Hypoxia Marker Carbonic Anhydrase IX Is Present in Abdominal Aortic Aneurysm Tissue and Plasma

Abdominal aortic aneurysms (AAA) are a significant cause of premature deaths worldwide. Since there is no specific treatment for reducing AAA progression, it is crucial to understand the pathogenesis leading to aneurysm wall weakening/remodeling and identify new proteins involved in this process which could subsequently serve as novel therapeutic targets. In this study, we analyzed the presence of the hypoxia-related proteins carbonic anhydrase IX (CA IX), hypoxia-inducible factor 1α (HIF-1α), and AKT as the key molecule in the phosphoinositide-3-kinase pathway in the AAA wall. Additionally, we used a blood-based assay to examine soluble CA IX (s-CA IX) levels in the plasma of AAA patients. Using western blotting, we detected CA IX protein in 12 out of 15 AAA tissue samples. Immunohistochemistry staining proved CA IX expression in the media of the aneurysmal wall. Evaluation of phosphorylated (p-AKT) and total AKT showed elevated levels of both forms in AAA compared to normal aorta. Using ELISA, we determined the concentration of s-CA IX >20 pg/mL in 13 out of 15 AAA patients. Results obtained from in silico analysis of CA9 and aneurysm-associated genes suggest a role for CA IX in aneurysmal wall remodeling. Our results prove the presence of hypoxia-related CA IX in AAA tissues and indicate a possible role of CA IX in hypoxia-associated cardiovascular diseases.

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Expression of carbonic anhydrase IX in craniopharyngiomas

Journal of Neurosurgery ◽

10.3171/2011.6.jns1168 ◽

2011 ◽

Vol 115 (4) ◽

pp. 796-801 ◽

Cited By ~ 10

Author(s):

Martin Proescholdt ◽

Marsha Merrill ◽

Eva-Maria Stoerr ◽

Annette Lohmeier ◽

Wolfgang Dietmaier ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Direct Sequencing ◽

Cyst Formation ◽

P53 Mutation ◽

Carbonic Anhydrase Ix ◽

Preoperative Imaging ◽

Normal Brain ◽

Tissue Samples ◽

Cyst Volume ◽

Ca Ix

Object In craniopharyngiomas, cystic growth causes pressure on vital structures of the adjacent brain, leading to significant morbidity. However, the molecular pathogenesis of this cyst formation remains unknown. Carbonic anhydrase IX (CA IX) is a tumor-associated, hypoxia-inducible enzyme, which can cause fluid production and development of cysts. The authors investigated CA IX expression in craniopharyngiomas and its correlation with the extent of cyst formation. In addition, the major pathways of CA IX regulation, hypoxia and p53 mutation, were analyzed. Methods Expression of CA IX was analyzed in 20 craniopharyngioma patients by means of in situ hybridization and immunohistochemistry. Preoperative imaging was used to quantify cyst volume. To analyze putative hypoxic induction of CA IX, immunohistochemical staining for HIF-1α and VEGF was performed. Since p53 negatively regulates CA IX expression, we also analyzed the tumors for p53 mutation by direct sequencing. Results Significant CA IX was found in 85% of the 20 cases. The extent of CA IX expression was significantly correlated with cyst volume. HIF-1α expression was largely absent in all tissue samples, whereas moderate VEGF expression was present in a subset of cases but without correlation to cyst volume. No p53 mutation was found in any of the analyzed tumors. Conclusions Carbonic anhydrase IX, which is virtually absent in normal brain, is significantly upregulated in craniopharyngiomas and shows a significant association with cyst size. The mechanisms of regulation remain unknown, since neither hypoxia nor p53 appears to play a role. These results indicate that inhibition of CA IX may be a potential target for the adjuvant treatment in patients with cystic craniopharyngiomas.

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Distinct Expression Patterns of Carbonic Anhydrase IX in Clear Cell, Microcystic, and Angiomatous Meningiomas

Journal of Neuropathology & Experimental Neurology ◽

10.1093/jnen/nlz091 ◽

2019 ◽

Vol 78 (12) ◽

pp. 1081-1088

Author(s):

Rati Chkheidze ◽

Patrick J Cimino ◽

Kimmo J Hatanpaa ◽

Charles L White ◽

Manuel Ferreira ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Clear Cell ◽

Expression Patterns ◽

Carbonic Anhydrase Ix ◽

World Health ◽

Loss Of Function ◽

Ca Ix ◽

Hypoxia Marker ◽

Other World ◽

Health Organization

Abstract Clear cell, microcytic, and angiomatous meningiomas are 3 vasculature-rich variants with overlapping morphological features but different prognostic and treatment implications. Distinction between them is not always straightforward. We compared the expression patterns of the hypoxia marker carbonic anhydrase IX (CA-IX) in meningiomas with predominant clear cell (n = 15), microcystic (n = 9), or angiomatous (n = 11) morphologies, as well as 117 cases of other World Health Organization recognized histological meningioma variants. Immunostaining for SMARCE1 protein, whose loss-of-function has been associated with clear cell meningiomas, was performed on all clear cell meningiomas, and selected variants of meningiomas as controls. All clear cell meningiomas showed absence of CA-IX expression and loss of nuclear SMARCE1 expression. All microcystic and angiomatous meningiomas showed diffuse CA-IX immunoreactivity and retained nuclear SMARCE1 expression. In other meningioma variants, CA-IX was expressed in a hypoxia-restricted pattern and was highly associated with atypical features such as necrosis, small cell change, and focal clear cell change. In conclusion, CA-IX may serve as a useful diagnostic marker in differentiating clear cell, microcystic, and angiomatous meningiomas.

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Inhibition of Carbonic Anhydrase IX Promotes Apoptosis through Intracellular pH Level Alterations in Cervical Cancer Cells

International Journal of Molecular Sciences ◽

10.3390/ijms22116098 ◽

2021 ◽

Vol 22 (11) ◽

pp. 6098

Author(s):

Ebru Temiz ◽

Ismail Koyuncu ◽

Mustafa Durgun ◽

Murat Caglayan ◽

Ataman Gonel ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Intracellular Ph ◽

Hela Cells ◽

Protein Level ◽

Caspase 3 ◽

Solid Tumours ◽

Carbonic Anhydrase Ix ◽

Parp Activation ◽

Ca Ix ◽

Cervical Cancer Cells

Carbonic anhydrase IX (CAIX) is a hypoxia-related protein that plays a role in proliferation in solid tumours. However, how CAIX increases proliferation and metastasis in solid tumours is unclear. The objective of this study was to investigate how a synthetic CAIX inhibitor triggers apoptosis in the HeLa cell line. The intracellular effects of CAIX inhibition were determined with AO/EB, AnnexinV-PI, and γ-H2AX staining; measurements of intracellular pH (pHi), reactive oxygen species (ROS), and mitochondrial membrane potential (MMP); and analyses of cell cycle, apoptotic, and autophagic modulator gene expression (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, caspase-12, Beclin, and LC3), caspase protein level (pro-caspase 3 and cleaved caspase-3, -8, -9), cleaved PARP activation, and CAIX protein level. Sulphonamide CAIX inhibitor E showed the lowest IC50 and the highest selectivity index in CAIX-positive HeLa cells. CAIX inhibition changed the morphology of HeLa cells and increased the ratio of apoptotic cells, dramatically disturbing the homeostasis of intracellular pHi, MMP and ROS levels. All these phenomena consequent to CA IX inhibition triggered apoptosis and autophagy in HeLa cells. Taken together, these results further endorse the previous findings that CAIX inhibitors represent an important therapeutic strategy, which is worth pursuing in different cancer types, considering that presently only one sulphonamide inhibitor, SLC-0111, has arrived in Phase Ib/II clinical trials as an antitumour/antimetastatic drug.

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Material Properties of Abdominal Aortic Aneurysm Wall From Uniaxial Tests

10.1115/imece2000-2541 ◽

2000 ◽

Author(s):

Mano J. Thubrikar ◽

Michel Labrosse ◽

Jihad Al-Soudi ◽

Brett Fowler ◽

Francis Robicsek

Keyword(s):

Experimental Data ◽

Material Properties ◽

Aortic Wall ◽

Abdominal Aortic Aneurysms ◽

Aortic Aneurysms ◽

Modeling Tools ◽

Aneurysm Wall ◽

Abdominal Aortic ◽

Abdominal Aortic Aneurysm Wall ◽

Mechanical Models

Abstract Abdominal aortic aneurysms (AAA) rupture when the aortic wall cannot withstand the stresses and strains induced by the pulsatile blood pressure. In recent years, different mechanical models of aneurysms have been presented (Vorp et al., 1998, Di Martino et al., 1998, Thubrikar et al., 1999). Although powerful modeling tools such as finite elements are available, there is still a need for experimental data concerning the mechanical properties of the aneurysm wall.

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Presence of NGAL/MMP-9 complexes in human abdominal aortic aneurysms

Thrombosis and Haemostasis ◽

10.1160/th06-11-0638 ◽

2007 ◽

Vol 98 (08) ◽

pp. 427-433 ◽

Cited By ~ 64

Author(s):

Chaoyong Zhu ◽

Angela Silveira ◽

Anne-Louise Hemdahl ◽

Anders Hamsten ◽

Ulf Hedin ◽

...

Keyword(s):

Elective Surgery ◽

Abdominal Aortic Aneurysms ◽

Aortic Aneurysms ◽

Intraluminal Thrombus ◽

Neutrophil Gelatinase Associated Lipocalin ◽

Reducing Conditions ◽

Aneurysm Wall ◽

Abdominal Aortic ◽

Aneurysm Growth ◽

Neutrophil Gelatinase

SummaryIt has been suggested that the intraluminal thrombus of abdominal aortic aneurysms (AAAs) predisposes for AAA enlargement and rupture.The growth of theAAA is dependent on proteolytic degradation of elastin. Here, we analysed whether the neutrophil gelatinase-associated lipocalin (NGAL) is expressed within the thrombus and the aneurysm wall. NGAL can bind to metalloproteinase- 9 (MMP-9) and inhibit its degradation,thereby preserving enzymatic activity. Biopsies were obtained from thrombus- free and thrombus-covered aneurysm wall and the intraluminal thrombus from patients undergoing elective surgery for AAA. Immunohistochemistry and real-time PCR were used to study NGAL and MMP-9 expression. Immunoprecipitation, gel zymography,Western blot and ELISA were used to detect and quantify NGAL/MMP-9 complexes. NGAL was detected in the thrombus, the interface between the thrombus and the underlying wall and in the wall itself.Double staining showed that neutrophils are the major source of NGAL expression. Immunoprecipitation of MMP-9 with antibody against NGAL showed that complexes of NGAL and active MMP-9 were present in thrombus, the interface fluid and the aneurysm wall.Western blot analyses using non-reducing conditions and gel zymography demonstrated that high-molecular-weight complexes of NGAL/MMP-9 were present within the different regions.The concentration of the NGAL/MMP-9 complex was highest in the luminal part of the thrombus. In conclusion, NGAL in complex with activated MMP-9 is present in AAA wall and thrombus. Neutrophil-derived NGAL could enhance the proteolytic activity associated with AAA, but the importance of this mechanism for aneurysm growth remains to be shown.

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A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells

Biochemical Journal ◽

10.1042/bcj20190177 ◽

2019 ◽

Vol 476 (10) ◽

pp. 1497-1513 ◽

Cited By ~ 8

Author(s):

Mam Y. Mboge ◽

Zhijuan Chen ◽

Daniel Khokhar ◽

Alyssa Wolff ◽

Lingbao Ai ◽

...

Keyword(s):

Breast Cancer ◽

Carbonic Anhydrase ◽

Cancer Cells ◽

Cancer Progression ◽

Expression Patterns ◽

Extracellular Ph ◽

Carbonic Anhydrase Ix ◽

Monocarboxylate Transporter ◽

Proton Efflux ◽

Ca Ix

AbstractThe most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pHe) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H+ shuttle system in CA IX to facilitate proton efflux through MCT4.

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Role of aryl hydrocarbon receptor in modulation of the expression of the hypoxia marker carbonic anhydrase IX

Biochemical Journal ◽

10.1042/bj20080952 ◽

2009 ◽

Vol 419 (2) ◽

pp. 419-425 ◽

Cited By ~ 11

Author(s):

Martina Takacova ◽

Tereza Holotnakova ◽

Jan Vondracek ◽

Miroslav Machala ◽

Katerina Pencikova ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Aryl Hydrocarbon Receptor ◽

Transcriptional Activation ◽

Hypoxia Inducible Factor ◽

Carbonic Anhydrase Ix ◽

Independent Manner ◽

Hypoxic Induction ◽

Aryl Hydrocarbon ◽

Ca Ix ◽

Tcdd Treatment

Tumour-associated expression of CA IX (carbonic anhydrase IX) is to a major extent regulated by HIF-1 (hypoxia-inducible factor-1) which is important for transcriptional activation and consists of the oxygen-regulated subunit HIF-1α and the partner factor ARNT [AhR (aryl hydrocarbon receptor) nuclear translocator]. We have previously observed that HIF-1α competes with the AhR for interaction with ARNT under conditions when both conditionally regulated factors are activated. We have therefore investigated whether TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin)-induced activation of the AhR pathway might interfere with CA IX expression. The results from the present study suggest that TCDD treatment reduces hypoxic induction of both CA IX mRNA and protein expression. Moreover, the transcriptional activity of the CA9 promoter was significantly reduced by expression of CAAhR (constitutively active AhR), which activates transcription in a ligand-independent manner. Finally, we found that ARNT is critical for both hypoxic induction and the TCDD-mediated inhibition of CA9 expression.

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Purification of small-size acidic proteoglycan-like domain of carbonic anhydrase IX fused with thioredoxine expressed in Escherichia coli for structural characterization

Biologia ◽

10.1515/biolog-2017-0156 ◽

2017 ◽

Vol 72 (11) ◽

Author(s):

Stanislava Bírová ◽

Zdenko Levarski ◽

Ivana Vidličková ◽

Silvia Pastoreková ◽

Ján Turňa ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Expression System ◽

Carbonic Anhydrase Ix ◽

Its Sequence ◽

Fusion Partner ◽

Aromatic Residues ◽

Acidic Domain ◽

Ca Ix ◽

Acidic Amino Acids ◽

Unusual Amino Acid

AbstractProteoglycan-like (PG) domain of carbonic anhydrase IX (CA IX) is a small-sized acidic domain (8.3 kDa; pI ∼ 3.5) with a very unusual amino acid composition. More than one third of its sequence consists of acidic amino acids and it contains no aromatic residues. It has been revealed that it holds one of the key roles in oncogenetic mechanisms, in which CA IX participates. However, it has not been structurally characterized yet. With these prospects, we developed an expression system of recombinant PG domain production in the form of a fusion protein using thioredoxine (Trx) as the fusion partner in

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Dual-functional Liposomes with Carbonic Anhydrase IX Antibody and BR2 Peptide Modification Effectively Improve Intracellular Delivery of Cantharidin to Treat Orthotopic Hepatocellular Carcinoma Mice

Molecules ◽

10.3390/molecules24183332 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3332 ◽

Cited By ~ 3

Author(s):

Zhang ◽

Lin ◽

Chan ◽

Liu ◽

Lu ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Carbonic Anhydrase ◽

Therapeutic Potential ◽

Intracellular Delivery ◽

Carbonic Anhydrase Ix ◽

Ca Ix ◽

Dual Functional ◽

Peptide Modification

Liposomal nanotechnology has a great potential to overcome the current major problems of chemotherapy. However, the lack of penetrability and targetability retards the successful delivery of liposomal carriers. Previously, we showed that BR2 peptide modification endowed cantharidin-loaded liposomes with intracellular penetration that enhanced the drug cytotoxic effects. Here, we aimed to improve the targeting delivery of drugs into cancer cells via highly expressed carbonic anhydrase IX (CA IX) receptors by modifying our previous catharidin-loaded BR2-liposomes with anti-CA IX antibody. A higher cellular uptake of dual-functional liposomes (DF-Lp) than other treatments was observed. Induction of CA IX over-expressing resulted in a higher cellular binding of DF-Lp; subsequently, blocking with excess antibodies resulted in a decreased cancer-cell association, indicating a specific targeting property of our liposomes towards CA IX expressed cells. After 3h tracking, most of the liposomes were located around the nucleus which confirmed the involvement of targeting intracellular delivery. Cantharidin loaded DF-Lp exhibited enhanced cytotoxicity in vitro and was most effective in controlling tumor growth in vivo in an orthotopic hepatocellular carcinoma model compared to other groups. Collectively, our results presented the advantage of the BR2 peptide and CA IX antibody combination to elevate the therapeutic potential of cantharidin loaded DF-liposomes.

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Biophysical Characterization of Cancer-Related Carbonic Anhydrase IX

International Journal of Molecular Sciences ◽

10.3390/ijms21155277 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5277

Author(s):

Katarina Koruza ◽

A. Briana Murray ◽

Brian P. Mahon ◽

Jesse B. Hopkins ◽

Wolfgang Knecht ◽

...

Keyword(s):

Mass Spectrometry ◽

Carbonic Anhydrase ◽

Small Angle ◽

Catalytic Domain ◽

Carbonic Anhydrase Ix ◽

Biophysical Characterization ◽

X Ray ◽

Ca Ix ◽

X Ray Scattering ◽

Ray Scattering

Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.

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