Numazawa S, Inoue N, Nakura H, Sugiyama T, Fujino E, Shinoki M, Yoshida T, Kuroiwa Y
Department of Biochemical Toxicology, School of Pharmaceutical Sciences, Showa University, Tokyo, Japan.
Biochem Pharmacol. 1996 Jul 26;52(2):321-9. doi: 10.1016/0006-2952(96)00210-9.
Human monocytic leukemia THP-1 cells were induced to differentiate into macrophage-like cells by treatment with cardiotonic steroid bufalin, which was previously shown to interact with the Na+, K+-ATPase with similar kinetics to ouabain, a specific inhibitor of the enzyme. This induction of differentiation was characterized by loss of proliferation, cell adherence, increased ability to reduce Nitro Blue tetrazolium (NBT), and increased expression of interleukin 1 beta (IL-1 beta). During this process, bufalin downregulated c-myb and c-myc expressions and induced c-fos and Egr-1 transcripts. Ouabain also caused similar changes in proto- oncogene expression and induced phenotypic markers of differentiated cells at concentrations comparable to bufalin. The 12-O-tetradecanoyl phorbol-13-acetate resistant THP-1 cell variant, which was unresponsive to this agent as to growth inhibition and proto-oncogene expression, responded to bufalin. The finding that protein kinase inhibitor H7 failed to bufalin-mediated c-fos induction further supports the theory that the signal transduction machinery caused by bufalin is separable from the phorbol ester. The cytotoxic effect of high doses of bufalin apparently disappeared in the medium where Na+ was replaced with choline ions. Furthermore, bufalin failed to induce c-fos expression and to downregulate c-myb transcripts in the low-Na+ medium. These findings indicate that an increased intracellular Na+ concentration resulting from the Na+, K(+)-ATPase inhibition possibly triggers the change in proto-oncogene expression evoked by bufalin.
人单核细胞白血病THP-1细胞通过强心甾类化合物蟾毒灵处理被诱导分化为巨噬细胞样细胞，此前研究表明蟾毒灵与Na+，K+-ATP酶相互作用的动力学与哇巴因（该酶的特异性抑制剂）相似。这种分化诱导的特征包括增殖丧失、细胞黏附、还原硝基蓝四氮唑（NBT）的能力增加以及白细胞介素1β（IL-1β）表达增加。在此过程中，蟾毒灵下调c-myb和c-myc的表达并诱导c-fos和Egr-1转录本。哇巴因在与蟾毒灵相当的浓度下也引起原癌基因表达的类似变化并诱导分化细胞的表型标志物。12-O-十四烷酰佛波醇-13-乙酸酯抗性THP-1细胞变体对该试剂的生长抑制和原癌基因表达无反应，但对蟾毒灵有反应。蛋白激酶抑制剂H7未能抑制蟾毒灵介导的c-fos诱导，这一发现进一步支持了蟾毒灵引起的信号转导机制与佛波酯可分离的理论。高剂量蟾毒灵的细胞毒性作用在Na+被胆碱离子取代的培养基中明显消失。此外，在低Na+培养基中，蟾毒灵未能诱导c-fos表达并下调c-myb转录本。这些发现表明，由Na+，K(+)-ATP酶抑制导致的细胞内Na+浓度升高可能触发了蟾毒灵引起的原癌基因表达变化。
