The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis

Abstract

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

RETRACTED: Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2

“…Consequently, p‐ANXA2 did not move to the plasma membrane as is shown in Figure 5, panels p–r. To validate the PKCα activation, besides the Gö6976, we further used the inhibitors chelerythrine and HBDDE (Alzamora et al, 2007; Ghashghaeinia et al, 2020; Mathur & Vallano, 2000). The results obtained are reported in Figure S7.…”

Association between nuclear factor of kappa B (NFκB) deficiency and induction of eryptosis in mouse erythrocytes

“…For each single sample a total of 50,000 cells were counted. Detailed information about the composition of the Ringer's solution, annexin wash buffer and the staining procedure have already been described by Ghashghaeinia et al [4]. Indeed, we observed a direct correlation between NFκBdeficiency and increased eryptosis, i.e.…”

Physcion Induces Hemolysis and Premature Phosphatidylserine Externalization in Human Erythrocytes

“…PKC isoforms are involved in a variety of cellular processes including growth, development, survival, and apoptosis. 27) In erythrocytes, PKC inhibition has been shown to be protective against energy exhaustion 28) as well as some toxic agents. 29,30) CK1α similarly mediates eryptosis secondary to energy exhaustion, oxidative stress, and calcium loading.…”