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Article

Carmustine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

by
Kashif Jilani
and
Florian Lang
*
Department of Physiology, University of Tuebingen, Gmelinstr. 5, Tuebingen D-72076, Germany
*
Author to whom correspondence should be addressed.
Toxins 2013, 5(4), 703-716; https://doi.org/10.3390/toxins5040703
Submission received: 20 February 2013 / Revised: 7 April 2013 / Accepted: 10 April 2013 / Published: 19 April 2013
Browse Figures
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Abstract

:
The nitrosourea alkylating agent, carmustine, is used as chemotherapeutic drug in several malignancies. The substance triggers tumor cell apoptosis. Side effects of carmustine include myelotoxicity with anemia. At least in theory, anemia could partly be due to stimulation of eryptosis, the suicidal death of erythrocytes, characterized by cell shrinkage and breakdown of phosphatidylserine asymmetry of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i). The present study tested whether carmustine triggers eryptosis. To this end [Ca2+]i was estimated from Fluo3 fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin V binding, and hemolysis from hemoglobin release. As a result a 48 h exposure to carmustine (≥25 µM) significantly increased [Ca2+]i, decreased forward scatter and increased annexin V binding. The effect on annexin V binding was significantly blunted in the absence of extracellular Ca2+. In conclusion, carmustine stimulates eryptosis at least partially by increasing cytosolic Ca2+ activity.

Graphical Abstract

1. Introduction

Carmustine (1,3-bis-(2-chloroethyl)-1-nitrosourea), a nitrosourea alkylating agent is widely used for the treatment of malignancies [1,2,3,4,5,6,7]. As systemic administration of nitrosoureas was poorly effective in the treatment of high grade glioma, carmustine wafers have been developed which deliver high local concentrations of the drug [1]. Carmustine is mainly effective by alkylating DNA and RNA [1] and inducing apoptosis [2,8,9,10,11,12]. Mechanisms involved in the triggering of apoptosis by carmustine include oxidative stress [2,8] at least in part by inhibition of glutathion reductase [13,14]. Carmustine induced oxidative stress is at least partially effective by increasing Ca2+ entry from extracellular space [15]. Side effects of systemic carmustine administration include anemia [2,8], which may at least partially result from erythrocyte death.
Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage and breakdown of phosphatidylserine (PS) asymmetry of the erythrocyte cell membrane [16,17]. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), which may result from Ca2+ entry through Ca2+ permeable cation channels [18,19]. The increase of [Ca2+]i results in cell shrinkage due to activation of Ca2+ sensitive K+ channels [20], K+ exit, hyperpolarization, Cl- exit and thus cellular loss of KCl with osmotically obliged water [21]. The increase of [Ca2+]i further leads to breakdown of PS asymmetry of the erythrocyte cell membrane with translocation of PS to the erythrocyte surface [22]. The Ca2+ sensitivity of eryptosis is enhanced by ceramide [23]. Additional stimulators of eryptosis include energy depletion [24], caspase activation [25,26,27,28,29] and dysregulation of AMP activated kinase AMPK [19], cGMP dependent protein kinase [30], Janus activated kinase JAK3 [31], casein kinase [32,33], p38 kinase [34], PAK2 kinase [35] as well as sorafenib [36] and sunitinib [37] sensitive kinases.
Eryptosis is stimulated by a myriad of xenobiotics [37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68] and excessive eryptosis participates in the pathophysiology of several clinical disorders [16], such as diabetes [29,69,70], renal insufficiency [71], hemolytic uremic syndrome [72], sepsis [73], malaria [74,75,76,77,78], sickle cell disease [79], Wilson’s disease [77], iron deficiency [80], malignancy [81], phosphate depletion [82], and metabolic syndrome [64].
The present study explored the effect of carmustine on erythrocyte [Ca2+]i, cell volume and PS exposure at the cell surface. As a result, carmustine increases [Ca2+]i, decreases erythrocyte volume and enhances the PS abundance at the erythrocyte surface.

2. Results and Discussion

The present study was designed to explore whether carmustine stimulates eryptosis, the suicidal death of erythrocytes. As eryptosis is triggered by increase of cytosolic Ca2+ activity ([Ca2+]i), Fluo3 fluorescence was employed to estimate [Ca2+]i. To this end, the erythrocytes were incubated in Ringer solution without or with carmustine (10–100 µM), loaded with Fluo3 AM and Fluo3 fluorescence quantified by FACS analysis. As illustrated in Figure 1, a 48 hours exposure of human erythrocytes to carmustine was followed by an increase of Fluo3 fluorescence, an effect reaching statistical significance at 25 µM carmustine concentration. Thus, carmustine treatment was followed by increase of [Ca2+]i in human erythrocytes.
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Figure 1. Effect of carmustine 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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of the Fluo3 fluorescence (arbitrary units) in erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) carmustine (10–100 µM). *** (p < 0.001) indicates significant difference from the absence of carmustine (ANOVA).
Figure 1. Effect of carmustine 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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of the Fluo3 fluorescence (arbitrary units) in erythrocytes exposed for 48 h to Ringer solution without (white bar) or with (black bars) carmustine (10–100 µM). *** (p < 0.001) indicates significant difference from the absence of carmustine (ANOVA).
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Figure 2. Effect of carmustine 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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) carmustine (10–100 µM). *** (p < 0.001) indicates significant difference from the absence of carmustine (ANOVA).
Figure 2. Effect of carmustine 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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of the normalized erythrocyte forward scatter (FSC) following incubation for 48 h to Ringer solution without (white bar) or with (black bars) carmustine (10–100 µM). *** (p < 0.001) indicates significant difference from the absence of carmustine (ANOVA).
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An increase of [Ca2+]i is expected to activate Ca2+ sensitive K+ channels leading to cellular loss of KCl together with osmotically obliged water and thus to cell shrinkage. Accordingly, cell volume was estimated from forward scatter in FACS analysis. As shown in Figure 2, a 48 h treatment with carmustine resulted in a decrease of forward scatter, an effect reaching statistical significance at 25 µM carmustine concentration. Accordingly, carmustine treatment was followed by erythrocyte shrinkage.
An increase of [Ca2+]i is further expected to trigger cell membrane scrambling with breakdown of PS asymmetry of the cell membrane and appearance of phosphatidsylserine at the cell surface. Accordingly, PS abundance at the cell surface was estimated utilizing annexin V binding in FACS analysis. As shown in Figure 3, a 48 h carmustine treatment increased the percentage of annexin V binding erythrocytes, an effect reaching statistical significance at 50 µM carmustine concentration. Accordingly, carmustine triggered cell membrane scrambling.
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Figure 3. Effect of carmustine on PS exposure and hemolysis. (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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of erythrocyte annexin V binding following incubation for 48 h to Ringer solution without (white bar) or with (black bars) presence of carmustine (10–100 µM). For comparison, arithmetic means ± SEM (n = 4) of the percentage of hemolysis is shown as grey bars. *** (p < 0.001) indicates significant differences from the absence of carmustine (ANOVA).
Figure 3. Effect of carmustine on PS exposure and hemolysis. (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 100 µM carmustine; (B) Arithmetic means ± SEM (n = 12) of erythrocyte annexin V binding following incubation for 48 h to Ringer solution without (white bar) or with (black bars) presence of carmustine (10–100 µM). For comparison, arithmetic means ± SEM (n = 4) of the percentage of hemolysis is shown as grey bars. *** (p < 0.001) indicates significant differences from the absence of carmustine (ANOVA).
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In a separate series of experiments, hemolysis was estimated by determination of hemoglobin in the supernatant. As illustrated in Figure 3, the percentage of hemolyzed erythrocytes tended to increase slightly following exposure of erythrocytes for 48 h to carmustine, an effect, however, not reaching statistical significance up to 100 µM carmustine concentration (Figure 3). In any case, the percentage of hemolyzed erythrocytes remained one magnitude lower than the percentage of erythrocytes exposing PS.
Further experiments were performed to test whether the stimulation of cell membrane scrambling following carmustine treatment was partially or even fully explained by Ca2+ entry from the extracellular space. To this end, erythrocytes were exposed to 100 µM carmustine for 48 h in either the presence of 1 mM extracellular Ca2+ or in the absence of extracellular Ca2+ and presence of the Ca2+ chelator EGTA (1 mM). As shown in Figure 4, removal of extracellular Ca2+ significantly blunted the effect of carmustine on annexin V binding. However, in the absence of extracellular Ca2+ the percentage annexin V binding erythrocytes was still slightly, but significantly, increased by carmustine treatment (Figure 4). Thus, carmustine induced cell membrane scrambling was, mainly but not completely, dependent on the presence of extracellular Ca2+.
Toxins 05 00703 g004 1024
Figure 4. Effect of Ca2+ withdrawal on carmustine induced annexin V binding. Arithmetic means ± SEM (n = 4) of the percentage of annexin V binding erythrocytes after a 48 h treatment with Ringer solution without (white bar) or with (black bars) 100 µM carmustine in the presence (left bars, +Calcium) and absence (right bars, −Calcium) of calcium. * (p < 0.05), *** (p < 0.001) indicate significant difference from the absence of carmustine (ANOVA), ### (p < 0.001) indicates significant difference from the respective values in the presence of Ca2+.
Figure 4. Effect of Ca2+ withdrawal on carmustine induced annexin V binding. Arithmetic means ± SEM (n = 4) of the percentage of annexin V binding erythrocytes after a 48 h treatment with Ringer solution without (white bar) or with (black bars) 100 µM carmustine in the presence (left bars, +Calcium) and absence (right bars, −Calcium) of calcium. * (p < 0.05), *** (p < 0.001) indicate significant difference from the absence of carmustine (ANOVA), ### (p < 0.001) indicates significant difference from the respective values in the presence of Ca2+.
Toxins 05 00703 g004
The present study explored whether carmustine triggers eryptosis, the suicidal death of erythrocytes. The results reveal that carmustine treatment of erythrocytes drawn from healthy volunteers is followed by erythrocyte shrinkage and by breakdown of PS asymmetry of the cell membrane, both hallmarks of eryptosis. The concentrations required for the stimulation of eryptosis were well in the range of the plasma concentrations encountered following in vivo application of carmustine [83]. When rats were given 12 mg/kg of carmustine i.p., the peak plasma concentration approached 28 µM [83]. The elimination half-time was about 16 min [83]. At least in theory, the effect of carmustine could be shared by other nitrosourea compounds.
The erythrocyte shrinkage following carmustine treatment is most likely the result of increased cytosolic Ca2+ activity, which activates Ca2+ sensitive K+ channels [20,84] leading to cell membrane hyperpolarization. The increased electrical driving force drives Cl exit and thus leads to cellular loss of KCl with osmotically obliged water [21].
The breakdown of PS asymmetry of the erythrocyte cell membrane was significantly blunted in the absence of extracellular Ca2+ and was again, at least in part, due to the increase of cytosolic Ca2+ activity ([Ca2+]i). An increase of [Ca2+]i is well known to stimulate cell membrane scrambling with PS translocation from the inner leaflet of the cell membrane to the outer leaflet of the cell membrane [16]. Mechanisms underlying Ca2+ entry include Ca2+ permeable nonselective cation channels involving the transient receptor potential channel TRPC6 [18]. The Ca2+ permeable erythrocyte cation channels are activated by oxidative stress [85], a well-known effect of carmustine [2,8].
Consequences of enhanced eryptosis include anemia. In vivo, eryptotic erythrocytes are mainly trapped in the spleen and thus rapidly removed from circulating blood [16]. As soon as the loss of erythrocytes by triggering of eryptosis is not matched by a similar enhancement of erythropoiesis, anemia develops [16]. During carmustine treatment the myelotoxic effect of the drug [2,8] is expected to impair erythropoiesis and thus to prevent compensatory increase of erythrocyte formation.
Consequences of enhanced eryptosis further include adhesion of PS, exposing erythrocytes to endothelial CXCL16/SR PSO [86]. The adhesion of erythrocytes to the vascular wall could at least in theory compromise microcirculation and thus interfere with blood flow [86,87,88,89,90,91]. The effect may be compounded by the stimulating effect of PS exposure on blood clotting, which may foster the development of thrombosis [87,92,93].

3. Experimental Section

3.1. Erythrocytes, Solutions and Chemicals

Leukocyte depleted erythrocytes were kindly provided by the blood bank of the University of Tübingen. The study is approved by the ethics committee of the University of Tübingen (184/2003V). Erythrocytes were incubated in vitro at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to carmustine (Enzo, Lörrach, Germany) at the indicated concentrations. Carmustine was dissolved in 50% ethanol. The final concentration of ethanol did not exceed 0.1%. Annexin V binding was not significantly different in the absence (1.8% ± 0.1%, n = 4) and presence of 0.1% ethanol (1.9% ± 0.2%, n = 4). In Ca2+ free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).

3.2. FACS Analysis of Annexin V Binding and forward Scatter

After incubation under the respective experimental condition, 50 µL cell suspension was washed in Ringer solution containing 5 mM CaCl2 and then stained with Annexin V FITC (1:200 dilution; ImmunoTools, Friesoythe, Germany) in this solution at 37 °C for 20 min under protection from light. In the following, the forward scatter (FSC) of the cells was determined, and annexin V fluorescence intensity was measured in FL 1 with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD, Heidelberg, Germany).

3.3. Measurement of Intracellular Ca2+

After incubation erythrocytes were washed in Ringer solution and then loaded with Fluo 3/AM (Biotium, Hayward, CA, USA) in Ringer solution containing 5 mM CaCl2 and 2 µM Fluo 3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo 3/AM loaded erythrocytes were resuspended in 200 µL Ringer. Then, Ca2+ dependent fluorescence intensity was measured in fluorescence channel FL 1 in FACS analysis.

3.4. Measurement of Hemolysis

For the determination of hemolysis the samples were centrifuged (3 min at 400g, room temperature) after incubation, and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.

3.5. Statistics

Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.

4. Conclusions

Exposure of erythrocytes from healthy volunteers to carmustine triggers Ca2+ entry with subsequent eryptosis, the suicidal erythrocyte death. Enhanced eryptosis may contribute to the development of anemia following carmustine treatment.

Acknowledgements

The authors acknowledge the meticulous preparation of the manuscript by Ali Soleimanpour. The study was supported by the Deutsche Forschungsgemeinschaft.

References

  1. Lin, S.H.; Kleinberg, L.R. Carmustine wafers: Localized delivery of chemotherapeutic agents in cns malignancies. Expert Rev. Anticancer Ther. 2008, 8, 343–359. [Google Scholar] [CrossRef]
  2. El-Sayed el, S.M.; Abdel-Aziz, A.A.; Helal, G.K.; Saleh, S.; Saad, A.S. Protective effect of n-acetylcysteine against carmustine-induced myelotoxicity in rats. Food Chem. Toxicol. 2010, 48, 1576–1580. [Google Scholar] [CrossRef]
  3. Ferreri, A.J.; Bruno Ventre, M.; Donadoni, G.; Cattaneo, C.; Fumagalli, L.; Foppoli, M.; Mappa, S.; Govi, S.; di Nicola, M.; Rossi, G.; et al. Safety and activity of a new intensive short-term chemoimmunotherapy in hiv-positive patients with burkitt lymphoma. Br. J. Haematol. 2012, 159, 252–255. [Google Scholar] [CrossRef]
  4. Geisler, C.H.; Kolstad, A.; Laurell, A.; Jerkeman, M.; Raty, R.; Andersen, N.S.; Pedersen, L.B.; Eriksson, M.; Nordstrom, M.; Kimby, E.; et al. Nordic mcl2 trial update: Six-year follow-up after intensive immunochemotherapy for untreated mantle cell lymphoma followed by beam or beac + autologous stem-cell support: Still very long survival but late relapses do occur. Br. J. Haematol. 2012, 158, 355–362. [Google Scholar] [CrossRef]
  5. Moore, S.; Kayani, I.; Peggs, K.; Qian, W.; Lowry, L.; Thomson, K.; Linch, D.C.; Ardeshna, K. Mini-beam is effective as a bridge to transplantation in patients with refractory or relapsed hodgkin lymphoma who have failed to respond to previous lines of salvage chemotherapy but not in patients with salvage-refractory dlbcl. Br. J. Haematol. 2012, 157, 543–552. [Google Scholar] [CrossRef]
  6. Wondergem, M.J.; Zijlstra, J.M.; de Rooij, M.; Visser, O.J.; Huijgens, P.C.; Zweegman, S. Improving survival in patients with transformed b cell non hodgkin lymphoma: Consolidation with (9)(0)yttrium ibritumomab tiuxetan-beam and autologous stem cell transplantation. Br. J. Haematol. 2012, 157, 395–397. [Google Scholar] [CrossRef]
  7. Linch, D.C.; Yung, L.; Smith, P.; Maclennan, K.; Jack, A.; Hancock, B.; Cunningham, D.; Hoskin, P.; Qian, W.; Holte, H.; et al. Final analysis of the uklg ly02 trial comparing 6–8 cycles of chop with 3 cycles of chop followed by a beam autograft in patients . Br. J. Haematol. 2010, 149, 237–243. [Google Scholar] [CrossRef]
  8. Thamilselvan, V.; Menon, M.; Thamilselvan, S. Carmustine enhances the anticancer activity of selenite in androgen-independent prostate cancer cells. Cancer Manag. Res. 2012, 4, 383–395. [Google Scholar] [CrossRef]
  9. Van Meter, T.E.; Broaddus, W.C.; Cash, D.; Fillmore, H. Cotreatment with a novel phosphoinositide analogue inhibitor and carmustine enhances chemotherapeutic efficacy by attenuating akt activity in gliomas. Cancer 2006, 107, 2446–2454. [Google Scholar] [CrossRef] [Green Version]
  10. Yamauchi, T.; Ogawa, M.; Ueda, T. Carmustine-resistant cancer cells are sensitized to temozolomide as a result of enhanced mismatch repair during the development of carmustine resistance. Mol. Pharmacol. 2008, 74, 82–91. [Google Scholar] [CrossRef]
  11. Papait, R.; Magrassi, L.; Rigamonti, D.; Cattaneo, E. Temozolomide and carmustine cause large-scale heterochromatin reorganization in glioma cells. Biochem. Biophys. Res. Commun. 2009, 379, 434–439. [Google Scholar] [CrossRef]
  12. El-Sayed el, S.M.; Abdel-Aziz, A.A.; Saleh, S.; Saad, A.S. The chemopreventive effect of dimethylthiourea against carmustine-induced myelotoxicity in rats. Food Chem. Toxicol. 2011, 49, 1965–1969. [Google Scholar] [CrossRef]
  13. Doroshenko, N.; Doroshenko, P. The glutathione reductase inhibitor carmustine induces an influx of Ca2+ in pc12 cells. Eur. J. Pharmacol. 2004, 497, 17–24. [Google Scholar] [CrossRef]
  14. Lock, J.T.; Sinkins, W.G.; Schilling, W.P. Effect of protein s-glutathionylation on Ca2+ homeostasis in cultured aortic endothelial cells. Am. J. Physiol. Heart Circ. Physiol. 2011, 300, H493–H506. [Google Scholar] [CrossRef]
  15. Doroshenko, N.; Doroshenko, P. Ion dependence of cytotoxicity of carmustine against pc12 cells. Eur. J. Pharmacol. 2003, 476, 185–191. [Google Scholar] [CrossRef]
  16. Lang, F.; Gulbins, E.; Lerche, H.; Huber, S.M.; Kempe, D.S.; Föller, M. Eryptosis, a window to systemic disease. Cell Physiol. Biochem. 2008, 22, 373–380. [Google Scholar] [CrossRef]
  17. Foller, M.; Mahmud, H.; Qadri, S.M.; Gu, S.; Braun, M.; Bobbala, D.; Hocher, B.; Lang, F. Endothelin b receptor stimulation inhibits suicidal erythrocyte death. FASEB J. 2010, 24, 3351–3359. [Google Scholar] [CrossRef]
  18. Foller, M.; Kasinathan, R.S.; Koka, S.; Lang, C.; Shumilina, E.; Birnbaumer, L.; Lang, F.; Huber, S.M. Trpc6 contributes to the Ca2+ leak of human erythrocytes. Cell Physiol. Biochem. 2008, 21, 183–192. [Google Scholar] [CrossRef]
  19. Foller, M.; Sopjani, M.; Koka, S.; Gu, S.; Mahmud, H.; Wang, K.; Floride, E.; Schleicher, E.; Schulz, E.; Munzel, T.; et al. Regulation of erythrocyte survival by amp-activated protein kinase. FASEB J. 2009, 23, 1072–1080. [Google Scholar] [CrossRef]
  20. Brugnara, C.; de Franceschi, L.; Alper, S.L. Inhibition of Ca2+-dependent K+ transport and cell dehydration in sickle erythrocytes by clotrimazole and other imidazole derivatives. J. Clin. Invest. 1993, 92, 520–526. [Google Scholar] [CrossRef]
  21. Lang, P.A.; Kaiser, S.; Myssina, S.; Wieder, T.; Lang, F.; Huber, S.M. Role of Ca2+-activated K+ channels in human erythrocyte apoptosis. Am. J. Physiol. Cell Physiol. 2003, 285, C1553–C1560. [Google Scholar]
  22. Berg, C.P.; Engels, I.H.; Rothbart, A.; Lauber, K.; Renz, A.; Schlosser, S.F.; Schulze-Osthoff, K.; Wesselborg, S. Human mature red blood cells express caspase-3 and caspase-8, but are devoid of mitochondrial regulators of apoptosis. Cell Death Differ. 2001, 8, 1197–1206. [Google Scholar] [CrossRef]
  23. Lang, F.; Gulbins, E.; Lang, P.A.; Zappulla, D.; Foller, M. Ceramide in suicidal death of erythrocytes. Cell Physiol. Biochem. 2010, 26, 21–28. [Google Scholar] [CrossRef]
  24. Klarl, B.A.; Lang, P.A.; Kempe, D.S.; Niemoeller, O.M.; Akel, A.; Sobiesiak, M.; Eisele, K.; Podolski, M.; Huber, S.M.; Wieder, T.; et al. Protein kinase c mediates erythrocyte “programmed cell death” following glucose depletion. Am. J. Physiol. Cell Physiol. 2006, 290, C244–C253. [Google Scholar]
  25. Bhavsar, S.K.; Bobbala, D.; Xuan, N.T.; Foller, M.; Lang, F. Stimulation of suicidal erythrocyte death by alpha-lipoic acid. Cell Physiol. Biochem. 2010, 26, 859–868. [Google Scholar] [CrossRef]
  26. Foller, M.; Huber, S.M.; Lang, F. Erythrocyte programmed cell death. IUBMB Life 2008, 60, 661–668. [Google Scholar] [CrossRef]
  27. Foller, M.; Mahmud, H.; Gu, S.; Wang, K.; Floride, E.; Kucherenko, Y.; Luik, S.; Laufer, S.; Lang, F. Participation of leukotriene c(4) in the regulation of suicidal erythrocyte death. J. Physiol. Pharmacol. 2009, 60, 135–143. [Google Scholar]
  28. Lau, I.P.; Chen, H.; Wang, J.; Ong, H.C.; Leung, K.C.; Ho, H.P.; Kong, S.K. In vitro effect of ctab- and peg-coated gold nanorods on the induction of eryptosis/erythroptosis in human erythrocytes. Nanotoxicology 2011, 6, 847–856. [Google Scholar]
  29. Maellaro, E.; Leoncini, S.; Moretti, D.; del Bello, B.; Tanganelli, I.; de Felice, C.; Ciccoli, L. Erythrocyte caspase-3 activation and oxidative imbalance in erythrocytes and in plasma of type 2 diabetic patients. Acta Diabetol. 2011. [Google Scholar] [CrossRef]
  30. Foller, M.; Feil, S.; Ghoreschi, K.; Koka, S.; Gerling, A.; Thunemann, M.; Hofmann, F.; Schuler, B.; Vogel, J.; Pichler, B.; et al. Anemia and splenomegaly in cgki-deficient mice. Proc. Natl. Acad. Sci. USA 2008, 105, 6771–6776. [Google Scholar] [CrossRef] [Green Version]
  31. Bhavsar, S.K.; Gu, S.; Bobbala, D.; Lang, F. Janus kinase 3 is expressed in erythrocytes, phosphorylated upon energy depletion and involved in the regulation of suicidal erythrocyte death. Cell Physiol. Biochem. 2011, 27, 547–556. [Google Scholar] [CrossRef]
  32. Kucherenko, Y.; Zelenak, C.; Eberhard, M.; Qadri, S.M.; Lang, F. Effect of casein kinase 1alpha activator pyrvinium pamoate on erythrocyte ion channels. Cell Physiol. Biochem. 2012, 30, 407–417. [Google Scholar] [CrossRef]
  33. Zelenak, C.; Eberhard, M.; Jilani, K.; Qadri, S.M.; Macek, B.; Lang, F. Protein kinase ck1alpha regulates erythrocyte survival. Cell Physiol. Biochem. 2012, 29, 171–180. [Google Scholar] [CrossRef]
  34. Gatidis, S.; Zelenak, C.; Fajol, A.; Lang, E.; Jilani, K.; Michael, D.; Qadri, S.M.; Lang, F. P38 mapk activation and function following osmotic shock of erythrocytes. Cell Physiol. Biochem. 2011, 28, 1279–1286. [Google Scholar] [CrossRef]
  35. Zelenak, C.; Foller, M.; Velic, A.; Krug, K.; Qadri, S.M.; Viollet, B.; Lang, F.; Macek, B. Proteome analysis of erythrocytes lacking amp-activated protein kinase reveals a role of pak2 kinase in eryptosis. J. Proteome Res. 2011, 10, 1690–1697. [Google Scholar] [CrossRef]
  36. Lupescu, A.; Shaik, N.; Jilani, K.; Zelenak, C.; Lang, E.; Pasham, V.; Zbidah, M.; Plate, A.; Bitzer, M.; Foller, M.; et al. Enhanced erythrocyte membrane exposure of phosphatidylserine following sorafenib treatment: An in vivo and in vitro study. Cell Physiol. Biochem. 2012, 30, 876–888. [Google Scholar] [CrossRef]
  37. Shaik, N.; Lupescu, A.; Lang, F. Sunitinib-sensitive suicidal erythrocyte death. Cell Physiol. Biochem. 2012, 30, 512–522. [Google Scholar] [CrossRef]
  38. Abed, M.; Towhid, S.T.; Mia, S.; Pakladok, T.; Alesutan, I.; Borst, O.; Gawaz, M.; Gulbins, E.; Lang, F. Sphingomyelinase-induced adhesion of eryptotic erythrocytes to endothelial cells. Am. J. Physiol. Cell Physiol. 2012, 303, C991–C999. [Google Scholar] [CrossRef]
  39. Abed, M.; Towhid, S.T.; Shaik, N.; Lang, F. Stimulation of suicidal death of erythrocytes by rifampicin. Toxicology 2012, 302, 123–128. [Google Scholar] [CrossRef]
  40. Bottger, E.; Multhoff, G.; Kun, J.F.; Esen, M. Plasmodium falciparum-infected erythrocytes induce granzyme b by nk cells through expression of host-hsp70. PLoS One 2012, 7, e33774. [Google Scholar] [CrossRef]
  41. Felder, K.M.; Hoelzle, K.; Ritzmann, M.; Kilchling, T.; Schiele, D.; Heinritzi, K.; Groebel, K.; Hoelzle, L.E. Hemotrophic mycoplasmas induce programmed cell death in red blood cells. Cell Physiol. Biochem. 2011, 27, 557–564. [Google Scholar] [CrossRef] [Green Version]
  42. Firat, U.; Kaya, S.; Cim, A.; Buyukbayram, H.; Gokalp, O.; Dal, M.S.; Tamer, M.N. Increased caspase-3 immunoreactivity of erythrocytes in stz diabetic rats. Exp. Diabetes Res. 2012, 2012, 316384. [Google Scholar]
  43. Ganesan, S.; Chaurasiya, N.D.; Sahu, R.; Walker, L.A.; Tekwani, B.L. Understanding the mechanisms for metabolism-linked hemolytic toxicity of primaquine against glucose 6-phosphate dehydrogenase deficient human erythrocytes: Evaluation of eryptotic pathway. Toxicology 2012, 294, 54–60. [Google Scholar] [CrossRef]
  44. Gao, M.; Cheung, K.L.; Lau, I.P.; Yu, W.S.; Fung, K.P.; Yu, B.; Loo, J.F.; Kong, S.K. Polyphyllin d induces apoptosis in human erythrocytes through Ca2+ rise and membrane permeabilization. Arch. Toxicol. 2012, 86, 741–752. [Google Scholar] [CrossRef]
  45. Ghashghaeinia, M.; Cluitmans, J.C.; Akel, A.; Dreischer, P.; Toulany, M.; Koberle, M.; Skabytska, Y.; Saki, M.; Biedermann, T.; Duszenko, M.; et al. The impact of erythrocyte age on eryptosis. Br. J. Haematol. 2012, 157, 606–614. [Google Scholar] [CrossRef]
  46. Ghashghaeinia, M.; Toulany, M.; Saki, M.; Bobbala, D.; Fehrenbacher, B.; Rupec, R.; Rodemann, H.P.; Ghoreschi, K.; Rocken, M.; Schaller, M.; et al. The nfkb pathway inhibitors bay 11–7082 and parthenolide induce programmed cell death in anucleated erythrocytes. Cell Physiol. Biochem. 2011, 27, 45–54. [Google Scholar] [CrossRef]
  47. Jilani, K.; Lupescu, A.; Zbidah, M.; Abed, M.; Shaik, N.; Lang, F. Enhanced apoptotic death of erythrocytes induced by the mycotoxin ochratoxin a. Kidney Blood Press Res. 2012, 36, 107–118. [Google Scholar] [CrossRef]
  48. Jilani, K.; Lupescu, A.; Zbidah, M.; Shaik, N.; Lang, F. Withaferin a-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes. Toxicol in Vitro 2012, 27, 52–58. [Google Scholar]
  49. Kucherenko, Y.V.; Lang, F. Inhibitory effect of furosemide on non-selective voltage-independent cation channels in human erythrocytes. Cell Physiol. Biochem. 2012, 30, 863–875. [Google Scholar] [CrossRef]
  50. Lang, E.; Jilani, K.; Zelenak, C.; Pasham, V.; Bobbala, D.; Qadri, S.M.; Lang, F. Stimulation of suicidal erythrocyte death by benzethonium. Cell Physiol. Biochem. 2011, 28, 347–354. [Google Scholar] [CrossRef]
  51. Lang, E.; Qadri, S.M.; Jilani, K.; Zelenak, C.; Lupescu, A.; Schleicher, E.; Lang, F. Carbon monoxide-sensitive apoptotic death of erythrocytes. Basic Clin. Pharmacol. Toxicol. 2012, 111, 348–355. [Google Scholar]
  52. Lang, F.; Qadri, S.M. Mechanisms and significance of eryptosis, the suicidal death of erythrocytes. Blood Purif. 2012, 33, 125–130. [Google Scholar] [CrossRef]
  53. Lupescu, A.; Jilani, K.; Zbidah, M.; Lang, E.; Lang, F. Enhanced Ca2+ entry, ceramide formation, and apoptotic death of erythrocytes triggered by plumbagin. J. Nat. Prod. 2012, 75, 1956–1961. [Google Scholar] [CrossRef]
  54. Lupescu, A.; Jilani, K.; Zbidah, M.; Lang, F. Induction of apoptotic erythrocyte death by rotenone. Toxicology 2012, 300, 132–137. [Google Scholar] [CrossRef]
  55. Lupescu, A.; Jilani, K.; Zelenak, C.; Zbidah, M.; Qadri, S.M.; Lang, F. Hexavalent chromium-induced erythrocyte membrane phospholipid asymmetry. Biometals 2012, 25, 309–318. [Google Scholar] [CrossRef]
  56. Polak-Jonkisz, D.; Purzyc, L. Ca influx versus efflux during eryptosis in uremic erythrocytes. Blood Purif. 2012, 34, 209–210. [Google Scholar] [CrossRef]
  57. Qadri, S.M.; Bauer, J.; Zelenak, C.; Mahmud, H.; Kucherenko, Y.; Lee, S.H.; Ferlinz, K.; Lang, F. Sphingosine but not sphingosine-1-phosphate stimulates suicidal erythrocyte death. Cell Physiol. Biochem. 2011, 28, 339–346. [Google Scholar] [CrossRef]
  58. Qadri, S.M.; Kucherenko, Y.; Lang, F. Beauvericin induced erythrocyte cell membrane scrambling. Toxicology 2011, 283, 24–31. [Google Scholar] [CrossRef]
  59. Qadri, S.M.; Kucherenko, Y.; Zelenak, C.; Jilani, K.; Lang, E.; Lang, F. Dicoumarol activates Ca2+-permeable cation channels triggering erythrocyte cell membrane scrambling. Cell Physiol. Biochem. 2011, 28, 857–864. [Google Scholar] [CrossRef]
  60. Qian, E.W.; Ge, D.T.; Kong, S.K. Salidroside protects human erythrocytes against hydrogen peroxide-induced apoptosis. J. Nat. Prod. 2012, 75, 531–537. [Google Scholar] [CrossRef]
  61. Shaik, N.; Zbidah, M.; Lang, F. Inhibition of Ca2+ entry and suicidal erythrocyte death by naringin. Cell Physiol. Biochem. 2012, 30, 678–686. [Google Scholar] [CrossRef]
  62. Vota, D.M.; Maltaneri, R.E.; Wenker, S.D.; Nesse, A.B.; Vittori, D.C. Differential erythropoietin action upon cells induced to eryptosis by different agents. Cell Biochem. Biophys. 2012, 65, 145–157. [Google Scholar]
  63. Weiss, E.; Cytlak, U.M.; Rees, D.C.; Osei, A.; Gibson, J.S. Deoxygenation-induced and Ca2+ dependent phosphatidylserine externalisation in red blood cells from normal individuals and sickle cell patients. Cell Calcium 2012, 51, 51–56. [Google Scholar] [CrossRef]
  64. Zappulla, D. Environmental stress, erythrocyte dysfunctions, inflammation, and the metabolic syndrome: Adaptations to Co2 increases? J. Cardiometab. Syndr. 2008, 3, 30–34. [Google Scholar] [CrossRef]
  65. Zbidah, M.; Lupescu, A.; Jilani, K.; Lang, F. Stimulation of suicidal erythrocyte death by fumagillin. Basic Clin. Pharmacol. Toxicol. 2012. [Google Scholar] [CrossRef]
  66. Zbidah, M.; Lupescu, A.; Shaik, N.; Lang, F. Gossypol-induced suicidal erythrocyte death. Toxicology 2012, 302, 101–1055. [Google Scholar] [CrossRef]
  67. Zelenak, C.; Pasham, V.; Jilani, K.; Tripodi, P.M.; Rosaclerio, L.; Pathare, G.; Lupescu, A.; Faggio, C.; Qadri, S.M.; Lang, F. Tanshinone iia stimulates erythrocyte phosphatidylserine exposure. Cell Physiol. Biochem. 2012, 30, 282–294. [Google Scholar] [CrossRef]
  68. Lang, E.; Qadri, S.M.; Lang, F. Killing me softly—Suicidal erythrocyte death. Int. J. Biochem. Cell Biol. 2012, 44, 1236–1243. [Google Scholar] [CrossRef]
  69. Calderon-Salinas, J.V.; Munoz-Reyes, E.G.; Guerrero-Romero, J.F.; Rodriguez-Moran, M.; Bracho-Riquelme, R.L.; Carrera-Gracia, M.A.; Quintanar-Escorza, M.A. Eryptosis and oxidative damage in type 2 diabetic mellitus patients with chronic kidney disease. Mol. Cell Biochem. 2011, 357, 171–179. [Google Scholar] [CrossRef]
  70. Nicolay, J.P.; Schneider, J.; Niemoeller, O.M.; Artunc, F.; Portero-Otin, M.; Haik, G., Jr.; Thornalley, P.J.; Schleicher, E.; Wieder, T.; Lang, F. Stimulation of suicidal erythrocyte death by methylglyoxal. Cell Physiol. Biochem. 2006, 18, 223–232. [Google Scholar] [CrossRef]
  71. Myssina, S.; Huber, S.M.; Birka, C.; Lang, P.A.; Lang, K.S.; Friedrich, B.; Risler, T.; Wieder, T.; Lang, F. Inhibition of erythrocyte cation channels by erythropoietin. J. Am. Soc. Nephrol. 2003, 14, 2750–2757. [Google Scholar] [CrossRef]
  72. Lang, P.A.; Beringer, O.; Nicolay, J.P.; Amon, O.; Kempe, D.S.; Hermle, T.; Attanasio, P.; Akel, A.; Schafer, R.; Friedrich, B.; et al. Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome. J. Mol. Med. 2006, 84, 378–388. [Google Scholar] [CrossRef]
  73. Kempe, D.S.; Akel, A.; Lang, P.A.; Hermle, T.; Biswas, R.; Muresanu, J.; Friedrich, B.; Dreischer, P.; Wolz, C.; Schumacher, U.; et al. Suicidal erythrocyte death in sepsis. J. Mol. Med. 2007, 85, 269–277. [Google Scholar]
  74. Bobbala, D.; Alesutan, I.; Foller, M.; Huber, S.M.; Lang, F. Effect of anandamide in plasmodium berghei-infected mice. Cell Physiol. Biochem. 2010, 26, 355–362. [Google Scholar] [CrossRef]
  75. Foller, M.; Bobbala, D.; Koka, S.; Huber, S.M.; Gulbins, E.; Lang, F. Suicide for survival—Death of infected erythrocytes as a host mechanism to survive malaria. Cell Physiol. Biochem. 2009, 24, 133–140. [Google Scholar] [CrossRef]
  76. Koka, S.; Bobbala, D.; Lang, C.; Boini, K.M.; Huber, S.M.; Lang, F. Influence of paclitaxel on parasitemia and survival of plasmodium berghei infected mice. Cell Physiol. Biochem. 2009, 23, 191–198. [Google Scholar] [CrossRef]
  77. Lang, P.A.; Schenck, M.; Nicolay, J.P.; Becker, J.U.; Kempe, D.S.; Lupescu, A.; Koka, S.; Eisele, K.; Klarl, B.A.; Rubben, H.; et al. Liver cell death and anemia in wilson disease involve acid sphingomyelinase and ceramide. Nat. Med. 2007, 13, 164–170. [Google Scholar] [CrossRef]
  78. Siraskar, B.; Ballal, A.; Bobbala, D.; Foller, M.; Lang, F. Effect of amphotericin b on parasitemia and survival of plasmodium berghei-infected mice. Cell Physiol. Biochem. 2010, 26, 347–354. [Google Scholar] [CrossRef]
  79. Lang, P.A.; Kasinathan, R.S.; Brand, V.B.; Duranton, C.; Lang, C.; Koka, S.; Shumilina, E.; Kempe, D.S.; Tanneur, V.; Akel, A.; et al. Accelerated clearance of plasmodium-infected erythrocytes in sickle cell trait and annexin-a7 deficiency. Cell Physiol. Biochem. 2009, 24, 415–428. [Google Scholar] [CrossRef]
  80. Kempe, D.S.; Lang, P.A.; Duranton, C.; Akel, A.; Lang, K.S.; Huber, S.M.; Wieder, T.; Lang, F. Enhanced programmed cell death of iron-deficient erythrocytes. FASEB J. 2006, 20, 368–370. [Google Scholar]
  81. Qadri, S.M.; Mahmud, H.; Lang, E.; Gu, S.; Bobbala, D.; Zelenak, C.; Jilani, K.; Siegfried, A.; Foller, M.; Lang, F. Enhanced suicidal erythrocyte death in mice carrying a loss-of-function mutation of the adenomatous polyposis coli gene. J. Cell Mol. Med. 2012, 16, 1085–1093. [Google Scholar] [CrossRef]
  82. Birka, C.; Lang, P.A.; Kempe, D.S.; Hoefling, L.; Tanneur, V.; Duranton, C.; Nammi, S.; Henke, G.; Myssina, S.; Krikov, M.; et al. Enhanced susceptibility to erythrocyte “apoptosis” following phosphate depletion. Pflugers Arch. 2004, 448, 471–477. [Google Scholar]
  83. Wong, K.H.; Wallen, C.A.; Wheeler, K.T. Biodistribution of misonidazole and 1,3-bis(2-chloroethyl)-1-nitrosourea (bcnu) in rats bearing unclamped and clamped 9l subcutaneous tumors. Int. J. Radiat. Oncol. Biol. Phys. 1989, 17, 135–143. [Google Scholar]
  84. Bookchin, R.M.; Ortiz, O.E.; Lew, V.L. Activation of calcium-dependent potassium channels in deoxygenated sickled red cells. Prog. Clin. Biol. Res. 1987, 240, 193–200. [Google Scholar]
  85. Brand, V.B.; Sandu, C.D.; Duranton, C.; Tanneur, V.; Lang, K.S.; Huber, S.M.; Lang, F. Dependence of plasmodium falciparum in vitro growth on the cation permeability of the human host erythrocyte. Cell Physiol. Biochem. 2003, 13, 347–356. [Google Scholar] [CrossRef]
  86. Borst, O.; Abed, M.; Alesutan, I.; Towhid, S.T.; Qadri, S.M.; Foller, M.; Gawaz, M.; Lang, F. Dynamic adhesion of eryptotic erythrocytes to endothelial cells via cxcl16/sr-psox. Am. J. Physiol. Cell Physiol. 2012, 302, C644–C651. [Google Scholar] [CrossRef]
  87. Andrews, D.A.; Low, P.S. Role of red blood cells in thrombosis. Curr. Opin. Hematol. 1999, 6, 76–82. [Google Scholar] [CrossRef]
  88. Closse, C.; Dachary-Prigent, J.; Boisseau, M.R. Phosphatidylserine-related adhesion of human erythrocytes to vascular endothelium. Br. J. Haematol. 1999, 107, 300–302. [Google Scholar] [CrossRef]
  89. Gallagher, P.G.; Chang, S.H.; Rettig, M.P.; Neely, J.E.; Hillery, C.A.; Smith, B.D.; Low, P.S. Altered erythrocyte endothelial adherence and membrane phospholipid asymmetry in hereditary hydrocytosis. Blood 2003, 101, 4625–4627. [Google Scholar] [CrossRef]
  90. Pandolfi, A.; di Pietro, N.; Sirolli, V.; Giardinelli, A.; di Silvestre, S.; Amoroso, L.; di Tomo, P.; Capani, F.; Consoli, A.; Bonomini, M. Mechanisms of uremic erythrocyte-induced adhesion of human monocytes to cultured endothelial cells. J. Cell Physiol. 2007, 213, 699–709. [Google Scholar] [CrossRef]
  91. Wood, B.L.; Gibson, D.F.; Tait, J.F. Increased erythrocyte phosphatidylserine exposure in sickle cell disease: Flow-cytometric measurement and clinical associations. Blood 1996, 88, 1873–1880. [Google Scholar]
  92. Chung, S.M.; Bae, O.N.; Lim, K.M.; Noh, J.Y.; Lee, M.Y.; Jung, Y.S.; Chung, J.H. Lysophosphatidic acid induces thrombogenic activity through phosphatidylserine exposure and procoagulant microvesicle generation in human erythrocytes. Arterioscler Thromb. Vasc. Biol. 2007, 27, 414–421. [Google Scholar]
  93. Zwaal, R.F.; Comfurius, P.; Bevers, E.M. Surface exposure of phosphatidylserine in pathological cells. Cell Mol. Life Sci. 2005, 62, 971–988. [Google Scholar] [CrossRef]

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Jilani, K.; Lang, F. Carmustine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane. Toxins 2013, 5, 703-716. https://doi.org/10.3390/toxins5040703

AMA Style

Jilani K, Lang F. Carmustine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane. Toxins. 2013; 5(4):703-716. https://doi.org/10.3390/toxins5040703

Chicago/Turabian Style

Jilani, Kashif, and Florian Lang. 2013. "Carmustine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane" Toxins 5, no. 4: 703-716. https://doi.org/10.3390/toxins5040703

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