We examined the expression of carbonic anhydrase IX (CA IX) by immunohistochemical staining using monoclonal antibody M75 (Institute of Virology, Slovak Academy of Sciences, Bratislava) in a group of 38 fibroadenomas and 55 carcinomas of the breast. In each case, the intensity of staining, percentage of labeled cells and subcellular localization of CA IX were assessed. CA IX was detected in 11/38 fibroadenomas (28.9%). Weak cytoplasmic positivity was dominant in these positive cases. Immunohistochemical analysis of 55 carcinomas showed CA IX expression in 34 cases (61.8%). Membrane staining alone was observed in 27/55 carcinomas (49.1%), while cytoplasmic positivity was found in 4/55 cases (7.3%). Combined membrane and cytoplasmic immunostaining of CA IX was detected in 3/55 carcinomas (5.4%). The intensity of immunoreactivity varied from weak to strong. Under 50% of reactive cells were found in 9/38 fibroadenomas (23.6%) and in 29/55 carcinomas (52.7%). More than 50% of reactive cells were found in 2/38 fibroadenomas (5.3%) and in 5/55 carcinomas (9.1%). Statistical analysis confirmed significant differences in the subcellular localization, intensity of immunoreactivity and percentage of labeled cells in fibroadenomas and carcinomas (p<0.05). Our results confirmed the hypothesis that expression of CA IX may represent a valuable tumor biomarker as well as a promising diagnostic and prognostic parameter in breast cancer.

• Klíčová slova
• Breast cancer, Carbonic anhydrase IX, Immunohistochemistry, Tumor biomarker,
• MeSH
• antigeny nádorové metabolismus   MeSH
• fibroadenom enzymologie   patologie   MeSH
• karboanhydrasa IX MeSH
• karboanhydrasy metabolismus   MeSH
• lidé MeSH
• nádorové biomarkery metabolismus   MeSH
• nádory prsu enzymologie   patologie   MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• karboanhydrasa IX MeSH
• karboanhydrasy MeSH
• nádorové biomarkery MeSH

The tumor microenvironment includes a complicated network of physiological gradients contributing to plasticity of tumor cells and heterogeneity of tumor tissue. Hypoxia is a key component generating intratumoral oxygen gradients, which affect the cellular expression program and lead to therapy resistance and increased metastatic propensity of weakly oxygenated cell subpopulations. One of the adaptive responses of tumor cells to hypoxia involves the increased expression and functional activation of carbonic anhydrase IX (CA IX), a cancer-related cell surface enzyme catalyzing the reversible conversion of carbon dioxide to bicarbonate ion and proton. Via its catalytic activity, CA IX participates in regulation of intracellular and extracellular pH perturbations that result from hypoxia-induced changes in cellular metabolism producing excess of acid. Through the ability to regulate pH, CA IX also facilitates cell migration and invasion. In addition, CA IX has non-catalytic function in cell adhesion and spreading. Thus, CA IX endows tumor cells with survival advantages in hypoxia/acidosis and confers an increased ability to migrate, invade and metastasize. Accordingly, CA IX is expressed in a broad range of tumors, where it is associated with prognosis and therapy outcome. Its expression pattern and functional implications in tumor biology make CA IX a promising therapeutic target, which can be hit either by immunotherapy with monoclonal antibodies or with compounds inhibiting its enzyme activity. The first strategy has already reached the clinical trials, whereas the second one is still in preclinical testing. Both strategies indicate that CA IX can become a clinically useful anticancer target, but urge further efforts toward better selection of patients for immunotherapy and deeper understanding of tumor types, clinical situations and synthetic lethality interactions with other treatment approaches.

• Klíčová slova
• Carbonic anhydrase IX, Hypoxia, Immunotherapy, Migration–invasion, pH regulation,
• MeSH
• antigeny nádorové imunologie   metabolismus   MeSH
• biokatalýza účinky léků   MeSH
• cílená molekulární terapie metody   MeSH
• hypoxie enzymologie   MeSH
• inhibitory enzymů terapeutické užití   MeSH
• karboanhydrasa IX MeSH
• karboanhydrasy imunologie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• monoklonální protilátky imunologie   terapeutické užití   MeSH
• nádory farmakoterapie   enzymologie   patologie   MeSH
• pohyb buněk * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• inhibitory enzymů MeSH
• karboanhydrasa IX MeSH
• karboanhydrasy MeSH
• monoklonální protilátky MeSH

Expression of CA IX is normally restricted to the mucosa of alimentary tract, but on the other hand, it takes place in a high percentage of human cancers derived from tissues which are normally CA IX-negative. It is a transmembrane protein with two extracellular domains: carbonic anhydrase (CA) with a high catalytic activity and a proteoglycan-like segment (PG), mediating cell-cell adhesion. Both CA and PG domains interact with the microenvironment and they could play a role in tumorigenesis, but their roles are poorly understood. The present work characterizes some newly recognized properties of the PG. One of them is a prevalently negative charge, caused by a high proportion of dicarboxylic amino acids. This is reflected by easy dissociation of complexes formed by PG either with monoclonal antibody M75 or with the cell surface receptor already at slightly acidic pH. This property might facilitate separation of cells from the primary tumor. Released cells may subsequently attach elsewhere in the organism and eventually start metastatic growth. Another aim of the present study was to identify human tumor cell lines which are expressing the presumed CA IX receptor molecule. The same cell lines were also tested for the presence of CA IX protein; we found that expression of CA IX and of the receptor is independent of each other. In addition, we examined the species specificity of CA IX receptors. The PG domain, which contains the epitope of mAb M75 -PGEEDLP- overlapping with the binding site for putative receptor is relatively conserved in evolution: human and rat CA IX cross-react with M75 antibody on western blots. Consistently with this, human and rat cells can attach to purified human CA IX protein. On the other hand, murine CA IX contains an entirely different equivalent of PG sequence and it does not react with M75 antibody or attach to human CA IX protein. This is suggestive of the co-evolution of CA IX protein together with its receptor.

• MeSH
• antigeny nádorové metabolismus   MeSH
• buněčná adheze MeSH
• buňky 3T3 MeSH
• ELISA MeSH
• HeLa buňky MeSH
• karboanhydrasa IX MeSH
• karboanhydrasy metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lidé MeSH
• monoklonální protilátky MeSH
• myši MeSH
• nádory enzymologie   MeSH
• referenční hodnoty MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• karboanhydrasa IX MeSH
• karboanhydrasy MeSH
• monoklonální protilátky MeSH

Carbonic anhydrase IX (CAIX) is an enzyme expressed on the surface of cells in hypoxic tumors. It plays a role in regulation of tumor pH and promotes thus tumor cell survival and occurrence of metastases. Here, derivatives of the cobalt bis(dicarbollide)(1-) anion are reported that are based on substitution at the carbon sites of the polyhedra by two alkylsulfonamide groups differing in the length of the aliphatic connector (from C1 to C4, n=1-4), which were prepared by cobalt insertion into the 7-sulfonamidoalkyl-7,8-dicarba-nido-undecaborate ions. Pure meso- and rac-diastereoisomeric forms were isolated. The series is complemented with monosubstituted species (n=2). Synthesis by a direct method furnished similar derivatives (n=2, 3), which are chlorinated at the B(8,8') boron sites. All compounds inhibited CAIX with subnanomolar inhibition constants and showed high selectivity for CAIX. The best inhibitory properties were observed for the compound with n= 3 and two substituents present in rac-arrangement with Ki =20 pM and a selectivity index of 668. X-ray crystallography was used to study interactions of these compounds with the active site of CAIX on the structural level.

• Klíčová slova
• anti-tumor agents, carbonic anhydrase IX, carboranes, cobalt, enzyme inhibitors,
• MeSH
• inhibitory karboanhydras chemie   metabolismus   terapeutické užití   MeSH
• karboanhydrasa IX antagonisté a inhibitory   metabolismus   MeSH
• katalytická doména MeSH
• kobalt chemie   MeSH
• komplexní sloučeniny chemie   metabolismus   terapeutické užití   MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• molekulární konformace MeSH
• nádory farmakoterapie   patologie   MeSH
• simulace molekulového dockingu MeSH
• sulfonamidy chemie   MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory karboanhydras MeSH
• karboanhydrasa IX MeSH
• kobalt MeSH
• komplexní sloučeniny MeSH
• sulfonamidy MeSH

Human carbonic anhydrase IX (CA IX), a protein specifically expressed on the surface of solid tumour cells, represents a validated target both for anticancer therapy and diagnostics. We recently identified sulfonamide dicarbaboranes as promising inhibitors of CA IX with favourable activities both in vitro and in vivo. To explain their selectivity and potency, we performed detailed X-ray structural analysis of their interactions within the active sites of CA IX and CA II. Series of compounds bearing various aliphatic linkers between the dicarbaborane cluster and sulfonamide group were examined. Preferential binding towards the hydrophobic part of the active site cavity was observed. Selectivity towards CA IX lies in the shape complementarity of the dicarbaborane cluster with a specific CA IX hydrophobic patch containing V131 residue. The bulky side chain of F131 residue in CA II alters the shape of the catalytic cavity, disrupting favourable interactions of the spherical dicarbaborane cluster.

• Klíčová slova
• Carbonic anhydrase IX, carborane, enzyme inhibitors, structure-activity relationship,
• MeSH
• antigeny nádorové genetika   MeSH
• HEK293 buňky MeSH
• hydrofobní a hydrofilní interakce MeSH
• inhibitory karboanhydras chemie   farmakologie   MeSH
• karboanhydrasa IX antagonisté a inhibitory   genetika   MeSH
• katalytická doména MeSH
• krystalografie rentgenová MeSH
• lidé MeSH
• protinádorové látky chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• sekvence aminokyselin MeSH
• sloučeniny boru chemie   MeSH
• sulfonamidy chemie   farmakologie   MeSH
• vazba proteinů MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• decaborane MeSH Prohlížeč
• inhibitory karboanhydras MeSH
• karboanhydrasa IX MeSH
• protinádorové látky MeSH
• sloučeniny boru MeSH
• sulfonamidy MeSH

A new series of s-triazine derivatives incorporating sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and piperazine or aminoalcohol structural motifs is reported. Molecular docking was exploited to select compounds from virtual combinatorial library for synthesis and subsequent biological evaluation. The compounds were prepared by using step by step nucleophilic substitution of chlorine atoms from cyanuric chloride (2,4,6-trichloro-1,3,5-triazine). The compounds were tested as inhibitors of physiologically relevant carbonic anhydrase (CA, EC 4.2.1.1) isoforms. Specifically, against the cytosolic hCA I, II and tumor-associated hCA IX. These compounds show appreciable inhibition. hCA I was inhibited with KIs in the range of 8.5-2679.1 nM, hCA II with KIs in the range of 4.8-380.5 nM and hCA IX with KIs in the range of 0.4-307.7 nM. As other similar derivatives, some of the compounds showed good or excellent selectivity ratios for inhibiting hCA IX over hCA II, of 3.5-18.5. 4-[({4-Chloro-6-[(4-hydroxyphenyl)amino]-1,3,5-triazin-2-yl}amino)methyl] benzene sulfonamide demonstrated subnanomolar affinity for hCA IX (0.4 nM) and selectivity (18.50) over the cytosolic isoforms. This series of compounds may be of interest for the development of new, unconventional anticancer drugs targeting hypoxia-induced CA isoforms such as CA IX.

• Klíčová slova
• 1,3,5-Triazine, Benzene sulfonamides, Carbonic anhydrase, Enzyme inhibition, Isoform selectivity,
• MeSH
• aminoalkoholy chemie   farmakologie   MeSH
• antigeny nádorové metabolismus   MeSH
• inhibitory karboanhydras chemická syntéza   chemie   farmakologie   MeSH
• karboanhydrasa I antagonisté a inhibitory   metabolismus   MeSH
• karboanhydrasa II antagonisté a inhibitory   metabolismus   MeSH
• karboanhydrasa IX antagonisté a inhibitory   metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• piperazin chemie   farmakologie   MeSH
• sulfonamidy chemie   farmakologie   MeSH
• triaziny chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminoalkoholy MeSH
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• inhibitory karboanhydras MeSH
• karboanhydrasa I MeSH
• karboanhydrasa II MeSH
• karboanhydrasa IX MeSH
• piperazin MeSH
• sulfonamidy MeSH
• triaziny MeSH

A novel library of human carbonic anhydrase (hCA) inhibitors based on the 2-sulfanilamido[1,2,4]triazolo[1,5-a]pyrimidine skeleton modified at its 7-position was prepared by an efficient convergent procedure. These derivatives were evaluated in vitro for their inhibition properties against a representative panel of hCA isoforms (hCA I, II, IV, IX, and XII). The target tumour-associated isoforms hCA IX and XII were potently inhibited with KIs in the low nanomolar range of 5-96 nM and 4-72 nM, respectively. Compounds 1d, 1j, 1v, and 1x were the most potent hCA IX inhibitors with KIs of 5.1, 8.6, 4.7, and 5.1 nM, respectively. Along with derivatives 1d and 1j, compounds 1r and 1ab potently inhibited hCA XII isoform with KIs in a single-digit nanomolar range of 8.8, 5.4, 4.3, and 9.0 nM, respectively. Compounds 1e, 1m, and 1p exhibited the best selectivity against hCA IX and hCA XII isoforms over off-target hCA II, with selectivity indexes ranging from 5 to 14.

• Klíčová slova
• Carbonic anhydrase inhibitors, [1,2,4]triazolo[15-a]pyrimidine, antiproliferative activity, structure–activity relationship, sulphanilamide,
• MeSH
• antigeny nádorové * MeSH
• inhibitory karboanhydras farmakologie   MeSH
• karboanhydrasa I metabolismus   MeSH
• karboanhydrasa II * metabolismus   MeSH
• karboanhydrasa IX metabolismus   MeSH
• lidé MeSH
• molekulární struktura MeSH
• protein - isoformy MeSH
• sulfanilamidy MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové * MeSH
• inhibitory karboanhydras MeSH
• karboanhydrasa I MeSH
• karboanhydrasa II * MeSH
• karboanhydrasa IX MeSH
• protein - isoformy MeSH
• sulfanilamidy MeSH

Carbonic anhydrase IX (CA IX), a tumor-associated metalloenzyme, represents a validated target for cancer therapy and diagnostics. Herein, we report the inhibition properties of isomeric families of sulfonamidopropyl-dicarba-closo-dodecaboranes group(s) prepared using a new direct five-step synthesis from the corresponding parent cages. The protocol offers a reliable solution for synthesis of singly and doubly substituted dicarba-closo-dodecaboranes with a different geometric position of carbon atoms. The closo-compounds from the ortho- and meta-series were then degraded to corresponding 11-vertex dicarba-nido-undecaborate(1-) anions. All compounds show in vitro enzymatic activity against CA IX in the low nanomolar or subnanomolar range. This is accompanied by clear isomer dependence of the inhibition constant (Ki ) and selectivity towards CA IX. Decreasing trends in Ki and selectivity index (SI ) values are observed with increasing separation of the cage carbon atoms. Interactions of compounds with the active sites of CA IX were explored with X-ray crystallography, and eight high-resolution crystal structures uncovered the structural basis of inhibition potency and selectivity.

• Klíčová slova
• carbonic anhydrase, crystallography, dicarba-closo-dodecaboranes, inhibitors, sulfonamide,
• MeSH
• antigeny nádorové chemie   metabolismus   MeSH
• inhibitory karboanhydras * farmakologie   MeSH
• izoenzymy MeSH
• karboanhydrasa I chemie   metabolismus   MeSH
• karboanhydrasa IX chemie   metabolismus   MeSH
• lidé MeSH
• nádory * MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• inhibitory karboanhydras * MeSH
• izoenzymy MeSH
• karboanhydrasa I MeSH
• karboanhydrasa IX MeSH

Carbonic anhydrase IX (CA IX) is a transmembrane enzyme overexpressed in hypoxic tumors, where it plays an important role in tumor progression. Specific CA IX inhibitors potentially could serve as anti-cancer drugs. We designed a series of sulfonamide inhibitors containing carborane clusters based on prior structural knowledge of carborane binding into the enzyme active site. Two types of carborane clusters, 12-vertex dicarba-closo-dodecaborane and 11-vertex 7,8-dicarba-nido-undecaborate (dicarbollide), were connected to a sulfonamide moiety via aliphatic linkers of varying lengths (1-4 carbon atoms; n = 1-4). In vitro testing of CA inhibitory potencies revealed that the optimal linker length for selective inhibition of CA IX was n = 3. A 1-sulfamidopropyl-1,2-dicarba-closo-dodecaborane (3) emerged as the strongest CA IX inhibitor from this series, with a Ki value of 0.5 nM and roughly 1230-fold selectivity towards CA IX over CA II. X-ray studies of 3 yielded structural insights into their binding modes within the CA IX active site. Compound 3 exhibited moderate cytotoxicity against cancer cell lines and primary cell lines in 2D cultures. Cytotoxicity towards multicellular spheroids was also observed. Moreover, 3 significantly lowered the amount of CA IX on the cell surface both in 2D cultures and spheroids and facilitated penetration of doxorubicin. Although 3 had only a moderate effect on tumor size in mice, we observed favorable ADME properties and pharmacokinetics in mice, and preferential presence in brain over serum.

• Klíčová slova
• Anti-tumor agents, Carbonic anhydrase IX, Carboranes, Dicarbollide, Drug penetration, Enzyme inhibitors, Multicellular spheroids,
• MeSH
• antigeny nádorové metabolismus   MeSH
• experimentální nádory farmakoterapie   metabolismus   MeSH
• inhibitory karboanhydras chemická syntéza   chemie   farmakologie   MeSH
• karboanhydrasa IX antagonisté a inhibitory   metabolismus   MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární struktura MeSH
• myši inbrední BALB C MeSH
• myši SCID MeSH
• myši MeSH
• nádory prsu farmakoterapie   metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   MeSH
• psi MeSH
• rekombinantní proteiny metabolismus   MeSH
• screeningové testy protinádorových léčiv MeSH
• sulfonamidy chemická syntéza   chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• inhibitory karboanhydras MeSH
• karboanhydrasa IX MeSH
• protinádorové látky MeSH
• rekombinantní proteiny MeSH
• sulfonamidy MeSH

Encapsulation is a well-established method of biomaterial protection, controlled release, and efficient delivery. Here we evaluated encapsulation of monoclonal antibody M75 directed to tumor biomarker carbonic anhydrase IX (CA IX) into alginate microbeads (SA-beads) or microcapsules made of sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) (PMCG). M75 antibody release was quantified using ELISA and its binding properties were assessed by immunodetection methods. SA-beads showed rapid M75 antibody release in the first hour, followed by steady release during the whole experiment of 7 days. In contrast, the M75 release from PMCG capsules was gradual, reaching the maximum concentration on the 7th day. The release was more efficient at pH 6.8 compared to pH 7.4. The released antibody could recognize CA IX, and target the CA IX-positive cells in 3D spheroids. In conclusion, SA-beads and PMCG microcapsules can be considered as promising antibody reservoirs for targeting of cancer cells.

• Klíčová slova
• Antibody delivery, cancer, carbonic anhydrase IX, controlled release, encapsulation,
• MeSH
• antigeny nádorové imunologie   metabolismus   MeSH
• buněčné sféroidy metabolismus   MeSH
• karboanhydrasa IX imunologie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• mikrosféry * MeSH
• monoklonální protilátky aplikace a dávkování   imunologie   farmakokinetika   MeSH
• nádorové biomarkery imunologie   metabolismus   MeSH
• nádorové buňky kultivované MeSH
• nádory metabolismus   patologie   MeSH
• PEG-DMA hydrogel * MeSH
• protinádorové látky aplikace a dávkování   MeSH
• uvolňování léčiv MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny nádorové MeSH
• CA9 protein, human MeSH Prohlížeč
• karboanhydrasa IX MeSH
• M75 monoclonal antibody MeSH Prohlížeč
• monoklonální protilátky MeSH
• nádorové biomarkery MeSH
• PEG-DMA hydrogel * MeSH
• protinádorové látky MeSH