scholarly journals UVRAG-Knockdown Regulates Mitochondrial Autophagy in Chronic Myeloid Leukaemia Cells by Targeting BNIP3L

2021 ◽  
Author(s):  
ChaoYong Liu ◽  
YanMin Ma ◽  
XiaoQin Zhang ◽  
Yang Liu ◽  
XiaoCheng Yin
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Chronic Myeloid Leukaemia ◽  
Western Blot ◽  
Myeloid Leukaemia ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
K562 Cells ◽  
Mitochondrial Mass ◽  
Related Proteins

Abstract Objective: To explore whether UVRAG regulates mitochondrial autophagy via BNIP3L in K562 cellsMaterial and methods: We designed various assays to verify the relation between UVRAG and BNIP3L, we estabilished a mitochondrial autophagy model of K562 cells by CCCP, a mitochondrial autophagy inducer, and regulated the expression of UVRAG by cells transfection. Then we detected the expression of the BINP3L and autophagy-related proteins LC3-II/LC3-Ⅰ and P62 by Western blot. The changes of ROS, mitochondrial mass, and mitochondrial membrane potential (MMP) were detected by flow cytometry technology.Results: We found that CCCP could induce K562 cells mitochondrial autophagy, along with the change of MMP, mitochondrial mass and accumulation of ROS, also our experiment proved that UVRAG-Knockdown could reverse this phenomenon. Investigating the pathway of mitochondrial autophagy revealed UVRAG knockdown was accompanied by a decrease in BNIP3L and LC3 expression, a increase in P62 during mitochondrial autophagy. Conclusion: In our study, the results suggested that UVRAG may regulate mitochondrial autophagy of K562 cells via targeting BINP3L, which may be a potential target for the treatment of CML.

Platelets apoptosis in rats after global brain ischemia

2018 ◽  
pp. 27-35
Author(s):  
А.А. Соколовская ◽  
Э.Д. Вирюс ◽  
В.В. Александрин ◽  
А.С. Роткина ◽  
К.А. Никифорова ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Ischemic Stroke ◽  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Brain Ischemia ◽  
Mitochondrial Membrane ◽  
Male Rats ◽  
Global Brain Ischemia ◽  
Platelet Apoptosis ◽  
Global Brain

Цель исследования. Ишемические повреждения головного мозга, являются одной из наиболее частой причин инвалидности и смертности во всем мире. Недавно была установлена роль апоптоза тромбоцитов в патофизиологии инсульта, однако его механизмы до сих пор остаются невыясненными. Несмотря на различные экспериментальные модели, направленные на мониторинг апоптоза тромбоцитов, результаты, относительно изучения и выявления апоптоза тромбоцитов при ишемии головного мозга у крыс, весьма немногочисленны. Цель исследования - анализ апоптоза тромбоцитов с помощью метода проточной цитофлуориметрии на модели глобальной ишемии мозга у крыс. Методика. В экспериментах использовано 6 крыс-самцов Вистар в возрасте от 5 до 6 мес., разделенных на 2 группы: интактный контроль (К) и глобальная ишемия головного мозга. Модель глобальной ишемии головного мозга у крыс воспроизводилась путём билатеральной окклюзии общих сонных артерий на фоне гипотензии. Уровень системного артериального давления снижали посредством кровопотери до 40-45 мм рт. ст. Суспензию тромбоцитов крыс получали методом гельфильтрации с использованием сефарозы 2B. Для анализа экстернализации фосфатидилсерина (ФС) тромбоциты крыс инкубировали с Аннексином V-PE в связывающем буфере. Для оценки митохондриального мембранного потенциала (ММП) тромбоциты инкубировали с катионным красителем JC-1. После инкубации образцы немедленно анализировали на проточном цитофлуориметре FACSCalibur (Becton Dickinson, США). Результаты. Согласно полученным данным, экстернализация ФС на тромбоцитах крыс, перенесших инсульт, была значительно выше (53,45 ± 4,21%), чем в контрольной группе крыс (5,27 ± 2,40%). Данный эффект подтверждается выраженной деполяризацией митохондриальных мембран (DYm). После экспериментальной ишемии мозга почти 40% тромбоцитов было деполяризовано. Заключение. Использованный в работе подбор методов и маркеров обеспечивает понимание механизмов апоптоза тромбоцитов как в экспериментальных, так и в клинических условиях. Полученные данные позволяют сделать заключение, что апоптоз тромбоцитов является одним из факторов развития глобальной ишемии головного мозга у крыс. Результаты могут быть использованы для понимания механизмов, участвующих в развитии ишемического повреждения, что, в свою очередь, может быть использовано при разработке новых терапевтических стратегий. Aim. Stroke is one of the most common causes of disability and mortality worldwide. Multiple experimental models of stroke have focused on monitoring of platelet apoptosis. However, studies on and detection of platelet apoptosis in rats with ischemic stroke are very scarce. We investigated platelet apoptosis in rats with global brain ischemia using flow cytometry. Methods. Experiments were carried out on healthy, adult Wistar male rats weighing 300-350 g. The rats were divided into the following 2 groups: intact rats and rats with global brain ischemia. Global brain ischemia was induced by two-vessel (2-VO) carotid occlusion in combination with hypotension. Systemic blood pressure was reduced by 40-45 mm Hg by inducing haemorrhage. Platelets were isolated by gel filtration on Sepharose 2B. For evaluation of phosphatidylserine (PS) externalization, platelets were incubated with Annexin V-PE and analyzed on FACSCalibur (BD Biosciences). Mitochondrial membrane potential (DY) was measured during platelets apoptosis using JC-1, a mitochondrial membrane potential indicator. Platelets were analyzed by flow cytometry immediately after the incubation. Results. PS externalization on platelets was significantly greater after global brain ischemia (53.45 ± 4.21%) than in the control group (5.27 ± 2.40%). Pronounced depolarization of mitochondrial membrane potential (DYm) confirmed this finding. In the rat group with experimental brain ischemia, almost 40% (35.24 ± 5.21%) of platelets were depolarized. Conclusion. Our results provide insight into mechanisms involved in platelet apoptosis during ischemic stroke and can be used in further development of new therapeutic strategies.

Anti-Glycophorin C Induces a Loss of Mitochondrial Membrane Potential but Fails To Activate Apoptotic Caspases.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4097-4097
Author(s):  
Gregory A. Denomme ◽  
Jonathan Micieli ◽  
Jenny Shu ◽  
Dan Wang ◽  
Bernard J. Fernandes
Keyword(s):  
Cell Death ◽  
Membrane Potential ◽  
Growth Inhibition ◽  
Mitochondrial Membrane Potential ◽  
K562 Cell ◽  
Mitochondrial Membrane ◽  
K562 Cells ◽  
Suppressive Effect ◽  
Glycophorin C

Abstract The human erythrocyte transmembrane sialoglycoprotein, glycophorin C (GYPC), plays a functional role in regulating red cell shape and mechanical stability. Antibodies to GYPC cause hemolytic disease of the fetus and newborn (HDFN) that is associated with classical Fcγ receptor-mediated phagocytosis. However, in vitro clonogenic studies with cord blood progenitor cells suggest that anti-GYPC also suppresses erythropoiesis, which is consistent with the observations of severe and early fetal anemia and late onset neonatal anemia [Transfus Med2005;15:125–32]. The mechanism of the suppressive effect on erythropoiesis is unknown. The K562 erythroleukemic cell line treated with anti-GYPC is a potential model system to study the suppressive effect of anti-GYPC. The present in vitro studies were designed to confirm the effect of anti-GYPC on K562 cell growth and viability, and to evaluate changes in mitochondrial membrane potential, phosphatidylserine (PS) expression, propidium iodide (PI) binding, and caspase activation. K562 cells fail to grow in the presence of anti-GYPC confirming earlier CFU-E/BFU-E studies [Brit J Haematol2006;133:443–4], and increased the exofacial expression of PS/PI over time. This process was caspase-independent as demonstrated by the failure of Z-VAD, a caspase inhibitor, to reverse growth inhibition and PS/PI expression. A loss of mitochondrial membrane potential was demonstrated using JC-1, a cationic dye that is sensitive to potential-dependent accumulation or loss in mitochondria. There was a 50% increase in K562 cell mitochondrial membrane potential disruption after 2 days of culture with anti-GYPC (see figure). Morphological examination of May Grunwalde Giemsa-stained K562 cells treated with anti-GYPC for 2 days showed a decrease in mitotic activity compared to isotype treated cells. By day 4, the anti-GYPC treated cells were showing evidence of plasma membrane damage and cell death resulting from fragmentation and dissolution of the cytoplasm. The addition of hemin, an oxidative form of iron protoporphyrin IX known to induce erythroid differentiation of K562 cells, to anti-GYPC treated cells reversed growth inhibition by 45% but did not prevent the loss of mitochondrial membrane potential. Overall, although caspases appear to be unimportant in anti-GYPC induced cell death, the mitchondria play an important role as the early events leading to antibody-mediated suppression of erythropoiesis. Mitochondrial Membrane Potential Disruption by Anti-GYPC Mitochondrial Membrane Potential Disruption by Anti-GYPC

Elevated Mitochondrial Membrane Potential in CLL Cells Is Associated with a more aggressive Natural History

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1765-1765
Author(s):  
Jeffrey R Gardner ◽  
Kristina Knapp ◽  
Mark G. Frattini ◽  
Nicole Lamanna ◽  
Renier Brentjens ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Natural History ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Doubling Time ◽  
Disease Course ◽  
Advisory Committees ◽  
Mitochondrial Mass ◽  
Cytogenetic Changes ◽  
Indolent Disease

Abstract Abstract 1765 Chronic lymphocytic leukemia (CLL) can be characterized by a variable natural history. While some patients have an aggressive disease course, others have an indolent course and never require treatment. Disease stage at diagnosis, cytogenetic changes, and mutation in the immunoglobulin hypervariable region, among other disease markers, have important prognostic contributions, but these prognostic markers do not reliably identify patients with an indolent natural history. We analyzed 174 individual patients with CLL and found that patients whose CLL cells retained a mitochondrial membrane potential (MMP) as measured by retention of the cyanine dye, JC-1, similar to normal B-lymphocytes had a particularly indolent disease course. While 73% of CLL patients had a substantial population of malignant lymphocytes with a higher mitochondrial membrane potential, 27% of patients had energetically normal CLL cells confirmed by co-immunostaining with anti-CD5 and anti-CD19. Elevated mitochondrial membrane potential is defined by the lymphocyte population having >50% of cells with a relative membrane potential (FL2/FL1=electrochemical potential/mitochondrial mass) > 2.0 (Figure 1). Patient samples from untreated patients were also organized into quintiles based upon the percentage of cells that populated the low mitochondrial membrane potential gate versus the percentage of cells that populated the high mitochondrial membrane potential gate (Figure 2). Patients in the lower quintile exhibited a predominance of cells in the low membrane potential gate across a broad range (.02%-42%) whereas patients in the upper quintile exhibited a predominance of cells in the high membrane potential gate across a much narrower range (88.6%-96.7%). With up to six years of follow up, no patient with normal MMP has required treatment or has advanced beyond Rai stage 0. Cytogenetic changes in this subgroup include normal cytogenetics, del13q, del11q, trisomy12, and del6q23. Of the 30 patients where immunoglobulin heavy chain variable gene mutations were measured, no patients with low MMP had a germline configuration. Lymphocyte doubling time was calculated for all patients for whom data is available and it is interesting to note that patients falling within the first quintile exhibited a median slope value of ∼1 with a median lymphocyte doubling time of ∼1.5 years, whereas patients in the upper quintile within the upper quintile exhibited a much steeper median slope value of 23 and an accelerated lymphocyte doubling time of approximately 7 months (see Figure 3 for patient examples). CLL cells with higher mitochondrial membrane potential have ultrastructural changes in the mitochondria typical of cells primarily utilizing glycolysis, but no difference in mitochondrial mass. CLL cells with a predominantly elevated MMP have increased lactate production and dramatically shortened survival when grown in media containing pyruvate as the primary energy substrate instead of glucose. These results indicate a significant difference in energy utilization and intracellular metabolism between malignant cells with normal and high MMP. Some patients with CLL cells that have elevated MMP appear to have an indolent disease course suggesting that elevated MMP does not necessarily portend an aggressive natural history, but to date, patients with CLL populations characterized by elevated MMP have encompassed the only patients in our study population who have required treatment directed at the CLL. The data indicate that analysis of the MMP in the CLL population can provide important prognostic information and may identify a population of patients with a benign course of disease. The prognostic significance of MMP in patients with CLL is independent of cytogenetics. The finding of low MMP appears to be confined to patients without a germline IGHV configuration. These results also indicate that the metabolic state of the malignant cell may play a central role in the clinical manifestation of disease and may point to the development of novel therapies that target mitochondrial respiration. Disclosures: Lamanna: Celgene Corporation:. Weiss:Celgene: Membership on an entity's Board of Directors or advisory committees. Scheinberg:Actinium Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

The Inhibitor of Cyclin-Dependent Kinase4a (INK4a) Signaling Pathway Induces Aging in Human Skeletal Muscle Myoblasts and Decreases the Mitochondrial Membrane Potential

2019 ◽  
Vol 9 (9) ◽  
pp. 1192-1198
Author(s):  
Dong Yan ◽  
Xiang Liao ◽  
Li-Xia Zhao ◽  
Chang-Hong Xiao
Keyword(s):  
Skeletal Muscle ◽  
Flow Cytometry ◽  
Membrane Potential ◽  
Signaling Pathway ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Human Skeletal Muscle ◽  
Lentiviral Vector ◽  
Accelerated Aging ◽  
Senescence Phenotype

The effect of the inhibitor of cyclin-dependent kinase4a (INK4a) signaling pathway on myoblastic aging was studied in this paper. Human skeletal muscle myoblasts were transfected with a recombinant lentiviral vector, pLVX-p16INK4a, encoding the p16INK4a gene, and RT-qPCR and western blotting were used to identify p16INK4a gene transcription and protein expression. The degree of cell senescence was assessed using Senescence-associated β-galactosidase staining. flow cytometry and JC-1 staining was used to analyze the mitochondrial membrane potential (MMP). The senescence phenotype was observed in myoblasts transfected with p16INK4a, the MMP was significantly decrease in p16INK4a-transfected myoblasts, while the MMP was decreased only slightly in control cells. Upregulation of the INK4a signaling pathway directly induced aging in human skeletal muscle myoblasts. Moreover, INK4a signaling pathway activated the mitochondrial pro-aging pathway by reducing the MMP, which indirectly accelerated aging in myoblasts.

Effect of thyroid hormone on mitochondrial properties and oxidative stress in cells from patients with mtDNA defects

2009 ◽  
Vol 296 (2) ◽  
pp. C355-C362 ◽  
Author(s):  
Keir J. Menzies ◽  
Brian H. Robinson ◽  
David A. Hood
Keyword(s):  
Oxidative Stress ◽  
Membrane Potential ◽  
Thyroid Hormone ◽  
Protein Expression ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Membrane ◽  
Ros Production ◽  
Mitochondrial Mass ◽  
Mtdna Mutations

Mitochondrial (mt)DNA mutations contribute to various disease states characterized by low ATP production. In contrast, thyroid hormone [3,3′,5-triiodothyronine (T3)] induces mitochondrial biogenesis and enhances ATP generation within cells. To evaluate the role of T3-mediated mitochondrial biogenesis in patients with mtDNA mutations, three fibroblast cell lines with mtDNA mutations were evaluated, including two patients with Leigh's syndrome and one with hypertrophic cardiomyopathy. Compared with control cells, patient fibroblasts displayed similar levels of mitochondrial mass, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), mitochondrial transcription factor A (Tfam), and uncoupling protein 2 (UCP2) protein expression. However, patient cells exhibited a 1.6-fold elevation in ROS production, a 1.7-fold elevation in cytoplasmic Ca2+ levels, a 1.2-fold elevation in mitochondrial membrane potential, and 30% less complex V activity compared with control cells. Patient cells also displayed 20–25% reductions in both cytochrome c oxidase (COX) activity and MnSOD protein levels compared with control cells. After T3 treatment of patient cells, ROS production was decreased by 40%, cytoplasmic Ca2+ was reduced by 20%, COX activity was increased by 1.3-fold, and ATP levels were elevated by 1.6-fold, despite the absence of a change in mitochondrial mass. There were no significant alterations in the protein expression of PGC-1α, Tfam, or UCP2 in either T3-treated patient or control cells. However, T3 restored the mitochondrial membrane potential, complex V activity, and levels of MnSOD to normal values in patient cells and elevated MnSOD levels by 21% in control cells. These results suggest that T3 acts to reduce cellular oxidative stress, which may help attenuate ROS-mediated damage, along with improving mitochondrial function and energy status in cells with mtDNA defects.

scholarly journals Neuroprotective Effects of Idebenone on hydrogen peroxide (H2O2)-induced Oxidative Damage in Retinal ganglion cell-5 (RGC-5) Cells

2019 ◽  
Author(s):  
Yuping Wang ◽  
Jing Wang ◽  
Xi Zhang ◽  
Yifan Feng ◽  
Yuanzhi Yuan
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Oxidative Damage ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Beclin 1 ◽  
Control Group ◽  
Related Protein ◽  
H2o2 Treatment ◽  
Cyt C

Abstract Purposes To investigated the neuroprotective effect of Idebenone against H2O2-induced oxidative damage in RGC-5 cells. Methods RGC-5 cells were treated with different concentrations (5, 10, 20μM) of idebenone for 12h prior to addition of 300µM H2O2 for 12 h. The apoptosis of RGC-5 cells were detected by flow cytometry. The changes of mitochondrial membrane potential were detected by JC-1 staining. The autophagy in RGC-5 cells was observed by transmission electron microscopy, and the expression level of autophagy-related protein light chain3, Beclin-1 and mitochondrial membrane potential-related protein Cyt-c in RGC-5 cells were measured by Western blot analysis. Results Flow cytometry showed that the apoptosis rates in control group, H2O2 group and H2O2-treatment with Idebenone pretreatment groups were (6.48±0.55)%, (27.34±0.51)%, (22.88±0.52)%, (15.45±0.81)%, (12.59±0.58)%, respectively(F = 559.7, P <0.0001). After incubation with H2O2, the number of autophagosomes increased significantly, while which was decreased in H2O2-treatment with Idebenone pretreatment groups. After incubation of RGC-5 cells with H2O2, the mitochondrial membrane potential was significantly decreased, while idebenone could prevent the decrease of MMP. Contrast with control group, LC3 II /I, the expression levels of Beclin-1 and Cyt-c in H2O2 group increased significantly(P<0.05); while contrast with H2O2 group, LC3 II/I, the expression of Beclin-1 and Cyt-c in H2O2-treatment with Idebenone pretreatment groups was significantly decreased(P<0.05). Conclusion Idebenone may have protective effects on RGC-5 cells suffering from oxidative damage induced by H2O2 through improving antioxidant capacity, reducing mitochondrial membrane potential decline and the activity of autophagy.

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