In-Vitro Characterization of the Anti-Cancer Activity of the Probiotic Bacterium Lactobacillus Fermentum NCIMB 5221 and Potential against Colorectal Cancer

Colorectal cancer (CRC) is a leading cause of mortality worldwide [1]. However, it is a type of cancer for which chemoprevention is considered a therapeutic and preventive strategy [2]. Probiotics have been used as biotherapeutics that reduce cancer recurrence and side effects in CRC patients [3-5]. When orally administered, probiotic along with intestinal microbial-produced metabolites (e.g. organic acids, peptides), that interact with cellular proliferation, differentiation, and intestinal inflammation, reduce the risk of CRC [6]. In the large intestine, short chain fatty acids (SCFAs) produced by bacterial fermentation have shown to exert anti-inflammatory [7] and anticarcinogenic actions. Studies have shown that fatty acids can mutually interact and protect against CRC. Nevertheless, the incorporation of fatty acids into CRC chemotherapies is still premature. The oral administration of specified probiotic bacteria remains the dominant method to increase the bio-production of these anti-tumorigenic compounds in the colon [8,9].


Introduction
Colorectal cancer (CRC) is a leading cause of mortality worldwide [1]. However, it is a type of cancer for which chemoprevention is considered a therapeutic and preventive strategy [2]. Probiotics have been used as biotherapeutics that reduce cancer recurrence and side effects in CRC patients [3][4][5]. When orally administered, probiotic along with intestinal microbial-produced metabolites (e.g. organic acids, peptides), that interact with cellular proliferation, differentiation, and intestinal inflammation, reduce the risk of CRC [6]. In the large intestine, short chain fatty acids (SCFAs) produced by bacterial fermentation have shown to exert anti-inflammatory [7] and anticarcinogenic actions. Studies have shown that fatty acids can mutually interact and protect against CRC. Nevertheless, the incorporation of fatty acids into CRC chemotherapies is still premature. The oral administration of specified probiotic bacteria remains the dominant method 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, an emphasis on the importance of well-established in vitro and in-vivo studies to select good candidates exists. Few studies have been found to establish a rigorous selection for new probiotic strains. Anticancer attributes, proper controls, and extensive evaluation of their anti-proliferative effect against cancer cells should be analyzed and compared with other established probiotic products. While studies have shown the ability of certain probiotics to affect SCFA levels, more evidence that the probiotic anti-cancer effect is solely due to the direct production of SCFAs [10][11][12] is needed. In this study the profile of L. fermentum NCIMB 5221, identified as a producer of ferulic acid (FA), was characterized. The latter is an anti-oxidant and anti-tumorigenic

Bacterial cultures
L. fermentum NCIMB 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) while 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 normal epithelial colon cell line, were purchased from ATCC American Type Cell Collection (ATCC, Manassas, VA). Caco-2 cells were maintained in (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% CO 2 ). Caco-2 cells were incubated in a CO 2 incubator at 37°C in air supplemented with 5% CO 2 for a maximum of two 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. The cell medium was then replaced by probiotic cell-free extracts mixed with serum/antibiotic-free DMEM.

Free fatty acid (FFA) analysis
In this analysis, the free fatty acids (FFAs) in the bacterial supernatant were converted to their CoA derivatives and then oxidized. This resulted in 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, a reaction mix was prepared. A 2 μl of ACS Reagent was added to all the standards (palmetic acid. sample wells were mixed and left to incubate (37°C, 30 min). The reaction mix (50 μl), f atty acid probe (2 μl), enzyme mix (2 μl), assay buffer (Igepal, 44 μl), and enhancer (N-ethylmaleimide, 2 μl) were mixed and vortexed briefly. Then, 50 μl of this reaction mixture was added to each well (standard or sample) and incubated (30 min, 37°C, away from light). Absorbance was measured at 570 nm for colorimetric assay in a microplate reader (Perkin Elmer, Victor 3, multi-label micro-plate reader, MA, USA).

Preparation of probiotic cell-free supernatants
Each probiotic bacterial colony 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. [14] and Kim et al [15]. For the preparation of the CM, bacterial cultures (16 h, 37 °C, 5% CO 2 ) were used to collect bacterial pellets, by centrifugation (4000 rpm, 15 -20 min, 4°C), to be washed (PBS) and resuspended in DMEM. The bacterial pellets in DMEM were maintained and incubated in a shaker incubator for two hours (37°C, 5% CO 2 , 100 rpm). After incubation, the culture medium was centrifuged (1000 x g, 15 min) and sterile-filtered (filter pore size 0.2 μM-pore-size filter). Prior to treating the cells, the CM of each Lactobacillus bacteria was combined 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 sterile-filtered (0.22 μm) and stored at −80 °C, until use.

Figure 1:
The mechanism behind the quantification of total free fatty acids using a coupled enzymatic reaction system (ACS-ACOD Method). ACS: Acyl CoA Synthetase. ACOD: Acyl CoA Oxidase. POD: peroxidase.

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 dissolve fully the substrate. The blank reaction (DMEM without cells), negative control (untreated cells in DMEM) and the assays (treated cells in CA+DMEM) were all reactions prepared to detect caspase-3 and caspase-7 activity in cell cultures in 96-well white opaque plates. After incubation with the treatment, the plate was removed from the incubator and allowed to equilibrate at RT. A 100 µL of the luminescent reagent was added to each well with pre-filled 100 µL of blank, negative control or treated cells in DMEM. The plate was gently mixed on a plate shaker (300 -500 rpm, 30 sec), followed by incubation at RT for 3 hours. Finally, the luminescence of each sample was measured in a plate-reading luminometer following the manufactures' instructions.

Probiotic effect on CRC cells vs. non-neoplastic colon cells
This assay was performed to demonstrate an anti-CRC effect of L. fermentum NCIMB 5221 by inhibiting CRC cell proliferation without affecting non-neoplastic colon cells. Caco-2 and CRL-1831 (4 -5×10 3 cells/ well) were seeded into 96-well culture plates (37°C, 5% CO 2 ) for 1, 2, 3, and 7 days. The cells in both populations were treated with probiotic CM. At each time point 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 [17,18]. The Model 1050 of 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. A 100 µl of sample was injected through an autosampler. A prepacked Rezex ROA-organic acid H+ (8%) (150mm × 7.80 mm, Phenomenex, Torrance, CA, USA) fitted with an ion-exclusion microguard refill cartridge was used. Data was acquired using ChemStation supported with LC3D software Rev A.03.02 (Agilent Technologies, CO, USA). The mobile phase (A) of H 2 SO 4 (0.05 M) and the mobile phase (B) of acetonitrile (2%) pumped isocratically at a flow rate of 0.8 -0.7 mL/ min, through a column heated to 35°C. Lactic, acetic, propionic, and butyric acids were used to prepare a standard solution at concentrations of 1, 10, 100, 500, and 1000 ppm (in triplicate). The concentrations of SCFAs were estimated using the linear regression equations (R 2 ≥ 0.99) generated from respective standard curves.

Role and efficacy of SCFAs: SCFA synthetic formulations vs. probiotic CM
This test intends to demonstrate the role and the relevance of naturally produced SCFAs by probiotic cells in their cell-free extracts (CM). SCFA synthetic formulations (SSF) with same composition of the naturally produced SCFAs in the probiotic CM were prepared (Table 1). Caco-2 cells (4 -5 × 10 3 cells/well), seeded onto 96-well plates (37°C, 5% CO 2 , 72 h) were used to determine the inhibitory actions of these compounds and compare it to the probiotic CM. SCFA synthetic mixtures with the same effect as the bacterial extract would suggest that the inhibitory effect against CRC cell is due to the concentration of SCFAs produced by L. fermentum. Another set of mixtures (SSF+LA) was used after the addition of lactic acid (similar to that produced by the bacteria) to investigate the effect of another bacterial component on the action of SCFAs.

Effect of SCFAs on CRC cell compared with normal cells
The objective of this step was to investigate the dose-dependent effect of SCFAs, pure or in a mixture, and the nature of their potential synergistic effect on both normal and cancerous colon cells. Different concentrations of lactate, acetate, 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 × 10 3 cells/ well, 37°C, 5% CO 2 ), left to stabilize and attach (24 -48 h), followed by incubation with treatment samples (37°C, 5% CO 2 , 72 h). SCFA treatments included increasing concentrations of lactate

L. fermentum NCIMB 5221 displays anti-proliferative activity against CRC cells in a time-dependent manner
The anti-proliferative and apoptotic effect of L. fermentum NCIMB 5221 against SW-480 CRC cells was compared with controls: L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103, using two types     Figure 3f). For the induction of cell death in SW-480 cancer cells by SP treatments, there was no significant difference between the treated groups (Figures 4a and 4b). However, for L. fermentum NCIMB 5221, the effect of CM in inducing apoptosis in CRC 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 result suggests that cancer cells might not be affected by bacteria-cell contact but by soluble bacterial factors or other microbial associated molecular patterns (MAMPs)

L. fermentum NCIMB 5221 inhibits CRC cells but not normal cells
It was necessary to elucidate and determine the mechanism by which bacterial symbionts affect cell growth in the epithelium in a tumor environment. To do so, CM prepared from probiotic cells of L. acidophilus ATCC 314, L. fermentum NCIMB 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 Figure 5d) showed no significant anti-proliferative effect, whereas L. fermentum NCIMB 5221 reduced cell growth by 59.4 ± 9.8.2% (p < 0.05), compared with 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 by SCFA composition, especially lactic, acetic, propionic, and butyric acids. The results as displayed 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 neither inhibit CRC growth nor produce detectable amounts of propionate in the media but had higher amounts of lactate, i.e. 1970.6 ± 9.6 and 3239.8 ± 9.9 mg/l, respectively, compared with L. fermentum NCIMB 5221 (480.6 ± 13.3 mg/l, Figure 6a). L. fermentum NCIMB 5221 produced the highest amount of acetate and butyrate, i.e. 224.2 ± 8.8 and 81.17 ± mg/L, respectively, (Figure 6d) compared with L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103 (p < 0.05, Figure  6b, 6.6d). L. fermentum NCIMB 5221 was the only probiotic bacterium to produce propionate (76.7 ± 7.9 mg/L, Figure 6c) compared with controls.

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 result of a specific SCFA, separate concentrations of SCFAs produced by the bacteria were tested. This revealed that acetic, propionic, and butyric acid concentration, quantified in Figure  6 have significantly less effect than the bacterial extract CM ( Figure  7a). Only L. fermentum NCIMB 5221 and L. acidophilus ATCC314, 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 bacterial SCFAs. 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 with 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), whereas after addition of lactic acid to the synthetic mixture, the SSF + LA corresponding to 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
To investigate and differentiate the effects of pure SCFAs and their mixtures, with or without lactic acid, on colon normal and cancer 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 more than 26% inhibition of cancer cells (Figure 8g), with no significant effect on normal cells (Figure 8b). For propionic acid, the inhibition was dose dependent, and 400 mg/L of propionate (Figure 8h) showed 43% inhibition with no inhibition on normal cells (Figure 8c). In the case of butyric acid, the inhibitory effect on CRC cells was dose-dependent and 300 mg/L of butyrate inhibited cell proliferation with a maximum inhibition of 93% (Figure 8i) with no significant effect on CRL 1831  (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 had no significant effect on the proliferation of both normal and cancer cells ( Figure 5.8a and 6.8f).

Discussion
This study demonstrated, for the first time, that L. fermentum NCIMB 5221 has a higher anti-proliferative effect against CRC cells related to a higher metabolic activity, than to other LAB bacteria (L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103) [19][20][21][22]. As a general characterization of this strain, L. fermentum NCIMB 5221 significantly (p < 0.01) 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 the following parameters: concentration of FFA in the bacterial supernatant (Figures 2a, 2b, and 2c), FFA/viable bacterial cell (Figure 2d) and FFA/g of bacterial pellet (Figure 2e). This reflected a significantly higher metabolic activity of this bacterium, compared with L. acidophilus ATCC 314 and L. rhamnosus ATCC 53103, additionally to its ability to produce more fatty acids, a feature that relates to the production of anticancer fatty acid compounds. For instance, SCFAs, linolenic acid [23] or conjugated linoleic acid (CLA) [24] are 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 long-chain (LCFA) fatty acids, demonstrated potential as chemotherapeutic agents for the treatment of colorectal cancer. For instance, lauric acid holds promise for preferential antineoplastic properties by higher induction of apoptosis in cancer cells [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 with 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 3f) and in inducing apoptosis at 24 h (p < 0.05, Figure 4e). As the supernatant (PS) contains sodium acetate (found in MRS broth), 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 greater potency compared with other Lactobacilli, in terms of soluble factors produced in the supernatant and not to the bacterial pellet itself [28]. This aligns with other studies where probiotic CM have shown effects similar to living bacteria [29] and supported the potential of probiotics against CRC [30]. To provide relevant evidence of the potential beneficial effect of probiotic bacteria L. fermentum NCIMB 5221 against colon cancer, 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 CRC cell viability in a time-dependent manner (Figure 5e) compared with controls ( Figures  5a and 5b). It also supported constant non-neoplastic cell growth in a serum-free media compared with 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 noncancerous colon cell growth. In order of potency, the tested probiotics were L. fermentum NCIMB 5221, L. rhamnosus ATCC 53103, and L. acidophilus ATCC 314. Those observations support the finding that an optimal anti-cancer 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 agrees with some if the reported actions of probiotic bacteria in producing factors that prevented tumor-initiating events 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]. SCFAs are defined as products of the anaerobic metabolism of mal-absorbed or nonabsorbed dietary carbohydrates by luminal bacteria and identified as the dominant ion species in the aqueous phase of feces (190 mM) [32]. In fact, it has been thusly proposed that to alter intestinal epithelial    [34]. In addition to its role as a fuel, butyrate is notable for its function as an inhibitor of histone deacetylases (HDACs), leading to hyperacetylation of chromatin, thereby influencing gene expression. During the concentrationdependent absorption of SCFAs, bicarbonate, salt, and water transport improves, maintaining a neutral or alkaline colonic pH [35]. In animals, they accelerated the restoration of colonic anastomoses and experimental colitis [36,37] and resulted in increased regional blood flow and oxygen uptake [38]. L. fermentum NCIMB 5221 is considered a fitter candidate based on its higher production of propionate and butyrate. Nonetheless, L. rhamnosus ATCC 53103 and L. acidophilus ATCC 314 may also have a beneficial effect through their elevated production of lactate (up 3200 mg/L, Figure 6a, p < 0.001). Here, lactate is a substrate for luminal lactate-utilizing bacteria that produce acetate and butyrate, as well as some propionate [39] and a regulator of epithelial proliferation in the gut through the repression of cyclin E1/ D1 gene transcription [29]. Other features that could support the prophylactic potential of L. fermentum NCIMB 5221 in colorectal health could be the transformation of LA to CLA and other compounds with antioxidant and anti-inflammatory properties [40]. When orally administered L. fermentum NCIMB 5221 was used to alleviate markers of metabolic syndrome in ZDF rats hypothetically through the release of FA, a phenolic acid found in foods [13].
Further analysis confirmed that L. fermentum NCIMB 5221 activity was not due to one of the SCFAs alone (Figure 7a). Since there was no significant difference between the bacterial extract and the SCFAs synthetic formulation (Figure 7b), L. fermentum NCIMB 5221 may owe its anti-cancer effect to SCFA release in total. After addition of lactic acid to the SCFA mixture, the effect of SSF+LA was significantly less than SSF or the CM. This implies that lactate may have repressed SCFA metabolism/intake in CRC cells and other non-anti-cancer factors produced by L. fermentum NCIMB 5221 that support the activity of secreted SCFAs in suppressing cancer cells. 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 500 µM 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 the 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 mechanisms that were repressed with the addition of lactate. L. fermentum NCIMB 5221 extract may have contained molecules playing a role in assuring the action of SCFAs involved with cell transporters. These include the monocarboxylated transporter 1 (MCT-1) and sodium-coupled monocarboxylate transporter (SMCT-1) receptor found on colonocytes. Their function is to transport SCFAs or SCFA receptors GPR41/ free fatty acid receptor 3 (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 the 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 alter the action of soluble factors that probiotic bacteria have produced. For example, a probiotic-derived polyphosphate have shown to inhibit progression of CRC, 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 pro-SCFA 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 against normal epithelial cells. The test also verified the effect of the 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 also concluded with SCFA synthetic formulations, as presented in this study (Figure 7b), Thus, this emphasize that the presence of another bacterial factor promoted 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% for L. fermentum NCIMB5221), then in the presence of lactate, only acetate (18. 6 ± 3.1%) will be responsible for the inhibitory effect that was closer to 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 when compared with cancer cells. Whereas with L. fermentum NCIMB 5221, there was a promotion of cell growth (Figure 5f), implying that, in addition to 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. It activates extracellular-signal-regulated kinases (ERKs), NF involved protein kinase C (PKC)-and mitogen-activated protein kinase (MAPK)dependent pathways, and inhibits cytokine-induced epithelial cell apoptosis and damage through a phosphoinositide 3-kinase-AKTdependent pathway [49]. Those proteins were demonstrated to present resistance against apoptosis and induce epithelial barrier fortification in intestinal epithelial cells by activating the p38 and ERK signaling pathways [50].