59668  />

Figure 2. Adenocarcinoma at 100× magnification. Histological picture at 100× magnification of an adenocarcinoma at the gastrointestinal anastomotic area.

Table 2. Gastric lesions following 50 weeks of duodno-gastric reflux.

Figure 3. Distribution of adenocarcinomas. Histogram showing distribution of adenocarcinomas in the various feeding groups. *p < 0.01.

different bioactive fatty acids were analyzed separately (data not shown).

3.2. Fatty Acid Profile and Anti-Inflammatory Fatty Acid Index (AIFAI) in Plasma

The plasma fatty acid composition is shown in Table 3. TTA reduced the level of EPA (C20:5n-3) and DHA (C22:6n-3) whereas the amount of dihomo-γ-linoleic acid (DGLA, C20:3n-6) increased. The level of arachidonic acid (AA, C20:4n-6) was not affected by TTA alone, but decreased in both groups given FO (Table 3). In the FO groups, the level of AA decreased and the levels of EPA and DHA increased significantly (p < 0.001) compared to the other treatment groups. The calculated plasma AIFAI was significantly increased in both groups receiving FO (Figure 4).

3.3. Immunohistochemical Findings

Immunohistochemical staining with polyclonal antibodyies against PCNA, COX-2 and p53 was performed in all cases where AC was observed (Figure 5 and Table 4). There was no significant difference between the groups in any of the three proteins (Table 4). In all groups, however, we observed signs of inflammation as evidenced by positive COX-2 staining in AC. Further, PCNA staining was positive in most AC indicating proliferation. Immunohistochemical analyses in rats without cancer revealed no differences between the various feeding groups. COX-2 always stained positive in the cytoplasm of the gastric mucosa with reduced activity in the upper middle layer of the gastric mucosa, interpreted as the area of the gut regenerative cell lineage (GRCL) [33]. PCNA stained positive in the cell nuclei in the area identified as the GRCL and the nuclei in the crypts of the intestine. p53 staining was negative. Illustration of immunohistochemical staining in normal gastric mucosa is shown in Figure 6.

4. Discussion

In this experiment we tested the hypothesis if bioactive fatty acids like n-3 PUFAs found in FO and/or TTA added to a HF diet could reduce the risk of developing AC in a DGR model. We also looked at the AC development in the HF compared to the LF diet group. We found that FO as well as TTA tended to decrease the occurrence of AC. However, when combining FO with

Figure 4. Anti inflammatory index in the various feeding groups. Histogram showing the anti inflammatory index for the specific treatment groups. a: treatment without FO; b: Treatment with FO. **p < 0.001.

Figure 5. Immunohistochemical staining of adenocarcinomas. Immunohistochemical staining of an adenocarcinoma shown at 100× magnification. A: COX2; B: PCNA and C: p53.

Figure 6. Immunohistochemical staining in normal gastric mucosa. Immunohistochemical staining of normal gastric mucosa tissue at 100× magnification. A: COX2; B: PCNA and C: p53.

Table 3. Plasma fatty acid profile.

Table 4. Immunohistochemical analysis of adenocarcinomas.

TTA, we observed a significantly lower development of ACs compared to the HF control group (p < 0.01). Our results thus demonstrate an additive effect of these bioactive lipids. When comparing the LF with the HF control group, we found a lower but not significant reduction in AC development in the LF group.

It is known that establishing a chronic gastric reflux of duodenal content in a rat model can induce gastric cancer without use of carcinogens [34]. Development of chronic inflammation is correlated to the amount of reflux. By increasing the reflux, the incidence of gastritis and cancer development increases [34,35]. The induction of adenocarcinomas follows the sequence of chronic inflammation, leading to metaplasia, dysplasia and finally cancer development. In our model we induced a moderate reflux and therefore we rarely found gastritis in other areas of the stomach than adjacent to the anastomosis.

There were significantly more adenocystic proliferations (ACP) in the HF + FO group and in the HF + FO + TTA treatment group (Table 2). Miwa et al. [35] has suggested that ACPs are precancerous lesions. According to these findings it might be considered whether TTA treatment alone is able to lower the frequency of ACP. FO seems to reduce the risk of ACP transforming into AC but this treatment did not fully prevent its occurrence. The underlying mechanisms of the anti-tumor activity of FO are not fully understood. There is some evidence indicating that its n-3 PUFAs, EPA and DHA, can reduce inflammation and promote apoptosis in cancer cells [20-22,36]. One of these mechanisms is believed to be that the n-6 PUFA, AA (20:4n-6) is the main substrate for eicosanoids like prostaglandins, leucotriens and thromboxans. These eicosanoids are mediators and regulators of inflammation [37,38]. The AA metabolism and its metabolites are known targets for anti-inflammatory drug therapy. This includes non-steroidal anti-inflammatory drugs (NSAIDs) that inhibit the COX-2 activity. This effect was documented in a similar reflux model where gastritis and cancer development was reduced signifycantly by treatment with meloxicam, a specific COX-2-inhibitor [13]. However, there was no effect of TTA and FO on the protein level of COX-2, as the COX-2 staining was equal in all treatment groups in specimens without AC lesions. An inhibition of the COX-2 activity instead of the protein level could not be excluded though. The same observation was seen on staining against PCNA and p53, where there was no difference between the groups.

It has been shown that supplementation of FO, which is rich in EPA and DHA, induce a time-dependent incorporation of these n-3 PUFAs into the cell membrane phospholipids [37,39,40]. Once present in the membranes, EPA and DHA will then dislodge AA available for synthesis of AA-derived eicosanoids. EPA and DHA are also able to act as a substrate for the cyclooxygenase and the 5-lipoxygenase [37]. These eicosanoids produced from EPA and DHA have a slightly modified structure and exert less inflammatory activity than AA-derived eicosanoids. EPA can also reduce proliferation by directly inducting apoptosis as demonstrated in cell culture models [41,42] .

The plasma fatty acid profile in each treatment group (Table 3) showed a significantly increase of the AIFAI (Figure 4) with a highly significant difference (p < 0.001) between the groups receiving FO compared to the groups without FO supplemented. This observation supports the hypothesis that EPA and DHA are able to exert anti-inflammatory effects in this model.

The rationale behind the combination of FO and TTA was that they theoretically might have additive antiproliferative effects, even if it has been reported that TTA reduce the EPA and DHA levels in plasma [43]. In the present study, the content of EPA and DHA in plasma increased after FO supplementation, whereas the content of dihomo-γ-linoleic acid (DGLA, C20:3n-6) was reduced. The synthesis of eicosanoids depends on the availability of the 20-carbon PUFAs, either arriving via circulation or arising from local tumor production catalyzed by delta 5 and delta 6 desaturases. FO reduced the content of AA in plasma, possibly making AA less available for eicosanoid synthesis, which could modulate inflammation [39]. After FO supplementation, the AIFAI increased in both groups (Figure 4). As shown in Figure 3, TTA reduced the incidence of AC even more than FO alone. As the AIFAI was unchanged after TTA treatment, we therefore hypothesize that the anti-cancer effect of TTA is different than for FO, and that is may be due to induction of apoptosis, possibly mediated by mitochondrial alterations and changes in inflammation signalling as outlined in the background section. Since no signifycant difference was found with the ANOVA test when treatments with different bioactive fatty acids were done separately, we assume that the pathways are different and that there is an additive effect of TTA and FO to reduce the occurrence of AC.

4. Conclusion

In conclusion, a diet containing both FO and TTA has an additive and reducing effect on the development of AC in a DGR rat model. The exact mechanism is not clear and further studies are needed to elucidate this.

5. Acknowledgements

The authors gratefully acknowledge Anne Aarsand, Inger Vikøyr, Christ Berge and Laila Vaardal for laboratory assistance and Åshild Åsebø for taking care of all the animals. This project was supported financially by Western Norway Regional Health Authority and the Nordic Centre of Excellence-MitoHealth.


  1. J. Ferlay, P. Autier, M. Boniol, M. Heanue, M. Colombet and P. Boyle, “Estimates of the Cancer Incidence and Mortality in Europe in 2006,” Annals of Oncology, Vol. 18, No. 3, 2007, pp. 581-592. doi:10.1093/annonc/mdl498
  2. D. M. Parkin, F. Bray, J. Ferlay and P. Pisani, “Global Cancer Statistics, 2002,” Cancer Journal for Clinicians, Vol. 55, No. 2, 2005, pp. 74-108. doi:10.3322/canjclin.55.2.74
  3. C. Prinz, S. Schwendy and P. Voland, “H Pylori and Gastric Cancer: Shifting the Global Burden,” World Journal of Gastroenterology, Vol. 12, No. 34, 2006, pp. 5458-5464.
  4. H. Brenner, D. Rothenbacher and V. Arndt, “Epidemiology of Stomach Cancer,” Methods in Molecular Biology, Vol. 472, 2009, pp. 467-477. doi:10.1007/978-1-60327-492-0_23
  5. K. D. Crew and A. I. Neugut, “Epidemiology of Gastric Cancer,” World Journal of Gastroenterology, Vol. 12, No. 3, 2006, pp. 354-362.
  6. S. Bahmanyarand and W. Ye, “Dietary Patterns and Risk of Squamous-Cell Carcinoma and Adenocarcinoma of the Esophagus and Adenocarcinoma of the Gastric Cardia: a Population-Based Case-control Study in Sweden,” Nutrition and Cancer, Vol. 54, No. 2, 2006, pp. 171-178. doi:10.1207/s15327914nc5402_3
  7. C. A. Gonzalez, P. Jakszyn, G. Pera, A. Agudo, S. Bingham and J. R. Quiros, “Meat Intake and Risk of Stomach and Esophageal Adenocarcinoma within the European Prospective Investigation into Cancer and Nutrition (EPIC),” Journal of the National Cancer Institute, Vol. 98, No. 5, 2006, pp. 345-354. doi:10.1093/jnci/djj071
  8. S. T. Mayne, H. A. Risch, R. Dubrow, W. H. Chow, M. D. Gammon and J. F. Fraumeni Jr., “Nutrient Intake and Risk of Subtypes of Esophageal and Gastric Cancer,” Cancer Epidemiology, Biomarkers & Prevention, Vol. 10, No. 10, 2001, pp. 1055-1062.
  9. S. A. Navarro Silvera, S. T. Mayne, H. Risch, M. D. Gammon, T. L. Vaughan and W. H. Chow, “Food Group Intake and Risk of Subtypes of Esophageal and Gastric Cancer,” International Journal of Cancer, Vol. 123, No. 4, 2008, pp. 852-860. doi:10.1002/ijc.23544
  10. P. A. van den Brandt, A. A. Botterweck and R. A. Goldbohm, “Salt Intake, Cured Meat Consumption, Refrigerator Use and Stomach Cancer Incidence: A Prospective Cohort Study (Netherlands),” Cancer Causes Control, Vol. 14, No. 5, 2003, pp. 427-438. doi:10.1023/A:1024979314124
  11. E. Riboli and T. Norat, “Cancer Prevention and Diet: Opportunities in Europe,” Public Health Nutrition, Vol. 4, No. 2B, 2001, pp. 475-484. doi:10.1079/PHN2001158
  12. C. Liu and R. M. Russell, “Nutrition and Gastric Cancer Risk: An Update,” Nutrition Reviews, Vol. 66, No. 5, 2008, pp. 237-249. doi:10.1111/j.1753-4887.2008.00029.x
  13. M. Oba, K. Miwa, T. Fujimura, S. Harada, S. Sasaki and T. Hattori, “Chemoprevention of Glandular Stomach Carcinogenesis through Duodenogastric Reflux in Rats by a COX-2 Inhibitor,” International Journal of Cancer, Vol. 123, No. 7, 2008, pp. 1491-1498. doi:10.1002/ijc.23742
  14. M. Oba, K. Miwa, T. Fujimura, S. Harada, S. Sasaki, K. Oyama and T. Hattori, “A Selective Cyclooxygenase-2 Inhibitor Prevents Inflammation-related Squamous Cell Carcinogenesis of the Forestomach via Duodenogastric Reflux in Rats,” Cancer, Vol. 115, No. 2, 2009, pp. 454-464. doi:10.1002/cncr.23990
  15. H. Chen, M. H. Ward, B. I. Graubard, E. F. Heineman, R. M. Markin and K. L. Tucker, “Dietary Patterns and Adenocarcinoma of the Esophagus and Distal Stomach,” American Journal of Clinical Nutrition, Vol. 75, No. 1, 2002, pp. 137-144.
  16. F. L. Haven, “The Effect of Cod-Liver Oil on Tumor Growth,” American Journal of Cancer, Vol. 27, 1936, pp. 95-98.
  17. V. Maillard, P. Bougnoux, P. Ferrari, M. L. Jourdan, M. Pinault and V. Chajes, “N-3 and N-6 Fatty Acids in Breast Adipose Tissue and Relative Risk of Breast Cancer in a Case-control Study in Tours, France,” International Journal of Cancer, Vol. 98, No. 1, 2002, pp. 78-83. doi:10.1002/ijc.10130
  18. P. Terry, P. Lichtenstein, M. Feychting, A. Ahlbom and A. Wolk, “Fatty Fish Consumption and Risk of Prostate Cancer,” Lancet, Vol. 357, No. 9270, 2001, pp. 1721-1812. doi:10.1016/S0140-6736(00)04889-3
  19. P. Terry, A. Wolk, H. Vainio and E. Weiderpass, “Fatty Fish Consumption Lowers the Risk of Endometrial Cancer: a Nationwide Case-control Study in Sweden,” Cancer Epidemiology, Biomarkers & Prevention, Vol. 11, No. 1, 2002, pp. 143-145.
  20. J. Cheng, K. Ogawa, K. Kuriki, Y. Yokoyama, T. Kamiya and S. Tokudome, “Increased Intake of n-3 Polyunsaturated Fatty Acids Elevates the Level of Apoptosis in the Normal Sigmoid Colon of Patients Polypectomized for Adenomas/Tumors,” Cancer Letters, Vol. 193, No. 1, 2003, pp. 17-24. doi:10.1016/S0304383502007176
  21. E. D. Courtney, S. Matthews, C. Finlayson, D. Di Pierro, A. Belluzzi and R. J. Leicester, “Eicosapentaenoic Acid (EPA) Reduces Crypt Cell Proliferation and Increases Apoptosis in Normal Colonic Mucosa in Subjects with a History of Colorectal Adenomas,” International Journal of Colorectal Disease, Vol. 22, No. 7, 2007, pp. 765-776. doi:10.1007/s00384-006-0240-4
  22. Y. Y. Fan, Y. Zhan, H. M. Aukema, L. A. Davidson, L. Zhou and R. S. Chapkin, “Proapoptotic Effects of Dietary (n-3) Fatty Acids Are Enhanced in Colonocytes of Manganese-Dependent Superoxide Dismutase Knockout Mice,” Journal of Nutrition, Vol. 139, No. 7, 2009, pp. 1328-1332. doi:10.3945/jn.109.106203
  23. K. Berge, K. J. Tronstad, P. Bohov, L. Madsen and R. K. Berge, “Impact of Mitochondrial Beta-Oxidation in Fatty Acid-mediated Inhibition of Glioma Cell Proliferation,” Journal of Lipid Research, Vol. 44, No. 1, 2003, pp. 118-127. doi:10.1194/jlr.M200312-JLR200
  24. K. Berge, K. J. Tronstad, E. N. Flindt, T. H. Rasmussen, L. Madsen, K. Kristiansen and R. K. Berge, “Tetradecylthioacetic Acid Inhibits Growth of Rat Glioma Cells ex vivo and in vivo via PPAR-dependent and PPAR-independent Pathways,” Carcinogenesis, Vol. 22, No. 11, 2001, pp. 1747-1755. doi:10.1093/carcin/22.11.1747
  25. K. J. Tronstad, O. Bruserud, K. Berge and R. K. Berge, “Antiproliferative Effects of a Non-Beta-Oxidizable Fatty Acid, Tetradecylthioacetic Acid, in Native Human Acute Myelogenous Leukemia Blast Cultures,” Leukemia, Vol. 16, No. 11, 2002, pp. 2292-2301. doi:10.1038/sj.leu.2402698
  26. K. J. Tronstad, B. T. Gjertsen, C. Krakstad, K. Berge, O. T. Brustugun and R. K. Berge, “Mitochondrial-Targeted Fatty Acid Analog Induces Apoptosis with Selective Loss of Mitochondrial Glutathione in Promyelocytic Leukemia Cells,” Chemistry & Biology, Vol. 10, No. 7, 2003, pp. 609-618. doi:10.1016/S1074-5521(03)00142-X
  27. P. O. Iversen, D. R. Sorensen, K. J. Tronstad, O. A. Gudbrandsen, A. C. Rustan and C. A. Drevon, “A Bioactively Modified Fatty Acid Improves Survival and Impairs Metastasis in Preclinical Models of Acute Leukemia,” Clinical Cancer Research, Vol. 12, No. 11, 2006, pp. 3525-3531. doi:10.1158/1078-0432.CCR-05-2802
  28. M. Suo, K. Mukaisho, A. Shimomura, H. Sugiha and T. Hattori, “Thioproline Prevents Carcinogenesis in the Remnant Stomach Induced by Duodenal Reflux,” Cancer Letters, Vol. 237, No. 2, 2006, pp. 256-262. doi:10.1016/j.canlet.2005.06.019
  29. E. G. Bligh and W. J. Dyer, “A Rapid Method of Total Lipid Extraction and Purification,” Canadian Journal of Biochemistry and Physiology, Vol. 37, No. 8, 1959, pp. 911-917. doi:10.1139/o59-099
  30. M. Kates, “General Analytical Procedures. In Techniques in Lipidology,” M. Dates Elsevier Science Publishers, Amsterdam, 1986, pp. 112-185.
  31. S. R. Chavali, W. W. Zhong, T. Utsunomiya and R. A. Forse, “Decreased Production of Interleukin-1-Beta, Prostaglandin-E2 and Thromboxane-B2, and Elevated Levels of Interleukin-6 and -10 Are Associated with Increased Survival during Endotoxic Shock in Mice Consuming Diets Enriched with Sesame Seed Oil Supplemented with Quil-A saponin,” International Archives of Allergy and Immunology, Vol. 114, No. 2, 1997, pp. 153-160. doi:10.1159/000237661
  32. T. Utsunomiya, S. R. Chavali, W. W. Zhong and R. A. Forse, “Effects of Sesamin-Supplemented Dietary Fat Emulsions on the ex Vivo Production of Lipopolysaccharide-Induced Prostanoids and Tumor Necrosis Factor Alpha in Rats,” American Journal of Clinical Nutrition, Vol. 72, No. 3, 2000, pp. 804-808.
  33. K. Mukaisho, K. Miwa, H. Kumagai, M. Bamba, H. Sugihara and T. Hattori, “Gastric Carcinogenesis by Duodenal Reflux through Gut Regenerative Cell Lineage,” Digestive Diseases and Sciences, Vol. 48, No. 11, 2003, pp. 2153-2158. doi:10.1023/B:DDAS.0000004519.26201.a4
  34. K. Miwa, T. Hattori and I. Miyazaki, “Duodenogastric Reflux and Foregut Carcinogenesis,” Cancer, Vol. 75, No. 6, 1995, pp. 1426-1432. doi:10.1002/1097-0142(19950315)75:6+<1426::AID-CNCR2820751506>3.0.CO;2-#
  35. K. Miwa, H. Hasegawa, T. Fujimura, H. Matsumoto, R. Miyata and T. Hattori, “Duodenal Reflux through the Pylorus Induces Gastric Adenocarcinoma in the Rat,” Carcinogenesis, Vol. 13, No. 12, 1992, pp. 2313-2316. doi:10.1093/carcin/13.12.2313
  36. S. P. Mehta, A. P. Boddy, J. Cook, V. Sams, E. K. Lund and M. Rhodes, “Effect of n-3 Polyunsaturated Fatty Acids on Barrett’s Epithelium in the Human Lower Esophagus,” American Journal of Clinical Nutrition, Vol. 87, No. 4, 2008, pp. 949-956.
  37. W. Fetterman Jr. and M. M. Zdanowicz, “Therapeutic Potential of n-3 Polyunsaturated Fatty Acids in Disease,” American Journal of Health-System Pharmacy, Vol. 66, No. 13, 2009, pp. 1169-1179. doi:10.2146/ajhp080411
  38. S. L. Tilley, T. M. Coffman and B. H. Koller, “Mixed Messages: Modulation of Inflammation and Immune Responses by Prostaglandins and Thromboxanes,” Journal of Clinical Investigation, Vol. 108, No. 1, 2001, pp. 15-23.
  39. M. J. James, R. A. Gibson and L. G. Cleland, “Dietary Polyunsaturated Fatty Acids and Inflammatory Mediator production,” American Journal of Clinical Nutrition, Vol. 71, No. 1, 2000, pp. 343S-348S.
  40. R. I. Sperling, A. I. Benincaso, C. T. Knoell, J. K. Larkin, K. F. Austen and D. R. Robinson, “Dietary Omega-3 Polyunsaturated Fatty Acids Inhibit Phosphoinositide Formation and Chemotaxis in Neutrophils,” Journal of Clinical Investigation, Vol. 91, No. 2, 1993, pp. 651-660. doi:10.1172/JCI116245
  41. H. Heimli, C. Giske, S. Naderi, C. A. Drevon and K. Hollung, “Eicosapentaenoic Acid Promotes Apoptosis in Ramos Cells via Activation of Caspase-3 and -9,” Lipids, Vol. 37, No. 8, 2002, pp. 797-802. doi:10.1007/s11745-002-0963-6
  42. H. Heimli, K. Hollung, and C. A. Drevon, “Eicosapentaenoic Acid-Induced Apoptosis Depends on Acyl CoASynthetase,” Lipids, Vol. 38, No. 3, 2003, pp. 263-268. doi:10.1007/s11745-003-1059-z
  43. D. K. Asiedu, L. Froyland, H. Vaagenes, O. Lie, A. Demoz and R. K. Berge, “Long-Term Effect of Tetradecylthioacetic Acid: A Study on Plasma Lipid Profile and Fatty Acid Composition and Oxidation in Different Rat Organs,” Biochimica et Biophysica Acta, Vol. 1300, No. 2, 1996, pp. 86-96.

Journal Menu >>