The Growth Inhibition Of Ouabain On Cancer Cells And The Effect Of Ouabain On A549Cells Migration

The cardiac glycoside, known ligands of Na~+/K~+-ATPase, have beenwidely used for the treatment of heart failure and atrial fibrillation. In the1980s, Stenkvist found that women on digitalis therapy have reduced risk ofbreast cancer, and the cancer cell were characterized by a series of morebenign features. Haux reported a link between the high the plasmaconcentration of digitoxin and reduced risk for leukemia, lymphoma andtumors of kidney or urinary tract in9271patients. Since then, some scholarsstarted to pay attention to the anti-tumor effect of the cardiac glycosides. Thecardiac glycosides such as Digoxin and Digitoxin can inhibit the growth oftumor cells. Na~+/K~+-ATPase α isoform may be the main target for the cardiacglycosides. The relationship between the different subtypes of Na~+/K~+-ATPaseα isoforms and the anti-tumor effect of cardiac glycosides is not yet clear.More attention is focused on α1，α2, α3subtypes in the four subtypes. Somestudies indicated that the anti-tumor effect of cardiac glycosides may berelated to their intracellular signal transduction. In this study, we study theeffect of Na~+/K~+-ATPase inhibitor on of the cancer cells and the relationshipbetween the growth inhibiton of ouabain on cancer cells and Na~+/K~+-ATPaseα1, α2, α3, and further study the effect of ouabain on Src/EGER pathway.The main feature of malignant tumors is tumor metastasis, which is theimportant factor leading to death in cancer patients. During the tumor cellsderived from epithelial tissue migrate to distant place, epithelial-mesenchymal(EMT) frequently occurs. Cell invasiveness are enhanced in EMT. E-cadherin,N-cadherin and vimentin are the important markers of EMT. Non-small celllung cancer A549has the important feature of the type II alveolar epithelialand can get invasiveness and migration power by EMT. In this study, wedetect the influence of ouabain on migration of A549cells by wound healing assay and transwell chamber migration assay and the effect of ouabain onthe expression of E-cadherin,N-cadherin,Vimentin,MMP-2and MMP-9.PART1Relationship between the growth inhibition of ouabain on thecancer cells and Na~+/K~+-ATPase α subunitObjective:1To study the growth inhibition of ouabain on tumor cells and the relationshipwith Na~+/K~+-ATPase α1,α2, α3subunit.2To study the effects of ouabain on EGFR,PI3K, Src expression.Methods: The effect of ouabain on the growth of cancer cells wasevaluated by MTT assay. The effect of ouabain on the expression ofNa~+/K~+-ATPase α subunits, Src, EGFR, PI3K was detected by western blotanalysis.Results:1Ouabain inhibited the growth of four cancer cells in a concentration-andtime-dependent manner.The growth inhibition of ouabain on A549cells wasthe most significant, followed by EC109cells and TE-1cells, the weakest wasthe SK-BR-3cells.2The expression of Na~+/K~+-ATPase α1protein in EC-109cells was thehighest, followed by SK-BR-3and A549cells. The least was the TE-1cells;The expression of Na~+/K~+-ATPase α2protein in A549cells was the highest,followed by the EC-109,TE-1,SK-BR-3; there was no significant difference inthe expression of Na~+/K~+-ATPase α3protein in the above four cells.3The Na~+/K~+-ATPase α1, α2and α3antibodies all could block the growthinhibition of ouabain on A549cells.4Ouabain down-regulated the expression of Na~+/K~+-ATPase α1, α2and α3protein in A549and SK-BR-3cells. The down-regulation effect of ouabainwas more significant on A549than that on SK-BR-3cells at the sameconcentration (100nM).5Ouabain decreased the expression of Src, EGFR, PI3K in A549andSK-BR-3cells. The down-regulation effect was more significant on A549thanthat in SK-BR-3cells at the same concentration (100nM). 6Src inhibitor PP2and PI3K inhibitor LY294002did not affect theNa~+/K~+-ATPase α subunit expressionConclusions: The different growth inhibition of ouabain on the cancercells is not related with the expression of Na~+/K~+-ATPase α1, α2, α3;Na~+/K~+-ATPase α1, α2and α3subunits are involved in the effect of ouabainon A549cells; The inhibition effect of ouabain might be related with thedownregulation of Na~+/K~+-ATPase α subunit, Src, EGFR, and PI3Kprotein.PART2The effects of ouabain on A549cells migrationObjective: To study the effects of ouabain on A549cells migrationMethods: The effects of ouabain on A549cell migration was detected bythe wound-healing assay and the transwell chamber imigration assay.Theeffect of ouabain on E-cadherin, N-cadherin, vimentin, MMP-2, and MMP-9proteins expression was detect by western blot analysis.Results:1After treated with EGF50ng/ml, the cells became long and narrow, grew outlonghorns and cell connections became loose. EGF significantly increased theexpression of vimentin protein and slightly increased the expression ofN-cadherin, and didn’n affect the expression of E-cadherin.2Ouabain inhibited the EGF-enhanced and basal migration of A549cells.3Ouabain down-regulated the expression of N-cadherin, Vimentin, MMP-2,MMP-9proteins, and didn’n affect the expression of E-cadherin.Conclusions: Ouabain inhibited the migration of A549cells and thiseffect may be due to the reducing expression of MMP-2and MMP-9.