Synthesis and evaluation of near-infrared fluorescent sulfonamide derivatives for imaging of hypoxia-induced carbonic anhydrase IX expression in tumors

A series of human carbonic anhydrase (hCA) IX inhibitors conjugated to various near-infrared fluorescent dyes was synthesized with the aim of imaging hypoxia-induced hCA IX expression in tumor cells in vitro, ex vivo and in vivo. The resulting compounds were profiled for inhibition of transmembrane hCA IX showing a range of potencies from 7.5 to 116 nM and up to 50-fold selectivity over the cytosolic form hCA II. Some of the compounds also showed inhibition selectivity for other transmembrane forms hCA XII and XIV as well. Compounds incubated in vitro with HeLa cells cultured under normoxic and hypoxic conditions detected upregulation of hCA IX under hypoxia by fluorescence microscopy. A pilot in vivo study in HT-29 tumor bearing mice showed significant accumulation of a fluorescent acetazolamide derivative in tumor tissue with little accumulation in other tissues. Approximately 10% of injected dose was non-invasively quantified in tumors by fluorescence molecular tomography (FMT), demonstrating the promise of these new compounds for quantitative imaging of hCA IX upregulation in live animals.     

Synthesis and antitumor activity evaluations of albumin-binding prodrugs of CC-1065 analog

An albumin-binding prodrug of the extremely potent CC-1065 analog, (+)-FDI-CBI, has been synthesized. This analog, (+)-FDI-CBIM, formed an albumin conjugate when added to human albumin in vitro. A greater amount (>3-fold) of the prodrug can be administered to animals compared to the free drug. The prodrug had significantly improved antitumor efficacy compared to the free drug in animal models using syngeneic animal tumors and human ovarian xenografted tumor cells. Antitumor drug delivery by in situ formation of drug-albumin conjugate is a promising strategy to improve antitumor efficacy.     

Improved in vivo anti-tumor effects of IgA-Her2 antibodies through half-life extension and serum exposure enhancement by FcRn targeting

Antibody therapy is a validated treatment approach for several malignancies. All currently clinically applied therapeutic antibodies (Abs) are of the IgG isotype. However, not all patients respond to this therapy and relapses can occur. IgA represents an alternative isotype for antibody therapy that engages FcαRI expressing myeloid effector cells, such as neutrophils and monocytes. IgA Abs have been shown to effectively kill tumor cells both in vitro and in vivo. However, due to the short half-life of IgA Abs in mice, daily injections are required to reach an effect comparable to IgG Abs. The relatively long half-life of IgG Abs and serum albumin arises from their capability of interacting with the neonatal Fc receptor (FcRn). As IgA Abs lack a binding site for FcRn, we generated IgA Abs with the variable regions of the Her2-specific Ab trastuzumab and attached an albumin-binding domain (ABD) to the heavy or light chain (HC_ABD/LC_ABD) to extend their serum half-life. These modified Abs were able to bind albumin from different species in vitro. Furthermore, tumor cell lysis of IgA-Her2-LC_ABD Abs in vitro was similar to unmodified IgA-Her2 Abs. Pharmacokinetic studies in mice revealed that the serum exposure and half-life of the modified IgA-Her2 Abs was extended. In a xenograft mouse model, the modified IgA1 Abs exhibited a slightly, but significantly, improved anti-tumor response compared to the unmodified Ab. In conclusion, empowering IgA Abs with albumin-binding capacity results in in vitro and in vivo functional Abs with an enhanced exposure and prolonged half-life.     

Chemotaxis of oral treponemes toward sera and albumin of rabbit

The motility and chemotaxis of human oral spirochetes Treponema denticola ATCC 35404, T. medium ATCC 700293, and T. vincentii ATCC 35580 were examined by a capillary assay method. Of five sera three human oral treponemes were dominantly chemoattractant to the rabbit serum. The checkerboard analysis of chemotaxis toward rabbit serum clearly showed that the motile T. denticola cells swam toward the culture media containing higher concentrations of the rabbit serum. T. denticola chemotaxis to the rabbit serum was clearly reduced by heating serum, and rabbit albumin contributed by 60 to 70% to its chemotaxis to the rabbit serum. Western blotting analysis demonstrated that these treponemes possessed rabbit albumin-binding polypeptides with approximate molecular sizes of 65 kDa and 70 kDa. Immunoelectron microscopy demonstrated that a 65 kDa rabbit albumin-binding polypeptide was located on the outer envelopes, suggesting that the rabbit albumin-binding polypeptide is responsible for chemotaxis toward rabbit serum.     

Stable overexpression of DNA fragmentation factor in T-47D cells: sensitization of breast cancer cells to apoptosis in response to acetazolamide and sulfabenzamide

Alterations in expression of the DFF40 gene have been reported in some cancers. This study is an in vitro study of the therapeutic effects of gene transfer that lead to elevation in DFF40 expression within T-47D cells in the presence of sulfonamide drugs. In this study, we have constructed a eukaryotic expression vector for DFF40 and transfected it into T-47D cancer cells. We used real time RT-PCR to detect the expression of DFF40 and the MTT assay to determine effects of the sulfonamide drugs acetazolamide, sulfabenzamide, sulfathiazole and sulfacetamide on cell viability in the presence of increased and normal DFF40 levels. Cell cycle distribution was assessed by propidium iodide (PI) staining and the rates of apoptosis by annexin V/PI staining. The DNA laddering analysis was employed to evaluate apoptosis. We observed that overexpression of DFF40 was only effective in decreasing viability in cells incubated with acetazolamide and sulfabenzamide. There was enhanced apoptosis in these groups, particularly with acetazolamide. The cell cycle distribution analysis showed that in the presence of sulfonamide drugs there were no substantial changes in empty-vector or DFF40-transfected cells, except for those cells treated with sulfabenzamide or sulfathiazole. There was no DNA laddering in cells that expressed the empty vector when incubated with sulfonamide drugs. In contrast, we observed DNA laddering in cells that expressed DFF40 in the presence of acetazolamide. Our results have demonstrated that combinatorial use of some sulfonamides such as acetazolamide along with increased expression of DFF40 can potently kill tumor cells via apoptosis and may be beneficial for treatment of some chemoresistant cancers.     

Characterization of albumin-binding properties of Peptostreptococcus magnus

A Peptostreptococcus magnus strain demonstrated binding activity for albumin preparations from humans, mice, and dogs, but not for rabbit or bovine albumin. The albumin binding site appeared to be heat stable and of protein nature. Treatment of P. magnus cells with trypsin under specified conditions enhanced this albumin binding. Electron micrographs and kinetic analyses revealed that this enhancement was the result of the removal of some cell wall associated proteins leading to a higher binding affinity without significant changes in binding site numbers. The albumin-binding proteins could be readily solubilized and purified by affinity chromatography. Upon gel electrophoresis the molecular mass of the albumin-binding proteins was estimated as 130 kilodaltons.     

In vitro effect of carbonic anhydrase inhibitor acetazolamide on cell viability,migration and colony formation of colorectal cancer cells

Acidification of extracellular medium in malignant tumors increases the invasive behaviors of cancer cells. In normal healthy tissues, acid production is catalyzed by carbonic anhydrases. Some of the carbonic anhydrase enzymes are overexpressed in certain types of cancer. The present study aimed to investigate the effect of acetazolamide, a potent carbonic anhydrase inhibitor, on in vitro cultivated cancer cells. Three different assays (MTT test, wound healing and clonogenic assay) were performed using human colorectal adenocarcinoma cells (SW620) to evaluate the suppressive effect of acetazolamide, on the colorectal cancer cells migration ability, colony formation and cell viability. The dose-dependent (1–1000 μM) reducing effect of acetazolamide on the cell viability was more significant within the first 48 h. This inhibitory effect of acetazolamide was found to be decreased at 72 h, and affects cells migration ability of cells at 24 and 48 h. Acetazolamide was observed to inhibit the cell viability, migration and colony formation ability of cells, depending on dose.     

Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.     

In Vivo Imaging and Quantification of Carbonic Anhydrase IX Expression as an Endogenous Biomarker of Tumor Hypoxia

Carbonic anhydrase IX (CA IX) is a transmembrane protein that has been shown to be greatly upregulated under conditions of hypoxia in many tumor cell lines. Tumor hypoxia is associated with impaired efficacy of cancer therapies making CA IX a valuable target for preclinical and diagnostic imaging. We have developed a quantitative in vivo optical imaging method for detection of CA IX as a marker of tumor hypoxia based on a near-infrared (NIR) fluorescent derivative of the CA IX inhibitor acetazolamide (AZ). The agent (HS680) showed single digit nanomolar inhibition of CA IX as well as selectivity over other CA isoforms and demonstrated up to 25-fold upregulation of fluorescent CA IX signal in hypoxic versus normoxic cells, which could be blocked by 60%–70% with unlabeled AZ. CA IX negative cell lines (HCT-116 and MDA-MB-231), as well as a non-binding control agent on CA IX positive cells, showed low fluorescent signal under both conditions. In vivo FMT imaging showed tumor accumulation and excellent tumor definition from 6–24 hours. In vivo selectivity was confirmed by pretreatment of the mice with unlabeled AZ resulting in >65% signal inhibition. HS680 tumor signal was further upregulated >2X in tumors by maintaining tumor-bearing mice in a low oxygen (8%) atmosphere. Importantly, intravenously injected HS680 signal was co-localized specifically with both CA IX antibody and pimonidazole (Pimo), and was located away from non-hypoxic regions indicated by a Hoechst stain. Thus, we have established a spatial correlation of fluorescence signal obtained by non-invasive, tomographic imaging of HS680 with regions of hypoxia and CA IX expression. These results illustrate the potential of HS680 and combined with FMT imaging to non-invasively quantify CA IX expression as a hypoxia biomarker, crucial to the study of the underlying biology of hypoxic tumors and the development and monitoring of novel anti-cancer therapies.     

Acetazolamide inhibits osmotic water permeability by interaction with aquaporin-1

Water channel proteins, known as aquaporins, are transmembrane proteins that mediate osmotic water permeability. In a previous study, we found that acetazolamide could inhibit osmotic water transportation across Xenopus oocytes by blocking the function of aquaporin-1 (AQP1). The purpose of the current study was to confirm the effect of acetazolamide on water osmotic permeability using the human embryonic kidney 293 (HEK293) cells transfected with pEGFP/AQP1 and to investigate the interaction between acetazolamide and AQP1. The fluorescence intensity of HEK293 cells transfected with pEGFP/AQP1, which corresponds to the cell volume when the cells swell in a hyposmotic solution, was recorded under confocal laser fluorescence microscopy. The osmotic water permeability was assessed by the change in the ratio of cell fluorescence to certain cell area. Acetazolamide, at concentrations of 1 and 10muM, inhibited the osmotic water permeability in HEK293 cells transfected with pEGFP/AQP1. The direct binding between acetazolamide and AQP1 was detected by surface plasmon resonance. AQP1 was prepared from rat red blood cells and immobilized on a CM5 chip. The binding assay showed that acetazolamide could directly interact with AQP1. This study demonstrated that acetazolamide inhibited osmotic water permeability through interaction with AQP1.     

Structure, dynamics, and stability variation in bacterial albumin binding modules: implications for species specificity

Protein G-related albumin-binding (GA) modules are frequently expressed on the surfaces of bacterial cells. The limited amino acid sequence variation among GA modules results in structural and functional differences with possible implications for bacterial pathogenesis and host specificity. In particular, the streptococcal G148-GA3 and F. magna ALB8-GA albumin-binding domains exhibit a degree of structural and dynamic diversity that may account for their varied affinities for different species of albumin. To explore the impact of GA module polymorphisms on albumin binding and specificity, we recently used offset recombinant PCR to shuffle seven artificially constructed representatives of the GA sequence space and scan the phage-displayed recombinant domains for mutations that supported binding to the phylogenetically distinct human and guinea pig serum albumins (HSA and GPSA) (Rozak et al. (2006) Biochemistry 45, 3263-3271). Surprisingly, phage selection revealed an overwhelming preference for a single recombinant domain (PSD-1, phage-selected domain-1) regardless of whether the phages were enriched for their abilities to bind one or both of these albumins. We describe here the NMR-derived structure, dynamics, and stability of unbound PSD-1. Our results demonstrate that increased flexibility is not a requirement for broadened specificity, as had been suggested earlier (Johansson et al. (2002) J. Mol. Biol. 316, 1083-1099), because PSD-1 binds the phylogenetically diverse HSA and GPSA even more tightly than G148-GA3 but is less flexible. The structural basis for albumin-binding specificity is analyzed in light of these new results.     

Localization of carbonic anhydrase in living osteoclasts with bodipy 558/568-modified acetazolamide, a thiadiazole carbonic anhydrase inhibitor.

We describe the synthesis of Bodipy 558/568-modified acetazolamide, a fluorescent inhibitor of carbonic anhydrase and its use to localize the enzyme in living cells. The modified acetazolamide, with its specific sulfonamide group intact, labeled cells at concentrations as low as 10(-9) M, with a minimal loading time of 5 min. The staining was decreased by 57.4% by preincubating cells with unaltered acetazolamide (1:100) or with trifluoromethane sulfonamide, 6-ethoxyzolamide, and 5-(3-hydroxybenzoyl)-thiophene-2-sulfonamide. The efficacy of the inhibitor was unchanged by the fluorescent label, as determined by an acridine orange assay that detects acidification of osteoclasts, the cell model used in this study. This compound should prove to be useful for studying carbonic anhydrase in many organisms because of the high degree of conservation of the active site of this enzyme. (J Histochem Cytochem 47:545-550, 1999)     

Crystal structure of a bacterial albumin-binding domain at 1.4 A resolution

The albumin-binding domain, or GA module, of the peptostreptococcal albumin-binding protein expressed in pathogenic strains of Finegoldia magna is believed to be responsible for the virulence and increased growth rate of these strains. Here we present the 1.4A crystal structure of this domain, and compare it with the crystal structure of the GA-albumin complex. An analysis of protein-protein interactions in the two crystals, and the presence of multimeric GA species in solution, indicate the GA module is "sticky", and is capable of forming contacts with a range of protein surfaces. This might lead to interactions with different host proteins.     

Synthesis and Antitumor Activity of Panaxadiol Pyrazole and Isooxazole Derivatives

In this study, we designed and synthesized 19 nitrogen-containing heterocyclic derivatives of panaxadiol (PD). We first reported the antiproliferative activity of these compounds against four different tumor cells. The results of the MTT assay showed that the PD pyrazole derivative (compound 12b ) had the best antitumor activity and could significantly inhibit the proliferation of four tested tumor cells. For A549 cells, the IC₅₀ value was as low as 13.44±1.23 μM. Western blot analysis showed that the PD pyrazole derivative was a bifunctional regulator. On the one hand, it can down-regulate the expression of HIF-1α by acting on PI3 K/AKT signaling pathway in A549 cells. On the other hand, it can induce the decrease of CDKs protein family and E2F1 protein expression levels, thus playing a crucial role in cell cycle arrest. According to the results of molecular docking, we found that multiple hydrogen bonds were formed between the PD pyrazole derivative and two related proteins, and the docking score of the derivative was also significantly higher than that of the crude drug. In summary, the study of the PD pyrazole derivative laid a foundation for the development of ginsenoside as an antitumor agent.     

Solid-phase purification and analysis of dicarboxylic porphyrins extracted from cultured tumor cells

We describe here a sensitive method for the purification and analysis of porphyrins present in hematoporphyrin derivative. Hematoporphyrin derivative is a solution containing a complex mixture of dicarboxylic porphyrins such as hematoporphyrin IX, monohydroxyethyl monovinyl deuteroporphyrin isomers, and protoporphyrin IX in addition to porphyrin aggregates of variable molecular sizes. This mixture is known for its ability to be selectively retained by tumor cells and for its cytotoxicity in the presence of light. In order to study the mechanisms of hematoporphyrin derivative uptake and its cellular metabolism, extraction methods are required that combine high recoveries with minimum changes of very labile components. Extraction with perchloric acid: methanol mixtures recovered only some 60% of the porphyrins taken up by tumor cells and artifactual fluorescent spots were seen on thin-layer chromatograms. Improved yields were obtained upon extraction with dimethyl sulfoxide or Triton X-100:4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) buffer mixture, but the extracts were not suitable for reverse-phase thin-layer chromatography (RTLC). The procedure described here consists of extracting porphyrins from cultured tumor cells with a buffered detergent followed by sequential chromatography on DEAE-cellulose columns and on reverse-phase octadecylsilyl cartridges. Identification of the isolated free dicarboxylic porphyrins is conveniently done by RTLC.     

Engineering bispecificity into a single albumin-binding domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.     

Hyaluronan as carrier of carboranes for tumor targeting in boron neutron capture therapy

Boron neutron capture therapy (BNCT) represents a promising approach for tumor therapy. A critical requirement for BNCT is tumor targeting, a goal that is currently addressed with the development of low and high molecular weight agents capable of interacting with receptors expressed by cancer cells. Here, we describe a new bioconjugate (HApCB) composed by n-propyl carborane linked to hyaluronan (HA) via an ester linkage for a degree of substitution of approximately 30%, leading to a water-soluble derivative. The structure and main physicochemical characteristics of the new HA derivative were determined by means of Fourier transform infrared, fluorescence, and 1H, 13C, and 10B NMR analysis and are herein reported in detail. As HA is recognized by the CD44 antigen, densely populating the surface of many tumor cells, HApCB is expected to deliver boron atoms from the locally released carborane cages directly to target cells for antitumor application in BNCT. In vitro biological experiments showed that HApCB was not toxic for a variety of human tumor cells of different histotypes, specifically interacted with CD44 as the native unconjugated HA, and underwent uptake by tumor cells, leading to accumulation of amounts of boron atoms largely exceeding those required for a successful BNCT approach. Thus, HApCB may be regarded as a promising new BNCT agent for specific targeting of cancer cells overexpressing the CD44 receptor.     

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.     

Comparison of vitamin E derivatives alpha-TEA and VES in reduction of mouse mammary tumor burden and metastasis

A novel nonhydrolyzable ether derivative of RRR-alpha-tocopherol, RRR-alpha-tocopherol ether acetic acid analog [2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chroman-6-yloxyacetic acid (alpha-TEA)], and a hydrolyzable ester derivative RRR-alpha-tocopheryl succinate (vitamin E succinate; VES) inhibited BALB/c mouse 66cl-4-GFP mammary tumor cell growth in vitro and in vivo. Treatment of 66cl-4-GFP cells in culture with alpha-TEA or VES induced dose-dependent DNA synthesis arrest and apoptosis and inhibited colony formation. Liposomal formulations of alpha-TEA delivered orally or by aerosol significantly reduced subcutaneous 66cl-4-GFP tumor burden and metastasis to lung and lymph nodes. Liposomal formulations of VES delivered by aerosol significantly reduced tumor burden and lung metastasis, but not lymph node metastasis. Unlike alpha-TEA, VES was ineffective in reducing tumor burden and metastasis to lungs and lymph nodes when administered orally. Analyses of tumor sections showed that alpha-TEA delivered by either method significantly reduced tumor cell proliferation as measured by Ki67, and increased apoptosis as measured by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL), whereas VES delivered by aerosol reduced tumor cell proliferation and increased apoptosis, but not significantly. In summary, the nonhydrolyzable ether vitamin E derivative alpha-TEA was effective in reducing tumor burden and metastasis when delivered either by aerosol or orally, whereas the hydrolyzable ester vitamin E derivative VES was effective only when delivered by aerosol.     

Mechanisms of the antitumor action of spirobromine in mice with leukemia P-388

The influence of new antitumor drug, spirobromine, a derivative of dispirotripiperazine, on DNA synthesis in tumor cells and organs at different times after its injection into mice with P388 leukemia has been studied. The duration of DNA synthesis inhibition in tumor cells was found to correlate with spirobromine antitumor activity. A certain selectivity of action of the studied compound on DNA synthesis in P 388 leukemia cells as compared to the action on DNA synthesis in bone marrow, small intestine, spleen and liver of tumor animals was observed.