关。有趣的是，加入抗氧化剂N-乙酰半胱氨酸几乎完全阻止了这些蛋白质的调节。蛋白质组学数据的验证证实谷胱甘肽S-转移酶P1是一种阿地福司汀抗性基因。随后对用1,4-二羟基苯或过氧化氢处理的HL60细胞的分析表明，细胞内过氧化物有类似增加，并且蛋白质组学图谱与阿地福司汀处理的图谱几乎相同。对一组细胞系进行蛋白质免疫印迹分析确定铜/锌超氧化物歧化酶（SOD）与阿地福司汀抗性相关。通过证明用二乙基二硫代氨基甲酸盐抑制SOD可增加阿地福司汀敏感性，而转染SOD I可减弱毒性，证实了SOD作为第二个阿地福司汀抗性基因的作用。重要的是，用1,4-二羟基苯或过氧化氢处理可复制阿地福司汀诱导的Bcr/abl多肽降解现象，这表明激酶抑制是一种活性氧依赖性现象。
阿地福司汀应归类为具有氧化还原活性取代基的二氢醌。
