Piperlongumine-induced phosphatidylserine translocation in the erythrocyte membrane

Abstract

Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca²⁺-activity ([Ca²⁺]i), formation of ceramide, oxidative stress and activation of p38 kinase.

Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca²⁺]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry.

Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca²⁺]i and the effect was not dependent on presence of extracellular Ca²⁺. Piperlongumine significantly increased ROS formation and ceramide abundance.

Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca²⁺ but at least partially due to ROS and ceramide formation.

Figure 1

Effect of piperlongumine on phosphatidylserine exposure (A) Original histogram of annexin V binding of erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 30 µM piperlongumine; (B) Arithmetic means ± SEM (n = 15) of erythrocyte annexin-V-binding following incubation for 48 h to Ringer solution without (white bar) or with (black bars) presence of piperlongumine (5–30 µM) or, for comparison, DMSO (0.1%) alone. * (p < 0.05), *** (p < 0.001) indicate significant differences from the absence of piperlongumine (ANOVA).

Figure 2

Effect of piperlongumine on erythrocyte forward scatter (A) Original histogram of forward scatter of erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 30 µM piperlongumine; (B) Arithmetic means ± SEM (n = 15) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) piperlongumine (5–30 µM) or, for comparison, DMSO (0.1%) alone. ** (p < 0.01), *** (p < 0.001) indicate significant differences from the absence of piperlongumine (ANOVA).

Figure 3

Effect of piperlongumine on erythrocyte cytosolic Ca²⁺ concentration (A) Original histogram of Fluo3 fluorescence in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 30 µM piperlongumine; (B) Arithmetic means ± SEM (n = 15) of the Fluo3 fluorescence (arbitrary units) in erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) piperlongumine (5–30 µM) or, for comparison, DMSO (0.1%) alone; (C) Arithmetic means ± SEM (n = 5) of the percentage of annexin-V-binding erythrocytes after a 48 h treatment with Ringer solution without (white bars) or with (black bars) 30 µM piperlongumine in the presence (left bars, +Calcium) and absence (right bars, −Calcium) of calcium. *** (p < 0.001) indicates significant difference from the respective values in the absence of piperlongumine (ANOVA).

Figure 4

Effect of piperlongumine on reactive oxygen species (A) Original histogram of 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence in erythrocytes following exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 30 µM piperlongumine; (B) Arithmetic means ± SEM (n = 5) of erythrocyte DCFDA fluorescence following incubation for 48 h to Ringer solution without (white bar) or with (black bar) presence of piperlongumine (30 µM). *** (p < 0.001) indicates significant difference from the absence of piperlongumine (ANOVA).

Figure 5

Effect of piperlongumine on ceramide abundance (A) Original histogram of anti-ceramide fluorescein isothiocyanate (FITC) fluorescence in erythrocytes after exposure for 48 h to Ringer solution without (grey shadow) and with (black line) presence of 30 µM piperlongumine; (B) Arithmetic means ± SEM (n = 9) of ceramide abundance at the erythrocyte surface following incubation for 48 h to Ringer solution without (white bar) or with (black bar) presence of piperlongumine (30 µM). ** (p < 0.01) indicates significant difference from the absence of piperlongumine (ANOVA).