Abstract: 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 CD_11b, 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 CD_11b, 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.

Key words: N, N '-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide, Cell line HL-60, apoptosis, differentiation