Geldanamycin Analog 17-DMAG Inhibits iNOS and Caspases in Gamma-Irradiated Human T Cells

Abstract

Inducible nitric oxide synthase (iNOS) expression and NO production increase after radiation exposure. We showed previously that inhibiting iNOS expression prevents hemorrhage injury; we therefore investigated whether inhibiting iNOS expression also limits radiation injury. Human Jurkat T cells were exposed to gamma radiation (2, 4, 6 or 8 Gy), and cell lysates were collected for analysis at selected times afterward. Radiation exposure increased iNOS expression within 4 h postirradiation by increasing the levels of the iNOS transcription factors NF-kappaB and KLF6. By 24 h postirradiation cell viability was reduced. In these cells, NO production, lipid peroxidation, protein nitration, apoptosomes (formed by cytochrome c, caspase 9 and Apaf-1), and caspase 3 activity were significantly elevated, suggesting that the iNOS pathway had been activated. Treatment with the iNOS inhibitors 17-DMAG or L-NIL-6 24 h prior to irradiation limited these changes, as did treatment with iNOS siRNA to silence the iNOS gene. These results suggest radiation injury involves the iNOS pathway, and iNOS-mediated NO produced endogenously in the T cell alters overall T-cell function and results in apoptosis and cell lethality. Control of iNOS expression may represent a useful approach for protecting T cells from radiation injury.

HSP90 inhibition by 17-DMAG reduces inflammation in J774 macrophages through suppression of Akt and nuclear factor-κB pathways

“…HSP90 has also been shown to complex with iNOS [63]. Inhibition of iNOS has been achieved with 17-DMAG which is a result of preventing HSP90 from forming a complex with iNOS as well as a result of increased HSP70 which suppresses iNOS activity [64]. We found that 17-DMAG significantly reduced immune-stimulated NO at a concentration (0.1 lM) that did not reduce cell viability.…”

Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans

“…For each acute dose of ionizing radiation, the Jurkat T cell trials (2 ml with 1×10 6 cells/ml) were performed in triplicate in 6-well microtiter plates. After irradiation, the microtiter plates were returned to the 5% CO 2 -incubator for the specified time (1–3 days), and cell viability (Figure 1E and S1) was determined with trypan blue [60]. For each trial, 50 µl of Jurkat T cells were mixed with 50 µl of 4% trypan blue; 20 µl of the cell suspensions were enumerated for viable cells by hemocytometer counts (Figure 1E and S1).…”

Geldanamycin-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

“…by up-regulating NF-κB [13,14]. As Hsp90 expression is particularly high in cancer cells and is associated with tumor cell progression, invasion and formation of metastases, as well as development of drug resistance [2], geldanamycin and its analogues have been considered for treament of cancer [2,3,12,15,16,17,18,19]. Geldanamycin has been shown to induce apoptosis [1,5,6,8,9,10,15,20,21,22,23], an effect paralleled by altered gene expression, downregulation of Akt, p38 MAPK activation, mitochondrial depolarization, reactive oxygen species formation, decline of reduced glutathion, lipid peroxidation and caspase activation [5,9,15,20,21].…”