Membrane-bound Carbonic Anhydrase Research Articles

Branchial CO2 and ammonia excretion in crustaceans: Involvement of an apical Rhesus-like glycoprotein.

To determine whether the crustacean Rh1 protein functions as a dual CO2 /ammonia transporter and investigate its role in branchial ammonia excretion and acid-base regulation. Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site-directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2 . Further, immunohistochemistry was used to assess sub-cellular localization of CmRh1 and a membrane-bound carbonic anhydrase (CmCAg). Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un-ionized ammonia/methylamine species. CmRh1 was co-localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. CmRh1 is the first identified apical transporter-mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates.

Multisite Labeling of Proteins Using the Ligand-Directed Reactivity of Triggerable Michael Acceptors.

Targeted modification of endogenous proteins without genetic manipulation of protein expression machinery has a range of applications from chemical biology to drug discovery. Despite being demonstrated to be effective in various applications, target-specific protein labeling using ligand-directed strategies is limited by stringent amino acid selectivity. Here, we present highly reactive ligand-directed triggerable Michael acceptors (LD-TMAcs) that feature rapid protein labeling. Unlike previous approaches, the unique reactivity of LD-TMAcs enables multiple modifications on a single target protein, effectively mapping the ligand binding site. This capability is attributed to the tunable reactivity of TMAcs that enable the labeling of several amino acid functionalities via a binding-induced increase in local concentration while remaining fully dormant in the absence of protein binding. We demonstrate the target selectivity of these molecules in cell lysates using carbonic anhydrase as the model protein. Furthermore, we demonstrate the utility of this method by selectively labeling membrane-bound carbonic anhydrase XII in live cells. We envision that the unique features of LD-TMAcs will find use in target identification, investigation of binding/allosteric sites, and studying membrane proteins.

Functional divergence of teleost carbonic anhydrase 4

The functional role of membrane-bound carbonic anhydrases (CAs) has been of keen interest in the past decade, and in particular, studies have linked CA in red muscle, heart, and eye to enhanced tissue oxygen extraction in bony fishes (teleosts). However, the number of purported membrane-bound CA isoforms in teleosts, combined with the imperfect system of CA isoform nomenclature, present roadblocks for ascribing physiological functions to particular CA isoforms across different teleost lineages. Here we developed an organizational framework for membrane-bound CAs in teleosts, providing the latest phylogenetic analysis of extant CA4 and CA4-like isoforms. Our data confirm that there are three distinct isoforms of CA4 (a, b, and c) that are conserved across major teleost lineages, with the exception of CA4c gene being lost in salmonids. Tissue distribution analyses suggest CA4a functions in oxygen delivery across teleost lineages, while CA4b may be specialized for renal acid-base balance and ion regulation. This work provides an important foundation for researchers to elucidate the functional significance of CA4 isoforms in fishes.

The Carbonic Anhydrase Inhibitor E7070 Sensitizes Glioblastoma Cells to Radio- and Chemotherapy and Reduces Tumor Growth.

Glioblastomas (GBMs), the most common and lethal primary brain tumor, show inherent infiltrative nature and high molecular heterogeneity that make complete surgical resection unfeasible and unresponsive to conventional adjuvant therapy. Due to their fast growth rate even under hypoxic and acidic conditions, GBM cells can conserve the intracellular pH at physiological range by overexpressing membrane-bound carbonic anhydrases (CAs). The synthetic sulfonamide E7070 is a potent inhibitor of CAs that harbors putative anticancer properties; however, this drug has still not been tested in GBMs. The present study aimed to evaluate the effects of E7070 on CA9 and CA12 enzymes in GBM cells as well as in the tumor cell growth, migration, invasion, and resistance to radiotherapy and chemotherapy. We found that E7070 treatment significantly reduced tumor cell growth and increased radio- and chemotherapy efficacy against GBM cells under hypoxia. Our data suggests that E7070 has therapeutic potential as a radio-chemo-sensitizing in drug-resistant GBMs, representing an attractive strategy to improve the adjuvant therapy. We showed that CA9 and CA12 represent potentially valuable therapeutic targets that should be further investigated as useful diagnostic and prognostic biomarkers for GBM tailored therapy.

Large pH oscillations promote host defense against human airways infection.

The airway mucosal microenvironment is crucial for host defense against inhaled pathogens but remains poorly understood. We report here that the airway surface normally undergoes surprisingly large excursions in pH during breathing that can reach pH 9.0 during inhalation, making it the most alkaline fluid in the body. Transient alkalinization requires luminal bicarbonate and membrane-bound carbonic anhydrase 12 (CA12) and is antimicrobial. Luminal bicarbonate concentration and CA12 expression are both reduced in cystic fibrosis (CF), and mucus accumulation both buffers the pH and obstructs airflow, further suppressing the oscillations and bacterial-killing efficacy. Defective pH oscillations may compromise airway host defense in other respiratory diseases and explain CF-like airway infections in people with CA12 mutations.

Carbonic Anhydrase Inhibitors of Different Structures Dilate Pre-Contracted Porcine Retinal Arteries.

Carbonic anhydrase inhibitors (CAIs), such as dorzolamide (DZA), are used as anti-glaucoma drugs to lower intraocular pressure, but it has been found that some of these drugs act as vasodilators of retinal arteries. The exact mechanism behind the vasodilatory effect is not yet clear. Here we have addressed the issue by using small vessel myography to examine the effect of CAIs of the sulfonamide and coumarin type on the wall tension in isolated segments of porcine retinal arteries. Vessels were pre-contracted by the prostaglandin analog U-46619, and CAIs with varying affinity for five different carbonic anhydrase (CA) isoenzymes found in human tissue tested. We found that all compounds tested cause a vasodilation of pre-contracted retinal arteries, but with varying efficacy, as indicated by the calculated mean EC50 of each compound, ranging from 4.12 µM to 0.86 mM. All compounds had a lower mean EC50 compared to DZA. The dilation induced by benzolamide (BZA) and DZA was additive, suggesting that they may act on separate mechanisms. No clear pattern in efficacy and affinity for CA isoenzymes could be discerned from the results, although Compound 5, with a low affinity for all isoenzymes except the human (h) CA isoform IV, had the greatest potency, with the lowest EC50 and inducing the most rapid and profound dilation of the vessels. The results suggest that more than one isozyme of CA is involved in mediating its role in controlling vascular tone in retinal arteries, with a probable crucial role played by the membrane-bound isoform CA IV.

Selective inhibition of carbonic anhydrase IX over carbonic anhydrase XII in breast cancer cells using benzene sulfonamides: Disconnect between activity and growth inhibition.

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.

Carbon acquisition mechanisms in Halophila johnsonii and Thalassia testudinum

Mechanisms for carbon uptake in the small-bodied Halophila johnsonii and large-bodied Thalassia testudinum were compared using photosynthesis measurements (oxygen flux) with, and without, the extracellular carbonic anhydrase inhibitor acetazolamide (AZ) and TRIS buffer. Our results indicated T. testudinum and H. johnsonii both utilize external membrane-bound carbonic anhydrase to facilitate dehydration of HCO3− into CO2(aq). T. testudinum also utilizes an active proton (H+) pump to create localized H+ gradients within the boundary layer and it derives a larger portion of photosynthetic carbon from bicarbonate than H. johnsonii. Stable carbon isotope composition (δ13C) of T. testudinum and H. johnsonii leaves under high and reduced irradiance were also compared to evaluate photosynthetic carbon use. Isotope ratios of H. johnsonii were significantly more negative than T. testudinum at a high-light field site (−10.44 ± 0.26‰ vs −9.25 ± 0.25‰). In contrast, there was no significant difference in isotope ratios between species for plants maintained in a greenhouse under reduced photosynthetically active radiation. This suggests photosynthesis of T. testudinum is more carbon limited or more reliant on bicarbonate for photosynthesis than H. johnsonii under high-light conditions. Although photosynthesis of both species is expected to increase in response to greater CO2(aq) availability, ocean acidification and its CO2(aq) enrichment of seawater may have greater benefits for H. johnsonii compared to T. testudinum, whose photosynthesis relies more heavily on HCO3− use.

Abstract 2931: Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype

Abstract Breast cancer is the second leading cause of cancer related deaths among women in the United States. Despite the tremendous progress that has been made towards treating localized tumors, nearly 40,000 women die each year, predominantly from metastatic drug resistance. The tumor microenvironment plays a pivotal role in determining tumor growth, invasion, metastasis, and therapeutic success or failure. Therefore, a therapy targeting the tumor microenvironment is needed to sufficiently preserve the quality of life of cancer patients, by inhibiting metastasis. Elevated levels of Carbonic anhydrase IX (CAIX) expression in primary breast cancers is a marker for highly aggressive and metastatic tumors, especially of the triple negative subtype (TNBC). It is also associated with hypoxia, extracellular acidification, and poor prognosis. Low pH (values of ~6.5-6.8) is toxic to normal cells in the tumor microenvironment while enhancing cancer cell proliferation and tumor growth. Our goal was to compare the structure of a CAIX-mimic bound to ureidosulfonamide inhibitors with the biological activity of these inhibitors in triple negative and estrogen receptor positive (ER+) breast cancer cell lines. CAIX is a reversible enzyme and at low pH (high proton concentration), the enzyme will consume protons, raising pH. Our hypothesis is that CAIX inhibition, in the context of an acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. In this study, we have shown the interaction of sulfonamide-based inhibitors with a CAIX-mimic using X-ray crystallography. These structures show that the inhibitors make multiple contacts within the active site cavity. This is consistent with the inhibitor-induced decrease in CAIX activity and to some extent expression. We have also investigated the effect of CA inhibition on breast cancer cell growth, proliferation, activation of cell death pathways and migration. This reveals that, although CA inhibition with the sulfonamide-based compounds inhibits cell growth and migration, it does not activate apoptotic pathways. In total, these observations indicate that CAIX is a viable small molecular drug target for the treatment of metastatic breast cancer. Citation Format: Mam Y. Mboge, Zhijuan Chen, Brian P. Mahon, Chingkkuang Tu, Alyssa S. Wolff, John V. Mathias, Fabrizio Carta, Claudiu T. Supuran, Rob McKenna, Susan C. Frost. Targeting membrane-bound carbonic anhydrases in breast cancer to intervene in the metastatic phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2931. doi:10.1158/1538-7445.AM2017-2931

Carbonic anhydrase inhibitors reduce cardiac dysfunction after sustained coronary artery ligation in rats.

Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.

Identification and expression of a novel carbonic anhydrase isozyme in the pufferfish Takifugu vermicularis

Carbonic anhydrase (CA) is a key element for maintaining acid base balance in fish. In our present experiment, novel CA isozymes were identified from the pear puffer (Takifugu vermicularis). Based on the high homology of two predicted CA sequences of the tiger puffer (Takifugu rubripes), a 1715bp novel cDNA was obtained from T. vermicularis. The open reading frame showed a complete coding sequence of 552bp with a deduced peptide sequence of 183 amino acids that exhibited highest (97%) identity with pufferfish putative CA III and CA IV-like sequences. In addition, this translated protein sequence showed 36–37% identity with zebrafish CA IV-like, CA XVa, CA XVb, and CA XVc proteins. Phylogenetic analysis revealed that the pufferfish novel protein (pCAn) was a membrane-bound CA protein. Alignment of multiple CA sequences illustrated that most of the putative active site residues of the pCAn isozyme were situated at highly conserved regions of the CA sequences. Examination of motif distribution suggested that the pCAn isozyme was very similar to the puffer predicted CA IV-like isozyme. Reverse transcription-polymerase chain reaction (PCR) analysis showed highly differential expression in the brain, gills, kidney, and muscle, whereas CA mRNA expression was almost absent in heart, liver, and intestine. Quantitative PCR expression of CA mRNA abundance suggested several-fold higher expression of pCAn isozymes in the gills compared to other tissues tested. Our results suggest that the pCAn isozyme might be related to CA IV-like isozymes. Further functional studies are needed to investigate the function of the pCAn isozyme in T. vermicularis.

Microsensor studies on Padina from a natural CO2 seep: implications of morphology on acclimation to low pH.

Low seawater pH can be harmful to many calcifying marine organisms, but the calcifying macroalgae Padina spp. flourish at natural submarine carbon dioxide seeps where seawater pH is low. We show that the microenvironment created by the rolled thallus margin of Padina australis facilitates supersaturation of CaCO3 and calcifi-cation via photosynthesis-induced elevated pH. Using microsensors to investigate oxygen and pH dynamics in the microenvironment of P.australis at a shallow CO2 seep, we found that, under saturating light, the pH inside the microenvironment (pHME ) was higher than the external seawater (pHSW ) at all pHSW levels investigated, and the difference (i.e., pHME - pHSW ) increased with decreasing pHSW (0.9 units at pHSW 7.0). Gross photosynthesis (Pg ) inside the microenvironment increased with decreasing pHSW , but algae from the control site reached a threshold at pH 6.5. Seep algae showed no pH threshold with respect to Pg within the pHSW range investigated. The external carbonic anhydrase (CA) inhibitor, acetazolamide, strongly inhibited Pg of P.australis at pHSW 8.2, but the effect was diminished under low pHSW (6.4-7.5), suggesting a greater dependence on membrane-bound CA for the dehydration of HCO3 (-) ions during dissolved inorganic carbon uptake at the higher pHSW . In comparison, a calcifying green alga, Halimeda cuneata f. digitata, was not inhibited by AZ, suggesting efficient bicarbonate transport. The ability of P.australis to elevate pHME at the site of calcification and its strong dependence on CA may explain why it can thrive at low pHSW .

Probing the ‘bipolar’ nature of the carbonic anhydrase active site: Aromatic sulfonamides containing 1,3-oxazol-5-yl moiety as picomolar inhibitors of cytosolic CA I and CA II isoforms

A series of potent inhibitors of human carbonic anhydrase (CA) isoforms I and II has been prepared via a direct, chemoselective sulfochlorination of a range of 1,3-oxazolyl benzenes and thiophenes, followed by primary sulfonamide synthesis. The latter functionality is a known zinc-binding group (ZBG) responsible for anchoring the inhibitors to the CA's zinc metal ion. The compound's periphery as well as the overall scaffold geometry was designed to enable optimal interactions with the two distinct sides of the enzyme's active site, one of which is lined with hydrophobic residues and while the other is predominantly hydrophilic. As a result, several compounds inhibiting the therapeutically important cytosolic CA I and CA II in picomolar range have been identified. These compounds are one of the most potent CA inhibitors identified to-date. Not only the remarkable (>10 000-fold), cytosolic CA I and CA II selectivity vs. the membrane-bound CA IX and CA XII isoforms, but also the pronounced CA II/I selectivity observed in some cases, allow considering this series as a set of isoform-selective chemical biology tools and promising starting points for drug candidate development.

Small molecules for immunomodulation in cancer: a review.

Small-molecule cytotoxic agents are already in use for cancer immunotherapy in the form of antibody conjugates containing these molecules linked covalently to antibodies or their fragments with the goal of targeting specific surface components of tumor cells. However, there are also reports of small molecules that act as antagonists to surface enzyme-linked receptors and receptors that interact with the tumor microenvironment, or that even inhibit metabolic enzymes. Such molecules have been shown to directly inhibit the signaling initiated by the respective ligands binding to their receptors, to recruit antibodies and other immunomodulatory molecules, or to promote or inhibit the proliferation of different immune cells to target specific types of cancer cells. This review will discuss immune response modifiers such as imiquimod, antibody-recruiting molecules that target prostate cancer, integrin receptor antagonists, indoleamine-2,3-dioxygenase inhibitors, emodin, RORɣt antagonists, ephrin receptor antagonists, membrane-bound carbonic anhydrase IX (CAIX) inhibitors, and selected protein kinase inhibitors. These small molecules can open up new ways to treat many types of cancers and possibly even other diseases that arise from immune dysregulation. Finally, the review will briefly discuss some additional targets that are being pursued to modify immune system responses in the tumor microenvironment.

Abstract 4935: Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer

Abstract Introduction: Optical molecular imaging is a novel strategy in diagnosis and therapy of breast cancer. Essential for succesful implementation of this technology is a high tumor-to-normal tissue ratio (TNR), which requires tumor-specific probes/tracers. However, most tumor markers are expressed in a minority of tumors only. Therefore, we have developed a novel probe binding to the membrane-bound protein carbonic anhydrase IX (CAIX), which is commonly expressed in solid tumors as a result of hypoxic conditions. The probe is based on nanobodies (15 kDa, variable domains obtained from heavy-chain antibodies present in animals from the Camelidae family), that has a faster tumor uptake and body clearance than conventional (monoclonal) antibodies, and can be applied for molecular imaging of pre-invasive breast cancer. Methods: High affinity CAIX-specific nanobodies were selected using a modified phage display technology and site-directed conjugated to IRDye800CW. Mice bearing orthotopically transplanted MCF10DCIS (DCIS) and CAIX-overexpressing MCF10DCIS (DCIS+CAIX) xenografts were injected with fluorescent CAIX-specific nanobody (B9-IR), or non-specific control nanobody (R2-IR) and imaged up to 48 hours non-invasively. Tumors were resected under image-guidance. Nanobody uptake was assessed in tissue sections and quantified using a biodistribution assay. Results: A highly specific nanobody for CAIX was obtained. In DCIS+CAIX xenografts, mean TNR was 4.3±0.6 (8 mice) already two hours after injection. In DCIS tumors, mean TNR was 1.8±0.1 (8 mice), which was higher than TNR obtained after injection with R2-IR (1.4±0.2, 4 mice). After three hours, probe accumulation was sufficient to allow imag-guided tumor resection. Biodistribution studies showed that B9-IR uptake was 4.6±0.8% of injected dose per gram in DCIS tumor tissue (%I.D./g), while 2.0±0.2 %I.D./g was found with R2-IR. Upon fluorescence imaging of tissue sections, B9-IR was present in perinecrotic areas, while R2-IR showed no accumulation in these regions. Conclusion: We have developed a novel IRDye800CW-labeled anti-CAIX-nanobody that can be used for rapid optical molecular imaging of (pre-)invasive breast cancer before and during surgery. Furthermore, the stability of the conjugate allowed for ‘molecular fluorescence pathology’, which might results in better contrast than conventional chromagen based immunohistochemistry at the pathology department. Acknowledgement: This research was supported by the Center for Translational Molecular Medicine (MAMMOTH). Citation Format: Aram SA van Brussel, Arthur Adams, Sabrina Oliveira, Mohamed El Khatabbi, Jeroen F. Vermeulen, Elsken Van der Wall, Willem PThM Mali, Patrick WB Derksen, Paul J. Van Diest, Paul MP Van Bergen en Henegouwen. Hypoxia targeting fluorescent nanobodies for optical molecular imaging of preinvasive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4935. doi:10.1158/1538-7445.AM2014-4935

Missense mutations and proximal RTA. Have we reached a new threshold? Focus on “Missense mutation T485S alters NBCe1-A electrogenicity causing proximal renal tubular acidosis”

THE REABSORPTION OF BICARBONATE by the kidney requires parallel operation of apical transporters that secrete H and corresponding basolateral transporters that absorb bicarbonate. Approximately 80% to 90% of the filtered bicarbonate is reabsorbed in the proximal tubule, where secretion of H into HCO3 -rich glomerular filtrate by the Na/H exchanger, NHE-3, results in the formation of H2CO3 that is rapidly converted, within the tubule lumen, by membrane-bound carbonic anhydrase (type IV), to H2O and CO2, which can freely diffuse into the proximal tubule and, via intracellular carbonic anhydrase (type II), is converted to intracellular H2CO3 and HCO3 . Bicarbonate exits the cell via the electrogenic Na-3HCO3 symporter (NBCe1-A), with the negative cell potential serving as the primary driving force. This process is summarized in the proximal tubule cell model displayed in Fig. 1. Renal tubular acidosis (RTA) is a rare group of disorders whether inherited or acquired, although the incidence and prevalence are not well defined. The phenotype, when fully expressed, is recognized clinically as a chronic non-gap metabolic acidosis in the face of evidence of an inability of the kidney to excrete net acid appropriately, and is often associated with abnor

Tumor hypoxia and metabolism – Towards novel anticancer approaches

The transcription factor hypoxia-inducible factor-1 (HIF-1) facilitates the induction of enzymes necessary for regulation of biological processes required for cell survival and the acquisition of an aggressive and invasive phenotype, such as regulation of the intracellular pH (pHi), anaerobic glycolysis, angiogenesis, migration/invasion... In this presentation, we will highlight some of the HIF-1-induced gene products – carbonic anhydrases IX and XII (CAs) and monocarboxylate transporters (MCTs) – which regulate the pHi by controlling export of metabolically-generated acids (carbonic and lactic acids). We reported that targeting these pHi-regulated processes through inhibition of either HIF-1-induced CAIX/CAXII or HIF-1-induced MCT4, MCT1 or Basigin/EMMPRIN/CD147 chaperone of MCTs, severely restricts glycolysis-generated ATP levels and tumor growth. In addition, we demonstrated that the Myc/HIF-1-targeted glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing a key step producing the NADH cofactor, activates the Akt pathway, thereby upregulating expression of the anti-apoptotic Bcl-xL. As a consequence, high expression of GAPDH contributes to tumor aggressiveness, in particular in the context Myc-driven B lymphomas. We propose that membrane-bound carbonic anhydrases (CAIX, CAXII), monocarboxylate transporters/chaperon Basigin (Myc-induced MCT1 and HIF-induced-MCT4) and GAPDH that are associated with exacerbated tumor metabolism, represent new potential targets for anticancer therapy.

Pharmacological inhibition of carbonic anhydrase XII interferes with cell proliferation and induces cell apoptosis in T-cell lymphomas

The membrane-bound carbonic anhydrase isoforms CAIX and CAXII, underpin a pH-regulating system that enables hypoxic tumor cell survival. Here, we observed for the first time an upregulation of CAXII in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LL) cells. First we showed that CAXII is overexpressed in thymocytes from tPTEN−/− mice suffering of T lymphoma and that its pharmacological inhibition decreased cell proliferation and induced apoptosis. The same results were observed with the SupT1 human T cell lymphoma line. In addition we observed an upregulation of CAXII in human T-ALL samples supporting the case that CAXII may represent a new therapeutic target for T-ALL/LL.

Differential acid–base regulation in various gills of the green crab Carcinus maenas: Effects of elevated environmental pCO2

Euryhaline decapod crustaceans possess an efficient regulation apparatus located in the gill epithelia, providing a high adaptation potential to varying environmental abiotic conditions. Even though many studies focussed on the osmoregulatory capacity of the gills, acid–base regulatory mechanisms have obtained much less attention. In the present study, underlying principles and effects of elevated pCO2 on acid–base regulatory patterns were investigated in the green crab Carcinus maenas acclimated to diluted seawater. In gill perfusion experiments, all investigated gills 4–9 were observed to up-regulate the pH of the hemolymph by 0.1–0.2 units. Anterior gills, especially gill 4, were identified to be most efficient in the equivalent proton excretion rate. Ammonia excretion rates mirrored this pattern among gills, indicating a linkage between both processes. In specimen exposed to elevated pCO2 levels for at least 7days, mimicking a future ocean scenario as predicted until the year 2300, hemolymph K+ and ammonia concentrations were significantly elevated, and an increased ammonia excretion rate was observed. A detailed quantitative gene expression analysis revealed that upon elevated pCO2 exposure, mRNA levels of transcripts hypothesized to be involved in ammonia and acid–base regulation (Rhesus-like protein, membrane-bound carbonic anhydrase, Na+/K+-ATPase) were affected predominantly in the non-osmoregulating anterior gills.

Specific Cell Surface Protein Imaging by Extended Self-Assembling Fluorescent Turn-on Nanoprobes

Visualization of tumor-specific protein biomarkers on cell membranes has the potential to contribute greatly to basic biological research and therapeutic applications. We recently reported a unique supramolecular strategy for specific protein detection using self-assembling fluorescent nanoprobes consisting of a hydrophilic protein ligand and a hydrophobic BODIPY fluorophore in test tube settings. This method is based on recognition-driven disassembly of the nanoprobes, which induces a clear turn-on fluorescent signal. In the present study, we have successfully extended the range of applicable fluorophores to the more hydrophilic ones such as fluorescein or rhodamine by introducing a hydrophobic module near the fluorophore. Increasing the range of available fluorophores allowed selective imaging of membrane-bound proteins under live cell conditions. That is, overexpressed folate receptor (FR) or hypoxia-inducible membrane-bound carbonic anhydrases (CA) on live cell surfaces as cancer-specific biomarkers were fluorescently visualized using the designed supramolecular nanoprobes in the turn-on manner. Moreover, a cell-based inhibitor-assay platform for CA on a live cell surface was constructed, highlighting the potential applicability of the self-assembling turn-on probes.