Interleukin-1 receptor antagonist (IL-1ra) is unable to reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130)

ELSEVIER

CancerLetters95 (1995)33-38

CANCER LETTERS

Interleukin-1 receptor antagonist (IL-lra) is unable to reverse cachexia in rats bearing an ascites hepatoma (Yoshida AH-130) Paola Costellia,Marta Lloverab,Neus Carb$, Cdia Garcia-Martinezb, Francisco J. L6pez-Sorianoqb,JosepM. ArgilW* aDipartimento di Medicina ed Oncologia Sperimentale, Sezione Patologia Generule, Universitd di Torino, Torino, Italy bDepartament de Bioquhica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain

Received14October1995;revisionreceived30May 1995;accepted30 May 1995

Abstract

The mechanismsleading to the developmentof cancer cachexia are still poorly understood. Recently, cytokines such as interleukin 1 and tumour necrosis factor-a have been involved as mediators of the tissue wasting consequent to tumour growth. The rat asciteshepatomaYoshida AH-130 is a highly anaplastictumour that causesin the host an early and marked depletion of both the skeletal muscle and the adiposetissue, mainly accountedfor by a hypercatabolic state.Profound hormonal alterations and the releaseof tumour necrosis factora and interleukin 1 by the tumour cells likely concur in forcing the metabolic balancetowards the catabolic side [ 11.In order to possibly achieve the correction of this wasting condition, the AH-130 bearing rats were administereda daily S.C.dose of interleukin 1 receptor antagonist (IL-lra; 2 mg/kg). This factor, however, was completely ineffective in either inhibiting tumour proliferation or in preventing the consequenttissue depletion and protein hypercatabolism.These observationssuggestthat interleukin 1 is not important, at least in this model system,for either the developmentof cachexia or tumour growth. Keywords: Cachexia; Tumour growth; Tumour necrosisfactor-a; Interleukin 1

1. Introduction Neoplastic patients frequently show a progressive wasting diathesis leading to body compositional changes associated with severe metabolic derangements. This syndrome, known as cachexia, is so * Correspondingauthor,Unitat de Bioquimicai Biologia Molecular B, Departamentde Bioquimicai Fisiologia,Facultatde Biologia, Universitat de Barcelona, Diagonal 645, 08071Barcelona,Spain.

common that 50% of the cancer patients show signs of it at the time of initial diagnosis. The hallmark of cancer cachexia is body weight loss, due to depletion of both skeletal muscle and adipose tissue [2]; this wasting pattern is mainly associated with a general hypercatabolic state [2,3]. Weight-losing cancer patients have a significantly reduced survival following cancer treatment when compared to non weight-losing individuals [4,5], thus it is important to design therapeutic strategies able to prevent tissue wasting.

0304-3835/95/$09.500 1995ElsevierScienceIrelandLtd. All rightsreserved SSDI 0304-3835(95)03858-T

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P. Costelli et al. I Cancer Letters 95 (1995) 33-38

Since the discovery of the parabiotic transfer of cachexia [6], a lot of work has been done to identify the humoral mediators involved in the development of tissue wasting [7-l 11. In the last decade, interest has focused on cytokines, a class of factors with regulatory function in both the immune and inflammatory responses [ 10,121. The growth of the Yoshida ascites hepatoma AH130 elicits in the host rat a rapid and progressive wasting pattern, particularly evident for the skeletal muscle and the adipose tissue [1,13-1.51. High rates of protein catabolism mainly account for muscle protein depletion [ 13,151. These alterations seem to be associated with detectable circulating tumour necrosis factor-a (TNF). In addition, we have previously demonstrated by blocking TNF in vivo by means of a polyclonal anti-murine specific antibody [ 161, that this cytokine is responsible, at least in part, for the enhanced muscle protein degradation encountered in rats bearing the AH- 130 hepatoma. However, in vitro studies have also shown that the cytokine has no direct action on protein turnover in incubated skeletal muscle [17]. In addition, the changes observed in the hormonal milieu of these animals, such as a hyperglucocorticoid state, do not seem to be responsible for the elevated muscle proteolytic rates, either [ 181. Bearing all these facts in mind, it was our aim to see if the indirect action of TNF on skeletal muscle proteolysis was mediated by another cytokine such as IL-l. This latter has also been reported to be involved in protein degradation [19]. In addition the AH-130 hepatoma cells themselves are able to release IL- 1. The experimental approach used to evaluate the role of IL-1 in mediating the TNF effects in the Yoshida AH-130 hosts was the administration of IL1 receptor antagonist (IL-lra), a protein which competes with IL-l for binding to the receptor, but is unable to activate the signal transduction pathway. 2. Materials and methods 2.1. Animals, tumour inoculation and treatment The experiment was performed using male Wistar rats (Interfauna, Barcelona, Spain) weighing about 100 g. They were maintained on a regular light-dark cycle (lights on from 0800 h to 2000 h) and had free access to food (Panlab, Barcelona, Spain) and water.

The tumour-bearing rats were inoculated intraperitoneally with lo* AH-130 Yoshida ascites hepatoma cells obtained from exponential tumours (for details see Ref. [13]). Half were then administered a daily S.C.dose of IL-lra (2 mglkg body wt., dissolved in physiological solution), while the other half received a corresponding volume of solvent. On days 0 and 4 after tumour transplantation the animals were weighed and anaesthetized. The tumour volume and cellularity was evaluated, and the blood was collected from the abdominal aorta into heparinized tubes and centrifuged (3500 x g, 10 min, 4’C) to obtain plasma. Many tissues were rapidly excised and weighed (cf. Ref. [15]; for the adipose tissues, cf. Ref. [20]); the gastrocnemius muscle was frozen in liquid nitrogen and stored at -80°C for protein turnover evaluation. 2.2. Protein turnover Protein turnover rates were determined as previously described [ 13,151. Briefly, the rats received an i.p. dose of sodium [14C]bicarbonate (250@Zilkg body wt.) 24 h before tumour transplantation. They were then killed at days 0 and 4 after the inoculum of the tumour, and the total and specific radioactivity determined. Fractional rates of protein synthesis (k,), degradation (kd), and accumulation (k,) were calculated as follows: kd = ln(tota1 protein radioactivity)lt k, = ln(specific protein radioactivity)lt k, = ln(tota1 protein)lt and expressed as %/day. Tissue protein was determined by the method of Lowry et al. [21], using bovine serum albumin as standard. For short term culture, the AH- 130 cells were harvested from the animals under sterile conditions, washed, resuspended (lo6 cells/ml) in DMEM containing 10% FCS, and incubated for 24 h at 37°C in an atmosphere of 5% CO2 and 95% 0,. In the middle of the incubation period, [3H]thymidine (10 &i/ml) was added to the culture medium. The cells were then harvested and washed with phosphate-buffered saline (PBS). The acid insoluble radioactivity incorporated into DNA was then meas-

P. Cosrelli et al. I Cancer Letters 95 (1995) 33-38

ured (cf. [ 131). DNA content was evaluated according to Boer [22], using herring sperm DNA as standard.

2.3. Data presentation Data are given as means + SD. Student’s t-test was used to calculate the significance of differences. For the fractional rates of protein turnover, significance of difference was calculated by analysis of variance on linear regressions [23].

2.4. Chemicals All reagents were obtained from Panreac (Barcelona, Spain), bovine serum albumin and herring sperm DNA from Sigma (St. Louis, MO, USA), sodium [14C]bicarbonate (53 mCi/mmol) and [3H]thymidine (85 Ci/mmol) from New England Nuclear (Boston, MA, USA). IL-lra was kindly provided by Dr. R.C. Thompson, Synergen, Boulder, CO, USA.

3.Results Table 1 showed the effect of IL-lra on body, carcass, and tissue weight. The AH-130 growth caused rapid carcass and body weight loss, which is paralleled by a decrease of gastrocnemius, soleus, and heart mass. In this experiment, probably because of Table 1 Effect of IL-

I ra on body and tissue weightin AH- 130 hosts

Initial b.w. (g) Final b.w. (g) carcass (g) Gastrocnemius Soleus Heart Liver Spleen Kidneys Adrenals WAT IBAT

Controls

AH-130 hosts

AH-130 hosts + IL-ln

102 k 10 133 * 12 92 f 0.4 625 f 61 48.1 f 4.1 535 f 40 6350+318 517+64 1192+39 25.5 f 1.2 485 f 100 234 + 23

108*8 121+ 12* 80 + 6** 514 f 45** 43.6 + 3.4** 431 f 35** 5764i436 351 f 39** 913 + 42** 22.6 zt 2.0** 504i93 164+27**

104i4 109 * 3** 82 f 4** 533 f 50** 40.2 + 1.9** 432 i 26** 5265?318** 296 + 22** 858*31** 26.4 + 3.9*** 421 f 82 183+38**

Data, expressed as mg (or g when indicated) per 100 g initial body weight, are means + SD. Significance of the differences: **P c 0.01 versus controls, ***P < 0.01 versus untreated AH-130 hosts; n = 5.

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the single time point selected, it was not possible to observe the transient hyperplastic/hypertrophic pattern usually shown by the parenchymal organs (liver, spleen, kidneys, and adrenals; cf. [13,15]), which were already reducing in size. The brown adipose tissue (IBAT) showed a degree of wasting comparable to that of the skeletal muscle, while the white adipose tissue (WAT) was not different from controls, further confirming our hypothesis that cancer cachexia is, at least at the very beginning, depletion of protein mass. The treatment with the IL-lra was not able to correct any of the alterations described above; the muscle examined, as well as the parenchymal organs and the IBAT, showed a weight loss similar to that of the untreated group (Table 1). Adrenals were the only exception, their weight becoming comparable to that of controls (Table 1). Muscle waste in the AH-130 bearing rats was mainly achieved through acceleration of protein catabolism rates, with virtually no changes in the protein synthesis rates (Table 2; cf. [13,15]). This unbalance resulted in negative protein accumulation rates (Fig. 1). The administration of the IL-lra to the tumour hosts did not modify the acceleration of muscle protein turnover observed in the untreated AH-l 30 bearers (Table 2, Fig. 1). The Yoshida ascites hepatoma AH- 130 cells in short term culture release IL- 1 and TNF [ 11.This fact led to the hypothesis that these cytokines may either function as autocrine growth factors for the AH-130 cells or have been produced by the cells in order to magnify the mobilization of substrates from the host tissues. However, when the AH-130 cells were cultured in the presence of escalating doses of IL-lra, the incorporation of [3H]thymidine into DNA was quite comparable among the doses selected (Fig. 2). Consistently, when the IL-lra was administered to Table 2 Gastrocnemius protein turnover

Controls AH-130 hosts AH-130 hosts + IL-lra

ks

kd

14.8 14.0 12.8

1.33 4.81* 3.61*

Fractional rates of protein synthesis (k,) and degradation (kd) are expressed as %/day. Significance of the differences: *P < 0.05 versus controls.

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P. Costelli et al. I Cancer Letters 95 (1995) 33-38 2500 --

1

AH-130

AH-130 + IL.-1ra

AH-130

An-130 + n-t,*

Fig. 3. AH-130 tumour volume and cellularity after in vivo treatment with the IL-lra. For details see Section 2.

AH-130

CO?MOlS

AH-130 + IL-1ra

Fig. 1. Gastrocnemiusprotein accumulation rates (k,) after treatment with the IL-lra. Significance of the differences: *p < 0.05 versus controls. For details see Section 2.

the AH- 130 bearing rats, no differences as for tumour volume and cellularity were observed (Fig. 3). 4. Discussion The role of IL-1 in mediating muscle protein catabolism has long been investigated, either in vitro or in vivo. An early report [24] indicated this cytokine as the factor responsible for the enhanced protein degradation observed when rat muscles were incubated in the presence of a purified macrophage culture supernatant. The same authors, however, were not able to confirm this observation using recombinant cytokines [25]. Repeated administrations of IL-l to rats induced significant anorexia, weight loss, and 1000

2

1 750 -

0 if 03% 2

500 -

x E b

250 -

0 0

0.5

1 IL-lra

10

20

200

Inglmll

Fig. 2. [3H]Thymidine incorporation into DNA in AH-130 cells in short term culture in the presence of escalating doses of IL-lra. For details see Section 2.

loss of body protein [26]. The observations concerning the presence of IL-l in the serum of cancer patients are inconsistent, however. Moldawer et al. [27] could not detect any IL-1 bioactivity in the plasma of weight-losing cancer patients. Similarly, we failed to detect any circulating IL-l in the AH-130 hosts, despite the fact that the tumour cells themselves release this factor [l]. In both these reports, however, the presence of biological inhibitors has not been taken into account, thus it is not really possible to exclude that IL-l is circulating. Other workers have made partially similar observations: TNF and IL-1 are produced by a tumour of non-lymphoid origin, the undifferentiated sarcoma MCG 101, which causes cachexia in mice and elicits an acute phase response, yet no plasma elevation of either cytokine could be demonstrated [28]. Recently, moreover, the local action of cytokines such as TNF and IL-1 has been gaining growing importance with respect to the systemic one. Bearing all these data in mind, we tested the effectiveness of IL-lra in rats bearing the ascites hepatoma AH-130, in order to identify whether or not IL1 was involved in the development of tissue waste, likely mediating TNF catabolic stimuli. The IL-lra is a pure antagonist that binds competitively to both type I and II IL-I receptors, and blocks the action of IL-l without exerting agonist activity [29-311. The in vivo activity of IL-lra was investigated in mice. In one study it was found that IL-lra inhibited the IL-l induced increase of corticosteroid levels [32]; in another study it was shown that the IL-lra was effective in blocking many in vivo inflammatory responses such as accumulation of polymorphonuclear leukocytes, elevation of serum concentrations of IL-6, and acute phase proteins [33]. Beneficial effects of the IL-lra have been observed in the treatment of rheumatoid arthritis, type I diabe-

P. Costelli et ul. I Cancer Letters 95 (1995) 33-38

tes, systemic lupus erythematosus, chronic myeloid leukemias [30]. The IL-lra has been shown to reduce the number of hepatic metastasis and their growth rate in mice bearing the B16 melanoma [34], and to decrease the enhanced tyrosine and 3-methylhistidine release from the incubated muscle of endotoxemic rats [ 351. The AH-130 tumour rapidly induces in the host a general hypercatabolic state, associated with marked hormonal derangements [ 1,14,18]. The multifactorial pathogenesis of this syndrome, at least in the AH130 model, has been previously demonstrated, since different kinds of treatment (anti-TNF antibodies, insulin, leupeptin, aspirin, clenbuterol), are able to prevent the development of tissue wasting and the acceleration of muscle protein degradation [ 16,18, 361. By contrast, the administration of IL-lra to the AH- 130 hosts did not result in the prevention of either tissue waste or muscle protein hypercatabolism. It is now well known that many cell types, either normal or neoplastic, release cytokines such as IL-l and/or TNF [1,28,37,38]. While the existence of an autocrine loop has been demonstrated for IL-2 and T cell clone expansion, it has not been clearly demonstrated for IL-6 and myeloma cells. The fact that the AH- 130 cells were able to release both TNF and IL- 1 in short term cultures suggested the possibility that an autocrine loop could also be identified in this case. However, culturing these cells in the presence of escalating doses of IL-lra did not result in a reduced incorporation of [3H]thymidine into DNA. Consistently, tumour growth was not reduced after in vivo treatment either. A quite comparable result was obtained treating the AH-130 hosts with a polyclonal anti-TNF antibody [16]. The lack of effect of either the IL-lra or the anti-TNF antibody in inhibiting tumour proliferation, however, did not exclude the involvement of both cytokines in regulating the AH130 growth. Little is known, in fact, about the possible effects exerted by these factors inside the cells, before they enter the secretion pathway. From these data it seems possible to conclude that IL- 1 is not the mediator through which TNF exerts its hypercatabolic effect on muscle protein, at least in the AH-130 model system. It is not likely, however, that the IL- 1 released by the tumour cells and by the immune system [l] is not exerting any biological effect. Other factors could mimic the IL-l bioactivity

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when it is blocked by the receptor antagonist, thus resulting in an apparent lack of effect of the IL-lra. Acknowledgements The authors would like to thank Dr. Robert C. Thompson, Synergen, Boulder, CO, USA, for kindly providing the IL-lra. Work supported by grants from the Fondo de Investigaciones Sanitarias de la Seguridad Social (94-1048) of the Spanish Health Ministry, the DGICYT (PB90-0497) the Spanish Ministry of Education and Science, the Minister0 dell’Universita e della Ricerca Scientifica e Tecnologica (60% and 40% funds), Roma, the Consiglio Nazionale delle Ricerche (Special project ACRO), Roma, the Associazione Italiana per la Ricerca sul Cancro, Milano. P.C. received a fellowship from the EC. References [l]

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