Article ID: WMC004323 ISSN 2046-1690

The Cardiac Glycosides Digitoxin, Digoxin and Ouabain Induce a Potent Inhibition of Glycolysis in Lung Cancer Cells

Corresponding Author: Dr. Miguel Lopez-Lazaro, Associate Professor, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Submitting Author: Dr. Miguel Lopez-Lazaro, Associate Professor, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Other Authors: Dr. José Manuel Calderón-Montaño, Postdoctoral Researcher, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain Ms. Estefanía Burgos-Morón, PhD Student, Department of Pharmacology, Faculty of Pharmacy, University of Seville - Spain

Article ID: WMC004323 Article Type: Research articles Submitted on:08-Jul-2013, 04:34:33 PM GMT Published on: 09-Jul-2013, 05:31:30 AM GMT Article URL: http://www.webmedcentral.com/article_view/4323 Subject Categories:CANCER Keywords:Cardiotonic steroids, Anticancer, Digitalis, Cisplatin, Dichloroacetate How to cite the article:Calderón-Montaño J, Burgos-Morón E, Lopez-Lazaro M. The Cardiac Glycosides Digitoxin, Digoxin and Ouabain Induce a Potent Inhibition of Glycolysis in Lung Cancer Cells. WebmedCentral CANCER 2013;4(7):WMC004323 Copyright: This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Source(s) of Funding: University of Seville.

Competing Interests: The authors declare that they do not have conflicts of interest.

WebmedCentral > Research articles Page 1 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

The Cardiac Glycosides Digitoxin, Digoxin and Ouabain Induce a Potent Inhibition of Glycolysis in Lung Cancer Cells

Author(s): Calderón-Montaño J, Burgos-Morón E, Lopez-Lazaro M

Abstract decades suggest that cardiac glycosides have anticancer potential [4-10]. Evidence suggests that some cardiac glycosides induce selective anticancer effects [4, 11-13], which may occur at concentrations Cardiac glycosides are promising anticancer drugs. commonly found in the plasma of patients treated with We have recently shown that the cardiac glycosides these drugs [13, 14]. Other reports suggest, however, digitoxin, digoxin and ouabain induce selective killing that cardiac glycosides-induced cytotoxicity is not of lung cancer cells, and that the cytotoxicity of selective for cancer cells [15]. Some cardiac digitoxin against these cells occurs at concentrations glycosides have shown potent and selective below those observed in the plasma of cardiac anticancer effects in mice xenografted with human patients treated with this drug (Oncogene, 2013. doi: malignant cells [16-19]; however, these results may be 10.1038/onc.2013.229). Here we report that digitoxin, an experimental artifact caused by the ability of digoxin and ouabain induce a potent inhibition of cardiac glycosides to kill human cells versus rodent glycolysis (glucose consumption and lactate cells rather than by their ability to kill cancer cells production) in A549 cells at nanomolar concentrations. versus normal cells [13, 20]. The cardiac drugs This inhibition was comparable to that observed with digitoxin and digoxin, the semi-synthetic cardiac millimolar concentrations of the glycolysis inhibitor glycoside UNBS1450, and two extracts from Nerium dichloroacetate, which is currently undergoing clinical oleander have entered clinical trials for the treatment trials for the treatment of cancer. Because platinum of cancer (see http://clinicaltrials.gov/ and references compounds are commonly used in the treatment of [6, 7, 9, 21]). lung cancer, we tested the cytotoxicity of several combinations of cisplatin with each cardiac glycoside; Recently, we observed that the cardiac glycosides these combinations induced synergistic, antagonistic digitoxin, digoxin and ouabain induced selective killing or additive effects mainly depending on the order at of lung cancer cells, and that the cytotoxicity of which the drugs were added to the cells. digitoxin against these cells occurred at concentrations below those commonly observed in the plasma of Introduction cardiac patients treated with this drug [13]. We have also recently observed that an extract from the cardenolide-containing plant Nerium oleander induced Cardiac glycosides, also known as cardiotonic steroids, a marked inhibition of glycolysis in lung cancer cells are a group of natural steroidal compounds that [22]. In this article, we report that the cardiac contain an unsaturated lactone ring in their structure glycosides digitoxin, digoxin and ouabain induce a and have the ability to inhibit the Na+/K+-ATPase pump. potent inhibition of glycolysis in lung cancer cells at This lactone moiety defines the two classes of cardiac nanomolar concentrations. We also report that specific glycoside: cardenolides (with the lactone 2-furanone) combinations of these cardiac glycosides with cisplatin and bufadienolides (with the lactone 2-pyrone). may increase the cytotoxicity of this commonly used Numerous cardiac glycosides have been isolated from anticancer drug in lung cancer cells. plants (e.g. digitoxin, digoxin and ouabain) and several have also been found in amphibians and mammals Material and Methods (e.g. digoxin, ouabain and bufalin). Some cardiac glycosides are used in cardiology for the treatment of cardiac congestion and some types of cardiac Chemicals and cell lines arrhythmias. The mechanism by which these drugs Digitoxin (97%), digoxin (98.6%), ouabain (95%), affect cardiac contractility is known to be mediated by cisplatin (99.9%), and dichloroacetate (98%), were a highly specific inhibition of Na+/K+-ATPase [1-3]. purchased from Sigma. The human A549 lung cancer Several reports published over the last several cell line and the human embryo lung fibroblastic MRC-5 cell line were purchased from the European

WebmedCentral > Research articles Page 2 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Collection of Cell Cultures. The cell lines were was calculated with the computer software Compusyn. maintained in DMEM supplemented with 2 mM A CI value <0.90 is considered to be synergism and is glutamine, 50 µg/mL penicillin, 50 µg/mL streptomycin represented by “+++++” for very strong synergism and 10% fetal bovine serum, and were cultured at (CI<0.10), “++++” for strong synergism (CI=0.10-0.30), 37°C in a humidified atmosphere containing 5% CO2. “+++” for synergism (CI=0.30-0.70), “++” for moderate Cell culture reagents were obtained from Life synergism (CI=0.70-0.85) and “+” for slight synergism Technologies. (CI=0.85-0.90). A CI value between 0.90 and 1.10 Assay for cytotoxic activity corresponds with additive effect and is indicated with “±”. A CI value >1.10 is considered to be antagonism The MTT assay is a colorimetric technique that allows and is represented by “-” for slight antagonism the quantitative determination of cell viability. It is (CI=1.10-1.20), “--” for moderated antagonism based on the capability of viable cells to transform the (CI=1.20-1.45), “---” for antagonism (CI=1.45-3.30), MTT salt (3-(4,5-dimethylthiazol-2-yl) “----” for strong antagonism (CI=3.30-10.00) and “-----” -2,5-diphenyltetrazolium bromide) into a formazan dye. for very strong antagonism (CI>10.00) [24]. Exponentially growing cells were seeded into 96-well plates and drugs were added 24 h later. Following the Statistical analysis incubation period specified in figure and table legends, All data were averaged from at least three medium was removed and 125 µL MTT (1 mg/mL in independent experiments and were expressed as medium) was added to each well for 4 hours. Then, 80 means ± standard error of the means (SEM). For µL 20% SDS in 0.02 M HCl were added, plates were statistical analysis we used the t-test (paired, incubated for 10 hours at 37 ºC, and optical densities two-tailed). A P-value >0.05 is not considered were measured at 540 nm on a multiwell plate statistically significant and is not represented by any spectrophotometer reader. Cell viability was symbol. A P-value <0.05 is considered to correspond expressed as percentage in relation to controls. All with statistical significance and is indicated with an data were averaged from at least three independent asterisk (*), a P-value <0.01 is indicated with a double experiments and were expressed as means ± asterisk (**), and a P-value <0.001 is indicated with a standard error of the means (SEM). triple asterisk (***). Inhibition of glycolysis Results and Discussion Glycolysis inhibition was assessed by measuring concentrations of glucose (initial product of glycolysis) and lactate (final product of glycolysis) in control and In a recent work we reported that the cardiac 6 treated cells. Briefly, 10 cells were exposed in 6-well glycosides digitoxin, digoxin and ouabain were more plates to the tested compounds for 8 h, and glucose cytotoxic on A549 lung cancer cells than on MRC5 and lactate concentrations were determined in cell non-malignant lung fibroblasts [13]. We showed that supernatants by using the Accutrend® Plus analyzer the IC50 values (means ± SEM, nM) for the cancer cell together with Accutrend glucose strips and BM-Lactate line and the normal cell line were, respectively, 7.39 ± Strips (Roche Diagnostics). After calibrating the 0.6 and 77.5 ± 12.4 for digitoxin, 8.0 ± 1.3 and 65.1 ± instrument with glucose and lactate calibration strips, 13.2 for digoxin, and 5.3 ± 0.4 and 29.4 ± 4.5 for test strips were used to determine glucose and lactate ouabain. These data are particularly relevant for levels via colorimetric-oxidase mediator reactions digitoxin, as this drug was cytotoxic on the cancer cell according to the manufacturer's instructions [23]. line at concentrations below those commonly Results are expressed as percentage of lactate observed in the plasma of cardiac patients treated with production and percentage of glucose consumption in this drug (20-33 nM). Here we provide graphs showing relation to untreated cells, and are shown as means ± how the viability of both cell lines was affected by standard error of the means (SEM) of three several concentrations of each cardiac glycoside and independent experiments. of the anticancer drug cisplatin (Illustration 1). Combination Index Cardiac glycosides are known inhibitors of the Na+/K+ A549 cancer cells were exposed for 44-48 h to several -ATPase pump. It has been known for some time that concentrations of each cardiac glycoside, cisplatin and glycolysis is coupled to sodium and potassium each cardiac glycoside in combination with cisplatin transport processes, and that some cardiac glycosides (the cardiac glycosides were added 4 h before (e.g., ouabain) can inhibit glycolysis in a variety of cisplatin or vice versa). Cell viability was assessed by non-malignant cells [25, 26]. It has recently been the MTT assay. The parameter Combination Index (CI) found that cancer cells are more reliant on glycolysis

WebmedCentral > Research articles Page 3 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

for their survival than non-malignant cells (reviewed in metastasized to other organs [29]. This suggests that [27, 28]). Based on these findings, we previously SGLT2 plays a role in glucose uptake in the metastatic proposed that cardiac glycosides might inhibit lesions of lung cancer, and that inhibition of the Na+/K+ glycolysis in cancer cells and that such inhibition might -ATPase pump by cardiac glycosides may inhibit play a critical role in the selective cytotoxicity of some glycolysis by reducing glucose entry into the cells. The cardiac glycosides (e.g. digitoxin) towards cancer cells inhibition of glycolysis induced by digitoxin, digoxin [5]. More recently, we showed that an extract from the and ouabain in A549 lung cancer cells may therefore leaves of the cardenolide-containing plant Nerium be caused by inhibition of the Na+/K+-ATPase pump, oleander induced a marked inhibition of glycolysis in which would lead to both the inhibition of the glycolytic lung cancer cells [22]. To test if digitoxin, digoxin and enzyme PFK and the inhibition of glucose entry into ouabain could inhibit glycolysis in A549 lung cancer the cells. cells, were exposed these cells to each cardiac The inhibition of glycolysis observed in lung cancer glycoside for 8 h, and glucose and lactate cells treated with digitoxin, digoxin and ouabain may concentrations were determined in cell supernatants. play a role in their cytotoxicity. The main cellular roles Results, represented in Illustration 2, show that A549 of glycolysis are to provide building blocks for cells exposed to nanomolar concentrations of all three biosynthesis and ATP, and evidence suggests that cardiac glycosides exhibited a marked reduction in inhibition of these processes may have a higher glucose consumption (A) and lactate production (B), impact on the viability of cancer cells than on that of comparable to those observed in cells exposed to an non-malignant cells. Indeed, glycolysis inhibition has equi-toxic concentration of the glycolysis inhibitor been proposed to be an anticancer strategy to dichloroacetate (DCA, 10 mM). No significant changes selectively kill cancer cells [27, 28]. Although here we in glucose consumption and lactate production were report that digitoxin, digoxin and ouabain induce µ observed in cells exposed to cisplatin 32 M. selective cytotoxicity against lung cancer cells and that Illustration 2C includes representative photographs of they induce a potent inhibition of glycolysis in these cells exposed for 8 h to each drug, where cell number cells, we cannot conclude that the inhibition of and morphology can be appreciated. These glycolysis is responsible for their selective anticancer photographs seek to show that the reduction in activity. As discussed previously [22], evidence glucose consumption and lactate production induced suggests that the expression and cellular location of by the cardiac glycosides is not caused by a reduction Na+/K+-ATPase alpha subunits in different types of in cell number (fewer cells would consume less cells (i.e., rodent cells, human cancer cells and human glucose and would produce less lactate). Knowing that non-malignant cells) may explain why different cells glucose and lactate are, respectively, the initial and are more or less susceptible to the cytotoxic activity of final products of glycolysis, our data indicate that cardiac glycosides [30-32]. The study of the possible digitoxin, digoxin and ouabain induce a marked association between the expression and cellular inhibition of glycolysis in A549 lung cancer cells at location of Na+/K+-ATPase alpha subunits and the nanomolar concentrations. ability of cardiac glycosides to inhibit glycolysis would The inhibition of glycolysis induced by digitoxin, help reveal whether the inhibition of glycolysis by digoxin and ouabain (Illustration 2) may be mediated these compounds plays a role in their selective by their known ability o inhibit the Na+/K+-ATPase cytotoxicity. pump. Indeed, several mechanisms have been Most cancer chemotherapy regimens include a + + proposed to explain how the inhibition of the Na /K combination of drugs. Since platinum compounds are -ATPase pump can lead to glycolysis inhibition [5]. It widely used in the treatment of lung cancer, we + + has been proposed that the inhibition of the Na /K assessed the cytotoxic activity of digitoxin, digoxin and -ATPase pump would decrease glycolysis activity via ouabain in combination with cisplatin in A549 lung inhibition of the key glycolytic enzyme cancer cells. Then, we calculated the parameter phosphofructokinase (discussed in reference [5]). In Combination Index (CI) with the computer software + + addition, inhibition of the Na /K -ATPase pump may Compusyn; this parameter is based on the restrict the activity of sodium glucose transporters Chou-Talalay method and offers quantitative definition (SGLTs), which couple glucose entry into some types for additive effect, synergism, and antagonism in drug of cells with the activity of this pump [5]. These combinations [24]. A549 cells were exposed for 44-48 transporters are typically found in small intestine and h to several concentrations of cisplatin, each cardiac renal epithelial cells; however, clinical data have glycoside and cisplatin in combination with each revealed that these transporters (SGLT2) are cardiac glycoside. Cell viability was then estimated overexpressed in lung cancer cells that have

WebmedCentral > Research articles Page 4 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

with the MTT assay. Results are collected in Steroids-Mediated Na+/K+-ATPase Targeting Could Illustration 3 (digitoxin), Illustration 4 (digoxin) and Circumvent Various Chemoresistance Pathways Illustration 5 (ouabain), and show that these Planta Med. 2013. combinations induced synergistic, antagonistic or 10. Mijatovic, T.; Dufrasne, F.; Kiss, R. Cardiotonic additive effects mainly depending on the steroids-mediated targeting of the Na(+)/K(+)-ATPase concentrations of the drugs and on the order at which to combat chemoresistant cancers Curr. Med. Chem. they were added to the cells. Evidence suggests that 2012, 19, 627-46. the Na+/K+-ATPase alpha1 subunit plays a role in the 11. Sreenivasan, Y.; Raghavendra, P.B.; Manna, S.K. intracellular accumulation of cisplatin and that cardiac Oleandrin-mediated expression of Fas potentiates glycosides can reduce its intracellular accumulation apoptosis in tumor cells J. Clin. Immunol. 2006, 26, [33]; this may help explain some antagonisms 308-22. observed in these Illustrations. The synergisms 12. Hallbook, H.; Felth, J.; Eriksson, A.; Fryknas, M.; observed in these illustrations with some combinations Bohlin, L.; Larsson, R.; Gullbo, J. Ex vivo activity of might be caused by the ability of cardiac glycosides to cardiac glycosides in acute leukaemia PLoS. One. circumvent a variety of resistance pathways discussed 2011, 6, e15718. elsewhere [9, 10]. 13. Calderon-Montano, J.M.; Burgos-Moron, E.; In summary, we report for the first time that the cardiac Lopez-Lazaro, M. The in vivo antitumor activity of glycosides digitoxin, digoxin and ouabain induce a cardiac glycosides in mice xenografted with human potent inhibition of glycolysis in lung cancer cells, cancer cells is probably an experimental artifact which may participate in their selective cytotoxicity. Oncogene. 2013, Jun 10. doi: 10.1038/onc.2013.229. We also show that specific combinations of these [Epub ahead of print]. cardiac glycosides may increase the cytotoxicity of 14. Lopez-Lazaro, M.; Pastor, N.; Azrak, S.S.; Ayuso, platinum compounds, which are commonly used in the M.J.; Austin, C.A.; Cortes, F. Digitoxin inhibits the treatment of lung cancer. growth of cancer cell lines at concentrations commonly found in cardiac patients J Nat. Prod. 2005, 68, 1642-5. References 15. Perne, A.; Muellner, M.K.; Steinrueck, M.; Craig-Mueller, N.; Mayerhofer, J.; Schwarzinger, I.; Sloane, M.; Uras, I.Z.; Hoermann, G.; Nijman, S.M.; 1. Rahimtoola, S.H. and Tak, T. The use of digitalis in Mayerhofer, M. Cardiac glycosides induce cell death in heart failure Curr. Probl. Cardiol. 1996, 21, 781-853. human cells by inhibiting general protein synthesis 2. Xie, Z. and Askari, A. Na(+)/K(+)-ATPase as a PLoS. One. 2009, 4, e8292. signal transducer Eur. J. Biochem. 2002, 269, 2434-9. 16. Mijatovic, T.; Op, D.B.; Van Quaquebeke, E.; 3. Schoner, W. and Scheiner-Bobis, G. Endogenous Dewelle, J.; Darro, F.; de Launoit, Y.; Kiss, R. The and exogenous cardiac glycosides: their roles in cardenolide UNBS1450 is able to deactivate nuclear hypertension, salt metabolism, and cell growth Am. J. factor kappaB-mediated cytoprotective effects in Physiol Cell Physiol. 2007, 293, C509-C536. human non-small cell lung cancer cells Mol. Cancer 4. Haux, J. Digitoxin is a potential anticancer agent for Ther. 2006, 5, 391-9. several types of cancer Med. Hypotheses. 1999, 53, 17. Zhang, H.; Qian, D.Z.; Tan, Y.S.; Lee, K.; Gao, P.; 543-8. Ren, Y.R.; Rey, S.; Hammers, H.; Chang, D.; Pili, R.; 5. Lopez-Lazaro, M. Digitoxin as an anticancer agent Dang, C.V.; Liu, J.O.; Semenza, G.L. Digoxin and with selectivity for cancer cells: possible mechanisms other cardiac glycosides inhibit HIF-1alpha synthesis involved Expert. Opin. Ther. Targets. 2007, 11, and block tumor growth Proc. Natl. Acad. Sci. U. S. A. 1043-53. 2008, 105, 19579-86. 6. Newman, R.A.; Yang, P.; Pawlus, A.D.; Block, K.I. 18. Hiyoshi, H.; Abdelhady, S.; Segerstrom, L.; Cardiac glycosides as novel cancer therapeutic agents Sveinbjornsson, B.; Nuriya, M.; Lundgren, T.K.; Mol. Interv. 2008, 8, 36-49. Desfrere, L.; Miyakawa, A.; Yasui, M.; Kogner, P.; 7. Prassas, I. and Diamandis, E.P. Novel therapeutic Johnsen, J.I.; Andang, M.; Uhlen, P. Quiescence and applications of cardiac glycosides Nat. Rev. Drug gammaH2AX in neuroblastoma are regulated by Discov. 2008, 7, 926-35. ouabain/Na,K-ATPase Br. J. Cancer. 2012, 106, 8. Mijatovic, T.; Van Quaquebeke, E.; Delest, B.; 1807-15. Debeir, O.; Darro, F.; Kiss, R. Cardiotonic steroids on 19. Tailler, M.; Senovilla, L.; Lainey, E.; Thepot, S.; the road to anti-cancer therapy Biochim. Biophys. Acta. Metivier, D.; Sebert, M.; Baud, V.; Billot, K.; Fenaux, 2007, 1776, 32-57. P.; Galluzzi, L.; Boehrer, S.; Kroemer, G.; Kepp, O. 9. Mijatovic, T. and Kiss, R. Cardiotonic Antineoplastic activity of ouabain and pyrithione zinc in

WebmedCentral > Research articles Page 5 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

acute myeloid leukemia Oncogene. 2012, 31, 3536-46. combat non-small cell lung cancers J. Pathol. 2007, 20. Lopez-Lazaro, M. Digoxin, HIF-1, and cancer Proc. 212, 170-9. Natl. Acad. Sci. U. S. A. 2009, 106, E26. 32. Yang, P.; Cartwright, C.; Efuet, E.; Hamilton, S.R.; 21. Rashan, L.J.; Franke, K.; Khine, M.M.; Kelter, G.; Wistuba, I.I.; Menter, D.; Addington, C.; Shureiqi, I.; Fiebig, H.H.; Neumann, J.; Wessjohann, L.A. Newman, R.A. Cellular location and expression of Characterization of the anticancer properties of Na(+) , K(+) -ATPase alpha subunits affect the monoglycosidic cardenolides isolated from Nerium anti-proliferative activity of oleandrin Mol. Carcinog. oleander and Streptocaulon tomentosum J. 2012 10. Ethnopharmacol. 2011, 134, 781-8. 33. Kishimoto, S.; Kawazoe, Y.; Ikeno, M.; Saitoh, M.; 22. Calderon-Montano, J.M.; Burgos-Moron, E.; Orta, Nakano, Y.; Nishi, Y.; Fukushima, S.; Takeuchi, Y. M.L.; Mateos, S.; Lopez-Lazaro, M. A Hydroalcoholic Role of Na+, K+-ATPase alpha1 subunit in the Extract from the Leaves of Nerium oleander Inhibits intracellular accumulation of cisplatin Cancer Glycolysis and Induces Selective Killing of Lung Chemother. Pharmacol. 2006, 57, 84-90. Cancer Cells Planta Med. 2013,Jul 3. doi:10.1055/s-0032-1328715. [Epub ahead of print]. 23. Cao, X.; Bloomston, M.; Zhang, T.; Frankel, W.L.; Jia, G.; Wang, B.; Hall, N.C.; Koch, R.M.; Cheng, H.; Knopp, M.V.; Sun, D. Synergistic antipancreatic tumor effect by simultaneously targeting hypoxic cancer cells with HSP90 inhibitor and glycolysis inhibitor Clin. Cancer Res. 2008, 14, 1831-9. 24. Chou, T.C. Drug combination studies and their synergy quantification using the Chou-Talalay method Cancer Res. 2010, 70, 440-6. 25. Paul, R.J.; Bauer, M.; Pease, W. Vascular smooth muscle: aerobic glycolysis linked to sodium and potassium transport processes Science. 1979, 206, 1414-6. 26. James, J.H.; Fang, C.H.; Schrantz, S.J.; Hasselgren, P.O.; Paul, R.J.; Fischer, J.E. Linkage of aerobic glycolysis to sodium-potassium transport in rat skeletal muscle. Implications for increased muscle lactate production in sepsis J Clin. Invest. 1996, 98, 2388-97. 27. Lopez-Lazaro, M. A new view of carcinogenesis and an alternative approach to cancer therapy Mol. Med. 2010, 16, 144-53. 28. Pelicano, H.; Martin, D.S.; Xu, R.H.; Huang, P. Glycolysis inhibition for anticancer treatment Oncogene. 2006, 25, 4633-46. 29. Ishikawa, N.; Oguri, T.; Isobe, T.; Fujitaka, K.; Kohno, N. SGLT gene expression in primary lung cancers and their metastatic lesions Jpn. J. Cancer Res. 2001, 92, 874-9. 30. Yang, P.; Menter, D.G.; Cartwright, C.; Chan, D.; Dixon, S.; Suraokar, M.; Mendoza, G.; Llansa, N.; Newman, R.A. Oleandrin-mediated inhibition of human tumor cell proliferation: importance of Na,K-ATPase alpha subunits as drug targets Mol. Cancer Ther. 2009, 8, 2319-28. 31. Mijatovic, T.; Roland, I.; Van Quaquebeke, E.; Nilsson, B.; Mathieu, A.; Van Vynckt, F.; Darro, F.; Blanco, G.; Facchini, V.; Kiss, R. The alpha1 subunit of the sodium pump could represent a novel target to

WebmedCentral > Research articles Page 6 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Illustrations Illustration 1

A549 human lung cancer cells are more sensitive than MRC5 human non-malignant lung fibroblasts to the cytotoxic activity of digitoxin, digoxin and ouabain (MTT assay). The anticancer drug cisplatin was used as positive control.

WebmedCentral > Research articles Page 7 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Illustration 2

Inhibition of glycolysis in lung cancer cells by digitoxin, digoxin and ouabain A) Percentage of glucose consumption and B) percentage of lactate production in A549 lung cancer cells exposed for 8 h to each cardiac glycoside, dichloroacetate (DCA) and cisplatin. C) Representative photographs of A549 cells exposed for 8 h to each drug.

WebmedCentral > Research articles Page 8 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Illustration 3

Cytotoxic activity of the cardiac glycoside digitoxin in combination with the anticancer drug cisplatin.

First compound added Second compound added Combination Concentration % Cell viability Concentration % Cell viability % Cell viability CI Effect 1 nM digitoxin 83,7 ± 2,8 76,9 ± 1,6 1,1 ± 1 µM cisplatin 85,9 ± 2,3 3 nM digitoxin 66,2 ± 2,7 63,4 ± 1,1 1,1 ± 10 nM digitoxin 32,1 ± 1,3 29,9 ± 4,2 0,9 + 1 nM digitoxin 83,7 ± 2,8 64,5 ± 0,1 0,9 + 3 µM cisplatin 72,6 ± 2,8 3 nM digitoxin 66,2 ± 2,7 56,8 ± 1,2 1,1 ± 10 nM digitoxin 32,1 ± 1,3 30,7 ± 3,4 1,0 ± 1 nM digitoxin 83,7 ± 2,8 45,7 ± 2,2 0,8 ++ 10 µM cisplatin 53,8 ± 0,2 3 nM digitoxin 66,2 ± 2,7 36,7 ± 2,1 0,7 ++ 10 nM digitoxin 32,1 ± 1,3 23,6 ± 3,5 0,8 ++ 1 nM digitoxin 83,7 ± 2,8 31,1 ± 0,1 1,0 ± 32 µM cisplatin 33,0 ± 2,6 3 nM digitoxin 66,2 ± 2,7 25,8 ± 0,0 0,8 ++ 10 nM digitoxin 32,1 ± 1,3 16,84 ± 0,2 0,7 ++ 1 nM digitoxin 83,0 ± 1,5 83,3 ± 1,9 1,2 - 3 nM digitoxin 63,0 ± 2,7 1 µM cisplatin 96,3 ± 2,6 63,1 ± 1,0 1,2 - 10 nM digitoxin 23,9 ± 0,7 23,9 ± 2,3 1,0 ± 1 nM digitoxin 83,0 ± 1,5 69,2 ± 3,1 0,8 ++ 3 nM digitoxin 63,0 ± 2,7 3 µM cisplatin 84,7 ± 3,8 57,5 ± 7,2 1,2 - 10 nM digitoxin 23,9 ± 0,7 14,2 ± 0,1 0,6 +++ 1 nM digitoxin 83,0 ± 1,5 57,1 ± 1,0 1,0 ± 3 nM digitoxin 63,0 ± 2,7 10 µM cisplatin 59,8 ± 2,4 45,6 ± 1,4 1,1 ± 10 nM digitoxin 23,9 ± 0,7 13,2 ± 3,0 0,6 +++ 1 nM digitoxin 83,0 ± 1,5 35,0 ± 0,7 1,1 ± 3 nM digitoxin 63,0 ± 2,7 32 µM cisplatin 40,7 ± 2,8 30,8 ± 1,5 1,2 - 10 nM digitoxin 23,9 ± 0,7 15,7 ± 0,6 1,0 ±

WebmedCentral > Research articles Page 9 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Illustration 4

Cytotoxic activity of the cardiac glycoside digoxin in combination with the anticancer drug cisplatin.

First compound added Second compound added Combination Concentration % Cell viability Concentration % Cell viability % Cell viability CI Effect 1 nM digoxin 88,8 ± 2,6 87,3 ± 2,6 1,9 --- 1 µM cisplatin 85,9 ± 2,3 3 nM digoxin 75,6 ± 4,8 78,4 ± 9,2 1,7 --- 10 nM digoxin 45,1 ± 3,9 46,0 ± 5,9 1,1 ± 1 nM digoxin 88,8 ± 2,6 72,2 ± 1,9 1,2 - 3 µM cisplatin 72,6 ± 2,8 3 nM digoxin 75,6 ± 4,8 61,7 ± 1,1 1,1 ± 10 nM digoxin 45,1 ± 3,9 41,1 ± 1,3 1,0 ± 1 nM digoxin 88,8 ± 2,6 52,2 ± 0,2 1,0 ± 10 µM cisplatin 53,8 ± 0,2 3 nM digoxin 75,6 ± 4,8 45,0 ± 0,8 0,9 ± 10 nM digoxin 45,1 ± 3,9 35,1 ± 0,7 1,0 ± 1 nM digoxin 88,8 ± 2,6 28,7 ± 0,2 0,3 +++ 32 µM cisplatin 33,0 ± 2,6 3 nM digoxin 75,6 ± 4,8 29,5 ± 0,0 0,4 +++ 10 nM digoxin 45,1 ± 3,9 21,4 ± 0,1 0,4 +++ 1 nM digoxin 91,8 ± 2,8 91,2 ± 0,0 1,4 -- 3 nM digoxin 79,9 ± 1,2 1 µM cisplatin 96,3 ± 2,6 70,23 ± 0,1 0,7 ++ 10 nM digoxin 51,8 ± 7,8 45,41 ± 0,2 0,8 ++ 1 nM digoxin 91,8 ± 2,8 76,3 ± 1,8 0,8 ++ 3 nM digoxin 79,9 ± 1,2 3 µM cisplatin 84,7 ± 3,8 60,8 ± 0,9 0,7 ++ 10 nM digoxin 51,8 ± 7,8 50,4 ± 4,8 1,1 ± 1 nM digoxin 91,8 ± 2,8 58,2 ± 1,0 0,9 + 3 nM digoxin 79,9 ± 1,2 10 µM cisplatin 59,8 ± 2,4 47,1 ± 0,2 0,7 ++ 10 nM digoxin 51,8 ± 7,8 41,7 ± 6,7 1,1 ± 1 nM digoxin 91,8 ± 2,8 36,1 ± 2,5 1,0 ± 3 nM digoxin 79,9 ± 1,2 32 µM cisplatin 40,7 ± 2,8 36,6 ± 1,1 1,2 - 10 nM digoxin 51,8 ± 7,8 26,1 ± 1,4 1,0 ±

WebmedCentral > Research articles Page 10 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Illustration 5

Cytotoxic activity of the cardiac glycoside ouabain in combination with the anticancer drug cisplatin.

First compound added Second compound added Combination Concentration % Cell viability Concentration % Cell viability % Cell viability CI Effect 1 nM ouabain 79,4 ± 0,5 72,0 ± 2,9 1,0 ± 1 µM cisplatin 85,9 ± 2,3 3 nM ouabain 47,4 ± 4,0 46,1 ± 4,5 1,2 - 10 nM ouabain 5,9 ± 0,8 4,8 ± 0,5 0,8 ++ 1 nM ouabain 79,4 ± 0,5 60,3 ± 0,5 1,0 ± 3 µM cisplatin 72,6 ± 2,8 3 nM ouabain 47,4 ± 4,0 42,9 ± 5,0 1,3 -- 10 nM ouabain 5,9 ± 0,8 3,8 ± 0,4 0,7 ++ 1 nM ouabain 79,4 ± 0,5 45,1 ± 0,2 1,0 ± 10 µM cisplatin 53,8 ± 0,2 3 nM ouabain 47,4 ± 4,0 34,0 ± 1,6 1,2 - 10 nM ouabain 5,9 ± 0,8 4,3 ± 0,3 0,7 ++ 1 nM ouabain 79,4 ± 0,5 26,7 ± 0,0 0,9 ± 32 µM cisplatin 33,0 ± 2,6 3 nM ouabain 47,4 ± 4,0 22,9 ± 3,3 1,2 - 10 nM ouabain 5,9 ± 0,8 3,1 ± 1,0 0,6 +++ 1 nM ouabain 93,6 ± 0,9 80,6 ± 4,8 0,7 ++ 3 nM ouabain 65,6 ± 4,5 1 µM cisplatin 96,3 ± 2,6 59,6 ± 3,2 1,0 ± 10 nM ouabain 12,6 ± 4,9 8,7 ± 2,0 0,9 + 1 nM ouabain 93,6 ± 0,9 74,3 ± 3,0 0,9 + 3 nM ouabain 65,6 ± 4,5 3 µM cisplatin 84,7 ± 3,8 56,0 ± 1,8 1,1 ± 10 nM ouabain 12,6 ± 4,9 13,0 ± 8,0 1,0 ± 1 nM ouabain 93,6 ± 0,9 53,2 ± 1,8 0,9 + 3 nM ouabain 65,6 ± 4,5 10 µM cisplatin 59,8 ± 2,4 46,4 ± 1,0 1,2 - 10 nM ouabain 12,6 ± 4,9 17,0 ± 1,0 1,3 -- 1 nM ouabain 93,6 ± 0,9 38,2 ± 0,7 1,3 -- 3 nM ouabain 65,6 ± 4,5 32 µM cisplatin 40,7 ± 2,8 32,0 ± 1 1,3 -- 10 nM ouabain 12,6 ± 4,9 15,4 ± 2,5 1,4 --

WebmedCentral > Research articles Page 11 of 12 WMC004323 Downloaded from http://www.webmedcentral.com on 09-Jul-2013, 05:31:30 AM

Disclaimer

This article has been downloaded from WebmedCentral. With our unique author driven post publication peer review, contents posted on this web portal do not undergo any prepublication peer or editorial review. It is completely the responsibility of the authors to ensure not only scientific and ethical standards of the manuscript but also its grammatical accuracy. Authors must ensure that they obtain all the necessary permissions before submitting any information that requires obtaining a consent or approval from a third party. Authors should also ensure not to submit any information which they do not have the copyright of or of which they have transferred the copyrights to a third party. Contents on WebmedCentral are purely for biomedical researchers and scientists. They are not meant to cater to the needs of an individual patient. The web portal or any content(s) therein is neither designed to support, nor replace, the relationship that exists between a patient/site visitor and his/her physician. Your use of the WebmedCentral site and its contents is entirely at your own risk. We do not take any responsibility for any harm that you may suffer or inflict on a third person by following the contents of this website.

WebmedCentral > Research articles Page 12 of 12