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.

Roles of the NFκB and glutathione pathways in mature human erythrocytes

“…As in the case of parthenolide, this reaction should decrease the level of GSH in erythrocytes. Indeed, treatment of erythrocytes with Bay 11-7082 completely depleted GSH from the cells with an IC 50 of approximately 2 µM [6]. Taken together, the NFκB pathway inhibitors Bay 11-7082 and parthenolide induce erythrocyte cell death by simultaneously targeting NFκB signaling and the antioxidative defense system of the cells (Fig.…”

“…These observations point to an important role of GSH in thrombocyte function. In line with this, Bay 11-7082 and parthenolide, with their capabilities to deplete reduced glutathione (GSH) and to inhibit NFκB activity [6], could control thrombocyte functions and blood clotting, and inhibit the risk of thrombosis. On the other hand, Bay 11-7082 and parthenolide also target NFκB and GSH in erythrocytes, and the challenge of erythrocytes with the inhibitors leads to increased PS exposure on the outer leaflet of the plasma membrane.…”

“…1). Further purification and dilution experiments clearly showed that the immuno-reactive bands did not originate from contaminating white blood cells or thrombocytes [6]. Mature erythrocytes expressed not only active NFκB but also the regulatory upstream elements of the NFκB signaling pathway IκB-α kinase (IKK) and IκB-α (Fig.…”

“…PS exposure and cell shrinkage are the hallmarks of eryptosis, the suicidal death of erythrocytes [14][15][16]. In control experiments, we excluded a non-specific lytic effect of the NFκB inhibitors at the concentrations used [6]. Thus, these results argued in favor of a functional role of NFκB signaling in erythrocyte survival.…”

“…NFκB is expressed in nucleus-free erythrocytes [6] and thrombocytes [22]. Thrombocytes play an essential role in hemostasis (blood clotting in response to injury) and in pathological conditions such as inflammation, immunity and tumor progression [23].…”