Study On The Mechanism Of Cardiac Glycoside-induced Suppression Of HIF-1α

Objectives:Recent studies have proposed putative anti-cancer effects of cardiac glycosides and HIF-1α has been recognized as the critical target of cardiac glycosides for cancer therapy, but the mechanism is still poorly understood. In present work, we investigate the potential mechanism of cardiac glycoside-mediated inhibition of HIF-1α protein. These observations not only raise the hope of using cardiac glycosides for cancer therapeutics more rational, but also provide the basis for further developing the novel HIF-1α inhibitors.Methods:(1) Western Blot and real-time RT-PCR were used to detect the expression levels of protein and mRNA after treatment of cardiac glycosides;(2) eIF-2a, Na+/K+ATPase and eIF-4E were silenced by small interfering RNA technique, Then measure HIF-1α protein after treatment of cardiac glycosides;(3) Cells were transfected with the plasmid PCMV-myc3-eIF4E to detect the expression levels of HIF-la protein after treatment of cardiac glycosides;(4) Molecular docking model showed the interaction between cardiac glycosides and translation factors;(5) To detect the effect of Ouabain on protein translation with a rabbit reticulocyte lysate in vitro translation assay;(6) we developed two dual reporter assays to determine the effect of cardiac glycosides on cap-dependent translation;(7) Immunoprecipitation was used to determine the interaction between eIF4E protein and HIF-1α mRNA or eIF-4G protein after treatment of Ouabain;(8) SRB assay was used to determine the effect of eIF4E protein on Proliferation inhibition of Ouabain.Results:1Cardiac glycosides inhibited the cellular expression of HIF-la protein.The different cancer cell lines (OVCAR8, HepG2, U2OS, AGS and Hela) were treated with variable concentrations of Ouabain (100,200,400,800nM) under hypoxic conditions for8h. Ouabain decreased hypoxia-induced HIF-1α protein expression in a concentration dependent manner in all cell lines. Similarly, Ouabain also blocked the accumulation of HIF-la induced by the cobalt chloride. Additionally, Proscillaridin A blocked HIF-la expression at a concentration significantly lower than Ouabain, while Digitonin failed to inhibit HIF-la expression.2Cardiac glycosides do not affect protein degradation, but decreases HIF-1α protein synthesis.Both the semi-quantity PCR and the Real-time PCR analysis indicated that HIF-la mRNA levels in U2OS cells were not significantly changed after Ouabain treatment. U2OS cells were treated with the ribosomal inhibitor, cycloheximide (CHX), under hypoxic conditions in the presence or absence of Ouabain at different time points. Western blotting analysis indicated that degradation rates of HIF-la were similar for both treated and untreated cells. Additionally, proteasomal inhibition MG132treatment did not reverse the Ouabain-triggered decrease of HIF-1α protein levels, implying the involvement of protein synthesis in HIF-lareducation. The rate of HIF-1α protein synthesis analysis indicated that Ouabain (l00nM) and Proscillaridin A (50nM) decreased the rate of HIF-1α protein synthesis effectively.3mTORC1, eIF2a and Na+/K+ATPase played a negligible role in the cardiac glycosides-triggered reduction of HIF-1αTreatment of U2OS cells with Ouabain at concentrations that potently inhibited HIF-1α protein expression, had no inhibitory effect on4E-BP1and mTOR phosphorylation. These results indicated that Ouabain inhibited HIF-1α translation by a mechanism that is independent of mTORC1. Although cardiac glycosides blocked the expression of HIF-1α accompanied while increasing the amount of phosphorylated eIF2a, Digitonin upregulated the phosphorylation of eIF2a without affecting the expression of HIF-1α.Moreover, the knockdown of eIF2α attenuated basal HIF-1α protein levels, but did not reverse the effects of cardiac glycosides on HIF-1αtranslation repression, implying that down-regulation of HIF-1α expression was independent of eIF2a phosphorylation. Knockdown of the Na+/K+-ATPase α1subunit had no effect on the levels of HIF-la protein but resulted in a robust accumulation of p-eIF2a, with no additional increase upon treatment with Ouabain.4cardiac glycosides inhibit the intracellular cap-dependent translationOuabain dramatically inhibited protein systhesis with a rabbit reticulocyte lysate in vitro translation assay. The molecular docking model exhibited that eIF4E is the most preferable target for Ouabain. We developed two dual reporter assays to determine the effect of cardiac glycosides on cap-dependent and cap-independent translation. Ouabain appears to inhibit cap-dependent translation, but had no effect on cap-independent translation. Moreover, we observed Ouabain inhibited the cellular expression of Cyclin D1, a protein regulated by eIF4E in post-transcriptional level. Thus, Ouabain inhibits eIF4E activity.5A critical role of eIF4E in Ouabain-driven HIF-1adownregulation.A positive correlation between the expression of eIF4E and HIF-1α among a panel of human cancer cell lines (R=0.882, P=0.004), including prostate (DU145), osteosarcoma (U2OS), liver (HepG2and SMMC-7721), lung (A549and95D), ovary (SKOV3) and cervical (HeLa) cancer cells was observed. U2OS cells were transfected with siRNA against eIF4E and knocking down the expression of eIF4E dramatically downregulated HIF-la.Consistent with these results, ectopic overexpressed eIF4E, U2OS cells resulted in increased expression of HIF-la. So We determined whether eIF4E is required for cardiac glycosides dependent inhibition of HIF-1α protein expression. The knockdown of eIF4E reversed the effects of Ouabain or Proscillaridin on HIF-1α expression under hypoxic conditions, while ectopic over-expression of eIF4E reduced cardiac glycosides driven HIF-1α downregulation in a concentration dependent manner. These results suggested that cardiac glycosides inhibited HIF-1α translation by disturbing the intracellular function of eIF4E. The immunoprecipitation analysis revealed that interaction between eIF4E and eIF4G was totally blocked after cells treated with Ouabain rather than the intracellular interaction between eIF4E and HIF-1α mRNA. In addition, ectopic over-expression of eIF4E reduced inhibition of tumor cell proliferation by Ouabain.Conclusion:In summary, the present study demonstrates for the first time that the interaction between eIF4E and eIF4G could serve as the molecular target of cardiac glycosides to mediate the inhibition of HIF-1α protein, rather than interaction between eIF4E and HIF-1α mRNA, the inhibition of the mTORC1, eIF2α signalings and Na+/K+ATPase. Cardiac glycosides blocked the participation of eIF4E in the formation of the cap-dependent translation initiation complex eIF4F to inhibit HIF-la translation.We believe these observations not only raise the hope of using cardiac glycosides for cancer therapeutics more rational, but also contribute a potential structure for further developing the novel HIF-la inhibitors.