Involvement of APOBEC3B in mutation induction by irradiation.

To better understand the cancer risk posed by radiation and the development of radiation therapy resistant cancer cells, we investigated the involvement of the cancer risk factor, APOBEC3B, in the generation of radiation-induced mutations. Expression of APOBEC3B in response to irradiation was determined in three human cancer cell lines by real-time quantitative PCR. Using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutation assay, mutations in the HPRT gene caused by irradiation were compared between APOBEC3B-deficient human hepatocellular carcinoma (HepG2) cells [APOBEC3B knocked out (KO) using CRISPR-Cas9 genome editing] and the parent cell line. Then, HPRT-mutated cells were individually cultured to perform PCR and DNA sequencing of HPRT exons. X-Irradiation induced APOBEC3B expression in HepG2, human cervical cancer epithelial carcinoma (HeLa) and human oral squamous cell carcinoma (SAS) cells. Forced expression of APOBEC3B increased spontaneous mutations. By contrast, APOBEC3B KO not only decreased the spontaneous mutation rate, but also strongly suppressed the increase in mutation frequency after irradiation in the parent cell line. Although forced expression of APOBEC3B in the nucleus caused DNA damage, higher levels of APOBEC3B tended to reduce APOBEC3B-induced γ-H2AX foci formation (a measure of DNA damage repair). Further, the number of γ-H2AX foci in cells stably expressing APOBEC3B was not much higher than that in controls before and after irradiation, suggesting that a DNA repair pathway may be activated. This study demonstrates that irradiation induces sustained expression of APOBEC3B in HepG2, HeLa and SAS cells, and that APOBEC3B enhances radiation-induced partial deletions.

Loss of EGF-dependent cell proliferation ability on radioresistant cell HepG2-8960-R.

Acquired radioresistance of cancer cells interferes with radiotherapy and increases the probability of cancer recurrence. HepG2-8960-R, which is one of several clinically relevant radioresistant (CRR) cell lines, has a high tolerance to the repeated clinically relevant doses of X-ray radiation. In this study, HepG2-8960-R had slightly lower cell proliferation ability than HepG2 in the presence of FBS. In particular, epidermal growth factor (EGF) hardly enhanced cell proliferation and DNA synthesis in HepG2-8960-R. Additionally, EGF could not induce the activation of Erk1/2, because the expression of EGF receptor (EGFR) protein decreased in HepG2-8960-R in accordance with the methylation of the EGFR promoter region. Therefore, cetuximab did not inhibit HepG2-8960-R cell proliferation. Our study showed that HepG2-8960-R had radioresistant and cetuximab-resistant abilities.

Radiosynthesis and initial evaluation of (18)F labeled nanocarrier composed of poly(L-lactic acid)-block-poly(sarcosine) amphiphilic polydepsipeptide.

INTRODUCTION: With the aim of developing radiotracers for in vivo positron emission tomography (PET) imaging of solid tumors based on the enhanced permeability and retention effect of nanocarriers, we have developed a polymer micelle named "Lactosome", which is composed of the amphiphilic polydepsipeptide, poly(L-lactic acid)-block-poly(sarcosine). This paper describes and evaluates the initial evaluation of the (18)F-labeled Lactosome as a novel contrast agent for the tumor PET imaging technique carried out. METHODS: (18)F-labeled Lactosomes were prepared by a film hydration method under sonication in water at 50°C from a mixture of 4-[(18)F]fluoro-benzoyl poly-L-lactic acid ((18)F-BzPLLA30) and the amphiphilic polydepsipeptide. For biodistribution studies, BALB/cA Jcl-nu/nu mice bearing HeLa cells in the femur region were used. We took both PET and near-infrared fluorescence (NIRF) images of tumor bearing mice after co-injection of (18)F-labeled Lactosome and NIRF-labeled Lactosome. RESULTS: (18)F-labeled Lactosomes were prepared at good yields (222-420MBq) and more than 99% of (18)F-BzPLLA30 was incorporated into (18)F-labeled Lactosome. The radioactivity of (18)F-labeled Lactosome was found to be stable and maintained at high level for up to 6h after injection into the blood stream. Tumor uptake increased gradually after the injection. The uptake ratio of tumor/muscle was 2.7 at 6h from the time of injection. Tumor PET imaging with (18)F-labeled Lactosome was as capable as tumor NIRF imaging with NIRF-labeled Lactosome. CONCLUSION: Tumor PET imaging using Lactosome as a nanocarrier may be therefore a potential candidate for a facile and general solid tumor imaging technique.

Pharmacokinetic change of nanoparticulate formulation "Lactosome" on multiple administrations.

Lactosome, which is a polymer micelle composed of poly(lactic acid)-b-poly(sarcosine), was applied successfully for solid tumor imaging. Lactosome is considered to escape from the reticuloendothelial system recognition, and shows prolonged in vivo blood clearance time. In vivo disposition of Lactosome, however, changed upon multiple dosages. Lactosome at the 2nd dosage was cleared from the blood stream by trapping at liver. This accelerated blood clearance (ABC) phenomenon is explained by production of anti-Lactosome IgM and IgG(3) through the immune response related with B-lymphocyte cells. The memory effect of B-lymphocyte cells lasted nearly for six months in mouse. The epitope moiety of Lactosome is concluded to be poly(sarcosine) based on the competitive inhibition assay. Since the ABC phenomenon was also reported with PEGylated liposome, nanoparticles in general may be potential in triggering the immune system.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes.

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

Control of in vivo blood clearance time of polymeric micelle by stereochemistry of amphiphilic polydepsipeptides.

Polymeric micelle, "Lactosome", is composed of amphiphilic polydepsipeptide with a hydrophobic block of helical poly(L-lactic acid) (PLLA) and a hydrophilic block of poly(sarcosine). Lactosome was labeled by incorporation of poly(lactic acid) having a near-infrared fluorescence (NIRF) chromophore, and studied on blood clearance and tumor imaging. In vivo blood clearance time of Lactosome was prolonged with incorporation of poly(D-lactic acid) (PDLA), but decreased with poly(D,L-lactic acid) (PDLLA). NIRF imaging with applying these Lactosomes to tumor-bearing mice revealed that the tumor/background intensity ratio increased with incorporation of PDLLA. Stereochemistry in the hydrophobic core of self-assemblies is thus an important factor for determining physical stability in the blood stream and consequently contrast in imaging.

5-O-(4-[125 I]Iodobenzyl)-L-ascorbic acid: electrophilic radioiodination and biodistribution in mice.

As a part of our efforts to develop potential imaging agents for ascorbate bioactivity, 5-O-(4-[(125)I]iodobenzyl)-L-ascorbic acid ([(125)I]1) was prepared through a two-step sequence which involved radioiodo-destannylation of a protected tributylstannyl precursor 6, followed by hydrolysis in acidic methanol of the protecting groups in 61% overall radiochemical yield, with a radiochemical purity of over 98% and a specific activity of more than 15.4 GBq/µmol. Tissue distribution of [(125)I]1 in tumor-bearing mice showed signs of distribution profiles similar to the reported results for 6-deoxy-6-[(18)F]fluoro-L-ascorbic (6-(18)FAsA) acid and 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) but with notable differences in the adrenal glands, in which considerably lower uptake of radioactivity and rapid clearance with time were observed. Pretreatment of mice with a known inhibitor of ascorbate transport, sulfinpyrazone, did not produce any significant change in the adrenal uptake of radioactivity after injection of [(125)I]1 compared to the control, suggesting that uptake in the adrenal glands is independent of the sodium-dependent vitamin C transporter 2 transport mechanism. Introduction of a bulky substituent at C-5 on AsA, such as an iodobenzyloxy group, may not be suitable for the design of analogs that may still be able to maintain characteristic distribution properties in vivo seen with AsA itself.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity.

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the alpha(1)-adrenergic receptor coupled with G protein, Galpha(h). However, the molecular mechanism is not well understood. Galpha(h) is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [(3)H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Galpha(h).

6-Deoxy-6-[131I]iodo-L-ascorbic acid for the in vivo study of ascorbate: autoradiography, biodistribution in normal and hypolipidemic rats, and in tumor-bearing nude mice.

Normal female rat distribution studies showed high and specific uptake of 6-deoxy-6-[(131)I]iodo-L-ascorbic acid (6-(131)IAsA) into the adrenal glands, known to highly express the ascorbate sodium-dependent vitamin C transporter-2 (SVCT-2), and the adrenal gland was clearly visualized by whole-body autoradiography. Preinjection of sulfinpyrazone, a known blocker of ascorbate transport, with 6-(131)IAsA resulted in decreased uptake of radioactivity in rat adrenal glands compared to the control group, seemingly illustrating the participation of the SVCT transporter (probably the SVCT-2 subtype) in the uptake process in vivo. 4-Aminopyrazolo[3,4-d]pyrimidine-induced hypolipidemic rats showed a 1.7-fold increase in adrenal uptake of radioactivity at 30 min postinjection of 6-(131)IAsA, compared to the control, with increased adrenal-to-liver and adrenal-to-kidney ratios. To further characterize 6-(131)IAsA for its tumor uptake properties, biodistribution studies were also performed using male nude mice implanted with either Y-1 adrenocortical tumor cells or adrenal medulla-derived PC12 cells. None of these tumors exhibited relevant uptake of 6-(131)IAsA while normal adrenal glands showed high uptake of radioactivity, suggesting that these tumors in this model have only a poor transport capacity for this agent. The present study demonstrates that the use of radioiodinated 6-IAsA may help to obtain information about functional alterations in diseased adrenal glands, but it does not exhibit desirable properties as a tumor-seeking agent for ascorbic acid bioactivity.

Design of Ga-DOTA-based bifunctional radiopharmaceuticals: two functional moieties can be conjugated to radiogallium-DOTA without reducing the complex stability.

From the X-ray crystal structures of Ga-DOTA chelates, we were able to deduce that two free carboxylate groups of the radiogallium-DOTA complex may be utilized for coupling to functional moieties that recognize molecular targets for in vivo imaging without reducing the radiogallium-complex stability. Thus, we designed 2,2'-[4,10-bis(2-{[2-(2-methyl-5-nitro-1H-imidazol-1-yl)ethyl]amino}-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,7-diyl]diacetic acid (DOTA-MN2) (7), employing a metronidazole moiety as the recognition site of hypoxic lesions, based on the drug design concept of bifunctional radiopharmaceuticals. Coupling of DOTA-bis(tert-butyl)ester 5 with 1-(2-aminoethyl)-2-methyl-5-nitroimidazole dihydrochloride, followed by deprotection, afforded the required 7 (DOTA-MN2). (67)Ga-labeling was carried out by reaction of DOTA-MN2 with (67)Ga-citrate. When (67)Ga-DOTA-MN2 was incubated in phosphate-buffered saline or mouse plasma, no measurable decomposition occurred over a 24-h period. In biodistribution experiments in NFSa tumor-bearing mice, (67)Ga-DOTA-MN2 displayed not only a significant tumor uptake, but also rapid blood clearance and low accumulations in nontarget tissues, resulting in high target-to-nontarget ratios of radioactivity. These results indicate the potential benefits of the drug design of (67)Ga-DOTA-MN2. The present findings provide helpful information for the development of radiogallium-labeled radiopharmaceuticals for SPECT and PET studies.

Synthesis of a beta-tetrapeptide analog as a mother compound for the development of matrix metalloproteinase-2-imaging agents.

Matrix metalloproteinase-2 (MMP-2) is an attractive target for the diagnosis of cancer and atherosclerosis in nuclear imaging. A cyclic decapeptide, cCTTHWGFTLC (cCTT), has been used as the mother compound for the development of MMP-2-imaging agents with high potency and selectivity. Most of radiolabeled derivatives of cCTT currently developed for in vivo studies of MMP-2, however, suffer from low accumulation in the target tissues, such as tumors. For enhanced in vivo stability and tissue penetration, we designed a linear beta-tetrapeptide analog, H-beta 3-Phe-beta-Ala-beta 3-Trp-beta 3-His-OH (1), to mimic cCTT. The component beta-amino acids were prepared by reduction of N-protected alpha-amino acid methyl esters to the alcohols, followed by conversion into the cyanides, and subsequent hydrolysis. Compound 1 was obtained from these beta-amino acids by the conventional solution method. In MMP-2 inhibition assay, compound 1 displayed desirably significant inhibition, which was comparable to cCTT. These findings suggest that compound 1 may serve as a mother compound in the design and development of in vivo MMP-2-imaging agents.

Radiosynthesis and evaluation of 11C-labeled diaryl-substituted imidazole and indole derivatives for mapping cyclooxygenase-2.

11C-labeled analogs of 4-chloro-5-(3-fluoro-4-methoxyphenyl)-1-(4-methylsulfonylphenyl)imidazole ([11C]1), 4-[4-chloro-5-(3-fluoro-4-methoxyphenyl)imidazol-1-yl]benzenesulfonamide ([11C]2) and 2-(4-aminosulfonylphenyl)-3-(4-methoxyphenyl)indole ([11C]3), which have been shown to have excellent potency and high selectivity for cyclooxygenase isoform 2 (COX-2) inhibiting activity, were prepared and evaluated in rats as potential radiopharmaceuticals for imaging the COX-2 enzyme by positron emission tomography. These 11C-labeled COX-2 inhibitors were synthesized in high radiochemical yields by O-[11C]methylation of phenolic precursors with [11C]methyl triflate in acetone containing NaOH as a base. In vivo evaluation in rats bearing AH109A hepatoma showed no specific binding of any tracer to COX-2 in any tissue such as the brain, heart, lung, kidney, and AH109A hepatoma. In ex vivo autoradiography, [11C]1 showed regionally different distribution in the brain, while [11C]2 and [11C]3 were not substantially taken up by the brain. In in vitro monolayer efflux assays, compound 3 was found to be a substrate for the P-glycoprotein (P-gp) efflux pump, but pretreatment of rats with the potent P-gp inhibitor, cyclosporine A, did not have any significant influence on the cerebral uptake of [11C]3. These results indicate that all three tracers were not suitable for in vivo imaging of COX-2. There seem to be some obstacles to finding a useful candidate for in vivo imaging application of COX-2 selective inhibitors only by standard consideration of in vitro affinity and selectivity, and the lipophilicity of the compound.

Radiosynthesis and in vivo evaluation of 11C-labeled 1,5-diarylpyrazole derivatives for mapping cyclooxygenases.

We prepared 11C-labeled 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole ([11C]1) and 4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-pyrazol-1-yl]benzenesulfonamide ([11C]2) for imaging COX-1 and COX-2 isoforms, respectively, by positron emission tomography. [11C]1 and [11C]2 were synthesized in high radiochemical yields by O-[11C]methylation with [11C]methyl triflate in acetone containing an equivalent of NaOH as a base with respect to the phenolic precursors. In vivo evaluation in rats bearing AH109A hepatoma demonstrated minimal specific binding of [11C] to COX-1 in peripheral organs, such as the spleen and small intestine. Carrier-saturable uptake of [11C]2 was found in the spleen, but COX-2-specific binding of [11C]2 was not identifiable in the brain, AH109A hepatoma or other peripheral organs, although ex vivo autoradiography showed regionally different distribution in the brain and AH109A. The results suggest that neither [11C]1 nor [11C]2 is a suitable radioligand for in vivo biomarkers of COX enzymes, mainly because of marked non-specific binding.

Decreased tissue accumulation of 6-deoxy-6-[18F]fluoro-L-ascorbic acid in glutathione-deficient rats induced by administration of diethyl maleate.

The relationship between in vivo biodistribution of 6-deoxy-6-[18F]fluoro-L-ascorbic acid (18F-DFA) and the content of tissue glutathione (GSH) was investigated in Wistar male rats. Following intravenous administration of 18F-DFA, the accumulation of radioactivity in most tissues, including the adrenal glands, liver and brain, was significantly reduced together with a decrease in the content of GSH by preloading of diethyl maleate (DEM) which depletes cellular GSH. Similar decreased uptake was also observed in the distribution of L-[1-14C]ascorbic acid (14C-AA) after DEM treatment. The possible biological mechanisms, including competition with endogenous AA and ascorbate recycling, that modulate the uptake and accumulation into tissues of 18F-DFA and 14C-AA in GSH-deficient rats are discussed.

Synthesis, in vitro and in vivo pharmacology of a C-11 labeled analog of CP-101,606, (+/-)threo-1-(4-hydroxyphenyl)-2-[4-hydroxy-4-(p-[11C]methoxyphenyl)piperidino]-1-propanol, as a PET tracer for NR2B subunit-containing NMDA receptors.

A carbon-11 labeled methoxyl analog of CP-101,606, (+/-)threo-1-(4-hydroxyphenyl)-2-[4-hydroxy-4-(p-[11C]methoxyphenyl)piperidino]-1-propanol [(+/-)[11C]1], was synthesized as a new subtype-selective PET radioligand for NMDA receptors. The in vitro binding studies using rat brain slices demonstrated that (+/-)[11C]1 shows an extremely high-specific binding to the NR2B subunit of NMDA receptors. In contrast to the in vitro binding, the in vivo binding to mouse and monkey brains showed no apparent specific localization of the radioactivity in any of the brain regions. Metabolism and physicochemical properties such as the lipophilicity of (+/-)[11C]1 seemed unlikely to affect the in vivo (+/-)[11C]1 binding. Among the various endogenous ligands acting at the NMDA receptors, polyamines (spermine and spermidine) and divalent cations (Mg(2+,) Zn(2+,) and Ca(2+)) strongly inhibited the in vitro (+/-)[11C]1 binding. Thus, the present studies point to the possibility that the polyamines and cations behave as endogenous inhibitors for (+/-)[11C]1 binding, leading to the loss of the specific binding in vivo.

Synthesis and evaluation of 4-bromo-1-(3-[18F]fluoropropyl)-2-nitroimidazole with a low energy LUMO orbital designed as brain hypoxia-targeting imaging agent.

In order to develop new imaging markers for brain hypoxia, 4-bromo-1-(3-fluoropropyl)-2-nitroimidazole (4-BrFPN) was designed based on molecular orbital calculations, synthesized and labeled with fluorine-18 as a lipophilic nitroimidazole analog with a lower energy LUMO orbital than those for fluoromisonidazole (FMISO) and 1-(3-fluoropropyl)-2-nitroimidazole (FPN). In an in vitro radiosensitization study, the sensitizer enhancement ratio for 4-BrFPN was found to be 1.65 at a I mM concentration, in comparison to 1.81 for FMISO. The preparation of 18F-labeled 4-BrFPN (4-Br18FPN) was achieved by [18F]fluoride ion displacement reaction of the tosylate precursor, in a reasonable radiochemical yield (33%, not corrected for decay). Metabolites in tumor and muscle extracts from methylcholanthrene-induced fibrosarcoma mice, as well as the tissue distribution of 4-Br18FPN in normal rats, were studied. The initial uptake into rat brain of 4-Br18FPN was significantly higher relative to 18F-labeled FMISO (18FMISO), followed by a rapid washout from the brain. The tumor uptake of 4-Br18FPN was somewhat enhanced compared to those obtained with 18FMISO and 18F-labeled FPN (18FPN), but with lower tumor localization than 18FMISO. Analyses of tumor and muscle extracts showed metabolites remaining base line on the radio-TLC plates, and they were produced to a greater extent in tumor than muscle. The use of two drugs which increase hypoxic cell fraction in tumor, hydralazine or nitro-L-arginine, produced a significant increase in tumor levels of 4-Br18FPN, suggestive of a hypoxic mechanism of accumulation. The results imply that lowering of the LUMO energy of a molecule alone is not sufficient to improve its biodistribution properties for better imaging of regions of hypoxia.

2-Amino-6-vinyl purine as a tool for post synthetic conjugation of DNA with radio-, spin-, fluorescence labels and peptides.

We have previously demonstrated that 2-amino-6-vinylpurine is an efficient cross-linking agent with high selectivity towards cytidine. In this study, we applied this nucleobase analog to versatile conjugation of oligodeoxynucleotides with radio-, spin-, fluorescence- and peptide labels. The oligodeoxynucleotides incorporating 2-amino-6-vinylpurine nucleoside were reacted with amino- bearing nucleophies in an excess amount to give the corresponding conjugates in good yield.