RESEARCH LETTERS

Modulation of Epidermal GrowthFactor-Induced Cell Proliferation byan v-3 Fatty-Acid-Containing LipidEmulsion on Human PancreaticCancer Cell Line Mia Paca-2v-3 Fatty acids derived from fish oil and eicosapentaenoic acid(EPA) in particular were found to have a growth-inhibitory effecton several human neoplastic cell lines in vitro.1,2 The underlyingmechanisms are not yet fully understood but likely occur at manylevels.v-3 Fatty acids are structural components of cellular mem-branes with modulating membrane fluidity and composition. Inaddition, there is increasing evidence thatv-3 fatty acids areinvolved in transmitting information from a variety of extracellularsignals (e.g., growth factors) across the membrane and thus actingat the second-messenger level by regulating signal-transducingmolecules.3,4

The epidermal growth factor (EGF) is a potent mitogen inmany epithelial systems, including the exocrine pancreas. EGFbinding to its receptor results in signal propagation to downstreamsecond messengers regulating the expression of a specific set ofgenes, leading to enhanced growth.5,6 Phosphorylation of EGFreceptor can initiate a degradation cascade of various membranelipid constituents.7 Recent studies have provided evidence thatEGF-mediated signal transduction can be modulated by polyun-saturated fatty acids.8

We investigated the effect of anv-3 fatty-acid–containing lipidemulsion on EGF-stimulated growth in human pancreatic cancercell line Mia Paca-2. The cells were grown at 37°C in a humidifiedatmosphere of 5% CO2 in Dulbecco’s modified Eagle mediumsupplemented with 10% fetal calf serum. Cells were cultured in96-well flat-bottomed tissue culture plates at a density of 53 103

cells per well in 100mL of medium and allowed to adhere for 24 hbefore test substances were added. Fish-oil–based lipid emulsion(Omegaven 10%, Fresenius-Kabi, Bad Homburg, Germany) con-taining 2.1 g/100 mL of EPA was added in concentrations rangingfrom 0.144 to 1.44mL z mL21, corresponding to an amount ofEPA ranging from 10 to 100mM. Control experiments werecarried out with a lipid emulsion based on soybean oil containingno v-3 fatty acids (Lipovenoes 10% PLR, Fresenius-Kabi) toexclude a non-specific effect of lipid emulsion on the cells. Growtheffects of EGF (20 ngz mL21) were determined by incubating thecells with EGF in serum-free medium for 12 h after 36 h ofpreincubation in 10% serum-supplemented medium alone or withthe additional lipid emulsions. Cell proliferation was determinedby bromodeoxyuridine uptake and cell counting by crystal-violetstaining.

We initially examined the effect of both lipid emulsions on thegrowth of Mia Paca-2-cells before treatment with EGF. Exposureof the cells with the fish-oil–based lipid emulsion inhibited growthin a dose- (Fig. 1A) and time- (data not shown) dependent manner.It induced 766 5% (P , 0.001) reduction of cell proliferationwithin 72 h at a concentration of 1.44mL z mL21, correspondingto 100 mM EPA. The control lipid emulsion with a similar lipidload had effects only in the highest concentrations tested. As inprevious studies,9 EGF promoted the growth of Mia Paca-2 cells ata concentration of 20 ngz mL21 (1126 6% of control;P , 0.01).Growth experiments with Mia Paca-2 cells incubated with thefish-oil–based lipid emulsion with or without EGF showed anabrogation of the growth-stimulatory effect of EGF and an en-hancement of the growth-inhibitory effect of the fish-oil–basedlipid emulsion alone (214 6 2%; P , 0.01); the control lipidemulsion had no effect (Fig. 1B).

This work was supported in part by the Else Kro¨ner-Fresenius-Foundation(Bad Homburg, Germany).

Correspondence to: Ju¨rgen Stein, MD, PhD, Professor of Gastroenterologyand Clinical Nutrition, 2nd Department of Medicine, J. W. Goethe Uni-versity, Theodor Stern Kai 7, D-60590 Frankfurt, Germany. E-mail:j.stein@em.uni-frankfurt.de

Date accepted: February 7, 2001.

FIG. 1. Effect of thev-3 fatty-acid–containing lipid emulsion with (A) orwithout (B) EGF on cell proliferation of Mia Paca-2-cells. (A) Cells wereexposed to increasing concentrations of lipid emulsions (0.144 to 1.44mL zmL21 corresponding to 10 to 100mM EPA in the fish-oil–based lipidemulsion) for 72 h. Results are expressed as percentages of the growth ofcells exposed to medium alone. (B) Cells were switched to serum-free mediumafter preincubation with the lipid emulsions in a concentration of 0.72mL zmL21, corresponding to 50mM EPA in the fish-oil–based lipid emulsion for36 h and further incubated with or without EGF (20 ngz mL21) for 12 h.Significance (A, treatment with the lipid emulsions versus control; B, treat-ment with versus treatment without EGF) was tested with the Mann–WhitneyU test (**P , 0.01, ***P , 0.001). Results represent the mean6 standarderror of six to eight determinations. Similar trends were observed in at leastthree replicating experiments. EGF, epidermal growth factor; FO, fish-oil–based lipid emulsion; SO, soybean-oil–based lipid emulsion.

Nutrition 17:474–479, 2001 0899-9007/01/$20.00©Elsevier Science Inc., 2001. Printed in the United States. All rights reserved.

In the present study, we demonstrated that preincubation withthe fish-oil–based lipid emulsion abrogated the growth-promotingeffect of EGF in human pancreatic cancer cells. Studies investi-gating the mechanism of this effect are currently underway.First, the EGF receptor is membrane bound, and the lipidenvironment of the surrounding membrane can influence EGFbinding to its receptor and its internalization. Second, afterbeing incorporated into the plasma membrane,v-3 fatty acidsmight change the composition or physical properties of themembrane so that EGF binding to its receptor is altered. Third,second messengers downstream from the EGF receptor that areinvolved in cell growth might intersect with signaling eventsinitiated by v-3 fatty acid after its incorporation into the cellmembrane.

In conclusion, our data show that nutrients present in fish oil,i.e., v-3 fatty acids, interfere with EGF-induced epithelial cellgrowth, which might provide a “natural” way to impair growth incancer cells, which overexpress the EGF receptor.10

ACKNOWLEDGMENT

The authors are indebted to Dr. Vladan Milovic for critical readingof the paper.

Angela Jordan, MScJurgen Stein, MD, PhD

2nd Department of MedicineJ. W. Goethe University

Frankfurt, Germany

REFERENCES

1. Begin ME, Das UN, Ells G, Horrobin DF. Differential killing of human carci-noma cells by n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst1986;77:1053

2. Falconer JS, Ross JA, Fearon KCH, et al. Effect of eicosapentaenoic acid andother fatty acids on the growth in vitro of human pancreatic cancer cell lines. Br JCancer 1994;69:826

3. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses.FASEB J 1995;9:484

4. Hwang D, Rhee SH. Receptor-mediated signalling pathways: potential targets ofmodulation by dietary fatty acids. Am J Clin Nutr 1999;70:545

5. Steel CM. Peptide regulatory factors and malignancy. Lancet 1989;8653:306. Milovic V, Deubner C, Zeuzem S, et al. EGF stimulates polyamine uptake in

Caco-2 cells. Biochem Biophys Res Commun 1995;206:9627. Ullrich A, Schlessinger J. Signal tranduction by receptors with tyrosine kinase

activity. Cell 1990;61:2038. Mollerup S, Haugen A. Differential effect of polyunsaturated fatty acids on

cell proliferation during human epithelial in vitro carcinogenesis: involve-ment of epidermal growth factor receptor tyrosine kinase. Br J Cancer1996;74:613

9. Liehr RM, Melnykovych G, Solomon TE. Growth effects of regulatory peptideson human pancreatic cancer cell lines PANC-1 and MIA PaCa-2. Gastroenter-ology 1990;98:1666

10. Barton CM, Hall PA, Hughes CM, Gullick WJ, Lemoine NR. Transforminggrowth factor alpha and epidermal growth factor in human pancreatic cancer.J Pathol 1991;163:111

PII S0899-9007(01)00535-4

Influence of Glutamine-EnrichedParenteral Nutrition on NitrogenBalance and Immunologic Status inPatients Undergoing Elective AorticAneurysm RepairAneurysm repairs comprise 10% of all vascular operative proce-dures. Aortic cross clamping and unclamping are key proceduresduring abdominal aortic reconstruction and thus risk factors forvisceral ischemic and reperfusion syndrome. These factors mightcause damage to the intestinal barrier.1–3 The breakdown of gas-trointestinal integrity results in increased gut permeability, micro-bial translocation that can lead to sepsis of intestinal origin, andmultiple organ failure.4,5

Several studies have indicated that glutamine-enriched totalparenteral nutrition (PN) improves nitrogen balance and immuno-logic status and reduces length of stay (LOS) of patients aftergeneral surgery and bone marrow transplantation.6

We investigated the effect of glutamine-enriched PN on thenitrogen balance, immunologic status, and LOS in patients under-going abdominal aortic repair.

PATIENTS

We studied 30 well-nourished male patients (average age6 stan-dard deviation: 656 8 y), undergoing elective aortic abdominalaneurysm reconstruction. Nutrition status was estimated for allpatients the day before their operations based on percentage ofoptimal body weight, body mass index, triceps skinfold thickness,mid–arm muscle circumference, albumin, transferrin, cholinester-ase, and total lymphocyte count. Patients with diabetes mellitus,renal failure, hepatic failure, or malignant disease were excluded.Patients were randomly assigned to a control group (group I) or anexperimental group (group II), in which glutamine-enriched PNwas used. Each group consisted of 15 patients who were compa-rable with regard to age, weight, height, and nutrition status (TableI). The study was approved by the local ethical committee.

METHODS

PN provided estimated requirements for energy (35 kcalz kg21 zd21) and protein (1.5 gz kg21 z d21). Energy requirements wereestimated based on the Harris–Benedict equation for men as mod-ified by Long.7,8 All patients had pulmonary artery catheters inplace to measure hemodynamic parameters and oxygen consump-tion (VO2 was used to calculate stress factors) on the day of theoperation and the first postoperative day. These measurementswere analyzed with hemodynamic-profile software HEMOPRO 2(Viggo-Spectramed, Helsingborg, Sweden).

Patients in both groups received the same amounts of PN(group II: 17.26 1.2 g N/d and 21006 185 kcal/d, group I:17.46 1.3 g N/d and 21306 168 kcal/d). PN (all-in-one mixture)was given continuously for the first 10 postoperative days. Allpatients received isonitrogenous and isoenergetic PN. Those ingroup I received standard amino-acid solution, and those in groupII received 0.202 g of glutamine per kilogram per day (Dipeptiven,

Correspondence to: Katarzyna A. Karwowska, MD, Karol MarcinkowskiUniversity of Medical Sciences, 1st Department of Anesthesiology, ul.Dluga 1/2, 61-848 Poznan´, Poland

Date accepted: February 16, 2001.

Nutrition Volume 17, Number 6, 2001 475Research Letters