Can hypoxia marker carbonic anhydrase IX serve as a potential new diagnostic marker and therapeutic target of non-small cell lung cancer?

Lung cancer represents the leading cause of cancer-related deaths. Non-small cell lung cancer (NSCLC), the most common form of lung cancer, is a molecularly heterogeneous disease with intratumoral heterogeneity and a significant mutational burden associated with clinical outcome. Tumor microenvironment (TME) plays a fundamental role in the initiation and progression of primary de novo lung cancer and significantly influences the response of tumor cells to therapy. Hypoxia, an integral part of the tumor microenvironment and a serious clinical phenomenon, is associated with increased genetic instability and a more aggressive phenotype of NSCLC, which correlates with the risk of metastasis. Low oxygen concentration influences all components of TME including the immune microenvironment. Hypoxia-inducible pathway activated in response to low oxygen supply mediates the expression of genes important for the adaptation of tumor cells to microenvironmental changes. A highly active transmembrane hypoxia-induced metalloenzyme - carbonic anhydrase IX (CAIX), as a part of transport metabolon, contributes to the maintenance of intracellular pH within physiological values and to the acidification of the extracellular space. CAIX supports cell migration and invasion and plays an important role in NSCLC tumor tissue and pleural effusion. Due to its high expression, it also represents a potential diagnostic differential biomarker and therapeutic target in NSCLC. To test new potential targeted therapeutic compounds, suitable models are required that more faithfully simulate tumor tissue, TME components, and spatial architecture.

Unlocking the paracrine crosstalk: adipocyte-derived factors affect carbonic anhydrase IX expression in colon and breast cancer cells.

Obesity is a major public health concern because it increases the risk of several diseases, including cancer. Crosstalk between obesity and cancer seems to be very complex, and the interaction between adipocytes and cancer cells leads to changes in adipocytes' function and their paracrine signaling, promoting a microenvironment that supports tumor growth. Carbonic anhydrase IX (CA IX) is a tumor-associated enzyme that not only participates in pH regulation but also facilitates metabolic reprogramming and supports the migration, invasion, and metastasis of cancer cells. In addition, CA IX expression, predominantly regulated via hypoxia-inducible factor (HIF-1), serves as a surrogate marker of hypoxia. In this study, we investigated the impact of adipocytes and adipocyte-derived factors on the expression of CA IX in colon and breast cancer cells. We observed increased expression of CA9 mRNA as well as CA IX protein in the presence of adipocytes and adipocyte-derived conditioned medium. Moreover, we confirmed that adipocytes affect the hypoxia signaling pathway and that the increased CA IX expression results from adipocyte-mediated induction of HIF-1α. Furthermore, we demonstrated that adipocyte-mediated upregulation of CA IX leads to increased migration and decreased adhesion of colon cancer cells. Finally, we brought experimental evidence that adipocytes, and more specifically leptin, upregulate CA IX expression in cancer cells and consequently promote tumor progression.

Functional consequences of altered glycosylation of tumor-associated hypoxia biomarker carbonic anhydrase IX.

Glycosylation is a posttranslational modification of proteins affecting numerous cellular functions. A growing amount of evidence confirms that aberrant glycosylation is involved in pathophysiological processes, including tumor development and progression. Carbonic anhydrase IX (CAIX) is a transmembrane protein whose expression is strongly induced in hypoxic tumors, which makes it an attractive target for anti-tumor therapy. CAIX facilitates the maintenance of intracellular pH homeostasis through its catalytic activity, which is linked with extracellular pH acidification promoting a more aggressive phenotype of tumor cells. The involvement of CAIX in destabilizing cell-cell contacts and the focal adhesion process also contributes to tumor progression. Previous research shows that CAIX is modified with N-glycans, O-glycans, and glycosaminoglycans (GAG). Still, the impact of glycosylation on CAIX functions has yet to be fully elucidated. By preparing stably transfected cells expressing mutated forms of CAIX, unable to bind glycans at their defined sites, we have attempted to clarify the role of glycan structures in CAIX functions. All three types of prepared mutants exhibited decreased adhesion to collagen. By surface plasmon resonance, we proved direct binding between CAIX and collagen. Cells lacking glycosaminoglycan modification of CAIX also showed reduced migration and invasion, indicating CAIX glycosaminoglycans' involvement in these processes. Analysis of signaling pathways affected by the loss of GAG component from CAIX molecule revealed decreased phosphorylation of c-Jun, of p38α kinase, focal adhesion kinase, and reduced level of heat shock protein 60 in cells cultured in hypoxia. Cells expressing CAIX without GAG exhibited increased metabolon formation and increased extracellular pH acidification. We also observed reduced CAIX GAG glycans in the inflammatory environment in hypoxia, pathophysiological conditions reflecting in vivo tumor microenvironment. Understanding the glycan involvement in the characteristics and functions of possible targets of cancer treatment, such as cell surface localized CAIX, could improve the therapy, as many drugs target glycan parts of a protein.

Propranolol, Promising Chemosensitizer and Candidate for the Combined Therapy through Disruption of Tumor Microenvironment Homeostasis by Decreasing the Level of Carbonic Anhydrase IX.

Resistance to chemotherapy represents a persisting medical problem, ranking among main causes of chemotherapy failure and cancer mortality. There is a possibility to utilize and repurpose already existing therapeutics which were not primarily intended for oncological treatment. Overactivation of adrenergic receptors and signaling dysregulation promotes tumor progression, metastatic potential, immune system evasion, tumor angiogenesis and drug resistance. The non-selective beta-blocker propranolol, approved in infantile haemangioma treatment, has a high potential for use in cancer therapy. We analyzed the effects of propranolol and 5-fluorouracil combination on sensitive and resistant cells derived from colorectal carcinoma in monolayers, single-component and co-culture spheroids and in vivo mouse models. Our results revealed that propranolol is able to exert its effect not only in chemosensitive colorectal cells, but also in 5-fluorouracil resistant cells. Propranolol disrupts the hypoxic adaptation machinery by inhibiting HIF1α, carbonic anhydrase IX, and activates apoptosis, which may be important in the management of chemo-resistant patients. We showed that propranolol slows down the growth of xenografts formed from colorectal cancer cells, even from cells already adapted to the ß-blocker. We provide clear evidence that blockade of ß-adrenergic receptors affects essential signaling pathways modulating tumor microenvironment and thus the response to anticancer therapy. Our findings indicate that propranolol could be repurposed to serve as chemosensitizer in combined therapy aimed at disrupting homeostasis of tumor microenvironment.

Carbonic Anhydrase IX in Tumor Tissue and Plasma of Breast Cancer Patients: Reliable Biomarker of Hypoxia and Prognosis.

Carbonic anhydrase IX (CA IX) is recognized as an excellent marker of hypoxia and an adverse prognostic factor in solid tumors, including breast cancer (BC). Clinical studies confirm that soluble CA IX (sCA IX), shed into body fluids, predicts the response to some therapeutics. However, CA IX is not included in clinical practice guidelines, possibly due to a lack of validated diagnostic tools. Here, we present two novel diagnostic tools-a monoclonal antibody for CA IX detection by immunohistochemistry and an ELISA kit for the detection of sCA IX in the plasma-validated on a cohort of 100 patients with early BC. We confirm that tissue CA IX positivity (24%) correlates with tumor grading, necrosis, negative hormone receptor status, and the TNBC molecular subtype. We show that antibody IV/18 can specifically detect all subcellular forms of CA IX. Our ELISA test provides 70% sensitivity and 90% specificity. Although we showed that this test could detect exosomes in addition to shed CA IX ectodomain, we could not demonstrate a clear association of sCA IX with prognosis. Our results indicate that the amount of sCA IX depends on subcellular CA IX localization, but more strictly on the molecular composition of individual molecular subtypes of BC, particularly on metalloproteinases inhibitor expression.

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.

Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells.

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.

Impairment of Hypoxia-Induced CA IX by Beta-Blocker Propranolol-Impact on Progression and Metastatic Potential of Colorectal Cancer Cells.

The coexistence of cancer and other concomitant diseases is very frequent and has substantial implications for treatment decisions and outcomes. Beta-blockers, agents that block the beta-adrenergic receptors, have been related also to cancers. In the model of multicellular spheroids formed by colorectal cancer cells we described a crosstalk between beta-blockade by propranolol and tumour microenvironment. Non-selective beta-blocker propranolol decreased ability of tumour cells to adapt to hypoxia by reducing levels of HIF1α and carbonic anhydrase IX in 3D spheroids. We indicated a double action of propranolol in the tumour microenvironment by inhibiting the stability of HIF1α, thus mediating decrease of CA IX expression and, at the same time, by its possible effect on CA IX activity by decreasing the activity of protein kinase A (PKA). Moreover, the inhibition of ß-adrenoreceptors by propranolol enhanced apoptosis, decreased number of mitochondria and lowered the amount of proteins involved in oxidative phosphorylation (V-ATP5A, IV-COX2, III-UQCRC2, II-SDHB, I-NDUFB8). Propranolol reduced metastatic potential, viability and proliferation of colorectal cancer cells cultivated in multicellular spheroids. To choose the right treatment strategy, it is extremely important to know how the treatment of concomitant diseases affects the superior microenvironment that is directly related to the efficiency of anti-cancer therapy.

PIMT Binding to C-Terminal Ala459 of CAIX Is Involved in Inside-Out Signaling Necessary for Its Catalytic Activity.

Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.

CAIX Regulates Invadopodia Formation through Both a pH-Dependent Mechanism and Interplay with Actin Regulatory Proteins.

Tumor metastasis is tightly linked with invasive membrane protrusions, invadopodia, formed by actively invading tumor cells. Hypoxia and pH modulation play a role in the invadopodia formation and in their matrix degradation ability. Tumor-associated carbonic anhydrase IX (CAIX), induced by hypoxia, is essential for pH regulation and migration, predisposing it as an active component of invadopodia. To investigate this assumption, we employed silencing and inhibition of CA9, invadopodia isolation and matrix degradation assay. Quail chorioallantoic membranes with implanted tumor cells, and lung colonization assay in murine model were used to assess efficiency of in vivo invasion and the impact of CAIX targeting antibodies. We showed that CAIX co-distributes to invadopodia with cortactin, MMP14, NBCe1, and phospho-PKA. Suppression or enzymatic inhibition of CAIX leads to impaired invadopodia formation and matrix degradation. Loss of CAIX attenuated phosphorylation of Y421-cortactin and influenced molecular machinery coordinating actin polymerization essential for invadopodia growth. Treatment of tumor cells by CAIX-specific antibodies against carbonic or proteoglycan domains results in reduced invasion and extravasation in vivo. For the first time, we demonstrated in vivo localization of CAIX within invadopodia. Our findings confirm the key role of CAIX in the metastatic process and gives rationale for its targeting during anti-metastatic therapy.

Haloperidol Affects Plasticity of Differentiated NG-108 Cells Through σ1R/IP3R1 Complex.

Haloperidol is an antipsychotic agent that primarily acts as an antagonist of D2 dopamine receptors. Besides other receptor systems, it targets sigma 1 receptors (σ1Rs) and inositol 1,4,5-trisphosphate receptors (IP3Rs). Aim of this work was to investigate possible changes in IP3Rs and σ1Rs resulting from haloperidol treatment and to propose physiological consequences in differentiated NG-108 cells, i.e., effect on cellular plasticity. Haloperidol treatment resulted in up-regulation of both type 1 IP3Rs (IP3R1s) and σ1Rs at mRNA and protein levels. Haloperidol treatment did not alter expression of other types of IP3Rs. Calcium release from endoplasmic reticulum (ER) mediated by increased amount of IP3R1s elevated cytosolic calcium and generated ER stress. IP3R1s were bound to σ1Rs, and translocation of this complex from ER to nucleus occurred in the group of cells treated with haloperidol, which was followed by increased nuclear calcium levels. Haloperidol-induced changes in cytosolic, reticular, and nuclear calcium levels were similar when specific σ1 blocker -BD 1047- was used. Changes in calcium levels in nucleus, ER, and cytoplasm might be responsible for alterations in cellular plasticity, because length of neurites increased and number of neurites decreased in haloperidol-treated differentiated NG-108 cells.

Sodium/calcium exchanger is upregulated by sulfide signaling, forms complex with the ß1 and ß3 but not ß2 adrenergic receptors, and induces apoptosis.

Hydrogen sulfide (H2S) as a novel gasotransmitter regulates variety of processes, including calcium transport systems. Sodium calcium exchanger (NCX) is one of the key players in a regulation calcium homeostasis. Thus, the aims of our work were to determine effect of sulfide signaling on the NCX type 1 (NCX1) expression and function in HeLa cells, to investigate the relationship of ß-adrenergic receptors with the NCX1 in the presence and/or absence of H2S, and to determine physiological importance of this potential communication. As a H2S donor, we used morpholin-4-ium-4-methoxyphenyl(morpholino) phosphinodithioate-GYY4137. We observed increased levels of the NCX1 mRNA, protein, and activity after 24 h of GYY4137 treatment. This increase was accompanied by elevated cAMP due to the GYY4137 treatment, which was completely abolished, when NCX1 was silenced. Increased cAMP levels would point to upregulation of ß-adrenergic receptors. Indeed, GYY4137 increased expression of ß1 and ß3 (but not ß2) adrenergic receptors. These receptors co-precipitated, co-localized with the NCX1, and induced apoptosis in the presence of H2S. Our results suggest that sulfide signaling plays a role in regulation of the NCX1, ß1 and ß3 adrenergic receptors, their co-localization, and stimulation of apoptosis, which might be of a potential importance in cancer treatment.

Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts.

BACKGROUND: Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (ß-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function. METHODS: The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively. RESULTS: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine. CONCLUSIONS: Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.

Characterization of carbonic anhydrase IX interactome reveals proteins assisting its nuclear localization in hypoxic cells.

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface.

Carbonic anhydrase IX interacts with bicarbonate transporters in lamellipodia and increases cell migration via its catalytic domain.

Carbonic anhydrase IX (CA IX) is a hypoxia-induced cell surface enzyme expressed in solid tumors, and functionally involved in acidification of extracellular pH and destabilization of intercellular contacts. Since both extracellular acidosis and reduced cell adhesion facilitate invasion and metastasis, we investigated the role of CA IX in cell migration, which promotes the metastatic cascade. As demonstrated here, ectopically expressed CA IX increases scattering, wound healing and transwell migration of MDCK cells, while an inactive CA IX variant lacking the catalytic domain (ΔCA) fails to do so. Correspondingly, hypoxic HeLa cells exhibit diminished migration upon inactivation of the endogenous CA IX either by forced expression of the dominant-negative ΔCA variant or by treatment with CA inhibitor, implying that the catalytic activity is indispensable for the CA IX function. Interestingly, CA IX improves cell migration both in the absence and presence of hepatocyte growth factor (HGF), an established inducer of epithelial-mesenchymal transition. On the other hand, HGF up-regulates CA IX transcription and triggers CA IX protein accumulation at the leading edge of lamellipodia. In these membrane regions CA IX co-localizes with sodium bicarbonate co-transporter (NBCe1) and anion exchanger 2 (AE2) that are both components of the migration apparatus and form bicarbonate transport metabolon with CA IX. Moreover, CA IX physically interacts with AE2 and NBCe1 in situ, as shown here for the first time. Thus, our findings suggest that CA IX actively contributes to cell migration via its ability to facilitate ion transport and pH control at protruding fronts of moving cells.

Isoproterenol accelerates apoptosis through the over-expression of the sodium/calcium exchanger in HeLa cells.

Apoptosis induction causes over-expression of the Na+/Ca2+ exchanger of type 1 (NCX1) in the HeLa cell line. During induction of apoptosis and in the presence of isoproterenol hydrochloride (I; ß-adrenergic agonist), increase in the NCX1 is even more pronounced. Anti-apoptotic Bcl-2 mRNA and protein is markedly reduced during apoptosis and in the presence of I, which causes a rapid increase in the Bax/Bcl-2 ratio. During apoptosis induction by apoptosis inducing kit (A), both with and without I, the active form of caspase-3, which is the executive enzyme in apoptosis, becomes visible on Western blots. Silencing NCX1 resulted in the reversal of the Bax/Bcl-2 ratio, it prevented a decrease in mitochondrial membrane potential compared to the AI group and it decreased the level of AI-induced apoptosis in HeLa cells. Based on the experiments with single apoptotic inducers camptothecin, cycloheximide and dexamethasone, it might be proposed that potentiated apoptotic effect in I-treated cells is due to the inhibition of nuclear topoisomerase. As illustrated in immunofluorescence and Western blot analysis, calnexin increased significantly during induction of the apoptosis in the presence of I. In addition, further decrease in sarco/endoplasmic ATPase 2 (SERCA2), decrease in reticular calcium and mitochondrial membrane potential was observed, which suggests development of the endoplasmic reticulum (ER) stress. Based on these results, we propose that I further enhanced NCX1 expression in apoptotic cells through the development of ER stress.

Selective Tumor Hypoxia Targeting Using M75 Antibody Conjugated Photothermally Active MoOx Nanoparticles.

Photothermal therapy (PTT) mediated at the nanoscale has a unique advantage over currently used cancer treatments, by being spatially highly specific and minimally invasive. Although PTT combats traditional tumor treatment approaches, its clinical implementation has not yet been successful. The reasons for its disadvantage include an insufficient treatment efficiency or low tumor accumulation. Here, we present a promising new PTT platform combining a recently emerged two-dimensional (2D) inorganic nanomaterial, MoOx, and a tumor hypoxia targeting element, the monoclonal antibody M75. M75 specifically binds to carbonic anhydrase IX (CAIX), a hypoxia marker associated with many solid tumors with a poor prognosis. The as-prepared nanoconjugates showed highly specific binding to cancer cells expressing CAIX while being able to produce significant photothermal yield after irradiation with near-IR wavelengths. Small aminophosphonic acid linkers were recognized to be more effective over the combination of poly(ethylene glycol) chain and biotin-avidin-biotin bridge in constructing a PTT platform with high tumor-binding efficacy. The in vitro cellular uptake of nanoconjugates was visualized by high-resolution fluorescence microscopy and label-free live cell confocal Raman microscopy. The key to effective cancer treatment may be the synergistic employment of active targeting and noninvasive, tumor-selective therapeutic approaches, such as nanoscale-mediated PTT. The use of active targeting can streamline nanoparticle delivery increasing photothermal yield and therapeutic success.

NF-κB inhibition significantly upregulates the norepinephrine transporter system, causes apoptosis in pheochromocytoma cell lines and prevents metastasis in an animal model.

Pheochromocytomas (PHEOs) and paragangliomas (PGLs) are specific types of neuroendocrine tumors that originate in the adrenal medulla or sympathetic/parasympathetic paraganglia, respectively. Although these tumors are intensively studied, a very effective treatment for metastatic PHEO or PGL has not yet been established. Preclinical evaluations of novel therapies for these tumors are very much required. Therefore, in this study we tested the effect of triptolide (TTL), a potent nuclear factor-kappaB (NF-κB) inhibitor, on the cell membrane norepinephrine transporter (NET) system, considered to be the gatekeeper for the radiotherapeutic agent 131I-metaiodobenzylguanidine (131I-MIBG). We measured changes in the mRNA and protein levels of NET and correlated them with proapoptotic factors and metastasis inhibition. The study was performed on three different stable PHEO cell lines. We found that blocking NF-κB with TTL or capsaicin increased both NET mRNA and protein levels. Involvement of NF-κB in the upregulation of NET was verified by mRNA silencing of this site and also by using NF-κB antipeptide. Moreover, in vivo treatment with TTL significantly reduced metastatic burden in an animal model of metastatic PHEO. The present study for the first time shows how NF-κB inhibitors could be successfully used in the treatment of metastatic PHEO/PGL by a significant upregulation of NET to increase the efficacy of 131I-MIBG and by the induction of apoptosis.

Acute stress differently modulates ß1, ß2 and ß3 adrenoceptors in T cells, but not in B cells, from the rat spleen.

OBJECTIVES: Stress-induced rise in circulating catecholamines (CAs), followed by modulation of ß-adrenergic receptors (adrenoceptors, ARs), is one of the pathways involved in the stress-mediated effects of immune functions. The spleen is an organ with a high number of lymphocytes and provides a unique microenvironment in which they reside. Thus, lymphocytes may respond differently to CAs in the spleen than in the circulation. No reports exist concerning the involvement of ß-ARs in stress-mediated effects on T and B cells isolated from the spleen. Therefore, our aim was to investigate the effect of single stress exposure on gene expression and cellular localization of ß-adrenoceptor subtypes in splenic T and B cells. We tried to correlate changes in adrenoceptors with the expression of apoptotic proteins. METHODS: Immobilization (IMMO) was used as a stress model. T and B cells were isolated from rat spleen using magnetically labeled antibodies. The gene expression of individual adrenoceptors and apoptotic proteins was evaluated by real-time PCR. Immunofluorescence was used to evaluate localization and adrenoceptor expression. RESULTS: We have found T cells to be more vulnerable to stress compared to B cells, because of increased ß1-, ß2- and ß3-ARs after a single IMMO. Moreover, ß2-ARs translocated from the nucleus to the plasma membrane in T cells after IMMO. The rise in ß-ARs most probably led to the rise of Bax mRNA and Bax to Bcl-2 mRNA ratio. This might suggest the induction of an apoptotic process in T cells. CONCLUSION: Higher susceptibility of T cells to stress via modulation of ß-ARs and apoptotic proteins might shift the immune responsiveness in the spleen.

Novel humanized monoclonal antibodies for targeting hypoxic human tumors via two distinct extracellular domains of carbonic anhydrase IX.

BACKGROUND: Hypoxia in the tumor microenvironment (TME) is often the main factor in the cancer progression. Moreover, low levels of oxygen in tumor tissue may signal that the first- or second-line therapy will not be successful. This knowledge triggers the inevitable search for different kinds of treatment that will successfully cure aggressive tumors. Due to its exclusive expression on cancer cells, carbonic anhydrase IX belongs to the group of the most precise targets in hypoxic tumors. CA IX possesses several exceptional qualities that predetermine its crucial role in targeted therapy. Its expression on the cell membrane makes it an easily accessible target, while its absence in healthy corresponding tissues makes the treatment practically harmless. The presence of CA IX in solid tumors causes an acidic environment that may lead to the failure of standard therapy. METHODS: Parental mouse hybridomas (IV/18 and VII/20) were humanized to antibodies which were subsequently named CA9hu-1 and CA9hu-2. From each hybridoma, we obtained 25 clones. Each clone was tested for antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) activity, affinity, extracellular pH measurement, multicellular aggregation analysis, and real-time monitoring of invasion with the xCELLigence system. RESULTS: Based on the results from in vivo experiments, we have selected mouse monoclonal antibodies VII/20 and IV/18. The first one is directed at the conformational epitope of the catalytic domain, internalizes after binding to the antigen, and halts tumor growth while blocking extracellular acidification. The second targets the sequential epitope of the proteo-glycan domain, does not internalize, and is able to block the attachment of cancer cells to the matrix preventing metastasis formation. In vitro experiments prove that humanized versions of the parental murine antibodies, CA9hu-1 and CA9hu-2, have preserved these characteristics. They can reverse the failure of standard therapy as a result of an acidic environment by modulating the TME, and both are able to induce an immune response and have high affinity, as well as ADCC and CDC activity. CONCLUSION: CA9hu-1 and CA9hu-2 are the very first humanized antibodies against CA IX that are likely to become suitable therapies for hypoxic tumors. These antibodies can be applied in the treatment therapy of primary tumors and suppression of metastases formation.