摘要
目的观察丙戊酸(VPA)对白血病细胞尤其是耐药白血病细胞的体外抗肿瘤作用,探讨细胞内谷胱甘肽(GSH)水平及抗氧化酶系活性与VPA诱导细胞凋亡之间的关系。方法采用体外细胞培养,通过MTT细胞活力测定、Caspase-3,8,9活性检测、细胞形态学观察、细胞周期分析及Annexin V/PI双染法检测细胞凋亡,观察VPA对白血病细胞的抗肿瘤作用;通过检测VPA作用前后细胞内总谷胱甘肽(tGSH)与GSH水平、谷胱甘肽还原酶(GSH-Rd)和谷胱甘肽过氧化物酶(GSH-Px)活性改变,以及GSH合成酶抑制剂丁胱亚磺酰亚胺(BSO)、GSH前体N-乙酰-半胱氨酸(NAC)和过氧化氢酶(CAT)对VPA诱导凋亡的影响,分析谷胱甘肽-氧化还原(GSH-redox)系统在VPA诱导细胞凋亡中的作用。结果VPA对所有受试细胞株均有增殖抑制作用,其IC50接近VPA抗癫痫有效血药浓度,VPA作用后细胞内Caspase-3,8,9活性增强,细胞出现凋亡形态学改变。VPA可使阿霉素耐药株K562/AO2及糖皮质激素(GC)耐药株Jurkat细胞周期阻滞于G0/G1期,在VPA作用下,Jurkat和K562/AO2细胞内tGSH和GSH均降低,以GSH降低为主,GSH-Rd和GSH-Px的活性亦显著降低。NAC和CAT减弱VPA诱导的凋亡,而BSO增强VPA诱导的凋亡。结论VPA抑制耐药白血病细胞增殖、诱导细胞周期阻滞与凋亡,这些作用与细胞内GSH-Rd、GSH-Px活性降低、GSH水平下调所导致的细胞内氧化应激反应激活有关。
Objective To investigate the anti-neoplastic effects of Valproic acid （VPA） on leukemic cells, especially drug-resistant lines, and to investigate whether modulation of GSH-redox status is involved in VPA- induced apoptosis. Methods After the treatment of VPA at various concentrations for indicated times, cellular proliferation of the Jurkat, CEM, HL-60, K562, K562/AO2 cells were evaluated via MTT assay. and the activities of Caspase-3, Caspase-8 and Caspase-9 were quantitatively analyzed by colorimetric assay. The morphological change and cell cycle distribution were also examined on Jurkat （Dexamethasone-resistant） and K562/AO2 （Doxorubicin-resistant） cell lines. The levels of intracellular glutathione/glutathione disulfide （GSH/GSSG） and the activities of the typical antioxidant enzymes, i. e. , glutathione reductase （GSH-Rd） and glutathione peroxidase （GSH-Px）, were measured on cell lysates of Jurkat and K562/AO2 cell lines prior to and after VPA treatment. Apoptosis rates of Jurkat and K562/AO2 cells treated with VPA along or in combination with N-aeetyl-1-cysteine （NAC）, eatalase （CAT） or DL-buthionine-（S,R）-sulfoximine （BSO） were determined by Annexin V/propidium iodide （PD staining with flow cytometry analysis. Results At concentrations comparable with that achieved at clinical settings, VPA inhibited cell proliferation, activated Caspase-3, 8, and 9, and induced cell cycle arrest in Jurkat and K562/AO2. A rapid decrease in GSH-Rd and GSH-Px activities and GSH content in Jurkat and K562/AO2 were detected after VPA treatment. Co-administration of NAC or CAT attenuated VPA-induced apoptosis. Conclusion VPA inhibit cell proliferation, induce cell cycle arrest and apoptosis in drug-resistant leukemic cells. Apoptosis correlates with down-regulation of intraeellular GSH and disruption of intracellular GSH-redox balance, possibly through inhibition of glutathione reductase and glutathione peroxidase.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2009年第1期133-137,共5页
Journal of Sichuan University(Medical Sciences)
