Effects of N, N'-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide on HL-60 leukemia cells in vitro
ZHOU Yong-lie, LV Ya-ping<, HU Wei-xiao<, QIU Lian-Nu, WANG Wen-Song, YANG Zhong-yu<, LIU Jian-dong
2006, 11(9):
1006-1012.
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AIM: To study the effect of N, N'-di-(m-methylphenyi) -3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation inhibition, differentiation and apoptosis in HL-60 human leukemia cell line and explore its possible mechanism.METHODS: Different concentration of ZGDHu-1 and the different time of cultivate were used to treat HL-60 cell.The proliferation inhibition was analyzed by cell counting,alive cell count and MTT assay.Cell apoptosis was analyzed by cell morphology, DNA agarose gel electrophoresis,DNA content and Annexin-V/PI.The TdT-mediated dUTpnick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis.The cell morphological analysis, expression of CD11b, CD13, CD14, CD64 and NBT reduction were performed to evaluate differentiation of HL-60 cells.The expressions of bcl-2, bax, Fas, P53 and mitochondrial membrane protein were analyzed by flow cytometry.While the mitochondrial transmembrance potential (ΔΧm) was labeled by dihydrorhodamin 123.RESULTS: ZGDHu-1 could inhibit HL-60 cell proliferation viability within a certain range of treating time and doses, with a 48 h IC50 of 180 ng·ml-1 and 72 h of 180 ng·ml-1.A majority of HL-60 cells were arrested in G2/M phase and a progressive decline in G0/1.The HL-60 cells apoposis was confirmed by type cell morphology,DNA fragment, sub-G1 phase, TUNEL and Annexin-Ⅴ/PI Labeling method with a time and dose related manner.The morphology of HL-60 cells cultured in the presence of 10 -100 ng·ml-1 ZGDHu-1 for three days was more mature with higher positive rate of NBT and up-regulated expressions of CD11b, CD13, CD14 and CD64 than those of control.The expression of bax and Fas was increased,and bcl-2 was unchanged by the treatment of ZGDHu-1.ZGDHu-1 could increase the expression of mitochondrial membrane protein in a dose-dependent manner while the ΔΧm was reduced.CONCLUSION: ZGDHu-1 can inhibit proliferation, induce differentiation and apoptosis of HL-60 cells.The mechanism may associate with its upregulation of bax, open the mitochondrial permeability transition pore and reduce ΔΧm.Furthermore, the Fas is activated during the apoptosis process.