Bufadienolides是在哺乳动物中鉴定的强心类固醇(CTS)。除了Na^/K^-ATPase抑制外，它们通过蛋白质-蛋白质相互作用激活信号转导。内源性bufadienolides的多样性和作用机制可能表明存在功能选择性和独特的细胞结果。我们评估了bufadienolidestelocinofifagin和marinofufagin是否诱导猪肾（LLC-PK1）细胞增殖或活力的变化以及这些变化的机制。在一些实验中,ouabain用作阳性对照。CTS表现出0.20的抑制IC50（telocinofagin），0.14(ouabain),在LLC-PK1细胞中测试了3.40μM（marinobufagin）对猪肾脏Na^/K^-ATPase活性和几乎不抑制它的浓度。CTS诱导快速ERK1/2磷酸化，但是对于marinobufagin和哇巴因而不是telocinobufagin观察到了相应的增殖反应。Telocinofagin增加Bax：Bcl-2表达率，亚G0细胞周期期和固缩核，表明细胞凋亡。Src和MEK1/2抑制剂减弱了marinofufagin的作用，但不减弱telocinofufagin的作用，这也不是由p38，JNK1/2和PI3K介导的。然而,BIO,GSK-3β抑制剂，减少扩散，作为telocinofagin，抑制Ser9磷酸化GSK-3β。两种药物的组合产生协同抗增殖作用。Wnt报告基因活性测定表明，端纤原蛋白通过在上游作用于β-连环蛋白稳定而损害Wnt/β-连环蛋白途径。我们的发现支持哺乳动物内源性bufadienolides可能表现出功能选择性。
Bufadienolides are cardiotonic steroids (CTS) identified in mammals. Besides Na⁺/K⁺-ATPase inhibition, they activate signal transduction via protein⁻protein interactions. Diversity of endogenous bufadienolides and mechanisms of action may indicate the presence of functional selectivity and unique cellular outcomes. We evaluated whether the bufadienolides telocinobufagin and marinobufagin induce changes in proliferation or viability of pig kidney (LLC-PK1) cells and the mechanisms involved in these changes. In some experiments, ouabain was used as a positive control. CTS exhibited an inhibitory IC50 of 0.20 (telocinobufagin), 0.14 (ouabain), and 3.40 μM (marinobufagin) for pig kidney Na⁺/K⁺-ATPase activity and concentrations that barely inhibited it were tested in LLC-PK1 cells. CTS induced rapid ERK1/2 phosphorylation, but corresponding proliferative response was observed for marinobufagin and ouabain instead of telocinobufagin. Telocinobufagin increased Bax:Bcl-2 expression ratio, sub-G0 cell cycle phase and pyknotic nuclei, indicating apoptosis. Src and MEK1/2 inhibitors blunted marinobufagin but not telocinobufagin effect, which was also not mediated by p38, JNK1/2, and PI3K. However, BIO, a GSK-3β inhibitor, reduced proliferation and, as telocinobufagin, phosphorylated GSK-3β at inhibitory Ser9. Combination of both drugs resulted in synergistic antiproliferative effect. Wnt reporter activity assay showed that telocinobufagin impaired Wnt/β-catenin pathway by acting upstream to β-catenin stabilization. Our findings support that mammalian endogenous bufadienolides may exhibit functional selectivity.
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