Research Of Anticancer Effect And The Relevant Target Of Ouabain

Objective:Research the effect of ouabain on human cancer cells to inhibit the proliferation and induce the apoptosis;To study the ouabain of different concentrations on Na+/K+-ATPase alphal isoform and alpha3isoform expression, Na+/K+-ATPase alphal and alpha3isoform was silenced by RNAi method, so that further confirmed the target of cardiac glycoside anticancer.Method:The growth inhibition of ouabain on OS-RC-2by MTT assay;The cell apoptosis of OS-RC-2was assayed by staining of cell with AO-EB, DNA fragmentation and AnnexinV-TITC/PI FCM,and OS-RC-2Cell cycle was detected by staining of cell with PI; The changes of intracellular reactive oxygen species and free calcium of OS-RC-2were determined with Laser confocal microscopy; Protein expression of Bax and Bcl-2in OS-RC-2was detected by western blot.The changes of drug sensitivity to ouabain on NCI-H446after Na+/K+-ATPase isoform RNAi silenced were analyzed by MTT assay; The protein expression of Na+/K+-ATPase alphal isoform and alpha3isoform of NCI-H446on different concentrations detected by Immunohistochemistry. The Na+/K+-ATPase alpha1isoform and alpha3isoform of NCI-H446was silenced by RNAi method; The mRNA expression of Na+/K+-ATPase alphal isoform and alpha3isoform of NCI-H446after silenced were identified by Real-time PCR and the protein expression of Na+/K+-ATPase alphal isoform and alpha3isoform of NCI-H446after silenced were identified by Immunohistochemistry.Result:Ouabain inhibited the OS-RC-2cells proliferation at20nmol·L-1.As shown by AO-EB fluorescence staining, DNA fragmentation assay and AnnexinV-TITC/PI FCM detection, OS-RC-2cells apoptosis could be induced by ouabain, PI staining assay indicated that the ratio of S phase of OS-RC-2cells was increased. Ouabain increased the level of intracellular reactive oxygen species and Ca2+obviously.The Bax protein expression increased, and the Bcl-2protein expression decreased in OS-RC-2cells after digitoxin treatment.The protein expression of Na+/K+-ATPase alphal isoform and alpha3isoform of NCI-H446are in the same.After Na+/K+-ATPase alphal and alpha3isoform of NCI-H446was silenced, the cells sensitivity to ouabain may be dependent on relative expression of Na+/K+-ATPase alpha3isoform.Conclusion:Ouabain can remarkably inhibite the OS-RC-2cells proliferation,and efficiently induce apoptosis in OS-RC-2cells. Confirmed the target of cardiac glycoside anticancer may be Na+/K+-ATPase alpha3isoform.