Mitochondrial toxin betulinic acid induces in vitro eryptosis in human red blood cells through membrane permeabilization

Betulinic acid (BA), a compound isolated from the bark of white birch ( Betula pubescens ), was reported to induce apoptosis in many types of cancer through mitochondrial dysfunction with low side effects in normal cells. Because of these features, BA is regarded as a potential anti-cancer agent. However, the effect of BA on the induction of cell death in human erythrocytes remains unknown. Given that BA is a mitochondrial toxin and mitochondria are the central cell death regulator, we hypothesized that BA is unable to elicit apoptosis (also known as eryptosis or erythroptosis) in human erythrocytes devoid of mitochondria. This study therefore tried to determine the in vitro effect of BA on the induction of eryptosis/erythroptosis. Contrary to our prediction, BA caused phosphatidylserine externalization, increase in cellular Ca 2+ ion concentration ([Ca 2+ ] i ) and eryptosis/erythroptosis in human erythrocytes with a lethal dose larger than that in cancer lines. Mechanistically, the rise of [Ca 2+ ] i seems not to be the only key mediator in the BA-mediated eryptosis/erythroptosis because depletion of external Ca 2+ and use of Ca 2+ channels blockers could not eliminate the BA’s effect. Also, BA was able to elicit discocyte–echinocyte transformation and release calcein from the RBC ghosts in a way similar to digitonin through membrane permeabilization. Collectively, we report here for the first time that BA induced eryptosis/erythroptosis in human erythrocytes through Ca 2+ loading and membrane permeabilization.