3-溴代丙酰胺苯甲酰脲对人白血病和淋巴瘤的抗肿瘤作用机理

Antitumor mechanism of 3-bromopropionylamino benzoylurea on leukemia and lymphoma

目的 研究新型微管微丝抑制剂 3 溴代丙酰胺苯甲酰脲 (JIMB0 1)抗人白血病和淋巴瘤的作用机制。方法 细胞毒测定用MTT法、细胞周期分析用流式细胞仪检测、DNA片断产生用琼脂糖凝胶电泳法、细胞Bcl 2蛋白磷酸化用Westernblot法等。结果 JIMB0 1体外对所测定的 9株人白血病和淋巴瘤细胞系均具有较强的抗增殖活性 ;可以使人白血病细胞 (CEM)阻滞于有丝分裂期 (G2 M期 ) ,并使细胞出现明显的凋亡形态变化和产生典型的DNA梯带 ;Westernblot结果表明JIMB0 1处理的CEM细胞表达的Bcl 2蛋白发生磷酸化或高磷酸化 ;JIMB0 1可以激活CEM细胞caspase 3,8和 9的催化活性 ,同时caspase 3抑制剂可以阻断JIMB0 1诱导CEM细胞凋亡。结论 微管微丝抑制剂JIMB0 1抗肿瘤作用机制为阻滞肿瘤细胞于M期 ,并通过Bcl 2的蛋白磷酸化 。

Aim To study the antitumor mechanism of 3-bromopropionylamino benzoylurea (JIMB01) on leukemia and lymphoma. Methods The antitumor effects of JIMB01 in cell culture was detected by MTT staining. JIMB01-induced apoptosis in leukemia and lymphoma cells was tested by Giemsa staining, fluorescent Hoechst33258 staining, as well as DNA gel electrophoresis. Cell cycle was analyzed by flow cytometry. JIMB01-induced Bcl-2 phosphorylation in CEM cell lines was detected by Western blot methods. The activities of caspase-3 and caspase-8 were determined by colorimetric protease assay and that of caspase-9 was determined by fluorescent intensity. Results This compound showed antiproliferative activities in a panel of nine human leukemia and lymphoma cell lines with IC 50 values in the range of 0.25 μmol·L -1 to 0.51 μmol·L -1. Morphological observation and cell cycle analysis indicated that CEM cells were blocked at mitosis phase by JIMB01. The fluorescent Hoechst33258 staining showed apoptotic nuclear degradation dispersed in the cytoplasm of CEM cells exposed to JIMB01 at 0.80 μmol·L -1 for 24 h. DNA degradation in the form of a multiple-unit DNA ladder was clearly demonstrated in CEM leukemia cells treated with JIMB01 at 0.15 μmol·L -1 or higher for 24 h using agarose gel electrophoresis. Bcl-2 phosphorylation became visible, in Western blot, within 6 h in CEM cells treated with JIMB01 at 0.15 μmol·L -1 or higher for 24 h. JIMB01 increased the activities of caspase-3, -8 and -9 in CEM cells; DEVD-fmk, a caspase-3 inhibitor, inhibited the cytotoxicity of JIMB01 in CEM leukemia cells. Conclusion The antitumor mechanism of JIMB01 is that JIMB01 may induce tumor cell apoptosis through Bcl-2 phosphorylation and then caspase passway.