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International Journal of Molecular... Oct 2022The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib's effects on cell...
The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib's effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells' TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy.
Topics: Acridine Orange; Apoptosis; Autophagy; Cell Death; Humans; Imatinib Mesylate; Protein Kinase Inhibitors; Stem Cells; Wortmannin
PubMed: 36233113
DOI: 10.3390/ijms231911812 -
Frontiers in Molecular Neuroscience 2022Intracellular amyloid β peptide (Aβ) accumulation has drawn attention in relation to the pathophysiology of Alzheimer's disease in addition to its extracellular...
Intracellular amyloid β peptide (Aβ) accumulation has drawn attention in relation to the pathophysiology of Alzheimer's disease in addition to its extracellular deposition as senile plaque. Cellular uptake of extracellular Aβ is one of the possible mechanisms by which intracellular Aβ deposits form. Given the relevance of Aβ inside cells, it is important to understand the mechanism by which it is taken up by them. In this study, we elucidated that Neuro2A and SH-SY5Y cells internalize specifically oligomerized Aβ in a time- and dose-dependent manner. The depletion of plasma membrane cholesterol with methyl-β-cyclodextrin or treatment with trypsin diminished the internalization of oAβ, suggesting that the oAβ uptake might be both a lipid raft-dependent and heparan sulfate proteoglycan-mediated process. Treatment with a macropinocytosis inhibitor (ethylisopropyl amiloride and wortmannin) also drastically reduced the uptake of oligomer-Aβ (oAβ). oAβ-treated cells exhibited an increase in Rac1 activity, indicating that macropinocytosis induced by oAβ is regulated by these small GTPases. These findings suggest that macropinocytosis is a major endocytic route through which oAβ42 enters cells.
PubMed: 36187354
DOI: 10.3389/fnmol.2022.804702 -
Aging Sep 2022Decreased BDNF and impaired TRKB signaling contribute to neurodegeneration in Alzheimer's disease (AD). We have shown previously that coumarin derivative LM-031 enhanced...
Decreased BDNF and impaired TRKB signaling contribute to neurodegeneration in Alzheimer's disease (AD). We have shown previously that coumarin derivative LM-031 enhanced CREB/BDNF/BCL2 pathway. In this study we explored if LM-031 analogs LMDS-1 to -4 may act as TRKB agonists to protect SH-SY5Y cells against Aβ toxicity. By docking computation for binding with TRKB using 7,8-DHF as a control, all four LMDS compounds displayed potential of binding to domain d5 of TRKB. In addition, all four LMDS compounds exhibited anti-aggregation and neuroprotective efficacy on SH-SY5Y cells with induced Aβ-GFP expression. Knock-down of TRKB significantly attenuated TRKB downstream signaling and the neurite outgrowth-promoting effects of these LMDS compounds. Among them, LMDS-1 and -2 were further examined for TRKB signaling. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in Aβ-GFP cells, implicating the neuroprotective effects are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. LMDS-1 and -2 are blood-brain barrier permeable as shown by parallel artificial membrane permeability assay. Our results demonstrate how LMDS-1 and -2 are likely to work as TRKB agonists to exert neuroprotection in Aβ cells, which may shed light on the potential application in therapeutics of AD.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Brain-Derived Neurotrophic Factor; Coumarins; Extracellular Signal-Regulated MAP Kinases; Humans; Membrane Glycoproteins; Membranes, Artificial; Neuroblastoma; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Receptor, trkB; Wortmannin
PubMed: 36170028
DOI: 10.18632/aging.204306 -
Cell Stress & Chaperones Nov 2022The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. RBM3, an exceptional cold shock protein, is...
RNA binding motif protein 3 (RBM3) promotes protein kinase B (AKT) activation to enhance glucose metabolism and reduce apoptosis in skeletal muscle of mice under acute cold exposure.
The main danger of cold stress to animals in cold regions is systemic metabolic changes and protein synthesis inhibition. RBM3, an exceptional cold shock protein, is rapidly upregulated in response to hypothermia to resist the adverse effects of cold stress. However, the mechanism of the protective effect and the rapid upregulation of RBM3 remains unclear. O-GlcNAcylation, an atypical O-glycosylation, is precisely regulated only by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) and participates in the signal transduction of multiple cellular stress responses as a "stress and nutrition receptor." Therefore, our study aimed to explore the mechanism of RBM3 regulating glucose metabolism and promoting survival in skeletal muscle under acute cold exposure. Meanwhile, our study verifies whether O-GlcNAcylation mediated by OGT rapidly upregulates RBM3. The blood and skeletal muscle of mice were collected at the end of cold exposure treatment for 0, 2, and 4 h. Changes in levels of RBM3, AKT, glycolysis apoptosis, and OGT were measured. The results show that acute cold exposure upregulated RBM3, OGT, and AKT phosphorylation and increased energy consumption, which enhanced glycolysis and prevent apoptosis. In the 32 °C mild hypothermia model in vitro, overexpression of RBM3 enhanced AKT phosphorylation. Meanwhile, inactivation of AKT by wortmannin resulted in increased apoptosis and decreased glucose metabolism in skeletal muscle under acute cold exposure. In addition, OGT-mediated O-GlcNAcylation of p65 was confirmed in mouse myoblast cell line (C2C12) cells at mild hypothermia. O-GlcNAcylation level affected p65 activity and nuclear translocation. Compared with wild type (WT) mice, RBM3 and p65 phosphorylation were decreased in specific skeletal muscle Ogt (KO) mice, whereas AKT phosphorylation, glycolysis, and apoptosis were increased. Taken together, O-GlcNAcylation of p65 upregulates RBM3 to promote AKT phosphorylation, enhance glucose metabolism, and reduce apoptosis in skeletal muscle of mice under acute cold exposure.
Topics: Mice; Animals; Proto-Oncogene Proteins c-akt; Hypothermia; N-Acetylglucosaminyltransferases; Apoptosis; Muscle, Skeletal; Glucose; RNA-Binding Motifs; RNA-Binding Proteins
PubMed: 36149580
DOI: 10.1007/s12192-022-01297-7 -
Journal of Cardiovascular Pharmacology Sep 2022This study aimed to explore whether vaspin could alleviate cardiac remodeling through attenuating oxidative stress in heart failure rats and to determine the associated...
This study aimed to explore whether vaspin could alleviate cardiac remodeling through attenuating oxidative stress in heart failure rats and to determine the associated signaling pathway. Cardiac remodeling was induced by myocardial infarction, transverse aortic constriction, or angiotensin (Ang) II infusion in vivo, and the neonatal rat cardiomyocytes (NRCMs) and neonatal rat cardiac fibroblasts (NRCFs) were treated with Ang II. Vaspin treatment alleviated fibrosis in myocardial infarction, transverse aortic constriction, and Ang II-treated rats. The Ang II-induced increases of atrial natriuretic peptide and brain natriuretic peptide in NRCMs and Ang II-induced increases of collagen I and collagen III in NRCFs were reduced after vaspin treatment. Vaspin administration inhibited the Ang II-induced increases of phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathway, superoxide anions, malondialdehyde, and NADPH oxidases activity in NRCMs and NRCFs. The overexpression of PI3K, Akt, or NADPH oxidases 1 reversed the attenuating effects of vaspin on Ang II-induced elevation of atrial natriuretic peptide and brain natriuretic peptide in NRCMs, as well as Ang II-induced increases of collagen I and collagen III in NRCFs. The administration of wortmannin (PI3K inhibitor) or MK2206 (Akt inhibitor) inhibited the oxidative stress induced by Ang II in NRCMs and NRCFs. The above results suggest that vaspin can alleviate cardiac dysfunction and remodeling in heart failure rats. Vaspin attenuates Ang II-induced hypertrophy of NRCMs and fibrosis of NRCFs through suppressing PI3K/Akt pathway to alleviate oxidative stress.
Topics: Angiotensin II; Animals; Atrial Natriuretic Factor; Collagen; Fibrosis; Heart Failure; Myocardial Infarction; Myocytes, Cardiac; NADPH Oxidases; Natriuretic Peptide, Brain; Oxidative Stress; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats; Ventricular Remodeling
PubMed: 36067399
DOI: 10.1097/FJC.0000000000001291 -
NPJ Microgravity Sep 2022The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity...
The spaceflight environment of the International Space Station poses a multitude of stresses on plant growth including reduced gravity. Plants exposed to microgravity and other conditions on the ISS display root skewing, changes in gene expression and protein abundance that may result in changes in cell wall composition, antioxidant accumulation and modification of growth anisotropy. Systematic studies that address the effects of microgravity on cellular organelles are lacking but altered numbers and sizes of vacuoles have been detected in previous flights. The prominent size of plant vacuoles makes them ideal models to study organelle dynamics in space. Here, we used Arabidopsis zigzag-1 (zig-1) as a sensitized genotype to study the effect of microgravity on plant vacuole fusion. Wortmannin was used to induce vacuole fusion in seedlings and a formaldehyde-based fixation protocol was developed to visualize plant vacuole morphology after sample return, using confocal microscopy. Our results indicate that microgravity enhances the zig-1 phenotype by reducing hypocotyl growth and vacuole fusion in some cells. This study demonstrates the feasibility of chemical inhibitor treatments for plant cell biology experiments in space.
PubMed: 36064795
DOI: 10.1038/s41526-022-00226-3 -
Cellular Physiology and Biochemistry :... Aug 2022In renal ischemia, the Na/K ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions....
BACKGROUND/AIMS
In renal ischemia, the Na/K ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na/K ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na/K ATPase using its analogue FTY720P and LLC-PK1 cells.
METHODS
The activity of the Na/K ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis.
RESULTS
FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na/K ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na/K ATPase in absence of free Ca revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular.
CONCLUSION
The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.
Topics: Animals; Calcium; Nitric Oxide; Organophosphates; Phosphatidylinositol 3-Kinases; Sodium-Potassium-Exchanging ATPase; Sphingosine; Swine; Wortmannin; rho-Associated Kinases
PubMed: 36041048
DOI: 10.33594/000000561 -
Autophagy Apr 2023Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease....
Macroautophagy/autophagy is a multistep degradative process that is essential for maintaining cellular homeostasis and is often dysregulated during disease. Systematically quantifying flux through this pathway is critical for gaining fundamental insights and effectively modulating this process. Established methods to quantify flux use steady-state measurements, which provide limited information about the perturbation and the cellular response. We present a theoretical and experimental framework to measure autophagic steps in the form of rates under non-steady-state conditions. We use this approach to measure temporal responses to rapamycin and wortmannin treatments, two commonly used autophagy modulators. We quantified changes in autophagy rates in as little as 10 min, which can establish direct mechanisms for autophagy perturbation before feedback begins. We identified concentration-dependent effects of rapamycin on the initial and temporal progression of autophagy rates. We also found variable recovery time from wortmannin's inhibition of autophagy, which is further accelerated by rapamycin. Furthermore, we applied this approach to study the effect of serum and glutamine starvation on autophagy. Serum starvation led to a rapid and transient increase in all the rates. Glutamine starvation led to a decrease in the rates on a longer timescale. In summary, this new approach enables the quantification of autophagy flux with high sensitivity and temporal resolution and facilitates a comprehensive understanding of this process.
Topics: Humans; Autophagy; Glutamine; Wortmannin; Lysosomes; Sirolimus
PubMed: 36026492
DOI: 10.1080/15548627.2022.2117515 -
In Vitro Cellular & Developmental... Sep 2022Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1 and R2) play important roles in the progression of malignant melanoma through tumor angiogenesis....
Vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1 and R2) play important roles in the progression of malignant melanoma through tumor angiogenesis. However, it is not clear whether the VEGF-A/VEGFR1 signaling pathway is involved in the proliferation and migration of melanoma cells. Thus, the effect of VEGF-A on cell migration was investigated in human melanoma cell lines. Of several splicing variants of VEGF-A, VEGF is the most abundant and responsible for VEGF-A biological potency. VEGF facilitated the migration of melanoma cells in both a chemotactic and chemokinetic manner, but cell proliferation was not affected by VEGF. VEGF also induced the phosphorylation of Akt. In addition, VEGF-induced cell migration was inhibited significantly by VEGFR1/2 or a VEGFR1-neutralizing antibody. Furthermore, the downregulation of VEGFR1 via the transfection of VEGFR1-targeting antisense oligonucleotides suppressed VEGF-induced cell migration. Moreover, wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI3K) in the PI3K/Akt pathway, suppressed VEGF-induced Akt phosphorylation and VEGF-induced cell migration. These findings suggest that the motility of melanoma cells is regulated by signals mediated through the PI3K/Akt kinase pathway with the activation of VEGFR1 tyrosine kinase by VEGF. Thus, the downregulation of signaling via VEGF-A/VEGFR1 might be an effective therapeutic approach that could prevent the progression of malignant melanoma.
Topics: Animals; Antibodies, Neutralizing; Cell Movement; Humans; Melanoma; Oligonucleotides, Antisense; Phosphatidylinositol 3-Kinase; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Proto-Oncogene Proteins c-akt; Signal Transduction; Vascular Endothelial Growth Factor A; Wortmannin
PubMed: 35997849
DOI: 10.1007/s11626-022-00717-3 -
Clinical, Cosmetic and Investigational... 2022Autophagy is an important process for maintaining intracellular homeostasis and is deregulated in ultraviolet B (UVB)-induced skin injury. Salidroside (SAL) is an active...
INTRODUCTION
Autophagy is an important process for maintaining intracellular homeostasis and is deregulated in ultraviolet B (UVB)-induced skin injury. Salidroside (SAL) is an active ingredient extracted from , which is a herbal medicine that has shown protection against ultraviolet (UV) radiation. Here, we investigated the functions and mechanisms of SAL on UVB-induced skin cell oxidative damage and autophagy.
METHODS
Human immortalized keratinocyte cell line HaCaT was used as a cell model of UV injury. HaCaT cells were exposed to UVB irradiation and then incubated with SAL to investigate cell viability, lactate dehydrogenase (LSD) in culture media, intracellular reactive oxygen species (ROS) level, oxidative stress, autophagy, and regulatory effects on SIRT1 protein.
RESULTS
SAL pretreatment (25, 50 and 100 μM) increased cell viability and inhibited LDH release in UVB-challenged HaCaT cells. SAL (100 μM) significantly reduced intracellular ROS level and suppressed oxidative stress, with increased MDA content and increased SOD activity. In addition, SAL pretreatment enhanced autophagy in UVB-irradiated HaCaT cells, increased protein expressions of Beclin-1 and ATG7, and decreased protein expression of P62. We also found that pretreatment with SAL increased the SIRT1 protein in irradiated HaCaT cells. SAL protected UVB-induced damage in a dependent manner on autophagy and SIRT1, as SAL-induced increase in viability was significantly attenuated by specific autophagy inhibitor Wortmannin (1 μM) or SIRT1 inhibitor EX-527 (100 nM).
DISCUSSION
The present study results speculate that SAL suppresses UVB-induced injury and autophagy by enhancing SIRT1 expression.
PubMed: 35941856
DOI: 10.2147/CCID.S367233