Induction of Suicidal Erythrocyte Death by Cantharidin.

Kousi Alzoubi,Florian Lang,Antonella Fazio,Caterina Faggio,Marilena Briglia,Jasmin Egler

doi:10.3390/toxins7082822

Kousi Alzoubi, Florian Lang + Show 4 more

Open Access

https://doi.org/10.3390/toxins7082822

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Journal: Toxins	Publication Date: Jul 28, 2015
Citations: 16	License type: CC BY 4.0

Affiliation: University of Tübingen, University of Messina

Abstract

The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 μg/mL), significantly decreased forward scatter (≥25 μg/mL), significantly increased [Ca2+]i (≥25 μg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 μM) and slightly decreased by p38 inhibitor skepinone (2 μM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone.

Highlights

Cantharidin, a traditional Chinese natural product, has been successfully used for the treatment of warts, molluscum contagiosum, and callus removal [1]
Erythrocytes lack mitochondria and nuclei, but are able to enter suicidal cell death or eryptosis, which is characterized by cell shrinkage [15] and translocation of phosphatidylserine to the outer surface of the erythrocyte cell membrane [16]
The present study explored whether cantharidin stimulates eryptosis

Summary

Introduction

Cantharidin, a traditional Chinese natural product, has been successfully used for the treatment of warts, molluscum contagiosum, and callus removal [1]. Signaling involved in the stimulation of eryptosis includes increased cytosolic Ca2+ activity ([Ca2+]i), ceramide [17], oxidative stress [16], caspase activation [16,18,19], activation of casein kinase 1α, Janus-activated kinase JAK3, protein kinase C, p38 kinase, and PAK2 kinase [16] or inhibition of AMP activated kinase AMPK, cGMP-dependent protein kinase, and sorafenib and sunitinib sensitive kinases [16]. The present study explored whether cantharidin stimulates eryptosis. To this end, erythrocytes from healthy volunteers were exposed to cantharidin, phosphatidylserine abundance at the erythrocyte surface determined using annexin-V-binding and cell volume estimated from forward scatter in flow cytometry. The involvement of phosphorylation was tested utilizing kinase inhibitors staurosporine and skepinone

Results and Discussion

Experimental Section

Hemolysis

Ceramide Abundance

Statistics

Conclusions