抗癌药物椭圆玫瑰树碱与组蛋白去乙酰化酶抑制剂丙戊酸和曲古抑菌素A联合使用，是一种治疗人类神经母细胞瘤的有效DNA损伤策略。

Anticancer agent ellipticine combined with histone deacetylase inhibitors, valproic acid and trichostatin A, is an effective DNA damage strategy in human neuroblastoma.

作者信息

Poljakova Jitka, Hrebackova Jana, Dvorakova Marketa, Moserova Michaela, Eckschlager Tomas, Hrabeta Jan, Göttlicherova Marketa, Kopejtkova Barbora, Frei Eva, Kizek Rene, Stiborova Marie

机构信息

Department of Biochemistry, Faculty of Science, Charles University, Prague, Czech Republic.

出版信息

Neuro Endocrinol Lett. 2011;32 Suppl 1:101-16.

PMID:22167207

Abstract

OBJECTIVES

Valproic acid (VPA) and trichostatin A (TSA) exert antitumor activity as histone deacetylase inhibitors, whereas ellipticine action is based mainly on DNA intercalation, inhibition of topoisomerase II and formation of cytochrome P450 (CYP)- and peroxidase-mediated covalent DNA adducts. This is the first report on the molecular mechanism of combined treatment of human neuroblastoma UKF-NB-3 and UKF-NB-4 cells with these compounds.

METHODS

HPLC with UV detection was employed for the separation and characterization of ellipticine metabolites formed by microsomes and peroxidases. Covalent DNA modifications by ellipticine in neuroblastoma cells and in incubations with microsomes and peroxidases were detected by 32P-postlabeling. Expression of CYP enzymes, peroxidases and cytochrome b5 was examined by Western blot.

RESULTS

The cytotoxicity of ellipticine to neuroblastomas was increased by pre-treating these cells with VPA or TSA. A higher sensitivity of cells to ellipticine correlated with an increase in formation of covalent ellipticine-derived DNA adducts in these cells. To evaluate the mechanisms of this finding, we investigated the modulation by VPA and TSA of CYP- and peroxidase-mediated ellipticine-derived DNA adduct formation in vitro. The effects of ellipticine in the presence of VPA and TSA on expression of CYPs and peroxidases relevant for ellipticine activation and levels of cytochrome b5 and P-glycoprotein in neuroblastoma cells were also investigated. Based on these studies, we attribute most of the enhancing effects of VPA and TSA on ellipticine cytotoxicity to enhanced ellipticine-DNA adduct formation caused by an increase in levels of cytochrome b5, CYP3A4 and CYP1A1 in neuroblastoma cells. A lower sensitivity of UKF-NB-4 cells to combined effects of ellipticine with VPA and TSA than of UKF-NB-3 cells is also attributable to high levels of P-glycoprotein expressed in this cell line.

CONCLUSION

The results found here warrant further studies and may help in the design of new protocols geared to the treatment of high risk neuroblastomas.

摘要

目的

丙戊酸（VPA）和曲古抑菌素A（TSA）作为组蛋白去乙酰化酶抑制剂发挥抗肿瘤活性，而椭圆玫瑰树碱的作用主要基于DNA嵌入、拓扑异构酶II抑制以及细胞色素P450（CYP）和过氧化物酶介导的共价DNA加合物的形成。这是关于用这些化合物联合处理人神经母细胞瘤UKF-NB-3和UKF-NB-4细胞的分子机制的首次报道。

方法

采用带紫外检测的高效液相色谱法分离和鉴定微粒体和过氧化物酶形成的椭圆玫瑰树碱代谢物。通过32P后标记法检测神经母细胞瘤细胞中以及与微粒体和过氧化物酶孵育时椭圆玫瑰树碱对DNA的共价修饰。通过蛋白质印迹法检测CYP酶、过氧化物酶和细胞色素b5的表达。

结果

用VPA或TSA预处理这些细胞可增加椭圆玫瑰树碱对神经母细胞瘤的细胞毒性。细胞对椭圆玫瑰树碱的更高敏感性与这些细胞中共价椭圆玫瑰树碱衍生的DNA加合物形成增加相关。为评估这一发现的机制，我们在体外研究了VPA和TSA对CYP和过氧化物酶介导的椭圆玫瑰树碱衍生的DNA加合物形成的调节作用。还研究了在VPA和TSA存在下椭圆玫瑰树碱对神经母细胞瘤细胞中与椭圆玫瑰树碱激活相关的CYPs和过氧化物酶表达以及细胞色素b5和P-糖蛋白水平的影响。基于这些研究，我们将VPA和TSA对椭圆玫瑰树碱细胞毒性的大部分增强作用归因于神经母细胞瘤细胞中细胞色素b5、CYP3A4和CYP1A1水平增加导致的椭圆玫瑰树碱-DNA加合物形成增强。UKF-NB-4细胞对椭圆玫瑰树碱与VPA和TSA联合作用的敏感性低于UKF-NB-3细胞，这也归因于该细胞系中高表达的P-糖蛋白。