Inhibition of BUB3 shunts glucose to glycolytic pathway by inducing PFKFB3 accumulation

Abstract Purpose: Metabolic reprogramming as a hallmark of cancer has countless connections with other biological behavior of tumor such as rapid mitosis. Mitotic checkpoint protein BUB3 as a key protein involved in the regulation of mitosis is modulated by PKM2, an important glycolytic enzyme. However the role of BUB3 in glucose metabolism remains unknown. Methods: We analyzed the TCGA data to evaluate BUB3 expression in certain tumors. The uptake of glucose and CO2 incorporation was tested by isotopic tracer methods. The lactate, NADPH, NADP and metabolic enzyme activities were tested by assay kits accordingly. Results: We show here that BUB3 is over expressed in cervical cancer and hepatocellular carcinoma. Interference of BUB3 increase the uptake of glucose and shunts the metabolic flux from pentose phosphate pathway to glycolytic pathway. The glycolysis metabolites lactate is increased by BUB3 interference whereas NADPH/NADP ratio is reduced. With regard to metabolic enzymes, interference of BUB3 increase PFKFB3 on protein level and enzyme activity, but not mRNA level. Moreover, the increasing of protein level is diminished when proteasome degradation pathway is blocked by MG132. Conclusions: BUB3 is a potential tumor promoter and plays certain roles in cancer cellular metabolic reprogramming.

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Functional Comparison between VP64-dCas9-VP64 and dCas9-VP192 CRISPR Activators in Human Embryonic Kidney Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22010397 ◽

2021 ◽

Vol 22 (1) ◽

pp. 397

Author(s):

Nasir Javaid ◽

Thuong L. H. Pham ◽

Sangdun Choi

Keyword(s):

Protein Level ◽

Reference Genes ◽

Mrna Level ◽

High Specificity ◽

Kidney Cells ◽

Human Embryonic Kidney ◽

Human Embryonic Kidney Cells ◽

Sox2 Expression

Reversal in the transcriptional status of desired genes has been exploited for multiple research, therapeutic, and biotechnological purposes. CRISPR/dCas9-based activators can activate transcriptionally silenced genes after being guided by gene-specific gRNA(s). Here, we performed a functional comparison between two such activators, VP64-dCas9-VP64 and dCas9-VP192, in human embryonic kidney cells by the concomitant targeting of POU5F1 and SOX2. We found 22- and 6-fold upregulations in the mRNA level of POU5F1 by dCas9-VP192 and VP64-dCas9-VP64, respectively. Likewise, SOX2 was up-regulated 4- and 2-fold using dCas9-VP192 and VP64dCas9VP64, respectively. For the POU5F1 protein level, we observed 3.7- and 2.2-fold increases with dCas9-VP192 and VP64-dCas9-VP64, respectively. Similarly, the SOX2 expression was 2.4- and 2-fold higher with dCas9-VP192 and VP64-dCas9-VP64, respectively. We also confirmed that activation only happened upon co-transfecting an activator plasmid with multiplex gRNA plasmid with a high specificity to the reference genes. Our data revealed that dCas9-VP192 is more efficient than VP64-dCas9-VP64 for activating reference genes.

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Dual Specificity Kinase DYRK3 Promotes Aggressiveness of Glioblastoma by Altering Mitochondrial Morphology and Function

International Journal of Molecular Sciences ◽

10.3390/ijms22062982 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2982

Author(s):

Kyeongmin Kim ◽

Sungmin Lee ◽

Hyunkoo Kang ◽

Eunguk Shin ◽

Hae Yu Kim ◽

...

Keyword(s):

Tumor Cells ◽

Metabolic Flux ◽

Mitochondrial Fission ◽

Mammalian Target Of Rapamycin ◽

Primary Brain Tumor ◽

Metabolic Reprogramming ◽

Migration And Invasion ◽

Mitochondrial Morphology ◽

Dual Specificity ◽

Novel Strategy

Glioblastoma multiforme (GBM) is a malignant primary brain tumor with poor patient prognosis. Although the standard treatment of GBM is surgery followed by chemotherapy and radiotherapy, often a small portion of surviving tumor cells acquire therapeutic resistance and become more aggressive. Recently, altered kinase expression and activity have been shown to determine metabolic flux in tumor cells and metabolic reprogramming has emerged as a tumor progression regulatory mechanism. Here we investigated novel kinase-mediated metabolic alterations that lead to acquired GBM radioresistance and malignancy. We utilized transcriptomic analyses within a radioresistant GBM orthotopic xenograft mouse model that overexpresses the dual specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3). We find that within GBM cells, radiation exposure induces DYRK3 expression and DYRK3 regulates mammalian target of rapamycin complex 1 (mTORC1) activity through phosphorylation of proline-rich AKT1 substrate 1 (PRAS40). We also find that DYRK3 knockdown inhibits dynamin-related protein 1 (DRP1)-mediated mitochondrial fission, leading to increased oxidative phosphorylation (OXPHOS) and reduced glycolysis. Importantly, enforced DYRK3 downregulation following irradiation significantly impaired GBM cell migration and invasion. Collectively, we suggest DYRK3 suppression may be a novel strategy for preventing GBM malignancy through regulating mitochondrial metabolism.

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A functional test for maternally inherited cadherin in Xenopus shows its importance in cell adhesion at the blastula stage

Development ◽

10.1242/dev.120.1.49 ◽

1994 ◽

Vol 120 (1) ◽

pp. 49-57 ◽

Cited By ~ 7

Author(s):

J. Heasman ◽

D. Ginsberg ◽

B. Geiger ◽

K. Goldstone ◽

T. Pratt ◽

...

Keyword(s):

Cell Adhesion ◽

Protein Level ◽

Mrna Level ◽

Functional Test ◽

Gastrula Stage ◽

Blastula Stage ◽

Xenopus Development ◽

Dose Dependent ◽

Embryonic Ectoderm ◽

E Cadherin

We report here on the consequences of reducing the expression of EP-cadherin at the earliest stages of Xenopus development. Injection of oligodeoxynucleotides antisense to maternal EP-cadherin mRNA into full-grown oocytes reduced the mRNA level in oocytes, and the protein level in blastulae. Adhesion between blastomeres was significantly reduced, as seen in whole embryos, and in assays of the ability of blastomeres to reaggregate in culture. This effect was especially conspicuous in the inner cells of the blastula and included the disruption of the blastocoel. The severity of the EP-cadherin mRNA depletion and of the disaggregation phenotype was dose dependent. This phenotype was rescued by the injection into EP-cadherin mRNA-depleted oocytes of the mRNA coding for a related cadherin, E-cadherin, that is normally expressed at the gastrula stage in the embryonic ectoderm.

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Status of the down-regulated canine testis using two different GNRH agonist implants in comparison with the juvenile testis

Reproduction ◽

10.1530/rep-13-0195 ◽

2013 ◽

Vol 146 (6) ◽

pp. 517-526 ◽

Cited By ~ 10

Author(s):

S Goericke-Pesch ◽

M Gentil ◽

A Spang ◽

M P Kowalewski ◽

K Failing ◽

...

Keyword(s):

Cytochrome P450 ◽

Gnrh Agonist ◽

Sertoli Cells ◽

Protein Level ◽

Limit Of Detection ◽

Mrna Level ◽

Cell Nuclei ◽

Chain Cleavage ◽

Cleavage Enzyme

Testicular function in the dog was down-regulated using two different GNRH agonist implants, with adult and juvenile testes serving as controls. Treatment resulted in an increased percentage of the interstitial area and decreased area of Leydig cell nuclei. Expression of StAR and the steroidogenic enzymes cytochrome P450 side-chain cleavage enzyme (P450scc, CYP11A1) and cytochrome P450 17α-hydroxylase-17,20-lyase (P450c17, CYP17A1) in Leydig cells was blocked at the mRNA and protein level, showing no differences between the two agonists. Staining for androgen receptor (AR) by immunohistochemistry was positive in Sertoli, Leydig and peritubular cells and some spermatogonia, with in situ hybridization confirming expression in Sertoli cells. At the mRNA level, expression of AR was not affected; however, translation was blocked (reduced percentage of AR-positive Sertoli cells), with the number of nuclei in basal position being decreased. In the juvenile testes, mRNA expression of StAR, CYP11A1 and CYP17A1 was higher compared with the other groups but distinctly lower for the AR. At the protein level, the expression was at the limit of detection for StAR; AR-positive Sertoli cells were not detected. Our observations show that the down-regulated testis is different from the juvenile one rather resembling the testicular status in seasonal breeders out of season.

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Expression and Cellular Localization of CXCR4 and CXCL12 in Human Carotid Atherosclerotic Plaques

Thrombosis and Haemostasis ◽

10.1160/th17-04-0271 ◽

2018 ◽

Vol 118 (01) ◽

pp. 195-206 ◽

Cited By ~ 22

Author(s):

Sophie Merckelbach ◽

Emiel van der Vorst ◽

Michael Kallmayer ◽

Christoph Rischpler ◽

Rainer Burgkart ◽

...

Keyword(s):

Protein Level ◽

Cellular Localization ◽

Mrna Level ◽

Plaque Morphology ◽

Atherosclerotic Plaques ◽

Cxcl12 Expression ◽

Carotid Plaques ◽

Unstable Plaques ◽

Human Carotid

Background and Aims The CXCR4/CXCL12 complex has already been associated with progression of atherosclerosis; however, its exact role is yet unknown. The aim of this study was to analyse the expression and cellular localization of CXCL12 and its receptor CXCR4 in human carotid atherosclerotic plaques. Methods Carotid plaques (n = 58; 31 stable, 27 unstable, based on histological characterization of plaque morphology) were obtained during carotid endarterectomy, and 10 healthy vessels were used as a control. Expression of cxcr4, cxcr7, cxcl12, ccl2/ccr2 and csf1/csf1r was analysed at mRNA, and level expression of CXCR4, CXCR7 and CXCL12 was analysed at protein level. Cellular localization was determined using consecutive and double immunohistochemical (IHC) staining and microdissection. Results At mRNA level, cxcr4, cxcr7 and cxcl12 were significantly higher expressed in stable carotid plaques compared with controls (p = 0.011, p < 0.001 and p < 0.001). Cxcl12 mRNA expression was successively augmented toward unstable plaques (p < 0.001). At protein level, CXCR4, CXCR7 and CXCL12 expression was significantly increased in both stable (p = 0.001, p < 0.001 and p = 0.035, respectively) and unstable (p = 0.003, p < 0.001 and p = 0.045, respectively) plaques compared with controls. Using IHC, CXCR4 was particularly localized in macrophages and small neovessels. Microdissection confirmed strongest expression of cxcr4 in macrophages within atherosclerotic plaques. Leukocytes and smooth muscle cells showed cxcr4 expression as well. For cxcl12, only microdissected areas with macrophages were positive. Conclusion Expression of CXCR4 and CXCL12 was significantly increased in both stable and unstable carotid atherosclerotic plaques compared with healthy vessels, both at mRNA and protein level. CXCR4 and CXCL12 were localized particularly in macrophages.

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Role of Metabolic Reprogramming in Epithelial–Mesenchymal Transition (EMT)

International Journal of Molecular Sciences ◽

10.3390/ijms20082042 ◽

2019 ◽

Vol 20 (8) ◽

pp. 2042 ◽

Cited By ~ 17

Author(s):

Hyunkoo Kang ◽

Hyunwoo Kim ◽

Sungmin Lee ◽

HyeSook Youn ◽

BuHyun Youn

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Molecular Mechanisms ◽

Cancer Metastasis ◽

Epithelial Mesenchymal Transition ◽

Metabolic Reprogramming ◽

Metabolic Enzyme ◽

Diverse Range ◽

Mesenchymal Transition ◽

Altered Glucose

Activation of epithelial–mesenchymal transition (EMT) is thought to be an essential step for cancer metastasis. Tumor cells undergo EMT in response to a diverse range of extra- and intracellular stimulants. Recently, it was reported that metabolic shifts control EMT progression and induce tumor aggressiveness. In this review, we summarize the involvement of altered glucose, lipid, and amino acid metabolic enzyme expression and the underlying molecular mechanisms in EMT induction in tumor cells. Moreover, we propose that metabolic regulation through gene-specific or pharmacological inhibition may suppress EMT and this treatment strategy may be applied to prevent tumor progression and improve anti-tumor therapeutic efficacy. This review presents evidence for the importance of metabolic changes in tumor progression and emphasizes the need for further studies to better understand tumor metabolism.

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Investigation of the Prognostic Role of Carbonic Anhydrase 9 (CAIX) of the Cellular mRNA/Protein Level or Soluble CAIX Protein in Patients with Oral Squamous Cell Carcinoma

International Journal of Molecular Sciences ◽

10.3390/ijms20020375 ◽

2019 ◽

Vol 20 (2) ◽

pp. 375 ◽

Cited By ~ 8

Author(s):

Alexander W. Eckert ◽

Susanne Horter ◽

Daniel Bethmann ◽

Johanna Kotrba ◽

Tom Kaune ◽

...

Keyword(s):

Squamous Cell Carcinoma ◽

Regression Analysis ◽

Oral Squamous Cell Carcinoma ◽

Carbonic Anhydrase ◽

Protein Level ◽

Normal Tissue ◽

Mrna Level ◽

Mrna Levels ◽

Cox's Regression ◽

Carbonic Anhydrase 9

s: Carbonic anhydrase 9 (CAIX) is an important protein that stabilizes the extracellular pH value and is transcriptionally regulated by hypoxia-inducible factor 1 (HIF1), but more stable than HIF1α. Here we show a comparative study that examines the prognostic value of CA9 mRNA, CAIX protein of tumor cells and secreted CAIX protein for oral squamous cell carcinoma (OSCC) patients. Tumor samples from 72 OSCC patients and 24 samples of normal tissue were analyzed for CA9 mRNA levels. A total of 158 OSCC samples were stained for CAIX by immunohistochemistry and 89 blood serum samples were analyzed by ELISA for soluble CAIX protein content. Survival analyses were performed by Kaplan–Meier and Cox’s regression analysis to estimate the prognostic effect of CA9/CAIX in OSCC patients. The CA9 mRNA and CAIX protein levels of tumor cells correlated with each other, but not with those of the secreted CAIX protein level of the blood of patients. ROC curves showed a significant (p < 0.001) higher mRNA-level of CA9 in OSCC samples than in adjacent normal tissue. Cox’s regression analysis revealed an increased risk (i) of death for patients with a high CA9 mRNA level (RR = 2.2; p = 0.02), (ii) of locoregional recurrence (RR = 3.2; p = 0.036) at higher CA9 mRNA levels and (iii) of death at high CAIX protein level in their tumors (RR = 1.7; p = 0.066) and especially for patients with advanced T4-tumors (RR = 2.0; p = 0.04). However, the secreted CAIX protein level was only as a trend associated with prognosis in OSCC (RR = 2.2; p = 0.066). CA9/CAIX is an independent prognostic factor for OSCC patients and therefore a potential therapeutic target.

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Fibrin Down-regulates LPS- and PMA-induced Tissue Factor Expression by Blood Mononuclear Cells

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614044 ◽

2000 ◽

Vol 84 (09) ◽

pp. 453-459 ◽

Cited By ~ 5

Author(s):

Maria Rossiello ◽

Alessandra Italia ◽

Anna Stramaglia ◽

Loreto Gesualdo ◽

Giuseppe Grandaliano ◽

...

Keyword(s):

Tissue Factor ◽

Incubation Time ◽

Mononuclear Cells ◽

Gradient Centrifugation ◽

Mrna Level ◽

Tumor Promoter ◽

Cellular Functions ◽

Blood Mononuclear Cells ◽

Tissue Plasminogen

SummarySeveral studies indicate that fibrin may play a functional role in inflammation by modulating a variety of cellular functions. We investigated the effect of fibrin on tissue factor (TF) production by blood mononuclear cells (MNC). Citrated human blood was recalcified and incubated at 37° C for 1-4 h. The resulting clot was lysed by the addition of tissue plasminogen activator (t-PA) and MNC were isolated by density gradient centrifugation. A control blood sample was processed in the same way but omitting calcium addition and clot formation. Clot-and blood-derived MNC did not express detectable TF activity and antigen whatever the incubation time. Clot-derived MNC, however, generated on average 5 fold less TF (activity and antigen) than control cells, when stimulated with lipopolysaccharide (LPS, 1 µg/ml) for 3 h at 37° C. A reduced TF response of clot-derived cells was also observed at mRNA level as indicated by RT-PCR and in situ hybridization. The effect was dependent on the incubation time within the clot, could not be reversed by enhancing LPS concentration or by adding serum, and was maintained if LPS was replaced by the tumor promoter PMA. A reduced TF response was also found when washed MNC were incorporated for 1 h at 37° C within purified fibrin but not when the cells were incubated with fibrinogen, thrombin or fibrin split products alone, indicating that contact with fibrin was responsible for the inhibition of TF production. Fibrin-induced down-regulation of TF response to LPS and PMA by MNC may represent a negative feed-back aimed at limiting excessive blood clotting activation in immuneinflammatory diseases.

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HIF-1α is required for disturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium

eLife ◽

10.7554/elife.25217 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 34

Author(s):

David Wu ◽

Ru-Ting Huang ◽

Robert B Hamanaka ◽

Matt Krause ◽

Myung-Jin Oh ◽

...

Keyword(s):

Vascular Inflammation ◽

Glycolytic Enzyme ◽

Metabolic Changes ◽

Metabolic Reprogramming ◽

Pyruvate Dehydrogenase Kinase ◽

Respiratory Capacity ◽

Disturbed Flow ◽

Bioenergetic Analysis ◽

Mitochondrial Respiratory

Hemodynamic forces regulate vascular functions. Disturbed flow (DF) occurs in arterial bifurcations and curvatures, activates endothelial cells (ECs), and results in vascular inflammation and ultimately atherosclerosis. However, how DF alters EC metabolism, and whether resulting metabolic changes induce EC activation, is unknown. Using transcriptomics and bioenergetic analysis, we discovered that DF induces glycolysis and reduces mitochondrial respiratory capacity in human aortic ECs. DF-induced metabolic reprogramming required hypoxia inducible factor-1α (HIF-1α), downstream of NAD(P)H oxidase-4 (NOX4)-derived reactive oxygen species (ROS). HIF-1α increased glycolytic enzymes and pyruvate dehydrogenase kinase-1 (PDK-1), which reduces mitochondrial respiratory capacity. Swine aortic arch endothelia exhibited elevated ROS, NOX4, HIF-1α, and glycolytic enzyme and PDK1 expression, suggesting that DF leads to metabolic reprogramming in vivo. Inhibition of glycolysis reduced inflammation suggesting a causal relationship between flow-induced metabolic changes and EC activation. These findings highlight a previously uncharacterized role for flow-induced metabolic reprogramming and inflammation in ECs.

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Downregulation of miRNA-205 Expression and Biological Mechanism in Prostate Cancer Tumorigenesis and Bone Metastasis

BioMed Research International ◽

10.1155/2020/6037434 ◽

2020 ◽

Vol 2020 ◽

pp. 1-17

Author(s):

Yu Sun ◽

Sheng-Hua Li ◽

Ji-Wen Cheng ◽

Gang Chen ◽

Zhi-Guang Huang ◽

...

Keyword(s):

Prostate Cancer ◽

Bone Metastasis ◽

Protein Level ◽

Target Genes ◽

Mrna Level ◽

Enrichment Analysis ◽

Functional Enrichment Analysis ◽

Functional Enrichment ◽

Forest Plot ◽

Biological Mechanism

Background. The expression and mechanism of microRNA-205 (miRNA-205) in prostate cancer (PCa) and its bone metastasis remain controversial. Materials and Methods. The expression and discriminating capability of miRNA-205 were assessed by drawing a forest plot and a summarized receiver operating characteristic (SROC) curve, using data available from 27 miRNA-array and miRNA-sequencing datasets. The miRNA-205 target genes were acquired from online prediction tools, differentially upregulated genes in PCa, and differentially expressed genes (DEGs) after miRNA-205 transfection into PCa cell lines. Functional enrichment analysis was conducted to explore the biological mechanism of miRNA-205 targets. Immunohistochemistry (IHC) was applied to verify the protein level of the hub gene. Results. The expression of miRNA-205 in the PCa group (1,461 samples) was significantly lower than that in the noncancer group (510 samples), and the downregulation of miRNA-205 showed excellent sensitivity and specificity in differentiating between the two groups. In bone metastatic PCa, the miRNA-205 level was further reduced than in nonbone metastatic PCa, and it showed a good capability in distinguishing between the two groups. In total, 153 miRNA-205 targets were screened through the three aforementioned methods. Based on the results of functional enrichment analysis, the targets of miRNA-205 were mainly enriched during chromosome segregation and phospholipid-translocating ATPase activity and in the spindle microtubule and the p53 signaling pathway. CDK1 had the highest connectivity in the PPI network analysis and was screened as one of the hub genes. A statistically significant negative correlation between miRNA-205 and CDK1 was observed. The expression of CDK1 in PCa samples was pronouncedly upregulated in terms of both the mRNA level and the protein level when compared with noncancer samples. Conclusion. miRNA-205 may play a vital role in PCa tumorigenesis and bone metastasis by targeting CDK1.

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