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Erythropoietin improves cardiac wasting and outcomes in a rat model of liver cancer cachexia
International Journal of Cardiology 218 (2016) 312–317
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International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Erythropoietin improves cardiac wasting and outcomes in a rat model of liver cancer cachexia Masakazu Saitoh a,⁎, Michiyoshi Hatanaka a,b, Masaaki Konishi a, Junichi Ishida a, Sandra Palus a, Nicole Ebner a, Wolfram Döhner c, Stephan von Haehling a, Stefan D. Anker a, Jochen Springer a a b c
Innovative Clinical Trials, Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany Medicinal Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan Center for Stroke Research Berlin, Charite´ Medical School, Berlin, Germany
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Article history: Received 8 April 2016 Accepted 12 May 2016 Available online 14 May 2016 Keywords: Cancer Cancer cachexia Yoshida hepatoma animal model Cardiac wasting Survival Erythropoietin
a b s t r a c t Background: Erythropoietin administration, which is clinically used in cancer patients with cancer-induced anemia, has also potentially beneficial effects on nonhematopoietic organs. We assessed the effects of erythropoietin on cancer cachexia progression and cardiac wasting compared with placebo using the Yoshida hepatoma model. Methods: Wistar rats were divided in a sham group (n = 10) and a tumor-bearing group (n = 60). The tumorbearing group was further randomized to placebo (n = 28), 500 Unit/kg/day (n = 16) or 5000 Unit/kg/day of erythropoietin (n = 16). Body composition was measured using nuclear magnetic resonance spectroscopy, cardiac function using echocardiography, physical activity using infrared monitoring system. Results: Tumor-bearing rats with high dose erythropoietin led to a significant improvement on survival compared with placebo (hazard ratio: 0.43, 95%CI: 0.20–0.92, p = 0.030), though low dose erythropoietin did not reach significance (hazard ratio: 0.46, 95%CI: 0.22–1.02, p = 0.056). Loss of body weight, wasting of lean mass, fat mass, and reduced physical activity were ameliorated in rats treated with both low and high doses of erythropoietin (p b 0.05, all). Moreover, reduced left ventricular mass and left ventricular systolic function were also ameliorated in rats treated with low and high doses of erythropoietin (p b 0.05, respectively). Conclusions: Overall, the present data support that cardiac wasting induced by cancer cachexia plays an important role which leads to impaired survival, provided that the erythropoietin could be an effective therapeutic approach for cancer cachexia progression and cardiac wasting. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer cachexia is a multifactorial syndrome of involuntary weight loss, fat or muscle loss and poor quality of life, and correlates with high mortality rate [1]. Moreover, it has been revealed that cachexia related clinical manifestation also includes wasting or dysfunction of tissue/organ such as brain, liver, gut and heart [2]. It has been reported that cardiac muscle wasting is associated with cancer cachexia leading to impaired cardiac function in animal models [3] and humans [4]. Previously our group showed that cardiac wasting was improved by treatments with either the β-blocker bisoprolol or the aldosterone antagonist spironolactone, resulting in improved quality of life and survival in tumor-bearing rats [5]. Cancer related clinical manifestations also include anemia, which contribute to fatigue, impaired quality of life, and poor survival [6–8]. ⁎ Corresponding author at: Innovative Clinical Trials, Department of Cardiology and Pneumology, University Medical Centre Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. E-mail address: [email protected] (M. Saitoh).
http://dx.doi.org/10.1016/j.ijcard.2016.05.008 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Erythropoietin administration, which is clinically used in patients with cancer-induced anemia [9,10], has also potentially beneficial effects on nonhematopoietic organs. Although specific receptor of erythropoietin is present in skeletal muscle, adipose tissue, and heart in murine models [11–13], beneficial effects of erythropoietin on cardiac wasting are not well known in cancer cachexia model. The Yoshida hepatoma model is well-established cancer cachexia model, is known to have cardiac wasting and high mortality rate [5, 14]. In this study, we investigate the effects of erythropoietin on cancer cachexia progression, cardiac wasting, and physical performance status using the Yoshida hepatoma model. 2. Materials and methods 2.1. Animals and cachexia model Male Wistar-Han rats were housed in a specific-pathogen-free facility under a 12 h light/dark cycle with food and water provided ad libitum. On the first day of experiment (D0), rats were injected intraperitoneally with 108 growing Yoshida AH-130 tumor cells, as described
M. Saitoh et al. / International Journal of Cardiology 218 (2016) 312–317
previously [15]. After 5 days, rats developed large tumors, and after 10 days, rats were severely cachectic. 2.2. Experimental design Rats were divided into two groups: sham group (n = 10) and tumor-bearing rats (n = 60). The tumor-bearing rats were further randomized to placebo (n = 28), 500 Units/day of erythropoietin (500 U EPO, n = 16) or 5000 Units/day erythropoietin (5000 U EPO, n = 16). 500 Units/kg/day or 5000 Units/kg/day were administered s.c. daily. Erythropoietin administration began on the next day of tumor inoculation. The placebo group received saline injections. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals. Study protocol and study procedures were approved by the local animal ethics committee. 2.3. Body composition On D0 and day 16 or day of animals' euthanasia (D16), nuclear magnetic resonance spectroscopy (EchoMRI-700TM, Echo Medical Systems, Houston, TX) was performed to assess body composition of each rat [16]. Body composition was recorded after removal of the tumor, or the respective days of killing, if rats had to be euthanized earlier due to reaching ethical endpoints. Weight of heart was recorded, and tumor cells were counted using a Neubauer chamber.
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Echocardiography was performed in M-mode to measure cardiac function and dimensions, and in B-mode to calculate functional parameters. Echocardiography was performed 1 day before starting the treatment and day 11 (D11). 2.5. Proteasome activity Proteasome activity was analyzed, as described previously [5]. Briefly, the gastrocnemius muscle was homogenized in an ice-cold buffer. Protein was incubated with fluorogenic substrates (benzoylVal-Gly-Arg-7-amidocoumarin for trypsin-like activity, succinylLeu-Leu-Val-Try-7-amido-4-methylcoumarin for chymotrypsin-like activity, and benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-methylcoumarin for peptidylglutamyl peptidase activity, Biomol, Hamburg, Germany). The fluorescence intensity was measured with a fluorometer (Twinkle LB 970, Berthold, Bad Wildbad, Germany) at 360 and 460 nm emission. The activity, expressed as nanomole per milligram per minute, was calculated by using free amidomethylcoumarin as a working standard. 2.6. Physical performance status Food intake and spontaneous physical activity were recorded on D11. Spontaneous physical activity was measured by an infrared monitoring system (Supermex, Muromachi, Tokyo, Japan) over a 24 h period as described previously [18].
2.4. Echocardiography
2.7. Statistical analysis
Rats were anesthetized using 1.5% isoflurane and laid in supine position on a heating pad to maintain body temperature at 39 °C. All hair was removed from the left chest. A high-resolution echocardiography system (Vevo 770; Visual Sonics Inc., Toronto, Canada) was used [17].
Data were analyzed with GraphPad PRISM 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Results are shown as mean ± standard error of mean. For the comparisons among groups, data were analyzed with analysis of variance followed by post hoc comparisons using Tukey's
Fig. 1. Total tumor cell numbers × 109 (A): there was no significant difference in tumor cell numbers between rats with placebo and rats with EPO groups. Change in body weight (B), lean mass (C), fat mass (D): average loss of body weight, lean mass, and fat mass was higher in placebo group than both 500 U EPO group and 5000 U EPO group at the end of the study (500 U EPO: p b 0.05 for body weight, p b 0.01 for lean mass, and p = 0.056) and (5000 U EPO: p b 0.05 for body weight, p b 0.01 for lean mass, and p b 0.05 for fat mass). Weight of heart at the end of the study (E): weight of heart was higher in rats with 500 U EPO than in rats with placebo (p b 0.05). Abbreviation: Erythropoietin, EPO. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
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test. Cox proportional hazard analysis was used to examine the effects of erythropoietin on survival. A value of p b 0.05 was considered to indicate statistical significance in all analysis.
1.4 g/day, p b 0.01, respectively; Fig. 1C). Average loss of fat was higher in the placebo group vs. the 5000 U EPO group (− 1.0 ± 0.2 g/day vs. − 0.8 ± 0.3 g/day, p b 0.05; Fig. 1D).
3. Results 3.2. Cardiac wasting The tumor growth was similar in all groups. At the end of the study, total tumor cell numbers were 2.365 ± 0.890 × 109 cell in placebo rats, 2.718 ± 0.995 × 109 cell in the 500 U EPO group and 2.153 ± 0.987 × 109 cell in rats treated with 5000 U EPO (all p N 0.2, Fig. 1A). Moreover, there were no observations of other malignancies during necropsy in each rat. 3.1. Body weight, body composition Average daily loss of body weight was higher in rats with placebo than in rats with 500 U EPO or 5000 U EPO (− 4.6 ± 0.9 g/day vs. − 3.2 ± 1.7 g/day or − 3.6 ± 1.9 g/day, p b 0.05, Fig. 1B). Average loss of lean mass was reduced in rats treated with 500 U EPO or 5000 U EPO (− 3.6 ± 0.8 g/day vs. − 2.4 ± 2.0 g/day or − 2.6 ±
Weight of the heart on the end of study in sham rats was 781 ± 52 mg, significantly higher than in rats with placebo (495 ± 67 mg, p b 0.001 vs. sham, Fig. 1E). The weight of the heart was significantly lower in rats with placebo than in rats with 500 U EPO (555 ± 69 mg, p b 0.05 vs. placebo, Fig. 1E). Echocardiographic parameters are shown in Fig. 2. The left ventricular (LV) mass on D11 was lower in rats with placebo than in rats with 500 U EPO or 5000 U EPO (445 ± 77 g vs. 587 ± 158 g and 507 ± 109 g, p b 0.001 and p b 0.05, respectively, Fig. 2A). LV end systolic diameter (LVESD) was higher in rats with placebo than in rats with 500 U EPO (4.0 ± 0.7 mm vs. 3.5 ± 0.7 mm; p b 0.05, Fig. 2B), even though LV end diastolic diameter (LVEDD) was comparable among 3 groups (Fig. 2C). LV ejection fraction (LVEF), LV fraction shortening (LVFS)
Fig. 2. Echocardiography: left ventricular mass (A) in D11 was more remarkable in rats with placebo than in rats with 500 U EPO (p b 0.001) or 5000 U EPO (p b 0.05). Left ventricular end systolic diameter (B) was significantly higher in rats with placebo than in rats with 500 U EPO (p b 0.05), Left ventricular end diastolic diameter (C) was comparable between rats with placebo and rats treated with EPO. Left ventricular ejection fraction (LVEF) (D), Left ventricular fractional shortening (LVFS) (E) and Left ventricular stroke volume (LVSV) (F) were significantly lower in rats with placebo than in rats with 500 U EPO (p b 0.01 for LVEF and LVFS, p b 0.05 for LVSV) and 5000 U EPO (p b 0.01 for LVEF, p b 0.05 for LVFS and LVSV). Cardiac output (G) was significantly lower in rats with placebo than in rats with 5000 U EPO (p b 0.01). Abbreviations: LV, left ventricular; Left ventricular end systolic diameter, LVESD; Erythropoietin, EPO; Left ventricular end diastolic diameter, LVEDD; Left ventricular ejection fraction, LVEF; Left ventricular fractional shortening, LVFS; Left ventricular stroke volume, LVSV; Cardiac output, CO. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
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Fig. 3. Skeletal muscle weight on D16: Muscle gastrocnemius (A), tibialis (B) and extensor digitalis longus (C) in rats with 500 U EPO were significantly higher than in rats with placebo (p b 0.01, p b 0.01 and p b 0.05). Catabolic signaling in the gastrocnemius muscle on Day 16: trypsin-like proteasome activity in rats with 500 U or 5000 U EPO were significantly lower than in rats with placebo (p b 0.001 and p b 0.01) (D). Chymotrypsin-like (E) and peptidyl-glutamyl protein-hydrolyzing (F) were comparable between rats with EPO (500 U and 5000 U EPO) and placebo. Abbreviations: Erythropoietin, EPO; extensor digitalis longus, EDL; peptidyl-glutamyl protein-hydrolyzing, PGPH. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
and LV stroke volume (LVSV) were also lower in rats with placebo than in treated rats Fig. 2D–G).
3.3. Muscle weight and catabolic signaling The weight of skeletal muscles is shown in Fig. 3A–C. At the end of the study, the weight of gastrocnemius (Fig. 3A), tibialis (Fig. 3B) and extensor digitalis longus (EDL) (Fig. 3C) in rats with 500 U EPO was significantly higher than in rats with placebo (p b 0.01, p b 0.01 and p b 0.05, respectively). Catabolic signaling is also shown in Fig. 3D–F. At the end of the study, trypsin-like proteasome activity was significantly lower in rats with 500 U or 5000 U EPO than in rats with placebo (191.4 ± 46.1 nmol/mg/min vs. 122.8 ± 76.8 nmol/mg/min and 116.1 ± 78.4 nmol/mg/min, p b 0.001 and p b 0.01, Fig. 3D), however chymotrypsin-like activity, peptidyl-glutamyl protein-hydrolyzing activity were similar among all groups.
3.4. Physical performance status On D11, food intake was 20.6 ± 3.1 g/24 h in sham rats vs. placebo rats (4.4 ± 3.4 g/24 h, p b 0.001 vs. sham, Fig. 4A). There were no significant differences among the placebo group and EPO groups. On D11, physical activity was 62,577 ± 9553 counts/day in sham rats and significantly reduced in the placebo rats (27,797 ± 13,269 counts/day, p b 0.05 vs. sham, Fig. 4B). Physical activity in rats with 500 U EPO or 5000 U EPO was higher than in rats with placebo (36,916 ± 15,418 counts/day and 40,343 ± 18524 counts/day, vs. placebo, both p b 0.05, Fig. 4B).
3.5. Survival On D16, the overall survival was 14.0% in rats with placebo, 37.5% in rats with 500 U EPO and 31.3% in rats with 5000 U EPO (Fig. 5). The
Fig. 4. Food intake (A) in D11 was comparable between rats with placebo and rats treated with EPO. Physical activity (B) in D11 was more remarkable in rats with placebo than in rats with 500 U EPO (p b 0.05) or 5000 U EPO (p b 0.05). Abbreviation: Erythropoietin, EPO. *p b 0.05 vs. placebo, and ***p b 0.001 vs. placebo.
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Fig. 5. Kaplan–Meier survival curves: EPO reduced mortality in comparison with placebo. Abbreviation: Erythropoietin, EPO.
median survival was 13 days in rats with placebo, 14 days in rats with 500 U EPO and 15 days in rats with 5000 U EPO. Treatment with 5000 U EPO reduced mortality (hazard ratio: 0.43, 95%CI: 0.20–0.92, p = 0.030), though 500 U EPO showed a trend for reduced mortality (hazard ratio: 0.46, 95%CI: 0.22–1.02, p = 0.056).
4. Discussion In the present study, we showed that erythropoietin treatment in tumor-bearing rats led to improvement in survival, weight loss, wasting of lean and fat mass, and reduction of proteasome activity in the skeletal muscle tissue and physical performance status. In addition, cardiac wasting was ameliorated in erythropoietin treated tumor-bearing rats. Erythropoietin administration reduced proteasome activity indicated by the trypsin-like activity in gastrocnemius muscle and improved muscle wasting. Ubiquitin-proteasome system signaling, which is responsible for the regulation of protein turnover and the elimination of abnormal protein, has been shown to be critical for coordinating muscle atrophy [19,20]. Some studies demonstrated a relationship between muscle wasting and poor physical performance status [21,22]. From our findings, it can be concluded that improved muscle wasting was associated with erythropoietin administration and leads to substantial improvement in physical performance status. Cardiac wasting, which was also observed in cancer cachexia rat and mouse models, has been described to induce cardiomyocyte remodeling and poor outcomes without treatment [2,23]. Previously our group reported that treatment with anti-heart failure drugs, the β-blocker bisoprolol or the aldosterone antagonist spironolactone, improved cardiac function and subsequently survival in Yoshida-hepatoma model [5]. This study suggested that amelioration of cardiac dysfunction in this model could influence on the improvement on the poor survival, though progressive cardiac wasting without erythropoietin treatment. The result suggested that cardiac wasting is related with survival in cancer cachexia. In the present study, erythropoietin also attenuated cardiac wasting to some extent, and improving cardiac dysfunction in cancer cachexia model. From the results presented here, it can be suggested that erythropoietin administration positively alter survival time, possibly by an improvement in cardiac wasting in tumor-bearing cachexia animals. In our intervention study, erythropoietin improved cardiac wasting in our model. It has been reported that the improvement of cardiac dysfunction in erythropoietin treated heart failure mice model was related to increased angiogenesis as well as near normalization of the myocardial levels of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and transforming growth factor β1 [14,24]. Pro-inflammatory cytokines, TNFs, IL-1 and IL6, are crucially involved in the mechanism of cancer cachexia [14,25,26] and which would be the one of the essential molecules to make better understanding of the mechanisms underlying in the therapeutic effect of erythropoietin in the cardiac and muscle dysfunction.
Erythropoietin administration is reported to improve the cancerinduced anemia, resulting in significantly improved physical performance status, quality of life and survival [27,28]. In addition to the amelioration of anemia, recent studies have shown that erythropoietin has the multiple tissue-protective efficacy such as an inhibition of apoptosis, attenuation of inflammatory responses in brain, kidney and heart [29, 30]. A tissue-protective receptor, complex consisting of the erythropoietin receptor and the beta common receptor (βcR) subunit, also known as CD131, which has much lower affinity of erythropoietin. βcR is confirmed exhibiting within the cardiomyocytes, and those of βcRknockout mice did not respond to erythropoietin, result in no improvement of apoptosis and survival [31]. These experiments are supportive of a role for βcR in the tissue-protective effects of erythropoietin in cardiomyocites. Dose response analyses showed that single dose of 150 to 500 Units/ kg is described the minimum effective dose of erythropoietin to achieve a tissue-protective effect in brain and heart disease model [32,33]. We also investigated the beneficial effects of both dose of erythropoietin (500 Units/kg/day and 5000 Units/kg/day/day) on weight loss, wasting, cardiac wasting. In our dose–response analysis showed that 500 Units/ kg of erythropoietin in this model provided the same efficacy as 5000 Units/kg/day administrated daily in rats with cancer cachexia. In terms from the dose setting, a tissue-protective effect could be one of the mechanisms for the therapeutic effects of erythropoietin in our study. In conclusion, the results of our Yoshida hepatoma rat model strongly support that cardiac wasting induced by cancer cachexia plays an important role which leads to impaired survival. Our findings suggested that the erythropoietin could be a novel therapeutic approach for cancer cachexia progression and cardiac wasting. Conflicts of interest SvH is consulting and has received honoraria from Vifor, Novartis, Thermo Fisher, Chugai, Roche, Sorin, Respicardia, and pfizer. WD is consulting and has received honoraria from Vifor. For the remaining authors none were declared. References [1] J.A. Norton, J.L. Peacock, S.D. Morrison, Cancer cachexia, Crit. Rev. Oncol. Hematol. 7 (1987) 289–327. [2] J.M. Argilés, B. Stemmler, F.J. López-Soriano, S. Busquets, Nonmuscle tissues contribution to cancer cachexia, Mediat. Inflamm. 2015 (2015) 182872. [3] S.M. Kazemi-Bajestani, H. Becher, K. Fassbender, Q. Chu, V.E. Baracos, Concurrent evolution of cancer cachexia and heart failure: bilateral effects exist, J. Cachex. Sarcopenia Muscle 5 (2014) 95–104. [4] L. Cramer, B. Hildebrandt, T. Kung, et al., Cardiovascular function and predictors of exercise capacity in patients with colorectal cancer, J. Am. Coll. Cardiol. 64 (2014) 1310–1319. [5] J. Springer, A. Tschirner, A. Haghikia, et al., Prevention of liver cancer cachexiainduced cardiac wasting and heart failure, Eur. Heart J. 35 (2014) 932–941. [6] S.E. Libretto, P.J. Barrett-Lee, K. Branson, et al., Improvement in quality of life for cancer patients treated with epoetin alfa, Eur. J. Cancer Care 10 (2001) 183–191. [7] I. Quirt, C. Robeson, C.Y. Lau, et al., Epoetin alfa therapy increases hemoglobin levels and improves quality of life in patients with cancer-related anemia who are not receiving chemotherapy and patients with anemia who are receiving chemo- therapy, J. Clin. Oncol. 19 (2001) 4126–4134. [8] H. Ludwig, S. Van Belle, P. Barrett-Lee, et al., The European Cancer Anaemia Survey (ECAS): a large, multinational, prospective survey defining the prevalence, incidence, and treatment of anaemia in cancer patients, Eur. J. Cancer 40 (2004) 2293–2306. [9] L. Repetto, K. Moeremans, L. Annemans, European Organisation for Research and Treatment of Cancer. European guidelines for the management of chemotherapyinduced anaemia and health economic aspects of treatment, Cancer Treat. Rev. 32 (Suppl. 2) (2006) S5–S9. [10] M. Aapro, J.L. Spivak, Update on erythropoiesis-stimulating agents and clinical trials in oncology, Oncologist 14 (Suppl. 1) (2009) 6–15. [11] F. Penna, S. Busquets, M. Toledo, et al., Erythropoietin administration partially prevents adipose tissue loss in experimental cancer cachexia models, J. Lipid Res. 54 (2013) 3045–3051. [12] L. Mille-Hamard, V.L. Billat, E. Henry, et al., Skeletal muscle alterations and exercise performance decrease in erythropoietin-deficient mice: a comparative study, BMC Med. Genet. 5 (2012) 29.
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Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
Erythropoietin improves cardiac wasting and outcomes in a rat model of liver cancer cachexia Masakazu Saitoh a,⁎, Michiyoshi Hatanaka a,b, Masaaki Konishi a, Junichi Ishida a, Sandra Palus a, Nicole Ebner a, Wolfram Döhner c, Stephan von Haehling a, Stefan D. Anker a, Jochen Springer a a b c
Innovative Clinical Trials, Department of Cardiology and Pneumology, University Medical Center Göttingen, Göttingen, Germany Medicinal Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan Center for Stroke Research Berlin, Charite´ Medical School, Berlin, Germany
a r t i c l e
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Article history: Received 8 April 2016 Accepted 12 May 2016 Available online 14 May 2016 Keywords: Cancer Cancer cachexia Yoshida hepatoma animal model Cardiac wasting Survival Erythropoietin
a b s t r a c t Background: Erythropoietin administration, which is clinically used in cancer patients with cancer-induced anemia, has also potentially beneficial effects on nonhematopoietic organs. We assessed the effects of erythropoietin on cancer cachexia progression and cardiac wasting compared with placebo using the Yoshida hepatoma model. Methods: Wistar rats were divided in a sham group (n = 10) and a tumor-bearing group (n = 60). The tumorbearing group was further randomized to placebo (n = 28), 500 Unit/kg/day (n = 16) or 5000 Unit/kg/day of erythropoietin (n = 16). Body composition was measured using nuclear magnetic resonance spectroscopy, cardiac function using echocardiography, physical activity using infrared monitoring system. Results: Tumor-bearing rats with high dose erythropoietin led to a significant improvement on survival compared with placebo (hazard ratio: 0.43, 95%CI: 0.20–0.92, p = 0.030), though low dose erythropoietin did not reach significance (hazard ratio: 0.46, 95%CI: 0.22–1.02, p = 0.056). Loss of body weight, wasting of lean mass, fat mass, and reduced physical activity were ameliorated in rats treated with both low and high doses of erythropoietin (p b 0.05, all). Moreover, reduced left ventricular mass and left ventricular systolic function were also ameliorated in rats treated with low and high doses of erythropoietin (p b 0.05, respectively). Conclusions: Overall, the present data support that cardiac wasting induced by cancer cachexia plays an important role which leads to impaired survival, provided that the erythropoietin could be an effective therapeutic approach for cancer cachexia progression and cardiac wasting. © 2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Cancer cachexia is a multifactorial syndrome of involuntary weight loss, fat or muscle loss and poor quality of life, and correlates with high mortality rate [1]. Moreover, it has been revealed that cachexia related clinical manifestation also includes wasting or dysfunction of tissue/organ such as brain, liver, gut and heart [2]. It has been reported that cardiac muscle wasting is associated with cancer cachexia leading to impaired cardiac function in animal models [3] and humans [4]. Previously our group showed that cardiac wasting was improved by treatments with either the β-blocker bisoprolol or the aldosterone antagonist spironolactone, resulting in improved quality of life and survival in tumor-bearing rats [5]. Cancer related clinical manifestations also include anemia, which contribute to fatigue, impaired quality of life, and poor survival [6–8]. ⁎ Corresponding author at: Innovative Clinical Trials, Department of Cardiology and Pneumology, University Medical Centre Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany. E-mail address: [email protected] (M. Saitoh).
http://dx.doi.org/10.1016/j.ijcard.2016.05.008 0167-5273/© 2016 Elsevier Ireland Ltd. All rights reserved.
Erythropoietin administration, which is clinically used in patients with cancer-induced anemia [9,10], has also potentially beneficial effects on nonhematopoietic organs. Although specific receptor of erythropoietin is present in skeletal muscle, adipose tissue, and heart in murine models [11–13], beneficial effects of erythropoietin on cardiac wasting are not well known in cancer cachexia model. The Yoshida hepatoma model is well-established cancer cachexia model, is known to have cardiac wasting and high mortality rate [5, 14]. In this study, we investigate the effects of erythropoietin on cancer cachexia progression, cardiac wasting, and physical performance status using the Yoshida hepatoma model. 2. Materials and methods 2.1. Animals and cachexia model Male Wistar-Han rats were housed in a specific-pathogen-free facility under a 12 h light/dark cycle with food and water provided ad libitum. On the first day of experiment (D0), rats were injected intraperitoneally with 108 growing Yoshida AH-130 tumor cells, as described
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previously [15]. After 5 days, rats developed large tumors, and after 10 days, rats were severely cachectic. 2.2. Experimental design Rats were divided into two groups: sham group (n = 10) and tumor-bearing rats (n = 60). The tumor-bearing rats were further randomized to placebo (n = 28), 500 Units/day of erythropoietin (500 U EPO, n = 16) or 5000 Units/day erythropoietin (5000 U EPO, n = 16). 500 Units/kg/day or 5000 Units/kg/day were administered s.c. daily. Erythropoietin administration began on the next day of tumor inoculation. The placebo group received saline injections. All animal manipulations were made in accordance with the European Community guidelines for the use of laboratory animals. Study protocol and study procedures were approved by the local animal ethics committee. 2.3. Body composition On D0 and day 16 or day of animals' euthanasia (D16), nuclear magnetic resonance spectroscopy (EchoMRI-700TM, Echo Medical Systems, Houston, TX) was performed to assess body composition of each rat [16]. Body composition was recorded after removal of the tumor, or the respective days of killing, if rats had to be euthanized earlier due to reaching ethical endpoints. Weight of heart was recorded, and tumor cells were counted using a Neubauer chamber.
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Echocardiography was performed in M-mode to measure cardiac function and dimensions, and in B-mode to calculate functional parameters. Echocardiography was performed 1 day before starting the treatment and day 11 (D11). 2.5. Proteasome activity Proteasome activity was analyzed, as described previously [5]. Briefly, the gastrocnemius muscle was homogenized in an ice-cold buffer. Protein was incubated with fluorogenic substrates (benzoylVal-Gly-Arg-7-amidocoumarin for trypsin-like activity, succinylLeu-Leu-Val-Try-7-amido-4-methylcoumarin for chymotrypsin-like activity, and benzyloxycarbonyl-Leu-Leu-Glu-7-amido-4-methylcoumarin for peptidylglutamyl peptidase activity, Biomol, Hamburg, Germany). The fluorescence intensity was measured with a fluorometer (Twinkle LB 970, Berthold, Bad Wildbad, Germany) at 360 and 460 nm emission. The activity, expressed as nanomole per milligram per minute, was calculated by using free amidomethylcoumarin as a working standard. 2.6. Physical performance status Food intake and spontaneous physical activity were recorded on D11. Spontaneous physical activity was measured by an infrared monitoring system (Supermex, Muromachi, Tokyo, Japan) over a 24 h period as described previously [18].
2.4. Echocardiography
2.7. Statistical analysis
Rats were anesthetized using 1.5% isoflurane and laid in supine position on a heating pad to maintain body temperature at 39 °C. All hair was removed from the left chest. A high-resolution echocardiography system (Vevo 770; Visual Sonics Inc., Toronto, Canada) was used [17].
Data were analyzed with GraphPad PRISM 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). Results are shown as mean ± standard error of mean. For the comparisons among groups, data were analyzed with analysis of variance followed by post hoc comparisons using Tukey's
Fig. 1. Total tumor cell numbers × 109 (A): there was no significant difference in tumor cell numbers between rats with placebo and rats with EPO groups. Change in body weight (B), lean mass (C), fat mass (D): average loss of body weight, lean mass, and fat mass was higher in placebo group than both 500 U EPO group and 5000 U EPO group at the end of the study (500 U EPO: p b 0.05 for body weight, p b 0.01 for lean mass, and p = 0.056) and (5000 U EPO: p b 0.05 for body weight, p b 0.01 for lean mass, and p b 0.05 for fat mass). Weight of heart at the end of the study (E): weight of heart was higher in rats with 500 U EPO than in rats with placebo (p b 0.05). Abbreviation: Erythropoietin, EPO. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
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test. Cox proportional hazard analysis was used to examine the effects of erythropoietin on survival. A value of p b 0.05 was considered to indicate statistical significance in all analysis.
1.4 g/day, p b 0.01, respectively; Fig. 1C). Average loss of fat was higher in the placebo group vs. the 5000 U EPO group (− 1.0 ± 0.2 g/day vs. − 0.8 ± 0.3 g/day, p b 0.05; Fig. 1D).
3. Results 3.2. Cardiac wasting The tumor growth was similar in all groups. At the end of the study, total tumor cell numbers were 2.365 ± 0.890 × 109 cell in placebo rats, 2.718 ± 0.995 × 109 cell in the 500 U EPO group and 2.153 ± 0.987 × 109 cell in rats treated with 5000 U EPO (all p N 0.2, Fig. 1A). Moreover, there were no observations of other malignancies during necropsy in each rat. 3.1. Body weight, body composition Average daily loss of body weight was higher in rats with placebo than in rats with 500 U EPO or 5000 U EPO (− 4.6 ± 0.9 g/day vs. − 3.2 ± 1.7 g/day or − 3.6 ± 1.9 g/day, p b 0.05, Fig. 1B). Average loss of lean mass was reduced in rats treated with 500 U EPO or 5000 U EPO (− 3.6 ± 0.8 g/day vs. − 2.4 ± 2.0 g/day or − 2.6 ±
Weight of the heart on the end of study in sham rats was 781 ± 52 mg, significantly higher than in rats with placebo (495 ± 67 mg, p b 0.001 vs. sham, Fig. 1E). The weight of the heart was significantly lower in rats with placebo than in rats with 500 U EPO (555 ± 69 mg, p b 0.05 vs. placebo, Fig. 1E). Echocardiographic parameters are shown in Fig. 2. The left ventricular (LV) mass on D11 was lower in rats with placebo than in rats with 500 U EPO or 5000 U EPO (445 ± 77 g vs. 587 ± 158 g and 507 ± 109 g, p b 0.001 and p b 0.05, respectively, Fig. 2A). LV end systolic diameter (LVESD) was higher in rats with placebo than in rats with 500 U EPO (4.0 ± 0.7 mm vs. 3.5 ± 0.7 mm; p b 0.05, Fig. 2B), even though LV end diastolic diameter (LVEDD) was comparable among 3 groups (Fig. 2C). LV ejection fraction (LVEF), LV fraction shortening (LVFS)
Fig. 2. Echocardiography: left ventricular mass (A) in D11 was more remarkable in rats with placebo than in rats with 500 U EPO (p b 0.001) or 5000 U EPO (p b 0.05). Left ventricular end systolic diameter (B) was significantly higher in rats with placebo than in rats with 500 U EPO (p b 0.05), Left ventricular end diastolic diameter (C) was comparable between rats with placebo and rats treated with EPO. Left ventricular ejection fraction (LVEF) (D), Left ventricular fractional shortening (LVFS) (E) and Left ventricular stroke volume (LVSV) (F) were significantly lower in rats with placebo than in rats with 500 U EPO (p b 0.01 for LVEF and LVFS, p b 0.05 for LVSV) and 5000 U EPO (p b 0.01 for LVEF, p b 0.05 for LVFS and LVSV). Cardiac output (G) was significantly lower in rats with placebo than in rats with 5000 U EPO (p b 0.01). Abbreviations: LV, left ventricular; Left ventricular end systolic diameter, LVESD; Erythropoietin, EPO; Left ventricular end diastolic diameter, LVEDD; Left ventricular ejection fraction, LVEF; Left ventricular fractional shortening, LVFS; Left ventricular stroke volume, LVSV; Cardiac output, CO. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
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Fig. 3. Skeletal muscle weight on D16: Muscle gastrocnemius (A), tibialis (B) and extensor digitalis longus (C) in rats with 500 U EPO were significantly higher than in rats with placebo (p b 0.01, p b 0.01 and p b 0.05). Catabolic signaling in the gastrocnemius muscle on Day 16: trypsin-like proteasome activity in rats with 500 U or 5000 U EPO were significantly lower than in rats with placebo (p b 0.001 and p b 0.01) (D). Chymotrypsin-like (E) and peptidyl-glutamyl protein-hydrolyzing (F) were comparable between rats with EPO (500 U and 5000 U EPO) and placebo. Abbreviations: Erythropoietin, EPO; extensor digitalis longus, EDL; peptidyl-glutamyl protein-hydrolyzing, PGPH. *p b 0.05 vs. placebo, **p b 0.01 vs. placebo, and ***p b 0.001 vs. placebo.
and LV stroke volume (LVSV) were also lower in rats with placebo than in treated rats Fig. 2D–G).
3.3. Muscle weight and catabolic signaling The weight of skeletal muscles is shown in Fig. 3A–C. At the end of the study, the weight of gastrocnemius (Fig. 3A), tibialis (Fig. 3B) and extensor digitalis longus (EDL) (Fig. 3C) in rats with 500 U EPO was significantly higher than in rats with placebo (p b 0.01, p b 0.01 and p b 0.05, respectively). Catabolic signaling is also shown in Fig. 3D–F. At the end of the study, trypsin-like proteasome activity was significantly lower in rats with 500 U or 5000 U EPO than in rats with placebo (191.4 ± 46.1 nmol/mg/min vs. 122.8 ± 76.8 nmol/mg/min and 116.1 ± 78.4 nmol/mg/min, p b 0.001 and p b 0.01, Fig. 3D), however chymotrypsin-like activity, peptidyl-glutamyl protein-hydrolyzing activity were similar among all groups.
3.4. Physical performance status On D11, food intake was 20.6 ± 3.1 g/24 h in sham rats vs. placebo rats (4.4 ± 3.4 g/24 h, p b 0.001 vs. sham, Fig. 4A). There were no significant differences among the placebo group and EPO groups. On D11, physical activity was 62,577 ± 9553 counts/day in sham rats and significantly reduced in the placebo rats (27,797 ± 13,269 counts/day, p b 0.05 vs. sham, Fig. 4B). Physical activity in rats with 500 U EPO or 5000 U EPO was higher than in rats with placebo (36,916 ± 15,418 counts/day and 40,343 ± 18524 counts/day, vs. placebo, both p b 0.05, Fig. 4B).
3.5. Survival On D16, the overall survival was 14.0% in rats with placebo, 37.5% in rats with 500 U EPO and 31.3% in rats with 5000 U EPO (Fig. 5). The
Fig. 4. Food intake (A) in D11 was comparable between rats with placebo and rats treated with EPO. Physical activity (B) in D11 was more remarkable in rats with placebo than in rats with 500 U EPO (p b 0.05) or 5000 U EPO (p b 0.05). Abbreviation: Erythropoietin, EPO. *p b 0.05 vs. placebo, and ***p b 0.001 vs. placebo.
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Fig. 5. Kaplan–Meier survival curves: EPO reduced mortality in comparison with placebo. Abbreviation: Erythropoietin, EPO.
median survival was 13 days in rats with placebo, 14 days in rats with 500 U EPO and 15 days in rats with 5000 U EPO. Treatment with 5000 U EPO reduced mortality (hazard ratio: 0.43, 95%CI: 0.20–0.92, p = 0.030), though 500 U EPO showed a trend for reduced mortality (hazard ratio: 0.46, 95%CI: 0.22–1.02, p = 0.056).
4. Discussion In the present study, we showed that erythropoietin treatment in tumor-bearing rats led to improvement in survival, weight loss, wasting of lean and fat mass, and reduction of proteasome activity in the skeletal muscle tissue and physical performance status. In addition, cardiac wasting was ameliorated in erythropoietin treated tumor-bearing rats. Erythropoietin administration reduced proteasome activity indicated by the trypsin-like activity in gastrocnemius muscle and improved muscle wasting. Ubiquitin-proteasome system signaling, which is responsible for the regulation of protein turnover and the elimination of abnormal protein, has been shown to be critical for coordinating muscle atrophy [19,20]. Some studies demonstrated a relationship between muscle wasting and poor physical performance status [21,22]. From our findings, it can be concluded that improved muscle wasting was associated with erythropoietin administration and leads to substantial improvement in physical performance status. Cardiac wasting, which was also observed in cancer cachexia rat and mouse models, has been described to induce cardiomyocyte remodeling and poor outcomes without treatment [2,23]. Previously our group reported that treatment with anti-heart failure drugs, the β-blocker bisoprolol or the aldosterone antagonist spironolactone, improved cardiac function and subsequently survival in Yoshida-hepatoma model [5]. This study suggested that amelioration of cardiac dysfunction in this model could influence on the improvement on the poor survival, though progressive cardiac wasting without erythropoietin treatment. The result suggested that cardiac wasting is related with survival in cancer cachexia. In the present study, erythropoietin also attenuated cardiac wasting to some extent, and improving cardiac dysfunction in cancer cachexia model. From the results presented here, it can be suggested that erythropoietin administration positively alter survival time, possibly by an improvement in cardiac wasting in tumor-bearing cachexia animals. In our intervention study, erythropoietin improved cardiac wasting in our model. It has been reported that the improvement of cardiac dysfunction in erythropoietin treated heart failure mice model was related to increased angiogenesis as well as near normalization of the myocardial levels of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and transforming growth factor β1 [14,24]. Pro-inflammatory cytokines, TNFs, IL-1 and IL6, are crucially involved in the mechanism of cancer cachexia [14,25,26] and which would be the one of the essential molecules to make better understanding of the mechanisms underlying in the therapeutic effect of erythropoietin in the cardiac and muscle dysfunction.
Erythropoietin administration is reported to improve the cancerinduced anemia, resulting in significantly improved physical performance status, quality of life and survival [27,28]. In addition to the amelioration of anemia, recent studies have shown that erythropoietin has the multiple tissue-protective efficacy such as an inhibition of apoptosis, attenuation of inflammatory responses in brain, kidney and heart [29, 30]. A tissue-protective receptor, complex consisting of the erythropoietin receptor and the beta common receptor (βcR) subunit, also known as CD131, which has much lower affinity of erythropoietin. βcR is confirmed exhibiting within the cardiomyocytes, and those of βcRknockout mice did not respond to erythropoietin, result in no improvement of apoptosis and survival [31]. These experiments are supportive of a role for βcR in the tissue-protective effects of erythropoietin in cardiomyocites. Dose response analyses showed that single dose of 150 to 500 Units/ kg is described the minimum effective dose of erythropoietin to achieve a tissue-protective effect in brain and heart disease model [32,33]. We also investigated the beneficial effects of both dose of erythropoietin (500 Units/kg/day and 5000 Units/kg/day/day) on weight loss, wasting, cardiac wasting. In our dose–response analysis showed that 500 Units/ kg of erythropoietin in this model provided the same efficacy as 5000 Units/kg/day administrated daily in rats with cancer cachexia. In terms from the dose setting, a tissue-protective effect could be one of the mechanisms for the therapeutic effects of erythropoietin in our study. In conclusion, the results of our Yoshida hepatoma rat model strongly support that cardiac wasting induced by cancer cachexia plays an important role which leads to impaired survival. Our findings suggested that the erythropoietin could be a novel therapeutic approach for cancer cachexia progression and cardiac wasting. Conflicts of interest SvH is consulting and has received honoraria from Vifor, Novartis, Thermo Fisher, Chugai, Roche, Sorin, Respicardia, and pfizer. WD is consulting and has received honoraria from Vifor. For the remaining authors none were declared. References [1] J.A. Norton, J.L. Peacock, S.D. Morrison, Cancer cachexia, Crit. Rev. Oncol. Hematol. 7 (1987) 289–327. [2] J.M. Argilés, B. Stemmler, F.J. López-Soriano, S. Busquets, Nonmuscle tissues contribution to cancer cachexia, Mediat. Inflamm. 2015 (2015) 182872. [3] S.M. Kazemi-Bajestani, H. Becher, K. Fassbender, Q. Chu, V.E. Baracos, Concurrent evolution of cancer cachexia and heart failure: bilateral effects exist, J. Cachex. Sarcopenia Muscle 5 (2014) 95–104. [4] L. Cramer, B. Hildebrandt, T. Kung, et al., Cardiovascular function and predictors of exercise capacity in patients with colorectal cancer, J. Am. Coll. Cardiol. 64 (2014) 1310–1319. [5] J. Springer, A. Tschirner, A. Haghikia, et al., Prevention of liver cancer cachexiainduced cardiac wasting and heart failure, Eur. Heart J. 35 (2014) 932–941. [6] S.E. Libretto, P.J. Barrett-Lee, K. Branson, et al., Improvement in quality of life for cancer patients treated with epoetin alfa, Eur. J. Cancer Care 10 (2001) 183–191. [7] I. Quirt, C. Robeson, C.Y. Lau, et al., Epoetin alfa therapy increases hemoglobin levels and improves quality of life in patients with cancer-related anemia who are not receiving chemotherapy and patients with anemia who are receiving chemo- therapy, J. Clin. Oncol. 19 (2001) 4126–4134. [8] H. Ludwig, S. Van Belle, P. Barrett-Lee, et al., The European Cancer Anaemia Survey (ECAS): a large, multinational, prospective survey defining the prevalence, incidence, and treatment of anaemia in cancer patients, Eur. J. Cancer 40 (2004) 2293–2306. [9] L. Repetto, K. Moeremans, L. Annemans, European Organisation for Research and Treatment of Cancer. European guidelines for the management of chemotherapyinduced anaemia and health economic aspects of treatment, Cancer Treat. Rev. 32 (Suppl. 2) (2006) S5–S9. [10] M. Aapro, J.L. Spivak, Update on erythropoiesis-stimulating agents and clinical trials in oncology, Oncologist 14 (Suppl. 1) (2009) 6–15. [11] F. Penna, S. Busquets, M. Toledo, et al., Erythropoietin administration partially prevents adipose tissue loss in experimental cancer cachexia models, J. Lipid Res. 54 (2013) 3045–3051. [12] L. Mille-Hamard, V.L. Billat, E. Henry, et al., Skeletal muscle alterations and exercise performance decrease in erythropoietin-deficient mice: a comparative study, BMC Med. Genet. 5 (2012) 29.
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