Apoptosis Inducible Factor P53 Decrease Glutamate-Associated Damage via Inhibiting Ferroptosis

2021 ◽  
Author(s):  
Zeyong Yang ◽  
Wenting Xuan ◽  
Yaru Jin ◽  
Yuanhai Li
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Protein Expression ◽  
P53 Protein ◽  
Staining Technique ◽  
Cell Counting ◽  
Ferrous Ions ◽  
Ht22 Cells ◽  
Expression Levels ◽  
Ht22 Cell

Abstract BACKGROUND: Ferroptosis, a pattern of programmed cell death decided by iron-associated lipid peroxidation, however, its role of p53-mediated xCT pathway in HT22 cell death remains obscure. Herein, this study is to investigate the potential mechanism of the effect of p53-mediated xCT pathway in HT22 cell lines in an iron-relevant mode. METHODS: The viability of HT22 cells were detected by Cell Counting Kit-8(CCK-8) and PI/Hoechst fluorescence double staining. The protein expression levels of p53 and xCT were determined by western boltting. DHE fluorescence staining technique supervised the intracellular reactive oxygen species (ROS), and intracellular lipid oxidant situation was confirmed by BODIPY 581/591 C11 lipid peroxidation sensor. Intracellular ferrous ions were monitored with FeRhoNox™-1 fluoresceent probe. RESULTS: The protein expression levels of p53 was obviously enhanced by tenovin-1 exposure. Accompanied with the upregulation of p53 protein, cell death was decreased significantly because of glutamate and erastin exposure with 8h, and p53-mediated xCT pathway was activated. Intracelluar ROS levels, lipid oxidant situations and ferrous ions were remarkably restricted from Glutamate-p53 groups in comparison with glutamate groups. CONCLUSIONS: Overall, P53-mediated xCT pathway could decrease the glutamate-associated neurotoxicity, which may be relevant to the inhibition of ferroptosis.

scholarly journals Pirfenidone attenuates synovial fibrosis and postpones the progression of osteoarthritis by anti-fibrotic and anti-inflammatory properties in vivo and in vitro

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Qilu Wei ◽  
Ning Kong ◽  
Xiaohui Liu ◽  
Run Tian ◽  
Ming Jiao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Counting ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fast Green ◽  
Expression Levels ◽  
Tnf Α ◽  
Anti Inflammatory ◽  
Safranin O ◽  
Tgf Β1

Abstract Background Osteoarthritis (OA) is a disease of the entire joint involving synovial fibrosis and inflammation. Pathological changes to the synovium can accelerate the progression of OA. Pirfenidone (PFD) is a potent anti-fibrotic drug with additional anti-inflammatory properties. However, the influence of PFD on OA is unknown. Methods Proliferation of human fibroblast-like synoviocytes (FLSs) after treatment with TGF-β1 or PFD was evaluated using a Cell Counting Kit-8 assay and their migration using a Transwell assay. The expression of fibrosis-related genes (COL1A1, TIMP-1, and ACTA-2) and those related to inflammation (IL-6 and TNF-α) was quantified by real-time quantitative PCR. The protein expression levels of COL1A1, α-SMA (coded by ACTA-2), IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay. A rabbit model of OA was established and then PFD was administered by gavage. The expression of genes related to fibrosis (COL1A1, TIMP-1, and ADAM-12) and inflammation (IL-6 and TNF-α) was measured using RNA extracted from the synovium. Synovial tissue was examined histologically after staining with H&E, Masson’s trichrome, and immunofluorescence. Synovitis scores, the volume fraction of collagen, and mean fluorescence intensity were calculated. Degeneration of articular cartilage was analyzed using a Safranin O-fast green stain and OARSI grading. Results The proliferation of FLSs was greatest when induced with 2.5 ng/ml TGF-β1 although it did not promote their migration. Therefore, 2.5 ng/ml TGF-β1 was used to stimulate the FLSs and evaluate the effects of PFD, which inhibited the migration of FLSs at concentrations as low as 1.0 mg/ml. PFD decreased the expression of COL1A1 while TGF-β1 increased both mRNA and protein expression levels of IL-6 but had no effect on α-SMA or TNF-α expression. PFD decreased mRNA expression levels of COL1A1, IL-6, and TNF-α in vivo. H&E staining and synovitis scores indicated that PFD reduced synovial inflammation, while Masson’s trichrome and immunofluorescence staining suggested that PFD decreased synovial fibrosis. Safranin O-Fast Green staining and the OARSI scores demonstrated that PFD delayed the progression of OA. Conclusions PFD attenuated synovial fibrosis and inflammation, and postponed the progression of osteoarthritis in a modified Hulth model of OA in rabbits, which was related to its anti-fibrotic and anti-inflammatory properties.

scholarly journals Pituitary Adenylate Cyclase Activating Polypeptide Elicits Neuroprotection Against Acute Ischemic Neuronal Cell Death Associated with NMDA Receptors

2018 ◽  
Vol 51 (4) ◽  
pp. 1982-1995 ◽  
Author(s):  
Yuji Kaneko ◽  
Julian P. Tuazon ◽  
Xunming Ji ◽  
Cesario V. Borlongan
Keyword(s):  
Cell Death ◽  
Adenylate Cyclase ◽  
Protein Expression ◽  
Cell Viability ◽  
Caspase 3 ◽  
High Mobility ◽  
High Mobility Group ◽  
Expression Levels ◽  
Pituitary Adenylate Cyclase ◽  
Nmdar Subunits

Background/Aims: The endogenous neurotrophic peptides pituitary adenylate cyclase-activating polypeptides (PACAP-27/38) protect against stroke, but the molecular mechanism remains unknown. Methods: Primary rat neural cells were exposed to PACAP-27 or PACAP-38 before induction of experimental acute ischemic stroke via oxygen-glucose deprivation-reperfusion (OGD/R) injury. To reveal PACAP’s role in neuroprotection, we employed fluorescent live/dead cell viability and caspase 3 assays, optical densitometry of mitochondrial dehydrogenase and cell growth, glutathione disulfide luciferase activity, ELISA for high mobility group box1 extracellular concentration, ATP bioluminescence, Western blot analysis of PACAP, NMDA subunits, apoptosis regulator Bcl-2, social interaction hormone oxytocin, and trophic factor BDNF, and immunocytochemical analysis of PACAP. Results: Both PACAP-27 and PACAP-38 (PACAP-27/38) increased cell viability, decreased oxidative stress-induced cell damage, maintained mitochondrial activity, prevented the release of high mobility group box1, and reduced cytochrome c/caspase 3-induced apoptosis. PACAP-27/38 increased the protein expression levels of BDNF, Bcl-2, oxytocin, and precursor PACAP. N-methyl-D-aspartate receptor (NMDAR)-induced excitotoxicity contributes to the cell death associated with stroke. PACAP-27/38 modulated the protein expression levels of NMDAR subunits. PACAP-27/38 increased the protein expression levels of the GluN1 subunit, and decreased that of the GluN2B and GluN2D subunits. PACAP-27, but not PACAP-38, increased the expression level of the GluN2C subunit. Conclusion: This study provides evidence that PACAP regulated NMDAR subunits, affording neuroprotection after OGD/R injury.

scholarly journals Melatonin Protects Band 3 Protein in Human Erythrocytes against H2O2-Induced Oxidative Stress

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2741 ◽  
Author(s):  
Rossana Morabito ◽  
Alessia Remigante ◽  
Angela Marino
Keyword(s):  
Oxidative Stress ◽  
Lipid Peroxidation ◽  
Protein Expression ◽  
Beneficial Effect ◽  
Anion Exchange ◽  
Rate Constant ◽  
Cell Shape ◽  
Band 3 ◽  
Band 3 Protein ◽  
Expression Levels

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 μM H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 µM have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 µM Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

Erythroid-Expression of HMGA2 Increases Fetal Hemoglobin in Human Adult Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2161-2161
Author(s):  
Jaira F. de Vasconcellos ◽  
Y. Terry Lee ◽  
Colleen Byrnes ◽  
Laxminath Tumburu ◽  
Antoinette Rabel ◽  
...  
Keyword(s):  
Protein Expression ◽  
Fetal Hemoglobin ◽  
Cell Counting ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Globin Mrna ◽  
Expression Levels ◽  
Gamma Globin ◽  
Empty Vector ◽  
Gene And Protein Expression

Abstract HMGA2 is a member of the high-mobility group A family and plays a role in the regulation of gene transcription and chromatin structure. HMGA2 is a validated target of the let-7 family of miRNAs. Let-7 miRNAs are highly regulated in erythroid cells during the fetal-to-adult developmental transition (1). Recent studies demonstrated that the LIN28 -let-7 axis mediated up-regulation of fetal hemoglobin (HbF) expression to >30% of the total globin levels in cultured erythroblasts from adult humans (2) and the amelioration of hypoxia-related sickling of cultured mature erythrocytes from pediatric patients with sickle cell disease (3). Interestingly, increased expression of endogenous HbF in a patient receiving gene therapy was also associated with truncated HMGA2 protein expression after lentiviral integration and disruption of let-7 targeting at the HMGA2 gene locus (4). Therefore, we hypothesized that HMGA2 may be involved in fetal hemoglobin regulation as a downstream target of the let-7 miRNAs. To study the effects of HMGA2 upon erythropoiesis and globin expression, lentiviral constructs were designed for let-7 resistant expression of HMGA2 driven by the erythroid-specific gene promoter region of the human SPTA1 gene (HMGA2 -SPTA1-OE), with a matched empty vector control. Transductions were performed in CD34+ cells from four adult healthy volunteers cultivated ex vivo in erythropoietin-supplemented serum-free media for 21 days. Overexpression of HMGA2 was confirmedby Q-RT-PCR (control: below detection limits; HMGA2 -SPTA1-OE: 2.51E+04 ± 3.44E+04 copies/ng) and Western blot analyses at culture day 14. Cell counting revealed no significant changes between HMGA2 -SPTA1-OE and control (empty vector) transductions at culture day 14. Terminal maturation with loss of CD71 from the erythroblast cell surface and enucleation assessed by thiazole orange staining were analyzed in the control and HMGA2 -SPTA1 -OE samples at the end of the culture period. Globin genes expression levels were evaluated for HMGA2 -SPTA1-OE by Q-RT-PCR. HMGA2 -SPTA1-OE caused a significant increase in gamma-globin mRNA expression levels compared to controls (control: 5.02E+05 ± 8.62E+04 copies/ng; HMGA2 -SPTA1-OE: 1.45E+06 ± 7.31E+05 copies/ng; p=0.037). Consistent with the increase in gamma-globin mRNA levels, HPLC analyses at culture day 21 demonstrated modest but significant increases in HbF levels in HMGA2 -SPTA1-OE compared to controls (HbF control: 5.41 ± 2.15%; HMGA2 -SPTA1-OE: 16.53 ± 4.43%; p=0.006). Possible effect(s) and downstream mechanism(s) triggered by HMGA2 -SPTA1-OE were investigated. Q-RT-PCR analyses demonstrated no significant changes in the let-7 family of miRNAs in HMGA2 -SPTA1-OE compared to controls. Expression patterns of several transcription factors such as BCL11A, KLF1, SOX6 and GATA1 were investigated by Q-RT-PCR and no significant changes were detected in HMGA2 -SPTA1-OE compared to controls. While BCL11A mRNA levels were decreased by HMGA2 -SPTA1 -OE, the differences did not reach statistical significance (control: 4.26E+02 ± 8.18E+01 copies/ng; HMGA2 -SPTA1 -OE: 2.84E+02 ± 1.48E+02 copies/ng; p=0.104). However, nuclear BCL11A protein levels assessed by Western analysis were suppressed in HMGA2 -SPTA1 -OE. In summary, these results demonstrate that HMGA2, a validated target of let-7 miRNAs, causes moderately increased gamma-globin gene and protein expression in human erythroblasts, and reduces levels of BCL11A protein. These data thus support the notion that suppression of let-7 miRNAs increases fetal hemoglobin, in part, by the targeting of erythroblast HMGA2 mRNA. (1) Noh SJ et al. J Transl Med. 7:98 (2009). (2) Lee YT et al. Blood. 122:1034-41 (2013). (3) Vasconcellos JF et al. PLoS One. 9:e106924 (2014). (4) Cavazzana-Calvo M et al. Nature. 467:318-22 (2010). Disclosures No relevant conflicts of interest to declare.

P53 Protein Expression Levels Are Prognostic in AML and Predict for Mutational Status.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2397-2397 ◽  
Author(s):  
Steven M. Kornblau ◽  
Joseph Barnett ◽  
YiHua Qiu ◽  
Wenjing Chen ◽  
Stefan Faderl ◽  
...  
Keyword(s):  
Protein Expression ◽  
P53 Protein ◽  
Leukemia Cell ◽  
Response To Therapy ◽  
Newly Diagnosed ◽  
P53 Expression ◽  
Mutation Status ◽  
Expression Levels ◽  
P53 Protein Expression ◽  
Very High

Abstract Mutation of the P53 gene in AML confers an adverse prognosis and is associated with unfavorable cytogenetics typically with complex karyoptyes. Since P53 function can also be modulated by MDM2 associated, ubiquitin dependent, destruction, the amount of P53 protein may also be relevant to leukemia blast biology. Previous studies have focused on the presence or absence of mutations, but have not examined the relevance of protein expression levels. We evaluated the levels of expression of P53 using high-throughput reverse phase protein array technology (RPPA) on 537 leukemia cell enriched samples from 423 patients (258 newly diagnosed, 47 primary refractory, 118 relapse 1, 2 or refractory). Levels of P53 were lower, equal and higher than that of normal CD34+ cells in 57%, 37% and 16% of cases, respectively. Almost all cases with very high P53 had unfavorable cytogenetics with complex karyotypes (15/16 newly diagnosed), and very high P53 was observed in 13% of patients with unfavorable cytogenetics compared to <1% with favorable or intermediate cytogenetics. Changes involving chromosome 5 (66% vs. 9%) and 7 (50% vs. 11%) were very common in those with very high P53 compared to those with normal or low P53. Levels of expression were similar regardless of disease status; newly diagnosed, primary refractory or relapsed We tested the hypothesis that patients with very high P53 were likely to have mutations by performing RT-PCR sequencing, covering exons 5–9 in 23 very high, and 11 low P53 expressing patients. Among very high expressers 11 missense mutations were found in 9 patients (39% overall), including 7 of 13 newly diagnosed cases (54%) compared to 0 of 11 low expressers (p=0.02 all cases, 0.05 newly diagnosed). Mutations occurred at known hot spots: including 5 in exon 8 [3 at codon 273], 3 in exon 7 and 2 in exon 5 and 6. High P53 expression was inversely correlated with PTEN expression (P=0.001, see accompanying abstract). Expression was unaffected by FLT3-ITD mutation status. For outcome analysis, martingale residuals were utilized to define an optimal cutpoint for dichotomization into groups with Higher (n=57, 50 treated) vs. lower (n=201, 165 treated) P53. Patients with higher P53 were significantly more likely to have an antecedent hematological disorder (37% vs. 17%, P=0.002) and unfavorable cytogenetics (65% vs. 39%, P=0.0002). Response to therapy and outcome were significantly worse in those with higher P53. The complete remission rate was significantly lower in those with higher P53 (46% vs. 66%, P=0.01). The relapse rate was significantly higher (74% vs. 47%, P=0.02) and the median remission duration was significantly shorter (26 vs. 43 weeks, P=0.02). The combination resulted in a significantly shorter median survival (26 vs. 44 weeks, P=0.0003). Analysis of MDM2 expression in these same cases is underway. This data demonstrates that cases of AML with very high levels of P53 protein expression, measured by RPPA, are frequently found in those with P53 mutations. Not all patients with high P53 levels have mutations, but high levels of P53 protein carry an adverse prognosis, regardless of mutation status.

Polyozellin, a key constituent of the edible mushroom Polyozellus multiplex, attenuates glutamate-induced mouse hippocampal neuronal HT22 cell death

2015 ◽  
Vol 6 (12) ◽  
pp. 3678-3686 ◽  
Author(s):  
Eun-Ju Yang ◽  
Kyung-Sik Song
Keyword(s):  
Cell Death ◽  
Edible Mushroom ◽  
Ht22 Cells ◽  
Ht22 Cell

Polyozellin, a key constituent of Polyozellus multiplex, was applied to glutamate-treated HT22 cells to evaluate its neuroprotective mechanisms.

Effect of Nitric Oxide Donor, a Modulator of Tumor Drug Resistance, on Cell Death and p53 Protein Expression

2009 ◽  
Vol 147 (4) ◽  
pp. 421-423
Author(s):  
T. A. Rajewskaya ◽  
S. A. Goncharova ◽  
N. P. Konovalova ◽  
A. A. Terent’ev ◽  
M. A. Lapshina
Keyword(s):  
Nitric Oxide ◽  
Drug Resistance ◽  
Cell Death ◽  
Protein Expression ◽  
P53 Protein ◽  
Nitric Oxide Donor ◽  
P53 Protein Expression ◽  
Tumor Drug Resistance

scholarly journals Tert-Butylhydroquinone Prevents Oxidative Stress-Mediated Apoptosis and Extracellular Matrix Degradation in Rat Chondrocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-12
Author(s):  
Bo Yang ◽  
Haisheng Huang ◽  
Qisong He ◽  
Wei Lu ◽  
Lu Zheng ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Matrix ◽  
Matrix Metalloproteinase ◽  
Protein Expression ◽  
Cell Viability ◽  
Cell Counting ◽  
Expression Levels ◽  
Extracellular Matrix Components ◽  
Mrna And Protein Expression

Oxidative stress-induced chondrocyte apoptosis and degradation of the extracellular matrix (ECM) play an important role in the progression of osteoarthritis (OA). In addition, tert-butylhydroquinone (TBHQ) is an activator of the nuclear factor erythroid derived-2-related factor 2 (Nrf2). The present study aimed to determine the effectiveness of TBHQ in preventing the apoptosis of chondrocytes and degradation of the extracellular matrix, induced by oxidative stress, in vitro. Therefore, rat chondrocytes were exposed to 20 μM tert-butyl hydroperoxide (TBHP) for 24 h to establish an oxidative damage model, in vitro. Thereafter, cell viability was evaluated using the Cell Counting Kit-8 assay. Moreover, the level of ROS was determined through 2′,7′-dichlorofluorescein diacetate staining. The mitochondrial membrane potential of chondrocytes was also measured using JC-1. Furthermore, cell apoptosis was assessed through Annexin V-fluorescein isothiocyanate/propidium iodide staining. The study also performed Western blotting and qPCR to evaluate the expression of extracellular matrix components, matrix catabolic enzymes, and changes in signalling pathways. The results showed that 2.5 and 5 μM of TBHQ reduced the TBHP-induced generation of excessive ROS and improved cell viability. Additionally, 2.5 and 5 μM of TBHQ prevented mitochondrial damage and apoptosis in rat chondrocytes. Treatment with TBHQ also increased the mRNA and protein expression levels of aggrecan and collagen II. However, TBHQ reduced the mRNA and protein expression levels of matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) in rat chondrocytes. In addition, treatment with TBHQ enhanced the protein expression levels of Nrf2, NADPH quinone oxidoreductase 1 (NQO-1), and hemeoxygenase-1 (HO-1) in rat chondrocytes. The current study showed that TBHQ was not only effective in protecting against TBHP-induced oxidative stress but also inhibited the apoptosis of rat chondrocytes and degradation of the ECM by activating the Nrf2 pathway. The results therefore suggest that TBHQ holds potential for use in the treatment of OA.

scholarly journals High Expression Levels of SLC38A1 Are Correlated with Poor Prognosis and Defective Immune Infiltration in Hepatocellular Carcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Yun Liu ◽  
Yong Yang ◽  
Linna Jiang ◽  
Hongrui Xu ◽  
Junwei Wei
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Protein Expression ◽  
Prognostic Significance ◽  
Functional Enrichment ◽  
High Expression ◽  
Expression Levels ◽  
Immune Infiltration ◽  
Mrna And Protein Expression

Solute Carrier Family 38 Member 1 (SLC38A1) is a principal transporter of glutamine and plays a crucial role in the transformation of neoplastic cells. However, the correlation between SLC38A1 expression, prognosis, and immune infiltration in hepatocellular carcinoma (HCC) has yet to be elucidated. We used two independent patient cohorts, namely, a Cancer Genome Atlas (TCGA) cohort and a Clinical Proteomic Tumor Analysis Consortium (CPTAC) cohort, to analyze the role of SLC38A1 in HCC at the mRNA and protein levels, respectively. In these two cohorts, SLC38A1 mRNA and protein expression levels were higher in HCC tissues than in adjacent nontumor tissues. Both SLC38A1 mRNA and protein expression were positively associated with clinicopathological characteristics (clinical stage, T stage, pathological grade, tumor size, and tumor thrombus), were negatively associated with survival, and were independent prognostic factors in HCC patients. Functional enrichment analyses further indicated that SLC38A1 was involved in multiple pathways related to amino acid metabolism, tumors, and immunity. High expression levels of SLC38A1 were inversely proportional to CD8+ T cells and directly proportional to macrophages M0, neutrophils, programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Moreover, we used immunohistochemical analysis of tissue samples and other online databases to further validate the expression levels and prognostic significance of SLC38A1 in HCC. Collectively, our study demonstrated that the upregulated expression of SLC38A1 was related to an unfavorable prognosis and defective immune infiltration in HCC.

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