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Pharmacological Reports
Copyright © 2013
2013, 65, 960–969
by Institute of Pharmacology
ISSN 1734-1140
Polish Academy of Sciences
Effect of infliximab on metabolic disorders
induced by Walker-256 tumor in rats
Daniele R. Miksza1, Camila O. de Souza1, Hely de Morais1,
Aline F. da Rocha1, Gláucia R. Borba-Murad1, Roberto B. Bazotte2,
Helenir M. de Souza1
Department of Physiological Sciences, State University of Londrina, 86051-990, Londrina, PR, Brazil
1
Department of Pharmacology and Therapeutics, State University of Maringá, 87020-900, Maringá, PR, Brazil
2
Correspondence:
Helenir Medri de Souza, e-mail: [email protected]
Abstract:
Background: The purpose of this study was to investigate the effect of infliximab, an anti-tumor necrosis factor a (TNFa) monoclonal antibody, on the progression of cachexia and several metabolic parameters affected by the Walker-256 tumor in rats.
Methods: Infliximab (0.5 mg/kg) was ip administered, twice a day, beginning at the day in which the Walker-256 tumor cells were inoculated. After 12 days of treatment, the tumor growth, some parameters of cachexia/anorexia, the blood levels of triacylglycerol,
glucose, lactate and urea, the peripheral response to insulin and the hepatic glycolysis and gluconeogenesis were investigated. The peripheral response to insulin was evaluated by the insulin tolerance test and the glycolysis and gluconeogenesis in isolated perfused liver.
Results: The treatment with infliximab did not alter the growth of the Walker-256 tumor, but attenuated (p < 0.05) the reduction of
body weight and prevented (p < 0.05) the loss of retroperitoneal adipose tissue induced by the tumor. Moreover, treatment with infliximab tended to minimize the loss of gastrocnemius muscle, the reduction in food intake, the peripheral response to insulin and the
liver gluconeogenesis from alanine, as well as the increased blood triacylglycerol, caused by the tumor. In contrast, treatment with
infliximab did not attenuate the reduction in hepatic glycolysis and glycemia, nor did it minimize the rise in blood levels of lactate
and urea induced by the tumor.
Conclusion: The treatment with infliximab ameliorated some changes associated with cachexia, such as the reduction of adipose tissue and body weight, suggesting that TNFa plays a significant role in mediating these changes induced by the tumor. In addition, infliximab tended to improve or had no effect on other metabolic parameters affected by the Walker-256 tumor, suggesting that other
mediators or tumor-related events are involved in these disorders.
Key words:
infliximab, cancer, Walker-256 tumor, anti-TNFa, metabolism
Introduction
Cancer cachexia is characterized by progressive
weight loss, caused by depletion of skeletal muscle
mass and fat tissue, associated with reduced food intake and several other related metabolic disorders [45,
46]. Cancer cachexia results in generalized weakness,
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Pharmacological Reports, 2013, 65, 960–969
debilitation of the patient, decreased tolerance and response to chemotherapy, and constitutes the main
cause of death [1].
Tumor necrosis factor a (TNFa), a proinflammatory cytokine produced by tumor and host tissues,
plays an important role in the development of cancer
cachexia. TNFa can induce anorexia, loss of body
Infliximab on metabolic parameters affected by Walker tumor
Daniele R. Miksza et al.
weight [21, 37], muscle mass [5] and adipose tissue
[55]. In addition, TNFa can alter the lipid profile
[50], the liver gluconeogenesis [31], the insulin sensitivity [39] and cause other metabolic disorders.
In this context, anti-TNFa therapy could be an important option in the treatment of cachexia and metabolic disorders associated with cancer. One of the
available anti-TNFa agents is infliximab, an IgG
monoclonal antibody that blocks the binding of TNFa
to its receptor with high specificity [22, 29].
Infliximab is used to treat inflammatory conditions
provoked by the proinflammatory effect of TNFa.
Such treatment with infliximab has had beneficial effects on intestinal inflammatory disease, psoriatic arthritis, rheumatoid arthritis [7, 42] and type 2 diabetes
[52, 53]. Moreover, infliximab treatment improved
the glycemic control of patients showing insulin resistance [23, 58], improved insulin signaling [9], restored glycemic homeostasis [2] and prevented the
liver glucose overproduction [26] in an animal model
of insulin resistance.
Nevertheless, the effect of infliximab on cachexia and
metabolic disorders associated with cancer has been investigated little and the available results are inconclusive
[6, 28, 56]. The present study investigated the effect of
infliximab on the development of cachexia/anorexia induced by the Walker-256 tumor and several metabolic
parameters affected by the tumor.
Materials and Methods
Chemicals
Infliximab was acquired from Schering-Plough (Rio
de Janeiro, Brazil) and regular insulin from Eli Lilly
(Rio de Janeiro, Brazil). Perfusion fluid salts, the gluconeogenic precursors and all other reagents were
purchased from Sigma Chemical Co. (St. Louis,
USA), Merck (Darmstadt, Germany) and Reagen (Rio
de Janeiro, Brazil).
Tumor implantation and treatment protocol
Male Wistar rats (220–230 g), fed with a standard
commercial laboratory diet (Nuvilab®), were used in
all experiments. The Walker-256 carcinosarcoma was
implanted in the rats as described previously in our
[13, 17] and in other studies [54]. For this purpose,
Walker-256 cells suspended in phosphate buffered saline (PBS: 16.5 mM phosphate, 137 mM NaCl,
2.7 mM KCl; pH 7.4) were subcutaneously inoculated, at 8 × 107 viable tumor cells per rat, into the
right rear flank (tumor-bearing rats), while healthy
rats were subcutaneously inoculated with PBS in the
same place.
The Walker tumor-bearing rats (WK) were divided
into two groups: untreated (WK group) and treated
with infliximab (WK + inflix group). The latter group
received intraperitoneally (ip) infliximab (0.5 mg/kg),
twice a day (8:00 a.m. and 5:00 p.m.), for 12 days,
starting on the day that the tumor cells were inoculated. The infliximab solution (100 µg in 100 µl saline) was prepared daily and the dose was based on
previous studies [2, 9, 26]. The untreated (WK) and
healthy (no tumor) rats received ip saline injections
instead of infliximab.
The experiments were done 12 days after infliximab or saline treatment. In all experiments, except
those in which the food intake was measured, the animals were fasted (24 h) to prevent the influence of the
reduced feeding and lower level of hepatic glycogen
of tumor-bearing rats, compared to healthy rats, in
some metabolic parameters such as blood glucose and
plasma triacylglycerols. The experimental protocols
were approved by the Animal Experimentation Ethics
Committee of the State University of Londrina.
Assessment of the tumor growth,
cachexia/anorexia and blood metabolic
parameters
After evaluation of the daily food intake, rats were
weighed and killed by decapitation to collect blood
samples for the analysis of plasma triacylglycerol,
glucose, lactate and urea. Afterwards, the tumor, the
retroperitoneal adipose tissue and gastrocnemius muscle were carefully removed and weighed. The change
in body weight was calculated as the difference between the final (day 12) and initial (day 1) weights.
Assessment of the peripheral response to
insulin
The glycemic response to insulin was evaluated
through the insulin tolerance test (ITT). In this test,
insulin (0.25 U/kg) was intravenously injected and the
blood samples to measure glycemia were collected
from the vena cava, 0, 5, 15, 30 min later.
Pharmacological Reports, 2013, 65, 960–969
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Liver perfusion to assess the glycolysis and
gluconeogenesis
The rats were anesthetized with sodium pentobarbital
(40 mg/kg, ip) and their livers were perfused in situ.
For this purpose, the portal vein and inferior vena
cava were cannulated. Krebs-Henseleit bicarbonate
(KH) perfusion fluid, at pH 7.4, 37°C and saturated
with O2 : CO2 (95 : 5%), was introduced into the portal vein at 4 ml/min per gram liver, as described previously [33, 36], and the outflowing perfusate was collected from the inferior vena cava at 2 min intervals,
to determine the concentrations of glucose, pyruvate,
lactate and urea. KH was composed of: 115 mM
NaCl, 25 mM NaHCO3, 5.8 mM KCl, 1.2 mM
Na2SO4, 1.18 mM MgCl2, 1.2 mM NaH2PO4 and
2.5 mM CaCl2. Livers were perfused with KH for the
first 10 min and then with KH + 2.5 mM alanine or
KH + 20 mM glucose for 30 min. The differences in
production of glucose, lactate, pyruvate and urea, before and during infusion of alanine or glucose were
used to calculate the areas under the curves (AUCs).
The AUCs were evaluated in time interval when the
liver was perfused with alanine or glucose, i.e., between 10 and 40 min using the program GraphPad
Prism 5. Hepatic glycolysis was calculated as the sum
of the hepatic production of pyruvate and lactate released into the liver perfusate.
Chemical analysis
The concentrations of glucose [10], lactate [25], pyruvate [16] and urea [24] were determined by enzymatic
methods. The assay of triacylglycerol in the plasma
was based on the Trinder [51] reaction.
Data analysis
Data were tested for normal distribution and homogeneous variance and the appropriate statistical test, indicated in the figure legends, was employed to analyze the results. Statistical analysis were carried out
with the programs Statistica 6.0 or GraphPad Prism
5.0, a difference being taken as significant when p <
0.05. Results are expressed as the mean ± standard error of the mean (SEM).
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Pharmacological Reports, 2013, 65, 960–969
Tumor mass in 24 h fasted Walker-256 tumor-bearing rats
treated with infliximab during 12 days (WK + inflix) or untreated (WK).
Data are the mean ± SEM of 9–12 experiments. Differences between
the means were compared by unpaired Student -test
Fig. 1.
t
Results
Treatment with infliximab for 12 days did not affect
the growth of the Walker-256 tumor. The mass of the
tumor in WK + inflix group was similar to that in WK
group, namely about 37 g, representing a considerable
fraction of the body weight of the rat (Fig. 1).
However, the infliximab treatment reduced (p <
0.05) the marked loss of body weight of the tumorbearing rats. While the healthy rats gained about 40 g
of body mass in 12 days, WK group lost (p < 0.05)
around 20 g, but the weight loss in WK + inflix group
was 45% lower (p < 0.05) than WK rats (Fig. 2A).
Similarly, infliximab treatment reduced (p < 0.05) the
accentuated loss (p < 0.05) of retroperitoneal adipose
tissue of WK rats. The fat mass was 41% greater (p <
0.05) in WK + inflix than in WK group and was similar to that of healthy rats (Fig. 2B).
Accordingly, WK group exhibited a reduced (p <
0.05) mass of gastrocnemius muscle (Fig. 2C) and
food intake (Fig. 2D), compared to the healthy rats,
whereas the gastrocnemius muscle mass (Fig. 2C) and
food intake (Fig. 2D) of WK + inflix group were not
significantly different from those of the healthy
group. Moreover, WK and WK + inflix groups had
Infliximab on metabolic parameters affected by Walker tumor
Daniele R. Miksza et al.
Changes in body mass (A), retroperitoneal adipose tissue mass (B), gastrocnemius muscle mass (C) and food intake (D), in 24 h fasted
healthy rats and Walker 256 tumor-bearing rats, treated with infliximab (WK# + inflix) during 12 days or untreated (WK). Data are the mean ± SEM
of 7–20 experiments. * p 0.05, ** p 0.01, *** p 0.001 healthy rats; p 0.05 WK (ANOVA One-Way followed by Newman-Keuls test)
Fig. 2.
<
<
<
vs.
higher (p < 0.05) plasma concentrations of triacylglycerol than the healthy rats, but the triacylglycerol levels tended to be lower in WK + inflix compared to WK groups (Fig. 3A). Nevertheless, the infliximab treatment was unable to reduce the fall in
glycemia (Fig. 3B), or the rise in blood lactate (Fig.
3C) and urea (Fig. 3D) of WK rats. WK and WK + inflix groups exhibited equally lower (p < 0.05) glucose
(Fig. 3B) and higher (p < 0.05) lactate (Fig. 3C) and
urea (Fig. 3D) levels in comparison with healthy rats.
In addition, insulin injections promoted lower glycemia reduction (p < 0.05) in WK and WK + inflix
groups than in the healthy group (Fig. 4A). However,
the percent of reduction in glycemia tended to be
<
vs.
greater, at all times points, in WK + inflix group than
in WK group (Fig. 4B). Furthermore, WK and WK +
inflix groups showed respectively lower (p < 0.05) or
tendency lower liver glucose production from alanine
than the healthy group (Fig. 5A). WK and WK + inflix groups also showed lower (p < 0.05) liver pyruvate (Fig. 5C) and urea (Fig. 5D) production and
tended to produce lower lactate (Fig. 5B) from alanine than the healthy group. However, according to
the AUCs, the reduction of liver glucose, urea and
lactate production tended to be less intense in WK +
inflix in comparison with WK group (Fig. 5).
In contrast, infliximab treatment did not attenuate
the accentuated inhibition of hepatic glycolysis in the
Pharmacological Reports, 2013, 65, 960–969
963
Plasma concentrations of triacylglycerol (A), glucose (B), lactate (C) and urea (D) in 24 h fasted healthy rats and Walker-256 tumorbearing rats, treated with infliximab during 12 days (WK + inflix) or untreated (WK). Data are the mean ± SEM of 10–16 experiments. ** p 0.01,
*** p 0.001 healthy rats (ANOVA One-Way followed by Newman-Keuls or Kruskal Wallis followed by Dunn’s tests)
Fig. 3.
<
<
vs.
tumor-bearing rats. WK and WK + inflix groups
showed similar sharp reduction (p < 0.05) in the production of lactate (Fig. 6A) and pyruvate (Fig. 6B)
and, consequently, in glycolysis (Fig. 6C) in comparison with healthy rats.
Discussion
Walker-256 tumor bearing rats are a good experimental model for the study of metabolic disorders and
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cancer cachexia [13, 17, 54]. Since TNFa has been
correlated to cachexia and many metabolic abnormalities associated with cancer and since Walker-256
cells produce TNFa [41] and increased levels of
TNFa in the liver [12] and increased production of
this cytokine by spleen lymphocytes [20] were found
in Walker-256 tumor bearing rats, we decided to
evaluate the effect of infliximab, an anti-TNFa, on
the growth of the tumor, progression of cachexia and
several metabolic parameters affected by the Walker256 tumor.
Although there are reports that TNFa produced by
the tumor enhances tumor growth [8], the treatment
Infliximab on metabolic parameters affected by Walker tumor
Daniele R. Miksza et al.
Fig. 4. Glycemia (A) and changes of glycemia in % of baseline (B),
following insulin administration in 24 h fasted healthy rats and
Walker-256 tumor-bearing rats, treated with infliximab (WK + inflix)
during 12 days or untreated (WK). Glycemia was measured at 0
(baseline), 5, 10, 15 and 30 min after the injection ( ) of insulin
(0.25 U/kg). Data are the mean ± SEM of 9-11 experiments. Differences within the same group relative to the baseline (A): * p 0.05,
*** p 0.001 (Friedman followed by Dunn’s tests). Differences between groups at fixed time (B): ** p 0.01, *** p 0.001 healthy
rats (ANOVA One-Way followed by Dunnett’s test)
iv
<
<
<
<
vs.
with infliximab for 12 days did not influence the
growth of the Walker-256 tumor (Fig. 1). In agreement with our results, the therapy involving other
anti-TNFa agents did not change the growth of the
Yoshida AH-130 tumor [15]. On the other hand, these
agents decreased the tumor growth induced by methylcholanthrene [21].
In spite of the fact that infliximab treatment did not
reduce the Walker-256 tumor growth, this drug decreased the pronounced fall in body weight (Fig. 2A)
and retroperitoneal fat weight (Fig. 2B) induced by
the tumor. The weight loss in cancer cachexia is due,
in large part, to the depletion of adipose tissue [3, 4,
45] and TNFa is considered the main mediator of this
process. This cytokine diminishes the mass of adipose
tissue by inhibiting lipogenesis and adipogenesis [55]
and by stimulating lipolysis [48] and apoptosis of adipocytes and preadipocytes [40, 55]. Although lipidmobilizing factor (LMF), produced by the tumor, may
be another mediator involved in the loss of adipose
tissue [47], the effect of infliximab preventing the loss
of fat mass suggests that the TNFa is the main mediator of this change in Walker-256 tumor bearing rats.
Corroborating our results, treatment with other
anti-TNFa agents diminished the weight loss caused
by lung adenocarcinoma or methylcholanthrene-induced sarcoma in mice, by reducing the depletion of
adipose tissue [43]. On the other hand, infliximab was
ineffective in the treatment of cachexia in patients
with advanced cancer of the pancreas [56] or in elderly patients with lung cancer [28].
Besides reducing the fall in body weight and preventing the loss of retroperitoneal adipose tissue, the
infliximab treatment tended to attenuate the tumorinduced loss of gastrocnemius muscle mass (Fig. 2C),
reduction in food intake (Fig. 2D) and rise in blood
triacylglycerol (Fig. 3A). Infliximab treatment also
tended to attenuate the decrease in the peripheral response to insulin (Fig. 4) and liver gluconeogenesis
(Fig. 5). The lack of significant effect of infliximab on
the reduction in skeletal mass induced by the tumor
(Fig. 2C) is consistent with its discreet effect on body
weight loss (Fig. 2A).
The overall lack of significant effect of infliximab
on the metabolic changes induced by the Walker-256
tumor suggests that TNFa is not the only mediator of
these changes in this model of cancer. In fact, the
metabolic disorders observed in cancer patients are of
multifactorial origin. The reduction of skeletal muscle
mass has been attributed not only to TNFa but also to
other cytokines (IL1, IL6 and INFg), proteolysisinducing factor (PIF) and toxohormones produced by
the tumor [34] which can inhibit protein synthesis, increase proteolysis [45, 48] and cell apoptosis [4].
Tumor-induced anorexia has been related to TNFa,
IL1 and IL6, all of which can inhibit the release of
neuropeptide Y (NPY) or stimulate the production of
corticotropin-releasing factor (CRF) in the hypothalamus [4, 6]. The raised triacylglycerol level has been
related to TNFa, IL6, IL1a, INFa and INFg and to
the increased blood concentration of free fatty acids
(FFA), which can inhibit lipoprotein lipase [18, 48,
55]. Insulin resistance has been related to TNFa, IL6
[30, 32, 39, 47, 60] and to the increased FFA in the
blood [18] all of which can influence the insulin sigPharmacological Reports, 2013, 65, 960–969
965
Production of glucose (A), lactate (B), pyruvate (C) and urea (D) and the respective areas under the curve (AUCs) in livers of 24 h fasted
healthy rats and Walker-256 tumor-bearing rats, treated with infliximab (WK + inflix) during 12 days or untreated (WK). Rat livers were submitted
to
perfusion as described in Materials and Methods. Alanine (2.5 mM) was infused between 10 and 40 min. Data are the mean ± SEM of
5–8 experiments. * p 0.05, ** p 0.01 healthy rats (ANOVA One-Way followed by Newman-Keuls or Kruskal Wallis followed by Dunn’s
tests)
Fig. 5.
in situ
<
<
vs.
naling. The inhibition of the hepatic gluconeogenesis
in Walker-256 tumor-bearing rats, indicated by the reduction of alanine catabolism (Fig. 5), also seems to
be related to cytokines. The administration of TNFa
and IL1b inhibited gluconeogenesis from alanine in
isolated perfused livers [31] and TNFa inhibited gluconeogenesis from lactate in hepatocyte culture [14].
Furthermore, TNFa [14], IL1b and IL10 [38, 59] reduced the activity of phosphoenolpyruvate carboxykinase (PEPCK).
In contrast with the discrete effect of infliximab
treatment on skeletal muscle (Fig. 2C) and food intake
(Fig. 2D), some other anti-TNFa agents have been
found to diminish the rate of protein breakdown and
loss of gastrocnemius muscle in rats bearing the
Yoshida AH-130 ascites hepatoma [15] and augment
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Pharmacological Reports, 2013, 65, 960–969
food intake in tumor-bearing anorexic rats [44, 49]. In
addition, neutralization of TNFa enhanced the autophosphorylation of the insulin receptor (IR) and the
phosphorylation of the IR substrate 1 (IRS-1), two important steps in the insulin signaling cascade, in both
adipose tissue and muscle [27]. This result is consistent
with the observations that infliximab promoted better
glycemic control in patients with insulin resistance [23,
58], improved the transduction of the insulin signal [9],
restored glucose homeostasis [2] and suppressed the
liver glucose overproduction in an animal model of insulin resistance induced by a high-fat diet [26].
Therefore, the lack of significant effect of infliximab on the metabolic parameters affected by the
Walker-256 tumor probably was not due to an insufficient dose of this antibody, since similar doses were
Infliximab on metabolic parameters affected by Walker tumor
Daniele R. Miksza et al.
Production of lactate (A) and pyruvate (B) and glycolysis (C)
and the respective areas under the curve (AUCs) in livers of 24 h
fasted healthy rats and Walker-256 tumor-bearing rats, treated with
infliximab (WK + inflix) during 12 days or untreated (WK). Rat livers
were submitted to
perfusion as described in Materials and
Methods. Glucose (20 mM) was infused between 10 and 40 min.
Data are the mean ± SEM of 5–10 experiments. * p 0.05, *** p
0.001 healthy rats (Kruskal Wallis followed by Dunn’s tests)
Fig. 6.
bearing rats. This lack of effect of infliximab suggests
that TNFa is not involved in these metabolic changes
induced by Walker-256 tumor.
The reduced rate of liver glycolysis in Walker-256
tumor-bearing rats seems to be related to inhibition of
glucokinase [54], an enzyme that phosphorylates glucose that has entered the hepatocyte favoring their
further uptake. A fall in glucokinase activity could be
due to decreased hepatic response to insulin or to low
blood insulin levels of tumor-bearing rats [19]. On the
other hand, the fall in glycemia and the rise of blood
lactate in tumor-bearing rats appear to be associated
with tumor cell metabolism. Fast-growing tumors
such as the Walker-256 become hypoxic and tend to
use the glycolytic pathway to produce energy, increasing greatly the consumption of glucose and the production of lactate [11, 35]. Similarly, it is possible that
the production of urea by the Walker-256 cells [41]
contributed significantly to the raised uremia in
tumor-bearing rats. The complete lack of effect of infliximab on the altered blood glucose, lactate and urea
levels is thus consistent with the inability of this antiTNFa treatment to slow tumor growth.
In conclusion, the infliximab treatment ameliorated
the fall in body weight and loss of adipose tissue and
tended to attenuate some other metabolic changes induced by the tumor. These results suggest that TNFa
plays a significant role in mediating the changes in adipose tissue and body weight elicited by tumor growth.
The discrete effect (not significant) and the inability of
infliximab treatment to prevent the metabolic changes
induced by the Walker-256 tumor imply that other mediators or tumor-related events are involved in these
metabolic disorders. Therefore, our results furnish new
information on the role of TNFa in the pathogenesis of
weight loss, depletion of body fat and other metabolic
disorders associated with the Walker-256 tumor.
in situ
<
<
vs.
effective in reducing TNFa levels [9, 57] and completely reverted some of these metabolic changes in
experimental models unrelated to cancer [2, 9, 26].
Finally, the infliximab treatment did not attenuate
the marked reduction of liver glycolysis (Fig. 6) and
glycemia (Fig. 3B), or the rise in the levels of lactate
(Fig. 3C) and urea (Fig. 3D) in the blood of tumor-
Acknowledgment:
This work was supported by Fundação Araucária.
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Received:
accepted:
July 5, 2012; in revised form: February 14, 2013;
March 11, 2013.
Pharmacological Reports, 2013, 65, 960–969
969