論文 - 曽我　朋義

p38α activates purine metabolism to initiate hematopoietic stem/progenitor cell cycling in response to stress

Karigane, D., Kobayashi, H., Morikawa, T., Ootomo, Y., Sakai, M., Nagamatsu, G., Kubota, Y., Goda, N., Matsumoto, M., Nishimura, E. K., Soga, T., Otsu, K., Suematsu, M., Okamoto, S., Suda, T., Takubo, K.

Cell Stem Cell 19 （ 2 ） 192 - 204 2016年08月

研究論文（学術雑誌）, 共著

　概要を見る

Hematopoietic stem cells (HSCs) maintain quiescence by activating specific metabolic pathways, including glycolysis. We do not yet have a clear understanding of how this metabolic activity changes during stress hematopoiesis, such as bone marrow transplantation. Here, we report a critical role for the p38MAPK family isoform p38α in initiating hematopoietic stem and progenitor cell (HSPC) proliferation during stress hematopoiesis in mice. We found that p38MAPK is immediately phosphorylated in HSPCs after a hematological stress, preceding increased HSPC cycling. Conditional deletion of p38α led to defective recovery from hematological stress and a delay in initiation of HSPC proliferation. Mechanistically, p38α signaling increases expression of inosine-5'-monophosphate dehydrogenase 2 in HSPCs, leading to altered levels of amino acids and purine-related metabolites and changes in cell-cycle progression in vitro and in vivo. Our studies have therefore uncovered a p38α-mediated pathway that alters HSPC metabolism to respond to stress and promote recovery.

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Mitochondrial Mg2+ homeostasis decides cellular energy metabolism and vulnerability to stress

Yamanaka, R., Tabata, S., Shindo, Y., Hotta, K., Suzuki, K., Soga, T., Oka, K.

Sci. Rep. 6   30027 2016年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Cellular energy production processes are composed of many Mg(2+) dependent enzymatic reactions. In fact, dysregulation of Mg(2+) homeostasis is involved in various cellular malfunctions and diseases. Recently, mitochondria, energy-producing organelles, have been known as major intracellular Mg(2+) stores. Several biological stimuli alter mitochondrial Mg(2+) concentration by intracellular redistribution. However, in living cells, whether mitochondrial Mg(2+) alteration affect cellular energy metabolism remains unclear. Mg(2+) transporter of mitochondrial inner membrane MRS2 is an essential component of mitochondrial Mg(2+) uptake system. Here, we comprehensively analyzed intracellular Mg(2+) levels and energy metabolism in Mrs2 knockdown (KD) cells using fluorescence imaging and metabolome analysis. Dysregulation of mitochondrial Mg(2+) homeostasis disrupted ATP production via shift of mitochondrial energy metabolism and morphology. Moreover, Mrs2 KD sensitized cellular tolerance against cellular stress. These results indicate regulation of mitochondrial Mg(2+) via MRS2 critically decides cellular energy status and cell vulnerability via regulation of mitochondrial Mg(2+) level in response to physiological stimuli.

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Serum metabolome profiles characterized by patients with hepatocellular carcinoma associated with hepatitis B and C

Saito, T., Sugimoto, M., Okumoto, K., Haga, H., Katsumi, T., Mizuno, K., Nishina, T., Sato, S., Igarashi, K., Maki, H., Tomita, M., Ueno, Y., Soga, T.

World J. Gastroenterol. 22 （ 27 ） 6224 - 6234 2016年07月

研究論文（学術雑誌）, 共著

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Aim: To clarify the characteristics of metabolite profiles in virus-related hepatocellular carcinoma (HCC) patients using serum metabolome analysis.
Methods: The serum levels of low-molecular-weight metabolites in 68 patients with HCC were quantified using capillary electrophoresis chromatography and mass spectrometry. Thirty and 38 of the patients suffered from hepatitis B virus-related HCC (HCC-B) and hepatitis C virus-related HCC (HCC-C), respectively.
Results: The main metabolites characteristic of HCC were those associated with glutathione metabolism, notably 13 γ-glutamyl peptides, which are by-products of glutathione induction. Two major profiles, i.e., concentration patterns, of metabolites were identified in HCC patients, and these were classified into two groups: an HCC-B group and an HCC-C group including some of the HCC-B cases. The receiver operating characteristic curve for the multiple logistic regression model discriminating HCC-B from HCC-C incorporating the concentrations of glutamic acid, methionine and γ-glutamyl-glycine-glycine showed a highly significant area under the curve value of 0.94 (95%CI: 0.89-1.0, P < 0.0001).

Conclusion: The serum levels of γ-glutamyl peptides, as well as their concentration patterns, contribute to the development of potential biomarkers for virus-related HCC. The difference in metabolite profiles between HCC-B and HCC-C may reflect the respective metabolic reactions that underlie the different pathogeneses of these two types of HCC.

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Profiling of plasma metabolites in postmenopausal women with metabolic syndrome

Iida, M., Harada, S., Kurihara, A., Fukai, K., Kuwabara, K., Sugiyama, D., Takeuchi, A., Okamura, T., Akiyama, M., Nishiwaki, Y., Suzuki, A., Hirayama, A., Sugimoto, M., Soga, T., Tomita, M., Banno, K., Aoki, D., Takebayashi, T.

Menopause 23 （ 7 ） 749 - 758 2016年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Objective: The aim of the study was to investigate the associations of amino acids and other polar metabolites with metabolic syndrome (MetS) in postmenopausal women in a lean Asian population.
Methods: The participants were 1,422 female residents enrolled in a cohort study from April to August 2012. MetS was defined according to the National Cholesterol Education Program Adult Treatment Panel III modified for Japanese women. Associations were examined between MetS and 78 metabolites assayed in fasting plasma samples using capillary electrophoresis-mass spectrometry. Replication analysis was performed to confirm the robustness of the results in a separate population created by random allocation.
Results: Analysis was performed for 877 naturally postmenopausal women, including 594 in the original population and 283 in the replication population. The average age, body mass index, and levels of high- and low-density lipoprotein cholesterol of the entire population were 64.6 years, 23.0 kg/m, 72.1 mg/dL, and 126.1 mg/dL, respectively. There was no significant difference in low-density lipoprotein cholesterol levels between women with and without MetS. Thirteen metabolites were significantly related to MetS: multiple plasma amino acids were elevated in women with MetS, including branched-chain amino acids, alanine, glutamate, and proline; and alpha-aminoadipate, which is generated by lysine degradation, was also significantly increased.
Conclusions: Our large-scale metabolomic profiling indicates that Japanese postmenopausal women with MetS have abnormal polar metabolites, suggesting altered catabolic pathways. These results may help to understand metabolic disturbance, including in persons with normal body mass index and relatively high levels of high-density lipoprotein cholesterol, and may have clinical utility based on further studies.

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p62/Sqstm1 promotes malignancy of HCV-positive hepatocellular carcinoma through Nrf2-dependent metabolic reprogramming

Saito, T., Ichimura, Y., Taguchi, K., Suzuki, T., Mizushima, T., Takagi, K., Hirose, Y., Nagahashi, M., Iso, T., Fukutomi, T., Ohishi, M., Endo, K., Uemura, T., Nishito, Y., Okuda, S., Obata, M., Kouno, T., Imamura, R., Tada, Y., Obata, R., Yasuda, D., Takahashi, K., Fujimura, T., Pi, J., Lee, MS., Ueno, T., Ohe, T., Mashino, T., Wakai, T., Kojima, H., Okabe, T., Nagano, T., Motohashi, H., Waguri, S., Soga, T., Yamamoto, M., Tanaka, K., Komatsu, M.

Nat. Commun. 7   12030 2016年06月

研究論文（学術雑誌）, 共著

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p62/Sqstm1 is a multifunctional protein involved in cell survival, growth and death, that is degraded by autophagy. Amplification of the p62/Sqstm1 gene, and aberrant accumulation and phosphorylation of p62/Sqstm1, have been implicated in tumour development. Herein, we reveal the molecular mechanism of p62/Sqstm1-dependent malignant progression, and suggest that molecular targeting of p62/Sqstm1 represents a potential chemotherapeutic approach against hepatocellular carcinoma (HCC). Phosphorylation of p62/Sqstm1 at Ser349 directs glucose to the glucuronate pathway, and glutamine towards glutathione synthesis through activation of the transcription factor Nrf2. These changes provide HCC cells with tolerance to anti-cancer drugs and proliferation potency. Phosphorylated p62/Sqstm1 accumulates in tumour regions positive for hepatitis C virus (HCV). An inhibitor of phosphorylated p62-dependent Nrf2 activation suppresses the proliferation and anticancer agent tolerance of HCC. Our data indicate that this Nrf2 inhibitor could be used to make cancer cells less resistant to anticancer drugs, especially in HCV-positive HCC patients.

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Smad2/3 are required for immobilization-induced skeletal muscle atrophy

Tando, T., Hirayama, A., Furukawa, M., Sato, Y., Kobayashi, T., Funayama1, A., Kanaji, A., Hao, W., Watanabe, R., Morita, M., Oike, T., Miyamoto, K., Soga, T., Nomura, M., Yoshimura, A., Tomita, M., Matsumoto, M., Nakamura, M., Toyama, Y., Miyamoto, T.

J. Biol. Chem. 291 （ 23 ） 12184 - 12194 2016年06月

研究論文（学術雑誌）, 共著, 査読有り

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Skeletal muscle atrophy promotes muscle weakness, limiting activities of daily living. However, mechanisms underlying atrophy remain unclear. Here, we show that skeletal muscle immobilization elevates Smad2/3 protein but not mRNA levels in muscle, promoting atrophy. Furthermore, we demonstrate that myostatin, which negatively regulates muscle hypertrophy, is dispensable for denervation-induced muscle atrophy and Smad2/3 protein accumulation. Moreover, muscle-specific Smad2/3-deficient mice exhibited significant resistance to denervation-induced muscle atrophy. In addition, expression of the atrogenes Atrogin-1 and MuRF1, which underlie muscle atrophy, did not increase in muscles of Smad2/3-deficient mice following denervation. We also demonstrate that serum starvation promotes Smad2/3 protein accumulation in C2C12 myogenic cells, an in vitro muscle atrophy model, an effect inhibited by IGF1 treatment. In vivo, we observed IGF1 receptor deactivation in immobilized muscle, even in the presence of normal levels of circulating IGF1. Denervation-induced muscle atrophy was accompanied by reduced glucose intake and elevated levels of branched-chain amino acids, effects that were Smad2/3-dependent. Thus, muscle immobilization attenuates IGF1 signals at the receptor rather than the ligand level, leading to Smad2/3 protein accumulation, muscle atrophy, and accompanying metabolic changes.

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Effects of fractionated radiation on murine glioma stem cell metabolism

Fujita, S., Osuka, S. Shibao, S., Igarashi, K., Soga, T., Sampetrean, O.

Toho J. Med. 2 （ 2 ） 45 - 52 2016年06月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Background: Glioma stem cells (GSCs) have an important role in tumor recurrence after treatment. We recently showed that, in a mouse model of glioma, GSCs adapt to repeated radiation exposures by changing
their secretory profile and modulating survival signaling. Irradiated GSCs also reduce their rate of proliferation. In the present study we examined whether these changes require a specific adaptation of GSC energy
metabolism.
Methods: Initial tumors were established in mice by orthotopic implantation of Ink4a/Arf-null neural stem
cells transduced with human HRASV12. GSCs isolated from the tumors were exposed (or not) to fractionated
radiation (12 fractions of 5 Gy), and five clones in each group of cells were assessed for glucose consumption,
lactate production, and content of intracellular metabolites.
Results: Rates of glucose consumption and lactate production were lower in clones exposed to radiation
than in nonirradiated clones, which suggests a reduction in glycolysis. Among intracellular metabolites, the
high concentrations of intermediates of glycolysis and nucleoside metabolism were reduced in irradiated
clones, whereas levels of essential amino acids were largely unchanged.
Conclusions: Repeated radiation changes the metabolic preferences of GSCs, in conjunction with their
slowed proliferation. Future studies should thus investigate intra- and extracellular metabolites, to identify
potential diagnostic and therapeutic targets for glioma recurrence.

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Identification of biomarkers for development of end-stage kidney disease in chronic kidney disease by metabolomic profiling

Kimura, T., Yasuda, K., Yamamoto, R., Soga, T., Rakugi, H., Hayashi, T., Isaka, Y.

Sci. Rep. 6 （ - ） 26138 - - 2016年05月

研究論文（学術雑誌）, 共著

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A critical issue in the management of chronic kidney disease (CKD) is to prevent patients from the progression to end-stage kidney disease (ESKD), however, there is only limited number of biomarkers for the discrimination of the high-risk CKD patients. We aimed to identify the metabolites which possess the ability to predict the earlier kidney deterioration. We performed capillary electrophoresis and liquid chromatography mass spectrometry (CE-MS)-based metabolic profiling in a prospective cohort, which consisted of referred 112 CKD patients with median follow-up period of 4.4 years. The association between the levels of candidate metabolites and the outcomes (progression to ESKD alone or in combination with death before ESKD) were assessed by multivariate Cox proportional hazard models after adjusting for the baseline covariates. A total of 218 metabolites were detected in the plasma of CKD patients. We identified 16 metabolites which have predictive values for the composite outcome: The risk for composite outcome was elevated from 2.0- to 8.0-fold in those with higher levels of 16 plasma metabolites. Our results suggest that the measurement of these metabolites may facilitate CKD management by predicting the risk of progression to ESKD.

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Metabolomic profiling reveals novel biomarkers of alcohol intake and alcohol-induced liver injury in community-dwelling men

Harada S., Takebayashi T., Kurihara A., Akiyama M., Suzuki A., Hatakeyama Y., Sugiyama D., Kuwabara K., Takeuchi A., Okamura T., Nishiwaki Y., Tanaka T., Hirayama A., Sugimoto M., Soga T., Tomita M.

Environ. Health Prev. Med. 21 （ 1 ） 18(283) - 26(284) 2016年01月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Objective: Metabolomics is a promising approach to the identification of biomarkers in plasma. Here, we performed a population-based, cross-sectional study to identify potential biomarkers of alcohol intake and alcohol-induced liver injury by metabolomic profiling using capillary electrophoresis-mass spectrometry (CE-MS).
Methods: Fasting plasma samples were collected from 896 Japanese men who participated in the baseline survey of the Tsuruoka Metabolomics Cohort Study, and 115 polar metabolites were identified and absolutely quantified by CE-MS. Information on daily ethanol intake was collected through a standardized, self-administered questionnaire. The associations between ethanol intake and plasma concentration of metabolites were examined. Relationships between metabolite concentrations or their ratios and serum liver enzyme levels in the highest ethanol intake group (>46.0 g/day) were then examined by linear regression analysis. Replication analysis was conducted in 193 samples collected from independent population of this cohort.
Results: Nineteen metabolites were identified to have an association with daily alcohol consumption both in the original and replication population. Three of these metabolites (threonine, glutamine, and guanidinosuccinate) were found to associate well with elevated levels of serum liver enzymes in the highest ethanol intake group, but not in the non-drinker group. We also found that the glutamate/glutamine ratio had a much stronger relation to serum γ-glutamyltransferase, aspartate transaminase, and alanine transaminase than glutamate or glutamine alone (standardized beta = 0.678, 0.558, 0.498, respectively).
Conclusions: We found 19 metabolites associated with alcohol intake, and three biomarker candidates (threonine, guanidinosuccinate and glutamine) of alcohol-induced liver injury. Glutamate/glutamine ratio might also be good biomarker.

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Effects of 3-styrylchromones on metabolic profiles and cell death in oral squamous cell carcinoma cells

Sakagami, H., Shimada, C., Kanda, Y., Amano, O., Sugimoto, M., Ota S., Soga, T., Tomita, M., Sato, A., Tanuma, S., Takao, K., Sugita, Y.

Toxicol. Rep.  2   1281 - 1290 2015年12月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

4H-1-benzopyran-4-ones (chromones) are important naturally-distributing compounds. As compared with flavones, isoflavones and 2-styrylchromones, there are only few papers of 3-styrylchromones that have been published. We have previously reported that among fifteen 3-styrylchromone derivatives, three new synthetic compounds that have OCH3 group at the C-6 position of chromone ring, (E)-3-(4-hydroxystyryl)-6-methoxy-4H-chromen-4-one (compound 11), (E)-6-methoxy-3-(4-methoxystyryl)-4H-chromen-4-one (compound 4), (E)-6-methoxy-3-(3,4,5-trimethoxystyryl)-4H-chromen-4-one (compound 6) showed much higher cytotoxicities against four epithelial human oral squamous cell carcinoma (OSCC) lines than human normal oral mesenchymal cells. In order to further confirm the tumor specificities of these compounds, we compared their cytotoxicities against both human epithelial malignant and non-malignant cells, and then investigated their effects on fine cell structures and metabolic profiles and cell death in human OSCC cell line HSC-2. Cytotoxicities of compounds 4, 6, 11 were assayed with MTT method. Fine cell structures were observed under transmission electron microscope. Cellular metabolites were extracted with methanol and subjected to CE-TOFMS analysis. Compounds 4, 6, 11 showed much weaker cytotoxicity against human oral keratinocyte and primary human gingival epithelial cells, as compared with HSC-2, confirming their tumor-specificity, whereas doxorubicin and 5-FU were highly cytotoxic to these normal epithelial cells, giving unexpectedly lower tumor-specificity. The most cytotoxic compound 11, induced the mitochondrial vacuolization, autophagy suppression followed by apoptosis induction, and changes in the metabolites involved in amino acid and glycerophospholipid metabolisms. Chemical modification of lead compound 11 may be a potential choice for designing new type of anticancer drugs.

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Effects of processing and storage conditions on charged metabolomic profiles in blood

Hirayama, A., Sugimoto, M. , Suzuki, A., Hatakeyama, Y., Enomoto, A., Harada, S., Soga, T., Tomita, M., Takebayashi, T.

Electrophoresis 36 （ 18 ） 2148 - 2155 2015年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The development of high-throughput metabolite measurement technologies has enabled the use of metabolomics for epidemiologic studies by profiling metabolite concentrations in large cohorts of human blood samples. Standard protocols are necessary to obtain unbiased profiles through multiple runs over long periods of time and to allow reliable statistical analyses. This study assessed the effects of sampling procedures and storage conditions on the stability of metabolomic profiles in plasma and serum. Charged metabolomic profiles were determined by capillary electrophoresis-mass spectrometry (CE-MS) and compared by multivariate analyses. The effects of pre-analytical procedures, including times for clotting and incubation of serum and plasma, respectively; incubation temperatures; and number of freeze-thaw cycles, were assessed. Overall, inter-individual differences in profiles were larger than intra-individual differences, and profiles in plasma showed better stability than those in serum. These quantified datasets of metabolites, along with their stability and variation, may help in interpreting data from long-term cohort studies.

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Metabolomic alterations in human cancer cells by vitamin C-induced oxidative stress

Uetaki, M., Tabata, S., Nakasuka, F., Soga, T., Tomita, M.

Sci. Rep. 5 （ - ） 13896 - - 2015年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Intravenous administration of high-dose vitamin C has recently attracted attention as a cancer therapy. High-dose vitamin C induces pro-oxidant effects and selectively kills cancer cells. However, the anticancer mechanisms of vitamin C are not fully understood. Here, we analyzed metabolic changes induced by vitamin C in MCF7 human breast adenocarcinoma and HT29 human colon cancer cells using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). The metabolomic profiles of both cell lines were dramatically altered after exposure to cytotoxic concentrations of vitamin C. Levels of upstream metabolites in the glycolysis pathway and tricarboxylic acid (TCA) cycle were increased in both cell lines following treatment with vitamin C, while adenosine triphosphate (ATP) levels and adenylate energy charges were decreased concentration-dependently. Treatment with N-acetyl cysteine (NAC) and reduced glutathione (GSH) significantly inhibited vitamin C-induced cytotoxicity in MCF7 cells. NAC also suppressed vitamin C-dependent metabolic changes, and NAD treatment prevented vitamin C-induced cell death. Collectively, our data suggests that vitamin C inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.

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Acquisition of chemoresistance to gemcitabine is induced by a loss-of-function missense mutation of DCK

Nakano, T., Saiki, Y., Kudo, C., Hirayama, A., Mizuguchi, Y., Fujiwara, S., Soga, T., Sunamura, M., Matsumura, N., Motoi, F., Unno, M., Horii, A.

Biochem. Biophys. Res. Commun. 464 （ 4 ） 1084 - 1089 2015年09月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The anti-tumor activity of gemcitabine (GEM) has been clinically proven in several solid tumors, including pancreatic cancer, biliary tract cancer, urinary bladder cancer, and non-small cell lung cancer. However, problems remain with issues such as acquisition of chemoresistance against GEM. GEM is activated after phosphorylation by deoxycytidine kinase (DCK) inside of the cell; thus, DCK inactivation is one of the important mechanisms for acquisition of GEM resistance. We previously investigated the DCK gene in multiple GEM resistant cancer cell lines and identified frequent inactivating mutations. In this study, we identified two crucial genetic alteration in DCK. (1) A total deletion of DCK in RTGBC1-TKB, an acquired GEM resistant cell line derived from a gall bladder cancer cell line TGBC1-TKB. (2) An E197K missense alteration of DCK in MKN28, a gastric cancer cell line; its acquired GEM resistant cancer cell line, RMKN28, showed a loss of the normal E197 allele. We introduced either normal DCK or altered DCK_E197K into RMKN28 and proved that only the introduction of normal DCK restored GEM sensitivity. Furthermore, we analyzed 104 healthy volunteers and found that none of them carried the same base substitution observed in MKN28. These results strongly suggest that (1) the E197K alteration in DCK causes inactivation of DCK, and that (2) loss of the normal E197 allele is the crucial mechanism in acquisition of GEM resistance in RMKN28.

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Alteration of the Intestinal Environment by Lubiprostone Is Associated with Amelioration of Adenine-Induced CKD

Mishima, E., Fukuda, S., Shima, H., Hirayama, A., Akiyama, Y., Takeuchi, Y., Fukuda, N. N., Suzuki, T., Suzuki, C., Yuri, A., Kikuchi, K., Tomioka, Y., Ito, S., Soga, T., Abe, T.

J. Am. Soc. Nephrol. 26 （ 8 ） 1787 - 1794 2015年08月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

The accumulation of uremic toxins is involved in the progression of CKD. Various uremic toxins are derived from gut microbiota, and an imbalance of gut microbiota or dysbiosis is related to renal failure. However, the pathophysiologic mechanisms underlying the relationship between the gut microbiota and renal failure are still obscure. Using an adenine-induced renal failure mouse model, we evaluated the effects of the ClC-2 chloride channel activator lubiprostone (commonly used for the treatment of constipation) on CKD. Oral administration of lubiprostone (500 µg/kg per day) changed the fecal and intestinal properties in mice with renal failure. Additionally, lubiprostone treatment reduced the elevated BUN and protected against tubulointerstitial damage, renal fibrosis, and inflammation. Gut microbiome analysis of 16S rRNA genes in the renal failure mice showed that lubiprostone treatment altered their microbial composition, especially the recovery of the levels of the Lactobacillaceae family and Prevotella genus, which were significantly reduced in the renal failure mice. Furthermore, capillary electrophoresis-mass spectrometry-based metabolome analysis showed that lubiprostone treatment decreased the plasma level of uremic toxins, such as indoxyl sulfate and hippurate, which are derived from gut microbiota, and a more recently discovered uremic toxin, trans-aconitate. These results suggest that lubiprostone ameliorates the progression of CKD and the accumulation of uremic toxins by improving the gut microbiota and intestinal environment.

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Alteration of metabolomic profiles by titanium dioxide nanoparticles in human gingivitis model

Garcia-Contreras, R., Sugimoto, M., Umemura, N., Kaneko, M., Hatakeyama, Y., Soga, T., Tomita, M., Scougall-Vilchis, R.J., Contreras-Bulnes, R., Nakajima, H., Sakagami, H.

Biomaterials 57   33 - 40 2015年07月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

Although nanoparticles (NPs) has afforded considerable benefits in various fields of sciences, several reports have shown their harmful effects, suggesting the necessity of adequate risk assessment. To clarify the mechanism of titanium dioxide nanoparticles (TiO2 NPs)-enhanced gingival inflammation, we conducted the full-scale metabolomic analyses of human gingival fibroblast cells treated with IL-1β alone or in combination with TiO2 NPs. Observation with transmission electron microscope demonstrated the incorporation of TiO2 NPs into vacuoles of the cells. TiO2 NPs significantly enhanced the IL-1β-induced prostaglandin E2 production and COX-1 and COX-2 protein expression. IL-1β reduced the intracellular concentrations of overall primary metabolites especially those of amino acid, urea cycle, polyamine, S-adenosylmethione and glutathione synthetic pathways. The addition of TiO2 NPs further augmented these IL-1β-induced metabolic changes, recommending careful use of dental materials containing TiO2 NPs towards patients with gingivitis or periodontitis. The impact of the present study is to identify the molecular targets of TiO2 NPs for the future establishment of new metabolic markers and therapeutic strategy of gingival inflammation.

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IDH2 and NPM1 mutations cooperate to activate Hoxa9/Meis1 and hypoxia pathways in acute myeloid leukemia

Ogawara, Y., Katsumoto, T., Aikawa, Y., Shima, Y., Kagiyama, Y., Soga, T., Matsunaga, H., Seki, T., Araki, K., Kitabayashi, I.

Cancer Res. 75 （ 10 ） 2005 - 2016 2015年05月

研究論文（学術雑誌）, 共著, 査読有り

　概要を見る

IDH1 and IDH2 mutations occur frequently in acute myeloid leukemia (AML) and other cancers. The mutant isocitrate dehydrogenase (IDH) enzymes convert α-ketoglutarate (α-KG) to the oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases. To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of AML in which mice were transplanted with nucleophosmin1 (NPM)(+/-) hematopoietic stem/progenitor cells cotransduced with four mutant genes (NPMc, IDH2/R140Q, DNMT3A/R882H, and FLT3/ITD), which often occur simultaneously in human AML patients. Conditional deletion of IDH2/R140Q blocked 2-HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. IDH2/R140Q was necessary for the engraftment or survival of NPMc(+) cells in vivo. Gene expression analysis indicated that NPMc increased expression of Hoxa9. IDH2/R140Q also increased the level of Meis1 and activated the hypoxia pathway in AML cells. IDH2/R140Q decreased the 5hmC modification and expression of some differentiation-inducing genes (Ebf1 and Spib). Taken together, our results indicated that IDH2 mutation is critical for the development and maintenance of AML stem-like cells, and they provided a preclinical justification for targeting mutant IDH enzymes as a strategy for anticancer therapy.
©2015 American Association for Cancer Research.

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Decreased miR122 in hepatocellular carcinoma leads to chemoresistance with increased arginine

Kishikawa, T., *Otsuka, M., Tan, P., Ohno, M., Sun, X., Yoshikawa, T., Shibata, C., Takata, A., Kojima, K., Takehana, K., Ohishi, M., Ota, S., Noyama, T., Kondo, Y., Sato, M., Soga, T., Hoshida, Y., Koike, K.

Oncotarget 6 （ 10 ） 8339 - 8352 2015年04月

研究論文（学術雑誌）, 共著

　概要を見る

Reduced expression of microRNA122 (miR122), a liver-specific microRNA, is frequent in hepatocellular carcinoma (HCC). However, its biological significances remain poorly understood. Because deregulated amino acid levels in cancers can affect their biological behavior, we determined the amino acid levels in miR122-silenced mouse liver tissues, in which intracellular arginine levels were significantly increased. The increased intracellular arginine levels were through upregulation of the solute carrier family 7 (SLC7A1), a transporter of arginine and a direct target of miR122. Arginine is the substrate for nitric oxide (NO) synthetase, and intracellular NO levels were increased in miR122-silenced HCC cells, with increased resistance to sorafenib, a multikinase inhibitor. Conversely, maintenance of the miR122-silenced HCC cells in arginine-depleted culture media, as well as overexpression of miR122 in miR122-low-expressing HCC cells, reversed these effects and rendered the cells more sensitive to sorafenib. Using a reporter knock-in construct, chemical compounds were screened, and Wee1 kinase inhibitor was identified as upregulators of miR122 transcription, which increased the sensitivity of the cells to sorafenib. These results provide an insight into sorafenib resistance in miR122-low HCC, and suggest that arginine depletion or a combination of sorafenib with the identified compound may provide promising approaches to managing this HCC subset.

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Cystathionine is a novel substrate of cystine/glutamate transporter: implications for immune function

Kobayashi, S., Sato, M., Kasakoshi, T., Tsutsui, T., Sugimoto, M., Osaki, M., Okada, F., Igarashi, K., Hiratake, J., Homma, T., Conrad, M., Fujii, J., Soga, T., Bannai, S., *Sato, H.

J Biol Chem . 290 （ 14 ） 8778 - 8788 2015年04月

研究論文（学術雑誌）, 共著

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The cystine/glutamate transporter, designated as system xc(-), is important for maintaining intracellular glutathione levels and extracellular redox balance. The substrate-specific component of system xc(-), xCT, is strongly induced by various stimuli, including oxidative stress, whereas it is constitutively expressed only in specific brain regions and immune tissues, such as the thymus and spleen. Although cystine and glutamate are the well established substrates of system xc(-) and the knockout of xCT leads to alterations of extracellular redox balance, nothing is known about other potential substrates. We thus performed a comparative metabolite analysis of tissues from xCT-deficient and wild-type mice using capillary electrophoresis time-of-flight mass spectrometry. Although most of the analyzed metabolites did not show significant alterations between xCT-deficient and wild-type mice, cystathionine emerged as being absent specifically in the thymus and spleen of xCT-deficient mice. No expression of either cystathionine β-synthase or cystathionine γ-lyase was observed in the thymus and spleen of mice. In embryonic fibroblasts derived from wild-type embryos, cystine uptake was significantly inhibited by cystathionine in a concentration-dependent manner. Wild-type cells showed an intracellular accumulation of cystathionine when incubated in cystathionine-containing buffer, which concomitantly stimulated an increased release of glutamate into the extracellular space. By contrast, none of these effects could be observed in xCT-deficient cells. Remarkably, unlike knock-out cells, wild-type cells could be rescued from cystine deprivation-induced cell death by cystathionine supplementation. We thus conclude that cystathionine is a novel physiological substrate of system xc(-) and that the accumulation of cystathionine in immune tissues is exclusively mediated by system xc(-).

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Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats

Kume, S., Yamato, M., Tamura, Y., Jin, G., Nakano, M., Miyashige, Y., Eguchi, A., Ogata, Y., Goda, N., Iwai, K., Yamano, E., Watanabe, Y., Soga, T., *Kataoka, Y.

PLoS ONE 10 （ 3 ） e0120106 2015年03月

研究論文（学術雑誌）, 共著

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In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days. A food-restricted group, in which rats were limited to 10 g/d of food (around 50% of the control group), was also assessed. The food-restricted group exhibited weight reduction similar to that of the fatigued group. CE-MS measurements were performed to evaluate the profile of food intake-dependent metabolic changes, as well as the profile in fatigue loading, resulting in the identification of 48 metabolites in plasma. Multivariate analyses using hierarchical clustering and principal component analysis revealed that the plasma metabolome in the fatigued group showed clear differences from those in the control and food-restricted groups. In the fatigued group, we found distinctive changes in metabolites related to branched-chain amino acid metabolism, urea cycle, and proline metabolism. Specifically, the fatigued group exhibited significant increases in valine, leucine, isoleucine, and 2-oxoisopentanoate, and significant decreases in citrulline and hydroxyproline compared with the control and food-restricted groups. Plasma levels of total nitric oxide were increased in the fatigued group, indicating systemic oxidative stress. Further, plasma metabolites involved in the citrate cycle, such as cis-aconitate and isocitrate, were reduced in the fatigued group. The levels of ATP were significantly decreased in the liver and skeletal muscle, indicative of a deterioration in energy metabolism in these organs. Thus, this comprehensive metabolic analysis furthered our understanding of the pathophysiology of fatigue, and identified potential diagnostic biomarkers based on fatigue pathophysiology.

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Hypoxia Induces a Lipogenic Cancer Cell Phenotype via HIF1α-Dependent and -Independent Pathways

*Valli, A., Rodriguez, M., Moutsianas, L., Fischer, R., Fedele, V., Huang, H.L., Van Stiphout, R., Jones, D., Mccarthy, M., Vinaxia, M., Igarashi, K., Sato, M., Soga, T., Buffa, F., Mccullagh, J., Yanes, O., Harris, A., Kessler. B.

Oncotarget 6 （ 4 ） 1920 - 1941 2015年02月

研究論文（学術雑誌）, 共著, 査読有り

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The biochemistry of cancer cells diverges significantly from normal cells as a result of a comprehensive reprogramming of metabolic pathways. A major factor influencing cancer metabolism is hypoxia, which is mediated by HIF1α and HIF2α. HIF1α represents one of the principal regulators of metabolism and energetic balance in cancer cells through its regulation of glycolysis, glycogen synthesis, Krebs cycle and the pentose phosphate shunt. However, less is known about the role of HIF1α in modulating lipid metabolism. Lipids serve cancer cells to provide molecules acting as oncogenic signals, energetic reserve, precursors for new membrane synthesis and to balance redox biological reactions. To study the role of HIF1α in these processes, we used HCT116 colorectal cancer cells expressing endogenous HIF1α and cells in which the hif1α gene was deleted to characterize HIF1α-dependent and independent effects on hypoxia regulated lipid metabolites. Untargeted metabolomics integrated with proteomics revealed that hypoxia induced many changes in lipids metabolites. Enzymatic steps in fatty acid synthesis and the Kennedy pathway were modified in a HIF1α-dependent fashion. Palmitate, stearate, PLD3 and PAFC16 were regulated in a HIF-independent manner. Our results demonstrate the impact of hypoxia on lipid metabolites, of which a distinct subset is regulated by HIF1α.

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