Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
1 THE “LITTLE” BUT POWERFUL PROBIOTICS AGAINST COLON CANCER Dokos Charalampos and Mironidou-Tzouveleki Maria Laboratory of Pharmacology, Medical School, Aristotle University of Thessaloniki, Greece Keywords: Colon Cancer (colon Ca), probiotics, diet, nutrition, anti-mutagenic agents. Correspondence author Dr. Maria Mironidou-Tzouveleki Anaesthesiologist Associate Professor of Pharmacology Komninon 4, 57010, Filyro, Thessaloniki, Greece [email protected] Tel: 00302310678385, Fax: 00302310678385. 2 Summary Colon cancer (Colon Ca) is a type of cancer that has great morbidity and mortality rate in western countries. Although the molecular and genetical basis of colorectal cancer is well established, environmental factors are still under research. Diet seems to be a major contributor to colon Ca, associated with the balance of colonic microflora. Probiotics, live microorganisms usually in dairy fragment cultures, are in the scope of current research as general anti-mutagenic agents in gastrointestinal diseases including colon Ca. Laboratory animal studies have shown that two species: Lactobacillus and Bifidobacteria have anti-mutagenic properties in colorectal cancer. However no direct evidence from dietary intervention or epidemiological studies exists that correlates diet and nutrition as contributors to colon Ca. Nevertheless research is still going on to throw light on the precise mechanisms that probiotics act as antimutagenic agents in colon Ca. 3 Introduction Colorectal cancer is a serious form of cancer, which attacks both men and women. Surprisingly it is the second most serious form of cancer in men, after lung cancer and women, following breast cancer. Over the past 20-30 years colorectal cancer has shown a little decrease in general population based on epidemiological studies. These studies imply that diet seems to be an important factor of increasing the risk of colorectal cancer.1- 4 On the other hand human gut is a natural habitat of a powerful bacterial community colonized by 300-500 different bacteria species.5,6 In experimental models, colonic microflora is a contributor to gastrointestinal diseases and also colon cancer (CCA).1,6-8 Laboratory animal studies also support that probiotics, prebiotics (nondigestible carbohydrates that stimulate lactic acid bacteria in gut) and synbiotics (agents that combine probiotics and prebiotics bacteria) have a positive effect on CCA.1,4,5,7 Although reports in current literature indicate that human diet is a contributor to CCA, there is no direct evidence for cancer suppression in humans. CCA’s molecular identity is well defined and described but epidemiological, animal and human dietary intervention studies must be accomplished because little is known about the potential mechanisms through which probiotic bacteria may inhibit CCA. Probiotics Human species live in synbiosis and the majority of the bacteria is found in colon.4 Colonic microflora starts immediately after birth (sterile infant colon) and within few days is well established. Naturally the type of delivery and diet might affect the rhythm of colonization and type of colonic microflora. After birth these bacteria are vulnerable to environmental factors. These factors vary from country to country according to economic and social status.4,5 Examples of great interest are low-birthweight premature infants delivered by caesarian section and antibiotic administration to infants which can dramatically change the colonization type of microflora.9 The term probiotic was derived from the Greek word “pro-biotikos” meaning “life”. Indeed probiotics are living organisms with the aim of benefiting health by balancing intestinal microflora. These valuable microorganisms were first characterized and described in modern times by Fuller10 as “live microbial feed supplements which beneficially affect the host animal by improving its microbial balance”. From the past until nowadays the key of success and admiration for probiotics in general population seems to be the huge effects of probiotics on gastrointestinal tract. In the past yoghurt, ferment milk products and cheese were favorite dairy products among consumers. A yoghurt per se, which is by the way a probiotic food product, was a favorite habit years ago. It is very important to remember that the Russian Nobel prizewinner Elie Metchnikoff observed a high life expectancy in Bulgarian shepherds who ate large amounts of ferment-milk products.3 In fact today experts still debate whether yoghurt starter cultures such as Lactobacillus bulgaricus and Streptococcus thermophilus should be considered as probiotics. Notice that the yoghurt starter cultures are sensitive to high pH conditions in the digestive tract and do not have a high colonization type in colon tract. Benefits of these starter cultures, which are still discussed, are the improvement of lactose digestion and reinforcement of immune system.5,11,12 Lactose maldigestion is a frequent disease that occurs in adults and in persons with bowel resection or enteritis. Persons with lactose maldigestion have better digestion and tolerance of lactose contained in yoghurt than in milk. Yoghurt bacteria deliver their 4 lactase enzyme and digest lactose. It is also believed that this intestinal delivery of lactose is much slower than that of milk.10,12 As a result a microorganism so as to be defined as an effective probiotic agent must have the following properties: (a) adhere to cells, (b) exclude or reduce pathogenic adherence, (c) persist and multiply, (d) produce acids, hyperoxide and bacteriocins (defined as local antibiotics) antagonist to pathogenic growth, (e) be safe, noninvase, noncarcinogenic, (f) coaggregate to form normal balanced flora.13 Although these characteristics have been proposed as to be named a specific strain “probiotic”, it seems they are not enough for public safety. FAO (Food and Agriculture Organization) and WHO (World Health Organization) have strict guidelines about candidate bacteria strains to be used as probiotics. Except for the above properties, functional and safety assessments need to be accomplished. Functional tests include in vitro and animal studies. Safety assessments include a minimum of series of tests including antibiotic resistance pattern, metabolic activities, toxin production, hemolytic activity and three phases of Clinical Studies each described by certain guidelines.12 Above any matter of public safety various studies indicate that probiotics have a wide range of general biological effects on human organism such as: (a) in vitro and in vivo enhance human immunity system reinforcement9-11,13-15 (b) reduce incidence or duration of specific diarrhea illnesses (e.g acute viral diarrhea)9,10,13,15 (c) contribute to the prevention and treatment of gastrointestinal conditions (e.g H. Pylori infection) and inflammatory bowel diseases (e.g Cronh’s disease)5,9,11,14,15 (d) improve asthmatic and food allergy status of children8 and (e) cholesterol lowering levels.9,13,16 Probiotics and Colon cancer (CCA) a) Laboratory animal studies Several animal models have been proposed for CCA research associated with probiotics, prebiotics and synbiotics. Probiotics are the most supportive evidence of the theory supposing that diet contributes to at least 75% of cases with CCA. Prebiotics are the “new entrance” that gains field in research but modern trends implicate that synbiotics are more effective than either pro- or prebiotics.1,7 The main two genera of probiotics that are widely used in cancer research are: Bifidobacterium and Lactobacillus species.1,13,14 These Gram-positive bacteria that are extensively used and named as probiotics have all the above mentioned characteristics so as to be probiotics according to FAO and WHO guidelines.9,11 Rodent model studies support the anti-carcinogenic properties for probiotics in CCA.1 These probiotics strains have the ability to modify colonic bacteria activities17 that take part in initation of CCA through a production of carcinogens, cocarninogens or procarcinogens.5 Oral administration in rats of these lactic acid bacteria effectively reduces DNA damage induced by chemical carcinogens in colonic mucosa1 such as Nmethyl-N’-nitro-N-nitrosoguanidine (MNNG) (Fig.1). Generally N-nitroso compounds are a group of genotoxic substances best known as initiators and promoters of CCA.5,17 The anti-mutagenic activities of Lactobacillus and Bifidobacteria species have been shown experimentally both in killed and alive bacterial cells. According to these data, live Lactobacillus and Bifidobacteria microorganisms showed a higher anti-mutagenic activity than killed bacterial cells of these species.13,14 Also it has been reported that oral administration in rats of the carcinogen 1,2-dimethyl hydrazine (DMH) in 5 combination with lactic acid bacteria (e.g bacteria from Lactobacillus species) or yoghurt, significantly have prevented DNA damage in cells of gastrointestinal tract (Fig.2).3 Another mutagen that has been reported in CCA rats is IQ (2-amino-3-methylo-3Hinidizo(4,5-f)quinoline). IQ is a heterocycle aromatic amine produced by food pyrolisis (Fig. 3). Food mutagen IQ has a multitarget organospecificity with specific cancer induction in Zymbal gland, skin, colon, oral cavity and mammary gland of rodents.18 It has been proved that lyophilized B. longum strains in male and female rats inhibited the formation of CCA tumors induced by IQ.1,18 Notice that IQ is not directly mutagenic but is metabolized by gut bacteria to active mutagen metabolite 7-OH IQ.19 Modern trends in laboratory animal studies imply the insertion of preneoplastic lesions such as the aberrant crypt foci (ACF) (Fig.4).1,20 ACF is a putative neoplastic lesion from adenomas and carcinomas that may develop in the colon of rodent models.13,20 It has been identified as a preneoplastic lesion visible in the colon of rodents as early as two weeks after carcinogen administration (e.g AOM, DMH). Also ACF has been identified in humans with easy quantify methods and because of its distinct preneoplastic nature. Normally few dysplastic ACF both in human subjects and rodents will be developed into cancers.17,20 Studies indicate that probiotics (L. acidophilus and B. longum) inhibit the formation of ACF induced by azoxymethane (AOM) (Fig.5).1,16 A decrease is also observed in β-glucoronidase activity 13 and heterocycle amines concentration.5,13 As mentioned above CCA is a well-defined type of cancer in both rodents and humans, with a series of precise histological changes in colonic tract with parallel mutations, activations and deletions of oncogenes and tumor suppression genes (Fig.6). At the same time members of colon microbial community promote CCA by producing specific enzymes such as β-glycosidase, β-glucuronidase, azoreductase, nitroreducatse. These enzymes are transformed into active carcinogens resulting in CCA.4,11 The question that now rises is when and how probiotics act in CCA process. In conclusion environmental factors such as diet have a major role in development of CCA in rodent models. However at this point it must be noticed that current literature indicated the differences between human and rodent caecum and colon. This could be a point of debate among researchers about the usefulness of those experiments using rodent models with CCA. Nevertheless laboratory rat models provide a supportive evidence for a laboratory based phenomenon and imply the association between nutritional and diet habits with CCA.17 b) Human-epidemiological studies Rodent model studies have proved the correlation between CCA and diet. However few epidemiological studies associate human diet with CCA. High consumption of dairy products containing Lactobacillus or/and Bifidobacterium may be related to lower risk of CCA.1 Epidemiological studies have related cancers in systemic organs, such as colon and stomach to inappropriate diet routine.19 Nevertheless more human epidemiological studies are needed so as to make definite conclusions about the beneficial effects of probiotics on the prevention or treatment of CCA. From the few epidemiological studies one that is of great interest has to do with the ingestion of L. acidophilus or best known as L. casei. Scientists have associated the activity of this probiotic bacterium with the significantly reduced levels of precarcinogen enzymes such as β-glucoronidase.5,11 Faecal and urine samples of healthy 6 volunteers showed low mutagenic activity after consumption of fried meat and L. casei.1,5,11,17 Probably the mutagenic compounds were taken up or degraded by the probiotic bacteria.15 L. casei Shirota (present in “Yakult” preparation) reduced also the recurrence rate of superficial bladder cancer. It has been reported to exhibit antitumor, immunostimulatory and antimicrobial activities. “Yakult” is a Japanese milk-based drink consumed by approximately 26 million people every day.9,11,21 Epidemiological studies are too difficult to be accomplished because human intervention studies lack well-validated biomarkers of CCA so risk biomarkers are under development.4 The SYNCAN project, funded by European Union (www.syncan.be), involves eight research centers in Europe so as to study the biochemical markers of CCA including colonic mucosal markers, faecal water markers and immunological markers. This 12 week randomized, double-blinded placebo controlled trial uses food supplement containing Lactobacillus GG, Bifidobacterium Bd12 and Raftilose Synergyl in adenoma patients of CCA (Fig.7).1,4 The few epidemiological studies that exist are contradictory. Some studies could not find association between ferment-milk products consumption and risk of CCA. Others show a significant but lower incidence of CCA with probiotics strains of ferment-milk products.1,3,12 So not all studies show a correlation between probiotics and microflora activities. Different probiotics may or may not induce significant changes in human colonic microflora.17,22 It is obvious that there is a problem both in accurate counting of the colonic microflora and a quantitative assessment for research purpose. Molecular biology’s methods such as fluorescent label and 16S RNA oligonucleotide probes designed as primers for polymerase chain reaction (PCR) have already been used for this purpose.17,23,24 Indeed in near future, genomics and proteomics will investigate the effect of pro- and prebiotics on gene expression and post-transciptional modifications. Data from molecular biology’s research will acquire further process so as to identify human groups responsive to pro- and prebiotics intervention. Using sequence analysis of clone libraries from amplified ribosomal DNA we can identify certain bacteria species suitable for colonic microflora.23,24 These new techniques of molecular biology like gene engineering may aid in designing and inserting new generations of probiotics in human colon. These new recombinant probiotics will be humanized and they are under development. They will produce predetermined therapeutic proteins for general gastrointestinal disease including CCA in humans (e.g subolin).13 Conclusions A lot has already been done in the area of laboratory animal studies. From these rodent studies there is indirect evidence that specific probiotics strains contribute to inhibition of CCA caused by carcinogenic compounds. Unfortunately few epidemiological studies give hope so as to associate probiotics and prevention of CCA in general population. However there are unanswered questions about how probiotics inhibit CCA development: is it an alteration of the metabolic activities of intestinal microflora? an alteration of physicochemical conditions in colon? a binding and degradation of potential carcinogens or production of antimutagenic compounds? Anyway work has to be done so as to have a definite and well trusted answer for the correlation between probiotics and risk of human CCA with the aid of molecular biology’s techniques. 7 Taking account of the results in current literature on both human and laboratory animal studies, the theory that states that specific probiotics strains may inhibit the development of human CCA seems to gain more and more researchers trust; 8 Figures 9 10 11 12 13 14 DIET INTERVENTION 12 Weeks 20 Patients/Group PATIENTS WITH HIGH RISK FOR COLON CANCER (POLYPECTOMISED) CONTROL DIET Blood, faeces and biopsy samples to BIOMARKER PATIENTS WITH HISTORY OF COLON CANCER (DUKES B) NETWORK SYNBIOTIC DIET 15 Legends Fig. 1: Chemical structure of N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Fig. 2: Chemical structure of 1,2-dimethyl hydrazine (DMH). Fig. 3: Chemical structure of 2-amino-3-methylo-3H-inidizo(4,5-f)quinoline (IQ). Fig. 4: Histological sample www.researchcanceruk.org). of aberrant crypt foci (ACF) (adapted from Fig. 5: Chemical structure of azoxymethane (AOM). Fig. 6: Sequential genetic changes that are associated with dysplasia and malignant neoplasia of the colon (adapted in Expert Review in Molecular Medicine ©1999 Cambridge University Press). Fig. 7: Simplified diagram of SYNCAN project. 16 References 1. Rafter J. Probiotics and colon cancer. Best Practice & Research Clinical Gastrenterology 2003; 17(5):894-859. 2. Rozen P. Prevention and public control in colon cancer. In: Fast Facts Gastroenterology Highlights 2001-02. Health Press Limited, Oxford, UK, Feb2002, p.77. 3. Wollowski I, Rechkemmer G. and Pool-Zobel LB. Protective role of probiotics and prebiotics in colon cancer. Am J Clin Nutr 2001; 73(suppl):451S-5S. 4. Karlsson CP. Thesis: Biomarkers for Colon Cancer, Applications in human and rat studies. Department of Medical Nutrition, Karolinska Institutet, Stokcholm, Sweden. Stockholm 2005. 5. Guraner F. and Malagelada RJ. Gut flora in health and disease. Lancet 2003; 361:512-19. 6. Floros I, Mironidou-Tzouveleki M, Pangalis A, Kalousis K. and Kokkas B. Multiple organ dysfunction / multiple organ failure syndrome. Review of Clinical Pharmacology and Pharmacokinetics (Greek edition). 2004; Suppl.22(3):149-173. 7. Le Leu KR, Brown LI, Hu Y. et al. A symbiotic combination of resistance starch and Bifidobacterium lactis facilitates apoptotic deletion of carcinogendamaged cells in rat colon. American Society for Nutritional Sciences 2005; 0022-3166/05. 8. Gurarner F. Enteric flora in health and disease. Digestion 2006; 73Suppl1:5-12. 9. Reid G, Jass J, Sebulsky TM and McCormick JK. Potential uses of probiotics in clinical practice. Clin Microb Rev Oct2003; 658-672. 10. Chermesh I., Eliakim R. Probiotics and gastrointestinal tract: where are we in 2005? World J Gastroenterology Feb2006; 12(6):853-857. 11. Senok AC, Ismaeel AY, Botta GA. Probiotics: facts and myths. Clin Microb Infect 2005; 11:958-966. 12. Philippe R. Marteau, Michæl de Vrese, Christophe J Cellier and Jurgen Schrezemmeir. Protection from gastrointestinal diseases with the use of probiotics. Am J Clin Nutr 2001; 73(suppl):430S-6S. 13. Kaur PI., Chopra K., Saini A. Probiotics: potential pharmaceutical applications. Eur J of Pharm Sciences 2002; 15:1-9. 14. Gagalides K, Kalousis K, Mironidou-Tzouveleki M. Probiotics the live drugs. Which is the role in gastrointestinal diseases. Scientific Annals of Medical School of Aristotle University of Thessaloniki (Greek Edition). 2005; 32(3):14. 15. Santosa S, Farnworth E, Jones PJ. Probiotics and their potential health claims. Nutr Rev Jun2006; 64(6):265-74. 16. David C. Lin. Probiotics as functional foods. Nutrition in Clinical Practice 2003; 18(6):497-506. 17. Commane D., Hughes R., Shortt C., Rowlan I. Fundamental and Molecular Mechanisms of Mutagenesis 2005; 591(1-2):276-289. 18. Reddy SB. Prevention of colon cancer by pre- and probiotics: evidence from laboratory studies. Br J Nutrition 1998; 80Suppl.2:S219-S223. 19. Ferguson RL, Philpott M, Karunasinghe N. Dietary cancer and prevention using antimutagens. Toxicology 2004; 198:147-159. 20. Caderni G, Femia PA, Giannini A. et al. Identification of mucin-depleted foci in the unsectioned colon of azoxymethane-treated rats: correlation with carcinogenersis. Cancer Research May2003; 63:2388-2392. 17 21. Sgouras D, Maragkoudakis P, Petraki K. et al. In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota. Appl and Env Microb Jan2004; 70(1):518-526. 22. Bergonzelli GE, Blum S, Brussow H., Corthesy-Theulaz I. Probiotics as treatment strategy for gastrointestinal diseases? Digestion 2005; 72(1):57-68. 23. Tlaskalova-Hogenova H, Stepankova R, Hudcevic T. et al. Commensal bacteria (normal microflora), mucosal immunity and chronic inflammatory and autoimmune diseases. Immunology Letters 2004; 93:97-108. 24. Gill HS and Guarner F. Probiotics and human health: a clinical prespective. PMJ 2004; 80(5):516-526.