- Email: [email protected]
A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain
Journal of Pharmacological Sciences 140 (2019) 109e112
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain Miaki Uzu a, Miki Nonaka a, Kanako Miyano a, Hiromi Sato b, Nagomi Kurebayashi c, Kazuyoshi Yanagihara d, Takashi Sakurai c, Akihiro Hisaka b, Yasuhito Uezono a, e, f, * a
Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo, 104-0045, Japan Laboratory of Clinical Pharmacology and Pharmacometrics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 260-8675, Japan Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, 113-8421, Japan d Division of Biomarker Discovery, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Chiba, 277- 8577, Japan e Division of Supportive Care Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Tokyo, 104-0045, Japan f Innovation Center for Supportive, Palliative and Psychosocial Care, National Cancer Center Hospital, Tokyo, 104-0045, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2019 Received in revised form 12 March 2019 Accepted 1 April 2019 Available online 4 May 2019
Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cancer cachexia Metabolome Xanthine oxidase
Cancer cachexia, a multifactorial metabolic syndrome characterized by weight loss, anorexia, muscle loss with or without loss of fat mass, and systemic inflammation, is observed in approximately 20% of patients with advanced cancer.1,2 This syndrome results in poor quality of life (QOL), highlighting the need for more effective treatments. However, clinical trials evaluating treatments for cancer cachexia have been disappointing. We previously established a new cancer cachexia model, which met the diagnostic criteria for this syndrome.3,4 We expect that evaluation of treatment strategies using our cachexia model will help us to overcome difficulties in drug development for treatment of cancer cachexia. Among many pre-clinical studies for cancer cachexia, a xanthine oxidase (XO) inhibitor febuxostat improved survival of rats inoculated with Yoshida hepatoma AH-130 cells, but did not improve food intake.5 In this study, we evaluated the therapeutic effect of febuxostat in our model and evaluated a potentially more effective therapeutic strategy targeting XO.
* Corresponding author. Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo, 104-0045, Japan. Fax: þ81 3 3542 0688. E-mail address: [email protected] (Y. Uezono). Peer review under responsibility of Japanese Pharmacological Society.
We first investigated the effect of oral administration of febuxostat on cachexic symptoms. Febuxostat was provided by Teijin Pharma Limited (Tokyo, Japan). Results are shown as the mean ± standard error of the mean (SEM). Statistical differences between groups were evaluated using Student's t-test, Welch's ttest, or one-way analysis of variance followed by the TukeyeKramer test. A value of P < 0.05 was considered statistically significant. Eight-week-old male BALB/c nu/nu mice (Clea-Japan, Tokyo, Japan) were anesthetized and subcutaneously inoculated into both flanks with 85As2 human gastric cancer cells (1.0 106 cells/site). Cachexic symptoms (weight loss, decreased food and water intake, and loss of skeletal muscle and fat) were observed 2 weeks after 85As2 implantation [Fig. 1(A)]. Febuxostat administration was initiated upon observation of cachexic symptoms. Febuxostat was added into drinking water at a concentration of 20 mg/mL (this concentration is almost equivalent to 5e7 mg/kg/day calculated by body weight and daily water intake of the used mice strain), and was administered continuously for 4 weeks. Administration of febuxostat showed a little improvement of daily water intake [Fig. 1(A)]; sustained water intake may be valuable because cancer cachexia causes decreased total body water.4 However, febuxostat
https://doi.org/10.1016/j.jphs.2019.04.005 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
110
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
did not improve other cachexia symptoms [Fig. 1(A) and (B)]. A pterin-based XO activity assay6 showed that febuxostat administration clearly inhibited XO in the liver, the organ in which XO mRNA expression level is the highest [Fig. 1(C)].7 In contrast, XO activity in the brain was unchanged after febuxostat treatment, suggesting that febuxostat effectively inhibited its target protein in peripheral organs but not in the central nervous system (CNS) due to low potential to cross the bloodebrain barrier (BBB). This result motivated us to investigate the possibility of targeting brain XO for treatment of cancer cachexia. Two weeks after 85As2 inoculation, forebrains were collected and quantitative alteration of low molecular weight metabolites was determined using a capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS) system.8 Ninety-six metabolites were detected in forebrains of both control and cachexic mice [Fig. 2(A)]. Levels of 18 metabolites were significantly different in forebrains of cachexic mice compared to those of control. Six metabolites related to purine nucleotide metabolism were among the metabolites altered in our cachexia model: adenosine monophosphate (AMP), guanosine monophosphate (GMP), inosine monophosphate (IMP), inosine, hypoxanthine, and adenosine triphosphate (ATP). Detailed analysis of the purine metabolic pathway revealed that breakdown of purine nucleotides was accompanied by increased uric acid [Fig. 2(B)]. XO converts hypoxanthine to xanthine, and xanthine to uric acid. As expected, XO activity in forebrains of cachexic mice was higher than in those of control [Fig. 3(A)]. Increased XO activity appeared to be a characteristic feature of early stage cancer cachexia, particularly since there was no difference in forebrain XO activity between the 2 groups 6 weeks after 85As2 implantation [Fig. 1(C)]. We then investigated protein expression levels of active glial cell markers in the hypothalamus in the forebrain. Previous studies have reported that both activated astrocytes and microglia suppress food intake by modulating activity of orexigenic or anorexigenic neurons.9,10 Western blot analysis showed that expression level of the astrocytic marker glial fibrillary acidic protein (GFAP) was significantly increased in the hypothalamus of cachexic mice compared to that of control [Fig. 3(B)]. Expression level of the microglial marker ionized calcium-binding adapter molecule (Iba)1 was also increased, but this increase was not significant. Both glial cells and XO contribute to the induction of inflammation.6 Therefore, the increase of their activity indicates the occurrence of inflammation in the CNS at the early stages of cancer cachexia. Elucidation of the mechanism by which glial cells involve the activation of XO will promote the understanding of the cachexic alteration in the CNS. In the current study, we observed purine nucleotide breakdown and activation of XO in brain in our cancer cachexia model. The inflammation in the CNS in cachexia has been known, however, we first found a specific enzyme XO which might be a candidate for therapeutic target in the CNS.11 As previously discussed, oral administration of febuxostat partially improved cachexic symptoms in the AH-130 cancer cachexia model.5 This result differed
Fig. 1. Oral administration of febuxostat partly improved daily water intake, while not other cachexic symptoms in 85As2-innoculated cachexia model. (A) Mice were
subcutaneously inoculated with 85As2 cells at week 0. Body weight, food intake, and water intake were measured once per week. (B) Six weeks after 85As2 implantation, mice were sacrificed and the gastrocnemius muscle and epididymal fat were weighed. (C) Six weeks after 85As2 implantation, mice were sacrificed and the liver and brain were homogenized in 0.05 M potassium phosphate buffer (pH ¼ 7.4), followed by incubation with 70 mM pterin solution at 37 C for 10 min. Then, fluorescent intensity of isoxanthopterin produced from pterin by xanthine oxidase (XO) was measured at 390 nm for up to 60 min. XO enzymatic activity was evaluated by the area under curve (AUC) of the fluorescent intensity normalized to homogenate protein concentration. Error bars indicate SEM (N ¼ 3e6). **P < 0.01, ***P < 0.001 corresponds to the statistical difference with respect to Control, and ###P < 0.001 corresponds to the statistical difference with respect to Cachexia.
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
111
Fig. 2. Purine nucleotide metabolism was altered in forebrains of mice 2 weeks after 85As2 implantation. (A) Detected 96 kinds of metabolites in forebrain were mapped on a volcano plot by log2 (mean value of cachexic mice/control) versus elog10 (P value of t-test of cachexic mice versus control). - represents metabolites with statistically significant fold changes (P < 0.05) and detailed information for each metabolite is listed. Metabolites related to purine metabolism are underlined. (B) The mean fold changes of 11 purine nucleotide metabolites are shown. Error bars indicate SEM (N ¼ 6e8). *P < 0.05, **P < 0.01 corresponds to the statistical difference with respect to Control. ADP: adenosine diphosphate, AMP: adenosine monophosphate, ATP: adenosine triphosphate, CMP: cytidine monophosphate, GDP: guanine diphosphate, GMP: guanine monophosphate, GTP: guanine triphosphate, IMP: inosine monophosphate, UMP: uridine monophosphate.
Fig. 3. Xanthine oxidase (XO) activity and protein expression levels of glial cells were enhanced in forebrains of mice 2 weeks after 85As2 implantation. (A) Two weeks after 85As2 implantation, mice were sacrificed and forebrains were homogenized in 0.05 M potassium phosphate buffer (pH ¼ 7.4), followed by incubation with 70 mM pterin solution at 37 C for 10 min. Fluorescent intensity of isoxanthopterin produced from pterin by XO was measured at 390 nm for up to 60 min. XO enzymatic activity was evaluated by the area under curve (AUC) of the obtained fluorescent intensity normalized to the homogenate's protein concentration. Error bars indicate SEM (N ¼ 4e6). (B) Two weeks after 85As2 implantation, mice were sacrificed and the hypothalamus was homogenized in CelLytic™ MT Cell Lysis Reagent (Sigma, St. Louis, MO, USA), followed by detection of glial fibrillary acid protein (GFAP) and ionized calcium-binding adapter molecule (Iba)-1 by western blotting. Each bar represents the mean intensity of the protein bands normalized to the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and is presented as a ratio relative to Control. Error bars indicate SEM (N ¼ 3e7). *P < 0.05 corresponds to the statistical difference with respect to Control.
from our study likely due to the beginning point of febuxostat administration and characteristics of the experimental models. Febuxostat administration was started simultaneously with cancer cell implantation in the AH-130 model, while it was started 2 weeks after cancer cell implantation in ours. There is a possibility that inhibition of peripheral XO could prevent the onset of cachexia, but not alleviate the already occurred cachexic symptoms. Moreover, 80% of rats inoculated with AH-130 cells died within 17 days after cell inoculation, while mice in our 85As2 model rarely died within 10 weeks after cell inoculation. The AH-130 model is an acute model; therefore, it is expected that febuxostat might prevent lethal damage to peripheral organs. While results using this model are valuable, long-term evaluation of cancer cachexia treatment is essential. The number of cancer survivors is increasing worldwide. Recently, the National Cancer Institute reported that in 2011 approximately 12 million people, roughly 4% of the U.S. population, were cancer survivors, an increase from approximately 4 million people, or 1.8% of the U.S. population, in the 1970s.12 Cancer survivors often suffer from multiple symptoms including pain, fatigue, and cachexia. Thus, efforts to improve QOL of cancer survivors should be increased and our 85As2 cancer cachexia model is suitable for this purpose. The data obtained from the present study are expected to provide new insights into treatment of cancer cachexia. Therapeutic effects of inhibiting XO in the CNS on cancer cachexia should be evaluated in the future.
Funding This study was supported by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grants Numbers JP201604170 and JP18K15037 to Uzu, JP16K21645 to Nonaka, and JP16K08568 to Miyano.
112
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
Conflict of interest disclosure None of the authors have a conflict of interest to declare. Acknowledgements We appreciate Prof. Chisato Mori (Center for Preventive Medical Science, Chiba University) for use of CE-TOFMS system. References 1. Evans WJ, Morley JE, Argiles J, et al. Cachexia: a new definition. Clin Nutr. 2008;27(6):793e799. 2. Lok C. Cachexia: the last illness. Nature. 2015;528:182e183. England. 3. Yanagihara K, Takigahira M, Mihara K, et al. Inhibitory effects of isoflavones on tumor growth and cachexia in newly established cachectic mouse models carrying human stomach cancers. Nutr Cancer. 2013;65(4):578e589. 4. Terawaki K, Sawada Y, Kashiwase Y, et al. New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line. Am J Physiol Endocrinol Metab. 2014;306(4): E373eE387.
5. Konishi M, Pelgrim L, Tschirner A, et al. Febuxostat improves outcome in a rat model of cancer cachexia. J Cachexia Sarcopenia Muscle. 2015;6(2):174e180. 6. Yisireyili M, Hayashi M, Wu H, et al. Xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice. Sci Rep. 2017;7(1):1266. 7. Wright RM, Vaitaitis GM, Wilson CM, Repine TB, Terada LS, Repine JE. cDNA cloning, characterization, and tissue-specific expression of human xanthine dehydrogenase/xanthine oxidase. Proc Natl Acad Sci USA. 1993;90(22): 10690e10694. 8. Hatakeyama H, Fujiwara T, Sato H, Terui A, Hisaka A. Investigation of metabolomic changes in sunitinib-resistant human renal carcinoma 786-O cells by capillary electrophoresis-time of flight mass spectrometry. Biol Pharm Bull. 2018;41(4):619e627. 9. Yang L, Qi Y, Yang Y. Astrocytes control food intake by inhibiting AGRP neuron activity via adenosine A1 receptors. Cell Rep. 2015;11(5):798e807. 10. Tu TH, Nam-Goong IS, Lee J, Yang S, Kim JG. Visfatin triggers anorexia and body weight loss through regulating the inflammatory response in the hypothalamic microglia. Mediat Inflamm. 2017;2017:1958947. 11. Burfeind KG, Michaelis KA, Marks DL. The central role of hypothalamic inflammation in the acute illness response and cachexia. Semin Cell Dev Biol. 2016;54:42e52. 12. Harrop JP, Dean JA, Paskett ED. Cancer survivorship research: a review of the literature and summary of current NCI-designated cancer center projects. Cancer Epidemiol Biomark Prev. 2011;20(10):2042e2047.
Contents lists available at ScienceDirect
Journal of Pharmacological Sciences journal homepage: www.elsevier.com/locate/jphs
Short Communication
A novel strategy for treatment of cancer cachexia targeting xanthine oxidase in the brain Miaki Uzu a, Miki Nonaka a, Kanako Miyano a, Hiromi Sato b, Nagomi Kurebayashi c, Kazuyoshi Yanagihara d, Takashi Sakurai c, Akihiro Hisaka b, Yasuhito Uezono a, e, f, * a
Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo, 104-0045, Japan Laboratory of Clinical Pharmacology and Pharmacometrics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, 260-8675, Japan Department of Cellular and Molecular Pharmacology, Juntendo University Graduate School of Medicine, Tokyo, 113-8421, Japan d Division of Biomarker Discovery, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Chiba, 277- 8577, Japan e Division of Supportive Care Research, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Tokyo, 104-0045, Japan f Innovation Center for Supportive, Palliative and Psychosocial Care, National Cancer Center Hospital, Tokyo, 104-0045, Japan b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 17 January 2019 Received in revised form 12 March 2019 Accepted 1 April 2019 Available online 4 May 2019
Cancer cachexia is a systemic wasting syndrome characterized by anorexia and loss of body weight. The xanthine oxidase (XO) inhibitor febuxostat is one of the promising candidates for cancer cachexia treatment. However, cachexic symptoms were not alleviated by oral administration of febuxostat in our cancer cachexia model. Metabolomic analysis with brains of our cachexic model showed that purine metabolism was activated and XO activity was increased, and thus suggested that febuxostat would not reach the brain. Accordingly, targeting XO in the brain, which controls appetite, may be an effective strategy for treatment of cancer cachexia. © 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).
Keywords: Cancer cachexia Metabolome Xanthine oxidase
Cancer cachexia, a multifactorial metabolic syndrome characterized by weight loss, anorexia, muscle loss with or without loss of fat mass, and systemic inflammation, is observed in approximately 20% of patients with advanced cancer.1,2 This syndrome results in poor quality of life (QOL), highlighting the need for more effective treatments. However, clinical trials evaluating treatments for cancer cachexia have been disappointing. We previously established a new cancer cachexia model, which met the diagnostic criteria for this syndrome.3,4 We expect that evaluation of treatment strategies using our cachexia model will help us to overcome difficulties in drug development for treatment of cancer cachexia. Among many pre-clinical studies for cancer cachexia, a xanthine oxidase (XO) inhibitor febuxostat improved survival of rats inoculated with Yoshida hepatoma AH-130 cells, but did not improve food intake.5 In this study, we evaluated the therapeutic effect of febuxostat in our model and evaluated a potentially more effective therapeutic strategy targeting XO.
* Corresponding author. Division of Cancer Pathophysiology, National Cancer Center Research Institute, Tokyo, 104-0045, Japan. Fax: þ81 3 3542 0688. E-mail address: [email protected] (Y. Uezono). Peer review under responsibility of Japanese Pharmacological Society.
We first investigated the effect of oral administration of febuxostat on cachexic symptoms. Febuxostat was provided by Teijin Pharma Limited (Tokyo, Japan). Results are shown as the mean ± standard error of the mean (SEM). Statistical differences between groups were evaluated using Student's t-test, Welch's ttest, or one-way analysis of variance followed by the TukeyeKramer test. A value of P < 0.05 was considered statistically significant. Eight-week-old male BALB/c nu/nu mice (Clea-Japan, Tokyo, Japan) were anesthetized and subcutaneously inoculated into both flanks with 85As2 human gastric cancer cells (1.0 106 cells/site). Cachexic symptoms (weight loss, decreased food and water intake, and loss of skeletal muscle and fat) were observed 2 weeks after 85As2 implantation [Fig. 1(A)]. Febuxostat administration was initiated upon observation of cachexic symptoms. Febuxostat was added into drinking water at a concentration of 20 mg/mL (this concentration is almost equivalent to 5e7 mg/kg/day calculated by body weight and daily water intake of the used mice strain), and was administered continuously for 4 weeks. Administration of febuxostat showed a little improvement of daily water intake [Fig. 1(A)]; sustained water intake may be valuable because cancer cachexia causes decreased total body water.4 However, febuxostat
https://doi.org/10.1016/j.jphs.2019.04.005 1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
110
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
did not improve other cachexia symptoms [Fig. 1(A) and (B)]. A pterin-based XO activity assay6 showed that febuxostat administration clearly inhibited XO in the liver, the organ in which XO mRNA expression level is the highest [Fig. 1(C)].7 In contrast, XO activity in the brain was unchanged after febuxostat treatment, suggesting that febuxostat effectively inhibited its target protein in peripheral organs but not in the central nervous system (CNS) due to low potential to cross the bloodebrain barrier (BBB). This result motivated us to investigate the possibility of targeting brain XO for treatment of cancer cachexia. Two weeks after 85As2 inoculation, forebrains were collected and quantitative alteration of low molecular weight metabolites was determined using a capillary electrophoresis-time of flight mass spectrometry (CE-TOFMS) system.8 Ninety-six metabolites were detected in forebrains of both control and cachexic mice [Fig. 2(A)]. Levels of 18 metabolites were significantly different in forebrains of cachexic mice compared to those of control. Six metabolites related to purine nucleotide metabolism were among the metabolites altered in our cachexia model: adenosine monophosphate (AMP), guanosine monophosphate (GMP), inosine monophosphate (IMP), inosine, hypoxanthine, and adenosine triphosphate (ATP). Detailed analysis of the purine metabolic pathway revealed that breakdown of purine nucleotides was accompanied by increased uric acid [Fig. 2(B)]. XO converts hypoxanthine to xanthine, and xanthine to uric acid. As expected, XO activity in forebrains of cachexic mice was higher than in those of control [Fig. 3(A)]. Increased XO activity appeared to be a characteristic feature of early stage cancer cachexia, particularly since there was no difference in forebrain XO activity between the 2 groups 6 weeks after 85As2 implantation [Fig. 1(C)]. We then investigated protein expression levels of active glial cell markers in the hypothalamus in the forebrain. Previous studies have reported that both activated astrocytes and microglia suppress food intake by modulating activity of orexigenic or anorexigenic neurons.9,10 Western blot analysis showed that expression level of the astrocytic marker glial fibrillary acidic protein (GFAP) was significantly increased in the hypothalamus of cachexic mice compared to that of control [Fig. 3(B)]. Expression level of the microglial marker ionized calcium-binding adapter molecule (Iba)1 was also increased, but this increase was not significant. Both glial cells and XO contribute to the induction of inflammation.6 Therefore, the increase of their activity indicates the occurrence of inflammation in the CNS at the early stages of cancer cachexia. Elucidation of the mechanism by which glial cells involve the activation of XO will promote the understanding of the cachexic alteration in the CNS. In the current study, we observed purine nucleotide breakdown and activation of XO in brain in our cancer cachexia model. The inflammation in the CNS in cachexia has been known, however, we first found a specific enzyme XO which might be a candidate for therapeutic target in the CNS.11 As previously discussed, oral administration of febuxostat partially improved cachexic symptoms in the AH-130 cancer cachexia model.5 This result differed
Fig. 1. Oral administration of febuxostat partly improved daily water intake, while not other cachexic symptoms in 85As2-innoculated cachexia model. (A) Mice were
subcutaneously inoculated with 85As2 cells at week 0. Body weight, food intake, and water intake were measured once per week. (B) Six weeks after 85As2 implantation, mice were sacrificed and the gastrocnemius muscle and epididymal fat were weighed. (C) Six weeks after 85As2 implantation, mice were sacrificed and the liver and brain were homogenized in 0.05 M potassium phosphate buffer (pH ¼ 7.4), followed by incubation with 70 mM pterin solution at 37 C for 10 min. Then, fluorescent intensity of isoxanthopterin produced from pterin by xanthine oxidase (XO) was measured at 390 nm for up to 60 min. XO enzymatic activity was evaluated by the area under curve (AUC) of the fluorescent intensity normalized to homogenate protein concentration. Error bars indicate SEM (N ¼ 3e6). **P < 0.01, ***P < 0.001 corresponds to the statistical difference with respect to Control, and ###P < 0.001 corresponds to the statistical difference with respect to Cachexia.
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
111
Fig. 2. Purine nucleotide metabolism was altered in forebrains of mice 2 weeks after 85As2 implantation. (A) Detected 96 kinds of metabolites in forebrain were mapped on a volcano plot by log2 (mean value of cachexic mice/control) versus elog10 (P value of t-test of cachexic mice versus control). - represents metabolites with statistically significant fold changes (P < 0.05) and detailed information for each metabolite is listed. Metabolites related to purine metabolism are underlined. (B) The mean fold changes of 11 purine nucleotide metabolites are shown. Error bars indicate SEM (N ¼ 6e8). *P < 0.05, **P < 0.01 corresponds to the statistical difference with respect to Control. ADP: adenosine diphosphate, AMP: adenosine monophosphate, ATP: adenosine triphosphate, CMP: cytidine monophosphate, GDP: guanine diphosphate, GMP: guanine monophosphate, GTP: guanine triphosphate, IMP: inosine monophosphate, UMP: uridine monophosphate.
Fig. 3. Xanthine oxidase (XO) activity and protein expression levels of glial cells were enhanced in forebrains of mice 2 weeks after 85As2 implantation. (A) Two weeks after 85As2 implantation, mice were sacrificed and forebrains were homogenized in 0.05 M potassium phosphate buffer (pH ¼ 7.4), followed by incubation with 70 mM pterin solution at 37 C for 10 min. Fluorescent intensity of isoxanthopterin produced from pterin by XO was measured at 390 nm for up to 60 min. XO enzymatic activity was evaluated by the area under curve (AUC) of the obtained fluorescent intensity normalized to the homogenate's protein concentration. Error bars indicate SEM (N ¼ 4e6). (B) Two weeks after 85As2 implantation, mice were sacrificed and the hypothalamus was homogenized in CelLytic™ MT Cell Lysis Reagent (Sigma, St. Louis, MO, USA), followed by detection of glial fibrillary acid protein (GFAP) and ionized calcium-binding adapter molecule (Iba)-1 by western blotting. Each bar represents the mean intensity of the protein bands normalized to the internal standard glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and is presented as a ratio relative to Control. Error bars indicate SEM (N ¼ 3e7). *P < 0.05 corresponds to the statistical difference with respect to Control.
from our study likely due to the beginning point of febuxostat administration and characteristics of the experimental models. Febuxostat administration was started simultaneously with cancer cell implantation in the AH-130 model, while it was started 2 weeks after cancer cell implantation in ours. There is a possibility that inhibition of peripheral XO could prevent the onset of cachexia, but not alleviate the already occurred cachexic symptoms. Moreover, 80% of rats inoculated with AH-130 cells died within 17 days after cell inoculation, while mice in our 85As2 model rarely died within 10 weeks after cell inoculation. The AH-130 model is an acute model; therefore, it is expected that febuxostat might prevent lethal damage to peripheral organs. While results using this model are valuable, long-term evaluation of cancer cachexia treatment is essential. The number of cancer survivors is increasing worldwide. Recently, the National Cancer Institute reported that in 2011 approximately 12 million people, roughly 4% of the U.S. population, were cancer survivors, an increase from approximately 4 million people, or 1.8% of the U.S. population, in the 1970s.12 Cancer survivors often suffer from multiple symptoms including pain, fatigue, and cachexia. Thus, efforts to improve QOL of cancer survivors should be increased and our 85As2 cancer cachexia model is suitable for this purpose. The data obtained from the present study are expected to provide new insights into treatment of cancer cachexia. Therapeutic effects of inhibiting XO in the CNS on cancer cachexia should be evaluated in the future.
Funding This study was supported by the Japan Society for the Promotion of Sciences (JSPS) KAKENHI Grants Numbers JP201604170 and JP18K15037 to Uzu, JP16K21645 to Nonaka, and JP16K08568 to Miyano.
112
M. Uzu et al. / Journal of Pharmacological Sciences 140 (2019) 109e112
Conflict of interest disclosure None of the authors have a conflict of interest to declare. Acknowledgements We appreciate Prof. Chisato Mori (Center for Preventive Medical Science, Chiba University) for use of CE-TOFMS system. References 1. Evans WJ, Morley JE, Argiles J, et al. Cachexia: a new definition. Clin Nutr. 2008;27(6):793e799. 2. Lok C. Cachexia: the last illness. Nature. 2015;528:182e183. England. 3. Yanagihara K, Takigahira M, Mihara K, et al. Inhibitory effects of isoflavones on tumor growth and cachexia in newly established cachectic mouse models carrying human stomach cancers. Nutr Cancer. 2013;65(4):578e589. 4. Terawaki K, Sawada Y, Kashiwase Y, et al. New cancer cachexia rat model generated by implantation of a peritoneal dissemination-derived human stomach cancer cell line. Am J Physiol Endocrinol Metab. 2014;306(4): E373eE387.
5. Konishi M, Pelgrim L, Tschirner A, et al. Febuxostat improves outcome in a rat model of cancer cachexia. J Cachexia Sarcopenia Muscle. 2015;6(2):174e180. 6. Yisireyili M, Hayashi M, Wu H, et al. Xanthine oxidase inhibition by febuxostat attenuates stress-induced hyperuricemia, glucose dysmetabolism, and prothrombotic state in mice. Sci Rep. 2017;7(1):1266. 7. Wright RM, Vaitaitis GM, Wilson CM, Repine TB, Terada LS, Repine JE. cDNA cloning, characterization, and tissue-specific expression of human xanthine dehydrogenase/xanthine oxidase. Proc Natl Acad Sci USA. 1993;90(22): 10690e10694. 8. Hatakeyama H, Fujiwara T, Sato H, Terui A, Hisaka A. Investigation of metabolomic changes in sunitinib-resistant human renal carcinoma 786-O cells by capillary electrophoresis-time of flight mass spectrometry. Biol Pharm Bull. 2018;41(4):619e627. 9. Yang L, Qi Y, Yang Y. Astrocytes control food intake by inhibiting AGRP neuron activity via adenosine A1 receptors. Cell Rep. 2015;11(5):798e807. 10. Tu TH, Nam-Goong IS, Lee J, Yang S, Kim JG. Visfatin triggers anorexia and body weight loss through regulating the inflammatory response in the hypothalamic microglia. Mediat Inflamm. 2017;2017:1958947. 11. Burfeind KG, Michaelis KA, Marks DL. The central role of hypothalamic inflammation in the acute illness response and cachexia. Semin Cell Dev Biol. 2016;54:42e52. 12. Harrop JP, Dean JA, Paskett ED. Cancer survivorship research: a review of the literature and summary of current NCI-designated cancer center projects. Cancer Epidemiol Biomark Prev. 2011;20(10):2042e2047.