alexa In-Vitro Characterization of the Anti-Cancer Activity of the Probiotic
ISSN: 1948-5956
Journal of Cancer Science & Therapy
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In-Vitro Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus Fermentum NCIMB 5221 and Potential against Colorectal Cancer

Imen Kahouli1,2, Meenakshi Malhotra1, Moulay Alaoui-Jamali3,4 and Satya Prakash1,2*
1Biomedical Technology and Cell Therapy Research Laboratory-Departments of Biomedical Engineering, Physiology, and Artificial Cells and Organs Research Center. Faculty of Medicine, McGill University, 3775 University Street, Montreal, Quebec, H3A 2B4, Canada
2Department of Experimental Medicine, Faculty of Medicine, McGill University, 1110 Pine Avenue West, Montreal, Quebec, H3A 1A3, Canada
3Departments of Medicine and Oncology, Faculty of Medicine, McGill University, Gerald Bronfman Centre, Room 210, 546 Pine Avenue West, Montreal, Quebec, H2W 1S6, Canada
4Lady Davis Institute for Medical Research and Segal Cancer Centre, Sir Mortimer B. Davis-Jewish General Hospital, 3755 Côte Ste-Catherine Road, Montreal, Quebec, H3T 1E2, Canada
Corresponding Author : Satya Prakash
Professor, Biomedical Engineering
Artificial Cells and Organs Research Center Member
Physiology and Experimental Medicine, Faculty of Medicine
McGill University, Montreal, Quebec, H3A 2B4, Canada
Tel: 514 398 3676
Fax: 514 398 7461
E-mail: [email protected]
Received: May 25, 2015; Accepted: July 15, 2015; Published: July 17, 2015
Citation: Kahouli I, Malhotra M, Alaoui-Jamali M, Prakash S (2015) 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 7:224-235. doi:10.4172/1948-5956.1000354
Copyright: © 2015 Kahouli I, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Objective: Lactic acid bacteria such as Lactobacillus fermentum, have shown to increase the levels of fecal short chain fatty acids known with its beneficial role in colonic health and were found to produce anti-carcinogenic compounds, suggesting a potential in colorectal cancer prevention. The aim of this study is to characterize the metabolic and anti-cancer features of L. fermentum NCIMB 5221 compared to two other Lactobacillus species.

Methods: A free fatty acid (FFA) profile was determined and the anti-proliferative and apoptotic effects of bacterial cell free extracts were investigated. The effect on the growth of colon cancer cells compared to nonneoplastic colon cells was determined. The production of different SCFAs by the probiotic bacteria and the efficacy of their composition were analyzed.

Results: The FFA profile of L. fermentum is distinctive FFA profile (~ 368 MAE, 16 h, p < 0.01) compared to L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103. L. fermentum extracts significantly inhibited cancer cell growth up to ~ 40% and induced apoptosis up to ~ 30% in SW-480 colon cancer cells (24 h, p < 0.05) compared to the untreated cells. However, L. fermentum did not inhibit CRL-1831 non-neoplastic colon cell growth but had a significant anti-proliferative effect against Caco-2 cancer cells (~ 60%, 72 h, p < 0.001) compared to control, which was related to the higher levels of SCFAs produced (~ 377 mg/L). Similar concentrations of SCFA formulations (that correspond to what was produced by L. fermentum) have shown the same inhibitory effect on Caco-2 cells.

Conclusion: L. fermentum NCIMB 5221 was more potent in suppressing colon cancer cells and promoting normal epithelial colon cell growth by the production of SCFAs and could be considered as biotherapeutic agent for the support of colonic health and the prevention of colorectal cancer.

L. fermentum; NCIMB 5221; Colorectal cancer; Proliferation; Short chain fatty acids; Apoptosis
Colorectal cancer (CRC) is a leading cause of mortality worldwide [1]. It is, however, a type of cancer for which chemoprevention is considered a therapeutic and preventive strategy [2]. Probiotics has been used as biotherapeutics that reduce cancer recurrence and side effects in CRC patients [3-5]. When orally administered, probiotic along with gut microbial-produced metabolites such as organic acids, peptides, etc… that interacts with cellular proliferation, differentiation, apoptosis, inflammation and reduce CRC risk [6]. Short chain fatty acids (SCFAs) produced by bacterial fermentation in the gut have been shown to exert anti-inflammatory [7] and anti-tumorigenic effects. Studies have shown that fatty acids can mutually interact and protect against colon cancer. Nevertheless, the incorporation of fatty acids into CRC chemotherapies is still premature and the oral administration of certain probiotic bacteria remains the prominent way to increase the bio-production of these anti-tumorigenic compounds in the colon [8,9].
While the selection criteria of probiotic bacteria originating from the gut or from traditionally fermented products are fairly empirical, there is an emphasis in the importance of well-established in vitro and in-vivo studies to select good candidates. Few studies have been found to establish a rigorous selection for new probiotic strains with anti-cancer attributes with proper controls and extensive evaluation of their anti-proliferative effect against cancer cells as compared to other established probiotic products. In addition, several studies have shown the ability of certain probiotics to affect SCFA levels, but it was infrequently proven that the probiotic anti-cancer effect is solely due to the direct production of SCFAs [10-12]. In this study, for L. fermentum NCIMB 5221, identified as a ferulic acid-producer, an anti-oxidant and an anti-tumorigenic compound [13], the ability to generate a stronger free fatty acid (FFA) profile was characterized, which was in comparison with L. acidophilus ATCC 314 and L. rhamnosus ATCC 51303, both characterized in previous studies for their potential anti-tumorigenic effect [14-17]. Notably, the proliferative effect of this bacterial strain was investigated by identifying the effects of different types of probiotic cell free extracts on the growth and apoptosis of colorectal cancer cells. Prior to this, study we conducted a previous preliminary comparative study and found this particular strain exhibited more potent characters associated with anti-cancer effects and survival to colon delivery when compared to other L. fermentum strains. To this end, to verify the noncytotoxic effect of the probiotic extracts, the same assay was performed in this study with non-neoplastic colon cells too. Here, a verification of a correlation between the levels of SCFAs produced and the anti cancer efficacy of the bacterium was performed. The production of SCFAs by L. fermentum NCIMB 5221 was determined in order to identify to which extent SCFAs (acetic acid, propionic acid and butyric acid) are responsible for the potential anti-cancer effect against colon cancer cells in vitro. To confirm the level of efficacy of naturally produced SCFAs, the assay was performed using only a pure mixture of synthetic SCFAs with similar composition to the probiotic ones.
Materials and Methods
Agar and De Man, Rogosa, Sharpe (MRS) broths were purchased from Fisher Scientific (Ottawa, ON, Canada). Dulbecco’s modified Eagle’s medium (DMEM) and Eagle’s Minimum Essential Medium (EMEM), phosphate-buffered saline (PBS), Roswell Park Memorial Institute medium (RPMI-1640), and fetal bovine serum (FBS) were purchased from Invitrogen. Water was purified with two systems: EasyPure reverse osmosis and NanoPure Diamond Life Science (UV/ UF) ultrapure water (Barnstead (Dubuque, IA, USA). Sodium L-Lactic acid, propionic, acetic and butyric was obtained from Sigma (St. Louis, MO, USA).
Bacterial cultures
L. fermentum NCMIB 5221 was purchased from the National Collection of Industrial and Marine Bacteria (NCIMB, Aberdeen, Scotland, UK). Bacterial strains of L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103 were obtained from Cedarlane Laboratories (Burlington, ON, Canada) and used as controls. Bacterial cultures were maintained by continuous subculturing in MRS broth at 1% (v/v) and bacterial growth was monitored with both OD at a wavelength of 620 nm (Perkin Elmer 1420 Multilabel Counter, USA) and by colony counting on agar plates.
Mammalian cultures
SW-480 colorectal cancer and Caco-2 epithelial colorectal cancer adenocarcinoma cells, as well as CRL-1831 (FHC) normal epithelial colon cell line were purchased from ATCC (Manassas, VA), Caco-2 cells were maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 20% FBS. SW-480 cells were maintained in RPMI- 1640 supplemented with 10% FBS and CRL-1831 was maintained in complete DMEM (10% FBS, 37°C, 5% CO2). Caco-2 cells were incubated in a CO2 incubator at 37°C in air supplemented with 5% CO2 for about 1-2 weeks for complete differentiation. For proliferation/ viability assays, all cells were left to attach in 96-well plates until 50-60% confluence (24-48 h) before experimentation. Cell medium was then replaced by probiotic cell free extracts mixed with serum/antibioticfree DMEM.
Free fatty acid analysis
In this analysis, the free fatty acids (FFAs) in the bacterial supernatant were converted to their CoA derivatives and then oxidized. This led to the formation of a color measured at 570 nm (Figure 1). The assay was performed based on the manufacturer’s instructions (Cell Biolabs Inc, CA, USA). For the induction of Acyl-CoA synthesis, 2 μl of ACS Reagent was added to all the standards (palmetic acid) and sample wells and all was mixed and left to incubate (37°C, 30 min). The reaction mix (50 μl), fatty acid probe (2 μl), enzyme mix (2 μl), assay buffer (Igepal, 44 μl), and enhancer (N-ethylmaleimide, 2 μl) were mixed and vortexed briefly. Following which 50 μl of the reaction mix was added to each well (standard/sample) and incubated (30 min, 37°C, away from light). Absorbance was measured at 570 nm for colorimetric assay in a micro-plate reader (Perkin Elmer, Victor 3, multi-label micro-plate reader, Massachusetts, USA).
Probiotic cell free supernatants
Each probiotic bacteria was grown anaerobically in MRS broth for 12-16 h. The conditioned medium (CM) and probiotic supernatant (PS) were prepared with slight modifications of protocols adapted from Grabig et al. [18] and Kim et al. [19]. For the CM, bacterial cultures (16 h, 37°C, 5% CO2) were used to collect bacterial pellets by centrifugation (4000 rpm, 15-20 min, 4°C), washed twice with PBS and re-suspended in DMEM. The bacterial pellets were kept on shaker incubator for 2 h (37°C, 5% CO2, 100 rpm). After incubation, the culture medium was centrifuged (1000 x g, 15 min) and sterile-filtered (0.2 μM-pore-size filter). Prior to the treatment on cells, the CM of each Lactobacillus bacteria was then mixed with DMEM at a ratio of 1:2. For the preparation of the PS, the bacterial pellet was removed by centrifugation (4000 rpm, 15 min, 4°C), then the recovered supernatant was a sterile filtered (0.22 μm) and stored at −80°C, until used.
Cell viability assay
Cell viability was determined using ATP bioluminescence assay (CellTi- ter-Glo Luminescent Cell Viability Assay; Promega), following the manufacturer’s protocol [20]. Colon cells (normal or cancer) were seeded onto 96-well culture plates (5-6×103 cells/well, 100 μl/ well) and stabilized for 24 - 48 h (37°C, 5% CO2) for cell attachment. After incubating the cells with the probiotic treatments (24, 48 and 72 h), the 96-well plate was left at room temperature (RT, 30 min) and a 100 μl of luminescent reagent was added to each well, followed by shaking (2 min, 200 rpm) and incubation at RT (10 min) to stabilize the luminescent signal. The signal was recorded using a spectrophotometer (Perkin Elmer, Victor 3, multi-label microplate reader, Massachusetts, USA).
Apoptosis assay
Apoptosis was determined by the assessment of caspase -3 and -7 using Caspase-Glo® 3/7 assay (Promega, USA). First, the buffer and the lyophilized substrate were equilibrated to RT before use. Both were mixed to fully dissolve the substrate. The blank reaction (cell culture medium without cells), negative control (untreated cells in medium) and the assays (treated cells in medium) were all reactions prepared to detect caspase-3 and -7 activity in cell cultures in 96-well plates compatible with the luminometer used (white opaque plates). After incubation with the treatment, the plate was removed from the incubator and allowed to equilibrate at RT. 100 μl of the luminescent reagent was added to each well of a white-walled 96-well plate containing 100 μl of blank, negative control or treated cells in culture medium. The plate was gently mixed using a plate shaker (300–500 rpm, 30 s), followed by incubation at RT for 3 h. Finally, the luminescence of each sample was measured in a plate-reading luminometer following manufactures’ instructions.
Probiotic effect on cancer colon cells vs. non-neoplastic colon cells
This assay was perfomed to show that L. fermentum NCIMB 5221 have an anti-colon cancer effect by inhibiting colon cancer cell proliferation without affecting non-neoplastic colon cells. Caco-2 and CRL-1831 (4–5×103 cells/ well) were seeded into 96-well culture plates (37°C, 5% CO2) for 1, 2, 3 and 7 days. The cells in both populations were treated with probiotic CM. At each time point (1, 2, 3 and 7 days) of incubation with probiotic treatments, cell proliferation was determined using an ATP bioluminescence assay.
Quantification of lactic acid and SCFAs
SCFAs produced by L. reuteri strains were measured during the growth of bacteria in SIF and after the preparation of corresponding CM. SCFAs were separated using a slightly modified HPLC method [21,22]. A Model 1050 UV HPLC system (Hewlett-Packard HP1050 series, Agilent Technologies, USA), equipped with a UV-vis detector and diode array detector (DAD) set at 210 ± 5 nm, was used. Briefly, a 100 μl of sample was injected through an autosampler. A prepacked Rezex ROA-organic acid H+ (8%) (150 × 7.80 mm, Phenomenex, Torrance, CA, USA) fitted with an ion-exclusion microguard refill cartridge was used. Data were acquired using ChemStation supported with LC3D software Rev A.03.02 (Agilent Technologies, CO, USA). The mobile phase (A) of H2SO4 (0.05 M) and the mobile phase (B) of acetonitrile (2%) were used with an isocratic gradient pumped at a flow rate of 0.8-0.7 mL/min, through a column heated to 35 °C. For the standard curves, lactic, acetic, propionic, and butyric acids were used to prepare a standard solution at different concentrations of 1, 10, 100, 500, 1000 ppm (in triplicate). The concentrations of SCFAs were estimated using the linear regression equations (R2 ≥ 0.99) generated from respective standard curves.
Role and efficacy of SCFAs: SCFA synthetic formulations vs. probiotic CM
This test was performed to demonstrate the role and the relevance of naturally produced SCFAs by probiotic cells. SCFA synthetic formulations (SSF) at the same composition of the SCFAs produced in the probiotic CM were prepared (Table 1). The growth of Caco-2 cells (4-5 × 103 cells/well), seeded onto 96-well plates (37°C, 5% CO2, 72 h) were used to determine the inhibitory effects of these compounds and were compared the probiotic CM. If SCFA synthetic mixtures had the same effect as the bacterial extract, this would suggest that the inhibitory effect against colon cancer cell is due to the concentration of SCFAs produced by L. fermentum. Another set of mixtures (SSF+LA) was used after addition of lactic acid (similar to what was produced by the bacteria) in order to investigate the effect of another soluble component on the action of SCFAs.
Effect of SCFAs on colon cancer cell compared to normal cells
The objective of this part of the study was to investigate the dosedependent effect of SCFAs, pure or in mixture, and the nature of their potential synergistic effect on both normal and cancerous colon cells. Different concentrations of lactic, acetic, propionate and butyrate were prepared (Table 2) and tested on Caco-2 and normal non-neoplastic colon cells CRL-1831 cells. Cells were seeded onto 96-well culture plates (4 - 5 × 103 cells/ well, 37°C, 5% CO2), left to stabilize and attach (24 - 48 h), followed by incubation with treatment samples (37°C, 5% CO2, 72 h). SCFA treatments included increasing concentrations of lactic acid (0, 325, 650, 1300 mg/l), propionic acid (0, 325, 650 and 1300 mg/l), lactic acid (0, 100, 200, 400 mg/l) and butyrate acid (0, 75, 150, 300 mg/l). For each degree of concentration, each of the SCFAs (lactic, acetic, propionate and butyrate) were mixed to prepare SC4 (325 mg/l of lactic acid, 325 mg/l of acetic acid, 100 mg/l of propionic acid and 75 mg/l of butyric acid), SC3 (325 mg/l of lactic acid, 650 mg/l of acetic acid, 200 mg/l of propionic acid, 150 mg/l of butyric acid), SC2 (650 mg/l of lactic acid, 1300 mg/l of acetic acid, 400 mg/l of propionic acid and 300 mg/l of butyric acid), and SC1 (1300 mg/l of lactic acid, 1300 mg/l of acetic acid, 400 mg/l of propionic acid and 300 mg/l of butyric acid).
Statistical analysis
Data are presented as means ± Standard Error of the Mean (SEM) of replicates. Correlations were determined using Pearson correlation. Statistical significance was generated for the treated groups as compared to each other by means of the one-way analysis of variances (ANOVA), with Tukey’s posthoc test using SPSS statistics software package (version 20.0, IBM Corporation, New York, NY, US). p-value of p < 0.05 were considered significant.
L. fermentum NCIMB 5221 produces more FFAs
L. fermentum NCIMB 5221 was characterized in terms of growth and production of FFAs in bacterial cultures. Data sets describing the total FFA concentration (μM PAE) in bacterial supernatant (Figure 2a, b, and c), FFA concentration per viable bacterial cell (Figure 2d) and FFA concentration per gram of bacterial pellet showed that L. fermentum NCIMB 5221 growth significantly increased FFA concentrations (367.8 ± 10.5 μM PAE) compared to L. acidophilus ATCC 314 (117.1 ± 3 μM PAE, p = 0.00096) and L. rhamnosus ATCC 53103 (87.4 ± 0.1 μM PAE, p = 0.00085, Figure 2b). The high FFA concentrations were maintained at 367.8 ± 10.5 μM PAE and 366.7 ± 6.6 μM PAE between 12 and 16 h of growth and then started dropping at the beginning of the stationary phase. Even at the end of the death phase (Figure 2c), L. fermentum NCIMB 5221 induced a significantly higher level of FFAs (320.8 ± 12.6 μM PAE) compared to L. acidophilus ATCC 314 (188.2 ± 6 9 μM PAE, p = 0.0161) and L. rhamnosus ATCC 53103 (281.32 ± 1.76 9 μM PAE, p = 0.0487). Values of FFA per viable bacterial cell, measured during both log phase and stationary phase (Figure 2d), remained significantly higher for L. fermentumNCIMB 5221 (6.7 ± 0.2×10-7 - 13.6 ± 0.2×10-7 μM PAE/cell, p < 0.05, Figure 2) compared to L. acidophilus ATCC 314 (3.3 ± 0.1×10-7 – 4,4 ± 0.2×10-7 μM PAE/cell) and L. rhamnosus ATCC 53103 (3.5 ± 0.1×10-7 – 5.8 ± 0.1×10-7 μM PAE/cell). In addition, in terms of bacterial weight, FFA generated per gram of the bacterial pellet was the highest for L. fermentum NCIMB 5221 at 12 h and 24 h (p < 0.001, Figure 2e).
L. fermentum NCIMB 5221 shows anti-proliferative activity against colon cancer cells in a time-dependent manner
The anti-proliferative and apoptotic effect of L. fermentum NCIMB 5221 against SW-480 colon cancer cells was compared to controls: L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103 using two types of probiotic cell free extracts, PS and CM. Thus, at 12 h, SW-480 cells treated with Lactobacilli PS showed no difference between groups in terms of proliferation and apoptosis (Figure 3a), while for Lactobacilli CM, L. fermentum NCIMB 5221 showed significant inhibition of colon cancer cell proliferation (12.9 ± 1.8%) compared to controls (p = 0.034, Figure 3d). At 24 h, Lactobacilli SP inhibited cancer cell proliferation with no significant difference between treatments (p = 0.0754, Figure 3d). While for Lactobacilli CM, L. fermentum NCIMB 5221 significantly killed colon cancer cells (38.1 ± 1.9% of inhibition (Figure 3e), and 29.8 ± 10,4% of apoptosis (Figure 3e) compared to all treatments (p = 0.0004, p = 0.0471, respectively). Interestingly, at 7 days, the SP of L. fermentum NCIMB 5221 significantly reduced cell growth by 42.6 ± 5.1% (p < 0.05), respectively, compared to control and L. acidophilus ATCC 314 (Figure 3c). Also, the CM of L. fermentum NCIMB 5221 significantly suppressed colon cancer cell growth by 67.7 ± 2% compared to untreated cells (p < 0.001, Figure 3f). For the induction of cell death in SW-480 cancer cells by SP treatments, there was no significant difference between groups (Figure 4a and b). However, for L. fermentum NCIMB 5221, the effect of CM in inducing apoptosis in colon cancer cells at 12 h (23.6 ± 7.5%, p < 0.05, Figure 4c) and 24 h (29.9 ± 10.4%, p < 0.05 Figure 4d) was shown to be significantly higher than controls. This suggested that cancer cells might not be affected by bacteria-cell contact but by soluble bacterial factors and Microbial- Associated Molecular Patterns (MAMPs).
L. fermentum NCIMB 5221 inhibits colon cancer cells but not normal cells
To elucidate the behavior through which bacterial symbionts affect cell growth in the epithelium in a tumor environment, the effect of the bacterial cell free cell extracts, CM, prepared from probiotic cells of L. acidophilus ATCC 314, L. fermentumNCIMB 5221 and L. rhamnosus ATCC 53103, was evaluated on the growth of both cancer (Caco-2) and non-cancerous (CRL-1831) colon cells (Figure 5). Results show that at 24 h of incubation with CM of L. fermentum NCIMB 5221 and L. rhamnosus ATCC 53103, cancer cell growth was inhibited by 28.6 ± 3.7% and 6.3 ± 1% (p < 0.01, Figure 5b and c), respectively, compared to untreated cells. At 48 h of incubation, cancer cell viability was reduced by 42.2 ± 2.2% and 11.4 ± 1.7% (p < 0.01), respectively, compared to untreated cells. At 72 h of incubation, L. acidophilus ATCC 314, L. fermentum NCIMB 5221 and L. rhamnosus ATCC 53103, inhibited cancer cell proliferation by 12.6 ± 1.9%, 59.4 ± 4.2% and 23.9 ± 2.5%, respectively, compared to untreated cells. Moreover, after 7 days, Caco-2 cell growth was reduced by L. fermentum NCIMB 5221 at 99.5 ± 0.1% (p < 0.05) compared to the control treatments (Figure 5 (a and b)). Interestingly, the data indicates that L. acidophilus ATCC 314, L.fermentum NCIMB 5221 and L. rhamnosus ATCC 53103 promoted the growth of CRL-1831 epithelial normal colon cells by 12.5 ± 5.3%, 11.9 ± 1% 32 ± 3.4%, respectively, compared to untreated cells. After 48 h of treatment by CM of L. acidophilus ATCC 314, L. fermentum NCIMB 5221 increased epithelial colon cell growth by 13 ± 8.4%, and 43.2 ± 3% (p < 0.05), respectively. At 72 h, L. acidophilus ATCC 314 (Figure 5d) showed no significant anti-proliferative effect, while L. fermentum NCIMB 5221 reduced cell growth by 84.2 ± 11.2% and 54.26 ± 2.8% (p < 0.05) respectively, at 7 d, compared to untreated cells (Figure 5f).
L. fermentum NCIMB 5221 produced higher levels of SCFAs
The effect of anti-proliferative activity induced by the cells incubated in CM, used to treat both cancer and normal cell lines, was characterized for their SCFA composition, especially lactic, acetic, propionic and butyric acids. The results as indicated in Figure 5 show levels of SCFAs produced by different strains of Lactobacillus bacteria in the media. , L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103, did not inhibit colon cancer growth and did not produce detectable amounts of propionic acid in the media but had higher amounts of lactic acid, i.e. 1970.6 ± 9.6 and 3239.8 ± 9.9 mg/l, respectively, compared to L. fermentum NCIMB 5221 (480.6 ± 13.3 mg/l, Figure 6a). L. fermentum NCIMB 5221 produced the highest amount of acetic and butyric acids, i.e. 224.2 ± 8.8 and 81.17 ± mg/l, respectively, (Figure 6d) compared to L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103 (p < 0.05, Figure 6b, d). L. fermentum NCIMB 5221 was the only probiotic bacterium to produce propionic acid (76.7 ± 7.9 mg/l, Figure 6c).
SCFAs produced by L. fermentum NCIMB 5221 are responsible for the inhibitory effect
To determine if the anti-proliferative effect of L. fermentum NCIMB 5221 is the production of a specific SCFA, separate concentrations of SCFAs produced by the bacteria were tested showing that acetic, propionic and butyric acid concentration quantified in Figure 6 have significantly less effect than the bacterial extract CM (Figure 7a). Both L. fermentum NCIMB 5221 and L. acidophilus ATCC314, but not L. rhamnosus ATCC 53103, produced SCFAs (Figure 6), thus only their corresponding synthetic SCFA formulations were used for this experiment to verify the role of SCFAs naturally produced. The results demonstrated that the synthetic SCFA formulation corresponding to L. fermentum NCIMB 5221 significantly decreased Caco-2 viability by 67.8 ± 7.2% compared to synthetic SCFA formulation corresponding to L. acidophilus ATCC 314 (22.6 ± 2.6%, p = 0.018). Thus, for L. fermentum NCIMB 5221, the synthetic SCFA formulation showed no significant difference with the probiotic CM (Figure 7b), while after addition of lactic acid to the synthetic mixture the SSF + LA corresponding L. fermentum NCIMB 5221 decreased Caco-2 viability only by 21.1 ± 2.9%.
Doses of SCFAs have differential effect on normal and cancer cells
In order to investigate and differentiate the effects of pure SCFAs and their mixtures, with or without lactic acid, on normal and cancer colon cells, increasing doses (Table 1) of acetic, propionic and butyric acids were tested on Caco-2 and CRL-1831 cells (Figure 8). Increasing concentrations of acetic acid to 1300 mg/l did not exceed of 26% inhibition of cancer cells (Figure 8g) with no effect on normal cells (Figure 8b). For propionic acid, the inhibition was dose dependent and 400 mg/l of this SCFA (Figure 8h) was less than 43% with none on normal cells (Figure 8c). In the case of butyric acid, the inhibitory effect on colon cancer cells was dose dependent and 300 mg/l of butyrate inhibited cell proliferation without exceeding a minimum of 93% (Figure 8i) with no significant effect on CRL 1831 normal cells (Figure 8d). Later, increasing doses of SCFAs were mixed to formulate synthetic SCFA mixtures: SSM1, SSM2, SSM3 and SSM4 (Table 1). The effect of each SCFA mixture was significantly higher (Figure 8j, p < 0.05)) than the total effect of separate doses of SCFAs, with no significant effect observed on CRL-1831 (Figure 8e). However, when the different concentrations of lactic acid were added to each mixture (+ LA), the anti-proliferative effect was significantly reduced (Figure 8j, p <0.001). When the doses of lactic acid were tested, they showed no significant effect on the proliferation of both normal and cancer cells (Figure 8a and f).
This study has demonstrated, for the first time, that L. fermentum NCIMB 5221 had a higher anti-proliferative effect against colon cancer cells in comparison to other probiotic bacteria (L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103) [14-17]. It started with a general characterization of this strain, in terms of the levels of total FFAs generated during growth in a lactobacilli MRS broth. L. fermentum NCIMB 5221 significantly affected the level of FFAs during most of the growth phases and surpassed both controls L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103, for all the following parameters: the concentration of FFA in the bacterial supernatant (μM, Figure 2a, b and c), FFA/ viable bacterial cell (Figure 2d) or FFA/ g of bacterial pellet (Figure 2e). This reflected a significant higher metabolic activity of this bacterium compared to L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103 and its ability to produce more fatty acids, a feature that relates to the ability of producing anti-cancer fatty acid compounds such as SCFAs, conjugated linolenic acid [23] or CLA [24] considered to be locally produced in the colon to target immune cell function and suppress the disease/inflammation [25,26]. Furthermore, fatty acids, classified as short-chain (SCFA), medium-chain (MCFA) or longchain (LCFA) fatty acids, displayed potential as chemotherapeutic agents for the treatment of colorectal cancer such as the case of lauric acid that holds promise for preferential antineoplastic properties and induction of apoptosis [27].
Investigating the anti-cancer effect of L. fermentum NCIMB 5221 was performed with a verification of the probable effect of its bacterial cell free extract on colon cancer cell proliferation (Figure 3) and cell death (Figure 4). The suppression of cancer cell growth and induction of apoptosis reflect a significant effect of those extracts against colon cancer cells. Each extract seemed to contain bacterial compounds with anti-proliferative effect expressed in different time points. Compared to SP, the CM bacterial extract was most effective in inhibiting cancer cell proliferation after 24 h and 7 days of treatment (p < 0.05, Figure 3e and f) and in inducing apoptosis at 24 h (p < 0.05, Figure 4e). As the supernatant (PS) contains sodium acetate (found in MRS), which may interfere with the efficacy of the test, more interest was focused on the conditioned medium (CM). Previous studies have shown that some L. fermentum strains have potency compared to other Lactobacilli, in terms of soluble factors produced in the supernatant [28] and not to the bacterial pellet itself [28], which is in agreement to other studies where probiotic conditioned media showed effects similar to living bacteria [29] and can be used to conclude the potential of probiotics against colorectal cancer [30]. To provide relevant evidence of the potential beneficial effect of probiotic bacteria L. fermentum NCIMB 5221 against colon cancer disease, the probiotic was tested on both Caco-2 cancer cells and CRL-1831 normal cells in vitro. L. fermentum NCIMB 5221 was shown to reduce colon cancer cell viability in a time dependent manner (Figure 5e) compared to controls (Figure 5a and b) and supported non-neoplastic cell growth constantly in a serum free media compared to untreated cells (Figure 5f). In fact, among all tested probiotic bacteria, the probiotics that inhibited the most cancer cells also showed the greatest proliferation of non-cancerous colon cell growth. Starting from the most potent, the tested probiotics were L. fermentum NCIMB 5221, L. rhamnosus ATCC 53103, and L. acidophilus ATCC 314. Those observations support the fact that an optimal anticancer drug would be one that destroys neoplastic cells but not healthy cells.
Interestingly, L. fermentum NCIMB 5221 produced the most SCFAs (p < 0.001, Figure 6) compared with L. rhamnosus ATCC 53103 and L. acidophilus ATCC 314. This observation goes hand in hand with what was reported on the role of probiotic bacteria in producing factors that suppress tumor in the colon while promoting a healthy epithelium. The considerable increase in the production of butyrate observed during the administration of L. fermentum was of importance in relation to colonic cancer [31] and it has been proposed to alter intestinal epithelial cell function, including colonic SCFA utilization, mainly butyrate, [32]. SCFAs are defined as products of anaerobic metabolism of malabsorbed or non-absorbed dietary carbohydrates by luminal bacteria and identified as the dominant ion species in the aqueous phase of feces (190 mM) [33]. SCFA concentration in the lumen is in the range of 70–130 mM, with molar ratios of acetate: Propionate: butyrate varying from approximately 75:15:10 to 40:40:20. It has been estimated that SCFAs can contribute to about 10% of the total caloric requirements in humans. Luminal SCFAs, especially butyrate, serve as the major energy source for human colonocytes, especially in the distal colon [34]. In addition to its role as a fuel, butyrate is notable for its function as an inhibitor of histone deacetylases, leading to hyperacetylation of chromatin, thereby influencing gene expression. The production of SCFAs prevents the osmotic cathartic effect of unaltered luminal carbohydrate. During the concentrationdependent absorption of SCFAs, salt and water transport improves and bicarbonate appears, maintaining a neutral or alkaline colonic pH [35]. In animals, they accelerated restorations in colonic anastomoses and experimental colitis [36,37] and they increased regional blood flow and oxygen uptake [38]. Propionate and butyrate are inhibitors of histone deacetylases (HDACs) that induce differentiation in normal colon cells but cause apoptosis in colon cancer cells. Nevertheless, in the context of this work, L. rhamnosus ATCC 53103 and L. acidophilus ATCC 314 may have a beneficial effect in the context of colon cancer by their superior production of lactate (up 3200 mg/l, Figure 6a, p < 0.001), a substrate for luminal bacteria known to utilize lactate and produce acetate and butyrate as well as some propionate [39]. Regarding the role of lactate, it was identified as a potential primary effector inducing transcriptional repression of the cyclin E1 gene in-vitro [29].
Other features that could support the prophylactic potential of L. fermentum NCIMB 5221 in colorectal health is the transformation of LA to CLA and other compounds with anti-oxidant and antiinflammatory properties [40] and the increased ability to produce ferulic acid (FA), a phenolic acid found in foods with antioxidant activity when orally administered L. fermentum NCIMB 5221 was used to alleviate markers of metabolic syndrome in ZDF rats [13].
Further analysis confirmed that L. fermentum NCIMB 5221 activity was not due to one of the SCFAs only (Figure 7a) and there was no significant difference between the bacterial extract and the SCFAs synthetic formulation (Figure 7b), which suggested that L. fermentum NCIMB 5221 may owe its anti-cancer effect to SCFAs and not to other compounds secreted in the media. However, after addition of lactic acid to the SCFA mixture, the effect of SSF+LA was significantly less than SSF or the CM, implying that lactate may have repressed SCFA metabolism/intake in colon cancer cells and there other factors produced by L. fermentum NCIMB 5221 that support the activity of secreted SCFAs in suppressing cancer cells. This could be explained by the fact that the transport of butyrate into cells is greatly inhibited by the presence of its analog, lactate, a monocarboxylic acid transported into cells via monocarboxylated transporter (MCT), or propionate that is found in the colonic lumen and structurally similar to butyrate [41]. Similarly, it was demonstrated that the uptake of 500μM butyrate in Caco–2 cells was reduced by 49.6% in the presence of propionate and by 57.2% in the presence of 10 mM L-lactate [42]. Under in vivo conditions where butyrate and propionate are present at >10 mM in colon, the transporter plays only a minor role in the entry of these compounds into colon cells. When SCFAs are at low concentrations, there is involvement of SLC5A8 as transporters of butyrate and propionate with a Michaelis constant of ~0.05 mM. However, at high concentrations, SCFAs diffuse into cells bypassing the transporter [43].
Importantly, the absorption and action of SCFAs within the extract of L. fermentum NCIMB 5221 on cancer cells could have involved other factors produced by the probiotic bacteria. L. fermetum NCIMB 5221 bacterial extract may have contained molecules playing a role in assuring the action of SCFAs involved with transporters such as the monocarboxylated transporter 1 (MCT-1) and sodium-coupled monocarboxylate transporter (SMCT-1) receptor found on colonocytes to transport SCFAs or SCFA receptors GPR41/FFAR3 and GPR43/ free fatty acid receptor 2 (FFAR2), expressed in a subpopulation of ghrelin and gastrin cells [44]. Recent studies have identified the plasma membrane transporter SLC5A8 and the cell-surface receptors GPR109A and GPR43 as essential for the biologic effects of SCFAs in colon [45]. Gpr109a was found crucial for butyrate-mediated induction of IL-18 in colonic epithelium. It was actively involved in promoting anti-inflammatory properties in colonic macrophages and dendritic cells and enabling them to induce differentiation of Treg cells and IL-10-producing T cells [46]. Several bacterial effectors may affect the action of soluble factors the probiotic bacteria have produced such as probiotic-derived polyphosphate shown to inhibit progression of colon cancer, inactivate the ERK pathway and induce cancer cell apoptosis [47] or cell-bound exopolysaccharides (c-EPS) with antitumor activity [46]. In certain cases, it was revealed that the inhibitory compound might be a macromolecule such as a protein, nucleic acid, or a polysaccharide [46].
Validation tests on the SCFAs’ effect on non-neoplastic cells and cancer cells were used to confirm the fact that different concentrations and mixtures of pure/synthetic SCFAs have significant suppressive effect on cancer cells but not on normal epithelial cells and verify the effect of addition of lactate with SCFAs on cancer cell proliferation (Figure 8). First, lactic acid did not affect cancer cell proliferation when tested at different doses (up to 1300 mg/l, Figure 8f), however, when added to SCFAs, they lost a significant part of the cancer-suppressing activity. This confirms that lactic acid could inhibit SCFA metabolism/ uptake in cancer cells as described in some studies [48] and as concluded with SCFA synthetic formulations (Figure 7b) and that the presence of another bacterial factor had a role in promoting the role of SCFAs to suppress cell growth. If we assume as concluded above that lactate and propionate inhibited the uptake of butyrate (by 31% in the case of L. fermemtum NCIMB5221), then in the presence of lactate, the acetate (18. 6 ± 3.1%) will be mainly responsible for the inhibitory effect on cells that is not significantly different from the SSF + LA (21.1 ± 2.9%, Figure 7). In the case of non-neoplastic colon cells, no significant effect was observed on CRL-1831 cell growth when treated with SCFAs and/or lactate. While with L. fermentum NCIMB 5221, there was a promotion of cell growth (Figure 5f), implying that, on top of SCFAs, other soluble or non-soluble bacterial compounds could have a beneficial action on normal cells. For example, lipoteichoic acid (LTA) was shown to induce signaling in colon epithelial cells through Toll-like receptor 2 (TLR2)–CD14 and/or TLR2–TLR6 heterodimers activating extracellular-signal-regulated kinases (ERKs) then NF involved protein kinase C (PKC)- and mitogen-activated protein kinase (MAPK)-dependent pathways, and inhibit cytokine-induced epithelial cell apoptosis and damage through a phosphoinositide 3-kinase–AKTdependent pathway [49]. Those proteins were demonstrated to present resistance to apoptosis and induce epithelial barrier fortification in intestinal epithelial cells by activating the p38 and ERK signaling pathways [50].
In this study, L. fermentum NCIMB 5221 showed the same colon cancer cell inhibitory effect as the SCFAs by themselves. This would suggest the use of this bacteria as a preventive agent but not only SCFAs, as was suggested in some studies where they were orally administered in the aim of producing a therapeutic effect [27, 51-53]. Notably, the use of the bacteria as a delivery mechanism for active compounds such as the SCFAs could be a better option, especially since L. fermentum NCIMB 5221 has the ability to produce anti-oxidant, anti-inflammatory and anti-carcinogenic in soluble and non-soluble components within the gut.
Here L. fermentum NCIMB 5221 was identified with an increased anti-proliferative effect against colon cancer cells in comparison with some other probiotic bacteria (L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103) characterized in previous studies for their potential anti-cancer effect [14-17]. Interestingly, L fermentum NCIMB 5221 exhibited a reverse effect on normal colon cells suggesting that this bacterium is harmful to cancer cells but beneficial to normal cells of the colon. These effects were strongly related in this work to the significant ability of L. fermentum to produce more FFAs and especially more acetic, propionic and butyric acids compared to other probiotics. This bacterium has also been shown to produce antioxidant and anti-cancer compounds that make it more suitable as an alternative bioprophylactic and biotherapeutic agent for colon cancer treatment.
Conflicts of interest
All authors do not have any conflicts of interest to disclose.

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