Evaluation of the activity of cardiac glycosides on RORγ and RORγT nuclear receptors

Cardiac glycosides, derived from plants and animals, have been recognized since ancient times. These substances hinder the function of the sodium-potassium pump within eukaryotic cells. Many reports have shown that these compounds influence the activity of nuclear receptors. Thus, we assessed the effects of various cardiac glycosides at nontoxic concentrations on ROR gamma and ROR gamma T. ROR gamma T is a crucial protein involved in the differentiation of Th17 lymphocytes. Sixteen analyzed cardiac glycosides exhibited varying toxicities in HepG2 cells, all of which demonstrated agonistic effects on ROR gamma, as confirmed in the ROR gamma-HepG2 reporter cell line. The overexpression of both the ROR gamma and ROR gamma T isoforms intensified the effects of these compounds. Additionally, these glycosides induced the expression of G6PC, a gene regulated by ROR gamma, in HepG2 cells. Subsequently, the effects of two endogenous cardiac glycosides (marinobufagenin and ouabain) and the three most potent glycosides (bufalin, oleandrin, and telecinobufagenin) were evaluated in Th17 primary lymphocytes. All of these compounds increased the expression of the IL17A, IL17F, IFNG, and CXCL10 genes, but they exhibited varying effects on GZMB and CCL20 expression. Molecular docking analysis revealed the robust binding affinity of cardiac glycosides for the ligand binding domain of the ROR gamma/ROR gamma T receptors. Thus, we demonstrated that at nontoxic concentrations, cardiac glycosides have agonistic effects on ROR gamma/ROR gamma T nuclear receptors, augmenting their activity. This potential can be harnessed to modulate the phenotype of IL17-expressing cells (e.g., Th17 or Tc17 lymphocytes) in adoptive therapy for combating various types of cancer.