BACKGROUND: Carbonic anhydrase IX (CA IX) is a hypoxia-induced enzyme regulating tumour pH and facilitating cell migration/invasion. It is primarily expressed as a transmembrane cell-surface protein, but its ectodomain can be shed by ADAM17 to extracellular space. This study aims to elucidate the impact of CA IX shedding on cancer cells. METHODS: We generated a non-shed CA IX mutant by deletion of amino acids 393-402 from the stalk region and studied its phenotypic effects compared to full-length, shedding-competent CA IX using a range of assays based on immunodetection, confocal microscopy, in vitro real-time cell monitoring and in vivo tumour cell inoculation using xenografted NMRI and C57BL/6J female mice. RESULTS: We demonstrated that the impairment of shedding does not alter the ability of CA IX to bind ADAM17, internalise, form oligomers and regulate pH, but induces cancer-promoting changes in extracellular proteome. Moreover, it affects intrinsic properties of cells expressing the non-shed variant, in terms of their increased ability to migrate, generate primary tumours and form metastatic lesions in lungs. CONCLUSIONS: Our results show that the ectodomain shedding controls pro-tumorigenic and pro-metastatic roles of the cell-associated CA IX and suggest that this phenomenon should be considered when developing CA IX-targeted therapeutic strategies.
- MeSH
- fenotyp MeSH
- invazivní růst nádoru patologie MeSH
- karboanhydrasa IX metabolismus MeSH
- karcinogeneze metabolismus patologie MeSH
- lidé MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory metabolismus patologie MeSH
- protein ADAM17 metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Diagnosis of oncological diseases remains at the forefront of current medical research. Carbonic Anhydrase IX (CA IX) is a cell surface hypoxia-inducible enzyme functionally involved in adaptation to acidosis that is expressed in aggressive tumors; hence, it can be used as a tumor biomarker. Herein, we propose a nanoscale graphene oxide (GO) platform functionalized with magnetic nanoparticles and a monoclonal antibody specific to the CA IX marker. The GO platforms were prepared by a modified Hummers and Offeman method from exfoliated graphite after several centrifugation and ultrasonication cycles. The magnetic nanoparticles were prepared by a chemical precipitation method and subsequently modified. Basic characterization of GO, such as the degree of oxidation, nanoparticle size and exfoliation, were determined by physical and chemical analysis, including X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), energy dispersive X-ray analysis (EDX), and atomic force microscopy (AFM). In addition, the size and properties of the poly-L-lysine-modified magnetic nanoparticles were characterized. The antibody specific to CA IX was linked via an amidic bond to the poly-L-lysine modified magnetic nanoparticles, which were conjugated to GO platform again via an amidic bond. The prepared GO-based platform with magnetic nanoparticles combined with a biosensing antibody element was used for a hypoxic cancer cell targeting study based on immunofluorescence.
- Publikační typ
- časopisecké články 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.
- MeSH
- antigeny nádorové imunologie metabolismus MeSH
- antitumorózní látky aplikace a dávkování MeSH
- buněčné sféroidy metabolismus MeSH
- karboanhydrasa IX imunologie metabolismus MeSH
- koncentrace vodíkových iontů 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
- systémy cílené aplikace léků metody MeSH
- uvolňování léčiv MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Carbonic anhydrase IX (CA IX) is a tumor-associated, highly active, transmembrane carbonic anhydrase isoform regulated by hypoxia and implicated in pH control and adhesion-migration-invasion. CA IX ectodomain (ECD) is shed from the tumor cell surface to serum/plasma of patients, where it can signify cancer prognosis. We previously showed that the CA IX ECD release is mediated by disintegrin and metalloproteinase ADAM17. Here we investigated the CA IX ECD shedding in tumor cells undergoing apoptosis in response to cytotoxic drugs, including cycloheximide and doxorubicin. METHODS: Presence of cell surface CA IX was correlated to the extent of apoptosis by flow cytometry in cell lines with natural or ectopic CA IX expression. CA IX ECD level was assessed by ELISA using CA IX-specific monoclonal antibodies. Effect of recombinant CA IX ECD on the activation of molecular pathways was evaluated using the cell-based dual-luciferase reporter assay. RESULTS: We found a significantly lower occurrence of apoptosis in the CA IX-positive cell subpopulation than in the CA IX-negative one. We also demonstrated that the cell-surface CA IX level dropped during the death progress due to an increased ECD shedding, which required a functional ADAM17. Inhibitors of metalloproteinases reduced CA IX ECD shedding, but not apoptosis. The CA IX ECD release induced by cytotoxic drugs was connected to elevated expression of CA IX in the surviving fraction of cells. Moreover, an externally added recombinant CA IX ECD activated a pathway driven by the Nanog transcription factor implicated in epithelial-mesenchymal transition and stemness. CONCLUSIONS: These findings imply that the increased level of the circulating CA IX ECD might be useful as an indicator of an effective antitumor chemotherapy. Conversely, elevated CA IX ECD might generate unwanted effects through autocrine/paracrine signaling potentially contributing to resistance and tumor progression.
- MeSH
- apoptóza účinky léků genetika MeSH
- cykloheximid aplikace a dávkování MeSH
- epitelo-mezenchymální tranzice genetika MeSH
- HeLa buňky MeSH
- hypoxie buňky genetika MeSH
- karboanhydrasa IX aplikace a dávkování genetika metabolismus MeSH
- lidé MeSH
- monoklonální protilátky aplikace a dávkování MeSH
- nádory genetika patologie MeSH
- protein ADAM17 genetika metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Východiská: Hypoxia lokálne pokročilých karcinómov hlavy a krku je jednou z hlavných príčin ich rádiorezistencie, ktorá sa klinicky manifestuje perzistenciou reziduálnej nádorovej choroby po liečbe rádioterapiou. Detekcia nádorovej hypoxie teda môže byť dôležitým prediktorom efektivity liečby. Karboanhydráza IX (carbonic anhydrase IX – CA IX) je proteín, kódovaný rovnomenným génom, ktorého expresia sa v nádorových tkanivách za hypoxických podmienok zvyšuje. CA IX preto predstavuje endogénny marker nádorovej hypoxie, identifikovateľný v nádorových tkanivách a jeho rozpustnú extracelulárnu doménu je možné detekovať aj v telesných tekutinách pacienta. Primárnym cieľom tejto štúdie bolo zistiť, či existuje korelácia medzi sérovou hladinou CA IX a reziduálnou nádorovou chorobou po liečbe. Sekundárnym cieľom bolo zistiť, ako sa mení sérová hladina CA IX počas frakcionovanej rádioterapie. Materiál a metódy: Táto prospektívna monocentrická klinická štúdia hodnotila súbor 30 pacientov s lokálne pokročilými skvamocelulárnymi karcinómami hlavy a krku, liečených rádioterapiou alebo konkomitantnou chemorádioterapiou s kuratívnym zámerom. Sérová koncentrácia rozpustnej formy CA IX sa vyšetrovala z venóznej krvi pomocou sendvičovej enzýmovej imunoanalýzy (ELISA) pred zahájením rádioterapie, v polovici rádioterapie, pri skončení rádioterapie a šesť týždňov po jej ukončení. Výsledky: Zistili sme výraznú variabilitu nameraných hladín CA IX v sledovanej populácii v rozsahu 0–1 696 pg/ml. Významné zmeny v strednej hodnote koncentrácie CA IX sme v priebehu rádioterapie a po nej nezistili. U 11 pacientov (36,7 %) sa liečbou dosiahla kompletná remisia ochorenia. U týchto pacientov sme zaznamenali v priemere nižšie predliečebné hodnoty CA IX v porovnaní s pacientmi s perzistenciou reziduálnej nádorovej choroby (37,57 vs 77,47; p = 0,154). Záver: Naše výsledky ukazujú, že sérová hladina CA IX sa u pacientov s lokálne pokročilými nádormi hlavy a krku v priebehu frakcionovanej rádioterapie významne nemení. Zistený vzťah medzi sérovou hladinou CA IX a reziduálnou nádorovou chorobou po rádioterapii je potrebné potvrdiť na väčšom súbore pacientov.
Background: Hypoxia of locally advanced head and neck cancers is one of the main causes of their radiation resistance that presents clinically as a persistence of residual tumor disease after radiation therapy. Therefore, detection of tumor hypoxia could be an important predictor of treatment efficacy. Carbonic anhydrase IX (CA IX) is a protein, coded by a homonymous gene, the expression of which increases in tumor tissues at hypoxic conditions. Hence, CA IX represents an endogenic marker of tumor hypoxia, identifiable in tumor tissues, and its soluble extracellular domain can also be detected in body fluids of the patient. The primary endpoint of this study was to explore whether a correlation exists between CA IX serum level and the residual tumor disease after therapy. The secondary endpoint was to find out how the serum concentration of CA IX changes during the course of fractionated radiation therapy. Materials and Methods: The presented prospective monocentric clinical study evaluated a population of 30 patients with locally advanced squamous cell head and neck cancers, treated by radiation therapy or concurrent chemo‑radiation therapy with a curative intent. The serum concentration of the soluble form of CA IX was examined from a venous blood sample, using sandwich enzyme‑linked immunosorbent assay (ELISA). The blood samples were obtained before the treatment initiation, in the middle of radiation therapy, at the time of finishing radiation therapy and six weeks after the treatment completion. Results: We found a substantial variability in the CA IX levels measured in the examined population, ranging 0–1,696 pg/ml. We found no significant changes in the mean value of CA IX concentration during the course of radiation therapy and after the treatment completion. In 11 patients (36.7%), the treatment resulted in complete remission of the disease. In these patients, lower average pre‑treatment levels of CA IX were noted when compared to patients with persistence of residual tumor disease (37.57 vs 77.47; p = 0.154). Conclusion: The results indicate that serum level of CA IX in patients with locally advanced head and neck cancers does not change significantly during the course of fractionated radiation therapy. The relation between CA IX serum level and residual tumor disease after radiation therapy requires verification on a larger population of patients. Key words: head and neck cancer – hypoxia – carbonic anhydrase IX – radiation therapy – residual tumor The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE “uniform requirements” for biomedical papers. Submitted: 10. 2. 2014 Accepted: 4. 3. 2014
- Klíčová slova
- karboanhydráza IX, radiorezistence,
- MeSH
- ELISA MeSH
- frakcionace dávky záření MeSH
- hypoxie buňky * účinky záření MeSH
- karboanhydrasy * krev MeSH
- lidé středního věku MeSH
- lidé MeSH
- nádorové biomarkery * krev MeSH
- nádorové proteiny * krev MeSH
- nádory hlavy a krku * enzymologie krev metabolismus radioterapie MeSH
- prospektivní studie MeSH
- reziduální nádor krev MeSH
- senioři MeSH
- spinocelulární karcinom enzymologie krev metabolismus radioterapie MeSH
- statistika jako téma MeSH
- transport proteinů MeSH
- výsledek terapie MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
Napriek tomu, že nové biologické a klinické poznatky o nádoroch obličiek neustále pribúdajú, manažment onkologických pacientov aj naďalej zostáva problematický. Významný prelom síce predstavuje zavedenie cielených terapeutík, ktoré majú výrazne lepší terapeutický efekt než konvenčná terapia, ďalší pokrok však možno dosiahnuť iba pomocou identifikácie a validácie relevantných biomarkerov na racionálnu stratifikáciu pacientov a voľbu vhodnej liečebnej stratégie. Cieľom prezentovanej štúdie je objasniť klinický význam anhydrázy IX kyseliny uhličitej (CA IX, z angl. carbonic anhydrase IX), ktorá je úzko viazaná s obličkovými nádormi a má zjavný diagnostický, prognostický, ako aj terapeutický potenciál.
Despite continuously increasing knowledge of biological and clinical features of kidney cancer, management of tumour patients remains problematic. Use of targeted therapeutics - which is much more effective compared to conventional therapy - is an important step forward; however, further progress can be achieved only through identification and validation of relevant biomarkers for rational stratification of patients and selection of proper treatment strategy. The aim of the proposed study is to clarify clinical importance of carbonic anhydrase IX that is tightly associated with kidney tumours and has a conclusive diagnostic, prognostic, as well as therapeutic potential.
