a potential anticancer drug Adriana Katz Cherniavsky Lev M Ainbinder E Tal D and Karlish SJD Weizmann Institute of Science Israel Ion channels and pumps as cancer targets ID: 1046024
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1. Na,K-ATPase isoform-selective cardiac glycosides-a potential anti-cancer drug ?Adriana Katz,Cherniavsky-Lev M., Ainbinder E., Tal D. and Karlish SJDWeizmann Institute of Science, Israel
2. Ion channels and pumps as cancer targets!Over the years, several reports have suggested that cardiacglycosides may have an anticancer utilization. In vitro and ex vivo experiments have revealed that some cardiac glycosides induce potent andselective anticancer effects, which may occur at concentrations commonly found in the plasma of patients treated with these drugs.Na+ /K+ -ATPase could be targeted to combat chemoresistant cancers.
3. Na,K ATPase is a vital protein in all mammalian cells.Subunit targets into the membrane, 3 isoforms: b1 b2 b3a subunitFXYDCatalytic moiety, 4 isoforms:a1, ubiqutous , all tissuesa2, skeletal muscle, heart, eyesa3, neurons braina4, spermsmembraneCytoplasmExtracellular spaceaccessory protein, regulatorDigoxinSpecific inhibitor. . Na,K -ATPase is an oligomeric transmembrane protein, localized to the basolateral plasma membrane in most epithelial cells.Na,/K-ATPase pumps Na+ and K+ against their physiological gradients.
4. Na,K pump is essential for many physiological processesRenal function, and regulation of hypertension .Cardiac contractionRegulation of intra ocular pressure, IOP.And many more….All of the isoforms are expressed in a tissue and functional specific manner.a1/b1a1b1a2/b2a3b1a3/b1a1/b1a2/b1Expression of isoforms in different tissues.a2/b3a1/b1
5. The cardiac glycosides are an important class of naturally occurring drugs whose actions include both beneficial and toxic effects on the heart. Cardiac glycosides (CG)Lactone ringSteroidGlycone
6. DigoxinWithering W (1785). “An account of the foxglove and some of its medical uses: with practical remarks on dropsy and other diseases.”Digitalis purpureaBufo bufo BufalinStrophanthus gratusOuabainCardiac glycosides, naturally occurring in plants and animals.
7. Cardiac glycosides have a long history of therapeutic application. Plants containing cardiac steroids have been used as poisons and heart drugs at least since 1500 B.C. The early understanding of their positive inotropic effects facilitated their use as effective drugs for the treatment of heart-related pathologies, yet their toxicity remains a serious problem.More recently, considerable in vitro, in vivo and epidemiological data support novel roles for CG’s such as inducing apoptosis and inhibit the growth of cancer cell lines. Control+10uM CG+20uM CGLiang‑Fei Ye,et al.2013 Oncology letters
8. Slingerland M. et al Na+,K+ ATPaseNa+/Ca+2exchang.Na+/H+ exchang.Proposed mode of action of cardiac glycosides, CGThe decrease in intracellular K+ and increase in intracellular Na+ and Ca2+ following inhibition of the Na+/K+-ATPase may induce apoptosis
9. Na,K-ATPase as a versatile signal transducer ? Na+/K+-ATPase mayalso act as a signal transducer. When intact cells are exposed to digitalis drugs (e.g., ouabain and digoxin) specific inhibitorsof this enzyme various cell signaling pathways are activated leading to highly cell-specific down-stream consequences.
10. Proliferation of CG-treated cells.Breast cancer cells are not more sensitive to CG’s cytotoxicity than are normal cells.Normal Cancer cellsClifford RJ, and Kaplan JH 2013 PLOS ONE 8,
11. Purification and stabilization of isoforms of human Na,K-ATPase expressed in Pichia pastoris
12. Isoforms of human Na,K-ATPase expressed in Pichia pastorisFXYD1Detergent , C12E8Phosphatidyl serine,SOPSPhosphatidyl choline, PCCholesterol Functional, stable, detergent-soluble FXYD complexEnables different combinations of isoformsa1a23MWHuman isomers/1 purified enzymes
13. Isoform selectivity is determined by the sugar component!Digoxin, is partially 2-selective CG, while Ouabain shows very low selectivityBound 3H-Ouabain, fraction of control00.20.40.60.811.20.0010.010.1100.20.40.60.811.20.0010.010.1110DigoxigeninDigoxinDigoxigenin, μMDigoxin, μMCardiac glycoside affinity and selectivity for the isoforms.
14. CGCalculated Kd±SEM, nMRatio of Kd’s, ± SEa12a3a1/a2a1/a3Ouabain9.8±0.3321.9±0.56P=0.000111.1±1.30.44±0.010.88±0.1Digoxin87±6.025.6±2.8P=0.00125±2.4P=0.0013.39±0.433.48±0.41Digoxigenin270±21332±11307±270.81±0.060.88±0.1-Methyl digoxin129±2043±7P=0.01831±4P=0.0093.0±0.674.16±0.84Digitoxin38±318.3±4P=0.0214±3.9P=0.0082.07±0.482.70±0.78Digitoxigenin101±13.4125±13.6134±15.60.8±0.130.75±0.13BufalinMarinobufagenin42.5±6.52240±13745±82470±4540±112430±2050.94±0.220.91±0.061.06±0.330.92 ±0.1Isoform selectivity of Cardiac glycosides
15. Na,K-ATPase structure with bound ouabainOuabainThe residues common to a2 and a3 and different in a1 are all located on the extracellular loops at the entrance to the Ouabain binding site, close to the sugar moiety.
16. In vitro growth inhibitory concentrations at 50% (IC50) in human cancer cells after three days of culture in the presence of the drug of interestInhibition of cancer cell proliferation at low concentrations is used to assess the therapeutic potential of drug candidates in preclinical studies.
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19. LLC-PKMCF-7HCT-116TK-10Cell lines: LLC-PK pig kidney, MCF-7 breast cancer , HCT-116 colon cancer, TK-10 renal cancer.In vitro growth inhibitory concentrations at 50% (IC50) in human cancer cells after two days of culture in presence of GBR, the most effective CG.1.
20. Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effectsDigoxinGBROleandrinHellebrig.OuabainHellebrin
21. 1234561. Digoxin2.Hellebrin3.Ouabain4.Oleandrin5.Hellebrigenin6. Gamabuf. Rhamnoside4 repetitions with 14 different cancer cells.Correlation of Ki for inhibition of human α1β1 by cardiac glycosides and the growth inhibition effectsCell growth is linearly correleted to the affinity of individual CG to the Na,K-ATPaes
22. Relative binding capacity (%of α1-GFP)Viability at 30 nM Ouabain (% of NT)α1-GFPSi-α1-8Si-α1-5Si-α1-3Viability is linearly correlated with number of active pumps in partially silenced H1299 cells
23. Viability (%of NT)Viability (%of NT)Bmax (pmol oub*mg protein-1)Total α1 (a.u.)hffh1299ovcar3hekpanc1helaskmel5mcf7lncapa549llcpk1mdamb231hffh1299ovcar3hekpanc1helaskmel5mcf7lncapa549llcpk1mdamb231h1299hffmda-mb-231llcpk1a549skmel5ovcar3lncapmcf7helahekpanc1anti α1anti α2anti α3 α1 α2 α350 ng Pur. Pr.ABCViability is linearly correlated with number of active pumps in a panel of cells
24. Although cardiac glycosides can inhibit the proliferation of cancer cells at very low concentrations (nM), they inhibit the proliferation of human nonmalignantcells at similar concentrations; this strongly suggests that their potential for cancer therapy is low.More experimental data are needed to further decipher the structure-activity relationship between CG’s and cancer cell cytotosicity.Viability is linearly correlated with number of active pumps– CG affect cancer cell viability only through binding and inhibition of NaK ATPase transport
25. Steven KarlishMarina Cherniavsky-Lev Elena Ainbinder Daniel TalYotam NadavMichael HabeckAcknowledgmentsThank you for your attention.