Limosilactobacillus fermentum UCO-979C exerts oncobiotic activity on human cancer cell lines

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Alarcon-Zapata Zapata, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6779348/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Probiotics are live microorganisms that, when administered appropriately, can confer beneficial effects on the host. Limosilactobacillus fermentum UCO-979C, a probiotic strain isolated from the human stomach, has demonstrated the ability to inhibit Helicobacte r pylori , a pathogen associated with gastric cancer. Recent studies have introduced the concept of "oncobiotics," referring to probiotics with anticancer properties. In this study, we aimed to investigate the cytotoxic effects of L. fermentum UCO-979C on human cancer cells. Our results revealed that the L. fermentum UCO-979C strain and its lysate exhibited significant cytotoxic activity against multiple tumor cell lines while demonstrating no cytotoxic effect on non-cancerous cells. Notably, gastrointestinal cancer cells displayed heightened sensitivity to the probiotic strain, showing a more significant decrease in cell viability compared to other tumor cells. These findings suggest that L. fermentum UCO-979C acts as an oncobiotic, specifically targeting gastrointestinal cancer cells. Understanding the cytotoxic mechanisms of L. fermentum UCO-979C on cancer cells holds promise for developing novel therapeutic strategies for gastrointestinal cancers. Further research is warranted to elucidate the underlying molecular pathways and evaluate the potential clinical applications of this probiotic strain in cancer treatment. Cancer cells Cytotoxicity Probiotics UCO-979C Oncobiotic Figures Figure 1 Figure 2 Figure 3 Figure 4 1. INTRODUCTION Humans coexist with various microorganisms, including fungi, viruses, and bacteria, in different parts of their bodies, such as the skin, mouth, female genitourinary system, and gastrointestinal tract. This collective population is the normal human microbiota ( 1 , 2 ). Probiotics, defined as live microorganisms that confer health benefits to the host, have been studied for their potential to regulate the microbiota ( 3 ). Gastrointestinal tract cancers account for 26% of all neoplasms and are responsible for 35% of cancer-related deaths worldwide ( 4 ). Strategies to prevent gastrointestinal cancers include polyp surveillance and chemoprevention, which involves using nutritional or pharmacological interventions to prevent, halt, or reverse the growth of neoplastic cells ( 5 – 7 ). Probiotics have emerged as potential chemopreventive agents, exerting anticancer effects through multiple mechanisms ( 8 ). These mechanisms encompass the binding and degradation of potentially carcinogenic substances, production of anti-tumorigenic compounds, enhancement of immune responses, suppression of microbiota growth associated with mutagenesis and carcinogenesis, and protection of cellular DNA against oxidative damage ( 9 – 11 ). Probiotics have demonstrated the ability to prevent and inhibit cancer progression while regulating cellular growth mechanisms ( 12 – 16 ). Moreover, different probiotic treatments have exhibited antiproliferative and cytotoxic effects on gastrointestinal cancer cells ( 17 – 19 ), including probiotic secreted factors or cell-free supernatants ( 17 , 20 – 23 ). Proposed mechanisms underlying the impact of probiotics on cancer and tumor processes include the maintenance of cell-cell junction integrity ( 24 , 25 ), antiproliferative and cytotoxic effects on tumor cells ( 26 – 30 ), inhibition of epithelial-mesenchymal transition and metastasis ( 31 – 33 ), and modification of the tumor microenvironment to inhibit tumor growth ( 34 – 36 ). Probiotics that display anticancer activity are now called "oncobiotics" and are defined as live microbes or their derived metabolites that exhibit ameliorative actions against cancer ( 37 ). Limosilactobacillus fermentum UCO-979C was isolated from a gastric biopsy obtained during upper digestive endoscopy. This strain possesses several probiotic characteristics, such as acid and bile tolerance and antibiotic susceptibility ( 38 ). The genome of strain UCO-979C has been sequenced, revealing the presence of genes associated with the binding of collagen and fibronectin proteins ( 39 ). L. fermentum UCO-979C modulates the inflammatory response in vitro ( 40 ). Importantly, L. fermentum UCO-979C has also shown inhibitory activity against Helicobacter pylori ( 41 , 42 ); this activity has also been observed in the consumption of gelatin and ice cream supplemented ( 43 , 44 ). To further characterize the properties of L. fermentum UCO-979C, now as an oncobiotic, this study aimed to investigate the cytotoxic effects of L. fermentum UCO-979C and its lysate on various human cancer cells. 2. METHODS 2.1 Bacteria strain and culture conditions. L. fermentum UCO-979C was a bacterial strain obtained from a human gastric biopsy and was maintained from the Bacterial Pathogenicity Laboratory repository at the Microbiology Department, University of Concepcion, Chile. A human gastric L. fermentum UCO-979C was cultured on Mann-Rogosa Sharpe broth (M.R.S.; BD Difco, Le Pont de Claix, France) in a microaerobic atmosphere at 37°C 24 h, followed by culturing on M.R.S. agar (BD Difco) under similar conditions for 24–48 h. For each assay, bacteria were obtained from the agar cultures, resuspended in liquid culture media, and their concentration adjusted to that required for each assay as described below. 2.2 Cells lines and growth conditions. 2.2.1 Normal Human Cells. This study used two types of normal human cells: Human Umbilical Cord Vein Endothelial Cells (HUVEC) and Human Dermal Fibroblast cells. HUVEC cells were obtained through the Jaffe Method ( 45 ). Under sterile conditions, the cord vein was cannulated and washed with 10 mM PBS. Subsequently, the vein was incubated for 10 minutes at 37°C with type II collagenase (Gibco, Life Technologies) at a concentration of 0.33 mg/mL for enzymatic digestion. The resulting cell-containing medium was collected in a sterile tube and centrifuged at 450 g for 10 minutes. The supernatant was discarded, and the pellet containing the HUVEC cells was resuspended in 5 mL of M-199 culture medium (Corning) supplemented with 20% fetal bovine serum (FBS) (Corning), 3.2 mM L-glutamine (Corning), penicillin/streptomycin (Biological Industries), and endothelial growth factor (Endothelial cell growth supplement, Merck). The cells were then cultured in 100 mm plates coated with 1% w/v gelatin and incubated in an atmosphere of 5% CO2 at 37°C until reaching 90% confluence. Human dermal fibroblasts HDF (106-05A, Cell Applications, Inc., USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries). The cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air. 2.2.1 Tumoral human cell lines. Human gastrointestinal tumoral cell lines, namely AGS (human stomach carcinoma cells, ECACC 89090402), HCT-116 (human colorectal carcinoma cells, ATCC CCL-247), and HT-29 (human colorectal carcinoma cells, ECACC 91072201), were cultured and expanded in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries), at 37°C in a 5% CO2 and 95% air atmosphere. Other tumoral cell lines used in this study included SK-MEL (human melanoma cells, ATCC HTB-67) and MCF-7 (human breast adenocarcinoma cells, ECACC 86012803). HaCaT (immortalized human keratinocytes) were kindly donated by Dr. Patricio Oyarzún at Universidad San Sebastián, Concepción, Chile. MCF-7 and HaCaT cells were cultured in DMEM (Gibco), while SK-MEL was cultured in Roswell Park Memorial Institute (RPMI 1640, Gibco). All culture media were supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries), and the cells were cultured at 37°C in a 5% CO2 and 95% air atmosphere. 2.2 Cell viability assay. The antiproliferative effects of L. fermentum UCO-979C on cells were assessed using the Sulforhodamine B (S.R.B.) method ( 46 ). Initially, the cells were seeded in 96-well microplates at a density of 2 × 104 cells per well. L. fermentum UCO-979C was adjusted to different McFarland (MF) standards (0.5, 1, 2, 4, and 6 MF) in DMEM culture medium or RPMI-1640, according to the culture medium used for each cell line, without antibiotics, and then added to the cells for 4 hours to promote bacterial adherence. After three washes with sterile 10 mM PBS, the viability assay was conducted at 24, 48, and 72 hours using the S.R.B. method. To perform the S.R.B. method, the cells were fixed with 10% (w/v) trichloroacetic acid (T.C.A.) from Winkler, Chile, and stained with 0.057% (w/v) Sulforhodamine B from Sigma-Aldrich in 1% (v/v) acetic acid from Winkler, Chile. The bound dye was dissolved in a 10 mM Tris base from Sigma-Aldrich, and the absorbance was measured at 510 nm using a multiplate reader (Synergy 2, BioTek Instruments). The percent cellular survival was calculated based on the absorbance values obtained. 2.4 Bacterial lysate extraction. L. fermentum UCO-979C was preserved in M.R.S. liquid media. An inoculum was cultured in 1000 mL of culture medium for 24 h at 37°C. The bacterial suspension was centrifuged for 8 min at 6000 x g and washed thrice with 10 mM PBS, pH 7.4. The pellet obtained was resuspended in 35 mL of sterile 10 mM PBS to get a bacterial lysate. The bacterial suspensions were lysate by a mechanical rupture in French Press with an approximate flow of 0.75 mL/minute at a pressure of 1000 psi. Subsequently, the bacterial lysate containing proteins and other soluble bacterial products was centrifuged at 12800 x g for 15 min at 4°C and filtered using 0.22 µm pore size (Millipore) ( 47 ). The supernatant was kept at -80°C. 2.5 Bacterial lysate effect on tumoral cells. AGS cells were seeded in 96-well plates at a 20.000 cells/well concentration for 24 hours in a DMEM culture medium. Then, the cells were washed twice with sterile 10 mM PBS and exposed for 24, 48, and 72 hours at concentrations of protein lysate of 10, 50, 100, 200, 800, and 1000 µg/mL in medium cell culture total bacterial lysate. Finally, AGS cells were seeded in conditions similar to those before. Viable cells were determined by the S.R.B. method as described previously. 2.5 Statistical analysis. Graphs and statistical data analysis were performed using GraphPad Prism 8 for Mac. Results are represented as the mean ± standard error of the mean (SEM). Student t-tests analyzed statistical differences. A p-value of less than 0.05 was considered statistically significant. 3. RESULTS 3.1 Effect of L. fermentum UCO-979C on normal cells. We investigated the potential cytotoxicity of L. fermentum UCO-979C on normal non-tumor cells, specifically HUVEC and HDF cells. The assay involved exposing the cells to different concentrations of the bacteria (ranging from 0.5 to 6 MF) for 24, 48, and 72 hours. Remarkably, no significant effects on cell viability were observed across different time points or concentrations (Fig. 1 ). These results are consistent with previous findings that demonstrate probiotics generally do not induce cytotoxicity in normal human cells. 3.2 Effect of L. fermentum UCO-979C on human gastrointestinal tumor cell lines. Considering the absence of cytotoxic activity of L. fermentum UCO-979C in normal cells, we evaluated its potential cytotoxicity in human gastrointestinal tumor cell lines. Since this bacterium originates from the gastric niche, we specifically examined its effects on AGS, HCT-116, and HT-29 cells. These cells were exposed to different concentrations of the resuspended bacteria and cultured without antibiotics for 24, 48, and 72 hours. Interestingly, when AGS cells were incubated with L. fermentum UCO-979C, a time- and concentration-dependent decrease in cell viability was observed. The cytotoxic effect was evident from 24 hours of incubation, with a reduction in cell viability observed even at the lowest concentration. This effect persisted from 1 MF, resulting in approximately 50% cell viability regardless of concentration. The same trend was observed at 48 and 72 hours of exposure, with cell viability dropping below 50% (Fig. 2 A). Similarly, HCT-116 cells displayed decreased cell viability with increasing concentrations of L. fermentum UCO-979C, up to 2 MF, at all time points. Unexpectedly, at 72 hours of exposure, a significant cytotoxic effect was observed at the highest concentrations, resulting in less than 10% cell viability (Fig. 2 B). In the case of HT-29 intestinal cells, no cytotoxic effect of L. fermentum UCO-979C was observed at the lowest concentrations after 24 hours. However, a marked cytotoxic effect was observed at higher concentrations, with cell viability dropping below 40%. Surprisingly, irrespective of bacterial concentration, a substantial reduction in cell viability was observed at 48 and 72 hours, with values below 25% (Fig. 2 C). These results demonstrate the pronounced cytotoxic effect of L. fermentum UCO-979C on the gastric and intestinal cell lines studied. 3.3 Effect of L. fermentum UCO-979C on other tumour cell lines and immortalized keratinocytes. To determine if the cytotoxic effect of L. fermentum UCO-979C on gastrointestinal cancer cells extends to other cell types, we investigated its impact on immortalized keratinocytes (HaCaT cells), melanocytes (SK-MEL cells), and breast cancer cells (MCF-7). A notable cytotoxic effect was observed despite HaCaT cells not being tumor cells. However, the cell viability remained relatively stable at around 75% across all concentrations and study durations, except at higher concentrations where L. fermentum UCO-979C demonstrated a cytotoxic effect of approximately 50% at 72 hours (Fig. 3 A). Interestingly, a cytotoxic effect was consistently observed in SK-MEL melanocytes, regardless of the bacterial concentration and exposure time spanning 24 to 72 hours. The cell viability ranged between 60% and 70% under these conditions, indicating that the impact of the bacterium on these cells is independent of concentration and time (Fig. 3 B). In the case of MCF-7 breast tumor cells, a slight decrease in cell viability was evident after 24 hours of exposure to L. fermentum UCO-979C. However, at 48 hours of analysis, a significant cytotoxic effect is observed, regardless of the bacterial concentration, resulting in a decrease in viability from 75–70%. As anticipated, this effect was sustained at 72 hours, with a 50% reduction in viability at the highest bacterial concentrations (Fig. 3 C). Therefore, L. fermentum UCO-979C demonstrated a cytotoxic effect in immortalized keratinocytes (HaCaT) and various non-gastrointestinal tract tumour cells, highlighting its importance in inducing cell death. 3.4. Effect of L. fermentum UCO-979C lysate on AGS cells. To investigate whether the cytotoxic effect is solely attributed to living bacteria, we examined whether the intracellular content of L. fermentum UCO-979C, obtained from its lysate, could also induce this effect. In this experiment, AGS cells were exposed to different concentrations of the bacterial lysate, determined by protein concentration, for 24, 48, and 72 hours. After 24 hours of incubation with the bacterial lysate, a slight cytotoxic effect on AGS cell viability was observed. This effect became more pronounced as the lysate concentration in the incubation medium increased. At a 1000 µg/mL concentration, the cell viability decreased to 70%. Surprisingly, after 48 hours of cell culture, the cytotoxic effect intensified at concentrations above 200 µg/mL, significantly reducing cell viability to 30% at the highest concentration. The same pattern was observed at 72 hours of exposure, with the cytotoxic effect becoming evident at a concentration of 100 µg/mL (Fig. 4 ). Remarkably, the intracellular content obtained from the L. fermentum UCO-979C lysate also exhibited a dose-dependent decrease in cell viability after 48 hours of exposure. These findings suggest that both live bacteria and their intracellular content, represented by the lysate, exert a cytotoxic effect on AGS cells. 4. DISCUSSION This study aimed to assess the cytotoxic potential of L. fermentum UCO-979C and its bacterial lysate on tumor cells as an initial exploration of their oncolytic properties. Previous research has reported selective cytotoxic behavior of Lactobacillus plantarum 5BL on various tumor cell lines, including AGS (gastric), HeLa (cervical), HT-29 (intestinal), and MCF7 (breast) while showing no cytotoxic effect on HUVEC cells, which served as the normal cell control ( 48 ). Other studies have demonstrated the antiproliferative effects of Lactobacillus strains on different cancer cell lines. For instance, L. acidophilus DSM9126 and L. lactis KX881782 have been shown to exhibit antiproliferative effects on intestinal tumoral CACO-2 cells, MCF7, and HeLa cells ( 49 ). Additionally, L. plantarum A7 and L. rhamnosus G.G. had a cytotoxic effect on CACO-2 cells, reducing cell viability to 60% and 80% after 48 hours of exposure. Similar results were observed in HT-29 cells ( 50 ). Moreover, L. rhamnosus GG at a concentration of 10 8 CFU/mL exhibited cytotoxic effects on CACO-2 cells after 48 hours of exposure, reducing viability to 60%. In contrast, no such effect was observed in HT-29 cells under the same conditions ( 51 ). Cytotoxic effects of probiotics have also been observed in colon carcinoma cells. In HT-29 cells, strains of L. delbreucki , L. plantarum , L. rhamnosus , L. plantarum , and L. brevis exhibited cytotoxic effects after 16 hours of exposure ( 52 ). In HGC-27 cells, cytotoxic effects were observed for L. rhamnosus L.G.G. and L. paracasei MPC2.1 at concentrations of 10 8 CFU/mL after 24 and 48 hours, suggesting L. paracasei IMPC2.1 as a potential probiotic for cancer prevention ( 53 ). A 20% inhibition of cell proliferation in HT-29 cells has been suggested as a significant antiproliferative effect ( 54 ). In the present study, L. fermentum UCO-979C did not affect the viability of HUVEC cells (derived from the human umbilical cord) and HDF cells (non-tumor cells). However, cell viability was decreased in tumor cell lines when exposed to L. fermentum UCO-979C, including AGS, SK-MEL, and HaCaT cells, which are not of gastrointestinal origin. A particular finding was the observed sensitivity in HaCaT cells, which are immortalized human keratinocytes but not oncogenically transformed ( 55 ). While the probiotic L. fermentum UCO-979C showed clear selectivity against cancer cells when compared to the primary non-tumour cells HUVEC and HDF, the cytotoxic effect on HaCaT cells warrants special consideration. This differential susceptibility may be attributed to HaCaT cells exhibiting considerably higher proliferation rates than primary cells and potentially harbouring certain alterations in cell signalling pathways ( 56 ). These features, such as an increased dependence on growth factors or changes in the expression of surface receptors, might render them more susceptible to the effects of L. fermentum UCO-979C or its metabolites, compared to the primary non-tumor cells that showed resistance. Future studies could explore these differences to better understand the determinants of cellular sensitivity to this probiotic. These selectivity findings are consistent with studies on a Lactobacillus fermentum strain. For example, extracts from Lactobacillus fermentum NCIMB 5221 were shown to significantly inhibit the growth of colorectal cancer cells SW-480 and Caco-2, and induce apoptosis, while not affecting non-neoplastic colon cells CRL-1831. This selectivity was linked to a higher production of short-chain fatty acids (SCFAs) by the bacterium ( 57 ). The observation that L. fermentum UCO-979C, isolated from the stomach, predominantly interacts with gastrointestinal epithelial cells, might contribute to its pronounced effects on gastrointestinal cancer lines. The mechanism of cell death induced by L. fermentum UCO-979C appears to be associated with apoptosis, a phenomenon also observed with other probiotic strains, including the L. fermentum NCIMB 5221. In the search for cytotoxic products secreted by bacteria, it has been found that metabolites secreted by L. lactis subspecies lactis 44 at a concentration of 40 µg/mL, after 48 hours of stimulation, exhibited a cytotoxic effect on AGS cells as well as on HT-29, MCF7, and HeLa tumor cells ( 29 ). In HT-29 cells, intracellular extracts of L. casei 01 demonstrated a cytotoxic effect, resulting in 89% viability reduction ( 58 ). Moreover, culture supernatants devoid of bacteria (at a concentration of 60 µg/mL) from L. casei , L. paracasei , L. rhamnosus , and L. plantarum strains showed cytotoxic effects, with an average viability reduction of 60% in CACO-2 and HT-29 cells after 48 hours of exposure ( 59 ). Additionally, cytoplasmic fractions of L. rhamnosus G.G., obtained through sonication, exhibited a cytotoxic effect with a 60% reduction in viability in gastric cancer cells (HGC-27) at a concentration of 1:1 v/v, corresponding to a bacterial concentration of 10 8 CFU/mL ( 60 ). When AGS cells were exposed to the lysate of L. fermentum UCO-979C containing soluble intracellular components of the bacteria, cytotoxic effects were also observed at 24, 48, and 72 hours of culture. 5. CONCLUSION In conclusion, the findings of this study provide evidence for the oncobiotic activity of L. fermentum UCO-979C. It demonstrates its ability to selectively induce cytotoxic effects in cancer cell lines, particularly those derived from the gastrointestinal tract, highlighting its potential antitumor properties. Notably, L. fermentum UCO-979C does not exhibit cytotoxicity in normal non-tumor cells, indicating its selective cytotoxic effect on cancer cells. These results further underscore its potential as a promising oncobiotic agent. It is important to note, however, that these initial findings are from in vitro assays, and the precise molecular mechanisms and active components responsible for the observed cytotoxicity require further elucidation. Furthermore, the cytotoxic effects observed in this study were not limited to live bacteria alone but were also evident in the bacterial lysate. This suggests the presence of intracellular components or metabolites in L. fermentum UCO-979C that contribute to its cytotoxic potential. Future investigations should focus on identifying and characterizing the molecules or groups responsible for the observed cytotoxic effects in the bacterial lysate. The precise mechanisms responsible for the antitumor effects of L. fermentum UCO-979C have yet to be fully understood. Thus, it is crucial to investigate the signalling pathways and cellular processes contributing to the cytotoxicity induced by L. fermentum UCO-979C. Such exploration will enhance our comprehension of its underlying mechanism of action. Furthermore, conducting in vivo models to evaluate the impact of L. fermentum UCO-979C on tumor growth and studying its interactions with the host immune system would yield valuable insights into its potential as a therapeutic agent, further solidifying its characterization as an oncobiotic. Declarations Disclosure statement The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author Contributions PA-Z: Conceptualization, Methodology, Software. PA-Z, FZ : Data curation, Writing- Original draft preparation. PA-Z., CP-S, BA-Z: Visualization, Investigation. FZ, AG-C, VO: Supervision.: PA-Z, CP-S, BA-Z: Software , Validation.: FZ, EN-L, AG-C, VO: Writing- Reviewing and Editing. Funding This work was supported by the Vicerrectoría de Investigación y Desarrollo (VRID) of the Universidad de Concepción under Grant VRID No 2023000895INT. References Biedermann, L., and Rogler, G. The intestinal microbiota: its role in health and disease. Eur J Pediatr. 2015;174:151-167. https://doi.org/10.1007/s00431-014-2476-2 Ruff, W. E., and Greiling, T. M., and Kriegel, M. A. Host-microbiota interactions in immune-mediated diseases. 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Faseb J. 2010;24: Tiptiri-Kourpeti, A., Spyridopoulou, K., Santarmaki, V., Aindelis, G., Tompoulidou, E., Lamprianidou, E. E., Saxami, G., Ypsilantis, P., Lampri, E. S., Simopoulos, C., Kotsianidis, I., Galanis, A., Kourkoutas, Y., Dimitrellou, D., and Chlichlia, K. Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells. PLoS One. 2016;11:e0147960. https://doi.org/10.1371/journal.pone.0147960 Escamilla, J., and Lane, M. A., and Maitin, V. Cell-free supernatants from probiotic Lactobacillus casei and Lactobacillus rhamnosus GG decrease colon cancer cell invasion in vitro. Nutr Cancer. 2012;64:871-878. https://doi.org/10.1080/01635581.2012.700758 Khoury, N., and El-Hayek, S., and Tarras, O., and El-Sabban, M., and El-Sibai, M., and Rizk, S. Kefir exhibits antiproliferative and proapoptotic effects on colon adenocarcinoma cells with no significant effects on cell migration and invasion. Int J Oncol. 2014;45:2117-2127. https://doi.org/10.3892/ijo.2014.2635 Haghshenas, B., and Abdullah, N., and Nami, Y., and Radiah, D., and Rosli, R., and Khosroushahi, A. Y. 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;30:51-59. https://doi.org/10.1016/j.anaerobe.2014.08.009 Nami, Y., and Abdullah, N., and Haghshenas, B., and Radiah, D., and Rosli, R., and Khosroushahi, A. Y. Probiotic potential and biotherapeutic effects of newly isolated vaginal Lactobacillus acidophilus 36YL strain on cancer cells. Anaerobe. 2014;28:29-36. https://doi.org/10.1016/j.anaerobe.2014.04.012 Bui, V. T., Tseng, H. C., Kozlowska, A., Maung, P. O., Kaur, K., Topchyan, P., and Jewett, A. Augmented IFN-gamma and TNF-alpha Induced by Probiotic Bacteria in NK Cells Mediate Differentiation of Stem-Like Tumors Leading to Inhibition of Tumor Growth and Reduction in Inflammatory Cytokine Release; Regulation by IL-10. Front Immunol. 2015;6:576. https://doi.org/10.3389/fimmu.2015.00576 Keith, B., and Simon, M. C. Hypoxia-inducible factors, stem cells, and cancer. Cell. 2007;129:465-472. https://doi.org/10.1016/j.cell.2007.04.019 Petrof, E. O., Claud, E. C., Sun, J., Abramova, T., Guo, Y., Waypa, T. S., He, S. M., Nakagawa, Y., and Chang, E. B. Bacteria-free solution derived from Lactobacillus plantarum inhibits multiple NF-kappaB pathways and inhibits proteasome function. Inflamm Bowel Dis. 2009;15:1537-1547. https://doi.org/10.1002/ibd.20930 Aragon, F., and Carino, S., and Perdigon, G., and de LeBlanc, A. D. Inhibition of Growth and Metastasis of Breast Cancer in Mice by Milk Fermented With Lactobacillus casei CRL 431. J Immunother. 2015;38:185-196. https://doi.org/Doi 10.1097/Cji.0000000000000079 Takagi, A., and Matsuzaki, T., and Sato, M., and Nomoto, K., and Morotomi, M., and Yokokura, T. Enhancement of natural killer cytotoxicity delayed murine carcinogenesis by a probiotic microorganism. Carcinogenesis. 2001;22:599-605. Vong, L., and Lorentz, R. J., and Assa, A., and Glogauer, M., and Sherman, P. M. Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol. 2014;192:1870-1877. https://doi.org/10.4049/jimmunol.1302286 Nataraj, B. H., and Shivanna, S. K., and Rao, P., and Nagpal, R., and Behare, P. V. Evolutionary concepts in the functional biotics arena: a mini-review. Food Sci Biotechnol. 2021;30:487-496. https://doi.org/10.1007/s10068-020-00818-3 Garcia, A., Navarro, K., Sanhueza, E., Pineda, S., Pastene, E., Quezada, M., Henriquez, K., Karlyshev, A., Villena, J., and Gonzalez, C. Characterization of Lactobacillus fermentum UCO-979C, a probiotic strain with a potent anti-Helicobacter pylori activity. Electron J Biotechn. 2017;25:75-83. https://doi.org/10.1016/j.ejbt.2016.11.008 Karlyshev, A. V., and Villena, J., and Gonzalez, C., and Albarracin, L., and Barros, J., and Garcia, A. Draft Genome Sequence of a Probiotic Strain, Lactobacillus fermentum UCO-979C. Genome Announc. 2015;3:https://doi.org/10.1128/genomeA.01439-15 Garcia-Castillo, V., Zelaya, H., Ilabaca, A., Espinoza-Monje, M., Komatsu, R., Albarracin, L., Kitazawa, H., Garcia-Cancino, A., and Villena, J. Lactobacillus fermentum UCO-979C beneficially modulates the innate immune response triggered by Helicobacter pylori infection in vitro. Benef Microbes. 2018;9:829-841. https://doi.org/10.3920/BM2018.0019 Merino, J. S., and Garcia, A., and Pastene, E., and Salas, A., and Saez, K., and Gonzalez, C. L. Lactobacillus fermentum UCO-979C strongly inhibited Helicobacter pylori SS1 in Meriones unguiculatus. Benef Microbes. 2018;9:625-627. https://doi.org/10.3920/BM2017.0160 Salas-Jara, M. J., and Sanhueza, E. A., and Retamal-Diaz, A., and Gonzalez, C., and Urrutia, H., and Garcia, A. Probiotic Lactobacillus fermentum UCO-979C biofilm formation on AGS and Caco-2 cells and Helicobacter pylori inhibition. Biofouling. 2016;32:1245-1257. https://doi.org/10.1080/08927014.2016.1249367 Parra-Sepulveda, C., Sanchez-Alonzo, K., Olivares-Munoz, J., Gutierrez-Zamorano, C., Smith, C. T., Carvajal, R. I., Saez-Carrillo, K., Gonzalez, C., and Garcia-Cancino, A. Consumption of a Gelatin Supplemented with the Probiotic Strain Limosilactobacillus fermentum UCO-979C Prevents Helicobacter pylori Infection in a Young Adult Population Achieved. Foods. 2022;11:https://doi.org/10.3390/foods11121668 Paucar-Carrion, C., Espinoza-Monje, M., Gutierrez-Zamorano, C., Sanchez-Alonzo, K., Carvajal, R. I., Rogel-Castillo, C., Saez-Carrillo, K., and Garcia-Cancino, A. Incorporation of Limosilactobacillus fermentum UCO-979C with Anti-Helicobacter pylori and Immunomodulatory Activities in Various Ice Cream Bases. Foods. 2022;11:https://doi.org/10.3390/foods11030333 Jaffe, E. A., and Nachman, R. L., and Becker, C. G., and Minick, C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973;52:2745-2756. https://doi.org/10.1172/JCI107470 Vichai, V., and Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc. 2006;1:1112-1116. https://doi.org/10.1038/nprot.2006.179 Cirpus, I. E., Geerts, W., Hermans, J. H., Op den Camp, H. J., Strous, M., Kuenen, J. G., and Jetten, M. S. Challenging protein purification from anammox bacteria. Int J Biol Macromol. 2006;39:88-94. https://doi.org/10.1016/j.ijbiomac.2006.02.018 Nami, Y., and Abdullah, N., and Haghshenas, B., and Radiah, D., and Rosli, R., and Khosroushahi, A. Y. Assessment of probiotic potential and anticancer activity of newly isolated vaginal bacterium Lactobacillus plantarum 5BL. Microbiol Immunol. 2014;58:492-502. https://doi.org/10.1111/1348-0421.12175 Ayyash, M., and Al-Dhaheri, A. S., and Al Mahadin, S., and Kizhakkayil, J., and Abushelaibi, A. In vitro investigation of anticancer, antihypertensive, antidiabetic, and antioxidant activities of camel milk fermented with camel milk probiotic: A comparative study with fermented bovine milk. J Dairy Sci. 2018;101:900-911. https://doi.org/10.3168/jds.2017-13400 Sadeghi-Aliabadi, H., and Mohammadi, F., and Fazeli, H., and Mirlohi, M. Effects of Lactobacillus plantarum A7 with probiotic potential on colon cancer and normal cells proliferation in comparison with a commercial strain. Iran J Basic Med Sci. 2014;17:815-819. Orlando, A., and Linsalata, M., and Russo, F. Antiproliferative effects on colon adenocarcinoma cells induced by co-administration of vitamin K1 and Lactobacillus rhamnosus GG. Int J Oncol. 2016;48:2629-2638. https://doi.org/10.3892/ijo.2016.3463 Hasannejad Bibalan, M., Eshaghi, M., Rohani, M., Esghaei, M., Darban-Sarokhalil, D., Pourshafie, M. R., and Talebi, M. Isolates of Lactobacillus plantarum and L. reuteri display greater antiproliferative and antipathogenic activity than other Lactobacillus isolates. J Med Microbiol. 2017;66:1416-1420. https://doi.org/10.1099/jmm.0.000591 Orlando, A., Refolo, M. G., Messa, C., Amati, L., Lavermicocca, P., Guerra, V., and Russo, F. Antiproliferative and proapoptotic effects of viable or heat-killed Lactobacillus paracasei IMPC2.1 and Lactobacillus rhamnosus GG in HGC-27 gastric and DLD-1 colon cell lines. Nutr Cancer. 2012;64:1103-1111. https://doi.org/10.1080/01635581.2012.717676 Grimoud, J., Durand, H., de Souza, S., Monsan, P., Ouarne, F., Theodorou, V., and Roques, C. In vitro screening of probiotics and synbiotics according to anti-inflammatory and anti-proliferative effects. Int J Food Microbiol. 2010;144:42-50. https://doi.org/10.1016/j.ijfoodmicro.2010.09.007 Boukamp, P., and Petrussevska, R. T., and Breitkreutz, D., and Hornung, J., and Markham, A., and Fusenig, N. E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line. J Cell Biol. 1988;106:761-771. https://doi.org/10.1083/jcb.106.3.761 Liu, Y., and Liu, Y., and Zeng, C., and Li, W., and Ke, C., and Xu, S. Concentrated Growth Factor Promotes Wound Healing Potential of HaCaT Cells by Activating the RAS Signaling Pathway. Front Biosci (Landmark Ed). 2022;27:319. https://doi.org/10.31083/j.fbl2712319 Kahouli, I., and Malhotra, M., and Alaoui-Jamali, M., and Prakash, S. In-vitro characterization of the anti-cancer activity of the probiotic bacterium Lactobacillus fermentum NCIMB 5221 and potential against colorectal cancer. J Cancer Sci Ther. 2015;7:224-235. Liu, C. T., and Chu, F. J., and Chou, C. C., and Yu, R. C. Antiproliferative and anticytotoxic effects of cell fractions and exopolysaccharides from Lactobacillus casei 01. Mutat Res. 2011;721:157-162. https://doi.org/10.1016/j.mrgentox.2011.01.005 Faghfoori, Z., and Pourghassem Gargari, B., and Saber, A., and Seyyedi, M., and Fazelian, S., and Khosroushahi, A. Y. Prophylactic effects of secretion metabolites of dairy lactobacilli through downregulation of ErbB-2 and ErbB-3 genes on colon cancer cells. Eur J Cancer Prev. 2020;29:201-209. https://doi.org/10.1097/CEJ.0000000000000393 Russo, F., and Orlando, A., and Linsalata, M., and Cavallini, A., and Messa, C. Effects of Lactobacillus rhamnosus GG on the cell growth and polyamine metabolism in HGC-27 human gastric cancer cells. Nutr Cancer. 2007;59:106-114. https://doi.org/10.1080/01635580701365084 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6779348","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":466004228,"identity":"1d75d364-6901-431b-9e0c-92552fcdfe65","order_by":0,"name":"Pedro Alarcon-Zapata","email":"","orcid":"","institution":"University of Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Pedro","middleName":"","lastName":"Alarcon-Zapata","suffix":""},{"id":466004232,"identity":"44d98a12-ac10-4121-a769-ec7478b39a57","order_by":1,"name":"Cristian Parra-Sepulveda","email":"","orcid":"","institution":"University of Concepcion","correspondingAuthor":false,"prefix":"","firstName":"Cristian","middleName":"","lastName":"Parra-Sepulveda","suffix":""},{"id":466004234,"identity":"d5ba0db6-4f8b-4bb2-9e73-70bb65b78c75","order_by":2,"name":"Barbara N. Alarcon-Zapata Zapata","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"N. Alarcon-Zapata","lastName":"Zapata","suffix":""},{"id":466004236,"identity":"516b0a3e-6479-4c61-808f-640672a828a6","order_by":3,"name":"Estefanía Nova-Lamperti","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Estefanía","middleName":"","lastName":"Nova-Lamperti","suffix":""},{"id":466004237,"identity":"d0bac237-74e2-4ac8-ae2c-37c3885bb90b","order_by":4,"name":"Apolinaria Garcia-Cancino","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Apolinaria","middleName":"","lastName":"Garcia-Cancino","suffix":""},{"id":466004240,"identity":"22be5440-ae4b-4cd6-bbc4-945a7a2a7610","order_by":5,"name":"Valeska Ormazabal","email":"","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":false,"prefix":"","firstName":"Valeska","middleName":"","lastName":"Ormazabal","suffix":""},{"id":466004242,"identity":"5ec4a2ac-6683-4374-ba65-9404ee293285","order_by":6,"name":"Felipe A. Zuniga","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAu0lEQVRIiWNgGAWjYBAC9gYgkcDAIAcmGRgsCGvhOQDRYgzVIkGkFiBIbCBeC/8ZswcPftmlbziefPADQw0xWhjOmBsk9iXnbjjzLFmC4RgRWuwZe8wkEnuYczfcyDFjYGwgxhZmHpCW+nSDG/nfiNTCBtSS8ONwgsGNHDYitfCwlUkkNhw3nHnmmbFEAjF+4eE/vE3yx59qeb7jyQ8/fKixIawFDBjboIwEIjUAwR/ilY6CUTAKRsEIBABpRTWMmahD1QAAAABJRU5ErkJggg==","orcid":"","institution":"Universidad de Concepción","correspondingAuthor":true,"prefix":"","firstName":"Felipe","middleName":"A.","lastName":"Zuniga","suffix":""}],"badges":[],"createdAt":"2025-05-29 20:53:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6779348/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6779348/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83940263,"identity":"3e7890cf-0701-44df-879a-96e1ab11506d","added_by":"auto","created_at":"2025-06-04 17:59:14","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":381730,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of normal non-tumor cells exposed to \u003cem\u003eL. fermentum \u003c/em\u003eUCO-979C. HUVEC and HDF cells were exposed to 0.5, 1, 2, 4, and 6 MF concentrations of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C. A) HUVEC cells exposed for 24, 48, and 72 h. B) HDF cells exposed for 24, 48, and 72 h. The error bars indicate the S.E.M. Experiment in triplicate.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6779348/v1/1c37490cc29e6fbf697d98c8.png"},{"id":83940265,"identity":"8bbb2cf6-f106-407c-9d22-6c1e1be73936","added_by":"auto","created_at":"2025-06-04 17:59:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":774083,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of gastrointestinal cancer cells exposed to \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C. AGS, HCT-116, and HT-29 cells were stimulated with concentrations of 0.5, 1, 2, 4, and 6 MF of \u003cem\u003eL. fermentum\u003c/em\u003eUCO-979C. A) AGS cells exposed for 24, 48, and 72 h. B) HCT-116 cells exposed for 24, 48, and 72 h. C) HT-29 cells exposed for 24, 48, and 72 h. The error bars indicate the S.E.M. Experiment in triplicate. (*) p \u0026lt;0.05, (**) p\u0026lt; 0.01 and (***) p\u0026lt;0.001\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6779348/v1/ff9bb28fb4c5f09366626373.png"},{"id":83940262,"identity":"3604c1f4-bb4c-418c-b178-f480f18802f6","added_by":"auto","created_at":"2025-06-04 17:59:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":872120,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of other cancer cells exposed to \u003cem\u003eL. fermentum \u003c/em\u003eUCO-979C. HaCaT, SK-MEL, and MCF-7 cells were stimulated with concentrations of 0.5, 1, 2, 4, and 6 MF of \u003cem\u003eL. fermentum \u003c/em\u003eUCO-979C. A) HaCaT cells were exposed for 24, 48, and 72 h. B) SK-MEL cells exposed for 24, 48, and 72 h. C) MCF-7 exposed for 24, 48, and 72 h. The error bars indicate the S.E.M. Experiment in triplicate. (*) p \u0026lt;0.05, (**) p\u0026lt; 0.01 and (***) p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6779348/v1/0e0b53580515055bf96f71f6.png"},{"id":83940438,"identity":"0ba6cf02-2e00-4360-9338-b3baea0b1a68","added_by":"auto","created_at":"2025-06-04 18:07:14","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":250371,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of AGS cells exposed to \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C lysates. AGS cells were stimulated with different concentrations of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C expressed in protein concentration lysate (10, 50, 100, 200, 800, and 1000 µg/mL) for 24, 48, and 72 h. The error bars indicate the S.E.M. Experiment in triplicate. (*) p \u0026lt;0.05 and (**) p\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6779348/v1/5dbb5117737e35e26184ef4b.png"},{"id":84422200,"identity":"00a87a88-6591-4a0d-a5c2-69bdc573e641","added_by":"auto","created_at":"2025-06-11 18:31:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3270451,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6779348/v1/cc1aa01f-26ee-4e4d-a1d8-be944e320e38.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e UCO-979C exerts oncobiotic activity on human cancer cell lines\u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eHumans coexist with various microorganisms, including fungi, viruses, and bacteria, in different parts of their bodies, such as the skin, mouth, female genitourinary system, and gastrointestinal tract. This collective population is the normal human microbiota (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Probiotics, defined as live microorganisms that confer health benefits to the host, have been studied for their potential to regulate the microbiota (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGastrointestinal tract cancers account for 26% of all neoplasms and are responsible for 35% of cancer-related deaths worldwide (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Strategies to prevent gastrointestinal cancers include polyp surveillance and chemoprevention, which involves using nutritional or pharmacological interventions to prevent, halt, or reverse the growth of neoplastic cells (\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Probiotics have emerged as potential chemopreventive agents, exerting anticancer effects through multiple mechanisms (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These mechanisms encompass the binding and degradation of potentially carcinogenic substances, production of anti-tumorigenic compounds, enhancement of immune responses, suppression of microbiota growth associated with mutagenesis and carcinogenesis, and protection of cellular DNA against oxidative damage (\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProbiotics have demonstrated the ability to prevent and inhibit cancer progression while regulating cellular growth mechanisms (\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Moreover, different probiotic treatments have exhibited antiproliferative and cytotoxic effects on gastrointestinal cancer cells (\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e), including probiotic secreted factors or cell-free supernatants (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Proposed mechanisms underlying the impact of probiotics on cancer and tumor processes include the maintenance of cell-cell junction integrity (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), antiproliferative and cytotoxic effects on tumor cells (\u003cspan additionalcitationids=\"CR27 CR28 CR29\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), inhibition of epithelial-mesenchymal transition and metastasis (\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), and modification of the tumor microenvironment to inhibit tumor growth (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Probiotics that display anticancer activity are now called \"oncobiotics\" and are defined as live microbes or their derived metabolites that exhibit ameliorative actions against cancer (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e UCO-979C was isolated from a gastric biopsy obtained during upper digestive endoscopy. This strain possesses several probiotic characteristics, such as acid and bile tolerance and antibiotic susceptibility (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The genome of strain UCO-979C has been sequenced, revealing the presence of genes associated with the binding of collagen and fibronectin proteins (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C modulates the inflammatory response \u003cem\u003ein vitro\u003c/em\u003e (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Importantly, \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C has also shown inhibitory activity against \u003cem\u003eHelicobacter pylori\u003c/em\u003e (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e); this activity has also been observed in the consumption of gelatin and ice cream supplemented (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). To further characterize the properties of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, now as an oncobiotic, this study aimed to investigate the cytotoxic effects of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C and its lysate on various human cancer cells.\u003c/p\u003e"},{"header":"2. METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Bacteria strain and culture conditions.\u003c/h2\u003e \u003cp\u003e \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C was a bacterial strain obtained from a human gastric biopsy and was maintained from the Bacterial Pathogenicity Laboratory repository at the Microbiology Department, University of Concepcion, Chile. A human gastric \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C was cultured on Mann-Rogosa Sharpe broth (M.R.S.; BD Difco, Le Pont de Claix, France) in a microaerobic atmosphere at 37\u0026deg;C 24 h, followed by culturing on M.R.S. agar (BD Difco) under similar conditions for 24\u0026ndash;48 h. For each assay, bacteria were obtained from the agar cultures, resuspended in liquid culture media, and their concentration adjusted to that required for each assay as described below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cells lines and growth conditions.\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Normal Human Cells.\u003c/h2\u003e \u003cp\u003eThis study used two types of normal human cells: Human Umbilical Cord Vein Endothelial Cells (HUVEC) and Human Dermal Fibroblast cells. HUVEC cells were obtained through the Jaffe Method (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Under sterile conditions, the cord vein was cannulated and washed with 10 mM PBS. Subsequently, the vein was incubated for 10 minutes at 37\u0026deg;C with type II collagenase (Gibco, Life Technologies) at a concentration of 0.33 mg/mL for enzymatic digestion. The resulting cell-containing medium was collected in a sterile tube and centrifuged at 450 g for 10 minutes. The supernatant was discarded, and the pellet containing the HUVEC cells was resuspended in 5 mL of M-199 culture medium (Corning) supplemented with 20% fetal bovine serum (FBS) (Corning), 3.2 mM L-glutamine (Corning), penicillin/streptomycin (Biological Industries), and endothelial growth factor (Endothelial cell growth supplement, Merck). The cells were then cultured in 100 mm plates coated with 1% w/v gelatin and incubated in an atmosphere of 5% CO2 at 37\u0026deg;C until reaching 90% confluence. Human dermal fibroblasts HDF (106-05A, Cell Applications, Inc., USA) were cultured in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries). The cells were maintained at 37\u0026deg;C in a humidified atmosphere of 5% CO2 and 95% air.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 Tumoral human cell lines.\u003c/h2\u003e \u003cp\u003eHuman gastrointestinal tumoral cell lines, namely AGS (human stomach carcinoma cells, ECACC 89090402), HCT-116 (human colorectal carcinoma cells, ATCC CCL-247), and HT-29 (human colorectal carcinoma cells, ECACC 91072201), were cultured and expanded in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM, Gibco) supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries), at 37\u0026deg;C in a 5% CO2 and 95% air atmosphere. Other tumoral cell lines used in this study included SK-MEL (human melanoma cells, ATCC HTB-67) and MCF-7 (human breast adenocarcinoma cells, ECACC 86012803). HaCaT (immortalized human keratinocytes) were kindly donated by Dr. Patricio Oyarz\u0026uacute;n at Universidad San Sebasti\u0026aacute;n, Concepci\u0026oacute;n, Chile. MCF-7 and HaCaT cells were cultured in DMEM (Gibco), while SK-MEL was cultured in Roswell Park Memorial Institute (RPMI 1640, Gibco). All culture media were supplemented with 10% Fetal Bovine Serum (Biological Industries) and 1% penicillin-streptomycin antibiotic mixture (Biological Industries), and the cells were cultured at 37\u0026deg;C in a 5% CO2 and 95% air atmosphere.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Cell viability assay.\u003c/h2\u003e \u003cp\u003eThe antiproliferative effects of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C on cells were assessed using the Sulforhodamine B (S.R.B.) method (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Initially, the cells were seeded in 96-well microplates at a density of 2 \u0026times; 104 cells per well. \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C was adjusted to different McFarland (MF) standards (0.5, 1, 2, 4, and 6 MF) in DMEM culture medium or RPMI-1640, according to the culture medium used for each cell line, without antibiotics, and then added to the cells for 4 hours to promote bacterial adherence. After three washes with sterile 10 mM PBS, the viability assay was conducted at 24, 48, and 72 hours using the S.R.B. method. To perform the S.R.B. method, the cells were fixed with 10% (w/v) trichloroacetic acid (T.C.A.) from Winkler, Chile, and stained with 0.057% (w/v) Sulforhodamine B from Sigma-Aldrich in 1% (v/v) acetic acid from Winkler, Chile. The bound dye was dissolved in a 10 mM Tris base from Sigma-Aldrich, and the absorbance was measured at 510 nm using a multiplate reader (Synergy 2, BioTek Instruments). The percent cellular survival was calculated based on the absorbance values obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Bacterial lysate extraction.\u003c/h2\u003e \u003cp\u003e \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C was preserved in M.R.S. liquid media. An inoculum was cultured in 1000 mL of culture medium for 24 h at 37\u0026deg;C. The bacterial suspension was centrifuged for 8 min at 6000 x g and washed thrice with 10 mM PBS, pH 7.4. The pellet obtained was resuspended in 35 mL of sterile 10 mM PBS to get a bacterial lysate. The bacterial suspensions were lysate by a mechanical rupture in French Press with an approximate flow of 0.75 mL/minute at a pressure of 1000 psi. Subsequently, the bacterial lysate containing proteins and other soluble bacterial products was centrifuged at 12800 x g for 15 min at 4\u0026deg;C and filtered using 0.22 \u0026micro;m pore size (Millipore) (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). The supernatant was kept at -80\u0026deg;C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Bacterial lysate effect on tumoral cells.\u003c/h2\u003e \u003cp\u003eAGS cells were seeded in 96-well plates at a 20.000 cells/well concentration for 24 hours in a DMEM culture medium. Then, the cells were washed twice with sterile 10 mM PBS and exposed for 24, 48, and 72 hours at concentrations of protein lysate of 10, 50, 100, 200, 800, and 1000 \u0026micro;g/mL in medium cell culture total bacterial lysate. Finally, AGS cells were seeded in conditions similar to those before. Viable cells were determined by the S.R.B. method as described previously.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical analysis.\u003c/h2\u003e \u003cp\u003eGraphs and statistical data analysis were performed using GraphPad Prism 8 for Mac. Results are represented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Student t-tests analyzed statistical differences. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effect of L. fermentum UCO-979C on normal cells.\u003c/h2\u003e \u003cp\u003eWe investigated the potential cytotoxicity of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C on normal non-tumor cells, specifically HUVEC and HDF cells. The assay involved exposing the cells to different concentrations of the bacteria (ranging from 0.5 to 6 MF) for 24, 48, and 72 hours. Remarkably, no significant effects on cell viability were observed across different time points or concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results are consistent with previous findings that demonstrate probiotics generally do not induce cytotoxicity in normal human cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Effect of L. fermentum UCO-979C on human gastrointestinal tumor cell lines.\u003c/h2\u003e \u003cp\u003eConsidering the absence of cytotoxic activity of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C in normal cells, we evaluated its potential cytotoxicity in human gastrointestinal tumor cell lines. Since this bacterium originates from the gastric niche, we specifically examined its effects on AGS, HCT-116, and HT-29 cells. These cells were exposed to different concentrations of the resuspended bacteria and cultured without antibiotics for 24, 48, and 72 hours.\u003c/p\u003e \u003cp\u003eInterestingly, when AGS cells were incubated with \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, a time- and concentration-dependent decrease in cell viability was observed. The cytotoxic effect was evident from 24 hours of incubation, with a reduction in cell viability observed even at the lowest concentration. This effect persisted from 1 MF, resulting in approximately 50% cell viability regardless of concentration. The same trend was observed at 48 and 72 hours of exposure, with cell viability dropping below 50% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eSimilarly, HCT-116 cells displayed decreased cell viability with increasing concentrations of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, up to 2 MF, at all time points. Unexpectedly, at 72 hours of exposure, a significant cytotoxic effect was observed at the highest concentrations, resulting in less than 10% cell viability (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). In the case of HT-29 intestinal cells, no cytotoxic effect of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C was observed at the lowest concentrations after 24 hours. However, a marked cytotoxic effect was observed at higher concentrations, with cell viability dropping below 40%. Surprisingly, irrespective of bacterial concentration, a substantial reduction in cell viability was observed at 48 and 72 hours, with values below 25% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results demonstrate the pronounced cytotoxic effect of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C on the gastric and intestinal cell lines studied.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effect of L. fermentum UCO-979C on other tumour cell lines and immortalized keratinocytes.\u003c/h2\u003e \u003cp\u003eTo determine if the cytotoxic effect of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C on gastrointestinal cancer cells extends to other cell types, we investigated its impact on immortalized keratinocytes (HaCaT cells), melanocytes (SK-MEL cells), and breast cancer cells (MCF-7).\u003c/p\u003e \u003cp\u003eA notable cytotoxic effect was observed despite HaCaT cells not being tumor cells. However, the cell viability remained relatively stable at around 75% across all concentrations and study durations, except at higher concentrations where \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C demonstrated a cytotoxic effect of approximately 50% at 72 hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eInterestingly, a cytotoxic effect was consistently observed in SK-MEL melanocytes, regardless of the bacterial concentration and exposure time spanning 24 to 72 hours. The cell viability ranged between 60% and 70% under these conditions, indicating that the impact of the bacterium on these cells is independent of concentration and time (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eIn the case of MCF-7 breast tumor cells, a slight decrease in cell viability was evident after 24 hours of exposure to \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C. However, at 48 hours of analysis, a significant cytotoxic effect is observed, regardless of the bacterial concentration, resulting in a decrease in viability from 75\u0026ndash;70%. As anticipated, this effect was sustained at 72 hours, with a 50% reduction in viability at the highest bacterial concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eTherefore, \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C demonstrated a cytotoxic effect in immortalized keratinocytes (HaCaT) and various non-gastrointestinal tract tumour cells, highlighting its importance in inducing cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Effect of L. fermentum UCO-979C lysate on AGS cells.\u003c/h2\u003e \u003cp\u003eTo investigate whether the cytotoxic effect is solely attributed to living bacteria, we examined whether the intracellular content of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, obtained from its lysate, could also induce this effect. In this experiment, AGS cells were exposed to different concentrations of the bacterial lysate, determined by protein concentration, for 24, 48, and 72 hours.\u003c/p\u003e \u003cp\u003eAfter 24 hours of incubation with the bacterial lysate, a slight cytotoxic effect on AGS cell viability was observed. This effect became more pronounced as the lysate concentration in the incubation medium increased. At a 1000 \u0026micro;g/mL concentration, the cell viability decreased to 70%. Surprisingly, after 48 hours of cell culture, the cytotoxic effect intensified at concentrations above 200 \u0026micro;g/mL, significantly reducing cell viability to 30% at the highest concentration. The same pattern was observed at 72 hours of exposure, with the cytotoxic effect becoming evident at a concentration of 100 \u0026micro;g/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Remarkably, the intracellular content obtained from the \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C lysate also exhibited a dose-dependent decrease in cell viability after 48 hours of exposure.\u003c/p\u003e \u003cp\u003eThese findings suggest that both live bacteria and their intracellular content, represented by the lysate, exert a cytotoxic effect on AGS cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eThis study aimed to assess the cytotoxic potential of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C and its bacterial lysate on tumor cells as an initial exploration of their oncolytic properties. Previous research has reported selective cytotoxic behavior of \u003cem\u003eLactobacillus plantarum\u003c/em\u003e 5BL on various tumor cell lines, including AGS (gastric), HeLa (cervical), HT-29 (intestinal), and MCF7 (breast) while showing no cytotoxic effect on HUVEC cells, which served as the normal cell control (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOther studies have demonstrated the antiproliferative effects of Lactobacillus strains on different cancer cell lines. For instance, \u003cem\u003eL. acidophilus\u003c/em\u003e DSM9126 and \u003cem\u003eL. lactis\u003c/em\u003e KX881782 have been shown to exhibit antiproliferative effects on intestinal tumoral CACO-2 cells, MCF7, and HeLa cells (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Additionally, \u003cem\u003eL. plantarum\u003c/em\u003e A7 and \u003cem\u003eL. rhamnosus\u003c/em\u003e G.G. had a cytotoxic effect on CACO-2 cells, reducing cell viability to 60% and 80% after 48 hours of exposure. Similar results were observed in HT-29 cells (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Moreover, \u003cem\u003eL. rhamnosus\u003c/em\u003e GG at a concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL exhibited cytotoxic effects on CACO-2 cells after 48 hours of exposure, reducing viability to 60%. In contrast, no such effect was observed in HT-29 cells under the same conditions (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCytotoxic effects of probiotics have also been observed in colon carcinoma cells. In HT-29 cells, strains of \u003cem\u003eL. delbreucki\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, \u003cem\u003eL. plantarum\u003c/em\u003e, and \u003cem\u003eL. brevis\u003c/em\u003e exhibited cytotoxic effects after 16 hours of exposure (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). In HGC-27 cells, cytotoxic effects were observed for \u003cem\u003eL. rhamnosus\u003c/em\u003e L.G.G. and \u003cem\u003eL. paracasei\u003c/em\u003e MPC2.1 at concentrations of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL after 24 and 48 hours, suggesting \u003cem\u003eL. paracasei\u003c/em\u003e IMPC2.1 as a potential probiotic for cancer prevention (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). A 20% inhibition of cell proliferation in HT-29 cells has been suggested as a significant antiproliferative effect (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C did not affect the viability of HUVEC cells (derived from the human umbilical cord) and HDF cells (non-tumor cells). However, cell viability was decreased in tumor cell lines when exposed to \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, including AGS, SK-MEL, and HaCaT cells, which are not of gastrointestinal origin. A particular finding was the observed sensitivity in HaCaT cells, which are immortalized human keratinocytes but not oncogenically transformed (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e). While the probiotic \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C showed clear selectivity against cancer cells when compared to the primary non-tumour cells HUVEC and HDF, the cytotoxic effect on HaCaT cells warrants special consideration. This differential susceptibility may be attributed to HaCaT cells exhibiting considerably higher proliferation rates than primary cells and potentially harbouring certain alterations in cell signalling pathways (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). These features, such as an increased dependence on growth factors or changes in the expression of surface receptors, might render them more susceptible to the effects of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C or its metabolites, compared to the primary non-tumor cells that showed resistance. Future studies could explore these differences to better understand the determinants of cellular sensitivity to this probiotic.\u003c/p\u003e \u003cp\u003eThese selectivity findings are consistent with studies on a \u003cem\u003eLactobacillus fermentum\u003c/em\u003e strain. For example, extracts from \u003cem\u003eLactobacillus fermentum\u003c/em\u003e NCIMB 5221 were shown to significantly inhibit the growth of colorectal cancer cells SW-480 and Caco-2, and induce apoptosis, while not affecting non-neoplastic colon cells CRL-1831. This selectivity was linked to a higher production of short-chain fatty acids (SCFAs) by the bacterium (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e). The observation that \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C, isolated from the stomach, predominantly interacts with gastrointestinal epithelial cells, might contribute to its pronounced effects on gastrointestinal cancer lines. The mechanism of cell death induced by \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C appears to be associated with apoptosis, a phenomenon also observed with other probiotic strains, including the \u003cem\u003eL. fermentum\u003c/em\u003e NCIMB 5221.\u003c/p\u003e \u003cp\u003eIn the search for cytotoxic products secreted by bacteria, it has been found that metabolites secreted by \u003cem\u003eL. lactis\u003c/em\u003e subspecies lactis 44 at a concentration of 40 \u0026micro;g/mL, after 48 hours of stimulation, exhibited a cytotoxic effect on AGS cells as well as on HT-29, MCF7, and HeLa tumor cells (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In HT-29 cells, intracellular extracts of \u003cem\u003eL. casei\u003c/em\u003e 01 demonstrated a cytotoxic effect, resulting in 89% viability reduction (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e). Moreover, culture supernatants devoid of bacteria (at a concentration of 60 \u0026micro;g/mL) from \u003cem\u003eL. casei\u003c/em\u003e, \u003cem\u003eL. paracasei\u003c/em\u003e, \u003cem\u003eL. rhamnosus\u003c/em\u003e, and \u003cem\u003eL. plantarum\u003c/em\u003e strains showed cytotoxic effects, with an average viability reduction of 60% in CACO-2 and HT-29 cells after 48 hours of exposure (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e). Additionally, cytoplasmic fractions of \u003cem\u003eL. rhamnosus\u003c/em\u003e G.G., obtained through sonication, exhibited a cytotoxic effect with a 60% reduction in viability in gastric cancer cells (HGC-27) at a concentration of 1:1 v/v, corresponding to a bacterial concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). When AGS cells were exposed to the lysate of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C containing soluble intracellular components of the bacteria, cytotoxic effects were also observed at 24, 48, and 72 hours of culture.\u003c/p\u003e"},{"header":"5. CONCLUSION","content":"\u003cp\u003eIn conclusion, the findings of this study provide evidence for the oncobiotic activity of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C. It demonstrates its ability to selectively induce cytotoxic effects in cancer cell lines, particularly those derived from the gastrointestinal tract, highlighting its potential antitumor properties. Notably, \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C does not exhibit cytotoxicity in normal non-tumor cells, indicating its selective cytotoxic effect on cancer cells. These results further underscore its potential as a promising oncobiotic agent. It is important to note, however, that these initial findings are from \u003cem\u003ein vitro\u003c/em\u003e assays, and the precise molecular mechanisms and active components responsible for the observed cytotoxicity require further elucidation. Furthermore, the cytotoxic effects observed in this study were not limited to live bacteria alone but were also evident in the bacterial lysate. This suggests the presence of intracellular components or metabolites in \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C that contribute to its cytotoxic potential.\u003c/p\u003e \u003cp\u003eFuture investigations should focus on identifying and characterizing the molecules or groups responsible for the observed cytotoxic effects in the bacterial lysate. The precise mechanisms responsible for the antitumor effects of \u003cem\u003eL. fermentum\u003c/em\u003eUCO-979C have yet to be fully understood. Thus, it is crucial to investigate the signalling pathways and cellular processes contributing to the cytotoxicity induced by \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C. Such exploration will enhance our comprehension of its underlying mechanism of action. Furthermore, conducting \u003cem\u003ein vivo\u003c/em\u003e models to evaluate the impact of \u003cem\u003eL. fermentum\u003c/em\u003eUCO-979C on tumor growth and studying its interactions with the host immune system would yield valuable insights into its potential as a therapeutic agent, further solidifying its characterization as an oncobiotic.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDisclosure statement\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAuthor Contributions\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePA-Z:\u003c/strong\u003e Conceptualization, Methodology, Software. \u003cstrong\u003ePA-Z, FZ\u003c/strong\u003e: Data curation, Writing- Original draft preparation. \u003cstrong\u003ePA-Z., CP-S, BA-Z:\u003c/strong\u003e Visualization, Investigation. \u003cstrong\u003eFZ, AG-C, VO:\u003c/strong\u003e Supervision.: \u003cstrong\u003ePA-Z, CP-S, BA-Z: Software\u003c/strong\u003e, Validation.: \u003cstrong\u003eFZ, EN-L, AG-C, VO:\u003c/strong\u003e Writing- Reviewing and Editing.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Vicerrector\u0026iacute;a de Investigaci\u0026oacute;n y Desarrollo (VRID) of the Universidad de Concepci\u0026oacute;n under Grant VRID No 2023000895INT.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBiedermann, L., and Rogler, G. 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Eur J Cancer Prev. 2020;29:201-209. https://doi.org/10.1097/CEJ.0000000000000393\u003c/li\u003e\n\u003cli\u003eRusso, F., and Orlando, A., and Linsalata, M., and Cavallini, A., and Messa, C. Effects of Lactobacillus rhamnosus GG on the cell growth and polyamine metabolism in HGC-27 human gastric cancer cells. Nutr Cancer. 2007;59:106-114. https://doi.org/10.1080/01635580701365084\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Cancer cells, Cytotoxicity, Probiotics, UCO-979C, Oncobiotic","lastPublishedDoi":"10.21203/rs.3.rs-6779348/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6779348/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProbiotics are live microorganisms that, when administered appropriately, can confer beneficial effects on the host. \u003cem\u003eLimosilactobacillus fermentum\u003c/em\u003e UCO-979C, a probiotic strain isolated from the human stomach, has demonstrated the ability to inhibit \u003cem\u003eHelicobacte\u003c/em\u003er \u003cem\u003epylori\u003c/em\u003e, a pathogen associated with gastric cancer. Recent studies have introduced the concept of \"oncobiotics,\" referring to probiotics with anticancer properties. In this study, we aimed to investigate the cytotoxic effects \u003cem\u003eof L. fermentum\u003c/em\u003e UCO-979C on human cancer cells. Our results revealed that the \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C strain and its lysate exhibited significant cytotoxic activity against multiple tumor cell lines while demonstrating no cytotoxic effect on non-cancerous cells. Notably, gastrointestinal cancer cells displayed heightened sensitivity to the probiotic strain, showing a more significant decrease in cell viability compared to other tumor cells. These findings suggest that \u003cem\u003eL. fermentum\u003c/em\u003eUCO-979C acts as an oncobiotic, specifically targeting gastrointestinal cancer cells. Understanding the cytotoxic mechanisms of \u003cem\u003eL. fermentum\u003c/em\u003e UCO-979C on cancer cells holds promise for developing novel therapeutic strategies for gastrointestinal cancers. Further research is warranted to elucidate the underlying molecular pathways and evaluate the potential clinical applications of this probiotic strain in cancer treatment.\u003c/p\u003e","manuscriptTitle":"Limosilactobacillus fermentum UCO-979C exerts oncobiotic activity on human cancer cell lines","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-04 17:59:09","doi":"10.21203/rs.3.rs-6779348/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"88fa09b7-593a-4d1f-984b-2b173816caee","owner":[],"postedDate":"June 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-06-11T18:23:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-04 17:59:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6779348","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6779348","identity":"rs-6779348","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}