Polyphyllin D induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization

Polyphyllin D (PD) is a potent anticancer agent isolated from a traditional medicinal herb Paris polyphylla that has been used in China for many years to treat cancer. PD is not a substrate of p-glycoprotein, and it can bypass the multi-drug resistance in cancer cell line R-HepG2. However, the effect of PD on the induction of cell death in human erythrocytes remains unknown. Given that PD is a small molecule that can depolarize the mitochondrial membrane potential and release apoptosis-inducing factor (AIF) in isolated mitochondria, we hypothesized that the apoptogenic effect of PD in human erythrocytes devoid of mitochondria would be minimal. This study therefore tried to evaluate the in vitro effect of PD on hemolysis and apoptosis in human erythrocytes. Apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, after PD treatment was determined by flow cytometry and confocal microscopy for the phosphatidyl-serine externalization and other apoptosis feature events. False to our prediction, PD caused hemolysis and eryptosis/erythroptosis in human RBCs. Mechanistically, elevation in the cytosolic Ca2+ ion level seems to be a key but not the only mediator in the PD-mediated eryptosis/erythroptosis because depletion of the external Ca2+ could not eliminate the PD effect. Also, PD was able to permeabilize the membrane of RBC ghosts in a way similar to digitonin. Taken together, we report here for the first time the toxicity of PD in human RBCs as well as its underlying mechanism for the hemolysis and eryptosis/erythroptosis.