scholarly journals 1α,25(OH) 2D3 Sensitive Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Ishikawa Cells

2017 ◽  
Vol 41 (2) ◽  
pp. 678-688 ◽  
Author(s):  
Ni Zeng ◽  
Yuetao Zhou ◽  
Shaqiu Zhang ◽  
Yogesh Singh ◽  
Bing Shi ◽  
...  
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Lactate Concentration ◽  
B Cell Lymphoma ◽  
Transport Processes ◽  
Glycolytic Flux ◽  
Protein Abundance ◽  
Cytosolic Ph ◽  
Transcript Levels ◽  
Ishikawa Cells

Background/Aims: Tumor cell proliferation is modified by 1,25-Dihydroxy-Vitamin D3 (1,25(OH)2D3), a steroid hormone predominantly known for its role in calcium and phosphorus metabolism. Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions and lactate, which is in part accomplished by Na+/H+ exchangers, such as NHE1 and monocarboxylate transporters, such as MCT4. An effect of 1,25(OH)2D3 on those transport processes has, however, never been reported. As cytosolic pH impacts on apoptosis, the study further explored the effect of 1,25(OH)2D3 on apoptosis and on the apoptosis regulating kinase AKT, transcription factor Forkhead box O-3 (FOXO3A) and B-cell lymphoma protein BCL-2. Methods: In human endometrial adenocarcinoma (Ishikawa) cells, cytosolic pH (pHi) was determined utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, NHE1 and MCT4 transcript levels using qRT-PCR, NHE1, MCT4, total & phospho AKT, total & phospho-FOXO3A and BCL-2 protein abundance by Western blotting, lactate concentration in the supernatant utilizing a colorimetric enzyme assay and cell death quantification using CytoTox 96®, Annexin V and Propidium Iodide staining. Results: A 24 hours treatment with 1,25(OH)2D3 (100 nM) significantly increased cytosolic pH (pHi), significantly decreased Na+/H+ exchanger activity, NHE1 and MCT4 transcript levels as well as protein abundance and significantly increased lactate concentration in the supernatant. Treatment of Ishikawa cells with 1,25(OH)2D3 (100 nM) further triggered apoptosis, an effect paralleled by decreased phosphorylation of AKT and FOXO3A as well as decreased abundance of BCL-2. Conclusions: In Ishikawa cells 1,25(OH)2D3 is a powerful stimulator of glycolysis, an effect presumably due to cytosolic alkalinization. Despite stimulation of glycolysis, 1,25(OH)2D3 stimulates slightly but significantly suicidal cell death, an effect presumably in part due to decreased activation of AKT with decreased inhibition of pro-apoptotic transcription factor FOXO3A and downregulation of the anti-apoptotic protein BCL-2.

scholarly journals Negative Effect of Ellagic Acid on Cytosolic pH Regulation and Glycolytic Flux in Human Endometrial Cancer Cells

2017 ◽  
Vol 41 (6) ◽  
pp. 2374-2382 ◽  
Author(s):  
Khalid N. M. Abdelazeem ◽  
Yogesh Singh ◽  
Florian Lang ◽  
Madhuri S. Salker
Keyword(s):  
Endometrial Cancer ◽  
Glucose Uptake ◽  
Tumor Cells ◽  
Ellagic Acid ◽  
Glycolytic Flux ◽  
Protein Abundance ◽  
Cytosolic Ph ◽  
Forward Scatter ◽  
Transcript Levels ◽  
Lactate Release

Background/Aims: Key properties of tumor cells include enhanced glycolytic flux with excessive consumption of glucose and formation of lactate. As glycolysis is highly sensitive to cytosolic pH, maintenance of glycolysis requires export of H+ ions, which is in part accomplished by Na+/H+ exchangers, such as NHE1. The carrier is sensitive to oxidative stress. Growth of tumor cells could be suppressed by the polyphenol Ellagic acid, which is found in various fruits and vegetables. An effect of Ellagic acid on transport processes has, however, never been reported. The present study thus elucidated an effect of Ellagic acid on cytosolic pH (pHi), NHE1 transcript levels, NHE1 protein abundance, Na+/H+ exchanger activity, and lactate release. Methods: Experiments were performed in Ishikawa cells without or with prior Ellagic acid (20 µM) treatment. NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance by Western blotting, pHi utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na+/H+ exchanger activity from Na+ dependent realkalinization after an ammonium pulse, cell volume from forward scatter in flow cytometry, reactive oxygen species (ROS) from 2’,7’-dichlorodihydrofluorescein fluorescence, glucose uptake utilizing 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose, and lactate concentration in the supernatant utilizing a colorimetric (570 nm)/ fluorometric enzymatic assay. Results: A 48 hour treatment with Ellagic acid (20 µM) significantly decreased NHE1 transcript levels by 75%, NHE1 protein abundance by 95%, pHi from 7.24 ± 0.01 to 7.02 ± 0.01, Na+/H+ exchanger activity by 77%, forward scatter by 10%, ROS by 82%, glucose uptake by 58%, and lactate release by 15%. Conclusion: Ellagic acid (20µM) markedly down-regulates ROS formation and NHE1 expression leading to decreased Na+/H+ exchanger activity, pHi, glucose uptake and lactate release in endometrial cancer cells. Those effects presumably contribute to reprogramming and growth inhibition of tumor cells.

scholarly journals Effect of MgCl2 and GdCl3 on ORAI1 Expression and Store-Operated Ca2+ Entry in Megakaryocytes

2021 ◽  
Vol 22 (7) ◽  
pp. 3292
Author(s):  
Kuo Zhou ◽  
Xuexue Zhu ◽  
Ke Ma ◽  
Jibin Liu ◽  
Bernd Nürnberg ◽  
...  
Keyword(s):  
Chronic Kidney Disease ◽  
Transcription Factor ◽  
Smooth Muscle ◽  
Kidney Disease ◽  
Protein Abundance ◽  
Rt Pcr ◽  
Calcium Sensing Receptor ◽  
Transcript Levels ◽  
Calcium Sensing ◽  
Store Depletion

In chronic kidney disease, hyperphosphatemia upregulates the Ca2+ channel ORAI and its activating Ca2+ sensor STIM in megakaryocytes and platelets. ORAI1 and STIM1 accomplish store-operated Ca2+ entry (SOCE) and play a key role in platelet activation. Signaling linking phosphate to upregulation of ORAI1 and STIM1 includes transcription factor NFAT5 and serum and glucocorticoid-inducible kinase SGK1. In vascular smooth muscle cells, the effect of hyperphosphatemia on ORAI1/STIM1 expression and SOCE is suppressed by Mg2+ and the calcium-sensing receptor (CaSR) agonist Gd3+. The present study explored whether sustained exposure to Mg2+ or Gd3+ interferes with the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. To this end, human megakaryocytic Meg-01 cells were treated with 2 mM ß-glycerophosphate for 24 h in the absence and presence of either 1.5 mM MgCl2 or 50 µM GdCl3. Transcript levels were estimated utilizing q-RT-PCR, protein abundance by Western blotting, cytosolic Ca2+ concentration ([Ca2+]i) by Fura-2 fluorescence and SOCE from the increase in [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, Mg2+ and Gd3+ upregulated CaSR and blunted or virtually abolished the phosphate-induced upregulation of NFAT5, SGK1, ORAI1,2,3, STIM1,2 and SOCE in megakaryocytes. In conclusion, Mg2+ and the CaSR agonist Gd3+ interfere with phosphate-induced dysregulation of [Ca2+]i in megakaryocytes.

scholarly journals NOXA genetic amplification or pharmacologic induction primes lymphoma cells to BCL2 inhibitor-induced cell death

2018 ◽  
Vol 115 (47) ◽  
pp. 12034-12039 ◽  
Author(s):  
Yuxuan Liu ◽  
Patrizia Mondello ◽  
Tatiana Erazo ◽  
Neeta Bala Tannan ◽  
Zahra Asgari ◽  
...  
Keyword(s):  
Cell Death ◽  
B Cell Lymphoma ◽  
Lymphoma Cell ◽  
Protein Abundance ◽  
Dependent Manner ◽  
Combination Strategies ◽  
Deacetylase Inhibitor ◽  
Bcl2 Protein

Although diffuse large B cell lymphoma (DLBCL) cells widely express the BCL2 protein, they rarely respond to treatment with BCL2-selective inhibitors. Here we show that DLBCL cells harboring PMAIP1/NOXA gene amplification were highly sensitive to BCL2 small-molecule inhibitors. In these cells, BCL2 inhibition induced cell death by activating caspase 9, which was further amplified by caspase-dependent cleavage and depletion of MCL1. In DLBCL cells lacking NOXA amplification, BCL2 inhibition was associated with an increase in MCL1 protein abundance in a BIM-dependent manner, causing a decreased antilymphoma efficacy. In these cells, dual inhibition of MCL1 and BCL2 was required for enhanced killing. Pharmacologic induction of NOXA, using the histone deacetylase inhibitor panobinostat, decreased MCL1 protein abundance and increased lymphoma cell vulnerability to BCL2 inhibitors in vitro and in vivo. Our data provide a mechanistic rationale for combination strategies to disrupt lymphoma cell codependency on BCL2 and MCL1 proteins in DLBCL.

scholarly journals Inhibition of Lithium Sensitive Orai1/ STIM1 Expression and Store Operated Ca2+ Entry in Chorea-Acanthocytosis Neurons by NF-κB Inhibitor Wogonin

2018 ◽  
Vol 51 (1) ◽  
pp. 278-289 ◽  
Author(s):  
Basma Sukkar ◽  
Stefan Hauser ◽  
Lisann Pelzl ◽  
Zohreh Hosseinzadeh ◽  
Itishri Sahu ◽  
...  
Keyword(s):  
Cell Death ◽  
Cortical Neurons ◽  
Lithium Treatment ◽  
Cell Types ◽  
Protein Abundance ◽  
Loss Of Function ◽  
Atpase Inhibitor ◽  
Patch Clamp Recording ◽  
Transcript Levels ◽  
Induced Pluripotent

Background/Aims: The neurodegenerative disease Chorea-Acanthocytosis (ChAc) is caused by loss-of-function-mutations of the chorein-encoding gene VPS13A. In ChAc neurons transcript levels and protein abundance of Ca2+ release activated channel moiety (CRAC) Orai1 as well as its regulator STIM1/2 are decreased, resulting in blunted store operated Ca2+-entry (SOCE) and enhanced suicidal cell death. SOCE is up-regulated and cell death decreased by lithium. The effects of lithium are paralleled by upregulation of serum & glucocorticoid inducible kinase SGK1 and abrogated by pharmacological SGK1 inhibition. In other cell types SGK1 has been shown to be partially effective by upregulation of NFκB, a transcription factor stimulating the expression of Orai1 and STIM. The present study explored whether pharmacological inhibition of NFκB interferes with Orai1/STIM1/2 expression and SOCE and their upregulation by lithium in ChAc neurons. Methods: Cortical neurons were differentiated from induced pluripotent stem cells generated from fibroblasts of ChAc patients and healthy volunteers. Orai1 and STIM1 transcript levels and protein abundance were estimated from qRT-PCR and Western blotting, respectively, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, SOCE from increase of [Ca2+]i following Ca2+ re-addition after Ca2+-store depletion with sarco-endoplasmatic Ca2+-ATPase inhibitor thapsigargin (1µM), as well as CRAC current utilizing whole cell patch clamp recording. Results: Orai1 and STIM1 transcript levels and protein abundance as well as SOCE and CRAC current were significantly enhanced by lithium treatment (2 mM, 24 hours). These effects were reversed by NFκB inhibitor wogonin (50 µM). Conclusion: The stimulation of expression and function of Orai1/STIM1/2 by lithium in ChAc neurons are disrupted by pharmacological NFκB inhibition.

Alteration of RhoH Expression Is Associated with Human Diffuse Large B-Cell Lymphoma.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4298-4298
Author(s):  
Yi Gu ◽  
Carmelita J. Alvares ◽  
Aparna C. Jasti ◽  
Michael Jansen ◽  
Judy Bean ◽  
...  
Keyword(s):  
Transcription Factor ◽  
B Cell ◽  
Somatic Mutations ◽  
Cell Lymphoma ◽  
Binding Protein ◽  
B Cell Lymphoma ◽  
Cell Development ◽  
Transcript Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract An increasing number of Rho GTPase family proteins have been demonstrated to play critical roles in blood and immune cell development and function. The newly defined RhoH gene has been previously demonstrated to be mutated in lymphoma samples (Dallery et al, 1995; Pasqualucci et al, 2001). These alterations include chromosomal rearrangements and a high frequency of somatic mutations (up to 46%) in human non-Hodgkin’s lymphomas and diffuse large B-cell lymphoma. The RhoH gene encodes a novel hematopoietic-specific member of the RhoE subfamily, which is GTPase deficient, remaining in the active, GTP-bound state. Thus the activity of RhoH is likely regulated by the level of the protein expressed in the cell. The somatic mutations in the RhoH gene have been mapped to a 1.6kb hypermutable region in the intron 1, suggesting the possibility of dysregulated RhoH expression. However, levels of RhoH expression have not been directly measured in these hematopoietic tumors and so it remains unclear whether these mutations translate into aberrant RhoH expression. We utilized quantitative real-time RT-PCR to measure RhoH transcript levels in primary DLBCL patient samples. Based on morphologic and immunophenotypic analysis, 17 DLBCL positive samples and 14 normal control samples were used for our study. The levels of TATA-box binding protein (TBP) and human phosphogycerate kinase (HPGK) cDNAs were also examined simultaneously for relative expression normalization. RhoH transcript levels in a subset of the DLBCL samples were markedly reduced. In particular, 6 of 17 (~35%) tested samples showed a greater than 3-fold reduction in RhoH expression based on both RhoH/TBP and RhoH/HPGK ratios when compared with the median RhoH expression level of 14 normal samples. Overall, RhoH expression levels of the DLBCL group were significantly altered (mainly decreased) as compared with those of the normal group (p < 0.04, student T-test). To further determine correlation of the abnormal RhoH expression with somatic mutations in the hypermutable region of the RhoH gene in the DLBCL samples, we performed genomic PCR amplification and sequencing analysis of this region from the normal and DLBCL samples. In addition, we utilized a computational approach (Trafac - http://trafac.cchmc.org) to identify evolutionarily conserved putative transcription factor binding sites (TFBS) between human and other species in the hypermutable region. 13 conserved TFBS between human and mouse were identified in the hypermutable region. Mutations in the DLBCL patients are localized in 6 of these predicted TFBS, including pancreatic and duodenal homeobox 1 (PDX1), zinc-finger binding protein-89 (ZBP-89), lymphoid enhancer factor 1 (LEF-1), BRIGHT, engrailed 1 and myelin transcription factor 1 (MyT1). Interestingly, LEF-1 and BRIGHT are B cell-specific transcription activators. These results suggest that RhoH expression is frequently altered in 35–40% of DLBCL samples and mutations in the hypermutable region of the RhoH gene in several cases encompass core binding sequences of transcription factors important in B cell development.

scholarly journals Role of Na+/Ca2+ Exchangers in Therapy Resistance of Medulloblastoma Cells

2017 ◽  
Vol 42 (3) ◽  
pp. 1240-1251 ◽  
Author(s):  
Lisann Pelzl ◽  
Zohreh Hosseinzadeh ◽  
Tamer al-Maghout ◽  
Yogesh Singh ◽  
Itishri Sahu ◽  
...  
Keyword(s):  
Annexin V ◽  
Therapy Resistance ◽  
Transport Processes ◽  
Protein Abundance ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Cell Current ◽  
Radiation Induced ◽  
Induced Apoptosis

Background/Aims: Alterations of cytosolic Ca2+-activity ([Ca2+]i) are decisive in the regulation of tumor cell proliferation, migration and survival. Transport processes participating in the regulation of [Ca2+]i include Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether medulloblastoma cells express Na+/Ca2+-exchangers, whether expression differs between therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells, and whether Na+/Ca2+-exchangers participate in the regulation of cell survival. Methods: In therapy sensitive D283 and therapy resistant UW228-3 medulloblastoma cells transcript levels were estimated by RT-PCR, protein abundance by Western blotting, cytosolic Ca2+-activity ([Ca2+]i) from Fura-2-fluorescence, Na+/ Ca2+-exchanger activity from the increase of [Ca2+]i (Δ[Ca2+]i) and from whole cell current (Ica) following abrupt replacement of Na+ containing (130 mM) and Ca2+ free by Na+ free and Ca2+ containing (2 mM) extracellular perfusate as well as cell death from PI -staining and annexin-V binding in flow cytometry. Results: The transcript levels of NCX3, NCKX2, and NCKX5, protein abundance of NCX3, slope and peak of Δ[Ca2+]i as well as Ica were significantly lower in therapy sensitive D283 than in therapy resistant UW228-3 medulloblastoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i, and augmented the ionizing radiation-induced apoptosis but did not significantly modify clonogenicity of medulloblastoma cells. Apoptosis was further enhanced by NCX3 silencing. Conclusions: Na+/Ca2+-exchanger activity significantly counteracts apoptosis but does not significantly affect clonogenicity after radiation of medulloblastoma cells.

scholarly journals Arabidopsis WRKY70 Is Required for Full RPP4-Mediated Disease Resistance and Basal Defense Against Hyaloperonospora parasitica

2007 ◽  
Vol 20 (2) ◽  
pp. 120-128 ◽  
Author(s):  
Colleen Knoth ◽  
Jon Ringler ◽  
Jeffery L. Dangl ◽  
Thomas Eulgem
Keyword(s):  
Cell Death ◽  
Transcription Factor ◽  
Salicylic Acid ◽  
Defense Mechanism ◽  
Wrky Transcription Factor ◽  
Reactive Oxygen Intermediates ◽  
Oxygen Intermediates ◽  
Transcript Levels ◽  
Hyaloperonospora Parasitica ◽  
Basal Defense

AtWRKY70, encoding a WRKY transcription factor, is co-expressed with a set of Arabidopsis genes that share a pattern of RPP4- and RPP7-dependent late upregulation in response to Hyaloperonospora parasitica infection (LURP) genes. We show that AtWRKY70 is required for both full RPP4-mediated resistance and basal defense against H. parasitica. These two defense pathways are related to each other, because they require PAD4 and salicylic acid (SA). RPP7 function, which is independent from PAD4 and SA, is not affected by insertions in AtWRKY70. Although AtWRKY70 is required for RPP4-resistance, it appears not to contribute significantly to RPP4-triggered cell death. Furthermore, our data indicate that AtWRKY70 functions downstream of defense-associated reactive oxygen intermediates and SA. Constitutive and RPP4-induced transcript levels of two other LURP genes are reduced in AtWRKY70 T-DNA mutants, indicating a direct or indirect role for AtWRKY70 in their regulation. We propose that AtWRKY70 is a component of a basal defense mechanism that is boosted by engagement of either RPP4 or RPP7 and is required for RPP4-mediated resistance.

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