Numazawa S, Shinoki M A, Ito H, Yoshida T, Kuroiwa Y
Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
J Cell Physiol. 1994 Jul;160(1):113-20. doi: 10.1002/jcp.1041600114.
Human leukemia K562 cell differentiation induction by naturally occurring bufadienolides purified from the Chinese drug Senso and synthetic bufalin derivatives was examined by a nitro blue tetrazolium reduction assay. Bufalin showed the strongest activity among all the bufadienolides tested in this study. The degree of the induction of nitro blue diformazan positive cells by the bufadienolides correlated well with their inhibitory activities against Na+,K(+)-ATPase prepared from K562 cells in vitro. N+,K(+)-ATPases from a variant K562 clone (ouabain resistant, OuaR) and murine leukemia cell line M1-T22, which were insensitive to the bufadienolides in terms of growth inhibition and cell differentiation, appeared to be refractory to bufalin in vitro. A binding study of 3H-bufalin and 3H-ouabain revealed that saturated levels of both ligands associated with K562 cells were virtually similar; however, affinity of 3H-bufalin was considerably higher than 3H-ouabain. The saturated level of 3H-bufalin observed in the OuaR cells was approximately half of that observed in K562 cells without a change in its affinity. Association of 3H-bufalin with K562 cells was completely blocked by pretreatment of the cells with cold ouabain at concentrations saturating the binding sites. These results suggest that bufalin acts on the cells by binding to sites on the cell membrane which also bind ouabain. It is thus proposed that N+,K(+)-ATPase inhibition is closely related to the initiation process in the induction of K562 cell differentiation induced by bufalin.
采用硝基蓝四氮唑还原试验，检测了从中药蟾酥中纯化得到的天然蟾毒配基及合成蟾毒灵衍生物对人白血病K562细胞的分化诱导作用。在本研究中检测的所有蟾毒配基中，蟾毒灵表现出最强的活性。蟾毒配基诱导硝基蓝二甲臜阳性细胞的程度与其体外对K562细胞制备的Na +,K(+)-ATP酶的抑制活性密切相关。来自K562变异克隆（哇巴因耐药，OuaR）和小鼠白血病细胞系M1-T22的Na +,K(+)-ATP酶，在生长抑制和细胞分化方面对蟾毒配基不敏感，在体外似乎对蟾毒灵也不敏感。3H-蟾毒灵和3H-哇巴因的结合研究表明，与K562细胞结合的两种配体的饱和水平实际上相似；然而，3H-蟾毒灵的亲和力明显高于3H-哇巴因。在OuaR细胞中观察到的3H-蟾毒灵饱和水平约为在K562细胞中观察到的一半，而其亲和力没有变化。用饱和结合位点浓度的冷哇巴因预处理细胞，可完全阻断3H-蟾毒灵与K562细胞的结合。这些结果表明，蟾毒灵通过与细胞膜上也结合哇巴因的位点结合而作用于细胞。因此，有人提出Na +,K(+)-ATP酶抑制与蟾毒灵诱导K562细胞分化的起始过程密切相关。
