Abstract 255: Cytoprotective Effects of Erythropoietin and Erythropoietin-derivatives in Ischaemic Human Myotubes and the Role of Pi3k/akt Signalling Pathway

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Rebekah Sian Hwee Yu ◽  
Daryll Baker ◽  
David Abraham ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Signalling Pathway ◽  
Critical Limb Ischaemia ◽  
Human Myotubes ◽  
Muscle Biopsies

Objectives Erythropoietin (Epo) has tissue-protective effects in response to injury, acting through the EpoR-βcR heteroreceptor. We have previously demonstrated the presence and interaction of the EpoR and βcR in human skeletal muscle. Here we aim to investigate the potential cytoprotective effects of Epo and an Epo-derivative (ARA-290) in a human in vitro model of skeletal muscle and establish a potential downstream signalling pathway utilised in protecting cells from apoptosis (including Jak-2, PI3k/Akt, NFkB). Methods Gastrocnemius muscle biopsies were obtained from patients with critical limb ischaemia and control samples were obtained from non-ischaemic patients. Human myoblasts were isolated from muscle biopsies, cultured, and allowed to differentiate into myotubes in order to investigate the cytoprotective effects of Epo and ARA-290 on myotubes subjected to simulated ischaemia. The PI3k inhibitors, LY294002 and wortmannin, were then used to determine the role of PI3k/Akt pathway in mediating cytoprotection. Following this, inhibitors against the upstreatm (Jak-2) and downstream (NFkB) molecules were also investigated. Western blot analysis, using the pro-apoptotic marker cleaved caspase-3 was performed and compared with levels of Akt and phosphorylated-Akt, using western blot analysis. Results Exogenous administration of Epo and ARA-290 were able to ameliorate the ischaemia-induced apoptosis on isolated human myotubes as shown by a significant reduction in cleaved caspase-3 expression. Addition of all inhibitors, to ARA-290 or Epo pre-treated cells, abolished the reduction in apoptosis. Conclusion The ability of ARA-290 to attenuate apoptosis in human myotubes undergoing ischaemic insult suggests a potential role in tissue protection in skeletal muscle injury. We propose that the PI3k/Akt signalling pathway is involved in mediating this cytoprotection.

Abstract 123: Ischemia Mediates Myogenic Progenitor Cell Dysfunction

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Matthew Fincher ◽  
David Abraham ◽  
Daryll Baker ◽  
Janice Tsui
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Satellite Cells ◽  
Progenitor Cell ◽  
Caspase 3 ◽  
Myogenic Differentiation ◽  
Critical Limb Ischaemia ◽  
Limb Ischaemia ◽  
Muscle Biopsies ◽  
Myogenic Progenitor

Introduction Treatment options for critical limb ischaemia (CLI) are limited. Recent evidence has suggested that even with successful revascularisation, patients often show little functional improvement. This has been attributed to a musculopathy that occurs in CLI. Myogenic progenitor satellite cells (SCs) provide skeletal muscle with an intrinsic ability to regenerate. It has been shown that there is an increase in SCs in ischaemic muscle, however their function in ischaemia is poorly understood and we hypothesize that ischaemia has a detrimental effect on SC function. Methods Gastrocnemius muscle biopsies were taken from CLI patients and compared with non ischaemic control biopsies. The phenotypical changes and frequency of satellite cells were investigated using PAX 7 immunohistochemistry and western blot. C2C12 myoblasts were used in vitro, to investigate the effect of ischaemia on muscle progenitor cell function. Myoblasts were exposed to simulated ischaemia for 24, 48 and 72hrs. Proliferation rates were assessed using an MTT assay. Differentiation and apoptosis were assessed by MYOD and cleaved caspase 3 western blotting respectively. Results There is an increased expression of PAX 7 in CLI muscle biopsies, shown by both immunostaining and western blot analysis, suggesting an increased number of SCs in ischaemic human skeletal muscle (p<0.05). Myoblasts cultured in ischaemic conditions demonstrated decreased cell proliferation, reduced myogenic differentiation (decreased MYOD expression), and increased apoptosis (increased cleaved caspase 3 expression). Conclusion Despite an upregulation of SCs in ischaemic tissue, their function is suppressed in ischaemic conditions and this may be contributing to the poor functional recovery of patients post revascularisation. Enhancement of muscle regeneration in ischaemia may be a useful therapeutic adjunct in the treatment of CLI.

scholarly journals Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy

2001 ◽  
Vol 47 (3) ◽  
pp. 451-458 ◽  
Author(s):  
Angelika Hammerer-Lercher ◽  
Petra Erlacher ◽  
Reginald Bittner ◽  
Rudolf Korinthenberg ◽  
Daniela Skladal ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Western Blot ◽  
Blot Analysis ◽  
Cardiac Troponin ◽  
Western Blot Analysis ◽  
Clinical Evidence ◽  
Mdx Mouse ◽  
Muscle Biopsies

Abstract Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

scholarly journals DIAPH1 Is Upregulated and Inhibits Cell Apoptosis through ATR/p53/Caspase-3 Signaling Pathway in Laryngeal Squamous Cell Carcinoma

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Jiechao Yang ◽  
Liang Zhou ◽  
Yanping Zhang ◽  
Juan Zheng ◽  
Jian Zhou ◽  
...  
Keyword(s):  
Squamous Cell Carcinoma ◽  
Western Blot ◽  
Cell Carcinoma ◽  
Squamous Cell ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Molecular Mechanisms ◽  
Caspase 3 ◽  
Laryngeal Squamous Cell Carcinoma ◽  
Poor Overall Survival

Cancer bioinformatics has been used to screen possible key cancer genes and pathways. Here, through bioinformatics analysis, we found that high expression of diaphanous related formin 1 (DIAPH1) was associated with poor overall survival in head and neck squamous cell carcinoma and laryngeal squamous cell carcinoma (LSCC). The effect of DIAPH1 in LSCC has not been previously investigated. Therefore, we evaluated the expression, function, and molecular mechanisms of DIAPH1 in LSCC. Immunohistochemistry and western blot analysis confirmed the significant upregulation of DIAPH1 in LSCC. We used DIAPH1 RNA interference to construct two DIAPH1-knockdown LSCC cell lines, AMC-HN-8 and FD-LSC-1, and validated the knockdown efficiency. Flow cytometry data showed that DIAPH1 inhibited apoptosis. Further, western blot analysis revealed that DIAPH1 knockdown increased the protein levels of ATR, p-p53, Bax, and cleaved caspase-3, -8, and -9. Thus, DIAPH1 is upregulated in LSCC and may act as an oncogene by inhibiting apoptosis through the ATR/p53/caspase-3 pathway in LSCC cells.

Dystrophin in skeletal muscle I. Western blot analysis using a monoclonal antibody

1989 ◽  
Vol 94 (1-3) ◽  
pp. 125-136 ◽  
Author(s):  
Louise V.B. Nicholson ◽  
Keith Davison ◽  
Gavin Falkous ◽  
Colin Harwood ◽  
Elizabeth O'Donnell ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Monoclonal Antibody ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis

scholarly journals The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 2158-2166 ◽  
Author(s):  
David Vicent ◽  
Eleftheria Maratos-Flier ◽  
C. Ronald Kahn
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Branch Point ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Geranylgeranyl Diphosphate ◽  
Protein Prenylation ◽  
Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

TIPE1 suppresses the invasion and migration of breast cancer cells and inhibits epithelial-to-mesenchymal transition primarily via the ERK signaling pathway

2019 ◽  
Vol 51 (10) ◽  
pp. 1008-1015 ◽  
Author(s):  
Shusheng Qiu ◽  
Wei Hu ◽  
Qiuhong Ma ◽  
Yi Zhao ◽  
Liang Li ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Western Blot ◽  
Cancer Cells ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Breast Cancer Cells ◽  
Mesenchymal Transition ◽  
Invasion And Migration ◽  
And Migration

Abstract Tumor necrosis factor α-induced protein 8-like-1 (TIPE1) functions as an activator or a repressor in a tumor cell type-specific manner. However, the role of TIPE1 in breast cancer, especially regarding metastasis, is unknown. In this study, we aimed to investigate the TIPE1 expression in breast cancer tissues, the biological functions, and the underlying mechanisms of TIPE1 regarding the metastatic properties of breast cancer cells. The results of immunohistochemical staining and western blot analysis indicated that TIPE1 expression was associated with tumor size and lymph node metastasis, and the expression of TIPE1 was downregulated in the tissues of patients with lymph node metastasis. Transwell and wound healing assay results showed that TIPE1 inhibited the invasive and migratory capacities of breast cancer cells. Moreover, the epithelial-mesenchymal transition (EMT) was suppressed in TIPE1-overexpressing cells, as demonstrated by western blot analysis. In addition, western blot analysis also showed that TIPE1 reduced the expression levels of MMP2 and MMP9 and decreased the phosphorylation level of ERK. These results suggested that TIPE1 might suppress the invasion and migration of breast cancer cells and inhibit EMT primarily via the ERK signaling pathway. Our findings revealed the anti-tumor metastasis role of TIPE1 in breast cancer and TIPE1 might be a new candidate prognostic indicator and a potential molecular target for the treatment of breast cancer.

Pan-BCL-2 Family Small Molecule Inhibitor GX015-070 (GeminX, Inc.) Exhibits Single Agent Cytotoxicity, Is Synergistic with Select Anti-Leukemia Cytotoxic Agents, and Induces Apoptosis in Cell Lines with t(4;11)(q21;q23) Translocations.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3368-3368 ◽  
Author(s):  
Jessicca M. Rege ◽  
Blaine W. Robinson ◽  
Manish Gupta ◽  
Jeffrey S. Barrett ◽  
Peter C. Adamson ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Family Member ◽  
Cell Lines ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Single Agent ◽  
Cytotoxic Agents ◽  
Response Surface Modeling

Abstract Background: Leukemias with MLL translocations, especially t(4;11), often are resistant to common chemotherapeutic agents, which may be due to abnormal apoptosis regulation. Pro- and anti-apoptotic BCL-2 family member interactions govern initiation of the intrinsic apoptosis pathway. GX015-070, which currently is in Phase I/IIA clinical trials, mimics the BH3 domain on pro-apoptotic BCL-2 family proteins and can bind the BH3 binding pocket of anti-apoptotic BCL-2 family members and modulate apoptosis. We performed comprehensive protein expression profiling of BCL-2 family member proteins and evaluated in vitro activity and mechanism of action of GX015-070 in cell lines with t(4;11). Methods: Baseline expression of BCL-2 family proteins was determined by Western blot analysis. Cytotoxicity was assessed by MTT after a 3 day exposure of RS4:11, SEM-K2 and MV4-11 cells in log phase growth to single agent GX015-070 at concentrations from 5 nM to 7.5 μM. Combined effects of fixed-concentration GX015-070 with cytotoxic agents over a range of concentrations were assayed by MTT, and the results were analyzed by pharmacostatistical response surface modeling. Disruption of specific pro- and anti-apoptotic BCL-2 family member interactions was investigated by co-immunoprecipitation/Western blot analysis. Flow cytometry and/or Western blot analysis of Caspase-3 activation, and a FACS TUNEL assay, were used to assess apoptosis in GX015-070 treated and untreated cells. Results: The three cell lines had similar baseline levels of expression of BCL-2 family proteins. BCL-2 and BAX were most abundant followed by PUMA, BAK, BCL-XL, BIM-EL, MCL-1, BIK and NOXA. Results of assays of GX015-070 activity and mechanism of action are in shown in the table. Conclusions: These data indicate that GX015-070 has potent cytotoxic activity in cell lines with t(4;11) as a single agent and that the cytotoxicity results from apoptosis. Response surface modeling in RS4:11 cells suggested ability to achieve effective doses with GX015-070 combined with cytosine arabinoside (Ara-C), dexamethasone (Dex) or doxorubicin (ADR) that are lower than projected from the single agents, but synergy was not suggested when GX015-070 was combined with etoposide, methotrexate or 6-thioguanine. The co-IP experiments give proof of principle that GX015-070 disrupts pro- and anti-apoptotic BCL-2 family protein interactions in cell lines with t(4;11). Additional pre-clinical experiments directed at overcoming drug resistance from abnormal cell death regulation in leukemias with t(4;11) using GX015-070 are in progress. These studies provide a framework to understand the cell death/survival machinery in primary leukemias with t(4;11) translocations more completely and manipulate that machinery to achieve better treatments. GX015-070 Activity and Mechanism Cell Line Single Agent Activity Synergy Inhibition Caspase-3 Activation TUNEL RS4:11 IC50=43.5 nM Ara-C, Dex, ADR Mcl1:Bak; Bcl2:Bak + + SEM-K2 IC50=156 nM In progress Mcl1:Bak; Bcl2:Bak + In Progress MV4-11 IC50=123 nM In progress Mcl1:Bak In progress +

scholarly journals Targeting To Hedgehog Signalling Pathway Increase Sensitivity Anticancer Effects of Arsenic Trioxide Mediated By miR-326

2021 ◽  
Author(s):  
Lei Chen ◽  
Qile Ye ◽  
Yue Su ◽  
Fei Yuan ◽  
Kaikun Yuan ◽  
...  
Keyword(s):  
Western Blot ◽  
Nude Mice ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Flow Cytometer ◽  
Signalling Pathway ◽  
Expression Level ◽  
Hedgehog Signalling ◽  
Hedgehog Signalling Pathway ◽  
Different Levels

Abstract Background: The radical cure of Glioblastoma multiforme (GBM) is a troublesome medical problem, owing to its resistance to temozolomide chemotherapy and very poor surgical results or high relapse rate. Resistance to temozolomide emerges from numerous signalling pathways that are altered in GBM, especially the hedgehog signalling pathway. Hence, further research is urgent needed to identify more effective treatment modalities. Methods: We evaluated the effect of ATO on viability, cell proliferation, colony formation production. Flow cytometer assesses the degree of apoptosis, and Western blot analysis the expression of hedgehog signalling pathway proteins(Gli1, Gli2 and SMO). Moreover, use database(CGGA and TCGA) to inquire the relationship between Arrb1 expression level and miR-326 expression level in different levels of gliomas. Finally, The methylation sequencing level of CpG in Arrb1 gene with the survival period of nude mice gives a good explanation to the results of the Immunohistochemica.Results: Flow cytometer showed that the ATO caused apoptosis increased in a dose-dependent manner. Western blot analysis revealed the low expression of Gli1, Gli2 and SMO as well as the mRNA levels(included FOXM1). Arrb1 expression level was positively related with miR-326 expression level in different levels of gliomas from databases(CGGA and TCGA). Immunohistochemical analysis showed that ATO downregulated the expression of SMO, GLI1and Arrb1. The methylation level of CpG in Arrb1 gene was significantly reduced and the survival period of nude mice was prolonged by ATO. Conclusion: Our results showed that the cytotoxicity of ATO could be regulated by the SMO via Hh signalling pathway as well as miR-326, presenting a promising potential therapy for patients with GBM.

scholarly journals Ovarian Cancer HO-8910 Cell Apoptosis Induced by Crocin in vitro

2015 ◽  
Vol 10 (2) ◽  
pp. 1934578X1501000 ◽  
Author(s):  
Dan Xia
Keyword(s):  
Ovarian Cancer ◽  
Flow Cytometry ◽  
Western Blot ◽  
Blot Analysis ◽  
Cell Apoptosis ◽  
Western Blot Analysis ◽  
Caspase 3 ◽  
Cell Cycle Distribution ◽  
Apoptotic Pathway ◽  
Apoptosis Rate

The effect and mechanism of ovarian cancer HO-8910 cell apoptosis induced by crocin. MTT assay was performed to detect the inhibitory action of crocin on the proliferation of HO-8910 cells. Flow cytometry was used to test the cell cycle distribution and apoptosis rate of ovarian cancer HO-8910 cells. Western blot analysis was utilized to measure the levels of apoptotic proteins such as p53, Fas/APO-1, and Caspase-3. MTT analysis revealed that crocin significantly inhibited the growth of HO-8910 cells. Additionally, flow cytometry illustrated that crocin raised the proportion of HO-8910 cells in the G0/G1 phase and increased their apoptosis rate. Furthermore, Western blot analysis revealed that crocin up-regulated the expression of p53, Fas/APO-1, and Caspase-3. The results of this study showed that crocin can significantly inhibit the growth of HO-8910 cells and arrest them in the G0/G1 phase. Crocin can also promote ovarian cancer HO-8910 cell apoptosis, most likely by increasing p53 and Fas/APO-1 expression, and then activating the apoptotic pathway regulated by Caspase-3.

scholarly journals Primary cilia respond to intermittent low-magnitude, high-frequency vibration and mediate vibration-induced effects in osteoblasts

2020 ◽  
Vol 318 (1) ◽  
pp. C73-C82 ◽  
Author(s):  
Yan-Hui Li ◽  
Dong Zhu ◽  
Zongbing Cao ◽  
Yanwei Liu ◽  
Jian Sun ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
High Frequency ◽  
Western Blot Analysis ◽  
Primary Cilia ◽  
Critical Role ◽  
Rt Pcr ◽  
High Frequency Vibration ◽  
Low Magnitude

Our objective was to investigate the role of primary cilia in low-magnitude, high-frequency vibration (LMHFV) treatment of MC3T3-E1 osteoblasts (OBs). We used chloral hydrate (CH), which has a well-characterized function in chemically removing primary cilia, to elucidate the role of primary cilia in LMHFV-induced OB osteogenic responses through cell viability assay, Western blot analysis, real-time quantitative RT-PCR, and histochemical staining methods. We observed a significant, 30% decrease in the number of MC3T3-E1 OBs with primary cilia (reduced from 64.3 ± 5%) and an approximately 50% reduction in length of primary cilia (reduced from 3 ± 0.8 μm) after LMHFV stimulation. LMHFV stimulation upregulated protein expression of the bone matrix markers collagen 1 (COL-1), osteopontin (OPN), and osteoclacin(OCN) in MC3T3-E1 OBs, indicating that LMHFV induces osteogenesis. High-concentration or long-duration CH exposure resulted in inhibition of MC3T3-E1 OB survival. In addition, Western blot analysis and RT-PCR revealed that CH treatment prevented LMHFV-induced osteogenesis. Furthermore, decreased alkaline phosphate activity, reduced OB differentiation, mineralization, and maturation were observed in CH-pretreated and LMHFV-treated OBs. We showed that LMHFV induces morphological changes in primary cilia that may fine-tune their mechanosensitivity. In addition, we demonstrated the significant inhibition by CH of LMHFV-induced OB mineralization, maturation, and differentiation, which might reveal the critical role of primary cilia in the process.

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