Continuous monitoring of postirradiation reoxygenation and cycling hypoxia using electron paramagnetic resonance imaging.

Reoxygenation has a significant impact on the tumor response to radiotherapy. With developments in radiotherapy technology, the relevance of the reoxygenation phenomenon in treatment efficacy has been a topic of interest. Evaluating the reoxygenation in the tumor microenvironment throughout the course of radiation therapy is important in developing effective treatment strategies. In the current study, we used electron paramagnetic resonance imaging (EPRI) to directly map and quantify the partial oxygen pressure (pO2 ) in tumor tissues. Human colorectal cancer cell lines, HT29 and HCT116, were used to induce tumor growth in female athymic nude mice. Tumors were irradiated with 3, 10, or 20 Gy using an x-ray irradiator. Prior to each EPRI scan, magnetic resonance imaging (MRI) was performed to obtain T2-weighted anatomical images for reference. The differences in the mean pO2 were determined through two-tailed Student's t-test and one-way analysis of variance. The median pO2 60 min after irradiation was found to be lower in HCT116 than in HT29 (9.1 ± 1.5 vs. 14.0 ± 1.0 mmHg, p = 0.045). There was a tendency for delayed and incomplete recovery of pO2 in the HT29 tumor when a higher dose of irradiation (10 and 20 Gy) was applied. Moreover, there was a dose-dependent increase in the hypoxic areas (pO2  < 10 mmHg) 2 and 24 h after irradiation in all groups. In addition, an area that showed pO2 fluctuation between hypoxia and normoxia (pO2  > 10 mmHg) was also identified surrounding the region with stable hypoxia, and it slightly enlarged after recovery from acute hypoxia. In conclusion, we demonstrated the reoxygenation phenomenon in an in vivo xenograft model study using EPRI. These findings may lead to new knowledge regarding the reoxygenation process and possibilities of a new radiation therapy concept, namely, reoxygenation-based radiation therapy.

Design of Nuclear Magnetic Resonance Molecular Probes for Hyperpolarized Bioimaging.

Nuclear hyperpolarization has emerged as a method to dramatically enhance the sensitivity of NMR spectroscopy. By application of this powerful tool, small molecules with stable isotopes have been used for highly sensitive biomedical molecular imaging. The recent development of molecular probes for hyperpolarized in vivo analysis has demonstrated the ability of this technique to provide unique metabolic and physiological information. This review presents a brief introduction of hyperpolarization technology, approaches to the rational design of molecular probes for hyperpolarized analysis, and examples of molecules that have met with success in vitro or in vivo.

Intratumoral evaluation of 3D microvasculature and nanoparticle distribution using a gadolinium-dendron modified nano-liposomal contrast agent with magnetic resonance micro-imaging.

The enhanced permeability and retention (EPR) effect is variable depending on nanoparticle properties and tumor/vessel conditions. Thus, intratumoral evaluations of the vasculature and nanoparticle distribution are important for predicting the therapeutic efficacy and the intractability of tumors. We aimed to develop a tumor vasculature evaluation method and high-resolution nanoparticle delivery imaging using magnetic resonance (MR) micro-imaging technology with a gadolinium (Gd)-dendron assembled liposomal contrast agent. Using the Gd-liposome and a cryogenic receiving coil, we achieved 50-µm isotropic MR angiography with clear visualization of tumor micro-vessel structure. The Gd-liposome-enhanced MR micro-imaging revealed differences in the vascular structures between Colon26- and SU-DHL6-grafted mice models. The vessel volumes and diameters measured for both tumors were significantly correlated with histological observations. The MR micro-imaging methods facilitate the evaluation of intratumoral vascularization patterns, the quantitative assessment of vascular-properties that alter tumor malignancy, particle retentivity, and the effects of treatment.

Design of a Hyperpolarized Molecular Probe for Detection of Aminopeptidase N Activity.

Aminopeptidase N (APN) is an important enzyme that is involved in tumor angiogenesis. Detection of APN activity can thus lead to early diagnosis and elucidation of tumor development. Although some molecular probes for APN have been developed, the detection of APN activity in opaque biological samples remains a challenge. To this end, we designed a hyperpolarized NMR probe [1-(13) C]Ala-NH2 which satisfies the prerequisites for APN detection, namely, sufficient retention of the hyperpolarized state, a high reactivity to APN, and an APN-induced chemical shift change. The [1-(13) C]Ala-NH2 probe allowed sensitive detection of APN activity using (13) C NMR spectroscopy.

Direct Monitoring of γ-Glutamyl Transpeptidase Activity In Vivo Using a Hyperpolarized (13) C-Labeled Molecular Probe.

The γ-glutamyl transpeptidase (GGT) enzyme plays a central role in glutathione homeostasis. Direct detection of GGT activity could provide critical information for the diagnosis of several pathologies. We propose a new molecular probe, γ-Glu-[1-(13) C]Gly, for monitoring GGT activity in vivo by hyperpolarized (HP) (13) C magnetic resonance (MR). The properties of γ-Glu-[1-(13) C]Gly are suitable for in vivo HP (13) C metabolic analysis since the chemical shift between γ-Glu-[1-(13) C]Gly and its metabolic product, [1-(13) C]Gly, is large (4.3 ppm) and the T1 of both compounds is relatively long (30 s and 45 s, respectively, in H2 O at 9.4 T). We also demonstrate that γ-Glu-[1-(13) C]Gly is highly sensitive to in vivo modulation of GGT activity induced by the inhibitor acivicin.

Ridaifen G, tamoxifen analog, is a potent anticancer drug working through a combinatorial association with multiple cellular factors.

Ridaifen-G (RID-G), a tamoxifen analog that we previously synthesized, has potent growth inhibitory activity against various cancer cell lines. Tamoxifen is an anticancer drug known to act on an estrogen receptor (ER) and other proteins. However, our previous studies interestingly suggested that the mechanism of action of RID-G was different from that of tamoxifen. In order to investigate the molecular mode of action of RID-G, we developed a novel chemical genetic approach that combined a phage display screen with a statistical analysis of drug potency and gene expression profiles in thirty-nine cancer cell lines. Application of this method to RID-G revealed that three proteins, calmodulin (CaM), heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1), and zinc finger protein 638 (ZNF638) were the candidates of direct targets of RID-G. Moreover, cell lines susceptible to RID-G show similar expression profiles of RID-G target genes. These results suggest that RID-G involves CaM, hnRNP A2/B1, and ZNF638 in its growth inhibitory activity.

Pimonidazole-alkyne conjugate for sensitive detection of hypoxia by Cu-catalyzed click reaction.

Hypoxia is involved in various diseases, such as cancers. Pimonidazole has often been used as the gold-standard marker to visualize hypoxic regions. Pimonidazole labels hypoxic regions by forming a covalent bond with a neighboring protein under hypoxic conditions in the body, which is detected by immunohistochemistry performed on tissue sections. To date, some pimonidazole-fluorophore conjugates have been reported as fluorescent probes for hypoxia imaging that do not require immunostaining. They are superior to pimonidazole because immunostaining can produce high background signals. However, large fluorophores in the conjugates may alter the original biodistribution and reactivity. Here, we report a new hypoxia marker, Pimo-yne, as a pimonidazole-alkyne conjugate. Pimo-yne has a similar hypoxia detection capability as pimonidazole because the alkyne tag is small and can be detected by Cu-catalyzed click reaction with azide-tagged fluorescent dyes. We successfully visualized hypoxic regions in tumor tissue sections using Pimo-yne with reduced background signals. The detected regions overlapped well with those detected by pimonidazole immunohistochemistry. To further reduce the background, we employed a turn-on azide-tagged fluorescent dye.

Click3D: Click reaction across deep tissues for whole-organ 3D fluorescence imaging.

Click chemistry offers various applications through efficient bioorthogonal reactions. In bioimaging, pretargeting strategies have often been used, using click reactions between molecular probes with a click handle and reporter molecules that make them observable. Recent efforts have integrated tissue-clearing techniques with fluorescent labeling through click chemistry, allowing high-resolution three-dimensional fluorescence imaging. Nevertheless, these techniques have faced a challenge in limited staining depth, confining their use to imaging tissue sections or partial organs. In this study, we introduce Click3D, a method for thoroughly staining whole organs using click chemistry. We identified click reaction conditions that improve staining depth with our custom-developed assay. The Click3D protocol exhibits a greater staining depth compared to conventional methods. Using Click3D, we have successfully achieved whole-kidney imaging of nascent RNA and whole-tumor imaging of hypoxia. We have also accomplished whole-brain imaging of hypoxia by using the clickable hypoxia probe, which has a small size and, therefore, has high permeability to cross the blood-brain barrier.

Whole-Body and Whole-Organ 3D Imaging of Hypoxia Using an Activatable Covalent Fluorescent Probe Compatible with Tissue Clearing.

Elucidation of biological phenomena requires imaging of microenvironments in vivo. Although the seamless visualization of in vivo hypoxia from the level of whole-body to single-cell has great potential to discover unknown phenomena in biological and medical fields, no methodology for achieving it has been established thus far. Here, we report the whole-body and whole-organ imaging of hypoxia, an important microenvironment, at single-cell resolution using activatable covalent fluorescent probes compatible with tissue clearing. We initially focused on overcoming the incompatibility of fluorescent dyes and refractive index matching solutions (RIMSs), which has greatly hindered the development of fluorescent molecular probes in the field of tissue clearing. The fluorescent dyes compatible with RIMS were then incorporated into the development of activatable covalent fluorescent probes for hypoxia. We combined the probes with tissue clearing, achieving comprehensive single-cell-resolution imaging of hypoxia in a whole mouse body and whole organs.

Ridaifen B, a tamoxifen derivative, directly binds to Grb10 interacting GYF protein 2.

Ridaifen B (RID-B) is a tamoxifen derivative that potently inhibits breast tumor growth. RID-B was reported to show anti-proliferating activity for a variety of estrogen receptor (ER)-positive human cancer cells. Interestingly, RID-B was also reported to possess higher potency than that of tamoxifen even for some ER-negative cells, suggesting an ER-independent mechanism of action. In this study, a T7 phage display screen and subsequent binding analyses have identified Grb10 interacting GYF protein 2 (GIGYF2) as a RID-B-binding protein. Using a cell-based assay, the Akt phosphorylation level mediated by GIGYF2 was found to have decreased in the presence of RID-B.

Activation of oxidoreductases by the formation of enzyme assembly.

Biological properties of protein molecules depend on their interaction with other molecules, and enzymes are no exception. Enzyme activities are controlled by their interaction with other molecules in living cells. Enzyme activation and their catalytic properties in the presence of different types of polymers have been studied in vitro, although these studies are restricted to only a few enzymes. In this study, we show that addition of poly-l-lysine (PLL) can increase the enzymatic activity of multiple oxidoreductases through formation of enzyme assemblies. Oxidoreductases with an overall negative charge, such as l-lactate oxidase, d-lactate dehydrogenase, pyruvate oxidase, and acetaldehyde dehydrogenase, each formed assemblies with the positively charged PLL via electrostatic interactions. The enzyme activities of these oxidoreductases in the enzyme assemblies were several-folds higher than those of the enzyme in their natural dispersed state. In the presence of PLL, the turnover number (kcat) improved for all enzymes, whereas the decrease in Michaelis constant (KM) was enzyme dependent. This type of enzyme function regulation through the formation of assemblies via simple addition of polymers has potential for diverse applications, including various industrial and research purposes.

EPR and Related Magnetic Resonance Imaging Techniques in Cancer Research.

Imaging tumor microenvironments such as hypoxia, oxygenation, redox status, and/or glycolytic metabolism in tissues/cells is useful for diagnostic and prognostic purposes. New imaging modalities are under development for imaging various aspects of tumor microenvironments. Electron Paramagnetic Resonance Imaging (EPRI) though similar to NMR/MRI is unique in its ability to provide quantitative images of pO2 in vivo. The short electron spin relaxation times have been posing formidable challenge to the technology development for clinical application. With the availability of the narrow line width trityl compounds, pulsed EPR imaging techniques were developed for pO2 imaging. EPRI visualizes the exogenously administered spin probes/contrast agents and hence lacks the complementary morphological information. Dynamic nuclear polarization (DNP), a phenomenon that transfers the high electron spin polarization to the surrounding nuclear spins (1H and 13C) opened new capabilities in molecular imaging. DNP of 13C nuclei is utilized in metabolic imaging of 13C-labeled compounds by imaging specific enzyme kinetics. In this article, imaging strategies mapping physiologic and metabolic aspects in vivo are reviewed within the framework of their application in cancer research, highlighting the potential and challenges of each of them.

The antitumor agent doxorubicin binds to Fanconi anemia group F protein.

Doxorubicin, a commonly used cancer chemotherapy agent, elicits several potent biological effects, including synergistic-antitumor activity in combination with cisplatin. However, the mechanism of this synergism remains obscure. Here, we employed an improved T7 phage display screening method to identify Fanconi anemia group F protein (FANCF) as a doxorubicin-binding protein. The FANCF-doxorubicin interaction was confirmed by pull-down assay and SPR analysis. FANCF is a component of the Fanconi anemia complex, which monoubiquitinates D2 protein of Fanconi anemia group as a cellular response against DNA cross-linkers such as cisplatin. We observed that the monoubiquitination was inhibited by doxorubicin treatment.

Exploration of the binding proteins of perfluorooctane sulfonate by a T7 phage display screen.

Perfluorooctane sulfonate (PFOS) is a pollutant widely found throughout nature and is toxic to animals. We created a PFOS analogue on a polyethylene glycol polyacrylamide copolymer and isolated peptides that preferentially bound the PFOS analogue using a T7 phage display system. Bioinformatic analysis using the FASTAskan program on the RELIC bioinformatics server showed several human proteins that likely bound PFOS. Among them, we confirmed binding between PFOS and a recombinant soluble form of monocyte differentiation antigen CD14 (sCD14) by a surface plasmon biosensor. Furthermore, PFOS inhibited TNF-α production induced by the sCD14 in mouse macrophage RAW264.7 cells.

The natural sulfoglycolipid derivative SQAP improves the therapeutic efficacy of tissue factor-targeted radioimmunotherapy in the stroma-rich pancreatic cancer model BxPC-3.

α-Sulfoquinovosylacyl-1,3-propanediol (SQAP) is a semi-synthetic derivative of natural sulfoglycolipid that sensitizes tumors to external-beam radiotherapy. How SQAP affects internal radiotherapy, however, is not known. Here, we investigated the effects of SQAP for radioimmunotherapy (RIT) targeting tissue factor (TF) in a stroma-rich refractory pancreatic cancer mouse model, BxPC-3. A low dose of SQAP (2 mg/kg) increased tumor uptake of the 111In-labeled anti-TF antibody 1849, indicating increased tumor perfusion. The addition of SQAP enhanced the growth-inhibitory effect of 90Y-labeled 1849 without leading to severe body weight changes, allowing for the dose of 90Y-labeled 1849 to be reduced to half that when used alone. Histologic analysis revealed few necrotic and apoptotic cells, but Ki-67-positive proliferating cells and increased vascular formation were detected. These results suggest that the addition of a low dose of SQAP may improve the therapeutic efficacy of TF-targeted RIT by increasing tumor perfusion, even for stroma-rich refractory pancreatic cancer.

Structure-guided design enables development of a hyperpolarized molecular probe for the detection of aminopeptidase N activity in vivo.

Dynamic nuclear polarization (DNP) is a cutting-edge technique that markedly enhances the detection sensitivity of molecules using nuclear magnetic resonance (NMR)/magnetic resonance imaging (MRI). This methodology enables real-time imaging of dynamic metabolic status in vivo using MRI. To expand the targetable metabolic reactions, there is a demand for developing exogenous, i.e., artificially designed, DNP-NMR molecular probes; however, complying with the requirements of practical DNP-NMR molecular probes is challenging because of the lack of established design guidelines. Here, we report Ala-[1-13C]Gly-d2-NMe2 as a DNP-NMR molecular probe for in vivo detection of aminopeptidase N activity. We developed this probe rationally through precise structural investigation, calculation, biochemical assessment, and advanced molecular design to achieve rapid and detectable responses to enzyme activity in vivo. With the fabricated probe, we successfully detected enzymatic activity in vivo. This report presents a comprehensive approach for the development of artificially derived, practical DNP-NMR molecular probes through structure-guided molecular design.

Characterization of marine X-family DNA polymerases and comparative analysis of base excision repair proteins.

While mammalian DNA polymerase ß (Pol ß), which is a member of the Pol X family, play important roles in base excision repair (BER) that efficiently removes DNA base lesions arising from both endogenous and exogenous agents, this protein has been found only a subset of animals. To understand natural evolution of this enzyme, we isolated and characterized Pol ß from jellyfish Aurelia sp.1. (AsPol ß). Despite of phylogenetic distance and environmental differences between jellyfish and mammals, in vitro assays showed biochemical characteristics of AsPol ß were very similar to those of a mammalian counterpart. We also searched two other homologs of mammalian genes that were involved in short patch (sp) BER in the nucleotide sequence database, and found that both of these homologs were encoded in the genomes of a lineage from Cnidarians through mammals and Arthropods. This study suggests that a DNA repair mechanism resembling mammalian sp-BER may be largely limited to a subset of animals. On the basis of our findings and previous reports, we discuss possible evolutional model of Pol ß and the other members of the Pol X family.

Foam fractionation of protein: correlation of protein adsorption onto bubbles with a pH-induced conformational transition.

A foam fractionation apparatus was prepared to aid protein separation at the gas-liquid interface. Using lysozyme as a model protein, we investigated the alteration of enzymatic and optical activities through foaming. The lysozyme transferred to the gaseous nitrogen phase after 5 min of bubbling with no exogenous detergent. The bacteriolytic and optical activities of lysozyme from the foamate were nearly equivalent to those of the original lysozyme. This result indicated that lysozyme did not irreversibly denature during foam fractionation. We then performed protein separation using binary mixtures of lysozyme and α-amylase. When the two proteins were dissolved in bulk solution of pH 10.5, which is close to the isoelectric point (pI) of lysozyme (10.7), selective fractionation of lysozyme from the foam was observed. Indeed, this fractionation was identical to that from a single component solution of lysozyme. Similarly, selective fractionation of α-amylase was achieved in pH 3.0 buffer. Furthermore, circular dichroism (CD) and subsequent model fitting revealed that the protein had a reduced or nearly complete absence of α-helical content, whereas the amount of ß-sheet structure and random coil was elevated in the buffer conditions that promoted protein adsorption. These results indicate that a pH-induced conformational transition might correlate with protein foaming.

Screening of a library of T7 phage-displayed peptides identifies alphaC helix in 14-3-3 protein as a CBP501-binding site.

CBP501 is a chemically modified peptide composed of twelve unnatural d-amino acids, which inhibits Chk kinase and abrogates G2 arrest induced by DNA-damaging agents. Here we identified an alphaC helix in 14-3-3 protein as a CBP501-binding site using T7 phage display technology. An affinity selection of T7 phage-displayed peptide using biotinylated CBP501 identified a 14-mer peptide NSDCIISRKIEQKE. This peptide sequence showed similarity to a portion of the alphaC helix of human 14-3-3ε, suggesting that CBP501 may bind to this region. Surface plasmon resonance (SPR) and ELISA demonstrated that CBP501 interacts with 14-3-3ε specifically at the screen-guided region. An avidin-agarose bead pull-down assay showed that CBP501 also binds to other 14-3-3 isoforms in Jurkat cells. Among the other known Chk kinase inhibitors tested, CBP501 showed the strongest affinity for 14-3-3ε. Thus, we conclude that in addition to the direct inhibition of Chk kinase activity, CBP501 directly binds to cellular 14-3-3 proteins through alphaC helix.

Camptothecin (CPT) directly binds to human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and inhibits the hnRNP A1/topoisomerase I interaction.

Camptothecin (CPT) is an anti-tumor natural product that forms a ternary complex with topoisomerase I (top I) and DNA (CPT-top I-DNA). In this study, we identified the direct interaction between CPT and human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) using the T7 phage display technology. On an avidin-agarose bead pull down assay, hnRNP A1 protein was selectively pulled down in the presence of C20-biotinylated CPT derivative (CPT-20-B) both in vitro and in vivo. The interaction was also confirmed by an analysis on a quartz-crystal microbalance (QCM) device, yielding a K(D) value of 82.7 nM. A surface plasmon resonance (SPR) analysis revealed that CPT inhibits the binding of hnRNP A1 to top I (K(D): 260 nM) in a non-competitive manner. Moreover, an in vivo drug evaluation assay using Drosophila melanogaster showed that the knockout of the hnRNP A1 homolog Hrb87F gene showed high susceptibility against 5-50 µM of CPT as compared to a wild-type strain. Such susceptibility was specific for CPT and not observed after treatment with other cytotoxic drugs. Collectively, our data suggests that CPT directly binds to hnRNP A1 and non-competitively inhibits the hnRNP A1/top I interaction in vivo. The knockout strain loses the hnRNP A1 homolog as a both CPT-binding partner and naïve brakes of top I, which enhances the formation of the CPT-top I-DNA ternary complexes and subsequently sensitizes the growth inhibitory effect of CPT in D. melanogaster.