Int J Clin Oncol (2009) 14:315–320 DOI 10.1007/s10147-008-0856-1
© The Japan Society of Clinical Oncology 2009
ORIGINAL ARTICLE Masanori Takahashi · Masanori Terashima Akinori Takagane · Kenichi Oyama · Hisataka Fujiwara Go Wakabayashi
Ghrelin and leptin levels in cachectic patients with cancer of the digestive organs
Received: August 17, 2007 / Accepted: October 24, 2008
Abstract Background. Cancer cachexia, a catabolic state characterized by weight loss, occurs frequently in patients with terminal-stage neoplastic diseases. Gastrointestinal hormones and cytokines may be associated with anorexia and wasting in cancer cachexia. Methods. This study aimed to examine the mechanism of anorexia in cachectic patients through a prospective investigation of plasma cytokines, ghrelin, and leptin in 16 cachectic patients with cancer of the digestive organs and 10 healthy volunteers. Results. Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1 receptor antagonist (IL-1Ra), and ghrelin levels were significantly higher in cachectic cancer patients than in the healthy volunteers, whereas leptin was significantly lower in the cachectic cancer patients. Plasma leptin levels and cytokine levels (TNF-α and IL-6) correlated significantly with body mass index (BMI), but plasma ghrelin levels did not correlate with BMI or with the grade of symptoms. Conclusion. Neither weight loss nor the grade of symptoms seemed to be directly associated with the increase in ghrelin levels. Hence, it is considered that the increase in ghrelin levels cannot simply be explained by an increase in ghrelin secretion, suggesting that other mechanisms, such as the decreased inactivation of ghrelin, may also play a role. Further studies are needed to clarify the mechanisms of the increase in ghrelin levels. Additionally, the changes in plasma cytokines (TNF-α and IL-6) and leptin in cachectic cancer patients suggest that these molecules may be useful markers for the evaluation of cancer cachexia.
M. Takahashi · K. Oyama · H. Fujiwara · G. Wakabayashi Department of Surgery, Iwate Medical University, Morioka, Japan M. Terashima (*) Department of Surgery, Fukushima Medical University, 1 Hikarigaoka, Fukushima 960-1295, Japan Tel. +81-24-548-2111; Fax +81-24-548-2735 e-mail:
[email protected] A. Takagane Department of Surgery, Hakodate Goryokaku Hospital, Hakodate, Japan
Key words Cachexia · Cancer of the digestive organs · Cytokine · Ghrelin · Leptin · Body mass index
Introduction Cancer cachexia is characterized by progressive emaciation involving depletion of host adipose tissue stores.1 Patients with this disorder suffer from numerous symptoms, including anorexia, nausea, and general fatigue. Various mechanisms have been implicated in the etiology of cancer cachexia, and several studies have shown that cachexia associated with advanced cancer is associated with an increase in plasma cytokine levels.1–3 Tumor necrosis factor (TNF)-α, interleukin (IL)-1, interferon (IFN)-γ, and IL-6 may play a role in many of the changes observed in cancer cachexia, including loss of body weight, loss of appetite, and induction of acute-phase proteins.4 It has also been reported that the intraperitoneal injection of IL-1β decreases food intake and induces weight loss in mice.5 Several gastrointestinal hormones may regulate food intake and energy balance by affecting the arcuate nucleus of the hypothalamus, which mediates changes in energy expenditure and appetite. Leptin is a peptide member of the cytokine receptor family and is produced primarily by fat cells. Leptin regulates fat mass by decreasing food intake (through decreasing the level of neuropeptide Y in the hypothalamus) and increasing resting energy expenditure,6 and the administration of recombinant leptin has been shown to decrease food intake and increase energy expenditure.7,8 Additionally, Aleman et al.9 found that in lung cancer patients with weight loss, circulating leptin concentrations were not elevated but were inversely related to the severity of the inflammatory response. Serum leptin concentrations in patients with advanced lung cancer depend only on the total amount of fat. Ghrelin is a 28-amino acid peptide that is secreted mainly from the stomach. It was first isolated in 1999 as an endogenous ligand for the growth hormone (GH) secretagogue receptor (GHS-R).10 In rodents11 and humans,12 several
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lines of evidence implicate a role of ghrelin in GH release, energy balance, food intake, and long-term regulation of body weight. Ghrelin, which is at present the only known circulating orexigenic hormone, exerts antagonistic effects on the leptin-induced decrease in food intake through activation of the hypothalamic neuropeptide Y-Y1 (NPYY1) pathway.13 Ghrelin administration can antagonize the effects of cytokines on appetite and body weight;14 moreover, Dixit et al.4 have reported that ghrelin exerts potent anti-inflammatory effects and attenuates endotoxin-induced anorexia in a murine endotoxemia model. Furthermore, elevation of the baseline plasma ghrelin level has been found in cachectic patients with lung cancer, with a further increase observed in patients with anorexia in follow up after chemotherapy.7 Cancer cachexia increases mortality and causes deterioration of the quality of life; therefore, improved understanding of the mechanisms that control energy balance in cancer cachexia may help in the development of new therapies for this pathological condition. The roles of cytokines, ghrelin, and leptin in cancer cachexia remain unclear, but we hypothesized that abnormal levels of these molecules might contribute to the disease. To test this hypothesis, we examined plasma cytokine and hormone levels prospectively in cachectic cancer patients and healthy volunteers.
Patients and methods The study protocol was approved by the Iwate Medical University Institutional Review Board, and the study was conducted between March 2002 and August 2003. Written informed consent was obtained from all patients. All clinical investigations described in the article were conducted in accordance with the guidelines of the Declaration of Helsinki. Patients Sixteen cachectic cancer patients who were hospitalized in Iwate Medical University were enrolled in the study (Table 1). Seven patients had undergone gastrectomy, of whom 3 had received total gastrectomy and 4 had undergone distal gastrectomy. The study was carried out at an average of 15 ± 5.32 months after gastric resection. Patients were selected according to the following eligibility criteria: (1) a diagnosis of stage IV15 cancer of the digestive organs (all patients had peritonitis carcinomatosa, and selected patients received the best supportive care); (2) the presence of subjective symptoms,16 as classified using the Cancer Therapy Evaluation Program, Common Toxicity Criteria (based on CTC version 2.0, 1998); (3) absence of organ failure or other primary cachectic states such as effects on the thyroid; (4) absence of severe liver disease (no evidence of cirrhosis on abdominal imaging, and no elevation of liver enzymes to more than three times the upper limit of the normal range); (5) absence of the need for ascites drainage;
Table 1. Patient characteristicsa Sex Male Female Age Cancer site Esophagus Stomach Colorectal Body mass index (kg/m2) Albumin (g/dl) Hemoglobin (g/dl) Symptoms Grade Anorexia 0 1 2 3 General fatigue 0 1 2 3 Nausea 0 1 2 3 a
12 4 63 ± 11 2 9 5 18.3 ± 0.6 3.2 ± 0.6 10.1 ± 1.7
0 11 3 2 2 9 4 1 8 7 1 0
Values are shown as means ± SD
and (6) the absence of mechanical intestinal obstruction. Additionally, patients were excluded from the study if they were being fed through a feeding tube; had a gastric fistula, an active peptic ulcer, or cancer involving the upper airway; or were undergoing treatment with corticosteroids and/or anabolic drugs. This study also included ten healthy volunteers. These subjects comprised five men and five women with a mean age ± SD of 30 ± 1.3 years (P = 0.003, compared with the cancer cachexia group) and a mean body mass index (BMI) ± SD of 23 ± 2.6 kg/m2 (P < 0.001, compared with the cancer cachexia group). The volunteers were healthy hospital personnel who had undergone no changes in body weight over the previous 6 months, had no acute or chronic disease, and were receiving no regular medication. The age and sex ratios of these two groups were quite different. However, this difference was not thought to be essential for further research; the data were thought to be necessary because this study also aimed to evaluate any attenuation of cancer cachexia. Parameters and hormone assays Blood samples were collected from the antecubital vein of volunteers and patients between 6 and 7 a.m. following an overnight fast. The blood was transferred into a chilled glass tube containing disodium ethylenediamine tetraacetic acid (EDTA) and stored at 4°C until centrifugation at 3000 rpm for 10 min. After processing, all sample aliquots were immediately stored at −80°C and only thawed immediately prior to performance of the assay. Plasma TNF-α, IFN-γ, IL-6,
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patients (P < 0.001, P < 0.001, and P = 0.006, respectively; Fig. 1); and the IFN-γ level was also higher in cachectic cancer patients than in healthy volunteers, but the difference was not statistically significant (P = 0.268). Figure 2 shows comparisons of the plasma levels of leptin and ghrelin between the two groups. The leptin level was significantly lower in cachectic cancer patients (P = 0.018), whereas the ghrelin level was significantly higher in cachectic cancer patients than in the healthy volunteers (P = 0.035). The plasma ghrelin levels were compared in patients who had and had not undergone gastrectomy, but no difference was observed between these two groups of patients (P = 0.340; data not shown). The plasma cytokine levels were then examined in relation to BMI. Plasma TNF-α and IL-6 showed significant correlations with BMI (R = −0.449, P = 0.021; and R = −0.534, P = 0.005, respectively). However, plasma IFN-γ and IL-1Ra did not correlate with BMI (R = −0.035, P = 0.866; and R = −0.550, P = 0.099, respectively; Fig. 3). Plasma leptin levels were significantly correlated with BMI (R = 0.570, P = 0.002), but plasma ghrelin levels did not correlate with BMI (R = −0.379, P = 0.056; Fig. 4). Furthermore, there was no correlation between plasma ghrelin and leptin levels (R = −0.233, P = 0.235). Plasma leptin levels did not correlate with plasma TNF-α, IFN-γ, IL-6, or IL-1Ra levels (R = −0.220, P = 0.280; R = 0.019, P = 0.923; R = −0.220, P = 0.280; and R = −0.241, P = 0.235, respectively), but a significant correlation was found between plasma ghrelin and plasma IL-1Ra levels (R = 0.637, P < 0.001). However, plasma
IL-1Ra, and leptin levels were measured using enzymelinked immunosorbent assay (ELISA) kits purchased from Funakoshi (Tokyo, Japan), and the plasma ghrelin level was measured using an ELISA kit purchased from Phoenix Pharmaceuticals (Belmont, CA, USA). All assays were performed according to the manufacturers’ instructions. Clinical parameters used in the study included gender, age, BMI, anorexia, fatigue, and nausea. Data analysis SPSS statistical software (version 11.0.J for Windows; SPSS, Chicago, IL, USA) was used for all statistical analyses. Quantitative parameters are expressed as means ± SD for baseline characteristics and means ± SEM for other data. Comparisons of the plasma levels of cytokines, ghrelin, and leptin in the different groups were performed by MannWhitney U-test. Regression analysis was used for other comparisons. A Kruskal-Wallis test was used for multiple comparisons. P values of less than 0.05 in two-tailed tests were regarded as statistically significant.
Results Plasma cytokine levels were first compared between cachectic cancer patients and the healthy volunteers. TNF-α, IL-6, and IL-1Ra were significantly elevated in cachectic cancer
0.5
IFN-g (pg/ml)
0.4 0.3 0.2 0.1 0
C
P < 0.001
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B 0.2
Cachexia Patients
P < 0.001
0.2 0.15 0.1 0.05
P = 0.268
0.15 0.1 0.05 0
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D IL-1Ra (pg/ml)
TNF-a (pg/ml)
A
IL-6 (pg/ml)
Fig. 1A–D. Comparisons of plasma cytokine levels in cachectic cancer patients (n = 16) and healthy volunteers (n = 10). Plasma levels of tumor necrosis factor-α (TNF-α; A) interleukin6 (IL-6; C), and interleukin 1 receptor antagonist (IL-1Ra; D) were significantly elevated in cachectic cancer patients compared with those in healthy subjects (P < 0.001, P < 0.001, and P = 0.006, respectively). Plasma levels of interferon-γ (IFN-γ; B) were higher in cachectic cancer patients than in healthy volunteers, but the difference did not reach statistical significance. The results are expressed as means ± SE (P < 0.05)
0.2
Cachexia Patients
Healthy Subjects
P = 0.006
0.16 0.12 0.08 0.04 0
Cachexia Patients
Healthy Subjects
Cachexia Patients
Healthy Subjects
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B
2
P = 0.018
1.6 1.2 0.8 0.4
0.5 0.4 0.3 0.2 0.1 0
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Cachexia Patients
Healthy Subjects
A 1 0.8
IFN-g (pg/ml)
TNF-a (pg/ml)
0.25
n = 26 R = -0.449 P < 0.05
0.6 0.4 0.2
0.2 0.15 0.1
n = 26 R = -0.035 P = 0.866
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15
20
25
30
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C
n = 26 R = -0.534 P < 0.05
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0.3 n = 26 R = -0.550 P = 0.099
0.25 0.2 0.15 0.1 0.05 0
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3.5 n = 26 3 R = 0.570 2.5 P < 0.05 2 1.5 1 0.5 0 10 15
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BMI (kg/m2)
BMI (kg/m2)
Leptin (pg/ml)
18
BMI (kg/m2)
BMI (kg/m2)
0
Fig. 4A,B. Regression analyses. A Regression analysis of leptin levels with BMI using data from the patient and volunteer groups (R = 0.57, P = 0.002). B Regression analysis of ghrelin levels with BMI using data from the patient and healthy volunteer groups (R = −0.379, P = 0.056). Filled circles, Cachectic cancer patients; open circles, healthy volunteers
Healthy Subjects
B 1.2
IL-6 (pg/ml)
Fig. 3A–D. Regression analyses. Regression analysis of TNF-α (A) and IL-6 (C) levels with body mass index (BMI) using data from the patient and healthy volunteer groups (R = −0.449, P = 0.021; and R = −0.534, P < 0.005, respectively). Each of the differences was statistically significant. Regression analysis of IFN-γ (B) and IL-1Ra (D) levels with BMI using data from the patient and volunteer groups (R = −0.035, P = 0.866; and R = −0.550, P = 0.099, respectively). Filled circles, Cachectic cancer patients; open circles, healthy volunteers
P = 0.035
0.7 0.6
Ghrelin (ng/ml)
A Leptin (pg/ml)
Fig. 2A,B. Comparisons of plasma ghrelin levels and plasma leptin levels in cachectic cancer patients (n = 16) and healthy volunteers (n = 10). Compared to healthy subjects, cachectic cancer patients showed significantly lower plasma leptin levels (A) and significantly elevated plasma ghrelin levels (B) (A; P = 0.035 and P = 0.018, respectively). The results are expressed as means ± SE (P < 0.05)
1
n = 26 R = -0.379 P = 0.056
0.8 0.6 0.4 0.2 0
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25
30
BMI (kg/m2)
ghrelin levels did not correlate with plasma TNF-α, IFN-γ, and IL-6 levels (R = 0.136, P = 0.506; R = 0.080, P = 0.697; and R = 0.006, P = 0.977, respectively). We also examined the levels of plasma cytokines (TNFα, IFN-γ, IL-6, and IL-1Ra), ghrelin, and leptin in relation to the grade of symptoms (anorexia, general fatigue, and nausea), but we found no correlations among these variables (data not shown).
10
15
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BMI (kg/m2)
Discussion A number of proinflammatory cytokines, including TNF-α, IL-6, and IL-1, have been proposed as mediators of the cachectic process, because high serum levels of TNF-α, IL6, and IL-1 have been found in cancer patients.17–19 Our results were consistent with these findings; and additionally,
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they demonstrated that plasma IFN-γ levels tended to be elevated in cachectic cancer patients, although the difference in plasma IFN-γ levels between cachectic cancer patients and healthy volunteers did not reach statistical significance. We also found an inverse correlation between BMI and IL-6, as shown previously by Mantovani et al.,20 and an inverse correlation between BMI and TNF-α. Collectively, these results suggest that high cytokine levels are related to weight decrease and may be a trigger for the development of cancer cachexia. Leptin levels are directly associated with weight loss after dieting,21,22 but the association between leptin and cancer cachexia is not fully understood. Leptin levels and weight loss have been shown to be associated in a cohort of lung23 and pancreatic cancer patients;24 furthermore, in our study, we found decreased leptin levels in cachectic cancer patients and a direct correlation with BMI. Leptin is predominantly expressed in the adipose tissue, and its level in plasma correlates well with body fat mass.7,8 Hence, we suggest that in cancer cachexia patients, the low plasma leptin level is related to both the decrease in adipose cells and the high level of inflammatory cytokines. We also found significant elevation of plasma ghrelin levels in cachectic patients. Hanada et al.25 have shown upregulation of ghrelin expression in cachectic nude mice bearing human melanoma cells, and elevated levels of total or active ghrelin in cancer cachexia have been reported in cohorts mainly comprising lung cancer patients;7,26 however, our results are the first to show elevated ghrelin in cachectic patients with cancer of the digestive organs. Most circulating ghrelin originates from the stomach, and depletion of plasma ghrelin has been reported immediately after gastrectomy;27 however, we did not find a difference in plasma ghrelin levels between the gastrectomy and nongastrectomy groups in the present study. Because T cells, spleen, thymus, small intestine, and placenta express ghrelin m-RNA,4 the reason for our finding of a lack of difference in plasma ghrelin levels between the gastrectomy and nongastrectomy groups may have been a potential compensatory production of ghrelin by other tissues over a long period. Therefore, we thought it is was mandatory to include the gastrectomy group in this examination. In the present study, we did not find a strong inverse correlation between plasma ghrelin and BMI, and a significant correlation was found between plasma ghrelin and plasma IL-1Ra. Wolf et al.22 reported a significant positive correlation between ghrelin levels and BMI loss over the previous 6 months in adult breast and colon cancer patients, and Garcia et al.26 found that both total and active ghrelin were inversely correlated with BMI in a population mainly comprising lung cancer patients. These results suggest that ghrelin has a potential role in the regulation of body weight in cachectic cancer patients. But it should be noted that, in our group of cachectic patients with cancer of the digestive organs, the increase in ghrelin levels was not correlated with weight loss or with the grade of symptoms. Hence, it is considered that the increase in ghrelin levels cannot simply be explained by an increase in ghrelin secretion, suggesting that other mechanisms, such as the decreased inactivation
of ghrelin, may also play a role. Further studies are needed to clarify the mechanisms of the increase in ghrelin levels. We consider the present study to be important because many factors (symptoms; various cytokines; gastrointestinal hormones [leptin and ghrelin]; and BMI) were analyzed simultaneously. Our study is the first to have analyzed these factors in cachectic patients with cancer of the digestive organs. However, in the present study, cachectic patients required drugs (such as analgesics and antiemetics) and parenteral hyperalimentation (two patients) for the control of various unpleasant symptoms. These drugs and parenteral hyperalimentation may have influenced the plasma concentrations of ghrelin, leptin, and the examined cytokines. Therefore, we think that further studies are required. There is a need for new therapies for patients with cancer cachexia; therefore, investigation of the mechanisms underlying cancer cachexia is important. Ghrelin and leptin have mutually antagonistic effects on the hypothalamus with regard to the control of food intake, and these molecules also have reciprocal regulatory effects on inflammatory cytokine expression in the immune system.7,8 In this context, our results suggest that the plasma levels of TNF-α, IL-6, and leptin are useful parameters for the evaluation of cancer cachexia. Various mechanisms have been implicated in the etiology of cancer cachexia, and the disease has been described as a GH-resistant state with high GH levels but low levels of insulin-like growth factor-I.28 A recent report showed that a higher dose of ghrelin was required to induce a larger increase in appetite, possibly due to ghrelin resistance, in cachectic cancer patients compared with controls; and ghrelin administration to a group of cachectic and noncachectic cancer patients induced a 30% increase in appetite and food intake.29 Therefore, the ghrelin level might show a significant inverse correlation with BMI. The high ghrelin levels we found in the cancer patients in our study were probably induced by ghrelin resistance. Based on these findings, we believe that ghrelin, GHS-R agonists, and cytokine antagonists may be useful as therapy for reducing anorexia and wasting in patients with cancer cachexia.
Conflict of interest statement Authors have no conflict of interest. Acknowledgments The authors are grateful to S. Fujiwara for technical assistance and to Drs. K. Ikeda, T. Higuchi, K. Otuka, and K. Aoki for collecting the samples.
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