Published papers

Salivary metabolomics have provided the potentials to detect both oral and systemic diseases. Capillary electrophoresis time-of-flight-mass spectrometry (CE-TOFMS) enables the identification and quantification of various charged metabolites. This method has been employed to biomarker discoveries using human saliva samples, especially for various types of cancers. The untargeted analysis contributes to finding new biomarkers. i.e., the analysis of all detectable signals including both known and unknown metabolites extends the coverage of metabolite to be observed. However, the observed data includes thousands of peaks. Besides, non-linear migration time fluctuation and skewed peaks are caused by the sample condition. The presented pretreatment protocols of saliva samples enhance the reproducibility of migration time drift, which facilitates the matching peaks across the samples and also results in reproducible absolute concentrations of the detected metabolites. The described protocols are utilized not only for saliva but for any liquid samples with slight modifications.

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid β-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane-selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

Background. Based on the theory of traditional Chinese medicine, Chinese herbs possess four different medicinal properties: hot, warm, cold, and cool. These serve as a reference guide for these herbal medicines. However, the molecular mechanisms supporting their relevance remain unclear. Methods. We performed metabolomics based on capillary electrophoresis-time-of-flight mass spectrometry (CE-TOF/MS) and multivariate data analysis for the structural identification of compounds of cold- and hot-natured Chinese herbs. Results. To this end, 30 selected herbs were analyzed and a total of 416 metabolites were identified via CE-TOF/MS, of which 193 compounds were detected in most herbs. The observed profiles offered the potential to understand the mechanism of association between the compounds and nature of the Chinese herbs. Comparison of the similarity in terms of chemical and molecular structures and content revealed that hot-natured herbs contained more nucleotides. In contrast, principal component analysis revealed the presence of more amino acid compounds in cold-natured herbs. Conclusion. Comparing the structural similarities between the samples using the Tanimoto coefficient revealed that a general non-specific structure was observed between cold- and hot-natured herbs; however, the distribution of the molecular groups seemed to contribute more toward the energy properties.

Extracellular vesicles (EVs) released from cancer cells contribute to various malignant phenotypes of cancer, including metastasis, cachexia, and angiogenesis. Although DNA, mRNAs, miRNAs, and proteins contained in EVs have been extensively studied, the function of metabolites in EVs remains unclear. In this study, we performed a comprehensive metabolomic analysis of pancreatic cancer cells, PANC-1, cultured under different oxygen concentrations, and small EVs (sEVs) released from them, considering the fact that hypoxia contributes to the malignant behavior of cells in pancreatic cancer, which is a poorly diagnosed cancer. sEVs were collected by ultracentrifugation, and hydrophilic metabolites were analyzed using capillary ion chromatography-mass spectrometry and liquid chromatography-mass spectrometry, and lipids were analyzed by supercritical fluid chromatography-tandem mass spectrometry. A total of 140 hydrophilic metabolites and 494 lipids were detected in sEVs, and their profiles were different from those in cells. In addition, the metabolomic profile of sEVs was observed to change under hypoxic stress, and an increase in metabolites involved in angiogenesis was also detected. We reveal the hallmark of the metabolites contained in sEVs and the effect of tumor hypoxia on their profiles, which may help in understanding EV-mediated cancer malignancy.

Large-scale metabolomic studies have become common, and the reliability of the peak data produced by the various instruments is an important issue. However, less attention has been paid to the large number of uncharacterized peaks in untargeted metabolomics data. In this study, we tested various criteria to assess the reliability of 276 and 202 uncharacterized peaks that were detected in a gathered set of 30 plasma and urine quality control samples, respectively, using capillary electrophoresis-time-of-flight mass spectrometry (CE-TOFMS). The linear relationship between the amounts of pooled samples and the corresponding peak areas was one of the criteria used to select reliable peaks. We used samples from approximately 3000 participants in the Tsuruoka Metabolome Cohort Study to investigate patterns of the areas of these uncharacterized peaks among the samples and clustered the peaks by combining the patterns and differences in the migration times. Our assessment pipeline removed substantial numbers of unreliable or redundant peaks and detected 35 and 74 reliable uncharacterized peaks in plasma and urine, respectively, some of which may correspond to metabolites involved in important physiological processes such as disease progression. We propose that our assessment pipeline can be used to help establish large-scale untargeted clinical metabolomic studies.

The increasing number of patients with osteoporosis and sarcopenia is a global concern among countries with progressively aging societies. The high medical costs of treating those patients suggest that prevention rather than treatment is preferable. We enrolled 729 subjects who attended both the second and third surveys of the Research on Osteoarthritis/Osteoporosis Against Disability (ROAD) study. Blood samples were collected from subjects at the second survey, and then a comprehensive metabolomic analysis was performed. It was found that 35 had newly developed osteoporosis at the third survey performed four years later, and 39 were newly diagnosed with sarcopenia at the third survey. In the second survey, we found that serum Gly levels were significantly higher even after adjustment for age, sex, and BMI in subjects with newly developed osteoporosis relative to those who remained osteoporosis-negative during the four-year follow-up. We also show that serum taurine levels were significantly lower at the second survey, even after adjustment for age, sex, and BMI in subjects with newly developed sarcopenia during the four-year follow-up compared with those not diagnosed with sarcopenia at the second or third surveys. Though our sample size and odds ratios were small, increased Gly and decreased taurine levels were found to be predictive of new development of osteoporosis and sarcopenia, respectively, within four years.

Epithelial-mesenchymal transition (EMT)-a fundamental process in embryogenesis and wound healing-promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.

Amino acids are indispensable nutrients for both normal and cancer cells. Cancer cells are unable to synthesize essential amino acids as well as some non-essential amino acids adequately to support rapid proliferation, and must take up amino acids from the surroundings. To meet the increased demand for the amino acid needed for proliferation, high levels of amino acid transporters are expressed on the surface of cancer cells. Cancer cells utilize amino acids to synthesize proteins and nucleotides, as well as to obtain energy. In addition, amino acids are known to play pathological roles in cancer cells. Interestingly, breast cancer cells limit the use of amino acids for cell proliferation based on amino acid availability, which depends on estrogen receptor status. Here, we present a summarized literature review of novel amino acid functions in cancer cells. This review organizes the available knowledge on 2 amino acid transporters, SLC7A5 and SLC7A11, which are considered essential for breast cancer cell growth in a cell-dependent manner. In particular, we propose the glutamine recycling model to clarify the mechanism underlying aberrant SLC7A5 activation. Finally, we overview the pathological significances of SLC7A5 and SLC7A11 in cancer tissues.

An effective combination of multi-omic datasets can enhance our understanding of complex biological phenomena. To build a context-dependent network with multiple omic layers, i.e., a trans-omic network, we perform phosphoproteomics, transcriptomics, proteomics, and metabolomics of murine liver for 4 h after insulin administration and integrate the resulting time series. Structural characteristics and dynamic nature of the network are analyzed to elucidate the impact of insulin. Early and prominent changes in protein phosphorylation and persistent and asynchronous changes in mRNA and protein levels through non-transcriptional mechanisms indicate enhanced crosstalk between phosphorylation-mediated signaling and protein expression regulation. Metabolic response shows different temporal regulation with transient increases at early time points across categories and enhanced response in the amino acid and nucleotide categories at later time points as a result of process convergence. This extensive and dynamic view of the trans-omic network elucidates prominent regulatory mechanisms that drive insulin responses through intricate interlayer coordination.

The PI3K/Akt/mTOR pathway has been well known to interact with the estrogen receptor (ER)-pathway and to be also frequently upregulated in aromatase inhibitor (AI)-resistant breast cancer patients. Intracellular levels of free amino acids, especially leucine, regulate the mammalian target of rapamycin complex 1 (mTORC1) activation. L-type amino acid transporters such as LAT1 and LAT3 are associated with the uptake of essential amino acids. LAT1 expression could mediate leucine uptake, mTORC1 signaling, and cell proliferation. Therefore, in this study, we explored amino acid metabolism, including LAT1, in breast cancer and clarified the potential roles of LAT1 in the development of therapeutic resistance and the eventual clinical outcome of the patients. We evaluated LAT1 and LAT3 expression before and after neoadjuvant hormone therapy (NAH) and examined LAT1 function and expression in estrogen deprivation-resistant (EDR) breast carcinoma cell lines. Tumors tended to be in advanced stages in the cases whose LAT1 expression was high. LAT1 expression in the EDR cell lines was upregulated. JPH203, a selective LAT1 inhibitor, demonstrated inhibitory effects on cell proliferation in EDR cells. Hormone therapy changed the tumor microenvironment and resulted in metabolic reprogramming through inducing LAT1 expression. LAT1 expression then mediated leucine uptake, enhanced mTORC1 signaling, and eventually resulted in AI resistance. Therefore, LAT1 could be the potential therapeutic target in AI-resistant breast cancer patients.

Mitochondrial electron transport chain complex I (ETCC1) is the essential core of cancer metabolism, yet potent ETCC1 inhibitors capable of safely suppressing tumor growth and metastasis in vivo are limited. From a plant extract screening, we identified petasin (PT) as a highly potent ETCC1 inhibitor with a chemical structure distinct from conventional inhibitors. PT had at least 1700 times higher activity than that of metformin or phenformin and induced cytotoxicity against a broad spectrum of tumor types. PT administration also induced prominent growth inhibition in multiple syngeneic and xenograft mouse models in vivo. Despite its higher potency, it showed no apparent toxicity toward nontumor cells and normal organs. Also, treatment with PT attenuated cellular motility and focal adhesion in vitro as well as lung metastasis in vivo. Metabolome and proteome analyses revealed that PT severely depleted the level of aspartate, disrupted tumor-associated metabolism of nucleotide synthesis and glycosylation, and downregulated major oncoproteins associated with proliferation and metastasis. These findings indicate the promising potential of PT as a potent ETCC1 inhibitor to target the metabolic vulnerability of tumor cells.

Patients with type 2 diabetes treated with Sodium glucose transporter 2 (SGLT2) inhibitors show reduced mortality and hospitalization for heart failure (HF). SGLT2 inhibitors are considered to activate multiple cardioprotective pathways; however, underlying mechanisms are not fully described. This study aimed to elucidate the underlying mechanisms of the beneficial effects of SGLT2 inhibitors on the failing heart. We generated a left ventricular (LV) pressure overload model in C57BL/6NCrSlc mice by transverse aortic constriction (TAC) and examined the effects of empagliflozin (EMPA) in this model. We conducted metabolome and transcriptome analyses and histological and physiological examinations. EMPA administration ameliorated pressure overload-induced systolic dysfunction. Metabolomic studies showed that EMPA increased citrulline levels in cardiac tissue and reduced levels of arginine, indicating enhanced metabolism from arginine to citrulline and nitric oxide (NO). Transcriptome suggested possible involvement of the insulin/AKT pathway that could activate NO production through phosphorylation of endothelial NO synthase (eNOS). Histological examination of the mice showed capillary rarefaction and endothelial apoptosis after TAC, both of which were significantly improved by EMPA treatment. This improvement was associated with enhanced expression phospho-eNOS and NO production in cardiac endothelial cells. NOS inhibition attenuated these cardioprotective effects of EMPA. The in vitro studies showed that catecholamine-induced endothelial apoptosis was inhibited by NO, arginine, or AKT activator. EMPA activates the AKT/eNOS/NO pathway, which helps to suppress endothelial apoptosis, maintain capillarization and improve systolic dysfunction during LV pressure overload.

Acute kidney injury (AKI) is defined as a rapid decline in kidney function. The associated syndromes may lead to increased morbidity and mortality, but its early detection remains difficult. Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we analyzed the urinary metabolomic profile of patients admitted to the intensive care unit (ICU) after invasive surgery. Urine samples were collected at six time points: before surgery, at ICU admission and 6, 12, 24 and 48 h after. First, urine samples from 61 initial patients (non-AKI: 23, mild AKI: 24, severe AKI: 14) were measured, followed by the measurement of urine samples from 60 additional patients (non-AKI: 40, mild AKI: 20). Glycine and ethanolamine were decreased in patients with AKI compared with non-AKI patients at 6–24 h in the two groups. The linear statistical model constructed at each time point by machine learning achieved the best performance at 24 h (median AUC: 89%, cross-validated) for the 1st group. When cross-validated between the two groups, the AUC showed the best value of 70% at 12 h. These results identified metabolites and time points that show patterns specific to subjects who develop AKI, paving the way for the development of better biomarkers.

Commensal microbiota colonize the surface of our bodies. The inside of the gastrointestinal tract is one such surface that provides a habitat for them. The gastrointestinal tract is a long organ system comprising of various parts, and each part possesses various functions. It has been reported that the composition of intestinal luminal metabolites between the small and large intestine are different; however, comprehensive metabolomic and commensal microbiota profiles specific to each part of the gastrointestinal lumen remain obscure. In this study, by using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome and 16S rRNA gene-based microbiome analyses of specific pathogen-free (SPF) and germ-free (GF) murine gastrointestinal luminal profiles, we observed the different roles of commensal microbiota in each part of the gastrointestinal tract involved in carbohydrate metabolism and nutrient production. We found that the concentrations of most amino acids in the SPF small intestine were higher than those in the GF small intestine. Furthermore, sugar alcohols such as mannitol and sorbitol accumulated only in the GF large intestine, but not in the SPF large intestine. On the other hand, pentoses, such as arabinose and xylose, gradually accumulated from the cecum to the colon only in SPF mice, but were undetected in GF mice. Correlation network analysis between the gastrointestinal microbes and metabolites showed that niacin metabolism might be correlated to Methylobacteriaceae. Collectively, commensal microbiota partially affects the gastrointestinal luminal metabolite composition based on their metabolic dynamics, in cooperation with host digestion and absorption.

18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) is usually used for staging or evaluation of treatment response rather than for cancer screening. However, 18F-FDG PET/CT has also been used in Japan for cancer screening in people with no cancer symptoms, and accumulating evidence supports this application of 18F-FDG PET/CT. Previously, we have observed a correlation between the saliva and tumor metabolomic profiles in patients with oral cancer. Hence, if salivary metabolites demonstrate a significant correlation with PET parameters such as the maximum standardized uptake value (SUVmax), they may have the potential to be used as a screening tool before PET/CT to identify patients with high SUVmax. Hence, in this study, we aimed to explore the relationship between salivary metabolites and SUVmax of 18F-FDG PET/CT using previously collected data. 18F-FDG PET/CT was performed for staging 26 patients with oral cancer. The collected data were integrated and analyzed along with quantified salivary hydrophilic metabolites obtained from the same patients with oral cancer and controls (n = 44). In total, 11 metabolites showed significant correlations with SUVmax in the delayed phases. A multiple logistic regression model of the two metabolites showed the ability to discriminate between patients with oral cancer and controls, with an area under the receiver operating characteristic curve of 0.738 (p = 0.001). This study uniquely confirmed a relationship between salivary metabolites and SUVmax of PET/CT in patients with oral cancer; salivary metabolites were significantly correlated with SUVmax. These salivary metabolites can be used as a screening tool before PET/CT to identify patients with high SUVmax, i.e., to detect the presence of oral cancer.

Uremic toxins are suggested to be involved in the pathophysiology of hemodialysis (HD) patients. However, the profile of uremic solutes in HD patients has not been fully elucidated. In this study using capillary electrophoresis mass spectrometry (CE-MS), we comprehensively quantified the serum concentrations of 122 ionic solutes before and after HD in 11 patients. In addition, we compared the results with those in non-HD patients with chronic kidney disease (CKD) to identify HD patient-specific solutes. We identified 38 solutes whose concentrations were higher in pre-HD than in CKD stage G5. Ten solutes among them did not significantly accumulate in non-HD CKD patients, suggesting that these solutes accumulate specifically in HD patients. We also identified 23 solutes whose concentrations were lower in both pre- and post-HD than in CKD stage G5. The serum levels of 14 solutes among them were not affected by renal function in non-HD patients, suggesting that these solutes tend to be lost specifically in HD patients. Our data demonstrate that HD patients have a markedly different profile of serum uremic solute levels compared to that in non-HD CKD patients. The solutes identified in our study may contribute to the pathophysiology of HD patients.