Mitsuyoshi Yoshimoto

Characterization of acetate metabolism in tumor cells in relation to cell proliferation: Acetate metabolism in tumor cells

To reveal the metabolic fate of acetate in neoplasms that may characterize the accumulation patterns of [1-(11)C]acetate in tumors depicted by positron emission tomography. Four tumor cell lines (LS174T, RPMI2650, A2780, and A375) and fibroblasts in growing and resting states were used. In uptake experiments, cells were incubated with[1-(14)C]acetate for 40 min. [(14)C]CO(2) was measured in the tight-air chamber, and the metabolites in cells were identified by thin layer chromatography and paper chromatography. The glucose metabolic rate of each cell line was measured with [2,6-(3)H]2-deoxy-glucose (DG), and the growth activity of each cell line was estimated by measuring the incorporation of [(3)H]methyl thymidine into DNA. Compared with resting fibroblasts, all four tumor cell lines showed higher accumulation of (14)C activity from [1-(14)C]acetate. These tumor-to-normal ratios of [1-(14)C]acetate were larger than those of DG. Tumor cells incorporated (14)C activity into the lipid-soluble fraction, mostly of phosphatidylcholine and neutral lipids, more prominently than did fibroblasts. The lipid-soluble fraction of (14)C accumulation in cells showed a positive correlation with growth activity, whereas the water-soluble and CO(2) fractions did not. These findings suggest that the high tumor-to-normal ratio of [1-(14)C]acetate is mainly due to the enhanced lipid synthesis, which reflects the high growth activity of neoplasms. This in vitro study suggests that [1-(11)C]acetate is appropriate for estimating the growth activity of tumor cells.

Putative Transport Mechanism and Intracellular Fate of Trans-1-Amino-3-18F-Fluorocyclobutanecarboxylic Acid in Human Prostate Cancer

Trans-1-amino-3-18F-fluorocyclobutanecarboxylic acid (anti–18F-FACBC) is an amino acid PET tracer that has shown promise for visualizing prostate cancer. Therefore, we aimed to clarify the anti–18F-FACBC transport mechanism in prostate cancer cells. We also studied the fate of anti–18F-FACBC after it is transported into cells. Methods: For convenience, because of their longer half-lives, 14C compounds were used instead of 18F-labeled tracers. Trans-1-amino-3-fluoro-1-14C-cyclobutanecarboxylic acid (14C-FACBC) uptake was examined in human prostate cancer DU145 cells with the following substrates of amino acid transporters: α-(methylamino) isobutyric acid (a system A–specific substrate) and 2-amino-2-norbornanecarboxylic acid (a system L–specific substrate). The messenger RNA expression of amino acid transporters in human prostate cancer specimens was analyzed by complementary DNA microarray and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Gene expression in DU145 cells was analyzed by qRT-PCR. We also examined the knockdown effect of the amino acid transporters system ASC transporter 2 (ASCT2) and sodium-coupled neutral amino acid transporter 2 (SNAT2) on 14C-FACBC uptake. In addition, the possibility of 14C-FACBC incorporation into proteins was examined. Results: 14C-FACBC uptake by DU145 cells was markedly decreased to approximately 20% in the absence of Na+, compared with that in its presence, indicating that Na+-dependent transporters are mainly responsible for the uptake of this tracer. Moreover, 2-amino-2-norbornanecarboxylic acid inhibited the transport of 14C-FACBC to the basal level in Na+-free buffer. In contrast, α-(methylamino) isobutyric acid did not inhibit 14C-FACBC accumulation in DU145 cells. Human prostate tumor specimens and DU145 cells had similar messenger RNA expression patterns of amino acid transporter genes. Although SNAT2 and ASCT2 are 2 major amino acid transporters expressed in prostate tumor tissues and DU145 cells, ASCT2 knockdown using small interfering RNA was more effective in lowering 14C-FACBC transport than SNAT2. Almost all intracellular 14C-FACBC was recovered from the nonprotein fraction. Conclusion: ASCT2, which is a Na+-dependent amino acid transporter, and to a lesser extent Na+-independent transporters play a role in the uptake of 14C-FACBC by DU145 cells. Among the Na+-independent transporters, system L transporters are also involved in the transport of 14C-FACBC. Moreover, 14C-FACBC is not incorporated into proteins in cells. These findings suggest a possible mechanism of anti–18F-FACBC PET for prostate cancer.

Intra-tumoral distribution of 64Cu-ATSM: A comparison study with FDG

(64)Cu-labeled diacetyl-bis(N(4)-methylthiosemicarbazone) ((64)Cu-ATSM) is a promising agent for internal radiation therapy and imaging of hypoxic tissues. In the present study, the intra-tumoral distribution of (64)Cu-ATSM was investigated by comparing it to that of [(18)F]FDG and histological findings. VX2 tumors were implanted into Japanese white rabbits subcutaneously. (64)Cu-ATSM and [(18)F]FDG were co-injected intravenously and the tumor was dissected and cut into 1 mm thick slices 1 h after the injection. The uptake of (64)Cu-ATSM and [(18)F]FDG was measured using a dual-tracer autoradiographic technique. Histological cell biology was estimated from the optical microscopy of tumor sections. The major accumulation of (64)Cu-ATSM was observed around the outer rim of the tumor masses which consisted mainly of active cells and expected to be hypoxic. [(18)F]FDG was distributed more widely with highest levels in the inner regions where pre-necrotic cells were mainly observed. (64)Cu-ATSM appears to be useful for the detection of hypoxic but active tumor cell regions in vivo.

Development of [90Y]DOTA-conjugated bisphosphonate for treatment of painful bone metastases

INTRODUCTION Based on the concept of bifunctional radiopharmaceuticals, we have previously developed (186)Re-complex-conjugated bisphosphonate analogs for palliation of painful bone metastases and have demonstrated the utility of these compounds. By applying a similar concept, we hypothesized that a bone-specific directed (90)Y-labeled radiopharmaceutical could be developed. METHODS In this study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as the chelating site, and DOTA was conjugated with 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. [(90)Y]DOTA-complex-conjugated bisphosphonate ([(90)Y]DOTA-HBP) was prepared by coordination with (90)Y, and its biodistribution was studied in comparison to [(90)Y]citrate. RESULTS In biodistribution experiments, [(90)Y]DOTA-HBP and [(90)Y]citrate rapidly accumulated and resided in the bone. Although [(90)Y]citrate showed a higher level of accumulation in the bone than [(90)Y]DOTA-HBP, the clearances of [(90)Y]DOTA-HBP from the blood and from almost all soft tissues were much faster than those of [(90)Y]citrate. As a result, the estimated absorbed dose ratios of soft tissues to osteogenic cells (target organ) of [(90)Y]DOTA-HBP were lower than those of [(90)Y]citrate. CONCLUSIONS [(90)Y]DOTA-HBP showed superior biodistribution characteristics as a bone-seeking agent and led to a decrease in the level of unnecessary radiation compared to [(90)Y]citrate. Since the DOTA ligand forms a stable complex not only with (90)Y but also with lutetium ((177)Lu), indium ((111)In), gallium ((67/68)Ga), gadolinium (Gd) and so on, complexes of DOTA-conjugated bisphosphonate with various metals could be useful as agents for palliation of metastatic bone pain, bone scintigraphy and magnetic resonance imaging.

Hypoxic but not ischemic neurotoxicity of free radicals revealed by dynamic changes in glucose metabolism of fresh rat brain slices on positron autoradiography.

Dynamic changes in the regional cerebral glucose metabolic rate induced by hypoxia/reoxygenation or ischemia/reperfusion were investigated with a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36°C, and serial two-dimensional time-resolved images of [18F]FDG uptake in the slices were obtained. In the case of loading hypoxia (oxygen deprivation)/pseudoischemia (oxygen and glucose deprivation) for various periods of time, the net influx constant (K) of [18F]FDG at preloading and after reoxygenation/pseudoreperfusion (post-loading) was quantitatively evaluated by applying the Patlak graphical method to the image data. Regardless of the brain region, with hypoxia lasting ≥20 minutes, the postloading K value was decreased compared with the unloaded control, whereas with pseudoischemia of ≤40 minutes, approximately the same level as the unloaded control was maintained. Next, the neuroprotective effect against hypoxia/pseudoischemia loading induced by the addition of a free radical scavenger or an N-methyl-D-aspartate (NMDA) antagonist was assessed by determining whether a decrease in the postloading K value was prevented. Whereas with 20-minute hypoxia, both agents exhibited a neuroprotective effect, in the case of 50-minute pseudoischemia, only the NMDA antagonist did so, with the free radical scavenger being ineffective. These results demonstrate that hypoxia causes irreversible neuronal damage within a shorter period than ischemia, with both free radicals and glutamate suggested to be involved in tandem in the neurotoxicity induced by hypoxia, whereas glutamate alone is involved in ischemic neurotoxicity.

αvβ3 Integrin-targeting radionuclide therapy and imaging with monomeric RGD peptide

The αvβ3 integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress αvβ3 integrin, as in tumor neovascularization, and αvβ3 integrin expression in other microvascular beds and organs is limited. Therefore, αvβ3 integrin is a suitable receptor for tumor‐targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to αvβ3 integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor‐targeting properties and the therapeutic potential of 111In‐ and 90Y‐labeled monomeric RGD peptides in BALB/c nude mice with SKOV‐3 human ovarian carcinoma tumors. DOTA‐c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to αvβ3 integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In‐DOTA‐c(RGDfK) and 90Y‐DOTA‐c(RGDfK) showed high uptake in tumors (7.3 ± 0.6% ID/g and 4.6 ± 0.8% ID/g, respectively) and gradually decreased over time (2.3 ± 0.4% ID/g and 1.5 ± 0.5% ID/g at 24 hr, respectively). High tumor‐to‐blood and ‐muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple‐dose administration of 90Y‐DOTA‐c(RGDfK) (3 × 11.1 MBq) suppressed tumor growth in comparison to the control group and a single‐dose administration (11.1 MBq). Monomeric RGD peptides, 111In‐DOTA‐c(RGDfK) and 90Y‐DOTA‐c(RGDfK), could be promising tracers for αvβ3 integrin‐targeting imaging and radiotherapy.

Predominant contribution of L-type amino acid transporter to 4-borono-2-18F-fluoro-phenylalanine uptake in human glioblastoma cells

INTRODUCTION 4-Borono-2-(18)F-fluoro-phenylalanine ((18)F-FBPA) has been used to anticipate the therapeutic effects of boron neutron capture therapy (BNCT) with 4-borono-L-phenylalanine (BPA). Similarly, L-[methyl-(11)C]-methionine ((11)C-MET), the most popular amino acid PET tracer, is a possible candidate for this purpose. We investigated the transport mechanism of (18)F-FBPA and compared it with that of (14)C-MET in human glioblastoma cell lines. METHODS Uptake of (18)F-FBPA and (14)C-MET was examined in A172, T98G, and U-87MG cells using 2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (a system L-specific substrate), 2-(methylamino)-isobutyric acid (a system A-specific substrate), and BPA. Gene expression was analyzed by quantitative real time polymerase chain reaction. RESULTS System L was mainly involved in the uptake of (18)F-FBPA (74.5%-81.1% of total uptake) and (14)C-MET (48.3%-59.4%). System A and ASC also contributed to the uptake of (14)C-MET. Inhibition experiments revealed that BPA significantly decreased the uptake of (18)F-FBPA, whereas 31%-42% of total (14)C-MET uptake was transported by BPA non-sensitive transporters. In addition, (18)F-FBPA uptake correlated with LAT1 and total LAT expressions. CONCLUSION This study demonstrated that (18)F-FBPA was predominantly transported by system L in human glioblastoma cells compared to (14)C-MET. Although further studies are needed to elucidate the correlation between (18)F-FBPA uptake and BPA content in tumor tissues, (18)F-FBPA is suitable for the selection of patients who benefit from BNCT with BPA.

Dynamic changes in glucose metabolism of living rat brain slices induced by hypoxia and neurotoxic chemical-loading revealed by positron autoradiography.

Summary. Fresh rat brain slices were incubated with 2-deoxy-2-[18F]-fluoro-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution at 36°C, and serial two-dimensional time-resolved images of [18F]FDG uptake were obtained from these specimens on imaging plates. The fractional rate constant (= k3*) of [18F]FDG proportional to the cerebral glucose metabolic rate (CMRglc) was evaluated by applying the Gjedde-Patlak graphical method to the image data. With hypoxia loading (oxygen deprivation) or glucose metabolism inhibitors acting on oxidative phosphorylation, the k3* value increased dramatically suggesting enhanced glycolysis. After relieving hypoxia ≤10-min, the k3* value returned to the pre-loading level. In contrast, with ≥20-min hypoxia only partial or no recovery was observed, indicating that irreversible neuronal damage had been induced. However, after loading with tetrodotoxin (TTX), the k3* value also decreased but returned to the pre-loading level even after 70-min TTX-loading, reflecting a transient inhibition of neuronal activity. This technique provides a new means of quantifying dynamic changes in the regional CMRglc in living brain slices in response to various interventions such as hypoxia and neurotoxic chemical-loading as well as determining the viability and prognosis of brain tissues.

Dynamic changes in glucose metabolism by lactate loading as revealed by a positron autoradiography technique using rat living brain slices

To demonstrate the preference of lactate over glucose as an energy substrate in normal brain tissue under normoxic condition, the dynamic changes in glucose uptake by lactate loading were investigated in living rat brain slices using a positron autoradiography technique. Fresh rat brain slices were incubated with [18F]2-fluoro-2-deoxy-D-glucose ([18F]FDG) in oxygenated Krebs-Ringer solution containing 10 mM glucose at 36 degrees C. During incubation, serial two-dimensional imaging of [18F]FDG uptake in the slices was constructed on the imaging plates. Lactate loading (20 mM) reversibly suppressed the [18F]FDG accumulation up to 80 min. Compared with the pre-loading and the unloaded control values, [18F]FDG uptake was suppressed to 25-45% in cerebral regions and 6-7% in cerebellum. The lactate concentration in the surrounding medium decreased after lactate loading. Hence brain tissue preferentially uses lactate over glucose under normoxic and euglycemic condition.

Influences of haemodialysis on the binding sites of human serum albumin: possibility of an efficacious administration plan using binding inhibition

BACKGROUND We have studied the possibility that low-dose treatment utilizing the inhibition that may occur between two drugs at the same site of human serum albumin (HSA) improves the pharmacological effects. The purpose is to elucidate the differences in the binding capacities of sites I and II of HSA between pre-haemodialysis (HD) and post-HD in patients with end-stage renal disease. METHODS We evaluated free fractions of site probes, (14)C-warfarin (site I) and (14)C-diazepam (site II), by ultrafiltration in serum between pre-HD and post-HD. To investigate effects on the binding capacities of HSA sites, free fractions of site probes were calculated from the radioactivities measured with a liquid scintillation counter. Endogenous uraemic toxins, 3-carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), indoxyl sulphate (IS) and hippurate (HA), were determined by HPLC. Free fatty acid (FFA) as an endogenous substance was determined with an automatic multi-item simultaneous analyser. RESULTS The concentrations of HSA and FFA increased significantly (post-HD/pre-HD ratio: 1.18 +/- 0.10, 5.46 +/- 4.91), the concentrations of IS and HA decreased significantly (post-HD/pre-HD ratio: 0.69 +/- 0.10, 0.33 +/- 0.15) and CMPF concentrations did not alter significantly (post-HD/pre-HD ratio: 0.97 +/- 0.12, P = 0.471). The free fractions of (14)C-warfarin decreased in all 14 patients at site I at post-HD compared to pre-HD (post-HD/pre-HD ratio: 0.59 +/- 0.13). The free fractions of (14)C-diazepam at site II remarkably decreased in 10 of 14 patients (post-HD/pre-HD ratio: 0.61 +/- 0.17) and unexpectedly increased in 4 (post-HD/pre-HD ratio: 1.08 +/- 0.06) post-HD compared to pre-HD. In these four patients, when we investigated the influences of these variation factors on the reduction of the binding capacities of site II, [FFA]/[HSA] increased significantly post-HD, compared to pre-HD (post-HD/pre-HD ratio: 6.91 +/- 6.58). ([FFA]/[HSA] ratios of the 4 patients were from 1.22 to 3.55, the highest for the 14 patients post-HD, but the ratios of the other 10 were below 1.2 post-HD.) CONCLUSION The binding capacity of site II was unexpectedly decremented by the effects of the remarkable elevation of FFA. Therefore, monitoring the binding capacity of site II in HD is important for patients with end-stage renal disease in the efficacious administration plan using the binding inhibition of HSA.