三氧化二砷对人神经母细胞瘤细胞凋亡作用的影响

Effect of arsenic trioxide on apoptosis of neuroblastoma cells

摘要:

目的：三氧化二砷(As2O3 )通过诱导细胞凋亡和诱导细胞分化效应治疗急性早幼粒细胞白血病(APL)患者已取得很好疗效。与APL细胞相似,神经母细胞瘤(NB)细胞也是分化成熟受阻,该文对As2O3 在体外对人神经母细胞瘤细胞凋亡作用的影响及可能的机制进行探讨。方法：比色法观察As2O3 对神经母细胞瘤SK N SH细胞增殖的影响;Giemsa染色观察细胞形态学改变;Annexinⅴ/PI双染色流式细胞术检测细胞凋亡;免疫细胞化学染色方法检测bcl 2蛋白表达变化。结果：As2O3 明显抑制SK N SH细胞增殖,抑制作用呈剂量时间依赖效应,并出现细胞凋亡的形态学改变;流式细胞仪检测4. 0μmol/LAs2O3 作用SK N SH细胞72h后,早期凋亡细胞比例( 9. 9±0. 8 )%,与对照组( 3. 7±0. 2 )%相比明显增多,差异有显著性(P< 0. 05 );分别以1. 0, 2. 0,4. 0μmol/L的As2O3 处理SK N SH细胞72h后,bcl 2表达随As2O3 浓度增加呈降低趋势,bcl 2表达阳性率分别为(26. 3±8. 0)%, (15. 0±4. 5)%和(4. 8±3. 2)%,与对照组(73. 6±6. 1)%相比,差异有显著性(P<0. 01)。结论：As2O3 可在体外抑制神经母细胞瘤细胞增殖,诱导凋亡,bcl 2参与了其调节作用。

Abstract:

OBJECTIVE: Recent clinical studies have shown that arsenic trioxide (As_2O_3) at low concentrations induces a complete remission with minimal toxicity in patients with refractory acute promyelocytic leukemia (APL). Studies suggest that As_2O_3 induces apoptosis and possible differentiation in APL cells. Like APL cells, neuroblastoma (NB) cells are thought to be arrested at an early stage of differentiation, and cells of highly malignant tumors fail to undergo spontaneous maturation. This study was designed to investigate the effect of (As_2O_3) on apoptosis of neuroblastoma cells in vitro and its mechanism. METHODS: The effect of a wide range of concentrations of As_2O_3 on the proliferation of neuroblastoma cells was measured by the colorimetric method. Morphological changes of the cells were observed under the optical microscope in Giemsa stain. Cell apoptosis was determined by the flow cytometry assay in Annexinⅴ/PI stain. Bcl-2 was detected by the immunocytochemical method. RESULTS: 0.5- 4.0 μmol/L As_2O_3 significantly inhibited the proliferation of the neuroblastoma cell line SK-N-SH and the inhibitory effect was dose- and time-dependent, which was accompanied by the appearance of morphologic characteristics of apoptosis. After 4.0 μmol/L As_2O_3 treatment for 72 hrs, the percentage of apoptotic cells [(9.9±0.8)%] increased significantly when compared with the untreated controls [(3.7±0.2)%] (P<0.05). The expression level of bcl-2 was down-regulated as the concentration of As_2O_3 increased. SK-N-SH cells treated with 1.0 μmol/L, 2.0 μmol/L, and 4.0 μmol/L As_2O_3, the percentage of bcl-2 positive cells [( 26.3±8.0)%, (15.0±4.5)% and (4.8±3.2)% respectively] decreased significantly when compared with the untreated controls [(73.6±6.1)%] (P<0.01). CONCLUSIONS: As_2O_3 may inhibit the proliferation and induce apoptosis of the neuroblastoma cells, in which bcl-2 is involved.