Triggering of Eryptosis, the Suicidal Erythrocyte Death, by Perifosine

Abstract

Background/Aims: The alkylphospholipid perifosine is used for the treatment of malignancy. The substance is effective by triggering suicidal tumor cell death or apoptosis. Side effects of perifosine include anemia. At least in theory, perifosine-induced anemia could result from stimulation of suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms participating in the orchestration of eryptosis include increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C and/or of SB203580 sensitive p38 kinase. The present study explored, whether perifosine induces eryptosis and, if so, whether its effect involves and/or requires Ca²⁺ entry, oxidative stress, ceramide and kinase activation. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 24 hours exposure of human erythrocytes to perifosine (2.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased average forward scatter, significantly increased the percentage of shrunken erythrocytes, and significantly decreased the percentage of swollen erythrocytes. Perifosine significantly increased the percentage of hemolytic erythrocytes. Perifosine significantly increased Fluo3-fluorescence, but decreased DCFDA fluorescence and ceramide abundance. The effect of perifosine on annexin-V-binding was significantly blunted by removal of extracellular Ca²⁺ and by addition of staurosporine (1 µM), but not by addition of SB203580 (2 µM). Conclusions: Perifosine triggers eryptosis, an effect at least in part due to Ca²⁺ entry and activation of staurosporine sensitive kinases.

Dual Gene Delivery Reagents From Antiproliferative Alkylphospholipids for Combined Antitumor Therapy

“…Though other authors have reported ca . 30% hemolysis after a 24-h exposure to 5.4 μM perifosine (Egler and Lang, 2017 ), a direct correlation with our results was difficult, due to significant differences in the experimental conditions.…”

Reprogramming of erythrocyte lifespan by NFκB‐TNFα naphthoquinone antagonist β‐lapachone is regulated by calcium overload and CK1α

“…In brief, control and experimental cells were stained with 1% Annexin-V-FITC in 5 mM CaCl 2 buffer (v/v) for 10 min at room temperature away from light, and the percentage of Annexin-positive cells was obtained at 488/520 nm excitation and emission spectra, respectively. FSC, as a reflection of cell size, was simultaneously recorded (Egler & Lang, 2017).…”

“…A number of chemotherapeutic agents have previously been shown to induce eryptosis (Egler & Lang, 2017;Lang et al, 2006) which may explain the anemia associated with treatment (Lang et al, 2017). pyran-5,6-dione, C 15 H 14 O 3 ; LAP), a naphthoquinone that naturally occurs in extracts of Lapacho tree (Lamberti et al, 2013) (Figure 1a), possesses an antitumor activity in vitro and in vivo (Ferraz da Costa et al, 2020).…”

Integrated Metabolomics and Network Pharmacology Approach to Explain Possible Action Mechanisms of Xin-Sheng-Hua Granule for Treating Anemia

“…During the process of anemia, the membrane of erythrocyte was injured, resulting in erythrocyte apoptosis. So far, more and more evidence has shown that organisms could initiate erythrocyte apoptosis to maintain homeostasis of anemia patients ( Knapp et al, 2012 ; Egler and Lang, 2017 ). Our research group have confirmed that herb pair Danggui-Chuanxiong could suppress erythrocyte apoptosis by inhibiting sphingolipids dependent signaling molecules ( Li W.X.…”