Stimulation of Suicidal Erythrocyte Death by the CDC25 Inhibitor NSC-95397

Abstract

Background/Aims: The CDC25B inhibitor NSC-95397 triggers apoptosis of tumor cells and is thus considered for the treatment of malignancy. The substance is effective in part by modification of gene expression. Similar to apoptosis of nucleated cells erythrocytes may undergo eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca²⁺ activity ([Ca²⁺]_i), oxidative stress, ceramide, as well as activation of protein kinases. The present study explored, whether NSC-95397 induces eryptosis and, if so, to shed some light on the mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca²⁺]_i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to NSC-95397 significantly increased the percentage of annexin-V-binding cells (≥ 1 µM), significantly decreased forward scatter (≥ 2.5 µM), and significantly increased Fluo3-fluorescence (≥ 1 µM), DCFDA fluorescence (5 µM) and ceramide abundance (≥ 5 µM). The effect of NSC-95397 (5 µM) on annexin-V-binding was slightly, but significantly blunted by removal of extracellular Ca²⁺ and by addition of the protein kinase C inhibitor staurosporine (1 µM). Conclusions: NSC-95397 triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part requiring entry of Ca²⁺ and activation of staurosporine sensitive kinase(s).

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death

“…Cytosolic Ca 2+ activity ([Ca 2+ ] i ) is determined utilizing Fluo3 fluorescence [35]. After cells staining with the Fluo-3 AM dye (Biotium, Hayward, USA) (excitation wave length of 488 nm and emission wavelength of 530 nm corresponding to the green channel FL1), the Geo mean and the shift of the signal between control and treated cells were evaluated on the FL1-log channel.…”

“…Examples: Cytosolic Ca 2+ activity ([Ca 2+ ] i ) is determined utilizing Fluo3 fluorescence [35]. A histogram and bar graph is shown (Fig.…”

“…3C and D). Moreover, the cells can be co-treated with the Ca 2+ chelator 1,2-Bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) BAPTA-AM (Thermo Fisher Scientific, Darmstadt, Germany) [35] and the difference of death with annexin-V between treated cells and co-treated with BATA-AM evaluated. The NSC-95397 and BAPTA-AM co-treatment decrease eryptosis due to the CDC25B phosphatase inhibitor (Fig.…”

“…Examples are provided by treatment of erythrocytes with the CDC25B phosphatase inhibitor NSC-95397 [35], the PLK1 kinase inhibitor BI-2536 [89], the protease inhibitor Lopinavir [22], the marine sponge-derived Fascaplycin [44], the selective histamine H1 receptor antagonist Terfenadine [58] and Fucoxanthin (a carotenoid from the chloroplasts of brown seaweeds) [25]. In parallel examples of substances interfering with eryptosis signalling, such as caspase inhibitor Z-VAD-FMK, p38 inhibitor SB203580, protein kinase C inhibitor staurosporine and casein kinase 1 CK1α inhibitor D4476 [9] are provided.…”

Inhibition of Colon Carcinoma Cell Migration Following Treatment with Purified Venom from Lesser Weever Fish (Trachinus Vipera)

“…?m-sensitive dye) (Molecular probes-Invitrogen, Eugene, OR, USA) or with Annexin-V-FITC (1: 200 dilution; ImmunoTools, Friesoythe, Germany) for 30 min at 37°C under protection from light. Cytofluorometric acquisitions were performed by means of a FACSCalibur (BD Biosciences) cytofluorometer, while data analysis was conducted using the CellQuestTM (BD Biosciences) and the Kaluza (Beckman Coulter) software [13, 14]. …”