The effect of camalexin and calyculin A on eryptosis

Abstract

Eryptosis, the suicidal death of erythrocytes (red blood cells), is known to be affected by xenobiotics. A characteristic hallmark of eryptosis is increased intracellular calcium (Ca2+), which opens Ca2+-dependent K+ channels resulting in cellular loss of KCl, and the associated osmotic efflux of water, resulting in cell shrinkage. Furthermore, molecular eryptotic changes culminate in cell membrane scrambling, exposing phosphatidylserine on the outer membrane leaflet. Here, we investigated the effect of two drugs, calyculin A and camalexin, on eryptosis. Fluorescence activated cell sorting (FACS) and light scat- tering were applied to measure changes in fluorescent properties and the size of erythro- cytes after treatment. Fluo3 fluorescence and annexin V-binding were used as a proxy for the intracellular Ca2+ concentration and phosphatidylserine exposure, respectively. Insights on the effects of calyculin A on eryptosis were investigated. Calyculin A was found to increase cytosolic Ca2+ activity and phosphatidylserine surface presentation, im- plicating it as an eryptotic inducer. The eryptotic induction effect of calyculin A was abolished in the absence of extracellular Ca2+ ions, highlighting its dependence on Ca2+ entry. On the other hand, additional experiments demonstrated that ceramide abundance, another property frequently occurring in eryptotic cells, was not significantly modified. Finally, staurosporine, SB203580, D4476, and zVAD, inhibitors of kinases and caspases, markedly blunted the effect of calyculin A on annexin V-binding. This indicated the in- volvement of the respective enzymes in the specific molecular pathways that mediate calyculin A-induced eryptosis. Similarly, camalexin was found to significantly increase cytosolic Ca2+ activity due to the activation of Ca2+ permeable cation channels, and cause subsequent cell shrinkage as well as increased membrane phosphatidylserine redistribution. However, the effect of camalexin was not abolished entirely upon the removal of extracellular Ca2+ ions, indi- cating the involvement of another mechanism. Camalexin-induced phosphatidylserine surface exposure has been previously shown to involve staurosporine- and chelerythrine- sensitive kinases, such as protein kinase C. Interestingly, cell membrane scrambling was abolished in the presence of staurosporine and chelerythrine but not the p38 kinase inhib- itor SB203580 and casein kinase inhibitor D4476, suggesting the involvement of protein kinase C in the molecular eryptotic pathway induced by camalexin. Additionally, general caspase inhibitors, such as Z-IETD-FMK, a caspase-8 inhibitor, and Z-DEVD-FMK, a caspase-3 inhibitor, were found to attenuate camalexin-stimulated eryptosis. In conclusion, the two studies presented in this work reveal that calyculin A and camalexin are eryptotic inducers as demonstrated by their ability to incite cell volume shrinkage and phospholipid scrambling on the plasma membrane. Moreover, this effect is dependent on Ca2+ influx in the case of calyculin A, and, at least, partially dependent on Ca2+ influx for camalexin. While resulting in the same phenotypic outcomes, calyculin A- and camalexin-induced eryptosis differed in the exact enzymes and pathways that transduce the eryptotic signal.