Induction of apoptosis by bufalin in human tumor cells is associated with a change of intracellular concentration of Na+ ions

In an attempt to characterize the mechanisms that are operative at the early stages of the induction of apoptosis by bufalin, a component of the traditional Chinese medicine chan'su, we examined the effects of bufalin on plasma membrane potential, as determined by monitoring the uptake by cells of rhodamine 123, Bufalin induced apoptosis in human monocytic leukemia THP-1 cells, in human lymphoblastic leukemia MOLT-3 cells, and in human colon adenocarcinoma COLO320DM cells but not in normal human leukocytes, for example, polymorphonuclear cells and lymphocytes, and not in murine leukemia P388D1 and M1 cells, Treatment for 3 h with bufalin at 10(-6) M caused a decrease in the plasma membrane potential in several lines of human tumor cells but not in murine leukemia cells, No changes in mitochondrial membrane potential, as monitored with the fluorescent dye JC-1, and no release of cytochrome c were observed within at least 6 h after the start of treatment with bufalin, Moreover, overexpression of bcl-2 in human leukemia HL60 cells that had been transfected with cDNA for bcl-2 prevented bufalin-induced apoptosis but, had no significant effect on the change in plasma membrane potential induced by bufalin, Since bufalin specifically inhibits the Na+,K+-ATPase of human but not murine tumor cells, and since this inhibition leads to a change in intracellular concentration of Na+ ions, our findings suggest that bufalin induces apoptosis in human tumor cells selectively via inhibition of the Na+,K+-ATPase, which acts upstream of the bcl-2 protein.