High glucose induces Nox4 expression and podocyte apoptosis through the Smad3/ezrin/PKA pathway

Podocyte is the major target in proteinuric kidney disease such as diabetic nephropathy. The underlying molecular mechanisms by which high glucose (HG) results in podocyte damage remain unclear. This study investigated the regulatory role of Smad3, ezrin, and protein kinase A (PKA) in NADPH oxidase (Nox4) expression, reactive oxidative species (ROS) production, and apoptosis in HG-treated podocytes. Human podocyte cell line was cultured and differentiated, then treated with 30 mM HG. Apoptosis and intracellular ROS level was assessed using TUNEL and DCF assay, respectively. Expressions of Nox4, phospho-Smad3Ser423/425, phospho-PKAThr197, and phospho-ezrinThr567 were evaluated using Western blotting. ELISA was used to quantify intracellular cAMP concentration and PKA activity. Knockdown assay was used to inhibit the expressions of Smad3, Nox4, and ezrin by lentiviral shRNA. In HG-treated podocytes, the level of phospho-Smad3Ser423/425 and phospho-ezrinThr567 was increased significantly, which was accompanied by the reduction of cAMP and phospho-PKAThr197. HG-induced apoptosis was significantly prevented by the Smad3 inhibitor SIS3 or shRNA-Smad3. In podocytes expressing shRNA-ezrin or shRNA-Nox4, apoptosis was remarkably mitigated following HG treatment. HG-induced upregulation of phospho-ezrinThr567 and downregulation of phospho-PKAThr197 was significantly prevented by SIS3, shRNA-ezrin or shRNA-Smad3. Forskolin, a PKA activator, significantly inhibited HG-mediated upregulation of Nox4 expression, ROS generation, and apoptosis. Additionally, an increase in the ROS level was prohibited in HG-treated podocytes with the knockdown of Nox4, Smad3, or ezrin. Taken together, our findings provided evidence that Smad3-mediated ezrin activation upregulates Nox4 expression and ROS production, by suppressing PKA activity, which may at least in part contribute to HG-induced podocyte apoptosis.

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SENP1-Mediated Desumoylation of DBC1 Inhibits Apoptosis Induced by High Glucose in Bovine Retinal Pericytes

Journal of Ophthalmology ◽

10.1155/2016/6392658 ◽

2016 ◽

Vol 2016 ◽

pp. 1-11

Author(s):

Jian Gao ◽

Xia Chen ◽

Qing Gu ◽

Xiaoxiao Liu ◽

Xun Xu

Keyword(s):

Diabetic Retinopathy ◽

High Glucose ◽

Molecular Mechanisms ◽

Characteristic Change ◽

High Concentration ◽

Retinal Pericytes ◽

Specific Protease ◽

Induced Apoptosis ◽

Pericyte Loss

Pericyte loss is an early characteristic change in diabetic retinopathy, but its precise molecular mechanisms have not been elucidated. This study investigated the role of SENP1 in pericyte loss in diabetic retinopathy. We demonstrated that a high concentration of glucose inhibited the expression of the Sentrin/SUMO-specific protease 1 (SENP1), which resulted in an increase in DBC1 sumoylation in bovine retinal pericytes (BRPCs). Furthermore, SENP1 overexpression attenuated hyperemia-induced apoptosis of BPRCs, and SENP1 knockdown aggravated this effect. We also provide evidence that DBC1 sumoylation/desumoylation is involved in the SENP1-regulated apoptosis of BRPCs under high glucose conditions. Understanding the role of SENP1 in the pathogenesis of high glucose induced pericyte loss could help elucidate important targets for future pharmacological interventions.

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Bufalin Induces Reactive Oxygen Species Dependent Bax Translocation and Apoptosis in ASTC-a-1 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1093/ecam/nep082 ◽

2011 ◽

Vol 2011 ◽

pp. 1-12 ◽

Cited By ~ 21

Author(s):

Lei Sun ◽

Tongsheng Chen ◽

Xiaoping Wang ◽

Yun Chen ◽

Xunbin Wei

Keyword(s):

Reactive Oxygen Species ◽

Cell Death ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Temporal Dynamics ◽

Reactive Oxygen ◽

Ros Generation ◽

Ros Production ◽

Oxygen Species ◽

Induced Apoptosis

Bufalin has been shown to induce cancer cell death through apoptotic pathways. However, the molecular mechanisms are not well understood. In this study, we used the confocal fluorescence microscopy (CFM) to monitor the spatio-temporal dynamics of reactive oxygen species (ROS) production, Bax translocation and caspase-3 activation during bufalin-induced apoptosis in living human lung adenocarcinoma (ASTC-a-1) cells. Bufalin induced ROS production and apoptotic cell death, demonstrated by Hoechst 33258 staining as well as flow cytometry analysis. Bax redistributed from cytosol to mitochondria from 12 to 48 h after bufalin treatment in living cells expressed with green fluorescent protein Bax. Treatment with the antioxidantN-acetyl-cysteine (NAC), a ROS scavenger, inhibited ROS generation and Bax translocation and led to a significant protection against bufalin-induced apoptosis. Our results also revealed that bufalin induced a prominent increase of caspase-3 activation blocked potently by NAC. Taken together, bufalin induced ROS-mediated Bax translocation, mitochondrial permeability transition and caspase-3 activation, implying that bufalin induced apoptosis via ROS-dependent mitochondrial death pathway in ASTC-a-1 cells.

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MiR-126 targets IL-17A to enhance proliferation and inhibit apoptosis in high-glucose-induced human retinal endothelial cells

Biochemistry and Cell Biology ◽

10.1139/bcb-2019-0174 ◽

2020 ◽

Vol 98 (2) ◽

pp. 277-283 ◽

Cited By ~ 1

Author(s):

Xiujuan Chen ◽

Xuequn Yu ◽

Xinxiang Li ◽

Li Li ◽

Fang Li ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Molecular Mechanisms ◽

Caspase 3 ◽

Vision Loss ◽

Retinal Endothelial Cells ◽

Interleukin 17A ◽

Novel Target ◽

Induced Apoptosis

Diabetic retinopathy (DR) is a common complication of diabetes mellitus (DM), which results in vision loss. This study explored the role of miR-126 in high-glucose-induced human retinal endothelial cells (HRECs) and its underlying molecular mechanisms. The results showed that the expression levels of miR-126 and interleukin-17A (IL-17A) in high-glucose-induced HRECs were downregulated and upregulated, respectively. Functionally, overexpression of miR-126 promoted proliferation and suppressed apoptosis in high-glucose-induced HRECs, while IL-17A reversed the effects induced by miR-126. However, overexpression of IL-17A inhibited the proliferation and induced apoptosis, while knockdown of IL-17A accelerated the proliferation and repressed apoptosis. In addition, miR-126 repressed the expression of IL-17A, Bax, and caspase-3, while promoting the expression of survivin and phosphorylation of PI3K and AKT; restoration of IL-17A rescued these effects. Furthermore, IL-17A was identified as a target of miR-126. This indicates that miR-126 enhances proliferation and inhibits apoptosis in high-glucose-induced HRECs by activating the PI3K–AKT pathway, increasing survivin levels, and decreasing Bax and caspase-3 expression by targeting IL-17A, suggesting that miR-126 could be a novel target for preventing DR.

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High Glucose-Induced ROS Production Stimulates Proliferation of Pancreatic Cancer via Inactivating the JNK Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2018/6917206 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 2

Author(s):

Jiao Luo ◽

Yukai Xiang ◽

Xiangxiang Xu ◽

Dazhang Fang ◽

Ding Li ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Cells ◽

High Glucose ◽

Ros Generation ◽

Cancer Cell Growth ◽

Jnk Pathway ◽

Pancreatic Cancer Cells ◽

Intracellular Ros ◽

Tumorigenesis And Progression

Aberrant glucose metabolism of diabetes mellitus or hyperglycemia stimulates pancreatic tumorigenesis and progression. Hyperglycemic environment can increase the ROS level of tumors, but the role of upregulation of ROS levels in pancreatic cancer (PC) still remains controversial. Here, the same as other reports, we demonstrate that high glucose promoted pancreatic cancer cell growth and resulted in an increase in the level of ROS. However, it is interesting that the phosphorylation of JNK was reduced. When treating PC cells with N-acetyl-L-cysteine (NAC), the intracellular ROS generation is repressed, but the expression of phosphorylation of JNK and c-Jun increased. Moreover, the JNK inhibitor SP600125 significantly promoted cell proliferation and suppressed cell apoptosis of pancreatic cancer cells under high glucose conditions. Collectively, high levels of ROS induced by high glucose conditions stimulated the proliferation of pancreatic cancer cells, and it may be achieved by inactivating the JNK pathway.

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Nonylphenol Induces Apoptosis through ROS/JNK Signaling in a Spermatogonia Cell Line

International Journal of Molecular Sciences ◽

10.3390/ijms22010307 ◽

2020 ◽

Vol 22 (1) ◽

pp. 307

Author(s):

Hyun-Jung Park ◽

Ran Lee ◽

Hyunjin Yoo ◽

Kwonho Hong ◽

Hyuk Song

Keyword(s):

Molecular Mechanisms ◽

Mapk Signaling ◽

Ros Generation ◽

Dependent Manner ◽

Jnk Signaling ◽

Apoptosis Markers ◽

Testicular Size ◽

Spermatogonia Cell ◽

Induced Apoptosis ◽

Dose Dependent

Nonylphenol (NP) is an endocrine-disruptor chemical that negatively affects reproductive health. Testes exposure to NP results in testicular structure disruption and a reduction in testicular size and testosterone levels. However, the effects of NP on spermatogonia in testes have not been fully elucidated. In this study, the molecular mechanisms of NP in GC-1 spermatogonia (spg) cells were investigated. We found that cell viability significantly decreased and apoptosis increased in a dose-dependent manner when GC-1 spg cells were exposed to NP. Furthermore, the expression levels of the pro-apoptotic proteins increased, whereas anti-apoptosis markers decreased in NP-exposed GC-1 spg cells. We also found that NP increased reactive oxygen species (ROS) generation, suggesting that ROS-induced activation of the MAPK signaling pathway is the molecular mechanism of NP-induced apoptosis in GC-1 spg cells. Thus, NP could induce c-Jun phosphorylation; dose-dependent expression of JNK, MKK4, p53, and p38; and the subsequent inhibition of ERK1/2 and MEK1/2 phosphorylation. The genes involved in apoptosis and JNK signaling were also upregulated in GC-1 spg cells treated with NP compared to those in the controls. Our findings suggest that NP induces apoptosis through ROS/JNK signaling in GC-1 spg cells.

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The p53/miR-34a/SIRT1 Positive Feedback Loop in Quercetin-Induced Apoptosis

Cellular Physiology and Biochemistry ◽

10.1159/000374024 ◽

2015 ◽

Vol 35 (6) ◽

pp. 2192-2202 ◽

Cited By ~ 48

Author(s):

Guohua Lou ◽

Yanning Liu ◽

Shanshan Wu ◽

Jihua Xue ◽

Fan Yang ◽

...

Keyword(s):

Cell Apoptosis ◽

Feedback Loop ◽

Molecular Mechanisms ◽

Rt Pcr ◽

Cycle Arrest ◽

Anticancer Effects ◽

Huh7 Cells ◽

Mirnas Expression ◽

Induced Apoptosis

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

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Evaluation of anticancer activity in vitro of a stable copper(I) complex with phosphine-peptide conjugate

Scientific Reports ◽

10.1038/s41598-021-03352-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Urszula K. Komarnicka ◽

Barbara Pucelik ◽

Daria Wojtala ◽

Monika K. Lesiów ◽

Grażyna Stochel ◽

...

Keyword(s):

A549 Cells ◽

Ros Generation ◽

Dependent Manner ◽

Hacat Cells ◽

Icp Ms ◽

Intracellular Ros ◽

M Phase ◽

Lung Adenocarcinoma A549 ◽

Induced Apoptosis

Abstract[CuI(2,9-dimethyl-1,10-phenanthroline)P(p-OCH3-Ph)2CH2SarcosineGlycine] (1-MPSG), highly stable in physiological media phosphino copper(I) complex—is proposed herein as a viable alternative to anticancer platinum-based drugs. It is noteworthy that, 1-MPSG significantly and selectively reduced cell viability in a 3D spheroidal model of human lung adenocarcinoma (A549), in comparison with non-cancerous HaCaT cells. Confocal microscopy and an ICP-MS analysis showed that 1-MPSG effectively accumulates inside A549 cells with colocalization in mitochondria and nuclei. A precise cytometric analysis revealed a predominance of apoptosis over the other types of cell death. In the case of HaCaT cells, the overall cytotoxicity was significantly lower, indicating the selective activity of 1-MPSG towards cancer cells. Apoptosis also manifested itself in a decrease in mitochondrial membrane potential along with the activation of caspases-3/9. Moreover, the caspase inhibitor (Z-VAD-FMK) pretreatment led to decreased level of apoptosis (more pronouncedly in A549 cells than in non-cancerous HaCaT cells) and further validated the caspases dependence in 1-MPSG-induced apoptosis. Furthermore, the 1-MPSG complex presumably induces the changes in the cell cycle leading to G2/M phase arrest in a dose-dependent manner. It was also observed that the 1-MPSG mediated intracellular ROS alterations in A549 and HaCaT cells. These results, proved by fluorescence spectroscopy, and flow cytometry, suggest that investigated Cu(I) compound may trigger apoptosis also through ROS generation.

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Abstract P241: Calpain-1 Is Increased in Mitochondria and Contributes to Mitochondrial ROS Generation in High Glucose--Stimulated Endothelial Cells and Endothelial Dysfunction in Mouse Models of Diabetes

Circulation Research ◽

10.1161/res.109.suppl_1.ap241 ◽

2011 ◽

Vol 109 (suppl_1) ◽

Author(s):

Qing Zhao ◽

Futian Tang ◽

Limei Shan ◽

Inga Cepinskas ◽

Gedas Cepinskas ◽

...

Keyword(s):

Endothelial Cells ◽

High Glucose ◽

Aortic Ring ◽

Ros Generation ◽

Type I ◽

Ros Production ◽

Diabetic Mice ◽

Calpain Activity ◽

Over Expression ◽

Mitochondrial Ros

Objectives: Elevated levels of reactive oxygen species (ROS) are the initial source of endothelial dysfunction in diabetes. Calpain has been implicated in diabetic vascular complications. The present study was to investigate the role of calpain in mitochondrial ROS generation in endothelial cells and vascular dysfunction in diabetic mice. Methods: Endothelial cells cultured from human umbilical vein (HUVEC) were stimulated with high glucose. Calpain activity and protein were determined in mitochondria of HUVEC. Intracellular and mitochondrial ROS generation as well as apoptosis were measured. Type I diabetic OVE 26 mice and type II diabetic db/db mice with calpastatin over-expression (OVE26/CAST and db/db-CAST) were generated, respectively. Type I diabetes was also induced in both wild-type and Tg-CAST mice by injection of streptozocin (STZ). The endothelium-dependent relaxation of aortic ring was measured. Results: High glucose significantly increased calpain-1 protein, calpain activity and ROS generation in mitochondria of HUVEC. Pharmacological inhibition of calpain or over-expression of calpastatin abrogated high glucose-induced intracellular ROS production, mitochondrial ROS generation and apoptosis in HUVEC. Incubation of isolated mitochondria with calpain-1 protein significantly induced its ROS generation and the membrane potential. In diabetic mice, calpain activity was induced in aortic vessels, which correlated with an increase in ROS production and protein tyrosine nitration. Over-expression of calpastatin prevented calpain activity, reduced ROS production and inhibited protein tyrosine nitration in diabetic mice. Aortic ring segments from diabetic mice exhibited a significant reduction in vascular relaxation to acetylcholine, which was reversed by over-expression of calpastatin in Tg-CAST, OVE26/CAST and db/db-CAST mice. Conclusions: This study has demonstrated a novel role of calpain in mitochondrial ROS generation, which contributes to apoptosis in endothelial cells during hyperglycemia. Thus, over-expression of calpastatin inhibits reduces ROS production and ameliorates endothelium-dependent vascular dysfunction in mouse models of diabetes.

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Abstract 263: Differential Effects of 17ß-Estradiol and 16a-Hydroxyestrone in Oxidative Stress and Proliferative Responses in Human Pulmonary Artery Smooth Muscle Cells - Implications in Pulmonary Arterial Hypertension

Hypertension ◽

10.1161/hyp.62.suppl_1.a263 ◽

2013 ◽

Vol 62 (suppl_1) ◽

Author(s):

Katie Y Hood ◽

Augusto C Montezano ◽

Margaret R MacLean ◽

Rhian M Touyz

Keyword(s):

Oxidative Stress ◽

Pulmonary Arterial Hypertension ◽

Cell Growth ◽

Arterial Hypertension ◽

Molecular Mechanisms ◽

Antioxidant Systems ◽

Ros Generation ◽

Ros Production ◽

Differential Effects ◽

Pulmonary Arterial

Women develop pulmonary arterial hypertension (PAH) more frequently than men. This may relate, in part, to metabolism of 17β-estradiol (E2), leading to formation of the deleterious metabolite, 16α-hydroxyestrone (16α OHE1), which plays a role in the remodelling of pulmonary arteries. Molecular mechanisms whereby 16αOHE1 influences PASMC remodelling are unclear but ROS may be important, since oxidative stress has been implicated in the pathogenesis of PAH. We hypothesised that E2 and 16αOHE1 leads to Nox-induced ROS production, which promotes PASMC damage. Cultured PASMCs were stimulated with either E2 (1nM) or 16αOHE1 (1nM) in the presence/absence of EHT1864 (100μM, Rac1 inhibitor) or tempol (antioxidant; 10μM). ROS production was assessed by chemiluminescence (O2-) and Amplex Red (H2O2). Antioxidants (thioredoxin, peroxiredoxin 1 and NQ01), regulators of Nrf2 (BACH1, Nrf2) and, marker of cell growth (PCNA) were determined by immunoblotting. E2 increased O2- production at 4h (219 ± 30% vs vehicle; p<0.05), an effect blocked by EHT1864 and tempol. E2 also increased H2O2 generation (152 ± 4%; p<0.05). Thioredoxin, NQ01 and peroxiredoxin1 (71 ± 6%; 78 ± 9%; 69 ± 8%; p<0.05 respectively) levels were decreased by E2 as was PCNA expression (72 ± 2%; p<0.05). 16αOHE1 exhibited a rapid (5 min) and exaggerated increase in ROS production (355 ± 41%; p<0.05), blocked by tempol and EHT1864. This was associated with an increase in Nox4 expression (139 ± 11% vs vehicle, p<0.05). 16αOHE1 increased BACH1, (129 ± 3%; p<0.05), a competitor of Nrf2, which was decreased (92 ± 2%). In contrast, thioredoxin expression was increased by 16aOHE1 (154 ± 22%; p<0.05). PCNA (150 ± 5%) expression was also increased after exposure to 16αOHE1. In conclusion, E2 and 16αOHE1 have differential effects on redox processes associated with PASMC growth. Whereas E2 stimulates ROS production in a slow and sustained manner without effect on cell growth, 16αOHE1 upregulates Nox4 with associated rapid increase in ROS generation and downregulation of antioxidant systems, affecting proliferation. Our findings suggest that E2 -derived metabolites may promote a pro-proliferative PASMC phenotype through Nox4-derived ROS generation. These deleterious effects may impact on vascular remodeling in PAH.

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The Roles of ROS Generation in RANKL-Induced Osteoclastogenesis: Suppressive Effects of Febuxostat

Cancers ◽

10.3390/cancers12040929 ◽

2020 ◽

Vol 12 (4) ◽

pp. 929

Author(s):

Mohannad Ashtar ◽

Hirofumi Tenshin ◽

Jumpei Teramachi ◽

Ariunzaya Bat-Erdene ◽

Masahiro Hiasa ◽

...

Keyword(s):

Bone Marrow Cells ◽

Tumor Lysis Syndrome ◽

Oxidase Inhibitor ◽

Ros Generation ◽

Ros Production ◽

Xanthine Oxidase Inhibitor ◽

Ovariectomized Mice ◽

Bone Damage ◽

Anticancer Treatment

Receptor activator of NF-κB ligand (RANKL), a critical mediator of osteoclastogenesis, is upregulated in multiple myeloma (MM). The xanthine oxidase inhibitor febuxostat, clinically used for prevention of tumor lysis syndrome, has been demonstrated to effectively inhibit not only the generation of uric acid but also the formation of reactive oxygen species (ROS). ROS has been demonstrated to mediate RANKL-mediated osteoclastogenesis. In the present study, we therefore explored the role of cancer-treatment-induced ROS in RANKL-mediated osteoclastogenesis and the suppressive effects of febuxostat on ROS generation and osteoclastogenesis. RANKL dose-dependently induced ROS production in RAW264.7 preosteoclastic cells; however, febuxostat inhibited the RANKL-induced ROS production and osteoclast (OC) formation. Interestingly, doxorubicin (Dox) further enhanced RANKL-induced osteoclastogenesis through upregulation of ROS production, which was mostly abolished by addition of febuxostat. Febuxostat also inhibited osteoclastogenesis enhanced in cocultures of bone marrow cells with MM cells. Importantly, febuxostat rather suppressed MM cell viability and did not compromise Dox’s anti-MM activity. In addition, febuxostat was able to alleviate pathological osteoclastic activity and bone loss in ovariectomized mice. Collectively, these results suggest that excessive ROS production by aberrant RANKL overexpression and/or anticancer treatment disadvantageously impacts bone, and that febuxostat can prevent the ROS-mediated osteoclastic bone damage.

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