Identification of a Novel Epigenetic Regulator That Acts As a Tumor Suppressor in the Development of MLL AML Leukemic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3923-3923
Author(s):  
Hangyu Yi ◽  
Halina Leung ◽  
Aliaksei Holik ◽  
Florida Voli ◽  
Marie-Liesse Asselin-Labat ◽  
...  
Keyword(s):  
Stem Cells ◽  
Tumor Suppressor ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Histone Demethylase ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Promising Therapeutic Approach

Abstract Acute myeloid leukemia (AML) is still a deadly form of leukemia due to frequent relapse caused by the persistence of drug-resistant leukemic stem cells (LSCs). We have previously demonstrated a crucial role for β-catenin signaling in regulating LSCs and identified GPR84 as an important β-catenin regulator in the maintenance of mixed-lineage leukemia (MLL) LSCs (Wang et al., Science 2010; Dietrich et al., Blood 2014). Hence, targeting LSCs by pharmacological inhibition of GPR84/β-catenin signaling represents a promising therapeutic approach. In collaboration with a pharmaceutical company that has developed a novel GPR84 antagonist (GP), we investigated the effect of GP in MLL pre-leukemic stem cell (pre-LSC) function. GP (20 μM) significantly inhibited the colony forming ability of MLL pre-LSCs (P < 0.0001) but had little effect on normal hematopoietic stem cells. Quantitative RT-PCR and western blot analysis confirmed GP-induced downregulation of GPR84 target genes, including Hoxa5, Hoxa7 and Meis1a, indicating GP-induced inhibition of GPR84 signaling. To further examine the mechanism of GPR84 inhibition on MLL pre-LSCs, we evaluated several epigenetic regulators (i.e. JMJD1c and EZH2) known to promote leukemogenesis (Zhu et al., ‎J Clin Invest 2016; Tanaka et al., Blood 2012). Western blot analysis showed that inhibition of GPR84 signaling did not alter the expression of JMJD1c or EZH2. However, we observed a significant increase in the expression of a novel and not-yet-characterized histone demethylase (HD) in AML. To investigate the role of HD in AML leukemogenesis, we overexpressed HD in MLL pre-LSCs and subsequent serial replating assay showed a marked reduction in colony forming ability (P < 0.005), indicating impaired self-renewal in vitro. Consistent with our in vitro observations, in vivo transplantation in syngeneic mice revealed a significant delay in leukemia onset and increase in mouse survival (P < 0.001). We next performed western blot analysis to examine the demethylase activity of HD, and our data revealed that HD overexpression caused a substantial reduction in global histone 3 lysine 36 dimethylation (H3K36me2), an epigenetic mark normally associated with transcriptional activation and elongation. In order to identify genes regulated by HD through demethylation of H3K36me2, we performed H3K36me2 ChIP-seq on HD overexpressing MLL pre-LSCs. Our analysis identified several genes including anti-apoptotic protein Mcl-1 and angiogenic receptor Nrp1, which are known to be involved in AML leukemogenesis, with decreased H3K36me2 mark on both the transcriptional start site and gene body. Subsequent western blot analysis confirmed the decreased expression of both Mcl-1 and Nrp1 in HD overexpressing pre-LSCs. Given the prominent roles of anti-apoptosis and angiogenesis in the development of hematologic malignancies such as leukemia, we are currently evaluating these mechanisms caused by HD overexpression in an important subtype of AML. Taken together, our study identifies a novel histone demethylase that acts downstream of GPR84 signaling to function as a potent tumor suppressor in the development of MLL LSCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Implication of Hypoxia-Inducible Factor-1 (HIF-1) As a New Therapeutic Target in JAK2V617F Positive Myeloproliferative Neoplasms (MPN)

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4318-4318 ◽  
Author(s):  
Julian Baumeister ◽  
Nicolas Chatain ◽  
Annika Hubrich ◽  
Caroline Küstermann ◽  
Stephanie Sontag ◽  
...  
Keyword(s):  
Western Blot ◽  
Cell Line ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Research Funding ◽  
Myeloproliferative Neoplasms ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Protein Levels

Abstract Myeloproliferative neoplasms (MPN) are a heterogeneous group of malignancies including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The JAK2V617F mutation can be found in 90% of PV and approximately 50% of ET and PMF patients. Hypoxia-inducible factors (HIFs) are master transcriptional regulators of the response to decreases in cellular oxygen levels. Unveiling the function of deregulated HIF-1 signaling in normal and malignant hematopoiesis was the aim of several recent publications, highlighting the importance of HIF-1 for the maintenance of leukemic stem cells (LSCs) in acute and chronic myeloid leukemia (AML/CML). In a JAK2V617F knock-in mouse model and in patients, JAK2V617F was shown to induce the accumulation of reactive oxygen species (ROS) in the hematopoietic stem cell compartment, leading to a stabilization of HIF-1α protein. Further, aberrant STAT5 and PI3K/AKT/mTOR signaling induced HIF-1α expression on the transcriptional and translational level. Ruxolitinib treatment inhibited growth and reduced the expression of HIF-1α and its target gene VEGF in the JAK2V617F human erythroleukemia cell line HEL. In several leukemic cell lines constitutive expression of HIF-1α was reported, even under normoxic conditions. However, it still remains unknown whether HIF-1α plays a role in JAK2V617F positive MPN. In this study, we investigated the role HIF-1α signaling in JAK2V617F positive MPN in vitro. We retrovirally transduced the murine bone marrow cell line 32D with JAK2V617F or JAK2WT. Western blot analysis revealed significant increases in HIF-1α protein levels in JAK2V617F positive cells compared to JAK2WT controls after cultivation in normoxic conditions and this effect was abrogated by treatment with the JAK1/JAK2 inhibitor ruxolitinib. Inhibition of HIF-1, binding to hypoxia response elements (HRE), by low doses of echinomycin (1 nM), significantly impaired proliferation and survival. Using an Annexin-V/7-AAD flow cytometry assay apoptosis was found to be selectively induced in JAK2V617F positive, but not JAK2WT cells after echinomycin treatment. Additionally, BrdU/7-AAD cell cycle analysis revealed that only JAK2V617F positive cells were significantly arrested in G0/1 phase. These findings were consistent with shRNA-mediated knockdown (KD) of HIF-1α in JAK2V617F transduced 32D cells in presence but not the absence of HIF-2 antagonist 2. Inhibition of HIF-2 was necessary due to a compensatory increase of HIF-2α protein levels, shown by Western Blot analysis, counteracting HIF-1α-KD mediated effects. We isolated PBMCs and BMMNCs from JAK2V617F positive patients or healthy controls using Ficoll density gradient centrifugation. Echinomycin significantly abrogated the colony formation ability alone and in combination with ruxolitinib. In vitro treatment with echinomycin significantly decreased cell number and viability of 8 JAK2V617F positive BMMNC samples (4 PV, 3 PMF, 1 preMF; p[1nM]=0.0169, p[5nM]=0.0009) and 7 PBMC samples (6 PV, 1 PMF; p[1nM]=0.0156, p[5nM]=0.0156) in a dose-dependent manner. In contrast, PBMCs from 6 healthy donors were unaffected by the treatment. The same effect was observed in heterozygous and homozygous iPS cell-derived progenitors from JAK2V617F positive PV patients, whereas JAK2WT cells were unaffected by the treatment. Collectively, our data indicate that targeting HIF-1 might represent a novel therapeutic approach in classical Philadelphia-chromosome-negative MPN. Disclosures Brümmendorf: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Janssen: Consultancy; Merck: Consultancy; Takeda: Consultancy.

Megakaryocytes and Functional Platelets Obtained from Human Subcutaneous Adipocytes in an In Vitro Differentiation System.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3702-3702
Author(s):  
Emi Saito ◽  
Yumiko Matsubara ◽  
Hidenori Suzuki ◽  
Yasuo Ikeda ◽  
Mitsuru Murata
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Culture System ◽  
Expression Vector ◽  
Variable Number Tandem Repeat ◽  
Flow Cytometric Analysis ◽  
Hematopoietic Stem ◽  
Cell Sample

Abstract Because of the difficulty in obtaining the sufficient amounts of hematopoietic stem cells from bone marrow, peripheral blood, and cord blood cells, studies of an in vitro experimental system that allows the production of abundant number of megakaryocytes (MKs) and platelets are currently the focus of research. Here, we present a novel system where human subcutaneous adipocytes were successfully differentiated into MK lineages in an in vitro liquid culture system. We also show that subcutaneous preadipocytes could be successfully transfected with vectors, to obtain modified MKs. Primary human subcutaneous preadipocytes (Cambrex Bio Science Walkersvile, Inc. Walkersville, MD, USA) were cultured in conditioned media to differentiate into mature adipocytes. Cells were cultured in serum-free media containing thrombopoietin for differentiation into MK lineages. The MKs or platelets were counted by flow cytometric analysis on day 14 using the relative value of CD41(+)/propidium iodide(+) cells or platelet size CD41(+) cells, respectively, versus 107 subcutaneous preadipocytes on day 0. The MK and platelet cell count was approximately 9600/ 107 and 2200/ 107, respectively. Morphological analysis with electron microscopy demonstrated that MKs, which had typical organelles such as granules, demarcation membrane, and nuclei, and platelets, which had typical contents such as granules, mitochondria, and open canalicular system, were successfully obtained from subcutaneous preadipocytes. We then attempted to transfect subcutaneous preadipocytes with vectors to obtain transformed MKs. Two glycoprotein (GP) Ib alpha polymorphisms, 145Thr/Met and 1–4 repeats of variable number tandem repeat of 13 amino-acid sequences, were used as the marker of gene transfer, which were detected by PCR-restricited fragment length polymorphism (RFLP) and Western blot analysis, respectively. Subcutaneous preadipocytes with the 145Thr/Thr and 1 repeat sequences were transfected with the expression vector carrying GPIb alpha with the 145Met and 4 repeats sequences. PCR-RFLP analysis with gel electrophoresis was performed on each RNA sample from the expression vector-transfected and non-transfected cells. Non-transfected cell sample had bands corresponding to the 145Thr sequence position, while the expression vector-transfected cell sample had bands corresponding to both the 145Thr and Met sequences. Western blot analysis with an anti-GPIb alpha monoclonal antibody, LJ-Ib alpha1 (a generous gift from Dr. ZM Ruggeri, The Scripps Research Institute, La Jolla, CA, USA), showed bands of the expected size corresponding to 1R and 1R/4R. In summary, we established an in vitro culture system to produce MKs and platelets from subcutaneous adipocytes. We were also able to obtain transfected MKs from subcutaneous preadipocytes.

scholarly journals JMJD1c, a Histone Demethylase, Is Required for the Maintenance of MLL-AF9 AML Leukemic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3530-3530 ◽  
Author(s):  
Halina Leung ◽  
Jianlong Wang ◽  
Jenny Yingzi Wang
Keyword(s):  
Stem Cells ◽  
Hox Genes ◽  
Conflicts Of Interest ◽  
Therapeutic Potential ◽  
Mrna Level ◽  
Histone Demethylase ◽  
Leukemic Cells ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem

Abstract While most leukemic cells are initially sensitive to chemo- and radiotherapy, leukemic stem cells (LSC) persist and are therefore considered to be the basis for disease relapse in acute myeloid leukemia (AML) (Nat Biotechnol, 25: 1315-1321, 2007). The discovery of epigenetic mutations being the cause of a number of cancers has increased interest in their therapeutic potential (Nat Rev Genet, 7:21-33, 2006; Cell, 128: 683-692, 2007). Although the clinical importance of epigenetic dysregulation has been recognised in various cancers, including MLL-rearranged AML, this topic is currently a novel area of study. Our microarray analysis comparing genes differentially expressed between LSC and normal hematopoietic stem cells (HSC) identified JMJD1c, a histone demethylase, as a potential LSC-specific target. An analysis of a comprehensive patient outcome database showed that high levels of JMJD1c were associated with significantly poorer survival in patients with AML (P = 0.0029), making it clinically relevant and emphasising the therapeutic potential of JMJD1c. Furthermore, the JMJD1c mRNA level was significantly higher in MLL-rearranged than in non-MLL AML patient xenograft samples. Western blot and quantitative PCR confirmed the microarray results, where JMJD1c was significantly upregulated in MLL-AF9 LSC compared to Hoxa9/Meis1a derived LSC and HSC. Overexpression of JMJD1c in Hoxa9/Meis1a pre-LSC significantly increased the clonogenic activity and proliferation compared to control cells. Subsequent microarray experiment identified Hox genes as downstream targets of this epigenetic regulator (fold change > 2, P < 0.05), suggesting the regulatory role of JMJD1c in the self-renewal pathway. Consistent with in vitro observations, there was a significant increase in disease progression as shown by leukemia onset in mice with Hoxa9/Meisa1 pre-LSC transduced with JMJD1c cDNA compared to control (P < 0.0001; at least 12 mice per cohort). Stable knockdown of JMJD1c in MLL-AF9 cells using shRNA significantly impaired the proliferation, clonogenic activity and induced cell differentiation in vitro. In vivo survival studies in mice showed that JMJD1c knockdown severely impaired the maintenance of MLL-AF9 induced leukemia (P < 0.0001; 12 mice per cohort). In conclusion, our studies have identified JMJD1c as an important driver in leukemia maintenance and strongly suggest that JMJD1c possesses an oncogenic role in MLL-AF9 leukemogenesis. Targeting this oncogene may enable us to directly target LSC as a strategy for overcoming drug resistance in AML patients. Disclosures No relevant conflicts of interest to declare.

Radiosensitivity of glioma to Gamma Knife treatment enhanced in vitro and in vivo by RNA interfering Ku70 that is mediated by a recombinant adenovirus

2010 ◽  
Vol 113 (Special_Supplement) ◽  
pp. 228-235 ◽  
Author(s):  
Qiang Jia ◽  
Yanhe Li ◽  
Desheng Xu ◽  
Zhenjiang Li ◽  
Zhiyuan Zhang ◽  
...  
Keyword(s):  
Western Blot ◽  
Gamma Knife ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cells ◽  
C6 Glioma ◽  
C6 Glioma Cells ◽  
C6 Cells

Object The authors sought to evaluate modification of the radiation response of C6 glioma cells in vitro and in vivo by inhibiting the expression of Ku70. To do so they investigated the effect of gene transfer involving a recombinant replication-defective adenovirus containing Ku70 short hairpin RNA (Ad-Ku70shRNA) combined with Gamma Knife treatment (GKT). Methods First, Ad-Ku70shRNA was transfected into C6 glioma cells and the expression of Ku70 was measured using Western blot analysis. In vitro, phenotypical changes in C6 cells, including proliferation, cell cycle modification, invasion ability, and apoptosis were evaluated using the MTT (3′(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay, Western blot analysis, and cell flow cytometry. In vivo, parental C6 cells transfected with Ad-Ku70shRNA were implanted stereotactically into the right caudate nucleus in Sprague-Dawley rats. After GKS, apoptosis was analyzed using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) method. The inhibitory effects on growth and invasion that were induced by expression of proliferating cell nuclear antigen and matrix metalloproteinase–9 were determined using immunohistochemical analyses. Results The expression of Ku70 was clearly inhibited in C6 cells after transfection with Ad-Ku70shRNA. In vitro following transfection, the C6 cells showed improved responses to GKT, including suppression of proliferation and invasion as well as an increased apoptosis index. In vivo following transfection of Ad-Ku70shRNA, the therapeutic efficacy of GKT in rats with C6 gliomas was greatly enhanced and survival times in these animals were prolonged. Conclusions Our data support the potential for downregulation of Ku70 expression in enhancing the radiosensitivity of gliomas. The findings of our study indicate that targeted gene therapy–mediated inactivation of Ku70 may represent a promising strategy in improving the radioresponsiveness of gliomas to GKT.

Regulation of Rhcg, an ammonia transporter, by aldosterone in the kidney

2021 ◽  
Author(s):  
Koji Eguchi ◽  
Yuichiro Izumi ◽  
Yukiko Yasuoka ◽  
Terumasa Nakagawa ◽  
Makoto Ono ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Membrane Fraction ◽  
Potassium Level ◽  
Collecting Duct ◽  
Subcutaneous Administration ◽  
Extracellular Potassium ◽  
Membrane Expression

Rh C glycoprotein (Rhcg), an ammonia transporter, is a key molecule in urinary acid excretion and is expressed mainly in the intercalated cells (ICs) of the renal collecting duct. In the present study we investigated the role of aldosterone in the regulation of Rhcg expression. In in vivo experiments using C57BL/6J mice, Western blot analysis showed that continuous subcutaneous administration of aldosterone increased the expression of Rhcg in membrane fraction of the kidney. Supplementation of potassium inhibited the effect of aldosterone on the Rhcg. Next, mice were subjected to adrenalectomy with or without administration of aldosterone, and then ad libitum 0.14M NH4Cl containing water was given. NH4Cl load increased the expression of Rhcg in membrane fraction. Adrenalectomy decreased NH4Cl-induced Rhcg expression, which was restored by administration of aldosterone. Immunohistochemical studies revealed that NH4Cl load induced the localization of Rhcg at the apical membrane of ICs in the outer medullary collecting duct. Adrenalectomy decreased NH4Cl-induced membrane localization of Rhcg, which was restored by administration of aldosterone. For in vitro experiments, IN-IC cells, an immortalized cell line stably expressing Flag-tagged Rhcg (Rhcg-Flag), were used. Western blot analysis showed that aldosterone increased the expression of Rhcg-Flag in membrane fraction, while the increase in extracellular potassium level inhibited the effect of aldosterone. Both spironolactone and Gӧ6983, a PKC inhibitor, inhibited the expression of Rhcg-Flag in the membrane fraction. These results suggest that aldosterone regulates the membrane expression of Rhcg through the mineralocorticoid receptor and PKC pathways, which is modulated by extracellular potassium level.

Interfering With Hyaluronic Acid Metabolism Suppresses Glioma Cell Proliferation by Regulating Autophagy

2020 ◽  
Author(s):  
Tao Yan ◽  
Xin Chen ◽  
Hua Zhan ◽  
Penglei Yao ◽  
Ning Wang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Hyaluronic Acid ◽  
Tumor Microenvironment ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Glioma Cell ◽  
Glioma Cells

Abstract BackgroundThe tumor microenvironment plays an important role in tumor progression. Hyaluronic acid (HA), an important component of the extracellular matrix in the tumor microenvironment, abnormally accumulates in a variety of tumors. Whereas the role of abnormal HA metabolism in glioma remains unclear. MethodsThe expression level of hyaluronic acid (HA) was analyzed by ELISA assay and proteins such as HAS3, CD44, P62, LC3, CCND1 and CCNB1 were measured with Western blot analysis. The cell viability and proliferation were measured by MTT and KI67 immunofluorescence staining respectively. Autophagic vesicles and autophagosomes were quantified by transmission electron microscopy (TEM) and GFP-RFP-LC3 fluorescence analysis respectively. Cell cycle was analyzed by flowcytometry and Western blot analysis. Immunohistochemical (IHC) staining was used to detect expression levels of HA, Ki67, HAS3 and CD44 in human and mouse tumor tissues. Lentivirus constructed HAS3 and CD44 knockout stable glioma cells were transplanted to BALB/C nude mice for in vivo experiments. 4-Methylumbelliferone (4MU) was also used to treat glioma bearing mice for verifing its anti-tumor ability. The expression curve of HAS3, CD44 and the disease-free survival (DFS) curves for HAS3, CD44 in patients with LGG and GBM was performed based on TCGA database. ResultsAs shown in the present study, HA, hyaluronic acid synthase 3 (HAS3) and a receptor of HA named CD44 are expressed at high levels in human glioma tissues and negatively correlated with the prognosis of patients with glioma. Silencing HAS3 or blocking CD44 inhibited the proliferation of glioma cells in vitro and in vivo. The underlying mechanism was attributed to the inhibition of autophagy flux and further maintaining glioma cell cycle arrest in G1 phase. More importantly, 4-Methylumbelliferone (4-MU), a small competitive inhibitor of UDP with the ability to penetrate the blood-brain barrier (BBB), also inhibited the proliferation of glioma cells in vitro and in vivo. ConclusionApproaches that interfere with HA metabolism by altering the expression of HAS3 and CD44 and the administration of 4-MU potentially represent effective strategies for glioma treatment.

scholarly journals A39 NOVEL GAIN OF FUNCTION NON-RECEPTOR TYROSINE KINASE MUTATIONS ARE LINKED TO THE PATHOGENESIS OF VEOIBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 46-48
Author(s):  
M Mehta ◽  
L Wang ◽  
C Guo ◽  
N Warner ◽  
Q Li ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Western Blot ◽  
Receptor Tyrosine Kinase ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Intestinal Inflammation ◽  
De Novo ◽  
Single Gene ◽  
Gain Of Function

Abstract Background Very early-onset inflammatory bowel disease (VEOIBD) is an emerging global disease, that results in inflammation of the digestive tract. Severe forms of VEOIBD can be caused by mutations in a single gene (monogenic variants) and, can result in death. A candidate gene which codes for a non-receptor tyrosine kinase (nRTK) has recently been implicated as a monogenic cause of IBD (unpublished). Whole exome sequencing was performed in two unrelated children who presented with symptoms of IBD identifying two distinct de novo gain of function mutations (S550Y and P342T). Both mutations are located in the highly conserved region of the nRTK, and were predicted to have similar downstream effects. Furthermore, four other patients with a variety of adult-onset immune disorders have recently been identified with rare variants in the same gene (M450I, R42P, A353T, V433M, S550F) but, their potential gain of function status remains to be determined. Studies show that this nRTK is an essential mediator in inflammation. It is expressed in both intestinal epithelial and immune cells however, its role in infantile IBD is unclear. This protein is first activated by phosphorylation and is linked to activating downstream transcription factors such as ERK and JNK. All these target proteins play a meaningful role in intestinal inflammation in patients with IBD. Aims Since we identified P342T and S550Y to be gain of function, we wanted to determine if the new variants exhibit a similar downstream impact on target protein expression levels when compared with S550Y and P342T. We also wanted to identify if all variants can be rescued with a known nRTK inhibitor. It is hypothesized that the new variants are gain of function and that all variants can be rescued with the inhibitor. Methods Using western blot analysis, the activation of ERK, JNK and nRTK was compared between wildtype (WT) and mutants. This in vitro method helped identify the degree of activation. For the second part of the study, HEK293T cells were treated with inhibitor to test for a rescue of phenotypes via western blot analysis. Results Results show an increased activation of nRTK, ERK and JNK in all variants with S550Y and S550F having the highest activation. Furthermore, pharmacological inhibition using small molecular kinase inhibitors resulted in decreased activation of nRTK, ERK and JNK suggesting a rescue of phenotypes. Conclusions Characterizing the downstream functional impact of these nRTK variants is an important first step to determine if gain of function nRTK mutations drive IBD. With a rising prevalence of IBD worldwide, these findings may lead to the development of pharmacological nRTK inhibitors as a novel personalized therapeutic approach for these patients and possibly for the broader IBD population. Funding Agencies CIHR

scholarly journals In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice

2020 ◽  
Vol 17 (23) ◽  
pp. 8811
Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon
Keyword(s):  
Lactate Dehydrogenase ◽  
Protein Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Biochemical Analysis ◽  
Positive Control ◽  
Exercise Endurance

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

The N-Terminally Truncated p53 Isoform Δ40p53 Influences Prognosis in Mucinous Ovarian Cancer

2012 ◽  
Vol 22 (3) ◽  
pp. 372-379 ◽  
Author(s):  
Gerda Hofstetter ◽  
Astrid Berger ◽  
Regina Berger ◽  
Arijana Zorić ◽  
Elena I. Braicu ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Confidence Interval ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Quantitative Polymerase Chain Reaction ◽  
Hazard Ratio ◽  
Histological Subtypes ◽  
Mutational Status

ObjectiveThe tumor suppressor p53 generates the N-terminally truncated isoforms Δ40p53 and Δ133p53 that possess the ability to modulate p53 function in vitro. The aim of the present study was to evaluate the clinical relevance of p53 isoforms in the main histological subtypes of ovarian cancer.MethodsΔ40p53, Δ133p53, and full-length p53 (FLp53) expression was determined in 45 mucinous, 30 endometrioid, and 91 serous ovarian cancer specimens as well as 42 normal ovarian tissues using reverse transcriptase–quantitative polymerase chain reaction. In a subgroup of mucinous ovarian cancer cases, Δ40p53 expression was examined using Western blot analysis. A functional yeast-based assay and subsequent sequencing were performed to analyze the p53 mutational status.ResultsIn endometrioid cancer specimens, Δ133p53 expression was significantly lower than in mucinous and serous cases (P = 0.016) or in normal tissues (P = 0.004). Mucinous cancer samples showed elevated Δ40p53 expression as compared with normal ovarian tissues (P = 0.003). In addition, high Δ40p53 expression constituted an independent prognostic marker for recurrence-free but not for overall survival in patients with mucinous ovarian cancer (hazard ratio, 0.267; 95% confidence interval, 0.094–0.756 [P = 0.013]; hazard ratio, 0.453, 95% confidence interval, 0.193–1.064 [P = 0.069]). Western blot analysis confirmed the presence of p53β and Δ40p53α in a subset of patients with mucinous ovarian cancer. Expression of p53 isoforms was not associated with p53 mutational status or clinicopathologic parameters.ConclusionsWe show that expression of p53 isoforms differs in histological subtypes, thus supporting the hypothesis that histological subtypes represent distinct disease entities. In addition, we provide first evidence for a favorable role of Δ40p53 in patients with mucinous ovarian cancer.

scholarly journals Expression and subcellular localization of BNIP3 in hypoxic hepatocytes and liver stress

2009 ◽  
Vol 296 (3) ◽  
pp. G499-G509 ◽  
Author(s):  
Mallikarjuna R. Metukuri ◽  
Donna Beer-Stolz ◽  
Rajaie A. Namas ◽  
Rajeev Dhupar ◽  
Andres Torres ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Ischemia Reperfusion ◽  
Redox Stress ◽  
No Donor ◽  
Interacting Protein

We have previously demonstrated that the Bcl-2/adenovirus EIB 19-kDa interacting protein 3 (BNIP3), a cell death-related member of the Bcl-2 family, is upregulated in vitro and in vivo in both experimental and clinical settings of redox stress and that nitric oxide (NO) downregulates its expression. In this study we sought to examine the expression and localization of BNIP3 in murine hepatocytes and in a murine model of hemorrhagic shock (HS) and ischemia-reperfusion (I/R). Freshly isolated mouse hepatocytes were exposed to 1% hypoxia for 6 h followed by reoxygenation for 18 h, and protein was isolated for Western blot analysis. Hepatocytes grown on coverslips were fixed for localization studies. Similarly, livers from surgically cannulated C57Bl/6 mice and from mice cannulated and subjected to 1–4 h of HS were processed for protein isolation and Western blot analysis. In hepatocytes, BNIP3 was expressed constitutively but was upregulated under hypoxic conditions, and this upregulation was countered by treatment with a NO donor. Surprisingly, BNIP3 was localized in the nucleus of normoxic hepatocytes, in the cytoplasm following hypoxia, and again in the nucleus following reoxygenation. Upregulation of BNIP3 partially required p38 MAPK activation. BNIP3 contributed to hypoxic injury in hepatocytes, since this injury was diminished by knockdown of BNIP3 mRNA. Hepatic BNIP3 was also upregulated in two different models of liver stress in vivo, suggesting that a multitude of inflammatory stresses can lead to the modulation of BNIP3. In turn, the upregulation of BNIP3 appears to be one mechanism of hepatocyte cell death and liver damage in these settings.

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