Effect of macrocalin A on NB4 leukemia cells in vitro.

Objective: To investigate the effect of macrocalin A (MA) on NB4 leukemia cell in vitro. Methods: In vitro, NB4 cells were incubated with MA, the cells’growth was tested by cell counting, MTT assay. Cell morphology, Hoechst 33258 fluorescence staining, DNA content, cell cycle analyzation and Annexin-V/PI were used to investigate the apoptosis of NB4. The differentiation of NB4 was analyzed by cell morphology, cytometry and nitroblue tetrazdium (NBT) test. The expressions of Bcl-2, bax, bad, Fas, p53 and mitochondrial membrane protein (APO2.7) were analyzed by flow cytometry. The mitochondrial transmembrancepotential (△Ψm) was labeled by dihydrorhodamin 123. There were negative control and ATRA control in this test. Results: Different concentrations of MA could obviously inhibit the growth of NB4 cells with time and dose dependence. The cells were differentiated by low concentration of MA 48 hours later, while apoptosis were significantly induced by higher concentration of MA. The expressions of Bcl-2, Fas were no signif icant differences, while the expressions of bax, bad, Bcl-2/bax, p53 and Apo2.7 increased and △Ψm decreased with time and dose dependent manner. Conclusion: In vitro, NB4 cells’proliferation inhibition and differentiation could obviously induced by MA. The mechanism probably associated with its up-regulation of bax, bad, having the effect of opening the mitochondrial permeabilitytransition pore, reducing △Ψm, finally inducing apoptosis.