scholarly journals Inhibition of TLR4 Signaling Affects Mitochondrial Fitness Overcoming Bortezomib Resistance in Myeloma Plasma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3073-3073
Author(s):  
Cesarina Giallongo ◽  
Daniele Tibullo ◽  
Giuseppina Camiolo ◽  
Fabrizio Puglisi ◽  
Daniela Cambria ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Er Stress ◽  
Mitochondrial Respiration ◽  
Research Funding ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Depolarization ◽  
Tlr4 Signaling ◽  
Mitochondrial Mass ◽  
Atp Production ◽  
Inflammatory Microenvironment

BACKGROUND Multiple myeloma (MM) is a B-cell malignancy critically dependent for survival and proliferation on signals coming from its inflammatory microenvironment in which toll-like receptors (TLR) may be potential linking elements between inflammation and cancer. It has been recently demonstrated that TLR4 pathway provides a protective effect against bortezomib (BTZ)-induced endoplasmic reticulum (ER) stress and pre-treatment of MM cells with LPS significantly reduces BTZ-induced apoptosis. AIM Since the acquisition of BTZ resistance is accompanied by an increased reliance on mitochondrial respiration, we investigated the role of TLR4 as stress-responsive mechanism that protect mitochondria during BTZ-induced ER stress as potential mechanism of drug resistance. RESULTS The activation of TLR4 signaling by LPS increased mitochondrial mass in human MM cell lines (HMCL: U266, MM1.S, OPM2, NCI-H929) and induced up-regulation of mitochondrial biogenesis markers (PGC1a, PRC and TFAM). After treatment with BTZ for 24h, all HMCL over-expressed TLR4 and its signaling was functional as suggested by up-regulation of MyD88 and MAPK activation. Compared to BTZ-sensitive cells (U266-S), U266-R showed higher levels of TLR4, p-p38 and p-ERK proteins and higher mitochondrial mass. Using a selective TLR4 inhibitor (TAK-242), we next treated U266-R cells with either 15nM BTZ, 20 μM TAK-242 or their combination. Combinatorial treatment significantly induced cell apoptosis (about 52%; p<0.001) that appeared to result from the deleterious effects of oxidative stress. Indeed, BTZ-induced intracellular ROS returned to normal levels after 3h and cells were able to up-regulate two anti-oxidant enzymes (GPX1 and GSTP1). On the contrary, TAK-242/BTZ activated a strong pro-oxidant status incresing ROS and RNS (reactive nitrogen species) levels, decreasing GSH ones and down-regulating GPX1 and GSTP1. Analyzing the effect of each treatment on mitochondrial polarization status, we observed about 6,7% of depolarized mitochondria after BTZ treatment, while TAK-242/BTZ combination induced a mitochondrial depolarization of about 69,3% (p<0.001). Moreover, cells treated with BTZ alone showed a compensatory up-regulation of the OXPHOS- (NDUFA-6 and MT-ND4) and mitochondrial fusion-related genes (mitofusin and OPA1) and TFAM. On the contrary, all these genes were down-regulated by TAK-242/BTZ combination. Also a dramatic drop in mitochondrial respiration was observed with a marked decrease in ATP production, consequent accumulation of AMP and a decreased NAD+/NADH and NADP+/NADPH ratio. Since high levels of oxidative stress and mitochondrial impairment activate mitophagy sensitizing cells to apoptosis, we evaluated co-localization of mitochondria (stained with MitoTracker) with the autophagosome marker LC3 using confocal microscopy. BTZ and TAK-242/BTZ increased Mitotracker/LC3 co-localization respectively of about 4,5 and 50 fold compared with control (BTZ vs combination: p<0.001). To evaluate whether TLR4 inhibition resensitizes resistant primary cells, CD138+ cells derived from 5 refractory/relapsed MM patients were treated with 5nM BTZ, 10mM TAK-242 or their combination. Compared to BTZ alone, combination treatment induced higher mitochondrial depolarization after 24h and significantly decreased viability of CD138+ cells after 48h. TLR4 inhibitor alone or in combination did never show cytotoxicity toward CD138- cells. CONCLUSION Taken together, these findings indicate thatTLR4 signaling is involved in the acquisition of bortezomib resistance protecting mitochondria during BTZ exposure and sustaining mitochondrial dynamics in BTZ-resitant cells. Inhibition of TLR4 may overcome bortezomib resistance in patients with relapsed/refractory MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Celgene: Honoraria; Amgen: Honoraria; Hospira: Honoraria; Teva: Honoraria; Novartis: Honoraria; Janssen: Honoraria. Di Raimondo:Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals Inhibition of TLR4 Signaling Affects Mitochondrial Fitness and Overcomes Bortezomib Resistance in Myeloma Plasma Cells

Cancers ◽  
2020 ◽  
Vol 12 (8) ◽  
pp. 1999 ◽  
Author(s):  
Cesarina Giallongo ◽  
Daniele Tibullo ◽  
Fabrizio Puglisi ◽  
Alessandro Barbato ◽  
Nunzio Vicario ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Plasma Cells ◽  
Mitochondrial Depolarization ◽  
Toll Like Receptor 4 ◽  
Tlr4 Signaling ◽  
Inflammatory Microenvironment ◽  
B Cell Malignancy ◽  
Severe Impairment ◽  
Cell Energy ◽  
Tlr4 Activation

Multiple myeloma (MM) is a B-cell malignancy requiring inflammatory microenvironment signals for cell survival and proliferation. Despite improvements in pharmacological tools, MM remains incurable mainly because of drug resistance. The present study aimed to investigate the implication of Toll-like receptor 4 (TLR4) as the potential mechanism of bortezomib (BTZ) resistance. We found that TLR4 activation induced mitochondrial biogenesis and increased mitochondrial mass in human MM cell lines. Moreover, TLR4 signaling was activated after BTZ exposure and was increased in BTZ-resistant U266 (U266-R) cells. A combination of BTZ with TAK-242, a selective TLR4 inhibitor, overcame drug resistance through the generation of higher and extended oxidative stress, strong mitochondrial depolarization and severe impairment of mitochondrial fitness which in turn caused cell energy crisis and activated mitophagy and apoptosis. We further confirmed the efficacy of a TAK-242/BTZ combination in plasma cells from refractory myeloma patients. Consistently, inhibition of TLR4 increased BTZ-induced mitochondrial depolarization, restoring pharmacological response. Taken together, these findings indicate that TLR4 signaling acts as a stress-responsive mechanism protecting mitochondria during BTZ exposure, sustaining mitochondrial metabolism and promoting drug resistance. Inhibition of TLR4 could be therefore be a possible target in patients with refractory MM to overcome BTZ resistance.

scholarly journals Mitochondria Targeting as an Effective Strategy for Cancer Therapy

2020 ◽  
Vol 21 (9) ◽  
pp. 3363 ◽  
Author(s):  
Poorva Ghosh ◽  
Chantal Vidal ◽  
Sanchareeka Dey ◽  
Li Zhang
Keyword(s):  
Cancer Therapy ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Mitochondrial Respiration ◽  
Mitochondrial Dynamics ◽  
Metastatic Tumor ◽  
Atp Production ◽  
Development Of Resistance ◽  
Mitochondrial Alterations ◽  
Mitochondria Targeting

Mitochondria are well known for their role in ATP production and biosynthesis of macromolecules. Importantly, increasing experimental evidence points to the roles of mitochondrial bioenergetics, dynamics, and signaling in tumorigenesis. Recent studies have shown that many types of cancer cells, including metastatic tumor cells, therapy-resistant tumor cells, and cancer stem cells, are reliant on mitochondrial respiration, and upregulate oxidative phosphorylation (OXPHOS) activity to fuel tumorigenesis. Mitochondrial metabolism is crucial for tumor proliferation, tumor survival, and metastasis. Mitochondrial OXPHOS dependency of cancer has been shown to underlie the development of resistance to chemotherapy and radiotherapy. Furthermore, recent studies have demonstrated that elevated heme synthesis and uptake leads to intensified mitochondrial respiration and ATP generation, thereby promoting tumorigenic functions in non-small cell lung cancer (NSCLC) cells. Also, lowering heme uptake/synthesis inhibits mitochondrial OXPHOS and effectively reduces oxygen consumption, thereby inhibiting cancer cell proliferation, migration, and tumor growth in NSCLC. Besides metabolic changes, mitochondrial dynamics such as fission and fusion are also altered in cancer cells. These alterations render mitochondria a vulnerable target for cancer therapy. This review summarizes recent advances in the understanding of mitochondrial alterations in cancer cells that contribute to tumorigenesis and the development of drug resistance. It highlights novel approaches involving mitochondria targeting in cancer therapy.

scholarly journals Effects of GH/IGF on the Aging Mitochondria

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1384 ◽  
Author(s):  
Sher Bahadur Poudel ◽  
Manisha Dixit ◽  
Maria Neginskaya ◽  
Karthik Nagaraj ◽  
Evgeny Pavlov ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Growth Hormone ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Function ◽  
Cellular Aging ◽  
Mitochondrial Mass ◽  
Atp Production ◽  
And Function

The mitochondria are key organelles regulating vital processes in the eukaryote cell. A decline in mitochondrial function is one of the hallmarks of aging. Growth hormone (GH) and the insulin-like growth factor-1 (IGF-1) are somatotropic hormones that regulate cellular homeostasis and play significant roles in cell differentiation, function, and survival. In mammals, these hormones peak during puberty and decline gradually during adulthood and aging. Here, we review the evidence that GH and IGF-1 regulate mitochondrial mass and function and contribute to specific processes of cellular aging. Specifically, we discuss the contribution of GH and IGF-1 to mitochondrial biogenesis, respiration and ATP production, oxidative stress, senescence, and apoptosis. Particular emphasis was placed on how these pathways intersect during aging.

scholarly journals LPS-mediated inflammation in cardiovascular diseases

2018 ◽  
pp. 14-22
Author(s):  
И.В. Федотов ◽  
Н.Ю. Русецкая ◽  
Е.В. Бобылева ◽  
В.Б. Бородулин
Keyword(s):  
Oxidative Stress ◽  
Heart Failure ◽  
Signaling Pathways ◽  
Active Oxygen Species ◽  
Atherosclerotic Lesion ◽  
Adaptor Protein ◽  
Bacterial Cells ◽  
Tlr4 Signaling ◽  
Atp Production ◽  
Atherosclerotic Process

Возникновение и развитие сердечной недостаточности как проявление атеросклеротического процесса или первичного поражения кардиомиоцитов при кардиомиопатиях может быть связано с накоплением в крови эндотоксинов - компонентов бактериальных клеток, в частности липополисахарида (ЛПС) клеточной стенки грамотрицательных бактерий. ЛПС при связывании с TOLL-подобными рецепторами (TLR) запускают сигнальные пути, вследствие чего происходит синтез провоспалительных цитокинов, выработка активных форм кислорода и азота, развитие окислительного стресса и воспаления в месте атеросклеротического поражения. Активация ЛПС-TLR4-сигнальных путей в тромбоцитах способствует развитию тромбоза. При ишемии миокарда и сердечной недостаточности наблюдается дисбаланс между окислительным стрессом и антиоксидантными механизмами. Миокард имеет эндогенные восстановительные механизмы, в том числе системы тиоредоксина (Trx) и глутатиона (GSH), которые направлены на удаление активных форм кислорода и восстановление окисленных белков, часть из которых участвует в ЛПС-TLR4-сигнальных путях. Например, GSH способен модифицировать адапторный белок MyD88, а Trx является прямым ингибитором протеинкиназы ASK1, способствуя подавлению проапоптотических сигнальных путей во время острого воспаления в кардиомиоцитах. Кроме того, Trx предотвращает дисфункцию митохондрий, увеличивает производство АТФ, ингибирует апоптоз и тем самым оказывает кардиопротекторное действие. Emergence and development of heart failure as a manifestation of atherosclerotic process or primary lesion to cardiomyocytes in cardiomyopathies can be caused by blood accumulation of endotoxins, components of bacterial cells, such as lipopolysaccharide (LPS) of gram-negative bacterial cell wall. LPS binding to TOLL-like receptors (TLR) triggers signaling pathways to induce synthesis of pro-inflammatory cytokines, production of reactive oxygen and nitrogen species, and development of oxidative stress and inflammation at the site of atherosclerotic lesion. Activation of LPS-TLR4-signaling pathways in platelets contributes to development of thrombosis. An imbalance between oxidative stress and antioxidant mechanisms is observed in myocardial ischemia and heart failure. The myocardium has endogenous restorative mechanisms, including the systems of thioredoxin (Trx) and glutathione (GSH) designed for removing active oxygen species and reducing oxidized proteins, some of which are involved in LPS-TLR4 signaling pathways. For example, GSH is able to modify the adaptor protein MyD88 while Trx is a direct inhibitor of protein kinase ASK1, to facilitate suppression of proapoptotic signaling pathways during acute inflammation in cardiomyocytes. In addition, Trx prevents mitochondrial dysfunction, increases ATP production, inhibits apoptosis and, thereby, exerts a cardioprotective effect.

scholarly journals The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

2019 ◽  
Author(s):  
Christina Wolf ◽  
Rahel Zimmermann ◽  
Osamah Thaher ◽  
Diones Bueno ◽  
Verena Wüllner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Mitochondrial Respiration ◽  
Culture Conditions ◽  
Mitochondrial Quality Control ◽  
Mitofusin 2 ◽  
Charcot Marie Tooth ◽  
Atp Production ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but similar mitochondrial oxygen consumption, membrane potential and ATP production. Mild oxidative stress procured by 100 &micro;M hydrogen peroxide applied 24 h before analysis, however, significantly increased respiration but decreased mitochondrial ATP production only in R94Q cells. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control.

scholarly journals MSI-1436 improves EMS adipose derived progenitor stem cells in the course of adipogenic differentiation through modulation of ER stress, apoptosis, and oxidative stress

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Lynda Bourebaba ◽  
Katarzyna Kornicka-Garbowska ◽  
Mohamad Al Naem ◽  
Michael Röcken ◽  
Jacek Łyczko ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Metabolic Syndrome ◽  
Adipose Tissue ◽  
Er Stress ◽  
Cellular Proliferation ◽  
Mitochondrial Dynamics ◽  
Adipogenic Differentiation ◽  
Tyrosine Phosphatase ◽  
Selective Inhibition ◽  
And Oxidative Stress

Abstract Background Protein tyrosine phosphatase 1B (PTP1B) is one of the major negative regulators of leptin and insulin signaling, and has been strongly implicated in insulin resistance development in the course of obesity and metabolic syndrome conditions; however, its exact role in controlling adipose tissue biogenesis is still poorly understood. Objectives This investigation aimed to elucidate whether selective inhibition of PTP1B using MSI-1436 compound may improve and restore the defective adipogenicity of ASCs isolated from EMS-affected horses. Methods Equine ASC EMS cells were cultured under adipogenic conditions in the presence of PTP1B inhibitor and were subsequently tested for expression of the main adipogenic-related genes using RT-qPCR, changes in free fatty acid profiles by means of GC-MS technique, and for mitochondrial dynamics improvement through the analysis of mitochondrial transmembrane potential and oxidative stress. Results Selective inhibition of PTP1B in equine ASC EMS cells improved substantially adipogenic differentiation by promoting cellular proliferation and normalizing expression of C/EBPalpha, PPARγ, and Adipoq markers that are critical for proper adipogenesis. Levels of secreted adiponectin and PPARγ were also shown to be increased in MSI-1436-conditioned cells, while total leptin levels markedly dropped under the same conditions. Moreover, MSI-1436 treatment enabled the regulation of metabolic-related transcripts that are crosslink to adipogenesis, namely Akt1, Akt2, and SHBG. The obtained results demonstrated also an obvious reduction in intracellular accumulated ROS and NO, as well as mitigated ER stress through the downregulation of Chop, Perk, Atf6, Ire1, and Xbp1 transcripts upon PTP1B inhibition. Furthermore, general fluctuations in FFA composition of all differentiated groups have been highlighted, where palmitic acid, palmitoleic acid, stearic acid, and linolelaidic acid that are known to be associated with the development of metabolic disorders were found to be normalized upon PTP1B inhibition during adipogenic differentiation. Conclusion The presented data provides the evidence that the use of PTP1B inhibitor may be successful in controlling and enhancing adipogenic differentiation of impaired equine ASCs affected by metabolic syndrome, and thus offers new insights for the management of obesity through the regulation of adipose tissue dynamics. Graphical abstract

Hemopexin Gene Therapy Inhibits Inflammation and Vaso-Occlusion in Transgenic Sickle Cell Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 412-412
Author(s):  
Gregory M Vercellotti ◽  
Ping Zhang ◽  
Chunsheng Chen ◽  
Julia Nguyen ◽  
Fuad Abdulla ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Therapy ◽  
Sickle Cell ◽  
Research Funding ◽  
Protective Role ◽  
Tlr4 Signaling ◽  
Total Rna ◽  
Genetics Research ◽  
In Trans

Abstract Hemolysis, oxidative stress, inflammation, vaso-occlusion, and organ infarction are hallmarks of sickle cell disease (SCD). Hemolysis releases free hemoglobin (Hb) and Hb-containing microparticles into the vasculature that upon oxidation to methemoglobin frees heme from the globin, which in turn can promote oxidative stress and activate toll-like receptor 4 (TLR4) signaling. Hemopexin (HPX), a plasma β1-glycoprotein, binds heme with a very high affinity (Kd < 10-12 M), and transports it to the liver for catabolism via CD91 receptor-mediated uptake. SCD patients have low serum HPX levels likely due to chronic hemolysis leading to increased HPX catabolism with insufficient compensatory increase in synthesis. We and others have shown that HPX transports heme to the liver, and inhibits heme toxicity and the activation of endothelial, leukocyte and platelet TLR4 signaling. Acute studies have shown HPX infusion prior to a heme challenge protects sickle mice from vaso-occlusion and developing acute pulmonary injury while chronic HPX infusion therapy modified heme toxicity to endothelium. We hypothesize that in SCD mice, hepatic overexpression of HPX will bind the proximal mediator of vascular activation, heme, and will inhibit inflammation and microvascular stasis (vaso-occlusion). To examine the protective role of HPX in SCD, we transplanted bone marrow from NY1DD SCD mice into HPX-/- or normal C57BL/6 mice. After 12 weeks, conversion to the HbS phenotype was confirmed by isoelectric focusing. Dorsal skin fold chambers (DSFC) were implanted in week 13 and microvascular stasis (% non-flowing venules) assessed in response to heme (3.2 µmol/kg) infusion. HPX-/- sickle mice had 34% ± 3% and 24% ± 2% at 1h and 4h post heme, significantly greater than HPX+/+ C57BL/6 sickle mice which had 21% ± 5% and 13% ± 8% at 1 and 4 h, (mean ± SD, p<.05), demonstrating the protective role of HPX in SCD. To further test our hypothesis, we utilized Sleeping Beauty (SB) transposon-mediated gene therapy to overexpress rat HPX in NY1DD and Townes-SS SCD mice. Rat HPX plasmid (pT2/Caggs-HPX) was delivered with an SB transposase plasmid (pK/CMV-SB 100X) and luciferase (LUC) plasmid (pT2/Caggs-LUC, as tracer) in trans into NY1DD or Townes-SS SCD mice by hydrodynamic tail vein injections. Control SCD mice were infused with the same volume of lactated Ringer's solution (LRS) or LUC plasmid with SB transposase plasmid in trans. One week later, the mice LUC bioluminescence imaging showed the liver was the primary location of expression. Four weeks later, the HPX SCD mice had marked increases in hepatic rat HPX mRNA (300-2000 copies/5ng total RNA) comparing to LRS and SB-LUC controls (0-44 copies/5ng total RNA). Plasma and hepatic HPX were significantly increased compared to LRS and SB-LUC controls. In vitro expression of the rat HPX plasmid in Chinese Hamster Ovary cells, and protein purification confirmed heme binding activity by spectroscopic scan absorbance shifts of rat HPX-heme complexes at 414nm. DSFCs were implanted 4 weeks after plasmid infusion and microvascular stasis was assessed in response to heme (3.2 µmol/kg) infusion. NY1DD and Townes-SS mice overexpressing rat HPX (SB-HPX) had significantly less stasis than LRS or SB-LUC treated SCD mice (Figure 1A and B). HPX overexpression markedly increased nuclear Nrf2 expression in the livers of sickle mice, presumably by promoting delivery of heme to the liver and activating the Keap1-Nrf2 axis. In addition, hepatic HO-1 activity and protein and CD91 protein were increased in sickle mice overexpressing HPX and NF-ĸB activation was markedly decreased as assessed by nuclear phospho-p65-NF-ĸB expression on western blots demonstrating the anti-inflammatory properties of HPX in sickle mice. In conclusion, supplementing HPX levels in transgenic sickle mice via gene therapy activates the Nrf2 anti-oxidant axis and ameliorates inflammation and vaso-occlusion. We speculate that plasma HPX supplementation may be beneficial in SCD especially during hemolytic crises or acute chest syndrome. Figure 1. Figure 1. Disclosures Vercellotti: Cydan: Research Funding; CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding. Belcher:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Biogen Idec: Research Funding.

scholarly journals Inhibition of Protein-Tyrosine Phosphatase PTP1B and LMPTP Promotes Palmitate/Oleate-Challenged HepG2 Cell Survival by Reducing Lipoapoptosis, Improving Mitochondrial Dynamics and Mitigating Oxidative and Endoplasmic Reticulum Stress

2020 ◽  
Vol 9 (5) ◽  
pp. 1294 ◽  
Author(s):  
Lynda Bourebaba ◽  
Jacek Łyczko ◽  
Michalina Alicka ◽  
Nabila Bourebaba ◽  
Antoni Szumny ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Hepg2 Cell ◽  
Mitochondrial Dynamics ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Public Health Problem ◽  
Alcoholic Fatty Liver ◽  
Protein Tyrosine

Objectives: Non-alcoholic fatty liver disease (NAFLD) is considered a well-known pathology that is determined without using alcohol and has emerged as a growing public health problem. Lipotoxicity is known to promote hepatocyte death, which, in the context of NAFLD, is termed lipoapoptosis. The severity of NAFLD correlates with the degree of hepatocyte lipoapoptosis. Protein–tyrosine phosphatases (PTP) including PTP1B and Low molecular weight PTP (LMPTP), are negative regulators of the insulin signaling pathway and are considered a promising therapeutic target in the treatment of diabetes. In this study, we hypothesized that the inhibition of PTP1B and LMPTP may potentially prevent hepatocyte apoptosis, mitochondrial dysfunction and endoplasmic reticulum (ER) stress onset, following lipotoxicity induced using a free fatty acid (FFA) mixture. Methods: HepG2 cells were cultured in the presence or absence of two PTP inhibitors, namely MSI-1436 and Compound 23, prior to palmitate/oleate overloading. Apoptosis, ER stress, oxidative stress, and mitochondrial dynamics were then evaluated by either MUSE or RT-qPCR analysis. Results: The obtained data demonstrate that the inhibition of PTP1B and LMPTP prevents apoptosis induced by palmitate and oleate in the HepG2 cell line. Moreover, mitochondrial dynamics were positively improved following inhibition of the enzyme, with concomitant oxidative stress reduction and ER stress abrogation. Conclusion: In conclusion, PTP’s inhibitory properties may be a promising therapeutic strategy for the treatment of FFA-induced lipotoxicity in the liver and ultimately in the management of the NAFLD condition.

scholarly journals Synergistic Effect of Carfilzomib and Metformin in Vascular Plasticity; The Emerging Role of Autophagy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3797-3797
Author(s):  
Panagiotis Efentakis ◽  
Ioanna Andreadou ◽  
Panagiota-Efstathia Nikolaou ◽  
Claudius Witzler ◽  
Svenja Siemer ◽  
...  
Keyword(s):  
Er Stress ◽  
Research Funding ◽  
Vascular Reactivity ◽  
Ros Production ◽  
Oral Gavage ◽  
Atp Production ◽  
Cellular Bioenergetics ◽  
The Impact

Introduction: Carfilzomib (Cfz) correlates with a risk of reversible cardiotoxicity in 5-10% of multiple myeloma (MM) patients. We have recently shown that metformin (Met) has a prophylactic role against the Cfz-induced cardiotoxicity in vivo, through activation of AMPKα signaling (Blood 2019;133:710-23). However, the impact of Cfz on vascular function is obscure. Therefore, we sought to investigate: i) the acute, ii) the sub-chronic effect of Cfz on the vascular reactivity, iii) the effect of metformin co-administration on the vascular phenotype and iv) the impact of Cfz and Met co-administration on aged Human Aortic Smooth Muscle Cells (HAoSMCs). Methods: Forty male C57Bl/6 mice were assigned as follows: Acute Protocol: i. Normal Saline (N/S 0.9%); ii. Cfz (8 mg/kg); iii. Met (140 mg/kg); iv. Cfz+Met (8 mg/kg; 140 mg/kg respectively). N/S and Cfz were injected intraperitoneally (IP), while Met was administered via oral gavage daily, for 2 days (n=5 per group). Mice were sacrificed 48h after the initiation of treatments. Sub-chronic Protocol: i. N/S 0.9%; ii. Cfz (8 mg/kg); iii. Met (140mg/kg); iv. Cfz+Met (8 mg/kg; 140mg/kg respectively) for 6 days. N/S and Cfz was injected IP, while Met was administered via oral gavage on alternate days for 6 days (n=5 per group). Mice were sacrificed on the 7th day after the initiation of treatments. At the endpoint of the experiments (48h and 7 days respectively), heparinized whole blood (WB) samples and aortic tissues were collected from the experimental animals. WB samples were used to determine leucocytes derived ROS production (oxidative burst) and aorta sections (3 mm) underwent ex vivo vascular studies (relaxants: Achetlyocholine (ACh), nitroglycerin (Gtn) contractant: PDGF2α). The remaining aortic tissue underwent molecular Western blot analysis for the elucidation of the underlying mechanism. For the in vitro experiments HAoSMCs Passage 7-8 were subjected to Cfz (0.1, 0.3μM), Met (10μM, 10mM) treatment and to the combination of the two compounds. Subsequently, cells were treated with the optimal concentrations of the compounds 24h after treatment with Angiotensin II (AngII, 100nM), CoCL2 (150μM) and 48h after Glucose (25μM) to simulate the hypertensive, hypoxic and diabetic stimuli respectively. Proliferation was assessed by MTT; LC3IIB (as a marker of autophagy) expression was analyzed by immunofluorescence (IF) and cellular bioenergetics and ATP production were investigated through Seahorse XF analyzer, facilitating the study of intracellular glycolysis and oxidative phosphorylation. Results: In the acute setting, Met did not ameliorate the ROS production induced by Cfz, while no effect of Cfz on vascular relaxation/contraction was evident. In the sub-chronic setting, Met limited WB macrophage-derived oxidative burst and, while Cfz did not affect vascular reactivity, the co-administration of Cfz and Met increased PDGF2α induced aortic contractility, deducing an increased plasticity of the vessel. Western blot analysis of the aortas revealed that Cfz led to a de-phosphorylation of AMPKα, an upregulation of mTOR and iNOS while induced endoplasmic reticulum (ER) stress as indicated by increased Calnexin, IRE1a and Bip expression. In the Cfz+Met group, Met restored AMPKα phosphorylation and partially abrogated ER stress markers calnexin and Bip. However, the co-administration of the agents led to a synergistic amplification of autophagy as shown by LC3IIB expression. In the aged HAoSMCs, only Met exhibited cytotoxic capacity, while Cfz failed to exhibit any cytotoxicity. Moreover, Met was found to totally abrogate mitochondrial oxidative phosphorylation and shift cellular ATP production to anaerobic glycolysis, while Cfz had no effect on cellular bioenergetics. In terms of cardiovascular risk factors, the co-treatment of Cfz and Met led to an additive cytotoxic effect in presence of CoCL2 and AngII. Finally, in all tested conditions Cfz and Met were found to have a synergistic effect in inducing autophagy, as shown by LC3IIB upregulation. Conclusions: Sub-chronic co-administration of CFz and Met increases vascular plasticity in vivo, through a converging AMPKα- and ER stress-dependent autophagy. In vitro, Cfz does not present any effect on aged cells, probably due to the decreased proteasome activity, while Met synergizes with Cfz to decrease proliferation through alterations in cellular bioenergetics and induction of autophagy. Disclosures Kastritis: Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Takeda: Honoraria; Pfizer: Honoraria; Prothena: Honoraria; Genesis: Honoraria. Dimopoulos:Sanofi Oncology: Research Funding. Terpos:Genesis: Honoraria, Other: Travel expenses, Research Funding; Amgen: Honoraria, Research Funding; Medison: Honoraria; Janssen: Honoraria, Other: Travel expenses, Research Funding; Celgene: Honoraria; Takeda: Honoraria, Other: Travel expenses, Research Funding.

scholarly journals The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

2019 ◽  
Author(s):  
Christina Wolf ◽  
Rahel Zimmermann ◽  
Osamah Thaher ◽  
Diones Bueno ◽  
Verena Wüllner ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Mitochondrial Respiration ◽  
Culture Conditions ◽  
Mitochondrial Quality Control ◽  
Mitofusin 2 ◽  
Charcot Marie Tooth ◽  
Atp Production ◽  
Charcot Marie Tooth Disease ◽  
Tooth Disease

Charcot-Marie-Tooth disease is a hereditary polyneuropathy caused by mutations in Mitofusin-2 (MFN2), a GTPase in the outer mitochondrial membrane involved in the regulation of mitochondrial fusion and bioenergetics. Autosomal-dominant inheritance of a R94Q mutation in MFN2 causes the axonal subtype 2A2A which is characterized by early onset and progressive atrophy of distal muscles caused by motoneuronal degeneration. Here, we studied mitochondrial shape, respiration, cytosolic and mitochondrial ATP content as well as mitochondrial quality control in MFN2-deficient fibroblasts stably expressing wildtype or R94Q MFN2. Under normal culture conditions, R94Q cells had slightly more fragmented mitochondria but a similar mitochondrial oxygen consumption, membrane potential and ATP production as wildtype cells. However, when inducing mild oxidative stress 24 h before analysis using 100 &micro;M hydrogen peroxide, R94Q cells exhibited significantly increased respiration but decreased mitochondrial ATP production. This was accompanied by increased glucose uptake and an upregulation of hexokinase 1 and pyruvate kinase M2 suggesting increased pyruvate shuttling into mitochondria. As these changes coincided with decreased levels of PINK1/Parkin-mediated mitophagy in R94Q cells, we conclude that the disease-causing R94Q mutation in MFN2 causes uncoupling of mitochondrial respiration from ATP production by a less efficient mitochondrial quality control triggered by oxidative stress.

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