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Inhibitory efficacy of bufadienolides on Na⁺,K⁺-pump activity versus cell proliferation.

ABSTRACT: Bufadienolides are cytotoxic drugs that may form the basis for anticancer agents. Due to structural and functional similarity to cardiotonic glycosides, application is restricted. We, therefore, investigated correlation of their putative anticancer effects with inhibition of Na⁺,K⁺pumps. The natural bufalin and three derivatives were tested. The anticancer effects of the drugs were checked by observing their inhibitory effects on proliferation of rat liver cancer cells using MTT assay. Inhibition of Na⁺,K⁺-pump was determined by measuring pump-mediated current of rat α1/β1 and α2/β1 Na⁺,K⁺pumps expressed in Xenopus oocytes. All tested bufadienolides inhibited cell proliferation and Na⁺,K⁺pump activity. An activity coefficient A=100xIC₅₀^{Na,K pump}/IC₅₀^pr^o^liferation was used to describe drug effectivity as anticancer drug. Natural bufalin exhibited lowest effectivity on cell proliferation, and also the A value for rat α1 isoform was the lowest (0.08), the α2 isoform was much less sensitive (A=1.00). The highest A values were obtained for the BF238 derivative with A=0.88 and 2.64 for the α1 and α2 isoforms, respectively. Therefore, we suggest that search for bufalin derivatives with high anticancer effect and low affinity for both Na⁺,K⁺pump isoforms may be a promising strategy for development of anticancer drugs.

SUBMITTER: Xu Y

PROVIDER: S-EPMC5600443 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Inhibitory efficacy of bufadienolides on Na+,K+-pump activity versus cell proliferation.

Xu Yinfang Y Liu Xuan X Schwarz Silvia S Hu Lihong L Guo Dean D Gu Quanbao Q Schwarz Wolfgang W

Biochemistry and biophysics reports 20160331

Bufadienolides are cytotoxic drugs that may form the basis for anticancer agents. Due to structural and functional similarity to cardiotonic glycosides, application is restricted. We, therefore, investigated correlation of their putative anticancer effects with inhibition of Na+,K+pumps. The natural bufalin and three derivatives were tested. The anticancer effects of the drugs were checked by observing their inhibitory effects on proliferation of rat liver cancer cells usin ...[more]

PMID: 28955873

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Project description:Na/K pumps build essential ion gradients across the plasmalemma of animal cells by coupling the extrusion of three Na+, with the import of two K+ and the hydrolysis of one ATP molecule. The mechanisms of selectivity and competition between Na+, K+, and inhibitory amines remain unclear. We measured the effects of external tetrapropylammonium (TPA+) and ethylenediamine (EDA2+) on three different Na/K pump transport modes in voltage-clamped Xenopus oocytes: 1) outward pump current (IP), 2) passive inward H+ current at negative voltages without Na+ or K+ (IH), and 3) transient charge movement reporting the voltage-dependent extracellular binding/release of Na+ (QNa). Both amines competed with K+ to inhibit IP. TPA+ inhibited IH without competing with H+, whereas EDA2+ did not alter IH at pH 7.6. TPA+ competed with Na+ in QNa measurements, reducing Na+-apparent affinity, evidenced by a ∼-75 mV shift in the charge-voltage curve (at 20 mM TPA+) without reduction of the total charge moved (Qtot). In contrast, EDA2+ and K+ did not compete with Na+ to inhibit QNa; both reduced Qtot without decreasing Na+-apparent affinity. EDA2+ (15 mM) right-shifted the charge-voltage curve by ∼+50 mV. Simultaneous occlusion of EDA2+ and Na+ by an E2P conformation unable to reach E1P was demonstrated by voltage-clamp fluorometry. Trypsinolysis experiments showed that EDA2+-bound pumps are much more proteolysis-resistant than Na+-, K+-, or TPA+-bound pumps, therefore uncovering unique EDA2+-bound conformations. K+ effects on QNa and IH were also evaluated in pumps inhibited with beryllium fluoride, a phosphate mimic. K+ reduced Qtot without shifting the charge-voltage curve, indicating noncompetitive effects, and partially inhibited IH to the same extent as TPA+ in non-beryllium-fluorinated pumps. These results demonstrate that K+ interacts with beryllium-fluorinated pumps inducing conformational changes that alter QNa and IH, suggesting that there are two external access pathways for proton transport by IH.

Project description:IntroductionHuman induced pluripotent stem cells (hiPSCs) harboring cardiac myosin heavy chain 6 promoter can differentiate into functional cardiomyocytes called "iCell cardiomyocytes" under blasticidin treatment condition. While iCell cardiomyocytes are expected to be used for predicting cardiotoxicity of drugs, their responses to antiarrhythmic agents remain to be elucidated. We first examined electrophysiological properties of iCell cardiomyocytes and mRNA levels of ion channels and Ca handling proteins, and then evaluated effects of class I antiarrhythmic agents on their Na+ and Ca2+ currents.MethodsiCell cardiomyocytes were cultured for 8-14 days (38-44 days after inducing their differentiation), according to the manufacturer's protocol. We determined their action potentials (APs) and sarcolemmal ionic currents using whole-cell patch clamp techniques, and also mRNA levels of ion channels and Ca handling proteins by RT-PCR. Effects of three class I antiarrhythmic agents, pirmenol, pilsicainide and mexiletine, on Na+ channel current (INa) and L-type Ca2+ channel current (ICaL) were evaluated by the whole-cell patch clamp.ResultsiCell cardiomyocytes revealed sinoatrial node-type (18%), atrial-type (18%) and ventricular-type (64%) spontaneous APs. The maximum peak amplitudes of INa, ICaL, and rapidly-activating delayed-rectifier K+ channel current were -62.7 ± 13.7, -8.1 ± 0.7, and 3.0 ± 1.0 pA/pF, respectively. The hyperpolarization-activated cation channel and inward-rectifier K+ channel currents were observed, whereas the T-type Ca2+ channel or slowly-activating delayed-rectifier K+ channel current was not detectable. mRNAs of Nav1.5, Cav1.2, Kir2.1, HCN4, KvLQT1, hERG and SERCA2 were detected, while that of HCN1, minK or MiRP was not. The class Ia antiarrhythmic agent pirmenol and class Ic agent pilsicainide blocked INa in a concentration-dependent manner with IC50 of 0.87 ± 0.37 and 0.88 ± 0.16 μM, respectively; the class Ib agent mexiletine revealed weak INa block with a higher IC50 of 30.0 ± 3.0 μM. Pirmenol, pilsicainide and mexiletine blocked ICaL with IC50 of 2.00 ± 0.39, 7.7 ± 2.5 and 5.0 ± 0.1 μM, respectively.ConclusionsIn iCell cardiomyocytes, INa was blocked by the class Ia and Ic antiarrhythmic agents and ICaL was blocked by all the class I agents within the ranges of clinical concentrations, suggesting their cardiotoxicity.

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Inhibitory efficacy of bufadienolides on Na⁺,K⁺-pump activity versus cell proliferation.

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Inhibitory efficacy of bufadienolides on Na<sup>+</sup>,K<sup>+</sup>-pump activity versus cell proliferation.

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