Search

Hypoxia-regulated protein carbonic anhydrase IX (CA IX) is up-regulated in different tumor entities and correlated with poor prognosis in breast cancer patients. Due to the radio- and chemotherapy resistance of solid hypoxic tumors, derivatives of betulinic acid (BA), a natural compound with anticancer properties, seem to be promising to benefit these cancer patients. We synthesized new betulin sulfonamides and determined their cytotoxicity in different breast cancer cell lines. Additionally, we investigated their effects on clonogenic survival, cell death, extracellular pH, HIF-1α, CA IX and CA XII protein levels and radiosensitivity. Our study revealed that cytotoxicity increased after treatment with the betulin sulfonamides compared to BA or their precursors, especially in triple-negative breast cancer (TNBC) cells. CA IX activity as well as CA IX and CA XII protein levels were reduced by the betulin sulfonamides. We observed elevated inhibitory efficiency against protumorigenic processes such as proliferation and clonogenic survival and the promotion of cell death and radiosensitivity compared to the precursor derivatives. In particular, TNBC cells showed benefit from the addition of sulfonamides onto BA and revealed that betulin sulfonamides are promising compounds to treat more aggressive breast cancers, or are at the same level against less aggressive breast cancer cells.

The main function of carbonic anhydrases (CAs) in cancer cells is the pH regulation through a conversion of H2 O and CO2 to H+ and HCO3 - . However, the data of in vitro and in vivo studies have demonstrated that transmembrane isoforms of CA IX and CA XII are involved in various steps of cancer cell migration, invasion and metastasis. According to literature, inhibition of these CAs can affect the expression of multiple proteins. Some scientific groups have reported the possible interactions between CA IX and E-cadherin-catenin system, CA IX and integrins, CA IX, CA XII and ion transporters, which all are highly involved in cell-to-cell adhesion, the formation of membrane protrusions and focal adhesions. Nevertheless, CA IX and CA XII have a high impact on tumor growth and metastases formation. The data discussed in this review highly supports the role of both transmembrane CAs in various cancer metastasis processes through interactions between CAs and other invasion proteins. Nevertheless, the exact mechanisms of these interactions are not yet determined and need further investigation. This article is protected by copyright.

In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68–56% identity with the large yellow croaker (Larimichthys crocea), tilapia (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.

Background: Although recent studies described important roles for carbonic anhydrase (CA) XII in epithelial carcinogenesis and tumor behavior, a consensus has not yet been reached regarding its clinicopathological significance in esophageal squamous cell carcinoma (ESCC). In the present study, we investigated its prognostic significance in ESCC. Materials and Methods: An immunohistochemical analysis was performed on 70 primary tumor samples obtained from ESCC patients who underwent esophagectomy, and the relationships between the expression of CA XII and various clinicopathological features or prognosis were analyzed. Results: Immunohistochemical staining showed that CA XII was primarily found in the cell membranes of carcinoma cells. Although the expression of CA XII was related to the pT category, it had no prognostic impact. We then examined the expression of CA XII according to the pT category. In pT2-3 ESCC, the 3-year survival rate of patients with the high grade expression of CA XII (29.1 %) was significantly lower than that of patients with the low grade expression of CA XII (70.3 %). Furthermore, a multivariate analysis demonstrated that the expression of CA XII was one of the most important independent prognostic factors following radical esophagectomy in pT2-3 ESCC. Conclusion: These results suggest that the expression of CA XII may be a valuable prognostic factor for patients with advanced ESCC. The results of the present study provide an insight into the role of CA XII as a biomarker in ESCC.

Ogļskābes anhidrāzes katalizē dzīvajos organismos būtisko bikarbonāta un ogļskābās gāzes savstarpējo atgriezenisko reakciju. Cilvēka organismā ir vairākas ogļskābes anhidrāžu izoformas, un dažu izoformu katalītiskā darbība ir nevēlama. Viena no šīm izoformām, ogļskābes anhidrāzes XII izoforma, saistīta ar vairāku ļaundabīgu audzēju attīstību. Maģistra darba iztrādē tika vairākkārtīgi uzlabota ogļskābes anhidrāzes XII izoformas ekspresijas un attīrīšanas sistēma un iegūtas vairākas ar ligandu saistītas ogļskābes anhidrāzes XII izoformas struktūras. Ar proteīnu rentgenstaru kristalogrāfijas palīdzību tika identificētas dažādu inhibitoru konformācijas ogļskābes anhidrāzes XII izoformas aktīvajā centrā, nodrošinot potenciālas iestrādnes selektīvi inhibējošu ligandu izveidei., Carbonic anhydrases catalyse an important bicarbonate and carbon dioxide reversible reaction in all life forms. There are many carbonic anhydrase isoforms in the human body, and some of them have an undesirable catalytic activity. Carbonic anhydrase isoform XII is one of them, and is connected with tumorigenesis. During the study, the expression and purification system for carbonic anhydrase XII was greatly improved and many ligand bound carbonic anhydrase XII structures were obtained. X-ray crystallography helped to identify different ligand conformations in the carbonic anhydrase XII active site, thus providing a forerun for potentially selective inhibitors.

Hiilihappoanhydraasi-entsyymit osallistuvat pH-tasapainon säätelyyn solujen, kudosten ja koko elimistön tasolla. Nämä entsyymit katalysoivat reaktiota, jossa hiilidioksidi ja vesi muuttuvat bikarbonaatiksi ja vetyioniksi. Kyseinen reaktio voi tapahtua myös käänteisesti vastakkaiseen suuntaan. Nisäkkäillä on 13 hiilihappoanhydraasia, jotka muodostavat isoentsyymiperheen. Kaikki nämä isoentsyymit katalysoivat yllä mainittua reaktiota, mutta niillä on myös eroja. Ne esiintyvät eri osissa solua ja toisaalta eri kudoksissa. Kullakin isoentsyymillä on myös ominainen aktiivisuus. Hiilihappoanhydraaseilla on tärkeä rooli useissa biologisissa prosesseissa, kuten hiilidioksidin ja veden kuljetuksessa, elimistössä olevien nesteiden tuotannossa, luun hajotuksessa ja aineenvaihduntareaktioissa. Kahden isoentsyymin, IX:n ja XII:n, on havaittu esiintyvän syöpäkasvaimissa ja vaikuttavan syövän mekanismeihin. Tämän väitöskirjatyön tavoitteena oli tutkia näiden kahden isoentsyymin säätelyä ja rooleja. Tutkimuksessa pyrittiin löytämään isoentsyymi IX:n ja XII:n säätelijöitä. Niiden säätelijät tunnetaan huonosti lukuun ottamatta hypoksiaa, eli vähentynyttä happipitoisuutta. Useat kasvutekijät nostivat isoentsyymi IX:n ja XII:n lähetti-RNA:n tasoja syöpäsolulinjoissa. Tämä vaikutus oli huomattavampi isoentsyymi XII:lla, joten näyttää siltä, että sen ilmentymisen tärkein säätelijä ei ole hypoksia, kuten isoentsyymi IX:n. Lisäksi selvitettiin isoentsyymi IX:n ja XII:n ilmentymistä proteiinitasolla hiiren sikiönkehityksen aikana. Kiinnostava löytö oli, että molempia isoentsyymejä ilmennettiin useissa kehittyvän hiiren kudoksissa ja osa näistä oli sellaisia, jotka eivät ilmennä kyseisiä proteiineja aikuisella hiirellä. Näin ollen on todennäköistä, että isoentsyymi IX:ää ja XII:ta tarvitaan tiettyjen kudosten muodostumiseen. Tutkimuksessa testattiin myös sitä, miten geenien ilmentyminen muuttuu hiirten mahalaukussa, kun isoentsyymi IX on poistettu. Mielenkiintoista oli, että tämän entsyymin puute muutti sellaisten geenien ilmentymistä, jotka ovat mukana kehitykseen liittyvissä prosesseissa, solujen erilaistumisessa ja immuunivasteissa. Tulokset vahvistavat käsitystä siitä, että isoentsyymi IX vaikuttaa useisiin fysiologisiin toimintoihin. Toisaalta näiden tulosten, ja muiden tutkijoiden aikaisempien havaintojen mukaan isoentsyymi IX:n ensisijainen tehtävä mahalaukussa ei liity pH:n säätelyyn tai kyseinen entsyymi on korvattavissa tätä toimintoa ajatellen. Tutkimuksessa tehdyt analyysit osoittivat, että NCCRP1-geenin ilmentymisen taso oli noussut tilastollisesti merkitsevästi niiden hiirten mahalaukussa, joiden isoentsyymi IX-geeni oli poistettu. Koska NCCRP1-geeni on huonosti tunnettu, se valittiin jatkoanalyyseihin. Tutkimuksen kuluessa kävi ilmi, ettei isoentsyymi IX säätele suoraan NCCRP1-geeniä. Tutkimus osoitti myös, että NCCRP1 on mitä ilmeisimmin alun perin väärin nimetty. Se ei olekaan solukalvoon kiinnittynyt immuunivasteita välittävä proteiini, vaan ilmentyy sytosolissa eli solun sisällä. Bioinformatiikka-analyysit ehdottivat NCCRP1-proteiinin kuuluvan F-box-proteiinien lektiini-tyypin alaperheeseen (FBXO-perhe). Kyseiset proteiinit osallistuvat viallisten glykoproteiinien hajotukseen. Näin ollen NCCRP1:n nimi tulisi muuttaa FBXO50:ksi. Väitöskirjan tulokset auttavat ymmärtämään paremmin kahden syöpään liittyvän hiilihappoanhydraasi-isoentsyymin säätelyä ja merkitystä erilaisissa fysiologisissa tilanteissa. Lisäksi tutkimuksessa karakterisoitiin NCCRP1-geeni, joka tulisi nimetä uudelleen. The carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate and a proton. The CAs are produced in a variety of tissues where they participate in several physiological processes, such as pH regulation, the production of body fluids, bone resorption, and metabolic processes. The CA family consists of 13 active isozymes in mammals: CA I, II, III, VII, and XIII are cytoplasmic; CA IV, IX, XII, XIV, and XV are anchored to plasma membranes; CA VA and VB are mitochondrial; and CA VI is the only secreted protein. This study had four specific aims. The first aim was to identify new regulators of CA IX and CA XII. This aim was proposed because, besides hypoxia, the regulation of these proteins is poorly characterized. The second goal was to examine the expression profiles of these isozymes during mouse embryonic development. The third aim was to investigate the whole-genome expression changes that occur in response to CA IX deficiency in the mouse stomach. These mice exhibit gastric hyperplasia, but the exact mechanisms leading to this disturbed cell lineage phenotype remain unknown. The fourth and final goal was to elucidate further the properties of some selected proteins that are dysregulated in the gastric mucosa of Car9?/? mice. Our studies demonstrate that the growth factors IGF-1, TGF-?, TGF-?1, and EGF increase CA9 and CA12 mRNA expression in cancer-derived cell lines as determined by quantitative real-time PCR (QRT-PCR). In the case of CA9, this effect was observed in only one cell line, whereas CA12 was elevated by growth factors in four cell lines. Thus, hypoxia may not be the primary regulator of CA12 in contrast to CA9. Immunohistochemistry was used to study CA IX and CA XII expression during mouse organogenesis. These isozymes were detected in several tissues of which a portion are negative for expression in adult mice. Therefore, it is possible that CA IX and CA XII have specific roles in the assembly of certain tissues. The changes caused by CA IX deficiency in the mouse stomach were explored using a cDNA microarray, which revealed the induction of 86 genes and the repression of 46 genes. Among the genes with altered regulation were those involved in developmental processes, cell differentiation and immune responses. These data and previous findings by others suggest that the primary role of CA IX in the stomach is not related to pH regulation or, alternatively, that its function in this tissue is compensated for by other CA isozymes. The microarray data indicated significant upregulation of non-specific cytotoxic cell receptor protein 1 (Nccrp1), a poorly characterized gene that was selected for further analyses. Silencing of CA9 in HeLa cells did not affect NCCRP1 levels, suggesting that NCCRP1 is not directly regulated by CA9 and that its upregulation is secondary to Car9 gene disruption. Moreover, it was demonstrated that human recombinant NCCRP1 is expressed intracellularly. Bioinformatic analyses revealed that NCCRP1 belongs to the lectin-type subfamily of F-box (FBXO) proteins, which are components of the E3 ubiquitin ligase complex. In conclusion, our data clearly demonstrate that NCCRP1 is not a transmembrane protein responsible for the cytolytic function of nonspecific cytotoxic cells as has been previously suggested. Therefore, the name of Nccrp1 should be changed to FBXO50.

The carbonic anhydrase (CA, EC 4.2.1.1) isozymes IX and XII are predominantly found in tumor cells and show a restricted expression in normal tissues. By efficiently hydrating carbon dioxide to protons and bicarbonate, these CAs contribute significantly to the extracellular acidification of solid tumors. CA IX and XII are overexpressed in many such tumors in response to the hypoxia inducible factor (HIF) pathway, and research on the involvement of these isozymes in cancer has progressed in recent years. The report of the X-ray crystal structure of CA IX, which is a dimeric protein with a quaternary structure not evidenced earlier for this family of enzymes, allows for structure-based drug design campaigns of inhibitors against this novel antitumor target. Indeed, it has been known for some time that aromatic/heterocyclic sulfonamides and sulfamates have good affinity for this isoform, but generally they do not show specificity for the inhibition of the tumor-associated isoform versus the remaining CA isozymes (CA I-VII, and XII-XV) found in mammals. Recently, we reported several classes of compounds with good selectivity for the tumor-associated CAs, being shown that CA IX/XII inhibition reverses the effect of tumor acidification, leading to inhibition of the cancer cells growth. CA IX/XII are now proposed as novel therapeutic antitumor targets. Furthermore, as some types of CA inhibitors (CAIs), such as the fluorescent sulfonamides accumulate only in hypoxic tumor cells overexpressing these enzymes, CAIs may be also used as diagnostic tools for imaging of hypoxic cancer cells. Work from several laboratories recently reported the proof-of-concept studies for the use of CA IX/XII inhibitors as well as antibodies both in the therapy and imaging of hypoxic tumors.

Özensoy, Özen (Balikesir Author), Metaloenzim karbonik anhidrazın [CA, EC 4.2.1.1] izoenzimi IX -CA IX- hipokside yüksek düzeyde ifade edilen genlerden birisidir. CA IX özellikle hipoksik tümör dokularında bulunup gastrointestinal yoldaki düşük düzeydeki ekspresyonu haricinde birçok normal dokuda bulunmayan yüksek aktiviteli ve tümörle ilişkili bir membran enzimidir. CA IX’un yeni antikanser tedavileri için ilaçlara hedef oluşturabileceği ve bir tümör ilerleme belirteci olduğu gösterilmiştir. Floresan ve membran geçirgenliğini etkileyen sülfanamidler gibi özgül inhibitörler kullanılarak bu tümörle ilişkili membran enzim aktivitesinin engellenmesinin tümör pH’sını değiştirip asiditeyi azalttığı ve normal pH değerlerine doğru 0.5-0.7 pH ünitesi kadar yaklaştırdığı gösterilmiştir. Bu nedenle, CA IX antikanser ilaç geliştirilmesi ve daha seçici ve etkin CA IX inhibitörlerinin kullanımıyla hipoksik kanserlerde pH dengesizliğinin kontrol edilmesi ve genellikle klasik kemo ve radyo terapiye cevap vermeyen bu tümörlerin tanı ve tedavisinde yararlı olabilecektir., One of the genes highly upregulated by hypoxia is that encoding isozyme IX of the metalloenzyme carbonic anhydrase [CA, EC 4.2.1.1], CA IX. CA IX is a high activity tumor-associated membrane enzyme predominantly found in hypoxic tumor tissues being absent from most normal tissues except for a low level of expression in the gastrointestinal tract. CA IX was demonstrated to be a druggable target for the development of novel anticancer therapies and as a tumor progression marker. Inhibition of this tumor associated membrane enzymatic activity by specific inhibitors, such as fluorescent- and membrane-impermeant sulfonamides, was shown to lead to changes in the tumor pH, with a reversal of the acidification towards more normal values by 0.5 #8211; 0.7 pH units. For this reason CA IX is an interesting target for anticancer drug development whereas more selective and powerful CA IX inhibitors could prove useful for elucidating the role of this protein in hypoxic cancers, for controlling the pH imbalance in tumor cells and for developing diagnostic or therapeutic applications for the management of hypoxic tumors, generally non-responsive to classical chemo-and radiotherapy.