Bifidobacterium animalis subsp. animalis-derived metabolites are implicated in microbiota–tumor crosstalk through dual anticancer and antimicrobial effects in colorectal cancer

Abstract

Colorectal cancer constitutes a considerable global health challenge due to its strong contribution to cancer-linked morbidity and mortality. Gut microbiota and their metabolites are increasingly recognized as modulators of tumor progression and the tumor microenvironment. This study examined the effects of extracellular metabolites from B. animalis subsp. animalis on human CRC cell lines (HCT-116 and HT-29) and on pathogenic bacteria (Escherichia coli and Staphylococcus aureus). Metabolite exposure produced dose-dependent inhibition of CRC cell viability, with IC₅₀ values of 63.92 µL/mL for HCT-116 and 46.86 µL/mL for HT-29, while causing minimal cytotoxicity in non-tumorigenic HUVEC cells. Flow cytometry indicated a modest rise in late apoptotic populations, suggesting activation of apoptotic pathways without extensive cell death. Antimicrobial assays showed effective, time- and dose-dependent suppression of E. coli and S. aureus growth under aerobic and anaerobic conditions. These findings suggest B. animalis-derived metabolites exert direct anticancer effects and indirectly modulate the tumor microenvironment by inhibiting pathogenic bacterial colonization.

Keywords

• Bifidobacterium animalis
• colorectal carcinoma
• Escherichia coli
• Staphylococcus aureus
• apoptosis
• probiotic metabolites

Supporting Institution

This research was supported by Istanbul Nişantaşı University BAP 2023/5.

Thanks

The authors are grateful to Istanbul Medipol University Health Science and Technologies Research Institute (SABITA) for the infrastructure and laboratory support they provided to conduct this study.

References

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.
2. Ferlay J, Colombet M, Soerjomataram I, Mathers C, Parkin DM, Piñeros M, et al. Estimating the global cancer incidence and mortality in 2018: GLOBOCAN sources and methods. Int J Cancer. 2019;144(8):1941-1953.
3. Verdina A, Garufi A, D’Orazi V, D’Orazi G. HIPK2 in colon cancer: a potential biomarker for tumor progression and response to therapies. Int J Mol Sci. 2024;25(14):7678.
4. Hossain MS, Karuniawati H, Jairoun AA, Urbi Z, Ooi DJ, John A, et al. Colorectal cancer: a review of carcinogenesis, global epidemiology, current challenges, risk factors, preventive and treatment strategies. Cancers (Basel). 2022;14(7):1732.
5. Rawla P, Sunkara T, Barsouk A. Epidemiology of colorectal cancer: incidence, mortality, survival, and risk factors. Gastroenterol Rev. 2019;14(2):89-103.
6. Cancer Genome Atlas Network. Comprehensive molecular characterization of human colon and rectal cancer. Nature. 2012;487(7407):330-337.
7. Lengauer C, Kinzler KW, Vogelstein B. Genetic instability in colorectal cancers. Nature. 1997;386(6625):623-627. Iacopetta B. TP53 mutation in colorectal cancer. Hum Mutat. 2003;21(3):271-276.
8. Iacopetta B, Russo A, Bazan V, Dardanoni G, Gebbia N, Soussi T, et al. Functional categories of TP53 mutation in colorectal cancer. Ann Oncol. 2006;17(5):842-847.

9. Kim GP, Colangelo LH, Wieand HS, Paik S, Kirsch IR, Wolmark N, et al. Prognostic and predictive roles of high-degree microsatellite instability in colon cancer. J Clin Oncol. 2007;25(7):767-772.
10. Samowitz WS, Holden JA, Curtin K, Edwards SL, Walker AR, Lin HA, et al. Inverse relationship between microsatellite instability and K-ras and p53 gene alterations in colon cancer. Am J Pathol. 2001;158(4):1517-1524.
11. Nešić D, Hsu Y, Stebbins CE. Assembly and function of a bacterial genotoxin. Nature. 2004;429(6990):429-433.
12. Johnson W, Lior H. A new heat-labile cytolethal distending toxin produced by Escherichia coli isolates from clinical material. Microb Pathog. 1988;4(2):103-113.
13. He Z, Gharaibeh RZ, Newsome RC, Pope JL, Dougherty MW, Tomkovich S, et al. Campylobacter jejuni promotes colorectal tumorigenesis through cytolethal distending toxin. Gut. 2019;68(2):289-300.
14. Graillot V, Dormoy I, Dupuy J, Shay JW, Huc L, Mirey G, et al. Genotoxicity of cytolethal distending toxin on human colorectal cell lines. Front Cell Infect Microbiol. 2016;6:34.
15. Xie YH, Chen YX, Fang JY. Comprehensive review of targeted therapy for colorectal cancer. Signal Transduct Target Ther. 2020;5(1):22.
16. Michire A, Anghel R, Draghia PM, Burlacu MG, Georgescu TF, Georgescu DE, et al. The microbiota and the relationship with colorectal cancer. Gastrointest Disord. 2022;4(2):66-76.
17. Bhattacharya S, Pal K, Jain S, Chatterjee SS, Konar J. Surgical site infection by methicillin resistant Staphylococcus aureus. J Clin Diagn Res. 2016;10(9):DC32.
18. Ma SC, Zhang JQ, Yan TH, Miao MX, Cao YM, Cao YB, et al. Novel strategies to reverse chemoresistance in colorectal cancer. Cancer Med. 2023;12(10):11073-11096.
19. Basch CH, Hillyer GC, Jacques ET. News coverage of colorectal cancer on Google News. JMIR Cancer. 2022;8(2):e39180.
20. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, et al. Expert consensus document: the International Scientific Association for Probiotics and Prebiotics consensus statement. Nat Rev Gastroenterol Hepatol. 2014.
21. Goldin BR, Gorbach SL. Effect of Lactobacillus acidophilus dietary supplements on intestinal cancer in rats. J Natl Cancer Inst. 1980;64(2):263-265.
22. Goldin BR, Gualtieri LJ, Moore RP. The effect of Lactobacillus GG on the initiation and promotion of DMH-induced intestinal tumors in the rat. Nutr Cancer. 1996;25:197-204.
23. Koo M, Rao AV. Long-term effect of bifidobacteria and neosugar on precursor lesions of colonic cancer in mice. Nutr Cancer. 1991;16(3-4):249-257.
24. Rao CV, Sanders ME, Indranie C, Simi B, Reddy BS. Prevention of colonic preneoplastic lesions by Lactobacillus acidophilus in rats. Int J Oncol. 1999;14(5):939-983.
25. Rowland I, Rumney C, Coutts J, Lievense L. Effect of Bifidobacterium longum and inulin on carcinogen-induced aberrant crypt foci. Carcinogenesis. 1998;19(2):281-285.
26. Singh J, Rivenson A, Tomita M, Shimamura S, Ishibashi N, Reddy BS. Bifidobacterium longum inhibits colon carcinogenesis. Carcinogenesis. 1997;18(4):833-841.
27. Ishikawa H, Akedo I, Otani T, Suzuki T, Nakamura T, Takeyama I, et al. Randomized trial of dietary fiber and Lactobacillus casei administration for prevention of colorectal tumors. Int J Cancer. 2005;116(5):762-767.
28. Rafter J, Bennett M, Caderni G, Clune Y, Hughes R, Karlsson PC, et al. Dietary synbiotics reduce cancer risk factors in colon cancer patients. Am J Clin Nutr. 2007;85(2):488-496.
29. O’Toole PW, Marchesi JR, Hill C. Next-generation probiotics: the spectrum from probiotics to live biotherapeutics. Nat Microbiol. 2017;2(5):1-6.
30. Nami Y, Abdullah N, Haghshenas B, Radiah D, Rosli R, Khosroushahi AY. Assessment of probiotic potential of Lactobacillus plantarum 5BL. Microbiol Immunol. 2014;58(9):492-502.
31. Tiptiri-Kourpeti A, Spyridopoulou K, Santarmaki V, Aindelis G, Tompoulidou E, Lamprianidou E, et al. Lactobacillus casei exerts antiproliferative effects in colon carcinoma cells. PLoS One. 2016;11(2):e0147960.
32. Wan Y, Xin Y, Zhang C, Wu D, Ding D, Tang L, et al. Fermentation supernatants of Lactobacillus delbrueckii inhibit colon cancer cells. Oncol Lett. 2014;7(5):1738-1742.
33. Wang SM, Zhang LW, Fan RB, Han X, Yi X. Induction of HT-29 apoptosis by lactobacilli. Res Microbiol. 2014;165(3):202-214.
34. Chung IC, OuYang CN, Yuan SN, Lin HC, Huang KY, Wu PS, et al. Heat-killed probiotic modulates NLRP3 inflammasome. Nutrients. 2019;11(3):516.
35. Karimi Ardestani S, Tafvizi F, Tajabadi Ebrahimi M. Heat-killed probiotic bacteria induce apoptosis of HT-29 cells. Hum Exp Toxicol. 2019;38(9):1069-1081.
36. Orlando A, Refolo M, Messa C, Amati L, Lavermicocca P, Guerra V, et al. Antiproliferative effects of Lactobacillus strains in gastric and colon cancer cells. Nutr Cancer. 2012;64(7):1103-1111.
37. Louis P, Hold GL, Flint HJ. The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev Microbiol. 2014;12(10):661-672.
38. Ishizeki S, Sugita M, Takata M, Yaeshima T. Effect of administration of bifidobacteria on intestinal microbiota in low-birth-weight infants and transition of administered bifidobacteria. Anaerobe. 2013;23:38-44.
39. Turroni F, Berry D, Ventura M. Bifidobacteria and their role in the human gut microbiota. Front Microbiol. 2017;7:2148.
40. Isticato R, Ricca E. Spore surface display. In: Driks A, Eichenberger P, editors. The bacterial spore: from molecules to systems. Washington (DC): ASM Press; 2016. p.349-366.
41. Kato K, Odamaki T, Mitsuyama E, Sugahara H, Xiao JZ, Osawa R. Age-related changes in the composition of gut Bifidobacterium species. Curr Microbiol. 2017;74:987-995.
42. Tojo R, Suárez A, Clemente MG, de los Reyes-Gavilán CG, Margolles A, Gueimonde M, et al. Intestinal microbiota in health and disease: role of bifidobacteria in gut homeostasis. World J Gastroenterol. 2014;20(41):15163-15174.
43. Bülbül MV, Özbek H, Acıkara OB, Ozbılgın S, Oz BE, Kurtul E, et al. Antiproliferative and apoptotic effects of Onobrychis albiflora extract on HCT-116 cells. J Exp Clin Med. 2018;35(4):101-109.
44. Acar TY, Bulbul MV, Vatandaslar E, Kurt A, Kalender SM, Keskin I, et al. Linarin inhibits survival and metastasis in triple-negative breast cancer cell line MDA-MB-231 by downregulating NF-κB p-p65/MMP-9 and enhancing caspase-3/7 expression. Nat Prod Res. 2025;1-10.
45. Hastings J, Kenealey J. Avenanthramide-C reduces the viability of MDA-MB-231 breast cancer cells through an apoptotic mechanism. Cancer Cell Int. 2017;17:1-12.
46. Aspiroz LS, Mennecozzi M, Batlle L, Corneo B, Healy L, Kotter M, et al. Promoting the adoption of best practices and standards to enhance quality and reproducibility of stem cell research. Stem Cell Rep. 2025.
47. Sobiesiak M, Fatyga N, Brzozowski A, Sikora A, Kupcewicz B, Maj M. Chromanone derivatives: evaluating selective anticancer activity across human cell lines. Eur J Med Chem. 2025;117771.
48. Faghfoori Z, Faghfoori MH, Saber A, Izadi A, Yari Khosroushahi A. Anticancer effects of bifidobacteria on colon cancer cell lines. Cancer Cell Int. 2021;21(1):258.
49. Koi M, Umar A, Chauhan DP, Cherian SP, Carethers JM, Kunkel TA, et al. Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability in colon tumor cells with homozygous hMLH1 mutation. Cancer Res. 1994;54(16):4308-4312.
50. Vilar E, Mukherjee B, Kuick R, Raskin L, Misek DE, Taylor JM, et al. Gene expression patterns in mismatch repair-deficient colorectal cancers highlight the therapeutic role of PI3K-AKT-mTOR pathway inhibitors. Clin Cancer Res. 2009;15(8):2829-2839.
51. Wang J, Liu Y, Cai J, Yang X, Xiong Z, Zou D, et al. Role of P53 mediated molecular regulation in starvation-induced autophagy in HCT-116 and HT-29 colorectal carcinoma cells. Biol Bull. 2023;50(Suppl 4):S522-S533.
52. Voutsadakis IA. Sensitivities and dependencies of BRAF mutant colorectal cancer cell lines with or without PIK3CA mutations for discovery of therapeutic vulnerabilities. Medicina (Kaunas). 2022;58(10):1498.
53. Wong CC, Yu J. Gut microbiota in colorectal cancer development and therapy. Nat Rev Clin Oncol. 2023;20(7):429-452.
54. Ghadimi D, Helwig U, Schrezenmeir J, Heller KJ, De Vrese M. Epigenetic imprinting by commensal probiotics inhibits the IL-23/IL-17 axis in an in vitro model of the intestinal mucosal immune system. J Leukoc Biol. 2012;92(4):895-911.
55. Zalila-Kolsi I, Dhieb D, Osman HA, Mekideche H. The gut microbiota and colorectal cancer: understanding the link and exploring therapeutic interventions. Biology (Basel). 2025;14(3):251.
56. de Oliveira Alves N, Dalmasso G, Nikitina D, Vaysse A, Ruez R, Ledoux L, et al. The colibactin-producing Escherichia coli alters the tumor microenvironment facilitating colorectal cancer progression and chemoresistance. Gut Microbes. 2024;16(1):2320291.
57. Devaux A, Villéger R, Roche G, Diémé B, Rodrigues M, Juban C, et al. L-serine promotes pro-carcinogenic effects of colibactin-producing E. coli. Gut Microbes. 2025;17(1):2515480.
58. Jans M, Kolata M, Blancke G, D'Hondt A, Gräf C, Ciers M, et al. Colibactin-driven colon cancer requires adhesin-mediated epithelial binding. Nature. 2024;635(8038):472-480.
59. Jian C, Yinhang W, Jing Z, Zhanbo Q, Zefeng W, Shuwen H. Escherichia coli on colorectal cancer: a two-edged sword. Microb Biotechnol. 2024;17(10):e70029.
60. Kengne MF, Mbaveng AT, Kuete V. Antibiotic resistance profile of Staphylococcus aureus in cancer patients. Biomed Res Int. 2024;2024:5859068.
61. Liu A, Garrett S, Hong W, Zhang J. Staphylococcus aureus infections and human intestinal microbiota. Pathogens. 2024;13(4):276.
62. Galanis A, Kiousi DE, Tzampazlis V, et al. Antimicrobial and antibiofilm activity of Lacticaseibacillus casei ATCC 393 against clinically relevant pathogens. Microorganisms. 2025;13:2667.
63. Moussadji N, Legba BB, Kokou K, Essone PN, Amana-Bokagne V, Tchadie EP, Baba-Moussa LS, Dougnon V, Agnandji ST, Lokossou G. Probiotic potential of Lactobacilli and Bifidobacteria found in breast milk in Gabon. Arch Microbiol Immunol. 2025;9:235-247.
64. Morsli DS, Tbahriti HF, Rahli F, Mahammi FZ, Nagdalian A, Hemeg HA, et al. Probiotics in colorectal cancer prevention and therapy: mechanisms, benefits, and challenges. Discov Oncol. 2025;16(1):406.