scholarly journals The Protective Effect of Autophagy on DNA Damage in Mouse Spermatocyte-Derived Cells Exposed to 1800 MHz Radiofrequency Electromagnetic Fields

2018 ◽  
Vol 48 (1) ◽  
pp. 29-41 ◽  
Author(s):  
Renyan Li ◽  
Mingfu Ma ◽  
Lianbing Li ◽  
Letian Zhao ◽  
Tianfeng Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Western Blot ◽  
Signaling Pathway ◽  
Electromagnetic Fields ◽  
Ros Generation ◽  
Molecular Signaling ◽  
Radiofrequency Electromagnetic Fields ◽  
Transmission Electron ◽  
Amp Activated Protein Kinase

Background/Aims: The effects of exposure to radiofrequency electromagnetic fields (RF-EMFs) on the male reproductive system have raised public concern and studies have shown that exposure to RF-EMFs can induce DNA damage and autophagy. However, there are no related reports on the role of autophagy in DNA damage in spermatocytes, especially after exposure to RF-EMFs. The aim of the present study was to determine the mechanism and role of autophagy induced by RF-EMFs in spermatozoa cells. Methods: Mouse spermatocyte-derived cells (GC-2) were exposed to RF-EMFs 4 W/kg for 24 h. The level of reactive oxygen species (ROS) was determined by ROS assay kit. Comet assay was utilized to detect DNA damage. Autophagy was detected by three indicators: LC3II/LC3I, autophagic vacuoles, and GFP-LC3 dots, which were measured by western blot, transmission electron microscopy, and transfection with GFP-LC3, respectively. The expression of the molecular signaling pathway AMP-activated protein kinase (AMPK)/mTOR was determined by western blot. Results: The results showed that RF-EMFs induced autophagy and DNA damage in GC-2 cells via ROS generation, and the autophagy signaling pathway AMPK/mTOR was activated by ROS generation. Furthermore, following inhibition of autophagy by knockdown of AMPKα, increased DNA damage was observed in GC-2 cells following RF-EMFs exposure, and overexpression of AMPKα promoted autophagy and attenuated DNA damage. Conclusions: These findings demonstrated that the autophagy which was induced by RF-EMFs via the AMPK/mTOR signaling pathway could prevent DNA damage in spermatozoa cells.

Role of autophagy in chemoresistance: Regulation of the ATM-mediated DNA-damage signaling pathway through activation of DNA–PKcs and PARP-1

2012 ◽  
Vol 83 (6) ◽  
pp. 747-757 ◽  
Author(s):  
Jung-Hoon Yoon ◽  
Sang-Gun Ahn ◽  
Byung-Hoon Lee ◽  
Sung-Hoo Jung ◽  
Seon-Hee Oh
Keyword(s):  
Dna Damage ◽  
Signaling Pathway ◽  
Dna Damage Signaling ◽  
Parp 1

scholarly journals The Role of Adenosine Monophosphate Activated Protein Kinase Alpha 1 (AMPK) in Gamma-Globin Regulation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 413-413
Author(s):  
Alicia Chang ◽  
Yankai Zhang ◽  
Nelda Itzep ◽  
Vivien A Sheehan
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Signaling Pathway ◽  
Insulin Signaling ◽  
Globin Gene ◽  
Insulin Signaling Pathway ◽  
Gamma Globin ◽  
Compound C ◽  
Globin Gene Expression

Abstract Background: Fetal hemoglobin (HbF, α2g2) induction has long been an area of investigation, as it is known to reduce the clinical complications of sickle cell disease (SCD) and beta thalassemia. Progress in identifying novel HbF inducing strategies has been stymied by an incomplete understanding of gamma-globin regulation. We used natural genetic variation to identify novel genes and pathways associated with HbF levels in patients with SCD. Our whole exome sequencing analysis of 1290 samples from patients with SCD identified the insulin signaling pathway to be related to HbF regulation. Functional studies performed in hematopoietic stem and progenitor cells (HSPCs) from patients with SCD established that FOXO3 is a positive regulator of HbF, and that metformin, a FOXO3 and AMPK activator, can induce HbF (Zhang et al, Blood 2018). We hypothesized that other proteins in the insulin signaling pathway, particularly AMPK, a direct activator of FOXO3, may contribute to HbF regulation and be a potential target for pharmacologic induction of HbF. Objectives: We now seek to determine the role of AMPK and AMPK activators such as piceatannol in HbF regulation through functional studies in HSPCs from patients with SCD. Methods: HSPCs from 3 unique patients with SCD were transduced with AMPK shRNA on day 5 of two phase primary erythroid culture. AMPK, FOXO3, gamma and beta globin gene expression were measured by RT-qPCR and HbF by HPLC respectively on day 14 of culture. HSPCs from 3 unique patients with SCD were treated with AICAR, piceatannol at 12.5µM and metformin at 100 µM on day 7 of erythroid culture. Cell lysate was collected on day 14, and AMPK, FOXO3, gamma and beta globin gene expression and protein levels measured by RT-qPCR and western blot respectively. Levels of pAMPK, at Thr172, were quantified by western blot. 1 µM Compound C was added with piceatannol and with metformin in separate erythroid cultures on day 7, and the effect on gamma globin and phosphorylation of AMPK at Thr172 was measured on day 14 by RT-qPCR and western blot respectively. Results: 70% knockdown of AMPK resulted in a 50% decrease in HbF (p<0.01) and a three-fold reduction in gamma-globin expression (p<0.001). HSPCs treated with metformin or piceatannol exhibited a 2-3 fold rise in AMPK, FOXO3 and gamma globin gene expression (p<0.001). HSPCs treated with piceatannol and metformin showed an increase in pAMPK at Thr172, the activated form of AMPK. In the presence of a specific AMPK inhibitor, Compound C, metformin and piceatannol, no induction of gamma globin was observed (Figure 1), and pAMPK was reduced to untreated levels. Conclusions: Knockdown of AMPK in HSPCs reduces gamma globin expression and %HbF, supporting the role of AMPK in gamma globin regulation. Drugs known to activate AMPK, metformin and piceatannol, increase gamma globin in SCD patient derived HSPCs. Pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction, and reduces the gamma globin induction of metformin and piceatannol to untreated levels. We therefore conclude that AMPK is a positive regulator of HbF, and that pharmacologic induction of HbF with metformin and piceatannol requires AMPK activity. Further work is needed to establish if FOXO3 and AMPK alone are instrumental in HbF regulation, or if other proteins in the insulin signaling pathway may play a role in HbF regulation. Disclosures No relevant conflicts of interest to declare.

scholarly journals EPAC1 Inhibition Protects the Heart from Doxorubicin-Induced Toxicity

2021 ◽  
Author(s):  
Marianne Mazevet ◽  
Maxance Ribeiro ◽  
Anissa Belhadef ◽  
Delphine Dayde ◽  
Anna Llach ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Death ◽  
Membrane Potential ◽  
Dilated Cardiomyopathy ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Dna Intercalation ◽  
Antitumoral Activity ◽  
Ros Generation

Rationale: The widely used chemotherapeutic agent Doxorubicin (Dox) induces cardiotoxicity leading to dilated cardiomyopathy and heart failure. This cardiotoxicity has been related to ROS generation, DNA intercalation, bioenergetic distress and cell death. However, alternative mechanisms are emerging, focusing on signaling pathways. Objective: We investigated the role of Exchange Protein directly Activated by cAMP (EPAC), key factor in cAMP signaling, in Dox-induced cardiotoxicity. Methods and Results: Dox was administrated in vivo (10 +/- 2 mg/kg, i.v.; with analysis at 2, 6 and 15 weeks post injection) in WT and EPAC1 KO C57BL6 mice. Cardiac function was analyzed by echocardiography and intracellular Ca2+ homeostasis by confocal microscopy in isolated ventricular cardiomyocytes. 15 weeks post-injections, Dox-treated WT mice, developed a dilated cardiomyopathy with decreased ejection fraction, increased telediastolic volume and impaired Ca2+ homeostasis, which were totally prevented in the EPAC1 KO mice. The underlying mechanisms were investigated in neonatal and adult rat cardiac myocytes under Dox treatment (1-10 uM). Flow cytometry, Western blot, BRET sensor assay, and RT-qPCR analysis showed that Dox induced DNA damage and cardiomyocyte cell death with apoptotic features rather than necrosis, including Ca2+-CaMKKβ-dependent opening of the Mitochondrial Permeability Transition Pore, dissipation of the Mitochondrial membrane potential, caspase activation, cell size reduction, and DNA fragmentation. Dox also led to an increase in both cAMP concentration and EPAC1 protein level and activity. The pharmacological inhibition of EPAC1 (CE3F4) but not EPAC2 alleviated the whole Dox-induced pattern of alterations including DNA damage, Mitochondrial membrane potential, apoptosis, mitochondrial biogenesis, dynamic, and fission/fusion balance, and respiratory chain activity, suggesting a crucial role of EPAC1 in these processes. Importantly, while preserving cardiomyocyte integrity, EPAC1 inhibition potentiated Dox-induced cell death in several human cancer cell lines. Conclusion: Thus, EPAC1 inhibition could be a valuable therapeutic strategy to limit Dox-induced cardiomyopathy without interfering with its antitumoral activity.

scholarly journals Bioactive nano-selenium antagonizes cobalt nanoparticle-mediated oxidative stress via the Keap1-Nrf2-ARE signaling pathway

2022 ◽  
Vol 24 (1) ◽  
Author(s):  
Siqi Wang ◽  
Chen Wang ◽  
Weinan Zhang ◽  
Wentao Fan ◽  
Fan Liu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Cell Activity ◽  
Inflammatory Factors ◽  
Ros Generation ◽  
Ros Production ◽  
Metal On Metal ◽  
Human Participant ◽  
Artificial Joint Replacement ◽  
Cobalt Nanoparticle

AbstractAt present, no effective treatment exists for the clinical toxicity of cobalt nanoparticles (CoNPs, 30 nm) after metal-on-metal (MOM) artificial joint replacement. As such, a better understanding of the CoNPs-toxicity mechanism is necessary and urgent for the development of effective and safe detoxification drugs. Our purpose was to explore the role of bioactive nano-selenium (BNS, > 97%) in antagonizing the toxicity of CoNPs and its mechanism through the Keap1-Nrf2-ARE signaling pathway. To examine BNS detoxification, we exposed HUVEC cells to CoNPs and BNS for 24 h, before measuring cell activity, reactive oxygen species (ROS), the GSH level, inflammatory factors, and KNA signaling pathway-related transcript and protein expression. CoNPs stimulate intracellular inflammation and ROS production to bring about significant downregulation of cellular activity and the GSH level. Conversely, BNS reduces ROS generation and suppresses inflammatory factors within cells to reduce CoNPs-mediated cytotoxicity, possibly via the KNA signaling pathway. Based on our results, BNS antagonizes CoNPs toxic effects by suppressing ROS production through the KNA pathway. Our research provides new insight into the clinical treatment of CoNPs toxicity and explores the potential of BNS in detoxification therapy. Trial registration: no human participant.

Dexmedetomidine Inhibited Proliferation and Invasion of Cervical Cancer Cells by Inhibiting the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription Signaling Pathway

2021 ◽  
Vol 11 (7) ◽  
pp. 1293-1304
Author(s):  
FenLan Xu ◽  
Liying Xu ◽  
Xiaoyan Xu ◽  
Zhenhua Huang ◽  
Liang Su
Keyword(s):  
Cervical Cancer ◽  
Cell Migration ◽  
Western Blot ◽  
Cell Viability ◽  
Signaling Pathway ◽  
Migration And Invasion ◽  
Cell Migration And Invasion ◽  
Stat Signaling ◽  
Related Proteins

The role of anesthetics in the treatment of cancer has been reported, but the role of Dexmedetomidine (Dex) in the treatment of cervical cancer (CC) has not been reported.In this study, cell viability and proliferation were determined by MTT and cloning formation assay. The expression of proliferation-related proteins ki67 and PCNA was detected by western blot. Wound healing and transwell detected cell migration and invasion, and western blot detected the expression of migration and invasion related proteins MMP4 and MMP9, and epithelial-mesenchymal transformation (ETM)-related proteins N-cadherin, Snail, Vimentin and E-cadherin. Western blot also detected the expression of pathway related proteins p-JAK2, p-STAT1, p-STAT3, JAK2, STAT1 and STAT3. It showed that Dex inhibited the cell viability and proliferation of Hela and siHa and the expression of ki67 and PCNA were also inhibited. Dex inhibited the cell migration and invasion, and inhibited the expression of MMP4 and MMP9. In addition, Dex inhibited the expression of N-cadherin, Snail and Vimentin, and promoted the expression of E-cadherin. Dex inhibited the expression of p-JAK2, p-STAT1 and p-STAT3. After the addition of JAK/STAT signaling pathway agonist IL-6, the inhibition of Dex on proliferation, migration and invasion of CC cells was reversed. And the addition of JAK/STAT signaling pathway inhibitor AG490 could counteract the excitatory effect of IL-6 on the pathway, at which time the cell proliferation, invasion and migration were significantly increased. In conclusion, our study demonstrated that Dex inhibited proliferation, migration, and invasion of cells in CC by blocking the JAK/STAT signaling pathway.

FGF-7 facilitates the process of psoriasis by inducing TNF-α expression in HaCaT cells

2019 ◽  
Vol 51 (10) ◽  
pp. 1056-1063 ◽  
Author(s):  
Jiaojiao Pu ◽  
Rui Wang ◽  
Guanglin Zhang ◽  
Ju Wang
Keyword(s):  
Western Blot ◽  
Signaling Pathway ◽  
Specific Antibody ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hacat Cells ◽  
Tnf Α

Abstract The purpose of this study was to uncover the mechanism of tumor necrosis factor (TNF)-α induction by fibroblast growth factor-7 (FGF-7) in human HaCaT cells and the potential role of FGF-7-specific antibody F-9 in psoriatic therapy. TNF-α expression in HaCaT cells induced by FGF-7 was analyzed by quantitative polymerase chain reaction, western blot analysis, and enzyme-linked immunosorbent assays. In vivo, the BALB/c mouse psoriasis model established by topical application of imiquimod (IMQ) was used to determine the role of FGF-7-specific antibody (F-9) in skin inflammation. We found that induction of TNF-α expression by FGF-7 in HaCaT cells was suppressed by FGF-7-specific antibody F-9. Western blot analysis results showed that FGF-7 induced TNF-α expression in HaCaT cells via the FGF receptor 2 (FGFR2)/AKT/NF-κB signaling pathway. In vivo, F-9 could significantly ameliorate the inflammations in a mouse psoriatic model evaluated by Psoriasis Area and Severity Index scores and ear thickness, which was consistent with the results of hematoxylin–eosin staining, immunohistochemistry assay, and western blot analysis. These results indicate that FGF-7 induces TNF-α expression in HaCaT cells and FGF-7 antibody F-9 alleviates IMQ-induced psoriasiform in mice. Therefore, FGF-7/FGFR2 signaling pathway is a potential target for psoriasis treatment.

scholarly journals DNA Methylation-Mediated Silencing of Mir-204 Enhances the Occurrence of T-Cell Acute Lymphoblastic Leukemia By Upregulating MMP-2 and MMP-9 through the NF-Κb Signaling Pathway

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 11-11
Author(s):  
Dabing Chen ◽  
Tingting Xiao ◽  
Dandan Lin ◽  
Haojie Zhu ◽  
Jingjing Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Matrix Metalloproteinase ◽  
Western Blot ◽  
Signaling Pathway ◽  
Promoter Region ◽  
Lymphoblastic Leukemia ◽  
Cell Acute Lymphoblastic Leukemia

Background : MicroRNAs (miR) are non-coding RNAs that play a role in regulation multiple functions in different cell types. Previous studies have shown that miR-204 is downregulated in T-ALL. We previously reported that matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) gene polymorphisms may be associated with the risk of T-cell acute lymphoblastic leukemia (T-ALL). The present study aims to decipher the role of miR-204 and MMP-2/MMP-9 in T-ALL occurrence to guide the diagnosis and treatment of T-ALL in the clinics. Methods: Expression of miR-204 was determined in the bone marrow and peripheral blood samples from 70 T-ALL patients and 70 healthy volunteers by real-time quantitative PCR (RT-qPCR). Bisulfite sequencing PCR (BSP) was used to detect the DNA methylation levels of the miR-204 promoter region in T-ALL patients and T-ALL cell lines.The effect of miR-204 on cell proliferation was evaluated with the cell counting kit-8 solution (CCK-8) assay and by Hoechst and PI double staining. The binding site of miR-204 on IRAK1 was predicted by the Primer Premier 5.0 and the defined binding sequences were used to construct luciferase-tag plasmids. The regulation of IRAK1 expression by miR-204 was evaluated by RT-qPCR and Western blot analysis. With the purpose to confirm the role of MMP-2 and MMP-9 in the occurrence of T-ALL, we investigated the effect of related proteins on T-ALL cells using Western blot. To determine that miR-204 affects the occurrence of T-ALL disease by regulating the NF-KB signaling pathway, RT-qPCR and Western Blot were used for verification. Results: DNA methylation directly affects the miR-204 expression in the promoter region when T-ALL developed. Moreover, overexpression of miR-204 inhibited the proliferation and enhanced the apoptosis of T-ALL cells. Notably, overexpression of miR-204 inhibited IRAK1, which in turn inhibited the proliferation and enhanced the apoptosis of T-ALL cells. Furthermore, IRAK1 enhanced the expression of MMP-2 and MMP-9 through phosphorylation of of p65 NF-κB, and miR-204 modulated MMP-2 and MMP-9 expression through the IRAK1/NF-κB signaling pathway. Conclusion s : Our results demonstrate that in T-ALL cells, DNA methylation-mediated silencing of miR-204 regulates the expression of MMP-2 and MMP-9 through increased transcription of IRAK1, and activation of the NF-κB signaling pathway. These data provide a potential mechanism for the role of MMP-2 and MMP-9 in the occurrence of T-ALL. Further studies will be needed to demonstrate whether demethylation of miR-204 may be a promising treatment for T-ALL. Disclosures No relevant conflicts of interest to declare.

scholarly journals Bioactive nano-selenium antagonizes cobalt nanoparticle- mediated oxidative stress via the Keap1-Nrf2-ARE signaling pathway

2021 ◽  
Author(s):  
Siqi Wang ◽  
Chen Wang ◽  
Weinan Zhang ◽  
Wentao Fan ◽  
Fan Liu ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Cell Activity ◽  
Inflammatory Factors ◽  
Ros Generation ◽  
Ros Production ◽  
Metal On Metal ◽  
Artificial Joint Replacement ◽  
Huvec Cells ◽  
Cobalt Nanoparticle

Abstract BackgroundAt present, no effective treatment exists for the clinical toxicity of cobalt nanoparticles (CoNPs) after metal-on-metal (MOM) artificial joint replacement. As such, a better understanding of the CoNPs-toxicity mechanism is necessary and urgent for the development of effective and safe detoxification drugs. Our purpose was to explore the role of bioactive nano-selenium (BNS) in antagonizing the toxicity of CoNPs and its mechanism through the Keap1-Nrf2-ARE signaling pathway. MethodsTo examine BNS detoxification, we exposed HUVEC cells to CoNPs (400μmol/l) and BNS (50μg/ml) for 24h, before measuring cell activity, reactive oxygen species (ROS), inflammatory factors, and KNA signaling pathway related transcript and protein expression. ResultsCoNPs stimulate intracellular inflammation and ROS production to bring about significant downregulation of cellular activity. Conversely, BNS reduces ROS generation and suppresses inflammatory factors within cells to reduce CoNPs-mediated cytotoxicity, possibly via the KNA signaling pathway. ConclusionsBased on our results, BNS antagonizes CoNPs toxic effects by suppressing ROS production through the KNA pathway. Our research provides new insight into the clinical treatment of CoNPs toxicity and explores the potential of BNS in detoxification therapy.

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