Silva Lilian N D, Pessoa Marco Tulio C, Alves Silmara L G, Venugopal Jessica, Cortes Vanessa F, Santos Herica L, Villar José A F P, Barbosa Leandro A
Laboratório de Bioquímica Celular, Universidade Federal de São João del Rei, Campus Centro-Oeste Dona Lindu, Divinópolis, MG, Brazil.
Laboratório de Síntese Orgânica e NanoEstruturas, Universidade Federal de São João Del Rei, Campus Centro Oeste Dona Lindu, Divinópolis, MG, Brazil.
Exp Cell Res. 2017 Oct 1;359(1):291-298. doi: 10.1016/j.yexcr.2017.07.017. Epub 2017 Jul 15.
Cardiotonic steroids (CTS) are compounds which bind to the Na,K-ATPase, leading to its inhibition and in some cases initiating signaling cascades. Long utilized as a treatment for congestive heart disease, CTS have more recently been observed to inhibit proliferation and cause apoptosis in several cancer cell lines. A synthetic derivative of the CTS digoxin, called 21-benzylidene digoxin (21-BD), activates the Na,K-ATPase rather than cause its inhibition, as its parent compound does. Here, the mechanism behind the unique effects of 21-BD are further explored. In HeLa cancer cells, low (5µM) and high (50µM) doses of 21-BD activated and inhibited the Na,K-ATPase, respectively, without altering the membrane expression of the Na,K-ATPase. While digoxin did not affect HeLa membrane cholesterol or phospholipid content, 50µM 21-BD increased both lipids via a mechanism reliant on an intact cell. Afterwards, the direct action of 21-BD was evaluated on erythrocyte membranes; however, no effect was observed. As CTS may generate reactive oxygen species (ROS) which can affect plasma membrane fluidity and therefore Na,K-ATPase activity, several markers involved in ROS generation were analyzed such as, lipid peroxidation (TBARS), reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD). GSH content and catalase activity were unaffected by digoxin or 21-BD. Surprisingly, TBARS and SOD activity was decreased with digoxin and with 50µM 21-BD. Thus, 21-BD and digoxin altered components involved in ROS generation and inhibition in a similar fashion. This study suggests alterations to the Na,K-ATPase and membrane lipids by 21-BD is not reliant on ROS generation.
强心甾类化合物（CTS）是一类能与钠钾ATP酶结合的化合物，可导致该酶受到抑制，在某些情况下还会引发信号级联反应。CTS长期以来一直被用作治疗充血性心脏病的药物，最近人们观察到它能抑制多种癌细胞系的增殖并诱导其凋亡。CTS地高辛的一种合成衍生物，称为21-亚苄基地高辛（21-BD），与其母体化合物不同，它能激活钠钾ATP酶而非抑制该酶。在此，我们进一步探究21-BD独特作用背后的机制。在HeLa癌细胞中，低剂量（5μM）和高剂量（50μM）的21-BD分别激活和抑制了钠钾ATP酶，且未改变钠钾ATP酶的膜表达。虽然地高辛不影响HeLa细胞膜胆固醇或磷脂含量，但50μM的21-BD通过一种依赖完整细胞的机制增加了这两种脂质的含量。之后，我们评估了21-BD对红细胞膜的直接作用；然而，未观察到任何影响。由于CTS可能产生活性氧（ROS），而ROS会影响质膜流动性，进而影响钠钾ATP酶的活性，因此我们分析了几种与ROS生成相关的标志物，如脂质过氧化（TBARS）、还原型谷胱甘肽（GSH）、过氧化氢酶（CAT）和超氧化物歧化酶（SOD）。地高辛或21-BD均未影响GSH含量和过氧化氢酶活性。令人惊讶的是，地高辛和50μM的21-BD均降低了TBARS和SOD活性。因此，21-BD和地高辛以相似的方式改变了参与ROS生成和抑制的成分。这项研究表明，21-BD对钠钾ATP酶和膜脂质的改变并不依赖于ROS的生成。
