Nutrition and cancer prevention

1. Nutrition and cancer prevention Zorica Juranić, Institut za onkologiju i radiologiju Srbije Beograd

2. The role of nutrition (and physical activity) in cancer prevention is in the focus of reasearch in modern medicine. Data from many epidemiological studies are constantly collected and analyzed. Undoubtedly, both of these approaches get their importance in programs for cancer prevention and it is published that their applications could diminish the incidence of various malignancies for 30% to 40%. Amin AR, Kucuk O, Khuri FR, Shin DM. Perspectives for cancer prevention with natural compounds. J Clin Oncol. 2009; 27:2712-2725.

3. The role of nutrition in prevention of malignant diseases Results from early epidemiological studies initiated new experimental studies in vitro and in vivo. It was proved that many of food extracts as well as of food constituents possess anti-oxidative/prooxidative action which is differently regulated in the dependence of redox potential of target cells. The bioactive pharmacophore of phytonutrients could inhibit signal transduction pathways related to the proliferation of malignant cells,1 but also could reactivate silenced tumor suppression genes, by targeting epigenetic regulators. According to this, food constituents could act as scavengers of ROS and additionally could have: - Anti-inflammatory action - Antitumor action - Immuno-modulating action.

4. Quercetin exhibited potential for the activation of histone acetyltransferase (HAT) and the inhibition of histone deacetyltransferase (HADC), both of which contributed to histone acetylation. • However, only the activation effect on HAT was associated with the ERK and JNK pathway. These results demonstrated that quercetin induced FasL-related apoptosis by transactivation through activation of c-jun/AP-1 and promotion of histone H3 acetylation in HL-60 cells. • Spagnuolo C, Russo M, Bilotto S, Tedesco I, Laratta B, Russo GL. Dietary polyphenols in cancer prevention: the example of the flavonoid quercetin in leukemia. Ann N Y Acad Sci. 2012 Jul;1259:95-103 • Russo M, Spagnuolo C, Tedesco I, Bilotto S, Russo GL. The flavonoid quercetin in disease prevention and therapy: facts and fancies. Biochem Pharmacol. 2012 Jan 1;83(1):6-15. • Lee WJ, Chen YR, Tseng TH. Quercetin induces FasL-related apoptosis, in part, through promotion of histone H3 acetylation in human leukemia HL-60 cells. Oncol Rep. 2011 Feb;25(2):583-91. • Román-Gómez J, Cordeu L, Agirre X, Jiménez-Velasco A, San José-Eneriz E, Garate L, Calasanz MJ, Heiniger A, Torres A, Prosper F. Epigenetic regulation of Wnt-signaling pathway in acute lymphoblastic leukemia. Blood. 2007 Apr 15;109(8):3462-9.

5. Promising results from experiments in vitro, as well as from studies on chemically -induced tumors in rodents in vivo, initiated well controlled epidemiological studies- clinical trials. Although some reports regarding cancer - preventive action of green tea catechins (Rizzi and Bettuzzi 2013) on the prevention of prostate cancer were confirmed, the other results were not always in accordance with the data obtained in vitro and in vivo. Even more some of them gave contradictory results (the incidence of some cancers was enhanced and these trials were finished before the planed end of the study). These are ATBC (alpha-tocopherol, and beta-caroten) and CARET (retinol efficacy trial). Participants (heavy smokers) were treated with high supplemental doses of beta-caroten. This study was prematurely ended as one analysis revealed a 28% increase in lung cancer incidence in relation to people using placebo. While epidemiological study showed prostate cancer risk reduction for 22% for vitamin E and for 63% with selenized yeast respectively, trial investigators revealed for 17% increased risk for prostate cancer for men using vitamin E supplements.

6. CONCLUSION I • Therefore, from scientific point of view, malignant diseases are very complex and it is too early to give some general recommendations regarding enforced use of large quantity of some food supplements in the aim of cancer prevention. • Only it could be said according to epidemiological studies, that moderate use of delicious food like various fruits and vegetables, olive oil, fish, may help in the prevention of some types of cancer and this also may be the pleasure for our taste.

7. II For secondary cancer prevention the action of food constituents might be mediated through immunomodulation?

8. In order to try to give the answer to this question regarding secondary cancer prevention, we determined and analyzed some of immune response parameters in patients with • melanoma

9. A B C Figure 1 (A) Serum IgM immunoreactivity to mushroom tyrosinase in healthy controls and in patients with melanoma or vitiligo, (B) Serum IgA immunoreactivity to mushroom tyrosinase in healthy controls and in patients with melanoma or vitiligo, (C) Serum IgG immunoreactivity to mushroom tyrosinase in healthy controls and in patients with melanoma or vitiligo. Red squares represent velues of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases. Đorđić M, et al., BMC Complement Altern Med 2012; 12:109.

10. A B C Figure 2 (A) Serum IgM immunoreactivity to melanin in healthy controls and in patients with melanoma or vitiligo, (B) Serum IgA immunoreactivity to melanin in healthy controls and in patients with melanoma or vitiligo, (C) Serum IgG immunoreactivity to melanin in healthy controls and in patients with melanoma or vitiligo. Red squares represent values of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases. Đorđić M, et al., BMC Complement Altern Med 2012; 12:109.

11. A B C D Figure 3(A) Percentage of CD16+CD56+ lymphocytes (NK cells) in healthy controls and in patients with melanoma or vitiligo, (B) Percentage of CD16+CD56+ overall white blood cells (NK cells) in healthy controls and in patients with melanoma or vitiligo, (C) Percentage of CD16+ overall white blood cells in healthy controls and in patients with melanoma or vitiligo, (D) Percentage of CD89+ overall white blood cells in healthy controls and in patients with melanoma or vitiligo. Red squares represent values of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases. Đorđić M, et al., BMC Complement Altern Med 2012; 12:109.

12. This set up a question is there any possibility that the oxidative burst reaction of granulocytes mediated by IgA auto-antibodies to melanoma -associated antigens tyrosinase or melanin could non-specifically damage neighboring lymphocytes? The answer could be: ”Very possible”, because Pleass et al.,2007. reported that cross-linking of IgA receptors (CD89) on neutrophils induced by immune complexes of antigen and specific IgA could induce significant not only eosinophil but also neutrophil NADPH oxidase activation and degranulation. Pleass RJ, Lang ML, Kerr MA, Woof JM. IgA is a more potent inducer of NADPHoxidase activation and degranulation in blood eosinophils than IgE. Mol Immunol. 2007 Feb;44(6):1401-8.

13. A B C Figure 5In vitro stimulation (G%) of PBMC of healthy controls, as well as of patients with malanoma or vitiligo, on proliferation by synthetic melanin (5mg/ml) (A), or by phytohemagglutinin (5mg/ml) from red beans (B), or by the mixture of melanin and phytohemagglutinin (C), in nutrient medium RPMI 1640 with 10% autologous plasma. Red squares represent values of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases. Đorđić M, et al., BMC Complement Altern Med 2012; 12:109.

14. Figure 1. Percentage of lymphocytes in healthy controls and in patients with melanoma or vitiligo Red squares represent values of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases.

15. Figure 4. Ratio of the percentage of granulocytes and the percentage of lymphocytes in healthy controls and in patients with melanoma or vitiligo. Red squares represent values of melanoma patients with metastatic disease, while brighter red squares with borders represent values of melanoma patients without metastases. Đorđić M, et al., BMC Complement Altern Med 2012; 12:109.

16. Therefore, consideration forthe use of oral immunisation (by thermically treated edible mushrooms, or black cuttlefish risotto) in the aim to enhance the IgM immunity to melanoma antigens melanin /or tyrosinase, could be taken in mind: • only in people with normal percentage of granulocytes, and normal or low levels of anti-melanoma IgA antibodies, • or in people to whom blockers of degranulation and of oxidative burst reaction of granulocytes were added. • Or in the presence of the IgM, or IgG synthesis stimulant

17. How to decrease enhanced and potentially dangerous IgA induced oxidative burst reaction and degranulation by neutrophils? • How to decrease IgA? • How to inhibit induction of oxidative burst reaction and degranulation? • How to decrease the percentage of granulocytes?

18. Group from Barcelona, from Spain, reported that a cocoa-enriched diet downregulate imunoglobulin (Ig) A (IgA) intestinal secretion in rats. • Massot-Cladera M, Pérez-Berezo T, Franch A, Castell M, Pérez-Cano FJ. Cocoa modulatory effect on rat faecalmicrobiota and colonic crosstalk. Arch Biochem. Biophys. 2012 Nov 15;527(2):105-12  • Pérez-Berezo T, Franch A, Castellote C, Castell M, Pérez-Cano FJ. Mechanisms involved in down-regulation of intestinal IgA in rats by high cocoa intake. J NutrBiochem. 2012 Jul;23(7):838-44.   • Pérez-Berezo T, Franch A, Ramos-Romero S, Castellote C, Pérez-Cano FJ, Castell M. Cocoa-enriched diets modulate intestinal and systemic humoral immune response in young adult rats. Mol Nutr Food Res. 2011 May;55 Suppl 1:S56-66.   • Ramiro-Puig E, Pérez-Cano FJ, Ramos-Romero S, Pérez-Berezo T, Castellote C, Permanyer J, Franch A, Izquierdo-Pulido M, Castell M. Intestinal immune system of young rats influenced by cocoa-enriched diet. J NutrBiochem. 2008 Aug;19(8):555-65. Caloric restriction reduces IgA levels in mouse small intestine • Lara-Padilla E, Campos-Rodríguez R, Jarillo-Luna A, Reyna-Garfias H, Rivera-Aguilar V, Miliar A, Berral de la Rosa FJ, Navas P, López-Lluch G. Caloric restriction reduces IgA levels and modifies cytokine mRNA expression in mouse small intestine. J NutrBiochem. 2011;22(6):560-6. • Viloria M, Lara-Padilla E, Campos-Rodríguez R, Jarillo-Luna A, Reyna-Garfias H, López-Sánchez P, Rivera-Aguilar V, Salas-Casas A, Berral de la Rosa FJ, García-Latorre E. Effect of moderate exercise on IgA levels and lymphocyte count in mouse intestine. Immunol Invest. 2011;40(6):640-56. • Suárez-Souto MA, Lara-Padilla E, Reyna-Garfias H, Viloria M, López-Sánchez P, Rivera-Aguilar V, Miliar-García Á, Kormanovski A, Domínguez-López ML, Campos-Rodríguez R. Caloric restriction modifies both innate and adaptive immunity in the mouse small intestine. J PhysiolBiochem. 2012 ; 68(2):163-73.

19. Conclusions II In addition to their direct anticancer action on malignant cells, some phytonutrients are blockers of damaging neutrophil degranulation and oxidative burst reaction. • It is reported that these properties have : quercetin, resveratrol, pterostilbene, and also piceatannol, pinosylvin, curcumin, N-feruloylserotonin.. . • And this is along with the results of earlier epidemiological studies that consumptions of berries have cancer protective action which is expressed on many levels. • Pečivová J et al.,Quercetin inhibits degranulation and superoxide generation in PMA stimulated neutrophils. Interdiscip Toxicol. 2012;5(2):81-3. • Mačičková et al., Effect of stilbene derivative on superoxide generation and enzyme release from human neutrophils in vitro. Interdiscip Toxicol. 2012;5(2):71-5  • Kanashiro et al.,. Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group. Z Naturforsch C. 2007 ;62(5-6):357-61.

20. III Immunomodulating effects of food proteins Some of food proteins we consume almost every day. Due to the insufficient activity of peptidases some of longer peptides are accumulated in small intestine. They have the potential to change some of functions of the organism; many of them are good for our health.

21. Proteins from: • wheat, • cow’s milk, • beans could induce well controlled immune stimulation which could (non-specifically) support the host immunity towards some infections, but also against some forms of malignancies.

22. New nomenclature and classifications of some of health disorders which could be induced by the ingestion of gluten is given at the meeting of 15 experts in London 2011. and this is the first time that it is accepted that person could have immunity to gliadin in the absence of celiac disease. Briefly, three main forms of gluten related health disorders were indentified: allergic (wheat allergy, IgE - mediated) autoimmune (celiac disease, dermatitis herpetiformis and gluten ataxia) non allergic, neither autoimmune but immune-mediated (gluten sensitivity, GS, characterized by definition: ”… to use gluten-free diet and to see if the symptom of disease resolve after the antigen withdrawn” ) Pathogenic, clinical and epidemiological differences between mentioned disorders also were outlined at this meeting. Sapone A, et al., BMC Med 2012;10:13.

23. Some of most recent references that reported the association between celiac disease and non-Hodgkin’s lymphoma • Schmitz F, Tjon JM, Lai Y, Thompson A, Kooy-Winkelaar Y, Lemmers RJ, Verspaget HW, Mearin ML, Staal FJ, Schreurs MW, Cupedo T, Langerak AW, Mulder CJ, van Bergen J, Koning F. Identification of a potential physiological precursor of aberrant cells in refractory coeliac disease type II. Gut. 2012. • Grainge MJ, West J, Solaymani-Dodaran M, Card TR, Logan RF. The long-term risk of malignancy following a diagnosis of coeliac disease or dermatitis herpetiformis: a cohort study. Aliment Pharmacol Ther 2012;35(6):730-9. • Malamut G, Meresse B, Cellier C, Cerf-Bensussan N. Refractory celiac disease: from bench to bedside. Semin Immunopathol 2012; 34(4):601-13. • Casella S, Zanini B, Lanzarotto F, Villanacci V, Ricci C, Lanzini A. Celiac disease in elderly adults: clinical, serological, and histological characteristics and the effect of a gluten-free diet. J Am Geriatr Soc 2012;60(6):1064-9. • Leslie LA, Lebwohl B, Neugut AI, Gregory Mears J, Bhagat G, Green PH. Incidence of lymphoproliferative disorders in patients with celiac disease. Am J Hematol 2012;87(8):754-9.

24. Could gluten - free diet help to prescribed oncological therapy to NHL patients?

25. A Japanese men who had intestinal diffuse large B-cell lymphoma associated with celiac disease was completely cured after prescribed oncological therapy and GFD. Makishima H, Ito T, Kodama R, Asano N, Nakazawa H, Hirabayashi K, Nakamura S, Ota M, Akamatsu T, Kiyosawa K, Ishida F. Intestinal diffuse large B-cell lymphoma associated with celiac disease: a Japanese case. Int J Hematol 2006; 83:63-65.

26. 23 H. pylori-negative untreated coeliac patients with gastric mucosa-associated lymphoid tissue (MALT) were included in the study. • Re-evaluation was 12 months after the beginning of GFD. • Regression of MALT was found in 9/13 (69%) of patients after gluten withdrawal. • In the control group (not on gluten - free diet), MALT disappeared in 2 of 10 (20%) patients (P = 0.0361). • The conclusion of the study was that antigens related to alimentary gluten may induce persistent stimuli for development of gastric MALT in coeliac patients. • Cuoco L, Cammarota G, Tursi A, Papa A, Certo M, Cianci RT et al. Disappearance of gastric mucosa-associated lymphoid tissue in coeliac patients after gluten withdrawal. Scand J Gastroenterol 1998;33 :401–405.

27. : Is there some connection between multiple myeloma and gluten?

28. Multiple myeloma is malignant disease which is characterized by the presence of monoclonal gamma globulins in serum. • It appears as the protein band of restricted mobility, “M” component, and is one of the diagnostic signs of the disease. • Disappearance of this component from the patient’s serum is one of the sign of the disease responsiveness to the oncological therapy.

29. Patient 1, IgG(kappa) as M component Patient 2 IgG(lambda) as M component Patient 3 IgG(kappa) as M component Figure 1.Three patients serum electrophoresis on 0.8% agarose gel .Upper slides are electrophoresis of patients serum, bottom slides show corresponding imunoprecipitation protein fraction with 0.4% gliadinin 1%SDS. The matched place of the M component and the antigliadinimmunoreactivity could be seen . Figure corresponds to the data presented in: Juranić Z, Radic J, Konic-Ristic A, Jelic S, Besu I, Mihaljevic B. Antibodies contained in "M" component of some patients with multiple myeloma are directed to food antigens? Leuk Res. 2006 Dec;30(12):1585-6

30. Note: It is very difficult to dissolve gliadin. Therefore in order to get solution of ( e.g.) 0.5% gliadin in 0.5% of SDS (sodium dodecylsulfate ), put 100mg of gliadin SIGMA in tube in one ml of 10% SDS at 37oC overnight and then gradually add 19 ml of distilled water with vigorous mixing (during 2h), in order to achieve homogenous suspension .

31. Nowadays, 2012, only 2 /18 randomly chosen patient’s sera with „M“ component showed immunoreactivity with gliadin. Patient (1) IgM(kappa), was with strong reactivity with gliadin while the other (3) was with IgG(λ), and had very mild reactivity.

32. Although the anti-gliadin immunoreactivity was found in patient’s sera after the immuno-electrophoresis, only IgA proteins showed reaction with gliadin in ELISA tests. May be that anti-gliadin IgG antibodies were hidden in immune complexes, or they are reactive with some other gliadin sequences? For this purpose we tested MM patient’s sera, as well as celiac disease (CD) patient’s sera for the immunoreaction with the native gliadin isolated from twelve wheat species usually used for bread and pasta making.

33. Results • In both patient’s groups IgA immunoreactivity to gliadin from different cultivars was almost homogenous and in correlation with results from commercial test (except for one patient with IgA (lambda)) myeloma, they were more then five times higher. • IgG immunoreactivity was more frequently inhomogeneous, and especially for few MM patients, it was more then five times higher and did not correlate with results obtained using Binding Site test. • This points to different content of immunogenic gliadin epitopes in various species of wheat.

34. Konic-Ristic A, et al. BMC Immunol2009; 10:32. The most active immunoreactivity of serum Ig of three myeloma patients was with durum wheat gliadin(and this is opposite to CD patients). It seems that gliadin isolated from durum wheat needs to be used as the antigen for testing anti-gliadin immunity in myeloma patients.

35. What does it mean for MM patients? • The possibility to decrease antigliadin immunity with gluten free diet is known in patients with celiac disease. • This possibility is in accordance with the data from only one report dealing with a possible link between disappearance of M component from serum of patient with celiac disease and gluten-free diet. • Pena et al., Gut 1976; 17:735-739.

36. Immunoreactivity to cow’s milk proteins was found in some in patients with NHL

37. A B C Figure 1. (A) Serum IgGimmunoreactivity to cow’s milk proteins (CMP) in healthy controls and in patients with B cell NHL or T cell NHL; (B) Serum IgAimmunoreactivity to cow’s milk proteins in healthy controls and in patients with B cell NHL or T cell NHL; (C) Serum IgMimmunoreactivity to cow’s milk proteins in healthy controls and in patients with B cell NHL or T cell NHL.

38. Results presented put the question: ”Could the use of CMP - free diet along with prescribed oncological therapy help more to patients with anti-CMP IgA antibodies?”

39. Could the use of appropriate diet help in the prevention of multiple myeloma? • Occurrence of “M” component in the sera of patients with psoriasis, or psoriatic arthritis, like in MGUS, has been reported , and it was stressed that these patients can sometimes develop multiple myeloma. Peltonen L, Nikoskelainen E, Kätkä K, Tyrkkö J. Monoclonal IgG immunoglobulinemia with subsequent myeloma in psoriatic arthritis. Dermatologica 1984; 169:207-210.

40. The results indicated much enhanced IgA and IgG immunoreactivity to CMP. Analysis of the patient’s serum by electrophoresis and immunofixation revealed the presence of “M” component (monoclonal IgG(lambda) immunoglobulin. When the patient heard about his enhanced immunoreactivity to CMP, he decided not to consume food with CMP. A two months after the start of CMP-free diet, analysis of serum proteins was carried out. At that time, the patient has not taken any immunosuppressive drugs.

41. Table 1. Proteins in patient’s serum before- and after CMP-free diet

42. Serum protein electrophoresis and immunofixation • Figure 1. left : before CMP-free diet; • right: after CMP-free diet. • “M” COMPONENT IgG(l) IS PRESENT IN THE SERUM BEFORE DIET (A). • “M” COMPONENT IS NOT PRESENT IN THE SERUM AFTER DIET (B). • Juranic Z, Besu I, Dajak M, Konic-Ristic A. Could the use of appropriate diet help in the prevention of multiple myeloma? J BUON 2009; 14(2):321-2. • Two months after the second test patient decided to try whether he could consume small quantities of milk, and after consummation of milk again, the M component reappeared in his serum.

43. CONCLUSIONS IIISensitivity of “M” component to specific antigen needs to be tested in laboratory before the start of the restriction diet. • It is very important to emphasize that one patient with multiple myeloma had high anti-cow’s milk IgG antibodies in the “M” component. • It could be serious mistake if somebody (who has “M” component’s immunoreactivity to CMP,) use gluten-free diet, or if somebody who has imunoreactivity to gluten use CMP- free diet. • Recommendations (done from the mentioned group of experts (Sapone et al.,2011) to use gluten-free diet and to see if the symptom of disease resolve after the antigen withdrawn is not so good in this case.

44. Food really contain anticancer agents whose action could be pronounced on many levels. • In general, it needs to be concluded that the role of nutrition in the cancer prevention even nowadays is developing field of science…

45. AcknowledgementsThis work is supported by Ministry of Education and Science of Serbia, grant No: 175011Many thanks to: • Aleksandra Konić-Ristić, MSc, PhD student • Ivana Matić, PhD student • Tatjana Stanojković, PhD • Aleksandra Erić-Nikolić, MD, MSc • Željko Žižak, MSc, PhD student • Irina Besu-Žižak,MD, PhD • Marija Ðorđić, PhD student • Ana Damjanović, PhD student • Branka Kolundžija, PhD student • Nađa Grozdanić, PhD student • Miomir Sasic, MD,PhD • Radan Dzodic, MD,PhD professor, Medical School, University of Belgrade • Svetislav Jelić, MDPhD professor, Medical School, University of Belgrade • Biljana Mihaljević, MD, PhD, professor, Medical School, University of Belgrade • Radmilo Krstic, MD, PhD professor of the Medical School, University of Belgrade • Janko Pralica MD • Marko Buta MD, MSc • Marko Jevric MD • Marijana Dajak, PhD • Suzana Matković, MD, MSc • For excellent technical assistance many thanks to: • Tatjana Petrovic,