Concurrent NRAS-BRAF variants in metastatic colorectal cancer: a Tunisian case report.

Target therapy for metastatic colorectal cancer needs the determination of KRAS, NRAS, and BRAF mutation status to identify patients resistant to anti-EGFR treatment. RAS genes (KRAS/NRAS) are mutated in 40-60% of metastatic colorectal cancer and BRAF in 5-10%. The presence of a double mutation in RAS and BRAF is rare. Therefore, RAS and BRAF mutations were considered exclusive. Herein, we describe a novel concomitant NRAS/BRAF mutation identified in a series of 865 colorectal cancer patients.

Tetraploidization Increases the Motility and Invasiveness of Cancer Cells.

Polyploidy and metastasis are associated with a low probability of disease-free survival in cancer patients. Polyploid cells are known to facilitate tumorigenesis. However, few data associate polyploidization with metastasis. Here, by generating and using diploid (2n) and tetraploid (4n) clones from malignant fibrous histiocytoma (MFH) and colon carcinoma (RKO), we demonstrate the migration and invasion advantage of tetraploid cells in vitro using several assays, including the wound healing, the OrisTM two-dimensional cell migration, single-cell migration tracking by video microscopy, the Boyden chamber, and the xCELLigence RTCA real-time cell migration. Motility advantage was observed despite tetraploid cell proliferation weakness. We could also demonstrate preferential metastatic potential in vivo for the tetraploid clone using the tail vein injection in mice and tracking metastatic tumors in the lung. Using the Mitelman Database of Chromosome Aberrations in Cancer, we found an accumulation of polyploid karyotypes in metastatic tumors compared to primary ones. This work reveals the clinical relevance of the polyploid subpopulation and the strategic need to highlight polyploidy in preclinical studies as a therapeutic target for metastasis.

Polo-like kinase inhibitor BI2536 induces eryptosis.

BI2536 is potent inhibitor of polo-like kinases PLK1, 2, and 3. The inhibition of PLKs in nucleated cells induces apoptosis by perturbing the cell cycle with consequent engagement of mitotic catastrophe. BI2536 is being tested as chemotherapy in various phase I/II/III clinical trials. Erythrocytes do not have a nucleus; however, they may undergo programmed suicide with characteristic hallmarks including cell shrinkage and phosphatidylserine translocation to the cell surface. This particular death is baptized eryptosis. Our study explored whether BI2536 induces eryptosis. We used flow cytometry to access death in red blood cells. We analyzed the cellular volume, the intracellular calcium concentration, the cell surface phosphatidylserine exposure, and the ceramide abundance. In addition, we analyzed the effect of BI2536 on hemolysis. Our investigation showed that after 48 h of incubation with PLK inhibitor BI2536, erythrocytes lost volume and were positive for annexin­V without any effect on hemolysis. Cells also showed an abundance of ceramide and an increase of intracellular calcium. All these finding suggest that BI2536 provokes eryptosis in red blood cells, ostensibly in part due to Ca2+ entry and ceramide accumulation.

Preferential Killing of Tetraploid Colon Cancer Cells by Targeting the Mitotic Kinase PLK1.

BACKGROUND/AIMS: Chromosomal instability is a well-known factor in the progression of different types of cancer, including colorectal cancer. Chromosomal instability results in severely rearranged karyotypes and aneuploidy. Tetraploidy constitutes an intermediate phase during the polyploidy/aneuploidy cascade in oncogenesis, and tetraploid cells are particularly resistant to chemotherapy. Whether inhibition of the mitotic protein polo-like kinase 1 (PLK1) prevents the survival of tetraploid colon cancer cells is unknown. METHODS: Diploid and tetraploid cells were transfected with siPLK1 or treated with PLK1 inhibitor Bi2536 in combination with spindle poison. Cell toxicity was assessed via crystal violet staining and clonogenic assay. Flow cytometry assessment analyzed numerous cell apoptotic parameters and cell cycle phases. Synergistic activity between Bi2536 and paclitaxel, vincristine or colchicine was calculated using the CompuSyn software. RESULTS: Inhibition or abrogation of PLK1 prevented the survival of colon cancer cells, specifically tetraploid cells. The cell death induced by PLK inhibition was due to mitotic slippage, followed by the activation of the intrinsic pathway of apoptosis. We further demonstrated that co-treatment of the tetraploid colon cancer cells with a PLK1 inhibitor and the microtubule polymerisation inhibitor vincristine or colchicine, but not the microtubule depolymerisation inhibitor paclitaxel, provoked a lethal synergistic effect. CONCLUSION: PLK1 inhibition together with microtubule-targeting chemicals, serve as a potent therapeutic strategy for targeting tetraploid cancer cells.

Gene Expression Signature of Acquired Chemoresistance in Neuroblastoma Cells.

Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.

Discovery of epi-Enprioline as a Novel Drug for the Treatment of Vincristine Resistant Neuroblastoma.

Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.

In Silico Identification and in Vitro Analysis of B and T-Cell Epitopes of the Black Turtle Bean (Phaseolus Vulgaris L.) Lectin.

BACKGROUND/AIMS: The incidence of lectin allergic disease is increasing in recent decades, and definitive treatment is still lacking. Identification of B and T-cell epitopes of allergen will be useful in understanding the allergen antibody responses as well as aiding in the development of new diagnostics and therapy regimens for lectin poisoning. In the current study, we mainly addressed these questions. METHODS: Three-dimensional structure of the lectin from black turtle bean (Phaseolus vulgaris L.) was modeled using the structural template of Phytohemagglutinin from P. vulgaris (PHA-E, PDB ID: 3wcs.1.A) with high identity. The B and T-cell epitopes were screened and identified by immunoinformatics and subsequently validated by ELISA, lymphocyte proliferation and cytokine profile analyses. RESULTS: Seven potential B-cell epitopes (B1 to B7) were identified by sequence and structure based methods, while three T-cell epitopes (T1 to T3) were identified by the predictions of binding score and inhibitory concentration. The epitope peptides were synthesized. Significant IgE binding capability was found in B-cell epitopes (B2, B5, B6 and B7) and T2 (a cryptic B-cell epitope). T1 and T2 induced significant lymphoproliferation, and the release of IL-4 and IL-5 cytokine confirmed the validity of T-cell epitope prediction. Abundant hydrophobic amino acids were found in B-cell epitope and T-cell epitope regions by amino acid analysis. Positively charged amino acids, such as His residue, might be more favored for B-cell epitope. CONCLUSION: The present approach can be applied for the identification of epitopes in novel allergen proteins and thus for designing diagnostics and therapies in lectin allergy.

Inhibition of Suicidal Erythrocyte Death by Reversine.

BACKGROUND/AIMS: The A3 adenosine receptor antagonist reversine (2-(4-morpholinoanilino)-6-cyclohexylaminopurine) influences cellular differentiation, inhibits cell proliferation, induces cell-cycle arrest, triggers apoptosis, causes cell swelling with polyploidy and stimulates autophagy. The effect on apoptosis involves mitochondria and caspases. Erythrocytes are lacking mitochondria but express caspases and are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), energy depletion and oxidative stress. The present study explored, whether reversine influences eryptosis. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), Ca2+ loading (30 minutes treatment with 1 µM Ca2+ ionophore ionomycin), or oxidative stress (15 min exposure to 0.3 mM tert-butylhydroperoxide). RESULTS: A 48 hours exposure of human erythrocytes to reversine (1-10 µM) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, Ca2+ loading, and oxidative stress were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of each, Ca2+ loading, energy depletion and oxidative stress on annexin-V-binding were significantly blunted in the presence of reversine (1-10 µM). The effect of ionomycin, but not the effects of energy depletion and oxidative stress on forward scatter were again significantly blunted in the presence of reversine (≥1 µM]. CONCLUSIONS: Reversine is a powerful inhibitor of cell membrane scrambling following energy depletion, Ca2+ loading and oxidative stress.

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

Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD), hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i) with Fluo3, oxidative stress with 2',7'-dichlorodihydrofuorescein diacetate (DCFDA), glutathione (GSH) with mercury orange 1(4-chloromercuryphenyl-azo-2-naphthol), lipid peroxidation with BODIPY 581/591 C11 fluorescence, and ceramide abundance with specific antibodies. The contribution of kinases and caspases is defined with the use of the respective inhibitors. It is hoped that the present detailed description of materials and methods required for the analysis of eryptosis encourages further scientists to enter this highly relevant research area.

Camalexin-Induced Cell Membrane Scrambling and Cell Shrinkage in Human Erythrocytes.

BACKGROUND/AIMS: The thaliana phytoalexin Camalexin has been proposed for the treatment of malignancy. Camalexin counteracts tumor growth in part by stimulation of suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms contributing to the complex machinery executing eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, protein kinase C and caspases. The present study explored, whether Camalexin induces eryptosis and, if so, to shed light on mechanisms involved. METHODS: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo-3 fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to Camalexin significantly increased the percentage of annexin-V-binding cells (≥ 10 µg/ml), significantly decreased forward scatter (≥ 5 µg/ml) and significantly increased Fluo-3-fluorescence (≥ 10 µg/ml), but did not significantly modify DCFDA fluorescence or ceramide abundance. The effect of Camalexin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by kinase inhibitors staurosporine (1 µM) and chelerythrine (10 µM), as well as by caspase inhibitors zVAD (10 µM) and zIETD-fmk (50 µM). CONCLUSIONS: Camalexin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part depending on Ca2+ entry, as well as staurosporine and chelerythrine sensitive kinase(s) as well as zVAD and zIETD-fmk sensitive caspase(s).

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

BACKGROUND: Injury by the sting of Lesser weever fish (Trachinus vipera) may lead to severe pain, edema or tissue necrosis. Cellular effects of the venom are still incompletely understood. Previous observations revealed that purified Lesser weever fish venom (LWFV) induces suicidal death of erythrocytes and HCT116 human colon carcinoma cells. The present study addressed the effect of the venom on colon carcinoma cell toxicity, shape and migration both in p53+/+ and/or p53-/- conditions. METHODS: Cells were exposed to medium without or with 500 µg/ ml LWFV. Cell shape, cell area and circularity were visualized and quantified by fluorescence microscopy. Cell volume, granularity and cells toxicity were assessed via the apoptotic parameters dissipation of mitochondrial inner transmembrane potential, phosphatidylserine surface exposure and cell membrane permeabilization were measured utilizing flow cytometry. Cell migration was evaluated using wound healing assay and two-dimensional migration assay. RESULTS: LWFV treatment was followed by a marked change of cell shape and size, significant decrease of cell area and circularity, significant impairment of cell migration, as well as induction of apoptosis after long exposition. CONCLUSIONS: LWFV exposure leads to cell shrinkage, increased granularity, apoptosis and impairment of cell migration, effects presumably contributing to LWFV-induced tissue injury.

Lithium Sensitivity of Store Operated Ca2+ Entry and Survival of Fibroblasts Isolated from Chorea-Acanthocytosis Patients.

BACKGROUND: The widely expressed protein chorein fosters activation of the phosphoinositide 3 kinase (PI3K) pathway thus supporting cell survival. Loss of function mutations of the chorein encoding gene VPS13A (vacuolar protein sorting-associated protein 13A) causes chorea-acanthocytosis (ChAc), a neurodegenerative disorder paralleled by deformations of erythrocytes. In mice, genetic knockout of chorein leads to enhanced neuronal apoptosis. PI3K dependent signalling upregulates Orai1, a pore forming channel protein accomplishing store operated Ca2+ entry (SOCE). Increased Orai1 expression and SOCE have been shown to confer survival of tumor cells. SOCE could be up-regulated by lithium. The present study explored, whether SOCE and/or apoptosis are altered in ChAc fibroblasts and could be modified by lithium treatment. METHODS: Fibroblasts were isolated from ChAc patients and age-matched healthy volunteers. Cytosolic Ca2+ activity ([Ca2+]i) was estimated from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarcoendoplasmatic Ca2+-ATPase (SERCA) inhibitor thapsigargin (1 µM), and apoptosis from annexin-V/propidium iodide staining quantified in flow cytometry. RESULTS: SOCE was significantly smaller in ChAc fibroblasts than in control fibroblasts. Lithium (2 mM, 24 hours) significantly increased and Orai1 blocker 2-Aminoethoxydiphenyl Borate (2-APB, 50 µM, 24 hours) significantly decreased SOCE. Annexin-V-binding and propidium iodide staining were significantly higher in ChAc fibroblasts than in control fibroblasts. In ChAc fibroblasts annexin-V-binding and propidium iodide staining were significantly decreased by lithium treatment, significantly increased by 2-APB and virtually lithium insensitive in the presence of 2-APB. CONCLUSIONS: In ChAc fibroblasts, downregulation of SOCE contributes to enhanced susceptibility to apoptosis. Both, decreased SOCE and enhanced apoptosis of ChAc fibroblasts can be reversed by lithium treatment.

Resveratrol and aspirin eliminate tetraploid cells for anticancer chemoprevention.

Tetraploidy constitutes a genomically metastable state that can lead to aneuploidy and genomic instability. Tetraploid cells are frequently found in preneoplastic lesions, including intestinal cancers arising due to the inactivation of the tumor suppressor adenomatous polyposis coli (APC). Using a phenotypic screen, we identified resveratrol as an agent that selectively reduces the fitness of tetraploid cells by slowing down their cell cycle progression and by stimulating the intrinsic pathway of apoptosis. Selective killing of tetraploid cells was observed for a series of additional agents that indirectly or directly stimulate AMP-activated protein kinase (AMPK) including salicylate, whose chemopreventive action has been established by epidemiological studies and clinical trials. Both resveratrol and salicylate reduced the formation of tetraploid or higher-order polyploid cells resulting from the culture of human colon carcinoma cell lines or primary mouse epithelial cells lacking tumor protein p53 (TP53, best known as p53) in the presence of antimitotic agents, as determined by cytofluorometric and videomicroscopic assays. Moreover, oral treatment with either resveratrol or aspirin, the prodrug of salicylate, repressed the accumulation of tetraploid intestinal epithelial cells in the Apc(Min/+) mouse model of colon cancer. Collectively, our results suggest that the chemopreventive action of resveratrol and aspirin involves the elimination of tetraploid cancer cell precursors.

Triggering of Erythrocyte Cell Membrane Scrambling by Emodin.

BACKGROUND/AIMS: The natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) is a component of several Chinese medicinal herbal preparations utilized for more than 2000 years. The substance has been used against diverse disorders including malignancy, inflammation and microbial infection. The substance is effective in part by triggering suicidal death or apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study aimed to test, whether emodin induces eryptosis and, if so, to elucidate underlying cellular mechanisms. METHODS: Phosphatidylserine abundance 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: Exposure of human erythrocytes for 48 hours to emodin (≥ 10 µM) significantly increased the percentage of annexin-V-binding cells, and at higher concentrations (≥ 50 µM) significantly increased forward scatter. Emodin significantly increased Fluo3-fluorescence (≥ 10 µM), DCFDA fluorescence (75 µM) and ceramide abundance (75 µM). The effect of emodin on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Emodin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry and paralleled by oxidative stress and ceramide appearance at the erythroctye surface.

Stimulation of Suicidal Erythrocyte Death by Phosphatase Inhibitor Calyculin A.

BACKGROUND/AIMS: The serine/threonine protein phosphatase 1 and 2a inhibitor Calyculin A may trigger suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+] i). Eryptosis is fostered by activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, and D4476 sensitive casein kinase. Eryptosis may further involve zVAD sensitive caspases. The present study explored, whether Calyculin A induces eryptosis and, if so, whether its effect requires Ca2+ entry, kinases and/or caspases Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, and [Ca2+] i from Fluo-3 fluorescence, as determined by flow cytometry. RESULTS: A 48 hours exposure of human erythrocytes to Calyculin A (≥ 2.5 nM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter and significantly increased Fluo-3 fluorescence. The effect of Calyculin A on annexin-V-binding was significantly blunted by removal of extracellular Ca2+, by staurosorine (1 µM), SB203580 (2 µM), D4476 (10 µM), and zVAD (10 µM). CONCLUSIONS: Calyculin A triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part requiring Ca2+ entry, kinase activity and caspase activation.

Ca2+ Entry, Oxidative Stress, Ceramide and Suicidal Erythrocyte Death Following Diosgenin Treatment.

BACKGROUND/AIMS: The bioactive steroid sapogenin diosgenin is considered for a wide variety of applications including treatment of malignancy. The substance counteracts tumor growth in part by stimulating apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether diosgenin induces eryptosis and, if so, to decipher cellular mechanisms involved. METHODS: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to diosgenin significantly increased the percentage of annexin-V-binding cells (≥ 5 µM), significantly decreased forward scatter (15 µM), significantly increased Fluo3-fluorescence (≥ 10 µM), significantly increased DCF fluorescence (15 µM), significantly increased ceramide abundance (15 µM) and significantly increased hemolysis (15 µM). The effect of diosgenin (15 µM) on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Diosgenin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry, oxidative stress and ceramide.

Triggering of Suicidal Erythrocyte Death by Fascaplysin.

BACKGROUND/AIMS: The bis-indole alkaloid Fascaplysin is effective against malignancy, an effect at least partially due to stimulation of tumor cell apoptosis. Similar to apoptosis of nucleated cells, erythrocytes could enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether Fascaplysin induces eryptosis and, if so, to shed light on the cellular mechanisms involved. METHODS: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface 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. Hemolysis was quantified from the hemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Fascaplysin (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, and significantly increased Fluo3-fluorescence, DCFDA fluorescence as well as ceramide abundance. The effect of Fascaplysin on annexin-V-binding and forward scatter was significantly blunted but not abolished by removal of extracellular Ca2+. CONCLUSIONS: Fascaplysin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress and ceramide.

Stimulation of Erythrocyte Cell Membrane Scrambling by Quinine.

BACKGROUND/AIMS: The analkaloid drug quinine is utilized mainly for the chemoprophylaxis of malaria. The multiple side effects of quinine include hemolytic anemia and hemolytic uremic syndrome, disorders involving suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide and D4476 sensitive casein kinase. The present study explored the putative effect of quinine on eryptosis and elucidated cellular 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 DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. RESULTS: A 48 hours exposure of human erythrocytes to quinine (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly affecting forward scatter. Quinine significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of quinine on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM). CONCLUSIONS: Quinine triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and D4476 sensitive casein kinase.

Stimulation of Suicidal Erythrocyte Death by Rottlerin.

BACKGROUND/AIMS: The phytochemical polyphenol rottlerin is a potent activator of diverse Ca2+ -sensitive K+ channels. Those channels play a decisive role in the execution of eryptosis, the suicidal death of erythrocytes, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i) and ceramide. The present study explored, whether rottlerin induces eryptosis and, if so, to test for the involvement of Ca2+ entry and ceramide. METHODS: Flow cytometry was employed to estimate phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was quantified by determination of haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to rottlerin (1 - 5 µM) significantly increased the percentage of annexin-V-binding cells, an effect paralleled by significant decrease of forward scatter. Up to 5 µM rottlerin failed to significantly increase average Fluo3-fluorescence. Rottlerin (5 µM) did, however, significantly increase the ceramide abundance. Rottlerin (5 µM) further significantly increased hemolysis. The effect of rottlerin (5 µM) on annexin-V-binding was virtually abolished by removal of extracellular Ca2+. CONCLUSIONS: Rottlerin stimulates eryptosis with erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by and at least in part due to Ca2+ entry and ceramide.

Down-Regulation of the Na+,Cl- Coupled Creatine Transporter CreaT (SLC6A8) by Glycogen Synthase Kinase GSK3ß.

BACKGROUND: The Na+,Cl- coupled creatine transporter CreaT (SLC6A8) is expressed in a variety of tissues including the brain. Genetic defects of CreaT lead to mental retardation with seizures. The present study explored the regulation of CreaT by the ubiquitously expressed glycogen synthase kinase GSK3ß, which contributes to the regulation of neuroexcitation. GSK3ß is phosphorylated and thus inhibited by PKB/Akt. Moreover, GSK3ß is inhibited by the antidepressant lithium. The present study thus further tested for the effects of PKB/Akt and of lithium. METHODS: CreaT was expressed in Xenopus laevis oocytes with or without wild-type GSK3ß or inactive K85RGSK3ß. CreaT and GSK3ß were further expressed without and with additional expression of wild type PKB/Akt. Creatine transport in those oocytes was quantified utilizing dual electrode voltage clamp. RESULTS: Electrogenic creatine transport was observed in CreaT expressing oocytes but not in water-injected oocytes. In CreaT expressing oocytes, co-expression of GSK3ß but not of K85RGSK3ß, resulted in a significant decrease of creatine induced current. Kinetic analysis revealed that GSK3ß significantly decreased the maximal creatine transport rate. Exposure of CreaT and GSK3ß expressing oocytes for 24 hours to Lithium was followed by a significant increase of the creatine induced current. The effect of GSK3ß on CreaT was abolished by co-expression of PKB/Akt. CONCLUSION: GSK3ß down-regulates the creatine transporter CreaT, an effect reversed by treatment with the antidepressant Lithium and by co-expression of PKB/Akt.