The NFĸB Pathway Inhibitors Bay 11-7082 and Parthenolide Induce Programmed Cell Death in Anucleated Erythrocytes

Abstract

The preclinical compounds Bay 11-7082 and parthenolide trigger apoptosis, an effect contributing to their antiinflammatory action. The substances interfere with the activation and nuclear translocation of nuclear factor NFĸB, by inhibiting NFĸB directly (parthenolide) or by interfering with the inactivation of the NFĸB inhibitory protein IĸB-α (Bay 11-7082). Beyond that, the substances may be effective in part by nongenomic effects. Similar to apoptosis of nucleated cells, erythrocytes may undergo apoptosis-like cell death (eryptosis) characterized by cell membrane scrambling with phosphatidylserine exposure, and cell shrinkage. Thus, erythrocytes allow the study of nongenomic mechanisms contributing to suicidal cell death, e.g. Ca²⁺ leakage or glutathione depletion. The present study utilized Western blotting to search for NFĸB and IĸB-α expression in erythrocytes, FACS analysis to determine cytosolic Ca²⁺ (Fluo3 fluorescence), phosphatidylserine exposure (annexin V binding), and cell volume (forward scatter), as well as an enzymatic method to determine glutathione levels. As a result, both NFĸB and IĸB-α are expressed in erythrocytes. Targeting the NFĸB pathway by Bay 11-7082 (IC₅₀ ≈ 10 µM) and parthenolide (IC₅₀ ≈ 30 µM) triggered suicidal erythrocyte death as shown by annexin V binding and decrease of forward scatter. Bay 11-7082 treatment further increased intracellular Ca²⁺ and led to depletion of reduced glutathione. The effects of Bay 11-7082 and parthenolide on annexin V binding could be fully reversed by the antioxidant N-acetylcysteine. In conclusion, the pharmacological inhibitors of NFĸB, Bay 11-7082 and parthenolide, interfere with the survival of erythrocytes involving mechanisms other than disruption of NFĸB-dependent gene expression.