The apoptotıc effects of SCFAs from Lactobacillus reuteri on (HT-29) human colon cancer cells

Öz

Colon cancer is one of the leading causes of death around the World. In colon or other types of cancer, chemical methods can be considered as a therapeutic strategy. However, probiotics can also be used as biotherapeutics to reduce recurrence and side effects in patients with colon cancer.

Short Chain Fatty Acids (SCFAs) the fermentation products of probiotic L. reuteri bacteria found in the gastrointestinal tract. There are several SCFAs including acetic, propionic, butyric and lactic acids that have been shown to have apoptosis inducing properties in human colon cancer cells. In this study, the anti-proliferative and pro-apoptotic activities of probiotics were explained and the effects of SCFAs produced by L. reuteri on mitochondrial apoptosis were observed with changes in ROS and LPO production levels on human colon cancer cells (HT-29).

Results suggested that SCFAs from L. reuteri increased the mortality rates of HT-29 cells, increased ROS and LPO production. In addition to these effects, downregulation of Bcl-2, elevation of cytochrome c and overproduction of caspase-3 protein in SCFAs-treated HT-29 cells were observed. It was concluded that SCFAs from L. reuteri have cytotoxic effects in HT-29 colon cancer cells.

Anahtar Kelimeler

• Colon cancer,Probiotics,L. reuteri,HT-29,SCFAs,ROS,LPO

Lactobacillus reuteri’den elde edilen KZYA’nin insan kolon kanser hücrelerinde (HT-29) apoptotik etkileri

Öz

Kolon kanseri, dünyadaki önde gelen ölüm nedenlerinden biridir. Kolon veya diğer kanser tiplerinde, kimyasal yöntemler terapötik bir strateji olarak düşünülebilir. Bununla birlikte, probiyotikler, kolon kanserli hastalarda hastalığın tekrarı ve yan etkilerini azaltmak için biyoterapötikler olarak kullanılabilir.

Kısa Zincirli Yağ Asitleri (KZYA), bağırsakta bulunan probiyotik L. reuteri bakterilerinin fermentasyon ürünleridir. İnsan kolon kanseri hücrelerinde apoptoz indükleyici özelliklere sahip olduğu gösterilen asetik, propiyonik, bütirik ve laktik asitleri içeren birçok KZYA vardır. Bu çalışmada, probiyotiklerin anti-proliferatif ve pro-apoptotik aktiviteleri açıklanmış ve L. reuteri'nin ürettiği KZYA'nin, insan kolon kanseri hücrelerinde (HT-29) mitokondriyal apoptoz yolağına etkileri ve ROS ile LPO üretim düzeylerindeki değişimleri gözlemlenmiştir.

Sonuçlara göre, L. reuteri'den elde edilen KZYA, HT-29 hücrelerinde mortalite oranını, ROS ve LPO üretimini anlamlı şekilde artırmıştır. Bu etkilere ilaveten KZYA ile tedavi edilen HT-29 hücrelerinde Bcl-2 seviyesinin azalması, sitokromun c ve kaspaz-3 miktarlarının yükselmesi gözlenmiştir. Tüm sonuçlar ışığında L. reuteri kaynaklı KZYA'nin kolon kanseri hücrelerinde sitotoksik etkileri olduğu sonucuna varılmıştır.

Anahtar Kelimeler

• Kolon kanseri,Probiyotikler,L. reuteri,HT-29,KZYA,ROS,LPO

Kaynakça

1. [1] Borinstein S.C., Conerly M., Dzieciatkowski S., Biswas S., Washington M.K., Trobridge P., Henikoff S., Grady W.M., Aberrant DNA Methylation Occurs in Colon Neoplasms Arising in the Azoxymethane Colon Cancer Model, Molecular Carcinogenesis, 49, 94-103, 2010.
2. [2] Tuynman J.B., Peppelenbosch M.P., Richel D.J., Cox-2 İnhibition as a Tool to Treat and Prevent Colorectal Cancer, Critical Reviews in Oncology/Hematology, 52, 81-101, 2004.
3. [3] Bogaert J., Prenen H., Molecular genetics of colorectal cancer, Ann Gastroenterol, 27 (1), 9–14, 2014.
4. [4] Guessous I., Dash C., Lapin P., Doroshenk M., Smith R.A., Klabunde C.N., Colorectal Cancer Screening Barriers and Facilitators in Older Persons, Preventive Medicine, 50, 3-10, 2010.
5. [5] Lakritz J.R., Poutahidis T., Levkovich T., Varian B.J., Ibrahim Y.M., Chatzigiagkos A., Mirabal S., Alm E.J., Erdman S.E., Beneficial bacteria stimulate host immune cells to counteract dietary and genetic predisposition to mammary cancer in mice, Int. J. Cancer, 135, 529–540, 2014.
6. [6] Fuller R., Probiotics in man and animals, The Journal of Applied Bacteriology, 66 (5), 365–378, 1989.
7. [7] Kahouli I., Malhotra M., Alaouijamali M., Prakash S., In-Vitro Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus Fermentum NCIMB 5221 and Potential against Colorectal Cancer, Journal of Cancer Science & Therapy, 07 (07), 224–235, 2015.
8. [8] Gianotti L., Morelli L., Galbiati F., Rocchetti S., Coppola S., Beneduce A., Braga M., A randomized double-blind trial on perioperative administration of probiotics in colorectal cancer patients, World Journal of Gastroenterology, 16 (2), 167–175, 2010.

9. [9] Liu Z., Qin H., Yang Z., Xia Y., Liu W., Yang J., Zheng Q., Randomised clinical trial: The effects of perioperative probiotic treatment on barrier function and post-operative infectious complications in colorectal cancer surgery - A double-blind study, Alimentary Pharmacology and Therapeutics, 33 (1), 50–63, 2011.
10. [10] Kahouli I., Tomaro-Duchesneau C., Prakash S., Probiotics in colorectal cancer (CRC) with emphasis on mechanisms of action and current perspectives, Journal of Medical Microbiology, 62 (8), 1107–23, 2013.
11. [11] Garagnani P., Pirazzini C., Franceschi C., Colorectal Cancer Microenvironment: Among Nutrition, Gut Microbiota, Inflammation and Epigenetics, Current Pharmaceutical Design, 19, 765–778, 2013.
12. [12] Besten G.D., Eunen K.V., Groen A.K., Venema K., Reijngoud D.J., Bakker B.M., The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism, J Lipid Res., 9, 2325–2340, 2013.
13. [13] VanZanten G.C., Knudsen A., Röytiö H., Forssten S., Lawther M., Blennow A., Lahtinen S.J., Jakobsen M., Svensson B., Jespersen L., The effect of selected synbiotics on microbial composition and short chain fatty acid production in a model system of the human colon, PLoS One, 7 (10), 1-11, 2012.
14. [14] Noverr M.C., Huffuagle G.B., Rationale of Candida albicans morphogenesis by fatty acid metabolites, Infection and immunity, 72, 6206-6210, 2004.
15. [15] Willett W.C., Diet and Cancer, The Oncologist, 5 (5), 393–404, 2000.
16. [16] Fichera G.A., Giese G., Non-immunologically-mediated cytotoxicity of Lactobacillus casei and its derivative peptidoglycan against tumor cell lines, Cancer Letters, 85 (1), 93–103, 1994.
17. [17] Biffi A., Coradini D., Larsen R., Riva L., Di Fronzo G., Antiproliferative effect of fermented milk on the growth of a human breast cancer cell line, Nutrition and Cancer, 28 (1), 93–99, 1997.
18. [18] Reddy B.S., Rivenson A., Inhibitory Effect of Bifidobacterium longum on Colon, Mammary, and Liver Carcinogenesis Induced by 2-Amino-3 methylimidazo[4,5-f]quinoline, a Food Mutagen, Cancer Research, 53, 3914-3418, 1993.
19. [19] Aso Y., Akaza H., Kotake T., Tsukamoto T., Imai K., Naito S., Preventive effect of a Lactobacillus casei preparation on the recurrence of superficial bladder cancer in a double-blind trial, The BLP Study Group European Urology, 27 (2), 104–9, 1995.
20. [20] Reuter G., The Lactobacillus and Bifidobacterium microflora of the human intestine: composition and succession, Current Issues in Intestinal Microbiology, 2 (2), 43–53, 2001.
21. [21] Ma D., Forsythe P., Bienenstock J., Live Lactobacillus reuteri Is Essential for the Inhibitory Effect on Tumor Necrosis Factor Alpha-Induced Interleukin-8 Expression, Infection and Immunity, 72 (9), 5308–5314, 2004.
22. [22] Kahouli I., Malhotra M., Tomaro-Duchesneau C., Saha S., Marinescu D., Rodes L., Prakash S., Screening and In-Vitro Analysis of Lactobacillus reuteri Strains for Short Chain Fatty Acids Production, Stability and Therapeutic Potentials in Colorectal Cancer, Bioequivalence & Bioavailability, 7 (1), 39–50, 2015.
23. [23] Jan G., Belzacq A.S., Haouzi D., Rouault A., Metivier D., Kroemer G., Brenner C., Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria, Cell Death Differ, 9 (2), 179–188, 2002.
24. [24] Zoratti M., Szabb I., The mitochondrial permeability transition, 1241, 139–176, 1995.
25. [25] Marzo I., Brenner C., Zamzami N., Susin S.A., Beutner G., Brdiczka D., Kroemer G., The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins, J Exp Med, 187 (8), 1261–1271, 1998.
26. [26] Decaudin D., Marzo I., Brenner C., Kroemer G., Mitochondria in chemotherapy-induced apoptosis: A prospective novel target of cancer therapy, International Journal of Oncology, 1998.
27. [27] Hofmanová J., Straková N., Vaculová A.H., Tylichová Z., Šafaříková B., Skender B., Kozubík A., Interaction of Dietary Fatty Acids with Tumour Necrosis Factor Family Cytokines during Colon Inflammation and Cancer, Mediators of Inflammation, 1-17, 2014.
28. [28] Hague A., Elder D.J., Hicks D.J., Paraskeva C., Apoptosis in colorectal tumour cells: induction by the short chain fatty acids butyrate, propionate and acetate and by the bile salt deoxycholate, International Journal of Cancer, 60 (3), 400–406, 1995.
29. [29] Hague A., Singh B., Paraskeva C., Butyrate acts as a survival factor for colonic epithelial cells: Further fuel for the in vivo versus in vitro debate, Gastroenterology, 112 (3), 1036–1040, 1997.
30. [30] Quiros A.R.B., Arias M.F., Hernández J.L., A screening method for the determination of ascorbic acid in fruit juices and soft drinks, Food Chemistry, 116 (2), 509-512, 2009.
31. [31] Shen H.M., Shi C.Y., Shen Y., Ong C.N., Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1, Free Radic. Biol. Med., 21 (2), 139–146, 1996.
32. [32] Smith M.T., Thor H., Hartzell P., Orrenius S., The measurement of lipid peroxidation in isolated hepatocytes, Biochem. Pharmacol., 31 (1), 19–26, 1982.
33. [33] Yuan J.S., Reed A., Chen F., Stewart C.N., Statistical analysis of real-time PCR data, BMC Bioinf., 7 85, 2006.
34. [34] Reed J.C., Double identity for proteins of the Bcl-2 family, Nature, 387, 773–776, 1997.
35. [35] Gui H., Shen Z., Concentrate diet modulation of ruminal genes involved in cell proliferation and apoptosis is related to combined effects of short-chain fatty acid and pH in rumen of goats, Journal of Dairy Science, 99 (8), 6627-6638, 2016.
36. [36] Thirabunyanon M., Hongwittayakorn P., Potential probiotic lactic acid bacteria of human origin induce antiproliferation of colon cancer cells via synergic actions in adhesion to cancer cells and short-chain fatty acid bioproduction, Applied Biochemistry and Biotechnology, 169, 511–525, 2013.
37. [37] Barrera G., Oxidative Stress and Lipid Peroxidation Products in Cancer Progression and Therapy, ISRN Oncology, 1-21, 2012.
38. [38] Jeong C.H., Joo S.H., Downregulation of Reactive Oxygen Species in Apoptosis, J Cancer Prev., 21 (1), 13–20, 2016.
39. [39] Darendelioglu E., Tartik M., Aykutoglu G., Baydas G., Turkish propolis protects human endothelial cells in vitro from homocysteine-induced apoptosis, Acta Histochemica, 118, 369-76, 2016.
40. [40] Simon H.U., Haj-Yehia A., Levi-Schaffer F., Role of reactive oxygen species (ROS) in apoptosis induction, Apoptosis. 5, 415–418, 2000.
41. [41] Jones R.M., Luo L., Ardita C.S., Richardson A.N., Kwon Y.M., Mercante J.W., Alam A., Gates C.L., Wu H., Swanson P.A., Lambeth J.D., Denning P.W., Neish A.S., Symbiotic lactobacilli stimulate gut epithelial proliferation via Nox-mediated generation of reactive oxygen species, EMBO J., 32 (23), 3017–3028, 2013.
42. [42] Kim A.D., Han X., Piao M.J., Hewage S.R., Hyun C.L., Cho S.J., Hyun J.W., Esculetin induces death of human colon cancer cells via the reactive oxygen species-mediated mitochondrial apoptosis pathway, Enviromental Toxicology and Pharmacology, 9 (12), 113257, 2015.
43. [43] Shabnam M.S., Srinivasan R., Wali A., Majumdar S., Joshi K., Behera D., Expression of p53 protein and the apoptotic regulatory molecules Bcl-2, Bcl-XL, and Bax in locally advanced squamous cell carcinoma of the lung, Lung Cancer, 45 (2), 181–188, 2004.
44. [44] Jurgensmeier J.M., Xie Z., Deveraux Q., Ellerby L., Bredesen D., Reed J.C., Bax directly induces release of cytochrome c from isolated mitochondria, Proceedings of the National Academy of Science, 95, 4997–5002, 1998.
45. [45] Ashkenazi A., Fairbrother W.J., Leverson J.D., Souers A.J., From basic apoptosis discoveries to advanced selective BCL-2 family inhibitors, Nature Reviews Drug Discovery, 16, 273–284, 2017.
46. [46] Ruemmele F.M., Schwartz S., Seidman E.G., Dionne S., Levy E., Lentze M.J., Butyrate induced Caco-2 cell apoptosis is mediated via the mitochondrial pathway, Gut, 52, 94–100, 2003.
47. [47] Medina V., Edmonds B., Young G.P., James R., Appleton S., Zalewski P.D., Induction of Caspase-3 Protease Activity and Apoptosis by Butyrate and Trichostatin A (Inhibitors of Histone Deacetylase): Dependence on Protein Synthesis and Synergy with a Mitochondrial Cytochrome c-dependent Pathway, Cancer Research, 57, 3697-3707, 1997.
48. [48] Lim S.H., Song K.S., Lee J., Butyrate and Propionate, Short Chain Fatty Acids, Attenuate Myocardial Damages by Inhibition of Apoptosis in a Rat Model of Ischemia-reperfusion, J. Korean Soc. Appl. Biol. Chem., 53 (5), 570-577, 2010.