Cardenolides are cardiac glycosides, mostly obtained from natural sources. They are well known for their inhibitory action on the Na,K-ATPase, an effect that regulates cardiovascular alterations such as congestive heart failure and atrial arrhythmias. In recent years, they have also sparked new interest in their anticancer potential. In the present study, the cytotoxic effects of the natural cardenolide convallatoxin (CON) were evaluated on non-small cell lung cancer (A549 cells). It was found that CON induced cytostatic and cytotoxic effects in A549 cells, showing essentially apoptotic cell death, as detected by annexin V-propidium iodide double-staining, as well as changes in cell form. In addition, it prompted cell cycle arrest in G2/M and reduced cyclin B1 expression. This compound also increased the number of cells in subG1 in a concentration- and time-dependent manner. At a long term, the reduction of cumulative population doubling was shown along with an increase of β-galactosidase positive cells and larger nucleus, indicative of senescence. Subsequently, CON inhibited the Na,K-ATPase in A549 cells at nM concentrations. Interestingly, at the same concentrations, CON was unable to directly inhibit the Na,K-ATPase, either in pig kidney or in red blood cells. Additionally, results of docking calculations showed that CON binds with high efficiency to the Na,K-ATPase. Taken together, our data highlight the potent anticancer effects of CON in A549 cells, and their possible link with non-classical inhibition of Na,K-ATPase.
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The Brazilian authors would like to thank the funding agencies CAPES /MEC (Ministry of Education) and CNPq/MCTI (Ministry of Science, Technology and Innovation) for their research scholarships. This work was also supported by the CNPq [Grants 472544/2013-6 and 490057/2011-0], the Marie Curie Foundation—IRSES/European Community [Grant 295251], and CAPES [Grant PNPD 2257/2011].
Koh PK, Faivre-Finn C, Blackhall FH, De Ruysscher D (2012) Targeted agents in non-small cell lung cancer (NSCLC): clinical developments and rationale for the combination with thoracic radiotherapy. Cancer Treat Rev 38:626–640. doi:10.1016/j.ctrv.2011.11.003CrossRefPubMedGoogle Scholar
Wang Y, Qiu Q, Shen J-J et al (2012) Cardiac glycosides induce autophagy in human non-small cell lung cancer cells through regulation of dual signaling pathways. Int J Dev Biol 44:1813–1824. doi:10.1016/j.biocel.2012.06.028Google Scholar
Felth J, Rickardson L, Rosén J et al (2009) Cytotoxic effects of cardiac glycosides in colon cancer cells, alone and in combination with standard chemotherapeutic drugs. J Nat Prod 72:1969–1974. doi:10.1021/np900210mCrossRefPubMedGoogle Scholar
Cerella C, Muller F, Gaigneaux A et al (2015) Early downregulation of Mcl-1 regulates apoptosis triggered by cardiac glycoside UNBS1450. Cell Death Dis 6:e1782. doi:10.1038/cddis.2015.134
Pongrakhananon V, Stueckle TA, Wang HL et al (2014) Monosaccharide digitoxin derivative sensitize human non-small cell lung cancer cells to anoikis through Mcl-1 proteasomal degradation. Biochem Pharmacol 88:23–35. doi:10.1016/j.bcp.2013.10.027CrossRefPubMedGoogle Scholar
Hong DS, Henary H, Falchook GS et al (2014) First-in-human study of pbi-05204, an oleander-derived inhibitor of akt, fgf-2, nf-κΒ and p70s6k, in patients with advanced solid tumors. Invest New Drugs 32:1204–1212. doi:10.1007/s10637-014-0127-0CrossRefPubMedGoogle Scholar
Schneider NFZ, Geller FC, Persich L et al (2016) Inhibition of cell proliferation, invasion and migration by the cardenolides digitoxigenin monodigitoxoside and convallatoxin in human lung cancer cell line. Nat Prod Res 30:1327–1331. doi:10.1080/14786419.2015.1055265CrossRefPubMedGoogle Scholar
Jorgensen P (1974) Purification and characterization of (Na + plus K+)-ATPase. 3. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim Biophys Acta 12:36–52CrossRefGoogle Scholar
Jensen BYJ, Nrby JG, Ottolenghi P (1984) Binding of sodium and potassium to the sodium pump of pig kidney evaluated from nucleotide-binding behaviour. J Physiol 346:219–241CrossRefPubMedPubMedCentralGoogle Scholar
Sousa L, Garcia IJP, Costa TGF et al (2015) Effects of iron overload on the activity of Na, K-ATPase and lipid profile of the human erythrocyte membrane. Plos ONE 10:e0132852. doi:10.1371/journal.pone.0132852
Fiske C, Subbarow Y (1825) The colorimetric determination of phosphorus. J Biol Chem 66:375–400Google Scholar
Noël F, Pimenta PHC, Dos Santos AR et al (2011) ∆2,3-ivermectin ethyl secoester, a conjugated ivermectin derivative with leishmanicidal activity but without inhibitory effect on mammalian P-type ATPases. Naunyn–Schmiedeberg’s Arch Pharm 383:101–107. doi:10.1007/s00210-010-0578-6CrossRefGoogle Scholar
Leu WJ, Chang HS, Chan SH et al (2014) Reevesioside A, a cardenolide glycoside, induces anticancer activity against human hormone-refractory prostate cancers through suppression of c-myc expression and induction of G1 arrest of the cell cycle. PLoS ONE 9:1–13. doi:10.1371/journal.pone.0087323Google Scholar
Lapenna S, Giordano A (2009) Cell cycle kinases as therapeutic targets for cancer. Nat Rev Drug Disc 8:547–566. doi:10.1038/nrd2907
Van Quaquebeke E, Simon G, RE A et al (2005) Identification of a novel cardenolide (2′’-oxovoruscharin) from Calotropis procera and the hemisynthesis of novel derivatives displaying potent in vitro antitumor activities and high in vivo tolerance: structure–activity relationship analyses. J Med Chem 48:849–856. doi:10.1021/jm049405aCrossRefPubMedGoogle Scholar
Feng B, Guo Y-W, Huang C-G et al (2010) 2′-epi-2′-O-acetylthevetin B extracted from seeds of Cerbera manghas L. induces cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells. Chem Biol Interact 183:142–153. doi:10.1016/j.cbi.2009.10.012CrossRefPubMedGoogle Scholar
Bielawski K, Winnicka K, Bielawska A (2006) Inhibition of DNA topoisomerases I and II, and growth inhibition of breast cancer MCF-7 cells by ouabain, digoxin and proscillaridin A. Biol Pharm Bull 29:1493–1497CrossRefPubMedGoogle Scholar
Weigand KM, Laursen M, Swarts HGP et al (2014) Na+,K+-ATPase isoform selectivity for digitalis-like compounds is determined by two amino acids in the first extracellular loop. Chem Res Toxicol 27:2082–2092. doi:10.1021/tx500290kCrossRefPubMedGoogle Scholar
Alves SLG, Paixão N, Ferreira LGR et al (2015) c-Benzylidene digoxin derivatives synthesis and molecular modeling†¯: evaluation of anticancer and the Na, K-ATPase activity effect. Bioorg Med Chem 23:4397–4404. doi:10.1016/j.bmc.2015.06.028CrossRefPubMedGoogle Scholar
Trenti A (2012) Analysis of the molecular mechanisms of the antineoplastic effect of ouabain. Università degli Studi di PadovaGoogle Scholar