Esin KIRAY, Serap YALÇIN AZARKAN, Ergin KARİPTAŞ

SERVİKOVAJİNAL MİKROFLORADAN İZOLE EDİLEN LACTOBACİLLUS PLANTARUM L4 SUŞUNUN HELA KANSER HÜCRE HATTI ÜZERİNDEKİ ANTİPROLİFERATİF ETKİSİ

Amaç: Lactobacillus spp. bakterilerinin çeşitli kanser hücrelerinin proliferasyonunu inhibe ettiği gösterilmiştir. Ancak, vajinal Lactobacillus’ların rahim ağzı kanseri hücreleri üzerindeki etkileri nadiren bildirilmiştir. Bu çalışmada Türkiye’de yaşayan sağlıklı kadınların servikovajinal florasından izole edilen L. plantarum L4 suşunun çeşitli probiyotik karakterleri ve HeLa kanser hücre hattı üzerindeki antiproliferatif etkisi araştırılmıştır. Gereç ve Yöntemler: Türün moleküler tanımlaması 16S rDNA analizi ile gerçekleştirilmiştir. L4 suşunun probiyotik özellikleri geleneksel yöntemlerle belirlenmiştir. L4 suşunun immun modilatör etkisini belirlemede insan Interleukin-10 (IL-10) ve Tümör Nekroz Factor-alfa (TNF-α) ELISA kitleri kullanılmıştır. HeLA hücre hattı üzerindeki antiproliferatif etkisi XTT kiti kullanılarak gerçekleştirilmiştir. Bulgular: L4 suşu güçlü probiyotik özellikler sergilemiştir. L4 suşunun, TNF-α üretimini azaltarak HeLa hücreleri üzerinde anti-inflamatuar bir etki gösterdi ve IL-10 üretiminin artırılmasını sağladı. L. plantarum L4 suşunun HeLa hücreleri üzerinde en büyük antiproliferatif etkinin metabolitin 0,0006 gr/ml’lik en yüksek dozunda %90-95 düzeyinde ölüm oranı gözlemlenirken canlı hücre sayısının %5-10 arasında olduğu görülmüştür. İnsan göbek ven endotel hücreleri (HUVEC) üzerinde herhangi bir antikanser etkisi olmamıştır. Sonuç: Güçlü probiyotik özelliklere sahip L. plantarum L4 suşu, immunmodilatör etkisi ve çok küçük dozlarda bile yüksek bir antiproliferatif etki göstermesi sebebiyle HPV kanseri için umut verici bir tedavi adayı olarak kabul edilebilir.

Anahtar Kelimeler:

Lactobacillus plantarum, antiproliferatif etki, HeLa, IL-10, TNF-α

ANTIPROLIFERATIVE EFFECT OF LACTOBACILLUS PLANTARUM L4 STRAIN ISOLATED FROM CERVICOVAGINAL MICROFLORA ON HELA CANCER CELL LINE

Objective: Lactobacillus has been shown to inhibit proliferation of various cancer cells, but the effects of vaginal Lactobacillus on cervical cancer cells have rarely been reported. The goal of this investigation was to assess the anti-proliferative effect on cancer cell line HeLa (Human Cervical Carcinoma Cell) and potential probiotic properties of Lactobacillus plantarum L4 isolated from cervicovaginal flora of healthy women in Turkey. Materials and Methods: Molecular identification of the species was performed by 16S rDNA analysis. Probiotic properties of the L4 strain were investigated by conventional methods. Human Interleukin-10 (IL-10) and Tumor Necrosis Factor- alpha (TNF-alpha) ELISA kits were used in the evelation of the immune modulator effect of the L4 strain. The antiproliferative effect of the L4 strain on the HeLA cell line was performed using the XTT kit. Results: L. plantarum L4 strain exhibited strong probiotic properties. The L4 strain showed an anti-inflammatory effect on HeLa by reducing the production of TNF-α and increased IL-10 production. The greatest antiproliferative effect of L. plantarum L4 strain on HeLa cells was observed at the highest dose of the metabolite 0.0006 gr/ml with a death rate of 90-95% while the number of living cells was found to be between 5-10%. The strain showed no anticancer effect on human umbilical vein endothelial cells (HUVEC). Conclusion: L. plantarum L4 strain, with strong probiotic properties, can be considered a promising treatment candidate for HPV cancer due to its immunomodulatory effect and high antiproliferative effect, even in very small doses.

Keywords:

Lactobacillus plantarum, Antiproliferative effect, IL-10, HeLa, TNF-α,

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1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2018;68(6):394-424. [CrossRef]
2. Bernard HU, Burk RD, Chen Z, van Doorslaer K, zur Hausen H, de Villiers EM. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 2010;401(1):70-9. [CrossRef]
3. Łaniewski P, Cui H, Roe DJ, Barnes D, Goulder A, Monk BJ, et al. Features of the cervicovaginal microenvironment drive cancer biomarker signatures in patients across cervical carcinogenesis. Sci Rep 2019;9(1):7333. [CrossRef]
4. Sudenga SL, Shrestha S. Key considerations and current perspectives of epidemiological studies on human papillomavirus persistence, the intermediate phenotype to cervical cancer. Int J Infect Dis 2013;17(4):e216-20. [CrossRef]
5. Alp Avcı G, Bozdayı G. İnsan papilloma virüsü, Kafkas J Med Sci 2013;3(3):136-44. [CrossRef]
6. Crosbie EJ, Einstein MH, Franceschi S, Kitchener HC. Human papillomavirus and cervical cancer. Lancet (London, England) 2013;382(9895):889-99. [CrossRef]
7. Gillet E, Meys JF, Verstraelen H, Bosire C, De Sutter P, Temmerman M, et al. Bacterial vaginosis is associated with uterine cervical human papillomavirus infection: a metaanalysis. BMC Infect Dis 2011;11:10. [CrossRef]
8. Gillet E, Meys JF, Verstraelen H, Verhelst R, De Sutter P, Temmerman M, et al. Association between bacterial vaginosis and cervical intraepithelial neoplasia: systematic review and meta-analysis. PLoS One 2012;7(10):e45201. [CrossRef]
9. Brusselaers N, Shrestha S, van de Wijgert J, Verstraelen H. Vaginal dysbiosis and the risk of human papillomavirus and cervical cancer: systematic review and meta-analysis. Am J Obstet Gynecol 2019;221(1):9-18-8. [CrossRef]
10. Tamarelle J, Thiébaut ACM, de Barbeyrac B, Bébéar C, Ravel J, Delarocque-Astagneau E. The vaginal microbiota and its association with human papillomavirus, Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium infections: a systematic review and metaanalysis. Clin Microbiol Infect 2019;25(1):35-47. [CrossRef]
11. Hickey RJ, Zhou X, Pierson JD, Ravel J, Forney LJ. Understanding vaginal microbiome complexity from an ecological perspective. Transl Res 2012;160(4):267-82. [CrossRef]
12. Nunn KL, Forney LJ. Unraveling the dynamics of the human vaginal microbiome. Yale J Biol Med 2016;89(3):331-7.
13. Martin DH, Marrazzo JM. The vaginal microbiome: current understanding and future directions. J Infect Dis 2016;15(214):36-41. [CrossRef] 14. Kıray E, Kariptas E. Probiyotikler, prebiyotikler ve sinbiyotiklerin kolorektal kanser ilişkisi. Elektronik Mikrobiyoloji Dergisi TR 2015;13(1):28-46.
15. Wang KD, Xu DJ, Wang BY, Yan DH, Lv Z1, Su JR. Inhibitory effect of vaginal lactobacillus supernatants on cervical cancer cells. Probiotics Antimicrob Proteins 2018,10(2):236- 42. [CrossRef]
16. Norenhag J, Du J, Olovsson M, Verstraelen H, Engstrand L, Brusselaers N. The vaginal microbiota, human papillomavirus and cervical dysplasia: a systematic review and network meta-analysis. BJOG 2020;127(2):171-80. [CrossRef]
17. Adebamowo SN, Ma B, Zella D, Famooto A, Ravel J, Adebamowo C. Mycoplasma hominis and Mycoplasma genitalium in the vaginal microbiota and persistent highrisk human papillomavirus infection. Front Public Health 2017;26(5):140. [CrossRef]
18. Shannon B, Yi TJ, Perusini S, Gajer P, Ma B, Humphrys MS, et al. Association of HPV infection and clearance with cervicovaginal immunology and the vaginal microbiota. Mucosal Immunol 2017;10(5):1310-19. [CrossRef]
19. Arokiyaraj S, Seo SS, Kwon M, Lee JK, Kim MK. Association of cervical microbial community with persistence, clearance and negativity of Human Papillomavirus in Korean women: a longitudinal study. Sci Rep 2018;19;8(1):15479. [CrossRef]
20. Champer M, Wong AM, Champer J, Brito IL, Messer PW, Hou JY, et al. The role of the vaginal microbiome in gynaecological cancer. BJOG 2018;125(3):309-15. [CrossRef]
21. Motevaseli E, Shirzad M, Akrami SM, Mousavi AS, Mirsalehian A, Modarressi MH. Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate, J Med Microbiol 2013;62(7):1065-72. [CrossRef]
22. Górska A, Przystupski D, Niemczura MJ, Kulbacka J. Probiotic bacteria: a promising tool in cancer prevention and therapy. Curr Microbiol 2019;76(8):939-49. [CrossRef]
23. Al Kassaa I, Hamze M, Hober D, Chihib NE, Drider D. Identification of vaginal lactobacilli with potential probiotic properties isolated from women in North Lebanon, Microb Ecol 2014;67(3):722-34. [CrossRef]
24. Charteris WP, Kelly PM, Morelli L, Collins JK. Quality control Lactobacillus strains for use with the API 50CH and API ZYM systems at 37 degrees C, J Basic Microbiol 2001;41(5):241- 51. [CrossRef]
25. Eryılmaz FT. Identification of potential probiotic properties of some lactic acid bacterial genera which is isolated from vaginal secretion, Ankara University, Biotechnology Institute, Ph.D. Thesis, Ankara, 2011.
26. Frank JA, Reich CI, Sharma S, Weisbaum JS, Wilson BA, Olsen GJ. Critical evaluation of two primers commonly used for amplification of bacterial 16S rRNA genes. Appl. Environ. Microbiol 2008;74(8):2461-70. [CrossRef]
27. Maragkoudakis PA, Zoumpopoulou G, Miaris C, Kalantzopoulos G, Pot B, Tsakalidou E. Probiotic Potential of Lactobacillus Strains Isolated from Dairy Products. Int. Dairy J 2006;16(3):189-99. [CrossRef]
28. Kaewnopparat S, Dangmanee N, Kaewnopparat N, Srichana T, Chulasiri M, Settharaksa S. In vitro probiotic properties of Lactobacillus fermentum SK5 isolated from vagina of a healthy woman. Anaerobe 2013;22:6-13. [CrossRef]
29. Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Khosroushahi AY. Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells. Anaerobe 2014;28:29-36. [CrossRef]
30. Maldonado NC, de Ruiz CS, Otero MC, Sesma F, Nader- Macías ME. Lactic acid bacteria isolated from young calvescharacterization and potential as probiotics. Res Vet Sci 2012;92(2):342-9. [CrossRef]
31. Juárez Tomás MS, Wiese B, Nader-Macías ME. Effects of culture conditions on the growth and auto-aggregation ability of vaginal Lactobacillus johnsonii CRL 1294. J Appl Microbiol 2005;99(6):1383-91. [CrossRef]
32. Younes JA, van der Mei HC, van den Heuvel E, Busscher HJ, Reid G. Adhesion forces and coaggregation between vaginal staphylococci and lactobacilli. PLoS One 2012;7(5):e36917. [CrossRef]
33. Santos CM, Pires MC, Leão TL, Hernández ZP, Rodriguez ML, Martins AK, et. al. Selection of Lactobacillus strains as potential probiotics for vaginitis treatment. Microb 2016;162(7):1195-207. [CrossRef]
34. Merghoub N, Benbacer L, Amzazi S, Morjani H, Mzibri ME. Cytotoxic effect of some Moroccan medicinal plant extracts on human cervical cell lines. J. Med. Plant Res 2009;3(12):1045-50.
35. Parsian M, Mutlu P, Yalçın S, Tezcaner A, Gündüz U. Half generations magnetic PAMAM dendrimers as an effective system for targeted gemcitabine delivery. Int J Pharm 2016;515(1-2):104-13. [CrossRef]
36. Haghshenas B, Abdullah N, Nami Y, Radiah D, Rosli R, Khosroushahi AY. Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines. Anaerobe 2014;30:51-9. [CrossRef]
37. Witkin SS, Alvi S, Bongiovanni AM, Linhares IM, Ledger WJ. Lactic acid stimulates interleukin-23 production by peripheral blood mononuclear cells exposed to bacterial lipopolysaccharide. FEMS Immunol Med Microbiol 2011;61(2):153-8. [CrossRef]
38. Ravel J, Gajer P, Abdo Z, Schneider GM, Koenig SS, McCulle SL, et al. Vaginal microbiome of reproductive-age women, Proc Natl Acad Sci U S A 2011;108(Suppl 1):4680-7. [CrossRef]
39. Lee J, Yun HS, Cho KW, Oh S, Kim SH, Chun T, et al. Evaluation of probiotic characteristics of newly isolated Lactobacillus spp.: immune modulation and longevity. Int J Food Microbiol 2011;148(2):80-6. [CrossRef]
40. Pan X, Chen F, Wu T, Tang H, Zhao Z. The acid, bile tolerance and antimicrobial property of Lactobacillus acidophilus NIT. Food Control 2009;20(6):598-602. [CrossRef]
41. Fernandez MF, Boris S, Barbes C. Probiotic properties of human lactobacilli strains to be used in the gastrointestinal tract. J Appl Microbiol 2003;94(3):449-55. [CrossRef]
42. Gupta R, Srivastava S. Antifungal effect of antimicrobial peptides (AMPs LR14) derived from Lactobacillus plantarum strain LR/14 and their applications in prevention of grain spoilage. Food Microbiol 2014;42:1-7. [CrossRef]
43. Martinez RCR, Wachsman M, Torres NI, Leblanc JG, Todorov SD, Franco BD. Biochemical, antimicrobial and molecular characterization of a noncytotoxic bacteriocin produced by Lactobacillus plantarum ST71KS. Food Microbiol 2013;34(2):376-81. [CrossRef]
44. Ryu EH, Yang EJ, Woo ER, Chang HC. Purification and characterization of antifungal compounds from Lactobacillus plantarum HD1 isolated from kimchi. Food Microbiol 2014;41:19-26. [CrossRef]
45. Bignoumba M, Onanga R, Bivigou Mboumba B, Gafou A, Mouanga Ndzime Y, et al. Vulvovaginal candidiasis among symptomatic women of childbearing age attended at a Medical Analysis Laboratory in Franceville, Gabon J Mycol Med 2019;29(4):317-9. [CrossRef]
46. Russo R, Superti F, Karadja E, De Seta F. Randomised clinical trial in women with Recurrent Vulvovaginal Candidiasis: Efficacy of probiotics and lactoferrin as maintenance treatment. Mycoses 2019;62(4):328-35. [CrossRef]
47. Swenson JM, Facklam RR, Thornsberry C. Antimicrobial susceptibility of vancomycin resistant Leuconostoc, Pediococcus and Lactobacillus species, Antimicrob Agents Ch 1990;34(4):543-9. [CrossRef]
48. Argyri AA, Zoumpopoulou G, Karatzas KA, Tsakalidou E, Nychas G-JE, Panagou EZ, et al. Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests. Food Microbiol 2013;33(2):282-91. [CrossRef]
49. Pino A, Bartolo E, Caggia C, Cianci A, Randazzo CL. Detection of vaginal lactobacilli as probiotic candidates. Sci Rep 2019;9(1):3355. [CrossRef]
50. Ammor MS, Flórez AB, Mayo B. Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria, Food Microbiol 2007;24(6):559-70. [CrossRef]
51. Knezević A, Stepanović S, Cupić M, Jevtović D, Ranin J, Jovanović T. Reduced quantity and hydrogen-peroxide production of vaginal lactobacilli in HIV positive women, Biomed Pharmacother 2005;59(9):521-33. [CrossRef]
52. Otero MC, Nader-Macías ME. Inhibition of Staphylococcus aureus by H2O2-producing Lactobacillus gasseri isolated from the vaginal tract of cattle. Anim Reprod Sci 2006;96(1- 2):35-46. [CrossRef]
53. Lin W, Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J. Clin. Investig 2007;117:1175-83. [CrossRef]
54. Sungur T, Aslim B, Karaaslan C, Aktas B. Impact of Exopolysaccharides (EPSs) of Lactobacillus gasseri strains isolated from human vagina on cervical tumor cells (HeLa). Anaerobe 2017;47:137-44. [CrossRef]
55. Wang Y, Liu XH, Li YH, Li O. The paradox of IL-10-mediated modulation in cervical cancer. Biomed. Rep 2013;1(3):347- 351. [CrossRef]
56. Kundu N, Fulton AM. Interleukin-10 inhibits tumor metastasis, downregulates MHC class I, and enhances NK lysis. Cell Immunol 1997;180(1):55-61. [CrossRef]
57. Rafter J. The effects of probiotic on colon cancer development. Nutr Rev 2004;17(2):277-84. [CrossRef]
58. 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. Appl Biochem Biotechnol 2013;169(2):511-25. [CrossRef]
59. Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Khosroushahi AY. Assessment of probiotic potential and anticancer activity of newly isolated vaginal bacterium Lactobacillus plantarum 5BL. Microbiol Immunol 2014;58(9):492-02. [CrossRef]