Tacrolimus(FK506) Induced Cytotoxicity Via TRAIL-R1 (DR4) or TRAIL-R2 (DR5) in Jurkat Cells.

Abstract

Purpose: To elucidate the mechanism of Tacrolimus(FK506) induced cytotoxicity via TRAIL(TNF related apoptosis inducing ligand)-R1(DR4) and TRAIL-R2(DR5) in FK506-treated Jurkat T cells. And signal transduction pathway of TNF-related events was studied. Methods: Viability of Jurkat T cells was measure by MTT assay. The catalytic activation of caspase-3 and caspase-9 proteases was determined by digestion of fluorogenic biosubstrates and western blot with anti-caspase-3 and anti-caspase-9 antibodies. The levels of mRNA and proteins for p53, Bax, PUMA, proline oxidase, TRAIL(TNF related apoptosis inducing ligand), TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, FasL, TNF-α, IL-6, and NKκB were measured by RT-PCR and western blot with specific antibodies. Also we further examined the localization of TRAIL family proteins using by fluorescent microscope with specific TRAIL family antibodies. Results: FK506 decreased the viability of Jurkat T cells dose- and time-dependently along with catalytic activation of caspase-3 and caspase-9, p53 phosphorylation, and changes in expression levels of Bax, PUMA, and proline oxidase protein. It caused an increase in expression of TRAIL, TRAIL-R1(DR4), TRAIL-R2(DR5), Fas, and FasL in the levels of mRNA and proteins of Jurkat T cells. Furthermore, FK506 increased extracellular release of TNF-α and IL-6 cytokines in Jurkat T cells. It also induced the transactivation of NKκB through the dephosphrylation of Ser486 residues in Jurkat t cells. Conclusion: These results suggest that FK506 induces apoptotic death of Jurkat cells through activation of caspase family protease, Bcl-2 family protein-related mitochondrial dysfunction, and activation of death-receptor mediated signaling pathways.