Starenki Dmitriy V, Namba Hiroyuki, Saenko Vladimir A, Ohtsuru Akira, Maeda Shigeto, Umezawa Kazuo, Yamashita Shunichi
Department of Molecular Medicine, Atomic Bomb Disease Institute, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.
Clin Cancer Res. 2004 Oct 15;10(20):6821-9. doi: 10.1158/1078-0432.CCR-04-0463.
The objective of the study was to determine the effects of a novel selective nuclear factor kappaB (NF-kappaB) inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), in thyroid carcinoma cells in vitro and in vivo and to additionally elucidate the molecular mechanisms underlying the action of this chemotherapeutic agent.
In the in vitro experiments, the induction of apoptosis by DHMEQ in various human thyroid carcinoma cell types was determined by flow cytometry analysis of annexin-V binding and the caspase activation by Western blotting. For the in vivo study, female nu/nu mice were xenografted with s.c. FRO thyroid tumors. DHMEQ solution was injected i.p. at a dose of 8 mg/kg/day for two weeks. Tumor dimensions were monitored twice weekly, and apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining.
Treatment with DHMEQ substantially inhibited the translocation of p65 and p50 NF-kappaB subunits to the nucleus, the DNA-binding activity of the RelA/p65, NF-kappaB-dependent expression of the inhibitor of apoptosis (IAP)-family proteins, cIAP-1, cIAP-2, and XIAP, and the de novo synthesis of inhibitor of nuclear factor kappaB alpha. At concentration levels ranging from 0.1 to 5 microg/ml, DHMEQ induced a caspase-mediated apoptotic response that could be abrogated by the c-Jun NH(2)-terminal kinase inhibitor SP600125 but not by either mitogen-activated protein/extracellular signal-regulated kinase kinase or p38 inhibitors. In contrast, normal human thyrocytes were resistant to DHMEQ-induced apoptosis. At higher doses of DHMEQ we observed the necrotic-like killing of both normal and malignant thyrocytes, which was resistant to mitogen-activated protein kinase inhibitors. In nude mice DHMEQ substantially inhibited tumor growth without observable side effects, and increased numbers of apoptotic cells were observed in the histologic sections of tumors treated with DHMEQ.
Our results show the potential usefulness of the novel NF-kappaB inhibitor, DHMEQ, in future therapeutic strategies for the treatment of thyroid cancers that do not respond to conventional approaches.
本研究的目的是确定新型选择性核因子κB（NF-κB）抑制剂去羟甲基环氧喹霉素（DHMEQ）在体外和体内对甲状腺癌细胞的作用，并进一步阐明这种化疗药物作用的分子机制。
在体外实验中，通过膜联蛋白-V结合的流式细胞术分析以及蛋白质免疫印迹法检测半胱天冬酶激活，来确定DHMEQ在各种人类甲状腺癌细胞类型中诱导凋亡的情况。在体内研究中，将雌性裸鼠皮下接种FRO甲状腺肿瘤。以8mg/kg/天的剂量腹腔注射DHMEQ溶液，持续两周。每周监测两次肿瘤大小，并通过末端脱氧核苷酸转移酶介导的缺口末端标记染色法确定肿瘤标本中的凋亡情况。
用DHMEQ处理可显著抑制p65和p50 NF-κB亚基向细胞核的转位、RelA/p65的DNA结合活性、凋亡抑制蛋白（IAP）家族蛋白cIAP-1、cIAP-2和XIAP的NF-κB依赖性表达以及核因子κBα抑制剂的从头合成。在0.1至5μg/ml的浓度范围内，DHMEQ诱导了半胱天冬酶介导的凋亡反应，该反应可被c-Jun氨基末端激酶抑制剂SP600125消除，但不能被丝裂原活化蛋白/细胞外信号调节激酶激酶或p38抑制剂消除。相反，正常人甲状腺细胞对DHMEQ诱导的凋亡具有抗性。在较高剂量的DHMEQ作用下，我们观察到正常和恶性甲状腺细胞均出现类似坏死的死亡，且这种死亡对丝裂原活化蛋白激酶抑制剂具有抗性。在裸鼠中，DHMEQ显著抑制肿瘤生长且无明显副作用，在用DHMEQ处理的肿瘤组织切片中观察到凋亡细胞数量增加。
我们的结果表明，新型NF-κB抑制剂DHMEQ在未来治疗对传统方法无反应的甲状腺癌的治疗策略中具有潜在的应用价值。
