Study On The Enhancing Mechanisms Of Nimodipine In HL-60 Apoptosis Induced By Cytarabine

Objective To explore the enhancing mechanisms of nimodipine in HL-60 apoptosis induced by Ara-c and provide some experimental evidences for the widely clinic use of nimodipine in the reversal of multidrug resistance of leukemia.Methods The methods of culturing standard HL-60 cell strain and observing development change through fluorescent invert microscope was used. Observing cell morphological changes stained with Giemsa method and DNA fragment through agarose gel electrophoresis were performed. The expression of apoptosis associated-gene bcl-2 and bax through immunohistochemistry was investigated.Results HL-60 leukemia cell in the control group has no change. While in the experimental groups, HL-60 leukemia cells treated with nimodipine, Ara-c and combination of them underwent apoptosis. Morphological changes of apoptosis cell and DNA ladder on agarose were clearly seen. From the moment when drug added in for 8 hours, the cells in the experimental groups underwent deformation, decreasing volume, bubbling, decreasing density, while the membranes of cell and bubble were still integral. The cells stained with Giemsa showed that the nuclear membrane were splitted, and the purplish red chromatins were divided and concintrated into pieces, approaching to nuclear membrane, gathering to the edge and forming standard apoptosis bodies. Protein expression of bcl-2, inhibitor of apoptosis, was obviously decreased in the experimental groups. Compared with the control group, there was a difference (P<0.05) in the D group in 8 hours, in B and C groups in 16 hour and significant difference (P<0.01) in D group in 16 hour. There was a difference among the D group and B, C groups in 16 hour and could still be seen between B and D in 24 hour. Protein expression of bax, inducer of apoptosis, was obviously elevated, being difference (P<0.05) between the C and D groups in 8 hour contrast to the control group. There was a difference (P<0.05) in the C group and a significant difference (P<0.01) in the D group in 16 hour compared with control group. The differences were all significant between every experimental groups and control group in 24 hour. Compared with the control group, the bcl-2/bax ratio was obviously decreased in the experimental groups, being of difference (P<0.05) in the B, C groups and significant difference (PO.01) in the D group at 8 hour. There was a difference (P<0.05) between every experimental group and control group at 16 hour and 24 hour. The ratio had no difference in the control group at every given time. There were no differences among all experimental groups at 0 hour, 8 hour and 24 hour. Compared with the D group, theratio was different in the B and C group at 16 hour.Conclusion Nimodipine and Ara-c could induce apoptosis of HL-60 cells, and the mechanism of apoptosis induced by them might down-regulate the expression of bcl-2 gene and up-regulate the expression of bax gene. The enhancing mechanism in HL-60 cell apoptosis induced by Ara-c might co-enhance the down-regulation of bcl-2 protein expression.