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 Effects of Leptin on Na+/Ca2+ Exchanger in PC12 Cells

2016 ◽  
Vol 40 (6) ◽  
pp. 1529-1537 ◽  
Author(s):  
Zijuan Zhang ◽  
Shuguang Sun ◽  
Caixia Du ◽  
Wei Li ◽  
Juan Zhang ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Intracellular Calcium ◽  
Pc12 Cells ◽  
Transcript Level ◽  
Protein Abundance ◽  
Rt Pcr ◽  
Transcript Levels ◽  
Distinct Role ◽  
Expression And Activity

Background/Aims: Alzheimer's disease (AD) is known to be related to alterations in neuronal intracellular calcium activity ([Ca2+]i). The present study revealed the distinct role of leptin in Na+/Ca2+-exchanger activity. Methods: [Ca2+]i was determined utilizing Fura-2 fluorescence. The activity of NCX was measured by removal of extracellular Na+ in the presence of external Ca2+. Na+/Ca2+-exchanger activity was further quantified from whole cell currents following removal of extracellular Na+. Na+/Ca2+-exchanger isoform NCX1 transcript levels and protein abundance were quantified by RT-PCR and Western blotting, respectively. Results: Exposure of PC12 cells to 30 µM amyloid (Aβ42) increased [Ca2+]i, an effect significantly blunted by 6 hours incubation with leptin before Aβ42 treatment. Moreover, leptin treatment significantly increased Na+/Ca2+-exchanger mediated Ca+ transport and current, NCX1 transcript level as well as NCX1 membrane protein abundance. Conclusion: We show that leptin blunts Aβ42-evoked [Ca2+]i increase by increasing expression and activity of Na+/Ca2+-exchanger NCX1.

scholarly journals Impact of Na+/Ca2+ Exchangers on Therapy Resistance of Ovary Carcinoma Cells

2015 ◽  
Vol 37 (5) ◽  
pp. 1857-1868 ◽  
Author(s):  
Lisann Pelzl ◽  
Zohreh Hosseinzadeh ◽  
Kousi Alzoubi ◽  
Tamer Al-Maghout ◽  
Sebastian Schmidt ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Therapy Resistance ◽  
Carcinoma Cells ◽  
Rt Pcr ◽  
Altered Expression ◽  
Transcript Levels ◽  
Cell Current ◽  
Channel Unit ◽  
Whole Cell Current

Background/Aims: According to previous observations, enhanced store-operated Ca2+-entry (SOCE) accomplished by the pore forming ion channel unit Orai1 and its regulator STIM1 contribute to therapy resistance of ovary carcinoma cells. Ca2+ signaling is further shaped by Ca2+ extrusion through K+-independent (NCX) and/or K+-dependent (NCKX) Na+/Ca2+-exchangers. The present study thus explored whether therapy resistance is further paralleled by altered expression and/or function of Na+/Ca2+-exchangers. Methods: In therapy resistant (A2780cis) and therapy sensitive (A2780sens) ovary carcinoma cells transcript levels were estimated from RT-PCR, 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 extracellular perfusate by Na+ free and Ca2+ containing (2 mM) extracellular perfusate, as well as cell death from PI -staining in flow cytometry. Results: The transcript levels of NCX3, NCKX4, NCKX5, and NCKX6, slope and peak of Δ[Ca2+]i as well as Ica were significantly higher in therapy resistant than in therapy sensitive ovary carcinoma cells. The Na+/Ca2+-exchanger inhibitor KB-R7943 (10 µM) significantly blunted Δ[Ca2+]i and significantly augmented the cisplatin-induced cell death of therapy resistant ovary carcinoma cells without significantly modifying cisplatin-induced cell death of therapy sensitive ovary carcinoma cells. Conclusion: Enhanced Na+/Ca2+-exchanger activity may contribute to the therapy sensitivity of ovary carcinoma 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 The p53/miR-34a/SIRT1 Positive Feedback Loop in Quercetin-Induced Apoptosis

2015 ◽  
Vol 35 (6) ◽  
pp. 2192-2202 ◽  
Author(s):  
Guohua Lou ◽  
Yanning Liu ◽  
Shanshan Wu ◽  
Jihua Xue ◽  
Fan Yang ◽  
...  
Keyword(s):  
Cell Apoptosis ◽  
Feedback Loop ◽  
Molecular Mechanisms ◽  
Rt Pcr ◽  
Cycle Arrest ◽  
Anticancer Effects ◽  
Huh7 Cells ◽  
Mirnas Expression ◽  
Induced Apoptosis

Background: The anti-tumor effects of quercetin have been reported, but the underlying molecular mechanisms remain to be elucidated. The aim of present study was to explore the role of miRNA in the anticancer effects of quercetin. Methods: The differential miRNAs expression between the HepG2 and Huh7 cells treated by quercetin were detected by microarray. The xCELLigence, Flow cytometry, RT-PCR and Western blot were used to analyze the cell proliferation, cell apoptosis, cell cycle arrest, anti-tumor genes, and protein expression. Results: miR-34a was up-regulated in HepG2 cells treated by quercetin exhibiting wild-type p53. When inhibiting the miR-34a, the sensitivity of the cells to quercetin decreased and the expression of the SIRT1 was up-regulated, but the acetylation of p53 and the expression of some genes related to p53 down-regulated. Conclusion: miR-34a plays an important role in the anti-tumor effects of querctin in HCC, miR-34a may be a tiemolecule between the p53 and SIRT1 and is composed of a p53/miR-34a/SIRT1 signal feedback loop, which could enhance apoptosis signal and significantly promote cell apoptosis.

The role of the stress-activated protein kinase (SAPK/JNK) signaling pathway in radiation-induced apoptosis

1998 ◽  
Vol 47 (3) ◽  
pp. 225-232 ◽  
Author(s):  
Marcel Verheij ◽  
Gerald A Ruiter ◽  
Shuraila F Zerp ◽  
Wim J van Blitterswijk ◽  
Zvi Fuks ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Jnk Signaling ◽  
Radiation Induced ◽  
Jnk Signaling Pathway ◽  
Induced Apoptosis

Activity of Targeted Molecular Therapeutics Against Primary AML Cells: Putative Role of the Bone Marrow Microenvironment.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2304-2304 ◽  
Author(s):  
Teresa McQueen ◽  
Marina Konopleva ◽  
Michael Andreeff
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Culture System ◽  
Hematological Malignancies ◽  
Annexin V ◽  
Leukemic Cells ◽  
Mek Inhibitors ◽  
Molecular Therapeutics ◽  
Induced Apoptosis

Abstract In hematological malignancies, there are reciprocal interactions between leukemic cells and cells of the bone marrow (BM) microenvironment such as mesenchymal stem cells (MSC). It is speculated that specific BM niches may provide a sanctuary for subpopulations of leukemic cells to evade chemotherapy-induced death and allow acquisition of a drug-resistant phenotype. In this study, we compared anti-leukemia effects of Ara-C and various signal transduction and apoptosis inhibitors in a co-culture system of primary AML and human bone marrow-derived MSC. AML blasts from 11 primary AML samples with high (>70%) blast count were co-cultured with MSC for 24 hours, after which they were exposed to the indicated concentrations of inhibitors for 48–96 hrs. Concentrations were selected on the basis of preliminary cell line studies which determined efficient inhibition of drug targets. Induction of apoptosis was analyzed by Annexin V flow cytometry after gating on the CD90 APC(−) (non-MSC) population. MSC protected leukemic blasts from spontaneous apoptosis in all 11 samples studied (mean annexinV positivity, 49.5±7.2% vs 25.3±4.8%, p<0.001) and from Ara-C-induced cytotoxicity in 6 out of 11 samples (p=0.02). No difference in the degree of protection was noted when MSC from older vs. younger donors were used (not shown). Co-culture of leukemic cells with MSC resulted in significant (p<0.03) suppression of inhibitor-induced apoptosis for all agents tested (Table 1), however PI3K/AKT inhibitors seemed to overcome MSC-mediated resistance. In addition, specific inhibitors of Bcl-2 and MDM2 induced apoptosis not only in suspension, but also in the MSC co-culture system, while Raf-1/MEK inhibitors were less effective. The AKT inhibitor A443654 caused apoptosis induction not only in leukemic cells, but also in MSC, which likely accounted for its high efficacy in stromal co-cultures (53±6% annexin V+). In a different study (Tabe et al, ASH 2005), we report that interactions of leukemic and BM stromal cells result in the activation of PI3K/ILK/AKT signaling in both, leukemic and stromal cells. We therefore propose that disruption of these interactions by specific PI3K/AKT inhibitors represents a novel therapeutic approach to eradicate leukemia in the BM microenvironment via direct effects on leukemic cells and by targeting activated BM stromal cells. Furthermore, Bcl-2 and MDM2 inhibitors appear to retain their efficacy in stroma-cocultured AML cells, while the efficacy of chemotherapy and Raf/MEK inhibitors in these conditions may be reduced. Further studies are aimed at the elucidation of the role of the BM microenvironment and its ability to activate specific signaling pathways in the pathogenesis of leukemias and on efforts to disrupt the MSC/leukemia interaction (Zeng et al, ASH 2005). Focus on this stroma-leukemia-stroma crosstalk may result in the development of strategies that enhance the efficacy of therapies in hematological malignancies and prevent the acquisition of a chemoresistant phenotype. Table 1. Leukemia Cell Apoptosis in a MSC/AML Co-Culture System Target Bcl-2/XL MDM2 PI3K AKT Raf-1 MEK Apoptosis was determined as percentage of Annexin V(+)CD90(−) cells, and calculated by the formula: % specific apoptosis = (test − control) x 100 / (100 − control). Compound, concentration Ara-C, 1 μM ABT-737, 0.1 μM Nutlin-3A, 2.5 μM LY294002, 10 μM A443654, 1 μM BAY43-9006, 2.5 μM CI1040, 3 μM AML 28±7 69±7 45±7 53.8±13.3 75±7 35±11 27±11 AML + MSC 16±4 38±6 28±6 31.2±6.9 53±6 18±8 15±5

2-Methoxyestradiol Sensitizes B-NHL Cells to TRAIL-Mediated Apoptosis Via Inhibition of HIF-1α, NF-κ B, YY1 and Induction of DR5 Expression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4642-4642
Author(s):  
Sara Huerta-Yepez ◽  
Mario I. Vega ◽  
Stavroula Baritaki ◽  
Eriko Suzuki ◽  
Theresa La Vallee ◽  
...  
Keyword(s):  
Annexin V ◽  
Nuclear Accumulation ◽  
Microtubule Disruption ◽  
Low Concentrations ◽  
Combination Studies ◽  
Apoptotic Activity ◽  
Induced Apoptosis ◽  
Patients Experience ◽  
First Time

Abstract There have been many advances in the treatment of B-NHL by both chemotherapy and immunotherapy. However, many patients experience recurrences and relapses and develop resistance to further treatments. Therefore, there is a need for new therapies. TRAIL and mAbs directed against DR4 or DR5 are currently being examined clinically, either alone or in combination with other therapies, for the treatment of resistant cancers. The objective of our study is to examine whether B-NHL can be sensitized to respond to TRAIL. 2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have apoptotic activity and anti-angiogenic activity and has been examined for its therapeutic efficacy, both preclinically and in humans. Further, we and others have shown that 2ME2 can sensitize solid tumors to TRAIL-induced apoptosis. Thus, we hypothesized that 2ME2 may also sensitize B-NHL cells to TRAIL-induced apoptosis and that 2ME2-induced microtubule disruption and inhibition of NF-κ B activity may be involved in 2ME2-induced sensitization. The present study examined the role of 2ME2 in TRAIL-sensitization of the B-NHL cell line, Ramos, as a model for B-NHL. Ramos cells were treated with 2ME2 (0.1, 1.0 μ m for 5 h) and then treated with TRAIL (2.5–10 ng/ml for 18 h). The cells were harvested and examined for apoptosis by Annexin V/PI and for activation of caspase-3. The findings demonstrate that, while single agents were not cytotoxic, the combination treatment resulted in significant cytotoxicity demonstrating synergy in apoptosis. The synergy was obtained with very low concentrations of 2ME2 (0.1μ m) and TRAIL (2.5 ng/ml), concentrations that were not effective in other tumor cell lines studied. We examined the potential mechanism involved in synergy induced by 2ME2 and TRAIL. Surface DR-5 expression was upregulated following treatment with 2ME2. In addition, the transcription repressor YY1 protein expression was inhibited as assessed by Western and immunohistochemistry (IHC). Microtubule disruption by 2ME2 results in inhibition of HIF-1α transcriptional activity through impairment of HIF-1α nuclear accumulation. Additionally, we have found that 2ME2 inhibits HIF-1α accumulation in the nucleus as assessed by IHC. Inhibition of HIF-1α has been shown to regulate apoptosis via upregulation of BID and phosphorylyzation of Bcl-2. The present findings demonstrate, for the first time, that 2ME2 sensitizes B-NHL cells to TRAIL-induced apoptosis via inhibition of both HIF-1α and YY1 and upregulation of DR5. The findings support potential therapeutic combination studies of 2ME2 with TRAIL for the treatment of resistant B-NHL In addition, we suggest that HIF-1α, YY1, and DR5 may serve as targets for therapeutic intervention and potentially as biomarkers for activity.

scholarly journals DR5 Knockout Mice Are Compromised in Radiation-Induced Apoptosis

2005 ◽  
Vol 25 (5) ◽  
pp. 2000-2013 ◽  
Author(s):  
Niklas Finnberg ◽  
Joshua J. Gruber ◽  
Peiwen Fei ◽  
Dorothea Rudolph ◽  
Anka Bric ◽  
...  
Keyword(s):  
Cell Death ◽  
Null Mouse ◽  
Organ Specific ◽  
Radiation Induced ◽  
Induced Apoptosis ◽  
The Brain ◽  
Necrosis Factor

ABSTRACT DR5 (also called TRAIL receptor 2 and KILLER) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (also called TRAIL and Apo2 ligand). DR5 is a transcriptional target of p53, and its overexpression induces cell death in vitro. However, the in vivo biology of DR5 has remained largely unexplored. To better understand the role of DR5 in development and in adult tissues, we have created a knockout mouse lacking DR5. This mouse is viable and develops normally with the exception of having an enlarged thymus. We show that DR5 is not expressed in developing embryos but is present in the decidua and chorion early in development. DR5-null mouse embryo fibroblasts expressing E1A are resistant to treatment with TRAIL, suggesting that DR5 may be the primary proapoptotic receptor for TRAIL in the mouse. When exposed to ionizing radiation, DR5-null tissues exhibit reduced amounts of apoptosis compared to wild-type thymus, spleen, Peyer's patches, and the white matter of the brain. In the ileum, colon, and stomach, DR5 deficiency was associated with a subtle phenotype of radiation-induced cell death. These results indicate that DR5 has a limited role during embryogenesis and early stages of development but plays an organ-specific role in the response to DNA-damaging stimuli.

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