Upregulation of Thymidylate Synthase Induces Pemetrexed Resistance in Malignant Pleural Mesothelioma.

Malignant pleural mesothelioma (MPM) is an invasive malignancy that develops in the pleural cavity, and antifolates are used as chemotherapeutics for treating. The majority of antifolates, including pemetrexed (PMX), inhibit enzymes involved in purine and pyrimidine synthesis. MPM patients frequently develop drug resistance in clinical practice, however the associated drug-resistance mechanism is not well understood. This study was aimed to elucidate the mechanism underlying resistance to PMX in MPM cell lines. We found that among the differentially expressed genes associated with drug resistance (determined by RNA sequencing), TYMS expression was higher in the established resistant cell lines than in the parental cell lines. Knocking down TYMS expression significantly reduced drug resistance in the resistant cell lines. Conversely, TYMS overexpression significantly increased drug resistance in the parental cells. Metabolomics analysis revealed that the levels of dTMP were higher in the resistant cell lines than in the parental cell lines; however, resistant cells showed no changes in dTTP levels after PMX treatment. We found that the nucleic acid-biosynthetic pathway is important for predicting the efficacy of PMX in MPM cells. The results of chromatin immunoprecipitation-quantitative polymerase chain reaction (ChIP-qPCR) assays suggested that H3K27 acetylation in the 5'-UTR of TYMS may promote its expression in drug-resistant cells. Our findings indicate that the intracellular levels of dTMP are potential biomarkers for the effective treatment of patients with MPM and suggest the importance of regulatory mechanisms of TYMS expression in the disease.

De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma.

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.

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

Metabolic Characterization of Antifolate Responsiveness and Non-responsiveness in Malignant Pleural Mesothelioma Cells.

Antifolates are a class of drugs effective for treating malignant pleural mesothelioma (MPM). The majority of antifolates inhibit enzymes involved in purine and pyrimidine synthesis such as dihydrofolate reductase (DHFR), thymidylate synthase (TYMS), and glycinamide ribonucleotide formyltransferase (GART). In order to select the most suitable patients for effective therapy with drugs targeting specific metabolic pathways, there is a need for better predictive metabolic biomarkers. Antifolates can alter global metabolic pathways in MPM cells, yet the metabolic profile of treated cells has not yet been clearly elucidated. Here we found that MPM cell lines could be categorized into two groups according to their sensitivity or resistance to pemetrexed treatment. We show that pemetrexed susceptibility could be reversed and DNA synthesis rescued in drug-treated cells by the exogenous addition of the nucleotide precursors hypoxanthine and thymidine (HT). We observed that the expression of pemetrexed-targeted enzymes in resistant MPM cells was quantitatively lower than that seen in pemetrexed-sensitive cells. Metabolomic analysis revealed that glycine and choline, which are involved in one-carbon metabolism, were altered after drug treatment in pemetrexed-sensitive but not resistant MPM cells. The addition of HT upregulated the concentration of inosine monophosphate (IMP) in pemetrexed-sensitive MPM cells, indicating that the nucleic acid biosynthesis pathway is important for predicting the efficacy of pemetrexed in MPM cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, and points to potential biomarkers for informing clinical decisions regarding the most effective therapies for patients with MPM.

Cancer mortality among atomic bomb survivors exposed as children.

OBJECTIVES: To compare cancer mortality among A-bomb survivors exposed as children with cancer mortality among an unexposed control group (the entire population of Japan, JPCG). METHODS: The subjects were the Hiroshima and Nagasaki A-bomb survivor groups (0-14 years of age in 1945) reported in life span study report 12 (follow-up years were from 1950 to 1990), and a control group consisting of the JPCG. We estimated the expected number of deaths due to all causes and cancers of various causes among the exposed survivors who died in the follow-up interval, if they had died with the same mortality as the JPCG (0-14 years of age in 1945). We calculated the standardized mortality ratio (SMR) of A-bomb survivors in comparison with the JPCG. RESULTS: SMRs were significantly higher in exposed boys overall for all deaths, all cancers, leukemia, and liver cancer, and for exposed girls overall for all cancers, solid cancers, liver cancer, and breast cancer. In boys, SMRs were significantly higher for all deaths and liver cancer even in those exposed to very low doses, and for all cancers, solid cancers, and liver cancer in those exposed to low doses. In girls, SMRs were significantly higher for liver cancer and uterine cancer in those exposed to low doses, and for leukemia, solid cancers, stomach cancer, and breast cancer in those exposed to high doses. CONCLUSIONS: We calculated the SMRs for the A-bomb survivors versus JPCG in childhood and compared them with a true non-exposed group. A notable result was that SMRs in boys exposed to low doses were significantly higher for solid cancer.

Cinnamon extract inhibits the postprandial overproduction of apolipoprotein B48-containing lipoproteins in fructose-fed animals.

We have reported previously that a cinnamon extract (CE), high in type A polyphenols, prevents fructose feeding-induced decreases in insulin sensitivity and suggested that improvements of insulin sensitivity by CE were attributable, in part, to enhanced insulin signaling. In this study, we examined the effects of CE on postprandial apolipoprotein (apo) B-48 increase in fructose-fed rats, and the secretion of apoB48 in freshly isolated intestinal enterocytes of fructose-fed hamsters. In an olive oil loading study, a water-soluble CE (Cinnulin PF, 50 mg/kg body weight, orally) decreased serum triglyceride (TG) levels and the over production of total- and TG-rich lipoprotein-apoB48. In ex vivo (35)S labeling study, significant decreases were also observed in apoB48 secretion into the media in enterocytes isolated from fructose-fed hamsters. We also investigated the molecular mechanisms of the effects of CE on the expression of genes of the insulin signaling pathway [insulin receptor (IR), IR substrate (IRS)1, IRS2 and Akt1], and lipoprotein metabolism [microsomal TG transfer protein (MTP), sterol regulatory element-binding protein (SREBP1c) in isolated primary enterocytes of fructose-fed hamsters, using quantitative real-time polymerase chain reaction. The CE reversed the expression of the impaired IR, IRS1, IRS2 and Akt1 mRNA levels and inhibited the overexpression of MTP and SREBP1c mRNA levels of enterocytes. Taken together, our data suggest that the postprandial hypertriglycerides and the overproduction of apoB48 can be acutely inhibited by a CE by a mechanism involving improvements of insulin sensitivity of intestinal enterocytes and regulation of MTP and SREBP1c levels. We present both in vivo and ex vivo evidence that a CE improves the postprandial overproduction of intestinal apoB48-containing lipoproteins by ameliorating intestinal insulin resistance and may be beneficial in the control of lipid metabolism.

Associations of periodontal damage and tooth loss with atherogenic factors among patients with type 2 diabetes mellitus.

OBJECTIVE: To clarify the associations of periodontal damage and tooth loss with atherogenic factors among diabetic patients. METHODS: We examined the correlations of age, sex, smoking, oral hygiene score, blood pressure, body mass index, and blood chemical data with the mean depth of periodontal pockets or the number of remaining teeth. PATIENTS: One hundred outpatients with type 2 diabetes aged 29 to 77 years. RESULTS: The mean depth of periodontal pockets was significantly associated with smoking, oral hygiene score, and HbA1c; the Spearman correlation coefficients (r) were 0.220, 0.417, and 0.260, respectively. Age, oral hygiene score, and HbA1c were inversely correlated with the number of remaining teeth (r=-0.306, -0.287, and -0.275, respectively). Serum total cholesterol was significantly correlated with the mean depth of pockets after adjustment for smoking, oral hygiene score, and HbA1c (r=0.211; P=0.044), while serum HDL cholesterol tended to be negatively associated with depth (r=-0.202; P=0.055). Serum HDL cholesterol was also associated with an increased number of teeth, which remained significant after adjustment for age, oral hygiene score, and HbA1c (r=0.202; P=0.048). The estimated glomerular filtration rate was significantly and positively correlated with the number of teeth in the univariate analysis, although consideration of the potential confounding factors somewhat weakened the association (r=0.186; P=0.069). CONCLUSIONS: We may expect better management of oral health in diabetic patients with control of dyslipidemia in addition to blood glucose.

Antioxidant small phenolic ingredients in Inonotus obliquus (persoon) Pilat (Chaga).

Inonotus obliquus (persoon) Pilat (Chaga, in Russia, kabanoanatake in Japan) is a fungus having been used as a folk medicine in Russia and said to have many health beneficial functions such as immune modulating and anti-cancer activities. In the present study, the antioxidant activity of hot water extract (decoction) of Chaga was precisely compared with those of other medicinal fungi (Agaricus blazei Mycelia, Ganoderma lucidum and Phellinus linteus) showing Chaga had the strongest antioxidant activity among fungi examined in terms of both superoxide and hydroxyl radicals scavenging activities. Further determination of the antioxidant potential of isolated fruiting body (brown part) and Sclerotium (black part) revealed the 80% MeOH extract of fruiting body had the highest potential as high as that of Chaga decoction. Finally, seven antioxidant components were isolated and purified from the 80% MeOH extract of Chaga fruiting body, and their chemical structures were determined as small phenolics as follows: 4-hydroxy-3,5-dimethoxy benzoic acid 2-hydroxy-1-hydroxymethyl ethyl ester (BAEE), protocatechic acid (PCA), caffeic acid (CA), 3,4-dihybenzaladehyde (DB), 2,5-dihydroxyterephtalic acid (DTA), syringic acid (SA) and 3,4-dihydroxybenzalacetone (DBL). Notably, BAEE was assigned as the new compound firstly identified from the natural source in the present study.

Role of hepatic AMPK activation in glucose metabolism and dexamethasone-induced regulation of AMPK expression.

To elucidate the role of AMPK in hepatic glucose metabolism, dominant negative (DN), constitutively active (CA) forms of the AMPKalpha1 subunit and control vector LacZ were overexpressed by means of adenovirus-mediated gene transfer. Five days after virus injection, hepatic AMPK activity was five-fold higher in CA mice than in DN mice. DN mice were apparently glucose intolerant with a higher fasting plasma glucose level (DN 82.3+/-0.7mg/dl, CA 42.5+/-4.8mg/dl and LacZ 54.3+/-2.4mg/dl). PEPCK, a gluconeogenic key enzyme, mRNA was increased 131.54% and 48.92% in DN mice compared to that of CA and LacZ, respectively. Thus, hepatic AMPK activation plays a role in the suppression of gluconeogenesis and this might be the cause of decreased fasting plasma glucose level in CA mice. We also investigated the effects of dexamethasone on hepatic AMPK expression and activity in rat liver, mice liver, as well as primary cultured hepatocytes. Subcutaneously injecting mice with dexamethasone (1mg/day) for 5 days significantly upregulated hepatic AMPKalpha1 and alpha2 expressions. Similarly, the treatment of primary cultured rat hepatocytes with dexamethasone (1microM) increased expression of the AMPKalpha1 subunit, AICAR-induced AMPK phosphorylation and kinase activity. Although increased AMPK expression cannot be attributed to dexamethasone-induced glucose intolerance, taken together our results raise the possibility that AMPK control liver glucose output and its expression in liver might be modulated by various hormones and growth factors.

Anthropometric, lifestyle and biomarker assessment of Japanese non-professional ultra-marathon runners.

BACKGROUND: Anthropometric characteristics, lifestyle, and baseline biological markers of Japanese non-professional ultra-marathon runners have not been fully assessed. METHODS: We evaluated anthropometric characteristics, lifestyle, and baseline biological markers of 180 Japanese amateur ultra-marathon runners (144 males [mean age: 50.5 +/- 9.4 (standard deviation) years] and 36 females [48.9 +/- 6.9]), and compared them with those of participants in a community heath check-up program and with the figures in the literature. We furthermore evaluated baseline blood indices according to monthly running distance with analysis of variance adjusted for age, body mass index, smoking and alcohol drinking habits. RESULTS: The ultra-marathon runners demonstrated more favorable values for body mass index and bone density, and the proportion of smoking, and undertaking physical activity (for both sexes), eating breakfast (for males), and having daily bowel movements (for females), while greater proportion of alcohol drinking habit (for both sexes), than the comparison group. Average monthly running distances and standard deviations (km) were 257.2 +/- 128.9 for males and 209.0 +/- 86.2 for females. Male runners possessed beneficial markers, including lowered triglyceride and elevated high-density lipoprotein cholesterol, and their values showed hockey-stick (or inverse hockey-stick) patterns depending on their monthly running distance. Some subjects running more than 300 km/month exhibited signs of an over-reaching/training syndrome, including somewhat lowered hemoglobin, ferritin and white blood cell count, and elevated creatine kinase and lactate dehydrogenase. CONCLUSIONS: Together with a desirable lifestyle, Japanese non-professional ultra-marathon runners with vigorous exercise habit demonstrated a preferable health status according to biological indices.

Estrogen controls branched-chain amino acid catabolism in female rats.

A diurnal rhythm occurs in the activity state of branched-chain alpha-keto acid dehydrogenase complex (BCKDC) in female but not male rats. We attempted to determine the role played by ovarian hormones in this difference in enzyme regulation. A series of experiments examined the effects of the 4-d estrous cycle, ovariectomy, and replacement of female sex steroids on the catabolism of BCAAs. A proestrous decrease in the activity state of the complex corresponded to an increase in the plasma 17beta-estradiol level. Withdrawal of gonadal steroids by ovariectomy resulted in an increase in the activity state of BCKDC and a decrease in the activity of the branched-chain alpha-keto acid dehydrogenase kinase (BDK). However, 17beta-estradiol reversed these effects, resulting in an increase in the BDK activity, thereby decreasing the activity of the complex. Progesterone administration was ineffective. The changes in the percentage of active BCKDC caused by 17beta-estradiol withdrawal and replacement resulted from changes in the amount of BDK protein associated with the complex and therefore its activity. Thus, the marked diurnal variation in the activity state of BCKDC exhibited by female rats involves estrogenic control of BDK activity. We hypothesize that the 17beta-estradiol-controlled feeding pattern produces these variations in BCKDC activity. This may function in female rats to conserve essential amino acids for protein synthesis.

Effects of sex, age and BMI on screening tests for impaired glucose tolerance.

The discriminating abilities of fasting plasma glucose (FPG) and HbA1c were compared on screening tests for impaired glucose tolerance (IGT) and IGT plus diabetes mellitus by the receiver operating characteristic (ROC) curve analysis. Furthermore, effects of sex, age and BMI were examined on sensitivity and specificity of the optimal cutoff points. This study included 997 subjects who were recruited for 75 g OGTT after the first screening of the Japan Diabetes Prevention Program. According to the 1997 criteria of the American Diabetes Association (ADA), 140 subjects were classified as diabetic and 256 as IGT. The areas under the ROC curves of FPG were significantly larger than those of HbA1c. The optimal cutoff points of FPG were 102 mg/dl for IGT and 105 mg/dl for IGT plus diabetes mellitus. Those of HbA1c were both 5.3%. In screening with FPG, females had significantly lower sensitivity and higher specificity than males, and the specificity for IGT plus diabetes mellitus was the lowest in the obese group. In screening with HbA1c, the specificity was low in the older and the obese groups. We concluded that FPG was superior to HbA1c for screening of IGT and IGT plus diabetes mellitus and the optimal cutoff point of FPG would be 102 mg/dl or greater.

Analysis of sex, age and disease factors contributing to prolonged life expectancy at birth, in cases of malignant neoplasms in Japan.

BACKGROUND: This study aimed to examine the contribution made by the change in mortality from malignant neoplasms to the life expectancy at birth, observed during the years 1965-1995 in Japan. METHODS: We used data on the population and number of deaths by cause, age and sex in 1965, 1975, 1985 and 1995. The contribution of different ages and causes of death to the change in life expectancy were examined with the method developed by Pollard. RESULTS: We found that, among all causes, the decrease of mortality from stomach cancer led to the greatest improvement in life expectancy for both sexes. On the other hand, negative contributions were seen with cancers of many sites, such as cancer of the intestine, liver and lung for males, and cancer of the intestine, gallbladder, lung and breast for females. Recently, the contributing years of all cancers have been negative because of the increase in mortality from malignant neoplasms. In addition, increase of death from malignant neoplasms in middle-aged and elderly people negatively influenced the life expectancy at birth. CONCLUSIONS: Female cancer influenced the improvement in life expectancy at birth. Cancer for males, however, contributed little to improvement of life expectancy at birth except for a little prolongation of life expectancy at birth during the years 1965-1975. To develop a public health policy, the contributing years to life expectancy at birth can be a useful indication in evaluating the impact of death from various diseases. It is necessary to analyze the contribution made by various causes of death to the changes of life expectancy at birth.

Effects of aging and denervation on the expression of uncoupling proteins in slow- and fast-twitch muscles of rats.

We investigated the effects of aging and denervation on the gene expression of uncoupling proteins (UCPs) in slow-twitch soleus and fast-twitch gastrocnemius muscles. In a comparison between the control limbs of 6- and 24-month-old rats, the mRNA levels of UCP3, heart-type fatty acid binding protein (HFABP), and glucose transporter-4 (GLUT4) were considerably lower in the gastrocnemius muscles of the older rats, whereas no significant differences in the mRNA levels of those genes as well as UCP2 and cytochrome oxidase subunit IV (COX-IV) were observed in the soleus muscles of young and old rats. The UCP3 and COX-IV protein levels were also reduced considerably in the aged gastrocnemius muscles with atrophy. Denervation of the sciatic nerve caused an increase in UCP3 mRNA levels in both muscles, but the regulation of other genes contrasted between the two types of skeletal muscles. In spite of the increased mRNA level, a remarkable reduction in UCP3 protein was found in the denervated gastrocnemius muscles. These results indicate that the effects of aging and denervation on the gene expression of UCPs, HFABP, GLUT4, and COX-IV are different between the muscle types. The reduction in the mitochondrial UCP3 and COX proteins in aged fast-twitch muscles may have a negative effect on energy metabolism and thermogenesis in old animals.

Mitochondrial ATPase subunit 6 and cytochrome B gene polymorphisms in young obese adults.

We hypothesized that the mutational strand asymmetry is more strongly exerted upon the mitochondrial cytochrome b (Cytb) gene, which is distant from the origin of the light-strand replication (Ori(L)), than upon the ATPase subunit 6 (ATP6) gene, which is close to the Ori(L). To test this hypothesis, we determined the sequences of these two genes in 96 Japanese young obese adults. The frequency of G-->A transitions was significantly higher than that of C-->T transitions in the Cytb gene, whereas the frequencies of G-->A and C-->T transitions were not significantly different in the ATP6 gene. The marked mutational strand asymmetry in the Cytb gene can be explained by the deamination of C to uracil in the long single-stranded state of the heavy strand during replication. The ratio of the nonsynonymous substitutions at the second codon positions to those at the first codon positions was significantly lower in the Cytb gene than in the ATP6 gene. The physicochemical differences between the standard and the replaced amino acid residues were significantly smaller in the Cytb gene than in ATP6 one. The present study indicates that amino acid sequences are less variable for Cytb than for ATP6 in spite of the strong mutational strand asymmetry for the Cytb gene.