Ubiquitin-Specific Protease 2 in the Ventromedial Hypothalamus Modifies Blood Glucose Levels by Controlling Sympathetic Nervous Activation.

Ubiquitin-specific protease 2 (USP2) participates in glucose metabolism in peripheral tissues such as the liver and skeletal muscles. However, the glucoregulatory role of USP2 in the CNS is not well known. In this study, we focus on USP2 in the ventromedial hypothalamus (VMH), which has dominant control over systemic glucose homeostasis. ISH, using a Usp2-specific probe, showed that Usp2 mRNA is present in VMH neurons, as well as other glucoregulatory nuclei, in the hypothalamus of male mice. Administration of a USP2-selective inhibitor ML364 (20 ng/head), into the VMH elicited a rapid increase in the circulating glucose level in male mice, suggesting USP2 has a suppressive role on glucose mobilization. ML364 treatment also increased serum norepinephrine concentration, whereas it negligibly affected serum levels of insulin and corticosterone. ML364 perturbated mitochondrial oxidative phosphorylation in neural SH-SY5Y cells and subsequently promoted the phosphorylation of AMP-activated protein kinase (AMPK). Consistent with these findings, hypothalamic ML364 treatment stimulated AMPKα phosphorylation in the VMH. Inhibition of hypothalamic AMPK prevented ML364 from increasing serum norepinephrine and blood glucose. Removal of ROS restored the ML364-evoked mitochondrial dysfunction in SH-SY5Y cells and impeded the ML364-induced hypothalamic AMPKα phosphorylation as well as prevented the elevation of serum norepinephrine and blood glucose levels in male mice. These results indicate hypothalamic USP2 attenuates perturbations in blood glucose levels by modifying the ROS-AMPK-sympathetic nerve axis.SIGNIFICANCE STATEMENT Under normal conditions (excluding hyperglycemia or hypoglycemia), blood glucose levels are maintained at a constant level. In this study, we used a mouse model to identify a hypothalamic protease controlling blood glucose levels. Pharmacological inhibition of USP2 in the VMH caused a deviation in blood glucose levels under a nonstressed condition, indicating that USP2 determines the set point of the blood glucose level. Modification of sympathetic nervous activity accounts for the USP2-mediated glucoregulation. Mechanistically, USP2 mitigates the accumulation of ROS in the VMH, resulting in attenuation of the phosphorylation of AMPK. Based on these findings, we uncovered a novel glucoregulatory axis consisting of hypothalamic USP2, ROS, AMPK, and the sympathetic nervous system.

Electron Paramagnetic Resonance Implemented with Multiple Harmonic Detections Successfully Maps Extracellular pH In Vivo.

Extracellular acidification indicates a metabolic shift in cancer cells and is, along with tissue hypoxia, a hallmark of tumor malignancy. Thus, non-invasive mapping of extracellular pH (pHe) is essential for researchers to understand the tumor microenvironment and to monitor tumor response to metabolism-targeting drugs. While electron paramagnetic resonance (EPR) has been successfully used to map pHe in mouse xenograft models, this method is not sensitive enough to map pHe with a moderate amount of exogenous pH-sensitive probes. Here, we show that a modified EPR system achieves twofold higher sensitivity by using the multiple harmonic detection (MHD) method and improves the robustness of pHe mapping in mouse xenograft models. Our results demonstrate that treatment of a mouse xenograft model of human-derived pancreatic ductal adenocarcinoma cells with the carbonic anhydrase IX (CAIX) inhibitor U-104 delays tumor growth with a concurrent tendency toward further extracellular acidification. We anticipate that EPR-based pHe mapping can be expanded to monitor the response of other metabolism-targeting drugs. Furthermore, pHe monitoring can also be used for the development of improved metabolism-targeting cancer treatments.

Eribulin improves tumor oxygenation demonstrated by 18F-DiFA hypoxia imaging, leading to radio-sensitization in human cancer xenograft models.

PURPOSE: Eribulin, an inhibitor of microtubule dynamics, is known to show antitumor effects through its remodeling activity in the tumor vasculature. However, the extent to which the improvement of tumor hypoxia by eribulin affects radio-sensitivity remains unclear. We utilized 1-(2,2-dihydroxymethyl-3-18F-fluoropropyl)-2-nitroimidazole (18F-DiFA), a new PET probe for hypoxia, to investigate the effects of eribulin on tumor hypoxia and evaluate the radio-sensitivity during eribulin treatment. METHODS: Mice bearing human breast cancer MDA-MB-231 cells or human lung cancer NCI-H1975 cells were administered a single dose of eribulin. After administration, mice were injected with 18F-DiFA and pimonidazole, and tumor hypoxia regions were analyzed. For the group that received combined treatment with radiation, 18F-DiFA PET/CT imaging was performed before tumors were locally X-irradiated. Tumor size was measured every other day after irradiation. RESULTS: Eribulin significantly reduced 18F-DiFA accumulation levels in a dose-dependent manner. Furthermore, the reduction in 18F-DiFA accumulation levels by eribulin was most significant 7 days after treatment. These results were also supported by reduction of the pimonidazole-positive hypoxic region. The combined treatment showed significant retardation of tumor growth in comparison with the control, radiation-alone, and drug-alone groups. Importantly, tumor growth after irradiation was inversely correlated with 18F-DiFA accumulation. CONCLUSION: These results demonstrated that 18F-DiFA PET/CT clearly detected eribulin-induced tumor oxygenation and that eribulin efficiently enhanced the antitumor activity of radiation by improving tumor oxygenation.

Ligand release from silicon phthalocyanine dyes triggered by X-ray irradiation.

Ligand release from silicon phthalocyanine (SiPc) dyes triggered by near-infrared (NIR) light is a key photochemical reaction involving caged compounds based on SiPc. Although NIR light is relatively permeable compared with visible light, this light can be attenuated by tissue absorption and scattering; therefore, using light to induce photochemical reactions deep inside the body is difficult. Herein, because X-rays are highly permeable and can produce radicals through the radiolysis of water, we investigated whether the axial ligands of SiPcs can be cleaved using X-ray irradiation. SiPcs with different axial ligands (alkoxy, siloxy, oxycarbonyl, and phenoxy groups) were irradiated with X-rays under hypoxic conditions. We found that the axial ligands were cleaved via reactions with hydrated electrons (e-aq), not OH radicals, generated from water in response to X-ray irradiation, and SiPc with alkoxy groups exhibited the highest cleavage efficiency. A quantitative investigation revealed that X-ray-induced axial ligand cleavage proceeds via a radical chain reaction. The reaction is expected to be applicable to the molecular design of X-ray-activatable functional molecules in the future.

Mitochondrial fission promotes radiation-induced increase in intracellular Ca2+ level leading to mitotic catastrophe in mouse breast cancer EMT6 cells.

Mitochondrial dynamics are crucial for cellular survival in response to various stresses. Previously, we reported that Drp1 promoted mitochondrial fission after x-irradiation and its inhibition resulted in reduced cellular radiosensitivity and mitotic catastrophe. However, the mechanisms of radiation-induced mitotic catastrophe related to mitochondrial fission remain unclear. In this study, we investigated the involvement of cellular ATP production, ROS generation, and Ca2+ levels in mitotic catastrophe in EMT6 cells. Knockdown of Drp1 and Fis1, which are mitochondrial fission regulators, resulted in elongated mitochondria and significantly attenuated cellular radiosensitivity. Reduced mitochondrial fission mainly decreased mitotic catastrophe rather than necrosis and apoptosis after irradiation. Cellular ATP contents in Drp1 and Fis1 knockdown cells were similar to those in control cells. N-acetylcysteine and 2-glucopyranoside ascorbic acid have no effect on mitotic catastrophe after irradiation. The cellular [Ca2+]i level increased after irradiation, which was completely suppressed by Drp1 and Fis1 inhibition. Furthermore, BAPTA-AM significantly reduced radiation-induced mitotic catastrophe, indicating that cellular Ca2+ is a key mediator of mitotic catastrophe induction after irradiation. These results suggest that mitochondrial fission is associated with radiation-induced mitotic catastrophe via cytosolic Ca2+ regulation.

Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the ß3 adrenergic stimulation.

Bone marrow provides progenitors of several types of cells, including muscle and white adipocytes, ensuring peripheral tissue homeostasis. However, the role of bone marrow-derived cells (BMCs) in induction of thermogenic adipocytes is unresolved. The purpose of this study is to examine whether BMCs are involved in the emergence of thermogenic adipocytes through adrenergic activation. Irradiation of mice with 8 Gy of X-ray-depleted BMCs and peripheral blood mononucleated cells (PBMCs), which in turn impaired induction of uncoupling protein 1 (UCP1) through administration of ß3 adrenergic receptor agonist, CL 316,243 (CL), in inguinal white adipose tissue (iWAT). In contrast, CL-induced UCP1 induction in brown adipose tissue was unaffected by BMC depletion. Transplantation of normal BMCs into mice depleted of BMCs recovered PBMC levels and rescued the ability of iWAT browning by CL. Furthermore, analyses of mice transplanted with green fluorescent protein (GFP)-labeled BMCs revealed that the number of GFP-positive BMCs and PBMCs were significantly decreased by CL and that GFP-positive stromal cells and GFP-positive UCP1-expressing multilocular adipocytes appeared in iWAT after CL administration, demonstrating differentiation of BMC-derived preadipocytes into UCP1-expressing thermogenic adipocytes. These results unveiled a crucial role of the BMC as a nonresident origin for a subset of thermogenic adipocytes, contributing to browning of white adipose tissue.-Yoneshiro, T., Shin, W., Machida, K., Fukano, K., Tsubota, A., Chen, Y., Yasui, H., Inanami, O., Okamatsu-Ogura, Y., Kimura, K. Differentiation of bone marrow-derived cells toward thermogenic adipocytes in white adipose tissue induced by the ß3 adrenergic stimulation.

Radiation-induced abnormal centrosome amplification and mitotic catastrophe in human cervical tumor HeLa cells and murine mammary tumor EMT6 cells.

Mitotic catastrophe is a form of cell death linked to aberrant mitosis caused by improper or uncoordinated mitotic progression. Abnormal centrosome amplification and mitotic catastrophe occur simultaneously, and some cells with amplified centrosomes enter aberrant mitosis, but it is not clear whether abnormal centrosome amplification triggers mitotic catastrophe. Here, to investigate whether radiation-induced abnormal centrosome amplification is essential for induction of radiation-induced mitotic catastrophe, centrinone-B, a highly selective inhibitor of polo-like kinase 4, was utilized to inhibit centrosome amplification, since polo-like kinase 4 is an essential kinase in centrosome duplication. When human cervical tumor HeLa cells and murine mammary tumor EMT6 cells were irradiated with 2.5 Gy of X-rays, cells with morphological features of mitotic catastrophe and the number of cells having >2 centrosomes increased in both cell lines. Although centrinone-B significantly inhibited radiation-induced abnormal centrosome amplification in both cell lines, such treatment did not change cell growth and significantly enhanced mitotic catastrophe in HeLa cells exposed to X-rays. In contrast, inhibition of centrosome amplification reduced cell growth and mitotic catastrophe in EMT6 cells exposed to X-rays. These results indicated that the role of radiation-induced abnormal centrosome amplification in radiation-induced mitotic catastrophe changes, depending on the cell type.

The Adjuvant Effect of Squalene, an Active Ingredient of Functional Foods, on Doxorubicin-Treated Allograft Mice.

Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E2 (PGE2) concentration (P < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response (P < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE2 production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.

In Vivo Extracellular pH Mapping of Tumors Using Electron Paramagnetic Resonance.

An electron paramagnetic resonance (EPR)-based method for noninvasive three-dimensional extracellular pH mapping was developed using a pH-sensitive nitroxyl radical as an exogenous paramagnetic probe. Fast projection scanning with a constant magnetic field sweep enabled the acquisition of four-dimensional (3D spatial +1D spectral) EPR images within 7.5 min. Three-dimensional maps of pH were reconstructed by processing the pH-dependent spectral information on the images. To demonstrate the proposed method of pH mapping, the progress of extracellular acidosis in tumor-bearing mouse legs was studied. Furthermore, extracellular pH mapping was used to visualize the spatial distribution of acidification in different tumor xenograft mouse models of human-derived pancreatic ductal adenocarcinoma cells. The proposed EPR-based pH mapping method enabled quantitative visualization of regional changes in extracellular pH associated with altered tumor metabolism.

Calmodulin-dependent protein kinase II (CaMKII) mediates radiation-induced mitochondrial fission by regulating the phosphorylation of dynamin-related protein 1 (Drp1) at serine 616.

Mitochondrial dynamics are suggested to be indispensable for the maintenance of cellular quality and function in response to various stresses. While ionizing radiation (IR) stimulates mitochondrial fission, which is mediated by the mitochondrial fission protein, dynamin-related protein 1 (Drp1), it remains unclear how IR promotes Drp1 activation and subsequent mitochondrial fission. Therefore, we conducted this study to investigate these concerns. First, we found that X-irradiation triggered Drp1 phosphorylation at serine 616 (S616) but not at serine 637 (S637). Reconstitution analysis revealed that introduction of wild-type (WT) Drp1 recovered radiation-induced mitochondrial fission, which was absent in Drp1-deficient cells. Compared with cells transfected with WT or S637A Drp1, the change in mitochondrial shape following irradiation was mitigated in S616A Drp1-transfected cells. Furthermore, inhibition of CaMKII significantly suppressed Drp1 S616 phosphorylation and mitochondrial fission induced by IR. These results suggest that Drp1 phosphorylation at S616, but not at S637, is prerequisite for radiation-induced mitochondrial fission and that CaMKII regulates Drp1 phosphorylation at S616 following irradiation.

Feasibility of in vivo three-dimensional T 2* mapping using dicarboxy-PROXYL and CW-EPR-based single-point imaging.

OBJECTIVES: The aim of this study was to demonstrate the feasibility of in vivo three-dimensional (3D) relaxation time T 2* mapping of a dicarboxy-PROXYL radical using continuous-wave electron paramagnetic resonance (CW-EPR) imaging. MATERIALS AND METHODS: Isotopically substituted dicarboxy-PROXYL radicals, 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-(1-15N)-1-oxyl (2H,15N-DCP) and 3,4-dicarboxy-2,2,5,5-tetra(2H3)methylpyrrolidin-(3,4-2H2)-1-oxyl (2H-DCP), were used in the study. A clonogenic cell survival assay was performed with the 2H-DCP radical using squamous cell carcinoma (SCC VII) cells. The time course of EPR signal intensities of intravenously injected 2H,15N-DCP and 2H-DCP radicals were determined in tumor-bearing hind legs of mice (C3H/HeJ, male, n = 5). CW-EPR-based single-point imaging (SPI) was performed for 3D T 2* mapping. RESULTS: 2H-DCP radical did not exhibit cytotoxicity at concentrations below 10 mM. The in vivo half-life of 2H,15N-DCP in tumor tissues was 24.7 ± 2.9 min (mean ± standard deviation [SD], n = 5). The in vivo time course of the EPR signal intensity of the 2H,15N-DCP radical showed a plateau of 10.2 ± 1.2 min (mean ± SD) where the EPR signal intensity remained at more than 90% of the maximum intensity. During the plateau, in vivo 3D T 2* maps with 2H,15N-DCP were obtained from tumor-bearing hind legs, with a total acquisition time of 7.5 min. CONCLUSION: EPR signals of 2H,15N-DCP persisted long enough after bolus intravenous injection to conduct in vivo 3D T 2* mapping with CW-EPR-based SPI.

Downregulation of the DNA repair enzyme apurinic/apyrimidinic endonuclease 1 stimulates transforming growth factor-ß1 production and promotes actin rearrangement.

The DNA repair enzyme apurinic/apyrimidinic endonuclease 1 (APE1) plays a central role in base excision repair and functions as a reductive activator of various transcription factors. Multiple other functionalities have been ascribed to APE1 in addition to these major functions. A recent study showed that APE1 knockdown upregulated the expression of a set of genes related to extracellular matrix (ECM) production, indicating an additional novel biological role for this enzyme. Based on this finding, we have investigated the effect of APE1 downregulation on ECM-related gene expression and its biological consequences. Endogenous APE1 expression was downregulated in human cervical carcinoma HeLa cells and human lung carcinoma A549 cells using siRNA. When the expression of six ECM-related genes (TGFB1, LAMC1, FN1, COL1A1, COL3A1, and COL4A1) was evaluated, we found that APE1 knockdown upregulated the expression of TGFB1 in both cell lines. APE1 downregulation promoted actin rearrangement, inducing F-actin accumulation in HeLa cells and the dissipation of stress fibers in A549 cells. We also discovered that APE1 knockdown enhanced cellular motility in A549 cells, which was suppressed by the inhibition of transforming growth factor (TGF)-ß1 signaling. These results suggested that APE1 controls the organization of actin cytoskeleton through the regulation of TGF-ß1 expression, providing novel insights into the biological significance of APE1.

Activation of eNOS in endothelial cells exposed to ionizing radiation involves components of the DNA damage response pathway.

In this study, the involvement of ataxia telangiectasia mutated (ATM) kinase and heat shock protein 90 (HSP90) in endothelial nitric oxide synthase (eNOS) activation was investigated in X-irradiated bovine aortic endothelial cells. The activity of nitric oxide synthase (NOS) and the phosphorylation of serine 1179 of eNOS (eNOS-Ser1179) were significantly increased in irradiated cells. The radiation-induced increases in NOS activity and eNOS-Ser1179 phosphorylation levels were significantly reduced by treatment with either an ATM inhibitor (Ku-60019) or an HSP90 inhibitor (geldanamycin). Geldanamycin was furthermore found to suppress the radiation-induced phosphorylation of ATM-Ser1181. Our results indicate that the radiation-induced eNOS activation in bovine aortic endothelial cells is regulated by ATM and HSP90.

In vivo tumour extracellular pH monitoring using electron paramagnetic resonance: the effect of X-ray irradiation.

The in vivo quantification of extracellular pH (pHe ) in tumours may provide a useful biomarker for tumour cell metabolism. In this study, we assessed the viability of continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy with a pH-sensitive nitroxide for the measurement of extracellular tumour pH in a mouse model. CW-EPR spectroscopy (750 MHz) of C3H HeJ mice hind leg squamous cell tumour was performed after intravenous tail vein injection of pH-sensitive nitroxide (R-SG, 2-(4-((2-(4-amino-4-carboxybutanamido)-3-(carboxymethylamino)-3-oxoproylthio)methyl)phenyl)-4-pyrrolidino-2,5,5-triethyl-2,5-dihydro-1Н-imidazol-1-oxyl) during stages of normal tumour growth and in response to a single 10-Gy dose of X-ray irradiation. An inverse relationship was observed between tumour volume and pHe value, whereby, during normal tumour growth, a constant reduction in pHe was observed. This relationship was disrupted by X-ray irradiation and, from 2-3 days post-exposure, a transitory increase in pHe was observed. In this study, we demonstrated the viability of CW-EPR spectroscopy using R-SG nitroxide to obtain high-sensitivity pH measurements in a mouse tumour model with an accuracy of <0.1 pH units. In addition, the measured changes in pHe in response to X-ray irradiation suggest that this may offer a useful method for the assessment of the physiological change in response to existing and novel cancer therapies.

Time-dependent changes in retinoids content in liver and adipose tissue after feeding of a vitamin A-deficient diet to mice.

Vitamin A is an important nutrient for multiple physiological functions. To elucidate the role of vitamin A in vivo, vitamin A-deficient diets have been often used in mice to establish a vitamin A-deficiency model. However, the information on the appropriate feeding periods and time course of changes in vitamin A content in organs after the start of vitamin A-deficient diet feeding is lacking. This study aimed to assess the retinoids levels in liver and white adipose tissue in mice fed a vitamin A-deficient diet for ≤8 weeks. High-performance liquid chromatography was used to measure the retinoids levels in liver and white adipose tissue every 2 weeks for ≤8 weeks. Vitamin A-deficient diet feeding significantly decreased retinol in the liver over 6 weeks, but retinyl palmitate, a main storage form of vitamin A, was not changed over 8 weeks. The plasma retinol level remained constant throughout the experiment. In white adipose tissue, retinyl palmitate gradually decreased over 8 weeks. These results indicate that vitamin A-deficient diet feeding longer than 6 weeks reduced retinol in liver and retinyl palmitate in white adipose tissue over 8 weeks, although it is not enough for the induction of a whole-body vitamin A deficiency.

Mechanism of the Radioresistant Colorectal Cancer Cell Line SW480RR Established after Fractionated X Irradiation.

Radioresistant cancer cells are risk factors for recurrence and are occasionally detected in recurrent tumors after radiotherapy. Intratumor heterogeneity is believed to be a potential cause of treatment resistance. Heterogeneity in DNA content has also been reported in human colorectal cancer; however, little is known about how such heterogeneity changes with radiotherapy or how it affects cancer radioresistance. In the present study, we established radioresistant clone SW480RR cells after fractionated X-ray irradiation of human colorectal cancer-derived SW480.hu cells, which are composed of two cell populations with different chromosome numbers, and examined how cellular radioresistance changed with fractionated radiotherapy. Compared with the parental cell population, which mostly comprised cells with higher ploidy, the radioresistant clones showed lower ploidy and less initial DNA damage. The lower ploidy cells in the parental cell population were identified as having radioresistance prior to irradiation; thus, SW480RR cells were considered intrinsically radioresistant cells selected from the parental population through fractionated irradiation. This study presents a practical example of the emergence of radioresistant cells from a cell population with ploidy heterogeneity after irradiation. The most likely mechanism is the selection of an intrinsically radioresistant population after fractionated X-ray irradiation, with a background in which lower ploidy cells exhibit lower initial DNA damage.

Partial Acquisition of Spectral Projections Accelerates Four-dimensional Spectral-spatial EPR Imaging for Mouse Tumor Models: A Feasibility Study.

PURPOSE: Our study aimed to accelerate the acquisition of four-dimensional (4D) spectral-spatial electron paramagnetic resonance (EPR) imaging for mouse tumor models. This advancement in EPR imaging should reduce the acquisition time of spectroscopic mapping while reducing quality degradation for mouse tumor models. PROCEDURES: EPR spectra under magnetic field gradients, called spectral projections, were partially measured. Additional spectral projections were later computationally synthesized from the measured spectral projections. Four-dimensional spectral-spatial images were reconstructed from the post-processed spectral projections using the algebraic reconstruction technique (ART) and assessed in terms of their image qualities. We applied this approach to a sample solution and a mouse Hs766T xenograft model of human-derived pancreatic ductal adenocarcinoma cells to demonstrate the feasibility of our concept. The nitroxyl radical imaging agent 2H,15N-DCP was exogenously infused into the mouse xenograft model. RESULTS: The computation code of 4D spectral-spatial imaging was tested with numerically generated spectral projections. In the linewidth mapping of the sample solution, we achieved a relative standard uncertainty (standard deviation/| mean |) of 0.76 µT/45.38 µT = 0.017 on the peak-to-peak first-derivative EPR linewidth. The qualities of the linewidth maps and the effect of computational synthesis of spectral projections were examined. Finally, we obtained the three-dimensional linewidth map of 2H,15N-DCP in a Hs766T tumor-bearing leg in vivo. CONCLUSION: We achieved a 46.7% reduction in the acquisition time of 4D spectral-spatial EPR imaging without significantly degrading the image quality. A combination of ART and partial acquisition in three-dimensional raster magnetic field gradient settings in orthogonal coordinates is a novel approach. Our approach to 4D spectral-spatial EPR imaging can be applied to any subject, especially for samples with less variation in one direction.

Theoretical Design and Synthesis of Caged Compounds Using X-Ray-Triggered Azo Bond Cleavage.

Caged compounds are frequently used in life science research. However, the light used to activate them is commonly absorbed and scattered by biological materials, limiting their use to basic research in cells or small animals. In contrast, hard X-rays exhibit high bio-permeability due to the difficulty of interacting with biological molecules. With the main goal of developing X-ray activatable caged compounds, azo compounds are designed and synthesized with a positive charge and long π-conjugated system to increase the reaction efficiency with hydrated electrons. The azo bonds in the designed compounds are selectively cleaved by X-ray, and the fluorescent substance Diethyl Rhodamine is released. Based on the results of experiments and quantum chemical calculations, azo bond cleavage is assumed to occur via a two-step process: a two-electron reduction of the azo bond followed by N─N bond cleavage. Cellular experiments also demonstrate that the azo bonds can be cleaved intracellularly. Thus, caged compounds that can be activated by an azo bond cleavage reaction promoted by X-ray are successfully generated.

Feasibility study of multimodal imaging for redox status and glucose metabolism in tumor.

Understanding the tumor redox status is important for efficient cancer treatment. Here, we noninvasively detected changes in the redox environment of tumors before and after cancer treatment in the same individuals using a novel compact and portable electron paramagnetic resonance imaging (EPRI) device and compared the results with glycolytic information obtained through autoradiography using 2-deoxy-2-[18F]fluoro-d-glucose ([18F]FDG). Human colon cancer HCT116 xenografts were used in the mice. We used 3-carbamoyl-PROXYL (3CP) as a paramagnetic and redox status probe for the EPRI of tumors. The first EPRI was followed by the intraperitoneal administration of buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis, or X-ray irradiation of the tumor. A second EPRI was performed on the following day. Autoradiography was performed after the second EPRI. After imaging, the tumor sections were evaluated by histological analysis and the amount of reducing substances in the tumor was measured. BSO treatment and X-ray irradiation significantly decreased the rate of 3CP reduction in tumors. Redox maps of tumors obtained from EPRI can be compared with tissue sections of approximately the same cross section. BSO treatment reduced glutathione levels in tumors, whereas X-ray irradiation did not alter the levels of any of the reducing substances. Comparison of the redox map with the autoradiography of [18F]FDG revealed that regions with high reducing power in the tumor were active in glucose metabolism; however, this correlation disappeared after X-ray irradiation. These results suggest that the novel compact and portable EPRI device is suitable for multimodal imaging, which can be used to study tumor redox status and therapeutic efficacy in cancer, and for combined analysis with other imaging modalities.

Radiation-induced nitric oxide mitigates tumor hypoxia and radioresistance in a murine SCCVII tumor model.

Tumor hypoxia, which occurs mainly as a result of inadequate tissue perfusion in solid tumors, is a well-known challenge for successful radiotherapy. Recent evidence suggests that ionizing radiation (IR) upregulates nitric oxide (NO) production and that IR-induced NO has the potential to increase intratumoral circulation. However, the kinetics of NO production and the responsible isoforms for NO synthase in tumors exposed to IR remain unclear. In this study, we aimed to elucidate the mechanism by which IR stimulates NO production in tumors and the effect of IR-induced NO on tumor radiosensitivity. Hoechst33342 perfusion assay and electron spin resonance oxymetry showed that IR increased tissue perfusion and pO2 in tumor tissue. Immunohistochemical analysis using two different hypoxic probes showed that IR decreased hypoxic regions in tumors; treatment with a nitric oxide synthase (NOS) inhibitor, L-NAME, abrogated the effects of IR. Moreover, IR increased endothelial NOS (eNOS) activity without affecting its mRNA or protein expression levels in SCCVII-transplanted tumors. Tumor growth delay assay showed that L-NAME decreased the anti-tumor effect of fractionated radiation (10Gy×2). These results suggested that IR increased eNOS activity and subsequent tissue perfusion in tumors. Increases in intratumoral circulation simultaneously decreased tumor hypoxia. As a result, IR-induced NO increased tumor radiosensitivity. Our study provides a new insight into the NO-dependent mechanism for efficient fractionated radiotherapy.