Vol. 44, No. 1, January 1998 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL Pages

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1 Vol. 44, No. 1, January 1998 Pages IN VIVO INHIBITION OF WALKER 256 TUMOUR CARNITINE PALMITOYLTRANSFERASE I BY SOYA OIL DIETARY SUPPLEMENTATION Alison Colquhoun 1, F~ibio E.P.de Mello* and Rui Curi*, Departamento de Histologia e Embriologia and *Departamento de Fisiologia e Biofisica, Instituto de CiSncias Biom6dicas, Universidade de Sao Paulo, CEP , S~o Paulo, SP, Brasil. Received October 27, 1997 The role of dietary fatty acids in the regulation of carnitine palmitoyltransferase (CPT) activity has been shown in liver but their role in the regulation of tumour CPT activity in vivo is unknown. The present study investigated the effects of several oils, given as dietary supplements, upon the activity of CPT I and II in the Walker 256 rat tumour and the inhibition or stimulation of tumour growth. CPT I activity was markedly inhibited by soya oil, rich in linoleic acid (70% inhibition vs control). CPT I mrna expression was not inhibited by any of the oils studied, indeed soya oil caused a marked increase (132% vs control) in expression. These results suggest that soya oil can modulate, in vivo, the 13-oxidative pathway of tumour tissue and further supports the hypothesis of tumour CPT I regulation by polyunsaturated fatty acids. Key Words:-carnitine palmitoyltransferase; fatty acids; soya oil; tumour; oxidation. INTRODUCTION Previous studies have reported the regulation of mrna expression of both lipogenic and glycolytic enzymes in rat liver by polyunsaturated fatty acids (1, 2). More recently, fatty acids have been reported to increase CPT I mrna expression in both foetal rat hepatocytes and the 1NS-1 pancreatic 13-cell line (3, 4). The regulation of CPT (E.C ) activity is largely unknown in tumour cells, both in vitro and in animal models. The present study has determined the in vivo effects of several oils on Walker 256 rat tumour cell proliferation and the 13-oxidative capacity of the tumour, by measurement of the maximal activity ofcpt I and II. The abbreviations used in the text are: CPT, carnitine palmitoyltransferase; PLC, phospholipase C. " 1 Corresponding author, Telephone , Fax /98/ ~ /0 Copyright by Academic Press Australia. All rights of reproduction in any form reserved.

2 Vol. 44, No. I, 1998 MATERIALS AND METHODS Animals - male Wistar rats weighing g were maintained at 23~ on a 12 hour light/dark cycle, with access to food (Purina chow) and water ad libitum. Animals received bilateral subcutaneous implants (100+3mg) of Walker 256 tumour in the flank under anaesthesia (chloral hydrate). The day of surgery was designated day 0 and, starting on day 1, the rats received daily dietary supplements ofoil by gavage, at a dose of 0.4% of body weight. Sham-treated rats received 0.9% saline at the same dose. Gavage was the chosen method of administration as this mimics the common form of human dietary supplementation with oils in the form of capsules in large, single doses. A minimum of fourteen rats were included in each group (minimum of 7 each in studies 1 and 2), and the studies were carried out for 12 days, after which the animals were killed and the tumour weight/volume measured. Rats were autopsied to check for macroscopic injury of the oesophagus, which may have limited food consumption~ but no animals presented such damage. Fatty acid compositions of the oils are given in Table 1. Mitoehondrial Preparation - tumours were fragmented and the mitochondria prepared for radiochemical measurement of maximal CPT I and II activity as in (5). Northern Blotting - Total RNA was extracted with Trizol (GIBCO), separated on 1% agarose gel and blotted onto Hybond-N nylon membrane (Amersham). Radioactive edna probe was prepared using the Rediprime system (Amersham) with [t~-32p]dctp. Membranes were hybridised with edna of rat CPT I and rat CPT II, generously provided by Prof. J.D. MeGarry, Dallas, USA and with edna of 13-actin, generously provided by Dr M. Hirata, S~o Paulo, Brazil. Results were normalised against 13-actin and quantified by scanning densitometry. Statistical significance was determined by Student's t-test, with significant difference being set at p<0.05. RESULTS Dietary supplementation was found to have no effect on mean body weight in all groups (Table 2). The presence of tumour prevented weight gain in all groups compared with normal rats of this age (gain of-30g/week). However, differences were found in food consumption, where almond oil caused a slight increase while soya oil caused a slight decrease in food consumption (Table 3). The final tumour weight was unchanged by the oils (Table 4), indicating a lack of effect on Walker 256 tumour cell proliferation by all of the oils studied. Tumour volume calculations correlated well (1"2=0.970) with measured tumour weights and are not presented here. Neither almond, macadamia, nor cod liver oil groups had altered CPT I activity compared with the control group (Table 5). However, CPT I activity was significantly decreased (p<0.01) in the soya oil group, with a decrease of 70% compared with the control group. In contrast to CPT I, CPT II activity was unchanged in all the groups. The effects of dietary supplementation upon CPT I and CPT II mrna expression were studied and the results were normalised by comparison with 13-actin mrna 152

3 TABLE 1 - Fatty acid composition of dietary oils (% of total) Fatty acid Almond Soya Macadamia Cod Liver 14: : : : : : : : : : t : : : : : TABLE 2 - Effect of dietary supplementation on rat body weight Dietary Group Mean body weight (g) Day 1 Day 12 Control Soya _+12.1 Almond Macadamia 155.2_ _+5.3 Cod Liver 161.3_ Values are mean + S.E.M. for eight rats per group. 153

4 TABLE 3 - Effects of dfetary supplementation on food consumption Dietary Group Mean food/rat/day (g) Week 1 Week 2 Control " Soya a 7, b Almond a b'~ Macadamia c Cod Liver C Values are mean + S.E.M. of six rats. Statistical significance p<0.05: a _ vs control week 1 b _ VS control week 2 ; c_ vs the same oil week 1 TABLE 4 - Effect of dietary supplementation on final tumour weight Dietary group Mean tumour weight (g) Study 1 Study 2 Combined data Control (7) (6) (13) Soya (6) (9) (15) Almond 1A (7) (7) (14) Macadamia (7) (6) (13) Cod liver (5) 1.56_+0.43 (7) (12) Values are mean + S.E.M., number of observations in parentheses. expression. CPT I mrna expression was increased in the soya oil group compared with the control group (Figure 1/Table 6). None of the other groups showed significant differences in expression. In the case of CPT II, mrna expression was increased, though not significantly, in the soya oil group and tended to increase in the other groups too. DISCUSSION These data show that soya oil, which is rich in linoleic acid, is able to modulate the maximal CPT I activity of the Walker 256 rat tumour in vivo. Long-chain polyunsaturated fatty acids derived from linoleic acid can readily be incorporated by tumour cells into various cellular compartments (6-8). In addition, they may be modified to produce eicosanoid derivatives, which may then be released to act in either an autocrine or 154

5 BIOCHEMISTRYond MOLECULAR BIOLOGY INTERNATIONAL TABLE 5 - Effects of dietary supplementation on Walker 256 tumour carnitine palmitoyltransferase I and li activity Dietary group CPT I CPT II (nmoles/rain/mg protein) Control S oya a Almond Macadamia Cod Liver Values are mean + S.E.M. for eight or nine observations. Statistical significance ~ - p<0.01 vs control CPT I (4.7Kb) CPT II (2,5Kb) 13-ACTIN (2.1Kb) FIGURE 1 - Northern blots of CPT I and CPT 1I mrna expression - Samples from left to right; 1-3 cod liver oil, 4-6 soya oil, 7-9 macadamia oil, almond oil, control paracrine way upon the tumour cells. Indeed, the Walker 256 tumour model has already been shown to have high plasma concentrations of prostaglandin E2 (9). While it remains to be identified how soya oil is able to decrease maximal CPT I activity of the tumour tissue, it does not appear to be via modifications in mrna expression (Figure 1/Table 6). The results show that CPT I mrna expression is, in fact, increased in response to the presence of soya oil, possibly in response to the inhibition of 155

6 TABLE 6 - Effects of dietary supplementation on Walker 256 tumour carnitine palmitoyltransferase I and II messenger RNA expression Dietary group % of control messenger RaNA CPT I CPT II Control Soya a Almond Macadamia Cod Liver _11.5 Values are mean + S.E.M. for four to six observations. Statistical significance a _ p<0.01 vs control enzyme activity in the mitochondria. The m vitro studies (see previous paper) have provided strong evidence in favour of the involvement of prostaglandins, derived from polyunsaturated fatty acids, in the regulation of tumour cell CPT I and, to some extent, CPT II activity. The mechanism by which these eicosanoids function in vivo remains to be determined, although it is reasonable to speculate on the involvement of second messenger systems linked to the prostaglandin E receptors, EP1-4, causing alterations in either camp or PLC/Ca 2+. (10, 11). Further studies are currently in progress to identify which mechanisms are involved in this novel regulation of tumour CPT activity. Preliminary data were presented at the 4th International Congress on Essential Fatty Acids and Eicosanoids, UK, July Funded by CNPq and FAPESP. REFERENCES 1. Jump,D.B., Clarke, S.D., Thelen,A.and Liimatta,M. (1994)d. LipidRes. 35, Clarke, S.D. and Jump, D.B. (1996) Lipids 31, $7-S Chatelain,F., Kohl,C., Esser, V., McGarry,J.D., Girard,L, Pegorier,J.P. (1996) Eur.d. Biochem. 235, Assimacopoulos-Jeannet,F., Thumelin, S., Roche,E, Esser, V., McGarry,J.D., Prentki,M. (1997)J.Biol.Chem. 272, Colquhoun,A. and Curi,R. (1995)Biochem.Mol.BioLlnt. 37, Rosenthal,M.D. (1987)Prog. LipidRes. 26, Sauer,L.A., Dauchy,R.T. and Blask,D.E. (1997) d.nutr. 127, Colquhoun,A. and Curi,R. (1997)Biochem.Mol.Biol.Int. 41, Siddiqui,R.A. and Williams,J.F. (1987)MedScLRes. 15, 45-46( 10. Chang,C.S., Negishi, M., Nishigaki, N. and Iehikawa, A. (1997) Biochem.d., 322, Schwaner,I. Offermans, S., Spicher,K., Seifert, R. and Schultz, G. (1995) Biochim.Biophys.Acta 1265,