二氟甲基鸟氨酸对T淋巴瘤Jurkat细胞生长的影响

Difluoromethylornithine suppresses growth and improves apoptosis of human leukemia Jurkat cells

目的研究二氟甲基鸟氨酸(difluromethylornithine,DFMO)对人T淋巴瘤Jurkat细胞的影响及其作用机制,为探讨DFMO能否用于治疗人白血病提供实验依据。方法DFMO(0～10mmol/L)处理人T淋巴瘤Jurkat细胞24～72h后,MTS法检测细胞的存活率,化学分析法检测精胺氧化酶(spermine oxidase,SMO)和乙酰多胺氧化酶(acetylpolyamine oxidase,APAO)活性,DNA片段化分析检测细胞凋亡,荧光染料法检测细胞线粒体膜电位变化,Western blot法检测细胞内Bax含量,分光光度法检测caspase-3酶活性。结果DFMO处理细胞能显著抑制Jurkat细胞的生长(P<0.01),抑制率随药物浓度的增加和作用时间的延长而加大,DFMO浓度为10mmol/L时,24h抑制率为33%,48h抑制率为38%,72h抑制率为49%。DFMO处理可导致Jurkat细胞DNA片段化,促进Bax由细胞质向线粒体转移,细胞内caspase-3活性增加(增加46%,P<0.05),并伴有线粒体膜电位的显著降低,DFMO浓液为2和5mmol/L时,分别降低29%和53%(P<0.01)。DFMO还能诱导细胞内SMO活性(增加1.1倍,P<0.05)但对APAO的活性无诱导性影响。结论DFMO对人T淋巴瘤Jurkat细胞具有生长抑制和诱导凋亡的作用。

Objective To investigate whether difluromethylornithine （DFMO） can be used in the treatment of human leukemia. Methods The cell proliferation was detected by MTS [3-（4,5-dimethylthiazol- 2-yl）-5-（3-carboxymethoxyphenyl）-2-（4-sulfophenyl）-2H-tetrazolium） ] assay after treatment of human lymphocyte Jurkat cells by DFMO （0 to 10 retool/L） for 24 to 72 h. Enzyme activity of spermine oxidase （SMO） and acetylpolyamine oxidase （PAO） was determined by chemiluminesence assay. DNA fragmentation assay was used to evaluate cell apoptosis. Fluorescent dye assay was performed to determine the changes in mitochondrial membrane potential. Western blotting was used to determine Bax content. Casepase-3 enzyme activity was measured by spectrophotometric method. Results DFMO treatment inhibited the proliferation of Jurkat cells significantly in a dosage- and time-dependent manner （P 〈 0.01 ）. When DFMO concentration was 10 mmol/L, the inhibitory rate for cell growth was 33% in 24 h, 38% in 48 h and 49% in 72 h respectively. DFMO treatment resulted in DNA fragmentation, promoted translocation of Bax from the cytoplasma to the mitochondria and increased caspase-3 activity by 46% （ P 〈 0. 05 ） accompanied by a significant reduction in the mitochondrial membrane potential in Jurkat cells, reducing 29% at 2 mmol/L DFMO and 53% at 5 mmol/L DFMO （P 〈0. 01 ）. DFMO promoted the activity of SMO by 1.1-fold （P 〈0.05） but no inducible effect on PAO activity in Jurkat cells. Conclusion DFMO can inhibit cell growth and induce apoptosis in human leukemia Jurkat cells.