新型多胺缀合物NNAMB诱导B16细胞凋亡及分化作用

NNAMB,a novel homospermidine conjugate,induces apoptosis and differentiation in B16 Melanoma cells

目的评价新型多胺缀合物NNAMB对B16黑色素瘤细胞诱导凋亡及分化作用。方法以MTT法、台盼蓝拒染法检测细胞活力;Hoechst33258染色观察细胞形态变化;流式细胞仪检测细胞周期变化、凋亡率及线粒体膜电位的变化;酶标仪检测caspase-3、-8、-9的活性及B16细胞内黑色素含量、酪氨酸酶活性等的变化。结果NNAMB在高剂量(>0.1μmol·L-1)下呈现剂量及时间依赖性的抑制B16黑色素瘤细胞的生长、诱导凋亡、降低线粒体膜电位、促进Caspase-3及-9的活化,但caspase-8活性与对照组细胞相比变化差异无显著性;NNAMB在低剂量(<0.1μmol·L-1)下通过增强酪氨酸酶活性,增加黑色素生成等诱导B16细胞分化。结论NNAMB在高剂量下通过线粒体/caspase-9/caspase-3途径诱导B16细胞凋亡;低剂量诱导细胞分化。

Aim To evaluate the mechanism of how NNAMB induces apoptosis and differentiation in B16 melanoma cells. Methods Cell viability was assessed by MTT assay and trypan blue dye exclusion method; morphological observation was achieved by fluorescence microscopy; cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry; the activity of caspase-3,-8, ：9 and tyrosinase, melanin content of the B16 cells were evaluated by Microboard reader. Results NNAMB was able to inhibit cells proliferation, induce apoptosis, lose mitochondrial membrane potential, increase the activity of caspase-3,-9 but not caspase-8 in a dose-and time-dependent manner at higher doses （ 〉 0. 1 μmol · L^-1 ）, inducing differentiation through enhancement of melanogenesis and increase of the activity of tyrosinase at lower doses （ 〈 0. 1 μmol · L^-1 ）. Conclusions NNAMB could induce apoptosis via mitochondria/ caspase-9/caspase-3 pathway at higher doses and induce differentiation at lower doses in B16 cells.