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Research in Nursing & Health, 2005, 28, 48–55 Conjugated Linoleic Acid Preserves Gastrocnemius Muscle Mass in Mice Bearing the Colon-26 Adenocarcinoma Erin Graves, 1 * Andrew Hitt, 1 * Michael W. Pariza, 2 Mark E. Cook, 2 Donna O. McCarthy 1z 1 National Institute of Nursing Research, NIH, Bethesda, MD 2 College of Agriculture, University of Wisconsin-Madison, WI Accepted 29 September 2004 Abstract: Cancer cachexia is a syndrome of weight loss, muscle wasting, fatigue, and anorexia that occurs in patients with advanced or recurrent solid tumor disease. Tumor necrosis factor-alpha (TNFa) and prostaglandin E2 (PGE2) have been implicated in the biology of cachexia and serve as possible targets for treatment of this condition. Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid that alters the synthesis of PGE2 and reduces the negative effects of TNF on body weight of healthy mice. We hypothesized that a diet supplemented with .5% CLA might reduce muscle wasting in mice bearing the colon-26 adenocarcinoma, an animal model of cancer cachexia. CLA preserved gastrocnemius muscle mass and reduced TNF receptors in muscle of tumor-bearing mice. These data suggest that CLA may preserve muscle mass by reducing the catabolic effects of TNF on skeletal muscle. ß 2004 Wiley Periodicals, Inc. Res Nurs Health 28:48–55, 2005 Keywords: colon-26 adenocarcinoma; cancer cachexia; mice; tumor necrosis factor; conjugated linoleic acid Cancer cachexia is a syndrome of progressive weight loss, skeletal muscle wasting, fatigue, and anorexia that occurs in patients with advanced or recurrent malignancies. Cachexia negatively affects quality of life and duration of survival in cancer patients (O’Gorman, McMillan, & McArdle, 2000). While our understanding of the biology of cancer cachexia is incomplete, there is clear evidence that pro-inflammatory cytokines, such as tumor necrosis factor-alpha (TNFa) and interleukin-6 (IL-6), are associated with the onset and progression of cancer cachexia (Argiles, Moore-Carrasco, Fuster, Busquets, & Lopez- Soriano, 2003). More recently, researchers have implicated the metabolism of arachidonic acid (AA) to prostaglandin E2 (PGE2) by cyclooxy- genase (COX) in the pathophysiology of cancer cachexia (Cahlin et al., 2000; Ross & Fearon, 2002). There are two isoforms of COX, which is the rate-limiting enzyme in the synthesis of PGE2 from AA. COX1 is constitutively expressed in cells, and expression of COX2 is increased by pro- inflammatory stimuli such as TNF (Perkins & Kniss, 1997; Said et al., 2002). PGE2, in turn, increases cell synthesis of pro-inflammatory cytokines (Williams, Pontzer, & Shacter, 2000). Treatment with nonspecific inhibitors of both Correspondence to Donna O. McCarthy, Room K6-326, 600 Highland Avenue, Madison, WI 53792-2455. *Research Technician. z Scientist. Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/nur.20052 48 ß 2004 Wiley Periodicals, Inc.

Conjugated linoleic acid preserves gastrocnemius muscle mass in mice bearing the colon-26 adenocarcinoma

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Research in Nursing & Health, 2005, 28, 48–55

Conjugated Linoleic AcidPreserves Gastrocnemius

Muscle Mass in Mice Bearingthe Colon-26 Adenocarcinoma

Erin Graves,1* Andrew Hitt,1* Michael W. Pariza,2 Mark E. Cook,2

Donna O. McCarthy1z

1National Institute of Nursing Research, NIH, Bethesda, MD2College of Agriculture, University of Wisconsin-Madison, WI

Accepted 29 September 2004

Abstract: Cancer cachexia is a syndrome of weight loss, muscle wasting,fatigue, and anorexia that occurs in patients with advanced or recurrent solidtumor disease. Tumor necrosis factor-alpha (TNFa) and prostaglandin E2(PGE2) have been implicated in the biology of cachexia and serve as possibletargets for treatment of this condition. Conjugated linoleic acid (CLA) is apolyunsaturated fatty acid that alters the synthesis of PGE2 and reduces thenegative effects of TNF on body weight of healthy mice. We hypothesized thata diet supplemented with .5% CLA might reduce muscle wasting in micebearing the colon-26 adenocarcinoma, an animal model of cancer cachexia.CLA preserved gastrocnemius muscle mass and reduced TNF receptors inmuscle of tumor-bearing mice. These data suggest that CLA may preservemuscle mass by reducing the catabolic effects of TNF on skeletal muscle.� 2004 Wiley Periodicals, Inc. Res Nurs Health 28:48–55, 2005

Keywords: colon-26 adenocarcinoma; cancer cachexia; mice; tumor necrosis factor;conjugated linoleic acid

Cancer cachexia is a syndrome of progressiveweight loss, skeletal muscle wasting, fatigue, andanorexia that occurs in patients with advancedor recurrent malignancies. Cachexia negativelyaffects quality of life and duration of survivalin cancer patients (O’Gorman, McMillan, &McArdle, 2000). While our understanding of thebiology of cancer cachexia is incomplete, there isclear evidence that pro-inflammatory cytokines,such as tumor necrosis factor-alpha (TNFa)and interleukin-6 (IL-6), are associated with theonset and progression of cancer cachexia (Argiles,Moore-Carrasco, Fuster, Busquets, & Lopez-Soriano, 2003). More recently, researchers have

implicated the metabolism of arachidonic acid(AA) to prostaglandin E2 (PGE2) by cyclooxy-genase (COX) in the pathophysiology of cancercachexia (Cahlin et al., 2000; Ross & Fearon,2002).

There are two isoforms of COX, which is therate-limiting enzyme in the synthesis of PGE2from AA. COX1 is constitutively expressed incells, and expression of COX2 is increased by pro-inflammatory stimuli such as TNF (Perkins &Kniss, 1997; Said et al., 2002). PGE2, in turn,increases cell synthesis of pro-inflammatorycytokines (Williams, Pontzer, & Shacter, 2000).Treatment with nonspecific inhibitors of both

Correspondence to Donna O. McCarthy, Room K6-326, 600 Highland Avenue,Madison, WI 53792-2455.

*Research Technician.zScientist.Published online in Wiley InterScience (www.interscience.wiley.com)

DOI: 10.1002/nur.20052

48 �2004 Wiley Periodicals, Inc.

isoforms of COX or with specific inhibitors ofCOX2 activity has been shown to slow thedevelopment of muscle wasting and weight lossin some animal models of cancer cachexia (Daviset al., 2004; Hussey & Tisdale, 2000; McCarthy,Whitney, Hitt, & Al-Majid, 2004; Strelkov, Fields,& Baracos, 1989) and to prolong survival andimprove functional status of cancer patients(Lundholm et al., 1994; McMillan et al., 1999).

Conjugated linoleic acid (CLA) describesgeometric and positional isomers of linoleic acid,an essential 18-carbon polyunsaturated fatty acid(PUFA) that is a precursor for synthesis of AA, a20 carbon PUFA. CLA is normally consumed inthe fat of red meat and dairy products. CLA, likeAA, is incorporated into the phospholipids of cellmembranes (Burdge et al., 2004). Supplementalintake of CLA reduces the relative ratio of AA toother fatty acids in the membrane (Ostrowska,Cross, Muralitharan, Bauman, & Dunshea, 2003)and may affect the efficiency with which COX1and COX2 metabolize AA to PGE2 (Belury, 2002;Levin et al., 2002). Tissues from antigen-sensi-tized animals fed CLA produce less PGE2 whenstimulated with antigen ex vivo (Whigham et al.,2001; Whigham et al., 2002). Cells incubated inmedium containing CLA express less COX2 andproduce less PGE2 when stimulated with lipopo-lysaccharide (LPS), a potent stimulus of TNFsynthesis (W.L. Cheng, Lii, Chen, Lin, & Liu,2004). Similarly, cells incubated with CLAproduced less TNF when stimulated with LPS(Yang & Cook, 2003b). Thus, CLA may beincorporated into the cell membrane reducingthe relative ratio of AA in membrane lipids, andsubsequently diminishing the pro-inflammatoryeffects of PGE2 and TNF.

Animal models may be useful in understandingthe biology of human clinical phenomena, and canbe used for developing or testing interventions thatmay be beneficial in treatment of the clinicalcondition. TNF was previously called cachectinbecause it was found in serum of tumor-bearinganimals with progressive weight loss and musclewasting. Purified forms of this serum factorinduced weight loss and muscle wasting wheninjected into healthy animals. However, mice fed adiet enriched with .5% CLA lost less weight wheninjected with TNF (Pariza, Park, & Cook, 2000) orwith LPS (C.C. Miller, Park, Pariza, & Cook,1994) and had lower serum levels of TNF fol-lowing injection of LPS (Yang & Cook, 2003b).These data suggest that the beneficial effects ofCLA on body weight following injection withTNF or LPS may be the result of decreased cellularresponses to TNF (Pariza et al.). In a mouse model

of spontaneous systemic lupus erythematosis,end-stage wasting of body mass was preventedand survival was prolonged in NZB/W F1 mice fed.5% CLA (Yang, Pariza, & Cook, 2000; Yang &Cook, 2003a). Thus, CLA might be beneficial inthe treatment of cancer cachexia.

The colon-26 adenocarcinoma is a murinetumor cell line that expresses COX2 and inducessignificant muscle wasting in tumor-bearinganimals without affecting food intake (Al-Majid& McCarthy, 2001; Zhou et al., 2003). Treat-ment with indomethacin, a nonspecific inhibitor ofCOX1 and COX2 activity, slowed weight loss andskeletal muscle wasting (McCarthy et al., 2004)and lowered serum levels of PGE2 (Tanaka,Tanaka, & Ishitsuka, 1989), TNF (Zhou et al.),and IL-6 (Davis et al., 2004) in this animal modelof cancer cachexia. We hypothesized that a dietcontaining .5% CLA might preserve body weightand gastrocnemius (gastroc) muscle mass intumor-bearing animals. We also examined theeffects of CLA on levels of TNF and TNF re-ceptor type I (TNFR1) in the gastroc musclehomogenates.

METHODS

Animal Model of Cancer Cachexia

The colon-26 adenocarcinoma was maintained inculture, and cells were harvested when confluentusing 1% trypsin. CLA was obtained from LodersCroklaan (Channahon, IL). Thirty-six pathogen-free female CD2F1 mice weighing 20–22 g werehoused three to a cage at 24�C and maintained on a12-hour light cycle (6 a.m. to 6 p.m.) with adlibitum access to food and water. This tumor cellline produces a similar degree of cachexia in maleand female mice (Davis et al., 2004); females wereused in this experiment because they have a slowergrowth curve and therefore are less susceptible tothe effects of diet on body weight gain. Othershave noted that the effects of CLA on lean bodymass are greater in males than in females (Park,Storkson, Albright, Liu, & Pariza, 1999). The useof female animals in the present study shouldprovide a more robust test of the effects of CLA onskeletal muscle wasting of cancer cachexia.

Two weeks before inoculation of tumor cells,12 animals were started on pulverized rodent chowsupplemented with .5% CLA (.5 mL/100 g diet),and 24 were maintained on pulverized chow withno added CLA (control diet). Body weights wereobtained weekly, and food intake of the three mice

CLA AND CACHEXIA/GRAVES ET AL. 49

per cage was measured three times a week. Twoweeks later, six of the mice on the CLA-supplemented diet and 12 of the animals on thecontrol diet were inoculated between the scapulaewith 5� 105 colon-26 adenocarcinoma cells in.1 mL phosphate buffered saline. Six of the tumor-inoculated animals on the control diet wereswitched to the diet supplemented with .5%CLA as were six animals without tumor. Oncestarted on the CLA (day�14 or day 0), animalswere maintained on the CLA supplemented dietfor the duration of the experiment. Twelveanimals, six tumor-bearing and six controls, weremaintained on the control diet for the duration ofthe experiment. Thus, there were six groups of sixanimals each; three groups were inoculated withtumor cells, and three served as healthy controlgroups (see Table 1). One group each of tumor-bearing and control animals was started on .5%CLA in the diet 14 days before inoculation oftumor cells (DTD, DtD), one group each wasstarted on CLA at the time of inoculation (dTD,dtD), and one group each of tumor-bearing andhealthy control animals was maintained on thecontrol diet (dTd, dtd) for the duration of theexperiment.

On day 21 of tumor growth, the animals wereeuthanized by cervical dislocation and weighed.The right and left gastroc muscles were removed,individually weighed, wrapped in aluminum foil,and buried in dry ice. The tumors were removedand weighed and did not exceed 5% of bodyweight in any of the animals. One animal in thedTD group did not develop a tumor and wassubsequently eliminated from the experiment.All muscles were stored at �80�C until analysis.Muscle mass was determined as the averageweight of the right and left gastroc muscles andis reported as mean muscle weight relative tobody weight. Data were analyzed using two-factor(tumor, diet) analysis of variance. When signi-

ficant main effects were found, the treatmentgroups were compared using one-way analysisof variance and Duncan’s post hoc pair wisecomparisons.

Gastroc Muscle Homogenates

The right gastroc from each animal was homo-genized in 1 mL of 50 mM Tris-HCl, 5 mM EDTA,1% (w/v) SDS, 0.3 mM aprotinin, 1 mM pepstatinA, 1 mM PMSF (phenylmethysulfonyl fluoride),and 1 mL phosphatase inhibitor cocktail I (Sigma,St. Louis, MO) per 100 mL of total homoge-nization buffer. The muscles were individuallyhomogenized in ice-cold homogenization bufferusing a PowerGen 125 tissue homogenizer (FisherScientific, Suwanee, GA). The homogenates werecentrifuged at 14 000g for 20 minutes at 4�C. Thesupernatants were quickly aliquoted into 50 mLvolumes and stored at �80�C. Protein concentra-tion of the supernatant (mg/mL) was determinedin duplicate using the DC protein assay kit ac-cording to manufacturer’s instructions (Bio-Rad,Hercules, CA) with bovine serum albumin as astandard. Samples were read at a 750-nm wave-length using a Spectracount microplate photo-meter (Packard, Meriden, CT).

ELISA for TNF and TNFR1

The gastroc muscle homogenates were probed forTNF or TNFR1 using a commercially availableenzyme-linked immunosorbance assay (ELISA)kit according to manufacturer’s instructions (R&DResearch, Minneapolis, MN). There were nosignificant differences in muscle weights of theanimals started on CLA on day �14 and day 0.Therefore, to accommodate all samples on oneplate and avoid inter-assay variability, only

Table 1. Experimental Design for Initiation of Diet Supplemented with .5%Conjugated Linoleic Acid (CLA) and Injection of Colon-26 Tumor Cells (T)

Day Treatment

Day �14 CLA CLA — — — —Day 0 T — T — T —

CLA CLA — —Day 21 All animals sacrificedDesignation DTD DtD dTD dtD dTd dtd

n¼ 6 n¼ 6 n¼ 5a n¼ 6 n¼ 6 n¼ 6

Once initiated, the CLA treatment was maintained for the duration of the

experiment. D¼CLA in diet, d¼ control diet, T¼ tumor cells, t¼no tumor cells.aOne animal failed to grow a tumor by day 21.

50 RESEARCH IN NURSING & HEALTH

samples from the day �14 and no CLA tumor-bearing and control animals (DTD, DtD, dTd, dtd)were tested for TNF or TNFR1. Homogenateswere diluted 1:4 in the buffer provided and assay-ed in duplicate. Data are reported as pg/mL ofhomogenate and as pg TNF or TNFR1 per mg oftotal protein in the homogenate.

RESULTS

Animal Model of Cancer Cachexia

Body weight gain is shown in Figure 1 as theaverage of the two cages of three mice each foreach of the six groups over the course of theexperiment. There were no significant group dif-ferences. One animal, inoculated with tumor cellsand switched to the CLA diet on day 0 (dTD), didnot develop a tumor by day 21 and was excludedfrom subsequent analyses. Data for muscle weightand muscle levels of TNF and TNFR1 is based on afinal number of 35 animals.

Mean weight of the gastroc muscle relative tobody weight at the time of sacrifice is shown inFigure 2. There was a main effect of tumor(F[1,29]¼ 5.8, p< .03) and of diet (F¼ 5.3,p< .02) on muscle mass, and no interaction effect

of diet and tumor. The effect of diet on musclemass in the tumor-bearing and healthy controlanimals was subsequently examined using one-way analysis of variance and Duncan’s post hoccomparisons. In the three groups of tumor-bearinganimals, mean muscle weight was significantlydifferent (F[2,14]¼ 4.4, p¼ .03). Muscles of themice given no CLAweighed significantly less thanmuscles of mice given CLA at day�14 or day 0 oftumor growth, and the day �14 and day 0 groupswere not significantly different from each other.There was no significant difference in the gastrocmuscle weight of the healthy control animals(p¼ .3). Pair wise comparisons of the tumor-bearing and control animals in each diet conditionrevealed that only in the animals maintained onthe control diet was gastroc muscle weight of thetumor-bearing mice significantly less than thenontumor-bearing control animals (t[10]¼ 2.86,p¼ .02). There was no difference in muscle weightbetween tumor-bearing and nontumor-bearingcontrol animals started on .5% CLA on day �14(p¼ .3) or day 0 (p¼ .7) of tumor growth. Thus,the administration of .5% CLA in the dietabrogated the effect of tumor growth on gastrocmuscle mass compared to animals given thecontrol diet with no CLA. Mean tumor weight inthe three groups of animals was not significantlydifferent (F[2,14]¼ .3, p¼ .7).

FIGURE 1. Average total grams body weight oftwo cages of three mice each over the course of theexperiment. D¼diet supplemented with .5% con-jugated linoleic acid (CLA), d¼ control diet,T¼ tumor cells, t¼no tumor cells. Animals werestarted and maintained on CLA 2 weeks before and3 weeks after inoculation of tumor cells (DTD,DtD), concurrent with inoculation of tumor cells(dTD, dtD) or were maintained on the control dietbefore and after the inoculation of tumor cells(dTd, dtd). n¼ 6 per group (includes one mouse indTD who did not develop a tumor over the course ofthe experiment). (Color figure can be viewed inissue online at www.interscience.wiley.com)

FIGURE 2. Gastroc muscle weight relative tobody weight in animals. D¼diet supplementedwith .5% CLA, d¼control diet, T¼ tumor cells,t¼no tumor cells. Animals were started andmaintained on CLA 2 weeks before and 3 weeksafter inoculation of tumor cells (DTD, DtD) orconcurrent with inoculation of tumor cells (dTD,dtD), or maintained on the control diet forthe duration of the experiment (dTd, dtd).�Significantly different from all other groups,p< .05.

CLA AND CACHEXIA/GRAVES ET AL. 51

ELISA for TNF and TNFR1

The mean levels of TNF in the muscle homo-genates of tumor-bearing and control animalswere below the level of detection (15 pg/mL).Mean levels of TNFR1 in the 4 groups of musclehomogenates are shown in Figure 3. There was amain effect of tumor (F[1,20]¼ 7.7, p< .02) andan interaction of tumor and diet (p< .01) on thelevel of the TNFR1 in the gastroc muscle homo-genates. Thus, the level of TNFR1 was signi-ficantly increased in the gastroc muscles of thetumor-bearing animals, and the level in musclesof tumor-bearing animals given CLA was signi-ficantly different from tumor-bearing animalsgiven no CLA.

DISCUSSION

Skeletal muscle wasting is a prominent feature ofcancer cachexia and contributes to the fatigueand weakness reported by patients with cachexia.The biological mechanisms of cancer cachexiaare complex, but a growing number of studies inboth animal models and cancer patients haveimplicated the pro-inflammatory cytokine TNF,and COX-mediated metabolism of AA to PGE2(Argiles et al., 2003). Cancer cachexia is now

regarded as a chronic inflammatory condition, andstrategies for treating cachexia include reducingthe activity of COX2 or TNF (Argiles, Almendro,Busquets, & Lopez-Soriano, 2004). CLA containspositional isomers of linoleic acid, a precursor ofAA, and is reported to alter the ratio of AA to otherfatty acids in the cell membrane (Ostrowska et al.,2003), or the efficiency of COX to metabolize AAto PGE2 (Belury, 2002). Animals treated withCLA also have lower circulating levels of TNF(Akahoshi et al., 2002; Yang & Cook, 2003b).

In the present study, gastroc muscle mass wassignificantly reduced in mice bearing the colon-26adenocarcinoma. When the diet of tumor-bearingmice was supplemented with .5% CLA, there wasno loss of gastroc muscle mass compared tonontumor-bearing controls. CLA was effective inblunting muscle wasting in mice whether started14 days before or on the same day as the ino-culation of tumor cells.

CLA has been reported to decrease body fat andincrease lean muscle mass when added to the dietof rodents and poultry (D’Orazi et al., 2003).We did not find that .5% CLA enhanced musclemass in the control animals. This may be due tothe lesser effects of CLA on lean body massin female mice compared to male mice (Parket al., 1999). It is also possible that muscles of thetumor-bearing mice were more susceptible to themetabolic effects of CLA than muscles of healthycontrol animals. In contrast, .5% CLA given in thediet of male rats inoculated with the Morris 7777hepatoma had no effect on total body proteincompared to animals given the control diet andthere was no effect of CLA on levels of TNFsecreted by splenocytes stimulated ex vivo withLPS (McCarthy-Beckett, 2002). Others havenoted that CLA has a greater effect on body fatand serum TNF in mice than in rats (Akahoshiet al., 2002). Alternatively, the body weight ofrats bearing the Morris 7777 hepatoma was notimproved with administration of indomethacin(McCarthy, 1999; Strelkov et al., 1989), suggest-ing that PGE2 synthesis may not play a significantrole in this rat model of cancer cachexia. Furtherstudies are needed to determine whether the bene-ficial effects of CLA on tumor-induced skeletalmuscle wasting are limited to tumors with in-creased expression of COX2 and/or synthesis ofPGE2.

Serum levels of TNF are elevated in micebearing the colon-26 adenocarcinoma (Zhou et al.,2003), and treatment of the tumor-bearing animalswith indomethacin preserved muscle mass andlowered serum levels of TNF. Others have shownthat drugs that block the synthesis or activity of

FIGURE3. Picograms (pg) of the tumor necrosisfactor-alpha (TNFa) type 1 receptor (TNFR1) permicrogram (mg) of soluble protein in homogenizedgastroc muscle of tumor-bearing (T) and controlmice (t) given conjugated .5% linoleic acid in theirdiet (D) or the control diet (d). Animals werestarted and maintained on CLA 2 weeks beforeand 3 weeks after inoculation of tumor cells(DTD, DtD) or maintained on the control diet forthe duration of the experiment (dTd, dtd).�Significantly different from all other groups,p< .05.

52 RESEARCH IN NURSING & HEALTH

TNF will also reduce skeletal muscle wasting intumor-bearing rats (Costelli et al., 2002). TNFexerts its biological effects by binding to twodistinct receptors, TNFR1 and TNFR2, and ex-posure to TNF will increase expression of TNFreceptors on cultured muscle cells (Zhang, Pilon,Marett, & Baracos, 2000). Levels of TNFR1mRNA were recently shown to be elevated in thegastroc muscle of tumor-bearing rats (Catalanoet al., 2003). In the present study, the level ofTNFR1 in gastroc muscles of mice bearingthe colon-26 adenocarcinoma was significantlygreater than in muscles of control animals.However, the level of TNFR1 in muscles oftumor-bearing animals given .5% CLA in theirdiet was similar to controls. These data suggestthat CLA may have blunted muscle wasting inthis animal model of cancer cachexia by reducingthe expression of TNFR1 in the muscle of thetumor-bearing animals.

There is a growing body of evidence that CLAin the diet inhibits tumorigenesis in rats treatedwith carcinogens (Belury, 2002; J.L. Cheng et al.,2003; Kim & Park, 2003). There are severalreports that CLA slows the growth of humanmammary tumor cells in culture (Chujo et al.,2003; Kemp, Jeffy, & Romagnolo, 2003; A.Miller, Stanton, Murphy, & Devery, 2003) and inimmune deficient mice given CLA in their diet(Visonneau et al., 1997). Others have found thatCLA reduced growth of human colon cancer cellsin culture (Kemp et al.; A. Miller et al.) but did notslow the growth of colon tumors in a strain of miceprone to colon polyps and tumors (Whelan,Petrick, McEntee, & Obukowicz, 2002). In thepresent study, CLA did not affect growth ofthe colon-26 adenocarcinoma in mice. Similarfindings were observed in mice inoculated withmammary tumors (Wong et al., 1997) and ratsinoculated with the Morris 7777 hepatoma(McCarthy-Beckett, 2002). While it is unclearwhether a diet enriched with CLA might slowtumor growth, the data from the present studysuggest that the anti-cachectic effects of CLA inmice bearing the colon-26 adenocarcinoma werenot secondary to suppressed tumor growth.

In summary, TNF and PGE2 have been impli-cated in the biology of cancer cachexia. Increaseddietary intake of CLA may alter the ratio of AA incell membranes, or affect its metabolism to PGE2.In the present study, a diet supplemented with .5%CLA reduced muscle wasting and muscle levelsof TNF receptors in mice bearing the colon-26adenocarcinoma. Indomethacin, a nonspecific in-hibitor of COX1 and COX2 activity, which re-duces the synthesis of PGE2, also prevents skeletal

muscle wasting in this mouse model of cancercachexia (McCarthy et al., 2004; Strelkov et al.,1989; Zhou et al., 2003). Further studies areneeded to determine if the beneficial effects ofCLA on tumor-induced skeletal muscle wastingare limited to tumors that express COX2 or TNF.The colon-26 adenomcarcinoma model of cancercachexia could be used to determine if the bene-ficial effects of CLA can be enhanced by co-administration of nonspecific or specific inhibitorsof COX2 activity. These studies may increase ourunderstanding of the biology of cancer cachexia,and contribute to the development of treatments toreduce the loss of skeletal muscle mass in cancerpatients with metastatic or progressive disease.

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