生物活性

Ouabain is a cardiac glycoside that canselectively inhibit the activity of Na⁺/K⁺-ATPase.

Ouabain inhibits rat kidney Na⁺/K⁺-ATPasewith an IC50 of 91μM.[1]

Ouabain inhibits DNA synthesis with an IC50 of 142 ± 2nM in MDA-MB-231 cells.[2]

Na⁺/K⁺-ATPase activityinhibition of ouabain

Cytotoxicityof ouabain[5]

体外研究

体内研究

激酶实验

Preparation of Na⁺,K⁺-ATPasefrom rat kidney[1]

A rat kidney preparation was used in orderto work with only one single isoform (α1β1) of Na⁺,K⁺-ATPase.Adult maleWistar rats were killed by decapitation and their kidneys wererapidly excised and stored at −80 °C. Preparations enriched in Na⁺,K⁺-ATPasewere obtained by chaotropic treatment with 2 M KI for 1 h and 0.1% sodium deoxicholateover-night, followed by differential centrifugation. The protein concentrationwas measured using bovine serum albumin as the standard.

Inhibitionof Na⁺,K⁺-ATPase activity

The Na⁺,K⁺-ATPaseactivity was determined as follow. The specific activity of the enzyme correspondsto the difference between the total ATPase activity and the activity measuredin the presence of 1mM ouabain. The preparation was incubated at 37 °C for 2 h,in a total volume of0.5 mL. The incubation was performed in the presence of84mM NaCl, 3mM KCl, 3mM MgCl₂, 1.2mM ATPNa₂, 2.5mM EGTA,10mM sodium azide and 20mM maleic acid buffered to pH 7.4 with Tris in theabsence or presence of inhibitor(s). Classical concentration–effect curves wereperformed with each of the three inhibitors, alone. For the construction of thecombination curves, we used increasing concentrations of the mixture ouabain:ouabageninor LQB93:ouabain in the fixed ratio 1:4 for determining the IC50 value and theconcentrations pair (E) leading to 50%inhibition, used in the isobolographicanalysis. For the construction of the isobolograms, we also performed another setof experiments, using increasing concentrations of LQB93 (or ouabain) in thepresence of two fixed concentrations of ouabain (or ouabagenin) in order todetermine two other pairs of concentrations with the same effect level.

细胞实验

Tissue culture[2]

All studies were performed on breast cancerMDA-MB-231 cells cultured in DMEM supplemented with 10% FBS, 50 U/mLpenicillin, 50 μg/mL streptomycin at 37^oC. Cells were cultured inCostar flasks and subconfluent cells were detached with 0.05% trypsin and 0.02%EDTA in calciumfree phosphate-buffered saline (PBS), counted in hemocytometers andplated at 5×10⁵ cells per well of six-well plates (Nunc) in 2 mL ofgrowth medium (DMEM without phenol red with 10% CPSR1). Cells reached about 80%of confluency at day 3 and in most cases such cells were used for the assays.

Cellviability assay

The assay was performed using 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Confluentcells, cultured for 24 h with various concentrations of studied compounds insix-well plates were washed three times with PBS and then incubated for 4 h in1 mL of MTT solution (0.5 mg/mL of PBS) at 37^oC. The medium wasremoved and 1 mL of 0.1 mol/L HCl in absolute isopropanol was added to attachedcells. Absorbance of converted dye in living cells was measured at a wavelengthof 570 nm. Cell viability of breast cancer MDA-MB-231 cells cultured in thepresence of studied compounds was calculated as a percent of control cells.After treatment of the cells with drug, the ratio of survived to dead cells in testedand control (untreated) cells was calculated for each drug concentration. Cellnumber was plotted versusdrug concentration, and IC50 values were calculatedfrom dose-response curves as the concentration of drugs that reduce the numberof viable cells to 50% of control using an Origin 7.5 software.

动物实验

Nude mouse xenograft assays[5]

Male athymic BALB/c nude mice at 4–5 weeksof age were used in the study. Mice were housed in wire-top cages with sawdustbedding in an isolated, clean, air-conditioned room at a temperature of 25–26°C and a relative humidity of ~50 %, lit 12 h/day. EC109/CDDP cells weresubcutaneously inoculated into athymic mice. Briefly, cells grown at logarithmphase were harvested, washed, and then resuspended in PBS at 2 × 10⁷ cells/mL. A cell resuspension of 200μL (4 × 10⁶ cells) was inoculateds.c. into the right flank of athymic mice each. For tumor growth analysis,tumor volume was calculated according to the formula V = 1/2ab²,where a and b represent the length and the width of tumor measured with slidingcaliper, respectively. The animals were monitored for tumor inspection everyother day. Drug treatment was not initiated until tumor volume reached about 50mm³. Tumor-bearing animals were randomly divided into three groupsof six animals each: four experimental groups including CDDP (3 mg/ kg) alone,ouabain (0.1 mg/kg) alone, CDDP (3 mg/kg) plus ouabain (0.1 mg/kg) groups, andcontrol group (PBS). The treatment schedule was a single injection (i.p.) ofCDDP or ouabain per animal every other day for 2 weeks (Table 2). Allprocedures were conducted in a laminar-flow biosafety hood. Inhibition rate = 1− [mean tumor weight of experimental group] / [mean tumor weight of controlgroup] × 100 %. After being treated for 2 weeks, tumor-bearing mice were sacrificedand the tumor from each mouse was harvested.

Mice livers were harvest for hematoxylin–eosin(HE) staining and aspartate transaminase (AST) and alanine transaminase (ALT)activities.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Pocas ES, Touza NA, da Silva AJ, Costa PR, Noel F. Synergistic interaction between ouabain and 8-methoxy-3,9-dihydroxy coumestan, a non-steroidal synthetic inhibitor of Na+,K+-ATPase. Life Sci. 2007;81(15):1199-1204.
[2] Winnicka K, Bielawski K, Bielawska A, Miltyk W. Apoptosis-mediated cytotoxicity of ouabain, digoxin and proscillaridin A in the estrogen independent MDA-MB-231 breast cancer cells. Archives of Pharmacal Research. 2007;30(10):1216-1224.
[3] O'Brien WJ LJ, Wallick ET. Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate. Arch Biochem Biophys. 1994 310(1):32-39.
[more]

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

相关化合物库

使用AMQUAR产品发表文献后请联系我们