Prostaglandin E and prostacyclin receptor expression in tumor and host tissues

from MCG 101-bearing mice: A model with prostanoid-related cachexia

Wenhua Wang, Marianne Andersson, Christina Lonnroth, Elisabeth Svanberg and Kent Lundholm*

Surgical Metabolic Research Laboratory at Lundberg Laboratory for Cancer Research, Department of Surgery,Sahlgrenska University Hospital, Goteborg, Sweden

Preclinical and clinical studies in our laboratory have suggestedthat prostaglandin (PG) E2 is involved in anorexia and cachexiadevelopment, although the role of COX pathways on the patho-genesis of cancer cachexia remains to be clarified. Expressions ofPGE (EP1, EP2, EP3a,b,d and EP4) and PGI (IP) receptors in thecentral nervous system (brain cortex, hypothalamus and brainstem), in peripheral (liver, white adipose tissue and skeletalmuscle) and tumor tissue from MCG-101-bearing mice with andwithout indomethacin treatment were investigated by RT-PCRand immunohistochemistry. Expression of EP1 in the liver andEP4 receptor in white adipose tissue were upregulated andresponded to indomethacin treatment, while downregulated ex-pression of EP3 in skeletal muscle from tumor-bearing mice wasunresponsive to indomethacin treatment despite improved carcassweight. Expression of EP and IP receptors in brain and tumor tis-sue from tumor-bearing mice were neither related nor responsiveto systemic PGE2 levels including increased IL-1b, IL-6 andTNF-a host activities. The expression IP receptor in CNS, periph-eral tissue and tumor tissue was unchanged by cachexia develop-ment. Our results suggest that transcription of EP receptors inliver, fat and skeletal muscle tissue may be a control level for hostmetabolic alterations during tumor progression, while overall EPand IP receptor expression in CNS did not indicate an importantcontrol level for appetite regulation in MCG 101-bearing micedespite prostanoid related anorexia.' 2005 Wiley-Liss, Inc.

Key words: anorexia; EP receptor; cachexia

Mediators of cancer cachexia are manifold and to some extentdependent on preclinical and clinical situations and the type ofmalignant tumors. The current MCG-101 tumor-bearing mousemodel is characterized by increased activities of inflammatorycytokines as IL-1b, IL-6 and TNF-a1–5 with increased tumor for-mation of eicosanoids, which results in elevated systemic PGE2

levels,6 while the lipooxygenase pathway is of minor importancecompared to other models.7 Accordingly, we have reported thatCOX inhibitors attenuate tumor growth with subsequentlyimproved food intake and body composition,1,6 where provisionof PGE2 to normal mice reduced food intake and body weight.8

However, the role of COX-pathways behind the pathogenesis ofcancer-induced anorexia and cachexia is still unprecise.5

Among active products of arachidonate metabolism, PGE2 islikely to have a key role to trigger populations of neurons for con-trol of neuroendocrine functions involved in restoring homeostaticbalance.9,10 The cellular response to PGE2 depends on the expres-sion of specific EP receptor subtypes or isoforms, which are trans-membrane G protein-coupled receptors and classified as EP1, EP2,EP3 and EP4 in target cells of both CNS and peripheral tissues.11,12

Each receptor is associated with a unique G protein and a secondmessenger system, as elevation of intracellular Ca2þ (EP1) andstimulation (EP2 and EP4) or inhibition (EP3) of adenylate cyclase.In rat brain, EP4 subtype has been proposed as a receptor media-ting the action of PGE2 on specific groups of neurons in responseto circulating proinflammatory cytokines as IL-1b,13 includingneurons of the paraventricular nucleus (PVN), nucleus of the soli-tary tract (NTS) and the caudal ventrolateral medulla (cVLM). Ithas been suggested that signal pathways across the blood brainbarrier may depend on activation of the local synthesis of PGE2

by cells of the microvasculature in PVN regions.14 Thus, PGE2

mediates fever in response to both exogenous and endogenous

pyrogens by acting at EP3 receptors in mice;15 EP3-like immuno-reactivity has also been observed in regions of the rostrocaudalaxis of the nervous system with sensory ganglia. Autonomic pre-ganglionic nuclei also contain neuronal cell bodies with immunor-eactivity, implying modulatory functions of EP3 in the centralautonomic nervous system.16 The brain itself is also an importantsource of PGE2 through the COX-pathways,17 although PGE2 canreadily cross the blood-brain barrier.18 In peripheral tissues, theexpression of EP1 and EP4 receptor mRNA was more pronouncedin preadipose cells than in adipocytes, while mRNAs encoding thea, b and g isoforms of the EP3 receptor were all exclusivelyexpressed in freshly isolated mature adipocytes.19 All these obser-vation suggest that prostanoid receptors may represent importantcontrol points for metabolism and cell function, in cancer cachexiasupported by our previous preclinical and clinical studies.6,20

Therefore, the present study evaluates PGE2 and prostacylinreceptor expression in the central nervous system, host peripheraltissues and tumors from MCG101-bearing mice with prostanoid-related cachexia.

Material and Methods

Animal experiments

The experimental protocol was approved by the Committee foranimal ethics at the Goteborg University. Adult, female age-matched C57BL/6 mice (20–23 g) (M & B A/S, Ejby, Denmark)were housed in plastic cages in a temperature controlled roomwith a 12 hr dark/light cycle. The animals received laboratoryrodent chow (B & K Universal AB, Stockholm, Sweden) and tapwater ad libitum for 14 days before the experiments. Mice wereseparated into groups of 3–4 mice per cage with a wire floor. Ani-mal groups were tumor-bearing mice with (TBþINDO) and with-out (TB) indomethacin treatment and nontumor-bearing freely fedcontrols (FF). All groups were allowed 3 days adaptation to wirefloors before the start of experiments, which was on day 0.

Tumor-bearing mice were implanted s.c. bilaterally in the flankwith a 3–5 mm3 of a transplantable MCG-101 methylcholan-threne-induced sarcoma21 under general anesthesia [a combinationof 100 mg/kg, i.p. Ketalar�R (Ketamine Hydrochloride, WarnerLarbert Nordic AB, Solna, Sweden) and 5 mg/kg, i.p Rompun�R

(Xylazine Hydrochloride, Bayer Sverige AB, Goteborg, Sweden)].Control mice were sham implanted. All animals had free access tochow and water ad libitum. Indomethacin was provided by thedrinking water at concentration of 6 mg/ml. Daily food intake andbody weight were registered between 0800 and 0900. All micewere sacrificed on day 10 upon implantation. Blood samples were

Grant sponsor: Swedish Cancer Society; Grant numbers: 2014, 4261;Grant sponsor: Swedish Research Council; Grant numbers: 08712, 13268,11611; Grant sponsor: Assar Gabrielsson Foundation (AB Volvo); Grantsponsor: Jubileumskliniken Foundation; Grant sponsor: IngaBritt and ArneLundberg Research Foundation; Grant sponsor: Swedish and GoteborgMedical Societies; Grant sponsor: the Medical Faculty, Goteborg University.*Correspondence to: Department of Surgery, Sahlgrenska University

Hospital, SE 413 45 Goteborg, Sweden.Fax:þ46-31-413892,þ46-31-826539. E-mail: kent.lundholm@surgery.gu.seReceived 9 March 2004; Accepted for revision 23 June 2004DOI 10.1002/ijc.20539Published online 9 February 2005 in Wiley InterScience (www.

interscience.wiley.com).

Int. J. Cancer: 115, 582–590 (2005)' 2005 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

obtained by cardiac puncture during general anesthesia. Plasmawas removed immediately for PGE2 and IL-6 determinations asdescribed.4,5 Liver, muscle, fat, tumor and brain (cortex, hypothal-amus and brain stem) were snap-frozen in liquid nitrogen follow-ing vascular bed rinsing with 20 ml saline through the left cardiacventricle and kept frozen (�808C) until RT-PCR analysis of EP1–4and IP receptors. For immunohistochemistry, liver, muscle, fat,tumor and brain tissues were rapidly removed and placed for20–24 hr of postfixation in paraformaldehyde at room temperaturefollowing 20 ml room-temperatured saline vascular bed rinsingand 4% paraformaldehyde perfusion. Tissue specimens were par-affin embedded and cut into 8 mm sections for use. Carcass weight(body weight minus tumor weight), wet and dry tumor weightwere determined as described.22

RNA isolation and quantification

Deep frozen tissues of liver, muscle, fat, tumor and brain tissuewere homogenized, and total RNA was either isolated by the acidguanidinium thiocyanate-phenol-chloroform method (ClonTech,ClonTech Laboratories, Inc., Palo Alto, CA) or extracted withGen EluteTM Mammalian Total RNA kit when it became commer-cially available (Sigma Chemical Co., St. Louis, MO). Total RNAconcentrations were estimated by spectrophotometry. Total RNAsamples were treated with DNAse 1 (ClonTech) prior to the RTreaction to remove genomic DNA contamination for RT-PCRanalysis. RNA was glyoxal treated for 1 hr at 508C and separatedin 1.2% agarose gel in a phosphate buffer system. Quality andquantity of RNA were always checked by an Agilent 2100 Bio-analyzer before quantification of EP receptor transcripts followingthe protocol of RNA 6000 Nano Assay Kit, where the ratio of 28Sand 18S rRNA was determined (Fig. 1).

cDNA synthesis and quantification

One microgram of total RNA was reversed transcribed by usingoligo d(T)-primer and Moloney Murine Leukemia Virus (MMLV)according to kit instructions (1st strand cDNA synthesis kit, Clon-Tech). The efficiency of the cDNA synthesis and the quality ofRNA were quantified in parallel by PCR of using ClonTech’s pos-itive control amplimer, glyceraldehyde 3-phosphate dehydrogen-ase (GAPDH), as housekeeping gene checked as constant amongthe animal groups, and control RNA from human placenta asexternal standard. For cDNA synthesis, a parallel reaction wasperformed with samples taken at random without reverse transcri-patase (RT) and utilized in PCR as a negative control. Analysis ofamplicons was run by Agilent 2100 Bioanalyzer following theprotocol of DNA 1000 Assay Kit to quantify the cDNA amount.The cDNA synthesis was optimized in all steps and strictly pro-portional to the amount of start RNA.

PCR amplification and quantification

Specific sense and antisense primers were selected from mouseEP1, EP2, EP3, EP4 and IP receptor cDNA sequences. With respectto the EP3 receptor, specific sense and antisense primers distinc-tive for the coding sequences of the a, b and g isoforms wereselected from their cDNA sequences.19 The 3 isoforms of EP3were included in 2 primer sets, i.e., EP3a,b in one set for PCR run-ning and EP3g in a separate running. Some of these primers (EP1,EP3a,b,g and EP4) have been used effectively for PCR amplifica-tion.23 Specific sense and antisense primers for EP2 and IP werenewly designed from the EP2 and IP receptor cDNA sequences(GenBank Accession Number NM008964 and D26157, respec-tively). All the primers except GAPDH were synthesized by Scan-dinavian Gene Synthesis AB, Sweden (Table I). Optimal numbersof amplification cycles were determined when analyzing the rela-tionship between PCR cycles and product accumulation in sepa-rate experiments to ensure that the PCR was within the area oflinear amplification. Different predicted long fragments wereamplified under various cycle conditions with different primer-sets(Table II) and standard cDNA (synthesized from kidney RNA)was always run in parallel to ensure quantitative measures ofEP1–4 amplification. It was repeatedly checked that both decreas-ing and increasing amounts of cDNA gave strictly proportionalamounts of the end products to ensure quantitative estimates ofthe amplicons (correlation coefficients > 0.92).

Most of the PCR reactions were performed using Master mix(Roche Diagnostics GmbH, Mannheim, Germany), with a concen-tration of 0.2 mM of each dNTP (dATP, dCTP, dGTP and dTTP),0.5 units of Taq DNA polymerase, 0.4 mM of each primer (senseand antisense), 1 ml cDNA and 1.5 mM MgCl2 to a final volume of20 ml. Formamide was added to a final concentration of 3% andMgCl2 to a final concentration of 2 mM to Master mix for EP4receptor reaction. Kidney cDNA was always used as a positive andquantitative control of the PCR reactions in all series of measure-ments. A negative control for the PCR reaction without cDNA wasalso used. PCR products were checked in Agilent 2100 Bioanalyzerfollowing the protocol of DNA 1000 Assay Kit for quantification(Fig. 2). The results were expressed by ratio to the value of GAPDH.

Immunohistochemical staining

Immunohistochemical staining was optimized: antigen retrievalwas accomplished by trypsination (EP1 and EP2 staining) or bymicrowave-radiation treatment in acid conditions (EP3 and EP4staining). Primary antibodies were diluted in 1% TBS-BSA con-taining 0.1% Saponin (1:100; Multiclonal Rabbit anti-rat EP1 andEP2 from Alpha Diagnostic International, Inc., TX; EP3 and EP4from Santa Cruz Biotechnology, Inc., CA), and incubated over-night (20 hr) at room temperature after blocking nonspecific pro-

FIGURE 1 – Electropherogram (Agilent 2100 Bioanalyzer) of ex-tracted RNA from tumor tissue used for PCR amplification. The ratioof 28S to 18S was 1.60.

TABLE I – THE SENSE AND ANTISENSE SEQUENCES FOR THEVARIOUS SPECIFIC PRIMERS

Message Primer Primer sequence Productsize

EP1 1.1 50-CGTGTCATTTCCTGGGTGGCT-30 5011.2 50- GTGGCTGTGGCTGAAGTGATG-30

EP2 2.1 50-TGCTGCCTGTCATCTAT-30 2012.2 50-TGAGGATAACGCTGATGT-30

EP3a 3.1 50-CTAATTGCAGTTCGCCTGGCT-30 4093.2 50-CGTCTCAAGTGCAGAGTCTTC-30

EP3b 3.3 50-CTAATTGCAGTTCGCCTGGCT-30 3203.4 50-CGTCTCAAGTGCAGAGTCTTC-30

EP3g 4.1 50-CTAATTGCAGTTCGCCTGGCT-30 5494.2 50-TGTGGCTTCATTCCTTGCCCA-30

EP4 5.1 50-CATCGACTGGACCACCAACGT-30 6645.2 50-TCTCCTTTAACTCCCGGGCGA-30

IP 6.1 50-AAGATGATGGCCAGCGATGGA-30 1736.2 50-ACTACACCAGCCACGAACATC-30

GAPDH 7.1 50-ACCACAGTCCATGCCATCAC-30 4527.2 50-TCCACCACCCTGTTGCTGTA-30

583PROSTAGLANDIN E RECEPTOR AND ANOREXIA

tein binding sites with 5% BSA for 30 min. The remainder of theimmunohistochemical procedures were performed as described.8

Secondary antibodies and developing system were from DakopattsAB, Alvsjo, Sweden. Parallel negative controls were confirmedfor each staining, using diluant instead of primary antibody insamples from the different experimental groups. Control stainingswere undertaken with either the first or the second antibody, aswell as by incubation with normal serum from the species inwhich the primary antibodies were raised. In addition, specificityof primary antibodies was tested by preincubation with corre-sponding blocking peptides.

Statistics

Time course changes of food intake and body weight amongdifferent groups were compared by 2-way ANOVA for repeatedmeasures. End point variables (prostanoid receptor expression,body composition, etc.) were compared by 1-way factorialANOVA. Results are presented as mean 6 SEM; p � 0.05 wasconsidered statistically significant.

Results

Food intake, body weight and net tumor growth

Food intake was not different among tumor-bearing mice andfreely fed controls before day 7. After that, food intake declined intumor-bearing mice and became significantly lower compared tofreely fed controls (p < 0.01). Food intake was significantly morepreserved in tumor-bearing mice treated with indomethacin com-pared to untreated tumor-bearing mice (p < 0.01) (Fig. 3). Animalweight increased to the some extent along tumor growth in bothindomethacin-treated and untreated mice. Thus, tumor weight waslower and carcass weight was significantly preserved by indome-thacin (p < 0.01; Fig. 4). Average plasma PGE2 concentrations intumor-bearing mice (1,132 6 287 pg/ml of plasma) decreased to846 62 pg/ml following indomethacin treatment (Fig. 5).

Tissue EP receptor content evaluated by immunohistochemistry

EP receptors in brain and fat tissue from normal freely fed miceare shown in representative microphotographs (Fig. 6). Histo-chemically, EP1 receptors were visualized in brain, liver, fat andtumor tissue but not in skeletal muscles. EP2 receptors were dem-onstrated in brain, liver and fat tissues but not in skeletal muscleand tumor tissue. EP3 receptors were confirmed in brain, muscleand fat but not in liver and tumor tissues, and EP4 receptors werefound in fat tissue only. Thus, fat tissue was the only site with all4 EP receptors by histochemical technique. Quantitative estimatesof EP-receptor protein expression were not performed.

Tissue expression of prostanoid receptors evaluated by RT-PCR

EP1, EP2 and EP3 receptors were expressed in brain cortex,hypothalamus, brain stem, liver, muscle and tumor tissue, whileall 4 EP receptors were expressed in fat tissue only. Overall pros-tanoid receptors expressions were not significantly different inbrain tissues (cortex, hypothalamus and brain stem) among tumor-bearing mice and freely fed controls (Fig. 7a–c), although EP3gshowed a trend to be increased in hypothalamus from tumor-bear-ing mice (p < 0.07). Indomethacin treatment had in general nosignificant effects on brain tissue expression but may have normal-ized hypothalamic EP3g expression in tumor-bearing mice(p < 0.10, Fig. 7b). EP1 expression was significantly increased inliver tissue from tumor-bearing mice, and this increase was signifi-cantly decreased by indomethacin (Fig. 8). A similar patternoccurred for EP2, but these changes did not reach statistical signif-icance (p < 0.10). By contrast, EP3 receptors expression (EP3a,b,g)

FIGURE 2 – Electropherogram of PCR products of a house keepinggene (GAPDH) (upper, peak 3) and EP2 (lower, peak 2) in tumor tis-sue are examined by an Agilent 2100 Bioanalyzer. Markers for orien-tation are also seen peak 1, 4 (upper) and 1, 3 (lower). Size andconcentration of samples are automatically calculated in relationshipto markers and a companying DNA-ladder (the latter not shown).Concentration of GAPDH was 6.7 ng/ml; 3.1 ng/ml was of EP2.

TABLE II – OPTIMAL PCR AMPLIFICATION CONDITIONS FOR VARIOUS RECEPTORS IN DIFFERENT TISSUES

Message Initialization Amplifiaction Cycle number Final extension

EP1 (liver, fat, tumor) 948C 2 min 948C 1 min, 53.98C 1 min 12 sec, 728C 1 min 30 728C 10 minEP1 (muscle) 948C 2 min 948C 1 min, 56.98C 1 min 12 sec, 728C 1 min 30 728C 10 minEP1 (brain) 948C 2 min 948C 1 min, 588C 1 min 12 sec, 728C 1 min 30 728C 10 minEP2 (liver, fat, tumor, muscle) 948C 2 min 948C 1 min, 55.38C 1 min 12 sec, 728C 1 min 32 728C 10 minEP2 (brain) 948C 2 min 948C 1 min, 588C 1 min 12 sec, 728C 1 min 34 728C 10 minEP3a,b (liver, fat, tumor, muscle) 948C 2 min 948C 1 min, 598C 1 min 12 sec, 728C 1 min 34 728C 10 minEP3g (fat, muscle) 948C 2 min 948C 1 min, 58.58C 1 min 12 sec, 728C 1 min 34 728C 10 minEP3g (liver, tumor) 948C 2 min 948C 1 min, 638C 1 min 12 sec, 728C 1 min 34 728C 10 minEP3a,b,g (brain) 948C 2 min 948C 1 min, 598C 1 min 12 sec, 728C 1 min 34 728C 10 minEP4 (fat) 948C 1 min, 588C 1 min 12 sec, 728C 1 min 36 728C 10 minIP (liver, fat, tumor, muscle) 948C 2 min 948C 45 sec, 62.68C 45 sec, 728C 1 min 32 728C 10 minIP (brain) 948C 2 min 948C 45 sec, 688C 45 sec, 728C 1 min 34 728C 10 minGAPDH (liver, fat, tumor,

muscle and brain)948C 1 min, 588C 1 min 12 sec, 728C 1 min 21 728C 10 min

584 WANG ET AL.

were significantly decreased in skeletal muscle tissue fromtumor-bearing mice compared to freely-fed controls without anyadditional change by indomethacin (Fig. 9). In fat tissue, EP4receptor expression was significantly increased in tumor-bearingmice, an effect that was normalized by indomethacin (Fig. 10).EP1 expression was most pronounced in tumor tissue (Fig. 11),but EP receptor expression was not high in tumor cells and wasrather confined to fat cells around the tumors (not shown). Indo-methacin had no significant effect on prostanoid receptors expres-sion in tumor tissue, although EP2, EP3g and IP expression showedtrends (p < 0.10) to increase following indomethacin treatment(Fig. 11).

Discussion

Experimental cancer cachexia is caused by decreased appetiteand energy dissipation that lead to redistribution of host tissues forsupport of tumor growth, which is also consistent in the currentmodel, where both adipose and lean tissues are lost.22 These alter-ations are mediated by classical hormones, cytokines and mostlikely by additional signals. Recent work in our laboratory has

pointed to the possibility that nitric oxide and eicosanoids are alsoinvolved.6 Of particular interest is the revival of information thateicosanoids,24 particularly PGE2, interfere with growth control ofmalignant tumors by altering COX transcription in some condi-tions.5,25 The potentials of such dynamics, both within and aroundtumor cells, are illustrated by provision of COX-inhibitors forattenuation of tumor growth in both clinical and experimentalstudies.2,20 Thus, our MCG 101 model has been used for years ondescriptive and mechanistic evaluations of the metabolic relation-ship between the host and a growing tumor.21 This tumor inducesanorexia, probably in part explained by tumor cell production ofPGE2, involving increased activities of cytokines, particularly out-side CNS,1,3,4,8,26 while the lipooxygenase pathway is of minorimportance.6 These mediators direct the loss of body fat andmuscle proteins by 68% and 32%, respectively, which are possibleto attenuate by indomethacin, anti TNFa and anti IL-1b.3,4,27

Thus, several lines of evidence support that prostanoid pathways

are significant in a number of changes in tumor host tissues as well

as in the control of tumor growth,5 while increased breakdown ofskeletal muscles28 as described for 15-HETE in a different modelis not quantitatively important.29

FIGURE 4 – Carcass weight (CW, body weight minus tumor weight)in tumor-bearing mice with (TBþINDO) and without (TB) indome-thacin treatment compared to body weight in freely fed controls (FF).Wet tumor weight (WTW), dry tumor weight (DTW) in tumor-bearingmice with (TBþINDO) and without (TB) indomethacin treatment.(ANOVA n ¼ 8 in each group. (a) p < 0.01 TB vs. TBþINDO; (b)p < 0.01 TB vs. FF).

FIGURE 5 – Plasma PGE2 concentration in tumor-bearing mice with(TBþINDO) and without (TB) indomethacin treatment compared tobody weight in freely fed controls (FF). (ANOVA n ¼ 8 in eachgroup. (a) p < 0.01 TB vs. TBþINDO; (b) p < 0.01 TB vs. FF).

FIGURE 3 – Time-course changes of food intake and animal weight in tumor-bearing mice with (TBþINDO) and without (TB) indomethacintreatment and freely fed controls (FF). (Food intake: between groups p < 0.01; over time p < 0.01; interaction between group and time p < 0.01.Body weight: between groups p < 0.01; over time p < 0.01; interaction between group and time p < 0.01) (8 animals in each observation point;ANOVA for repeated measures).

585PROSTAGLANDIN E RECEPTOR AND ANOREXIA

In our study, we analyzed EP and IP receptors in both the cen-tral nervous system and peripheral tissues from tumor-bearingmice with and without indomethacin treatment, since it wasreported that EP receptor expression in cells along the blood brainbarrier can be upregulated by peripheral provision of LPS, circu-

lating IL-1b and activated COX pathways as a signal transmissionmechanism between cytokines in the periphery and neurotransmit-ters in the central nervous system.13,30 Usually, anorexia inducedby IL-1b, LPS and PGE2 administration has been suggested tobe in part mediated by serotonin containing neurons,8,31,32

FIGURE 6 – Immunoreactivities of anti-EP1–4 receptor in brain stem and white adipose tissue from normal freely fed mice are shown in repre-sentative microphotographs (original amplification �40, brown color is receptor staining). (a) Negative control in brain stem. (b) Negative con-trol in white adipose tissue. (c) Anti-EP1 immunoreactivity in neuron of brain stem. (d) Anti-EP3 in brain stem. (e) Anti-EP2 in white adiposetissue. ( f ) Anti-EP4 in white adipose tissue.

586 WANG ET AL.

although we could not confirm a role of serotonergic mechanismsbehind anorexia in the current tumor model.33

Prostanoids are a group of lipid derived mediators that consistof the prostaglandins and thromboxanes. In response to cell stimu-lation, prostanoids are synthesized by the cycloocygenase path-ways from arachidonic acid released from membrane phos-pholipids by the actions of phospholipases. The prostanoids, onceformed, are quickly released to the outside of cells. Owing tochemical and metabolic instability, prostanoids are most effectivein the vicinity of their production. Thus, they act as ‘‘local short-range hormones’’ in maintenance of local homeostasis in a varietyof tissues and cells. Therefore, it can be anticipated that control ofprostanoid receptor expression should be an important regulatorystep in many tissues. Each receptor is specifically distributed inthe body and expression levels are variable among organs and tis-sues. Thus, the cellular responses to prostanoids depend in part onthe expression of specific prostanoid receptor subtypes via aunique second messenger system in target cells including theamount of receptor transcripts.11,12

FIGURE 7 – (a) Expression of PGE2 (EP1, EP2, EP3) and PGI2 (IP)receptors in brain cortex from tumor-bearing mice with (TBþINDO)and without (TB) indomethacin treatment compared to freely fed con-trols (FF) (n ¼ 7 in each group). GAPDH (Glyceraldehyde 3-Phos-phate Dehydrogenase). (b) Expression of PGE2 (EP1, EP2, EP3) andPGI2 (IP) receptors in brain hypothalamus from tumor-bearing micewith (TBþINDO) and without (TB) indomethacin treatment com-pared to freely fed controls (FF) (n ¼ 7 in each group). GAPDH(Glyceraldehyde 3-Phosphate Dehydrogenase). (c) Expression ofPGE2 (EP1, EP2 and EP3) and PGI2 (IP) receptors in brain stem fromtumor-bearing mice with (TBþINDO) and without (TB) indomethacintreatment compared to freely fed controls (FF) (n ¼ 7 in each group).GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase).

FIGURE 8 – Expression of PGE2 (EP1, EP2 and EP3) and PGI2 (IP)receptors in liver tissue from tumor-bearing mice with (TBþINDO)and without (TB) indomethacin treatment compared to freely fed con-trols (FF) (n ¼ 5 in each group; (a) p < 0.01 TB vs. TBþINDO;(b) p < 0.01 TB vs. FF). GAPDH (Glyceraldehyde 3-PhosphateDehydrogenase).

FIGURE 9 – Expression of PGE2 (EP1, EP2 and EP3) and PGI2 (IP)receptors in skeletal muscle tissue from tumor-bearing mice with(TBþINDO) and without (TB) indomethacin treatment compared tofreely fed controls (FF) (n ¼ 5 in each group; (b) p < 0.01 TB vs. FF;(c) p < 0.01 TBþINDO vs. FF). GAPDH (Glyceraldehyde 3-Phos-phate Dehydrogenase).

587PROSTAGLANDIN E RECEPTOR AND ANOREXIA

Differential expression of the EP and IP receptors occurs in thecentral nervous system.34 EP3 mRNA is widely expressed in neu-rons of cortex, hippocampus, thalamus, hypothalamus, midbrainand the lower brain stem. Among subregions in hypothalamus,EP3 mRNA is expressed in neurons of the medial and median pre-optic nuclei, especially in neurons surrounding the organum vas-culosa lamina terminalis (OVLT), with EP3 receptor expressionupregulated following peripheral IL-1 administration.30 In con-trast, expression of EP4 and EP2 mRNA was only reported insmall groups of neuron and nonneuronal cells, with increasedexpression in some of these following peripheral administration ofLPS.13 Also, EP1, EP3 and EP4 mRNAs are expressed in neuronsof the dorsal root ganglion (DRG),35,36 while IP mRNA was mostabundantly expressed in neurons of DRG.35 Thus, IP mRNA wascoexpressed with transcripts for EP receptor subtypes in someneurons, which suggests that EPs and IP may play either overlap-ping or different roles in neuron transmission.

In our study, EP1, EP2 and EP3 were detected in different brainareas (cortex, hypothalamus and brain stem) by RT-PCR, as con-firmed by immunoreactivities in neurons (Fig. 6), although it wasrecently reported that EP1 is not expressed in brain.37 However,estimates did not show significant net overall differences amongtumor-bearing mice with or without indomethacin treatment andfreely fed controls in any evaluated area of the brain, which doeshowever not exclude highly local differences. Our results suggestthat expression of EP and IP receptors in brain tissue of MCG101tumor-bearing mice was not particularly responsive to elevatedsystemic PGE2 levels or to host IL-1b, IL-6 and TNF-a activitiesin this model. So, EP receptor induction in the central nervous sys-tem was not directly related to increased COX activities outsideCNS, and CNS expression was not sensitive to indomethacin treat-ment that never the less caused improved appetite.38 However,expression of EP4 in brain tissue could not be detected by RT-PCR, so it remains to evaluate its role in future experiments ifpresent.

EP receptors are widely distributed in peripheral organ andtissues, consequently with different physiological roles. Forexample, mRNA expression of both EP2 and EP4 in a murinemacrophage-like cell line (RAW 264.7) were variable uponLPS exposure,39 and the upregulation of EP2 by LPS wasinhibited by simultaneous administration of interferon-g.40,41.Also, the abundance and localization of mRNAs for EP2, EP3and EP4 changed considerably in mice during pseudopreg-nancy.42 In our study, peripheral tissue examination revealedincreased expression of EP1 and probably of EP2 (borderline)

in liver tissue and of EP4 in fat tissue from tumor-bearingmice, which were all normalized by systemic administration ofindomethacin, while depressed EP expressions in skeletalmuscles were not altered by indomethacin. Thus, it seems as ifboth feed-forward and feed-back mechanisms may occur forEP-receptors as described for cytokines and cytokine receptorsin skeletal muscles.43,44 Also it was reported that EP2 receptorexpression was effectively induced by LPS treatment with atime course to that of COX-2, an induction which was inhibitedby interferon-g.40

PGs contribute not only to the regulation of triglyceride metab-olism in mature adipocytes,45–47 but also to adipose tissue matura-tion and differentiation.48–50 Major PGs produced in adiposetissue of both rodents and humans are PGE2 and PGI2. These PGsappear to have separate roles on targeting cells in adipose tissue.PGI2 is primarily active in triggering adipocyte differentiation bymeans of elevation of intracellular cyclic adenosine 30, 50-mono-phosphate and free calcium, whereas PGE2 has been found to bewithout any adipogenic activity. In mature adipocytes, PGE2 has apronounced antilipolytic effect mediated through inhibition ofadenylate cyclase, seemingly inconsistent with loss of body fat inMCG 101-bearing mice22 with elevated systemic PGE2. This con-stellation suggests that decreased lipogenesis is a major explana-tion behind the pronounced loss of body fat in this model.22

Likewise depressed protein synthesis is the most importantexplanation to the loss of skeletal muscles.28 It has also beenreported that IP, EP1 and EP4 receptors were considerably moreabundant in preadipocytes than in matured cells, whereas EP3receptors were exclusively expressed in mature adipocytes.19

Thus, loss of body fat in cancer cachexia is most likely a mix ofaltered lipogenesis/lipolysis and changed adipocyte maturationbased on present and previous findings.22,51,52

EP3 is one of the most widely distributed EP receptors in thebody including skeletal muscles.53 The distribution and functionof EP3 for regulation in smooth muscle cells have been intensivelystudied,6,42,54,55, but the function of EP3 receptor in skeletalmuscle tissue is still unclear. In our study, EP3 expression wasdownregulated in skeletal muscles of tumor-bearing mice and wasnot normalized by indomethacin treatment, which indicates thatEP3 expression in skeletal muscle is not directly or proportionallyrelated to systemic PGE2 levels or to elevated IL-1b, IL-6 andTNF-a activities in the host. Therefore, it is evident that netmuscle protein catabolism in MCG 101-bearing mice is notdirectly related to EP receptor control since indomethacinimproved dry carcass weight of which skeletal muscle tissue is a

FIGURE 10 – Expression of PGE2 (EP1, EP2, EP3 and EP4) and PGI2(IP) receptors in fat tissue from tumor-bearing mice with (TBþINDO)and without (TB) indomethacin treatment compared to freely fed controls(FF) (n¼5 in each group; (a) p < 0.01 TB vs. TBþINDO; (b) p < 0.01TB vs. FF). GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase).

FIGURE 11 – Expression of PGE2 (EP1, EP2 and EP3) and PGI2 (IP)receptors in tumor tissue from tumor-bearing mice with (TBþINDO)and without (TB) indomethacin treatment (n ¼ 5 in each group).GAPDH (Glyceraldehyde 3-Phosphate Dehydrogenase).

588 WANG ET AL.

major component, where increased PGE2 has been proposed toinduce net breakdown of skeletal muscle tissue.56,57 Expression ofEP1, EP2, EP3 and IP in tumor tissue was not responsive to indo-methacin, which however attenuated tumor growth. This observa-tion further supports our previous indications that indomethacineffects on tumor progression are not entirely mediated via COX-related pathways,5 although we are not aware of any well-described nonreceptor mediated effect by prostaglandins.

In summary, expressions of EP and IP receptors in brain andtumor tissue of MCG101-bearing mice were not directly relatedto systemic or tissue levels of PGE2. So, EP receptor regulationin host tissues is more complex than just related to host and

tumor COX activities. Our results suggest that net overall EPand IP receptor expression in CNS is not a major mechanismfor the control of anorexia in MCG101-bearing mice, whilefocal alterations in defined neurons may be. By contrast, overallEP receptor expression in liver, fat and skeletal muscle tissueseems to be a control mechanism for metabolic alterations intumor-induced cachexia provided that transcripts are related tofunctional receptors as confirmed in our initial studies on PGEreceptor knock-out mice, where it was demonstrated that thelack of EP1 and EP3 in host tissues promoted and attenuatedtumor growth, respectively, with corresponding alterations in bodycomposition.58

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