Screening of biologically active monosaccharides

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Mar 29, 2016 - lulose (D-Alu, also known as D-psicose), the C3 epimer of D-Fru, while anticancer4,5) and neuroprotective6,7) effects have been identified for ...
Bioscience, Biotechnology, and Biochemistry

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Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans Hirofumi Sakoguchi, Akihide Yoshihara, Ken Izumori & Masashi Sato To cite this article: Hirofumi Sakoguchi, Akihide Yoshihara, Ken Izumori & Masashi Sato (2016) Screening of biologically active monosaccharides: growth inhibitory effects of d-allose, d-talose, and l-idose against the nematode Caenorhabditis elegans, Bioscience, Biotechnology, and Biochemistry, 80:6, 1058-1061, DOI: 10.1080/09168451.2016.1146069 To link to this article: http://dx.doi.org/10.1080/09168451.2016.1146069

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Date: 10 May 2016, At: 21:37

Bioscience, Biotechnology, and Biochemistry, 2016 Vol. 80, No. 6, 1058–1061

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Screening of biologically active monosaccharides: growth inhibitory effects of D-allose, D-talose, and L-idose against the nematode Caenorhabditis elegans Hirofumi Sakoguchi1, Akihide Yoshihara2, Ken Izumori2 and Masashi Sato1,* 1 2

Faculty of Agriculture, Department of Applied Biological Science, Kagawa University, Miki, Japan; Rare Sugar Research Center, Kagawa University, Miki, Japan

Received December 4, 2015; accepted January 12, 2016

Downloaded by [Kagawa University] at 21:37 10 May 2016

http://dx.doi.org/10.1080/09168451.2016.1146069

We compared the growth inhibitory effects of all aldohexose stereoisomers against the model animal Caenorhabditis elegans. Among the tested compounds, the rare sugars D-allose (D-All), D-talose (DTal), and L-idose (L-Ido) showed considerable growth inhibition under both monoxenic and axenic culture conditions. 6-Deoxy-D-All had no effect on growth, which suggests that C6-phosphorylation by hexokinase is essential for inhibition by D-All. Key words:

growth inhibitory effect; aldohexose; Caenorhabditis elegans; rare sugar

Monosaccharides exist in a variety of stereoisomer forms. With the exception of isomers such as D-glucose (D-Glc) and D-fructose (D-Fru), which exist in great abundance, the vast majority of the stereoisomers are rare in nature. It is therefore difficult to isolate them from natural sources due to their scarcity, and there have been no systematic methods for the production of such rare sugars. As a result, the biological activities of these rare sugars have largely remained unstudied until recently. Izumori developed a novel method, called the Izumo-ring strategy, for the systematic production of rare sugars using three enzyme classes: aldose-ketose isomerases, epimerases, and oxidoreductases.1) This approach facilitates the study of the biological activities of these rare sugars. For example, antihyperglycemic2) and anti-obesity3) effects have been identified for D-allulose (D-Alu, also known as D-psicose), the C3 epimer of D-Fru, while anticancer4,5) and neuroprotective6,7) effects have been identified for D-allose (D-All), the C3 epimer of D-Glc. Biological activities of these rare sugars can be attributed to the properties of antimetabolites that mimic the structures of metabolizable sugars, including D-Glc and D-Fru. In order to investigate biological activities of these rare sugars, a simple bioassay is necessary due to the sheer number of monosaccharide stereoisomers.

The nematode Caenorhabditis elegans is an attractive multicellular animal model for biological research. It has been widely used as a model system for studying development, aging, metabolism, and other physiological processes. We developed growth assay methods using C. elegans larvae in monoxenic conditions where animals are cultured in liquid medium containing Eschericha coli as a food source.8) Compared to other bioassay methods using cancer cells, the monoxenic assay has several advantages, including short test period (3 days), cost-effectiveness, and no need for high-level aseptic techniques. The assay also enabled us to identify sugars that have weak biological activities, as C. elegans is able to withstand the osmotic stress of high sugar concentrations (~300 mM). For these reasons, we believe that the growth assay is a convenient and useful primary screening method to search for biologically active sugars. In our previous study,8) the growth inhibitory effects of all ketohexose stereoisomers against C. elegans were examined under monoxenic conditions. Of these ketohexose stereoisomers, only D-Alu strongly inhibited nematode growth.8) We performed further screening of aldohexose stereoisomers including D- and L-forms of All, altrose (Alt), Glc, mannose (Man), gulose (Gul), idose (Ido), galactose (Gal), and talose (Tal) using the monoxenic assay. Here, we report a comparison of the growth inhibitory effects of these aldohexoses against C. elegans, and that among these, D-All, D-Tal, and LIdo exerted considerable growth inhibition. The inhibitory effect of these bioactive rare sugars was also confirmed under axenic conditions, where animals were cultured in the chemically defined medium CeMM (C. elegans maintenance medium)9) to exclude the possible influence of E. coli. In addition, we discuss the mechanisms responsible for D-All-induced inhibition based on the bioassay results of a deoxy derivative of D-All. The structures of aldohexoses are shown in Fig. 1. Details of the sugars used in this study are described in the Supplementary material, which is available online. The wild-type C. elegans strain N2 (var. Bristol) was provided by the Caenorhabditis Genetic Center at

*Corresponding author. Email: [email protected] Abbreviations: IC50, half maximal inhibitory concentration; CI, confidence intervals; SD, standard deviation. © 2016 Japan Society for Bioscience, Biotechnology, and Agrochemistry

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Fig. 1. Chemical structures of aldohexose stereoisomers (Fischer projection). Notes: All, allose; Alt, altrose; Glc, glucose; Man, mannose; Gul, gulose; Ido, idose; Gal, galactose; Tal, talose.

University of Minnesota. Worms were maintained at 20 °C on nematode growth medium (NGM) plate seeded with E. coli OP50.10) Eggs of C. elegans were collected by treating egg-bearing adults with alkaline hypochlorite solution, and were shaken in S basal liquid medium at 20 °C for 24 h, to prepare first-stage larvae (L1).10) Monoxenic assay was performed as follows: About 20 L1 larvae worms were transferred into each well of 24-well culture plate (4846-24FS; Watson Co., Ltd., Kobe, Japan) that held 200 μL of complete S liquid medium10) containing E. coli OP50 (2.8 mg (wet)/mL, ca 1.7 × 109 cells/mL) with one of the sugars at a concentration of 167 mM. After incubation at 20 °C for 72 h, worms were anesthetized with 25 mM sodium azide. Individual images of each worm (n = 10), which were selected at random, were taken with a CCD digital camera (Olympus DP70) attached to an Olympus SMZ9 microscope, followed by analysis using ImageJ to calculate the area of the worm’s projection. The area was used as an index of body size. Each experiment repeated two times. Control worms were incubated in medium without sugar. Results were compared by one-way analysis of variance (ANOVA), followed by a Tukey-Kramer multiple comparison test using GraphPad Prism 6 software. IC50 values were estimated using the Probit method.11) Values were calculated as the concentrations needed to inhibit growth by 50% relative to controls. The axenic assay was performed in the same way as the monoxenic assay, except for the use of CeMM9) as a medium without E. coli, and the incubation period was 7 days. The original CeMM contained 180 mM as a carbon source, but we changed the concentration of D-Glc to 90 mM to avoid osmotic toxicity when a sugar was added. First, we compared the growth inhibitory effects of all the aldohexose stereoisomers in the C. elegans model under monoxenic culture conditions. In our previous report,8) we evaluated the growth of the nematodes in terms of their egg-bearing rate; for this report, however, in order to evaluate growth more precisely, we used the projected area of the nematodes as the

index. Each stereoisomer was evaluated at a concentration of 167 mM with the growth assay using L1 larvae. Body sizes (mean area of worm’s projections) of animals treated with each sugar are shown in Fig. 2. No deaths were observed in the tested animals in all experiments during the 72-h period. L1 larvae were incubated for 3 days in S liquid medium with E. coli food and without any sugar to give egg-bearing mature adults, which were used as untreated controls (mean body size, 5.86 × 104 μm2). Among the 16 aldohexose stereoisomers, D-All, D-Tal, and L-Ido showed significant inhibition of nematode growth (Fig. 2). Treatment with D-All, D-Tal, and L-Ido gave relative body sizes of 60.8, 64.4, and 49.5%, respectively, when compared to untreated controls. No changes were observed in motility of the animals treated with the three sugars, compared with controls. Bright-field images of typical C. elegans treated with the three sugars, and an untreated control animal (egg-bearing adult), are shown in Supplementary Figure S1. The body sizes and morphology of the treated animals were nearly the same as those of L4 larvae or young adults bearing no eggs. Mature adults of C. elegans have well-developed gonadal cells and eggs in the uterus, but the treated animals in question lacked these characteristics (Fig. S1). Egg-bearing rates of C. elegans treated with each aldohesose are also shown in Supplementary Table S1. These findings indicate that D-All, D-Tal, and L-Ido delayed development of C. elegans. Inhibition was not due to osmotic effects; when D-mannitol was substituted for these sugars, no effects on growth were observed at a concentration of 167 mM (mean body size, 5.50 × 104 μm2; 93.9% to the untreated controls). The other 13 stereoisomers did not affect growth to a statistically significant extent, compared with untreated controls (Fig. 2). The animals treated with these inactive sugars grew up to become mature adults during the test period. We estimated the IC50 values for D-All, D-Tal, and L-Ido from each individual dose-response curve (42– 278 mM). The IC50 values for D-All, D-Tal, and L-Ido were estimated to be 200, 197, and 121 mM (95% CIs, 190–209, 53–315, and 107–134 mM), respectively.

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Fig. 2. Growth inhibition of C. elegans by aldohexoses under monoxenic conditions. Notes: Body sizes (mean area of worm’s projections) of C. elegans exposed to each sugar at a concentration of 167 mM are shown (n = 20 each). Error bars represent SD. Statistical analysis of data was performed by one-way ANOVA, followed by a Tukey–Kramer multiple comparison test. Statistical difference (p < 0.01) is indicated by different letters. Abbreviations are as in Fig. 1.

In the axenic medium CeMM, L1 larvae were incubated for 7 days to give egg-bearing mature adults, which were as untreated controls. Treatment with 167 mM D-All, D-Tal, and L-Ido gave relative body sizes of 50.3, 35.9, and 34.8%, respectively compared with untreated controls (Fig. 3). As the inhibitory effects of D-All, D-Tal, and L-Ido were also observed in animals cultured under axenic conditions, this excluded the possibility that the inhibitory effects were caused by metabolites that E. coli produced from these sugars. Next, we focused on a deoxy derivative of D-All to examine the mechanisms responsible for aldohexose-induced growth inhibition. D-Glc taken up by cells is first phosphorylated by hexokinase (HXK) to form D-Glc-6phosphate via glycolysis. D-All is also phosphorylated by HXK, but the resulting D-All-6-phosphate (D-All-6P) cannot be fully metabolized in rice.12) D-All-6-P accumulates in the cell and interferes with carbohydrate metabolism by inhibiting glycolytic enzymes.12) To examine whether nematode growth inhibition by D-All

Fig. 3. Growth inhibition of C. elegans by D-All, D-Tal and L-Ido under axenic conditions. Notes: Body sizes (mean area of worm’s projections) of C. elegans exposed to each sugar at a concentration of 167 mM are shown (n = 20 each). Error bars represent SD. Statistical analysis of data was performed by one-way ANOVA, followed by a Tukey–Kramer multiple comparison test. Statistical difference (p < 0.01) is indicated by different letters. Abbreviations are as in Fig. 1.

is dependent on C6-phosphorylation by HXK, 6-deoxyD-All was tested for growth inhibitory effects against C. elegans. 6-Deoxy-D-All is a structural derivative of D-All, which does not undergo HXK phosphorylation because it lacks a hydroxyl group at C6. 6-Deoxy-DAll showed no significant effects on C. elegans growth, even at a concentration of 167 mM (mean body size, 5.69 × 104 μm2; 97.1% to the untreated controls) under monoxenic conditions. This suggests that C6phosphorylation by HXK is essential for nematode growth inhibition by D-All. D-All and L-Ido are the C3 and C5 epimers of D-Glc, respectively, and D-Tal is the C2 epimer of D-Gal or the C4 epimer of D-Man, which indicates that these bioactive rare sugars may act as antimetabolites in carbohydrate metabolism due to their structural similarity to natural substrates such as D-Glc, D-Gal, and D-Man. Recently, antiproliferative effects of D-All4,5) on cancer cells have been reported. Yamaguchi et al. reported that D-All strongly induced thioredoxin-interacting protein (TXNIP) in hepatocellular carcinoma cells.5) TXNIP is a potent negative regulator of D-Glc uptake, and could be involved in inhibition of cell proliferation.13,14) In cancer cells, D-All probably acts as an antimetabolite in a wider sense, which includes not only inhibitors of metabolic pathways, but also signal transduction modulators in energy metabolism. The inhibition by D-All against C. elegans might be mediated via the TXNIP signaling. D-All has also been reported to have neuroprotective effects against cerebral ischemia/reperfusion injury. The protective effects were caused by the antioxidative6) and anti-inflammatory7) properties of D-All. In conclusion, this exploratory study provides new information on the biological activities of aldohexose stereoisomers. Among the stereoisomers, the rare sugars D-All, D-Tal, and L-Ido showed considerable inhibition of C. elegans growth under both monoxenic and axenic culture conditions. 6-Deoxy-D-All had no effect on growth, which suggests that HXK is essential for inhibition by D-All. D-All has already been reported to have various biological activities such as anticancer

Growth inhibitory effects of aldohexose isomers

and neuroprotective effects; based on our results, D-Tal and L-Ido are also expected to have valuable biological activities. However, the structure–activity relationship between the aldohexose stereoisomers and inhibition of C. elegans growth is unclear at this time. Further studies are in progress to identify the mechanisms responsible for growth inhibition induced by these bioactive rare sugars, which may lead to the development of a new class of antimetabolite drugs.

Disclosure statement No potential conflict of interest was reported by the authors.

Funding

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This work was supported by the JSPS KAKENHI [grant number 22580122].

Supplemental material The supplemental material for this paper is available at http:// dx.doi.org/10.1080/09168451.2016.1146069

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[3] Ochiai M, Nakanishi Y, Yamada T, et al. Inhibition by dietary D-psicose of body fat accumulation in adult rats fed a high-sucrose diet. Biosci. Biotechnol. Biochem. 2013;77:1123–1126. [4] Sui L, Dong Y, Watanabe Y, et al. Growth inhibitory effect of D-allose on human ovarian carcinoma cells in vitro. Anticancer Res. 2005;25:2639–2644. [5] Yamaguchi F, Takata M, Kamitori K, et al. Rare sugar D-allose induces specific up-regulation of TXNIP and subsequent G1 cell cycle arrest in hepatocellular carcinoma cells by stabilization of p27kip1. Int. J. Oncol. 2008;32:377–385. [6] Nakamura T, Tanaka S, Hirooka K, et al. Anti-oxidative effects of D-allose, a rare sugar, on ischemia-reperfusion damage following focal cerebral ischemia in rat. Neurosci. Lett. 2011;487:103– 106. [7] Gao D, Kawai N, Nakamura T, et al. Anti-inflammatory effect of D-allose in cerebral ischemia/reperfusion injury in rats. Neurol. Med. Chir. (Tokyo). 2013;53:365–374. [8] Sato M, Kurose H, Yamasaki T, et al. Potential anthelmintic: Dpsicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans. J. Nat. Med. 2008;62:244– 246. [9] Szewczyk NJ, Kozak E, Conley CA. Chemically defined medium and Caenorhabditis elegans. BMC Biotechnol. 2003;3:19. [10] Lewis JA, Fleming JT. Basic culture methods. Methods Cell Biol. 1995;48:3–29. [11] Litchfield JT Jr, Wilcoxon F. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 1949;96:99– 113. [12] Kano A, Fukumoto T, Ohtani K, et al. The rare sugar D-allose acts as a triggering molecule of rice defence via ROS generation. J. Exp. Bot. 2013;64:4939–4951. [13] Stoltzman CA, Kaadige MR, Peterson CW, et al. MondoA senses non-glucose sugars: regulation of thioredoxin-interacting protein (TXNIP) and the hexose transport curb. J. Biol. Chem. 2011;286:38027–38034. [14] Stoltzman CA, Peterson CW, Breen KT, et al. Glucose sensing by MondoA:Mlx complexes: a role for hexokinases and direct regulation of thioredoxin-interacting protein expression. Proc. Natl. Acad. Sci. USA. 2008;105:6912–6917.