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Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage a
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Hanru Wang , Susan E. P. Bastian , Andrew Lawrence & Gordon S. Howarth
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School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia b
School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Adelaide, Australia c
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Microbiology Department, SA Pathology at Women's and Children's Hospital, Adelaide, Australia d
School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia and Microbiology Department, SA Pathology at Women's and Children's Hospital, Adelaide, Australia Published online: 27 Jan 2015.
To cite this article: Hanru Wang, Susan E. P. Bastian, Andrew Lawrence & Gordon S. Howarth (2015): Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage, Nutrition and Cancer, DOI: 10.1080/01635581.2015.990570 To link to this article: http://dx.doi.org/10.1080/01635581.2015.990570
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Nutrition and Cancer, 0(0), 1–11 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.990570
Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage Hanru Wang School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia
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Susan E. P. Bastian School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Adelaide, Australia
Andrew Lawrence Microbiology Department, SA Pathology at Women’s and Children’s Hospital, Adelaide, Australia
Gordon S. Howarth School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia and Microbiology Department, SA Pathology at Women’s and Children’s Hospital, Adelaide, Australia
We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LBC) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer. Received 4 April 2014; accepted in final form 29 October 2014. Address correspondence to Gordon S. Howarth, School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, South Australia, 5371, Australia. Tel: C61 8 8313 7885. Fax: C61 8 8313 7972. E-mail: [email protected]
INTRODUCTION Colorectal cancer is the fourth most frequently diagnosed and the second most deadly type of cancer worldwide, with approximately 100,000 new cases diagnosed and 50,000 estimated deaths each year in the United States (1). To date, colon cancer cannot be treated effectively. Clinical trials have shown 5-fluorouracil (5-FU) to be effective as adjuvant chemotherapy for high-risk Stage II and III colon cancer (2). Nevertheless, chemotherapy is currently used as an effective method to treat colon cancer, although it commonly results in debilitating side-effects (e.g., mucositis). The severity of mucositis varies from mild to severe, characterized by pain, ulceration, and bleeding along the entire digestive tract. Indeed, mucositis may be so severe that death can result (3). Chemotherapy induces the generation of reactive oxygen species (ROS), which cause tissue damage (4). Chemotherapy also damages the DNA of epithelial basal cells leading to cell death (4). Combined, these biological reactions are responsible for the pathogenesis of mucositis (4). The development of new agents, which would not compromise the efficacy of existing chemotherapy regimens but would 1
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attenuate the intestinal damage induced by chemotherapy, could hold great potential for the future treatment of colon cancer. Investigations into the clinical application of probiotics such as lactobacillus and bifidobacterium for the potential prevention or treatment of intestinal disorders have been growing steadily (5,6). Mechanisms of probiotic action described to date include adhesion to the intestinal–lumen interface; enhancement of mucosal barrier function; competition with pathogens for receptor binding, nutrients and colonization; promotion of immune responses; elaboration of bacteriocins; and modulation of cell kinetics (6,7). Escherichia coli Nissle 1917 (EcN) was the first bacterium to be identified as a probiotic (8). EcN has been reported to exert therapeutic activity against several intestinal disorders such as inflammatory bowel disease (9), particularly the ulcerative colitis variant (9) and other forms of gut inflammation in humans (10). Moreover, EcN has been reported to improve mucosal integrity by modulating the tight junction molecule, zonula occludens-1 (ZO-1), thereby reducing intestinal permeability in the colon in mice following dextran sodium sulfate (DSS)-induced damage (11). In recent years, factors released from probiotics have created a new frontier of disease therapy as a result of their capacity to combat intestinal disorders in the absence of living probiotic bacteria (12,13). Probiotic-derived factors encompass proteins and other molecules released from living probiotics into their respective culture media (supernatants [SN]) and have been shown to exert beneficial biological and physiological properties in certain circumstances; although studies have been limited (6). For example, bacteriocins and reuterin released from probiotics have been shown to inhibit the adhesion and viability of known enteric pathogens such as grampositive and gram-negative bacteria as well as yeasts, fungi, and protozoa (7,14). Probiotic factors released from Lactobacillus rhamnosus GG (LGG) were reported to prevent alcoholinduced Caco-2 cell monolayer barrier dysfunction (15). To this end, 2 new proteins, p75 and p40, from LGG supernatants have been shown to play pivotal roles in reducing tumor necrosis factor alpha (TNF-a)-induced epithelial cell apoptosis and promoting the growth of epithelial cells (16). In addition, EcN SN partially protected the intestine from 5-FU-induced damage in rats and in IEC-6 cells (12,13). EcN SN has also revealed promise in the treatment of human gastrointestinal motility disorders (17). The potential utility of probiotic-derived factors in cancer chemotherapy is revealing some promise. In human studies, it has been reported that EcN SNs, as well as other metabolic factors derived from the probiotic formulation VSL#3 (Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus), significantly decreased cyclooxygenase-2 (COX-2) gene expression
induced by gastrin or the proinflammatory cytokine TNF-a (18). The inhibition of COX-2 expression is reported to be an important therapeutic approach in the prevention of colon carcinogenesis (18,19). Moreover, Propionibacterium freudenreichii ITG P9 strain fermented milk SN induced apoptosis of HGT-1 cancer cells (20). Short chain fatty acids (SCFAs) produced from certain bacterial strains such as Clostridium butyricum, have also been shown to exert anti-carcinogenic properties (6,21). The SCFA butyrate, has also been demonstrated to impose cell cycle arrest, differentiation and apoptosis in many tumor cell lines (21). In addition, butyrate has been found to inhibit the action of histone deacetylases, involved in tumorigenesis (21). Importantly, however, the composition and bioactivities of factors from the same bacterial strain could vary, depending on the composition of the growth medium. The resultant factors could, therefore, have differential and possibly disparate efficacies in the context of cancer treatment. For example, Streptococcus thermophilus grown in milk produced high levels of enzymes such as BCAA aminotransferase, ketol-acid reductoisomerase, and pyruvate formate-lyase (22). However, these enzymes were barely detectable when the same cells were grown in M17 medium. The primary aim of the current study was therefore to investigate the potential for EcN when grown in different growth media to produce SN derived factors able to differentially kill Caco-2 cells. Specifically, effects on Caco-2 cell viability, ROS generation, and cell monolayer permeability were sought. The second aim was to identify a mechanism of action for EcN SNs by determining effects on these end-points in the presence and absence of the antimetabolite chemotherapy drug, 5-FU.
METHODS EcN SN Preparation The method for preparing bacterial SNs has previously been described by Prisciandaro et al. (12,23). Briefly, E. coli Nissle 1917 (EcN) was purchased from Ardeypharm (Herdecke, Germany). EcN [25 £ 107 colony forming units (CFU)] was grown separately in 4 different broth growth media; Luria-Bertani (LB) (GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia), tryptone soya (TSB), de Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (the last 3 all from Oxoid, Adelaide, South Australia, Australia). EcN (80 mL) was incubated at 37 C for 24 h and reached a concentration of approximately 109 CFU/mL. EcN mixtures were then centrifuged at 6000 rpm for 20 min (HeraeusÒ MegafugeÒ 1.0, Thermo Fisher Scientific, Waltham, MApH). SN were then collected and buffered with HEPE to a final concentration of 20 mM. The pH was adjusted to 7.2 using HCl (3 M)/NaOH (3 M). SNs were then filtered through a 0.22-mm low protein-binding
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EcN FACTORS AND 5-FU INDUCED CELL DAMAGE
syringe driven filter (MillexÒ Syringe Filter Units, Merck KGaA, Darmstadt, Germany) and stored at -20 C. Each SN (0.5 mL) was transferred to a 1.5-mL Eppendorf tube and the total moisture content was removed by using a Savant SpeedVacÒ Concentrator (Savant SpeedVac SC110Ò , Thermo Fisher Scientific, Waltham, MA,) for at least 24 h. Dried SNs were then weighed and suspended in serum free Dulbecco’s Modified Eagle Medium (DMEM) to reach a final concentration of 100 mg/mL. All SNs were refiltered through a 0.22-mm syringe driven filter (MillexÒ Syringe Filter Units, Merck KGaA, Darmstadt, Germany) prior to use. All control growth media SNs were prepared the same way as EcN SNs without previously growing EcN. The resultant treatment groups were as follows: DMEM (control), LB, TSB, MRS, and M17; EcN SNs were LBC, TSBC, MRSC, and M17C. The “C” symbol indicates that each broth had been used to grow EcN.
Cell Culture Caco-2 human colorectal adenocarcinoma cells (passage 25-35) were cultured in medium containing DMEM supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) antibiotics (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia). All cells were maintained at 37 C in a humidified incubator with 5% CO2 and grown in 75 cm2 vented tissue culture flasks (CELLSTARÒ , Greiner Bio-One, Frankenhausen, Germany). Culture medium was changed twice each week and passaged when 80% confluence had been reached.
Cell Viability Caco-2 cell viability was examined by MTT assay (24,25). Cells (5 £ 103 cells/mL; 100 uL) were placed in each well of a 96-well tissue culture plate for 48 h for attachment to the bottom of the well. After 48 h, the culture medium was replaced with serum-free DMEM (control) and media (100 mg/mL), EcN SNs (100 mg/mL) and/or 5-FU (0.5 mM). The cells were then cultured for either 24 or 48 h. MTT powder [3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; Molecular ProbesÒ , Life Technologies, Mulgrave, Victoria, Australia] was dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS) (GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) to reach a final concentration of 1 mg/mL. The MTT solution (50 mL) was then sterilized by 0.22-mm filtration and added to each well. After the cells were incubated at 37 C for 4 h, the medium was then replaced with 100 mL of dimethyl sulfoxide (Sigma-Aldrich, Sydney, Australia) for 15 min with minor shaking. Absorbance was read at 570 nm by a spectrophotometer (Tecan infinite 200Ò PRO, M€annedorf, Switzerland). Data were expressed as number of viable cells compared to the percentage of control cells treated with serum-free DMEM (25). Treatments were performed in
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triplicate, whereas the entire experiment was conducted 3 times (n D 9). Detection of ROS Through Flow Cytometry A modified assay using a CellROXÒ Oxidative Stress Reagent was performed to detect ROS by flow cytometry (C10444, Life Technologies, Mulgrave, Victoria, Australia). The cell-permeable CellROXÒ reagent is nonfluorescent while in a reduced state but exhibits a strong fluorescent signal upon oxidation, which can be detected with excitation and emission values of 508 and 525 nm (C10444 manual, Life Technologies, Mulgrave, Victoria, Australia). Caco-2 cells were grown in 12-well culture plates at a concentration of 7 £ 104 cells/mL for 48 h to allow attachment. Broths (LB, TSB, MRS, and M17) and EcN SNs including LBC, TSBC, MRSC, and M17C at a final concentration of 100 mg/mL and/or 5-FU at a final concentration of 2 mM were used to replace DMEM for a further 24 or 48 h. Cell preparation followed the method recommended by the manufacturer (C10444, Life Technologies, Mulgrave, Victoria, Australia) with minor adjustments based on other relevant publications (26). Briefly, after 24 or 48 h, all media were aspirated and cells harvested by trypsinization. Cells were then washed with 3 mL of DPBS and centrifuged at 1500 rpm for 2 min at room temperature (Eppendorf Centrifuge 5810, Hamburg, Germany). Supernatants were then discarded and cell pellets were resuspended with 1 mL of DPBS containing CellROXÒ reagent (1 mM) for 30 min at 37 C in the dark. Next, each cell suspension was gently mixed before analysis by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, CA), using a 488-nm laser with a 530/30 BP filter. Ten thousand events were collected for each treatment. Results were presented by histogram and data were analyzed by BD CellQuest ProTM software Ó 2002 (Becton Dickinson Biosciences, San Jose, CA).
Transepithelial Electrical Resistance Assay Caco-2 cells were harvested from a 75 cm2 vented tissue culture flask. Cell suspensions (200 mL) were added to the upper layer of 0.33 cm2 Transwell clear polyester permeable membranes (Corning Costar, Tewksbury, MA) at a density of 1 £ 105 cells/Transwell. Next, 600 mL of DMEM supplemented with 10% FCS and 1% antibodies (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) were added to the bottom well. Cells were incubated at 37 C in a humidified incubator with 5% CO2 for the duration of the study. Cells were cultured with DMEM supplemented with 10% FCS and 1% antibodies (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) for the first 23 days to allow sufficient time to form a healthy monolayer barrier. Media were changed every 2 to 3 days prior to the beginning of the
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experiment. On the 23rd day, for control treatment groups, media were replaced with DMEM control, broth controls (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) at a concentration of 100 mg/mL on the upper layer of the Transwell and DMEM at the lower layer for all control groups. For 5-FU treatment groups, all media were replaced with DMEM control, broth controls (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) at a concentration of 100 mg/mL on the upper layer of the Transwell and 5-FU (2 mM) at the lower layer for all 5-FU treated groups. Transepithelial electrical resistance (TER) measurements were recorded prior to changes of media for the first 23 days and 24, 48, 72, and 96 h after adding treatments. Treatments were carried out in duplicate and repeated 3 times for the entire experiment (n D 6). Monolayer resistance was measured by a millicell-ERS volt-ohm meter (Millipore, Billerica, MA) with electrodes. Values were expressed as ohms per square centimeter (V/cm2) (12).
Ferric Reducing Antioxidant Power Assay The antioxidant activity of all treatments including DMEM control, broths (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) was measured by ferric reducing antioxidant power (FRAP) assay (27,28). Total moisture content in each treatment was removed by using a Speedvac (Servant Speedvac SC110). Dried samples were then weighed and suspended in Milli-Q water to reach a final concentration of 50 mg/mL. Ascorbic acid (0.1–1.0 mg/mL) was dissolved in Milli-Q water as a positive control. Ferrous sulphate prepared in Milli-Q water (0.1–1.0 mM) was used to generate a standard curve. FRAP reagent was prepared with 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-Tripyridyl-s-Triazine) in 40 mM HCL, and 20 mM ferrous chloride at a ratio of 10:1:1 and kept in the dark. All chemicals were purchased from Sigma Aldrich (Sydney, Australia). Each sample (15 mL) was added in triplicate into each well of a nonsterile 96-well plate (Grenier Bio-one, Basel, Switzerland). FRAP reagent (150 mL) was immediately added into each sample. The plate was then kept in the dark with minor shaking for 4 min and read at 593 nm using a spectrophotometer (Tecan infinite 200Ò PRO, M€annedorf, Switzerland). A linear equation was generated based on the ferrous sulphate standard curve and Fe2C concentration (mM) in a sample was calculated based on this equation. FRAP values of test samples were expressed as mmol Fe2C per gram of sample weight (mmol/g). Treatments were performed in three replicates while the entire experiment was conducted 3 times (n D 9). Statistical Analysis Results are expressed as mean § SEM. IBM SPSS Statistics for Windows, version 20.0 (SPSS, Armonk, NY) was used for all statistical analyses. One-way analysis of variance with a
Tukey’s post hoc test was performed to determine the difference between means, with statistical significance set at P < 0.05.
RESULTS Effects of Different Broths and EcN SNs on Cell Viability of Caco-2 Cells in the Presence and Absence of 5-FU The cell viability of Caco-2 cells cultured for 24 h in broth LB and EcN SNs M17C was not significantly different compared to DMEM controls (100%) (Fig. 1A). However, the number of viable cells was significantly reduced to approximately 60–87% when Caco-2 cells were cultured in TSB, MRS, and M17 broths and EcN SNs (LBC, TSBC, and MRSC) (Fig. 1A; P < 0.05). Surprisingly, EcN SNs (LBC and MRSC) and broth (TSB and MRS) reduced the cell viability to approximately 60% compared to DMEM controls, with LBC producing the greatest decrease in cell viability (61.1 § 5.7%) among all treatments (Fig. 1A; P < 0.001). The cytotoxic effects were also observed in broths and EcN SNs at 48 h (Fig. 1B). Broths (LB, TSB, MRS and M17) and EcN SNs (LBC, MRSC and M17C), excluding EcN SN TSBC, significantly reduced cell viability to 51–78%, compared to DMEM controls (100%) (Fig. 1B; P < 0.05); whereas only TSBC did not significantly reduce cell viability, compared to DMEM controls (P > 0.05). More importantly, EcN SNs (LBC and MRSC) and MRS broth decreased cell viability to almost 50% compared to DMEM controls (P < 0.001) at 48 h (Fig. 1B). 5-FU treatment reduced viable cell numbers in all groups compared to DMEM treated controls. The cell viability of 5FU treated Caco-2 cells (5-FU control) was significantly reduced, to 79.3 § 1.0% at 24 h and 63.8 § 5.2% at 48 h, compared to DMEM control (100%; Fig. 1A and B; P < 0.05). All broths (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) together with 5-FU significantly decreased Caco-2 cell viability to approximately 55– 78% at 24 h and 38–60% at 48 h compared to DMEM controls (Fig. 1A and B; P < 0.05). TSB and MRS broths, excluding LB and M17, in combination with 5-FU further reduced cell viability to approximately 38–50%, compared to 5-FU controls (79.3 § 1.0%) at 24 h (Fig 1A; P < 0.05). Meanwhile, EcN SNs (LBC and MRSC), excluding TSBC and M17C, together with 5-FU further decreased the proportion of viable cells to approximately 37–60% compared to 5-FU controls (79.3 § 1.0%) at 24 h (Fig 1A; P < 0.001). At 48 h, LB, TSB, and M17 broths and EcN SNs (TSBC and M17C), together with 5-FU, tended to decrease Caco-2 cell viability to approximately 50–63%, consistent with 5-FU control (63.8 § 5.2%), although statistical significance was not attained (Fig. 1B; p D 1.00). However, EcN SNs (LBC and MRSC) and MRS broth, together with 5-FU, further reduced cell viability to approximately 37–43% compared
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FIG. 1. Combined effects of media, Escherichia coli Nissle 1917 (EcN) supernatants (SNs), and/or 5-fluorouracil (5-FU; mM) on the viability of Caco-2 cells for 24 h (Fig. 1A) or 48 h (Fig. 1B). Cells were treated with serum free Dulbecco’s Modified Eagle Medium (control), bacterial media alone [Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17)] and EcN SNs (LBC, TSBC, MRSC, and M17C) at a final concentration of 100 mg/mL, either alone or in combination with 5-FU (500 mM). Data are expressed as percentage of viable cells relative to untreated cell controls. Data are presented as means § SEM of three independent experiments (n D 9). Bar data not sharing the same letter are significantly different (P < 0.05). # indicates a significant difference compared to DMEM control in the groups without 5-FU. *indicates a significant difference compared to 5-FU control in all 5-FU treatment groups (P < 0.05).
with 5-FU control (63.8 § 5.2%) at 48 h (P < 0.001). LBC together with 5-FU produced the lowest proportion of viable cells (37.4 § 3.4%) among all the treatments tested at 48 h (P < 0.001). Effects of Different Broths and EcN SNs on the Generation of ROS from Caco-2 Cells in the Presence and Absence of 5-FU The intensity histogram of ROS generation under treatments of DMEM (control) and 5-FU (5-FU control) for 24 h and 48 h is shown in Fig. 2A and 2B, respectively. The histogram was gated as M1. The percentage of ROS generation was
calculated within the area of gate M1 by BD CellQuest ProTM software Ó 2002 (Becton Dickinson Biosciences, San Jose, CA). The percentage difference (PD) in ROS generation from Caco-2 cells cultured in different broths and EcN SNs and/or 5-FU as compared to Caco-2 cells cultured in DMEM, is shown in Fig. 3A (24 h) and B (48 h). ROS generation from Caco-2 cells cultured in broths (LB, TSB, MRS, and M17) and EcN SNs (TSBC and MRSC) generated similar levels of ROS (approximately minus 5%) compared to DMEM controls at 24 h (Fig. 3A). However, EcN SNs (LBC and M17C) reduced ROS generation (approximately minus 9%) compared to the DMEM control at 24 h (Fig. 3A; P < 0.05) There were no significant difference in
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DMEM, broths, EcN SNs, and/or 5-FU. TER values 24, 48, 72, and 96 h after adding treatments are shown in Fig. 5. The TER value of the DMEM treatment, broths and EcN SNs decreased gradually over time from 9.8 § 0.1 £ 103 V/cm2 at 24 h to 4.9 § 0.1 £ 103 V/cm2 at 96 h (Fig. 5A). The resistance value of all broths and EcN SNs did not significantly change, compared to DMEM controls at all time-points measured (Fig. 5A; P > 0.05). 5-FU control, broths C 5-FU and EcN SNs C 5-FU did not change the resistance value at 24 h compared to DMEM control (Fig. 5B; P > 0.05). However, the TER values of 5-FU control significantly decreased over time to 5.5 § 0.1 £ 103 V/cm2 at 48 h, 3.4 § 0.1 £ 103 V/cm2 at 72 h, and 2.0 § 0.1 £ 103 V/cm2 at 96 h compared to DMEM controls (Fig. 5B; P < 0.05). All broths and EcN SNs together with 5-FU did not significantly change the resistance value compared to 5-FU control at all time-points measured (P > 0.05). FIG. 2. Representative histogram of reactive oxygen species (ROS) density demonstrating the gating of Dulbecco’s Modified Eagle Medium and 5-FU (2 mM) treated Caco-2 cells for 24 h (A) and 48 h (B) assessed by flow cytometry.
ROS generation between broths or EcN SNs and DMEM control at 48 h (Fig. 3B; P > 0.05). At 24 h the PD in ROS generation from Caco-2 cells treated with 5-FU increased significantly (12.3 § 1.7%) compared to the DMEM control (Fig. 3A; P < 0.05). This became more apparent at 48 h with 22.5 § 2.9% of PD in ROS generation resulting from 5-FU treatment (Fig. 3B; P < 0.05). All broths and EcN SNs (LBC, TSBC, MRSC, and M17C) in combination with 5-FU produced similar values of PD for ROS generation compared to 5-FU control with no significant difference among treatments at 24 h (Fig. 3B; P > 0.05). Broths (LB, TSB, and M17) and EcN SNs (LBC, TSBC, and M17C) cultured Caco-2 cells together with 5-FU increased PD in ROS generation compared to DMEM control to approximately 20%, which was consistent with 5-FU control (22.5 § 2.9%) (Fig. 3B). Moreover, broth MRS and EcN SNs MRSC together with 5-FU significantly reduced PD in ROS generation to approximately 10% compared to 5-FU control (Fig. 3B; P < 0.05).
Effects of Different Broths and EcN SNs on the Cell Barrier of Caco-2 Cells in the Presence and Absence of 5-FU Caco-2 cells were cultured in Transwells for 23 days prior to treatment to allow cells to form a tight cell barrier (Fig. 4). On day 23, TER values (9.5 § 0.3 £ 103 V/cm2) were significantly increased compared to day 1 (3.8 § 0.2 £ 103 V/cm2) (Fig. 4; P < 0.05). On Day 23, cells were treated with either
Antioxidant Capacity of Broths and EcN SNs The FRAP values of broths and EcN SNs are shown in Fig. 6. The ascorbic acid, FRAP value of 13.2 § 1.1, was used as a positive control. All media (DMEM, LB, TSB, MRS and M17) and all EcN SNs (LBC, TSBC, MRSC and M17C) recorded extremely low FRAP values (
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Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage a
b
c
Hanru Wang , Susan E. P. Bastian , Andrew Lawrence & Gordon S. Howarth
d
a
School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia b
School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Adelaide, Australia c
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Microbiology Department, SA Pathology at Women's and Children's Hospital, Adelaide, Australia d
School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia and Microbiology Department, SA Pathology at Women's and Children's Hospital, Adelaide, Australia Published online: 27 Jan 2015.
To cite this article: Hanru Wang, Susan E. P. Bastian, Andrew Lawrence & Gordon S. Howarth (2015): Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage, Nutrition and Cancer, DOI: 10.1080/01635581.2015.990570 To link to this article: http://dx.doi.org/10.1080/01635581.2015.990570
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Nutrition and Cancer, 0(0), 1–11 Copyright Ó 2015, Taylor & Francis Group, LLC ISSN: 0163-5581 print / 1532-7914 online DOI: 10.1080/01635581.2015.990570
Factors Derived From Escherichia Coli Nissle 1917, Grown in Different Growth Media, Enhance Cell Death in a Model of 5-Fluorouracil-Induced Caco-2 Intestinal Epithelial Cell Damage Hanru Wang School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia
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Susan E. P. Bastian School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Adelaide, Australia
Andrew Lawrence Microbiology Department, SA Pathology at Women’s and Children’s Hospital, Adelaide, Australia
Gordon S. Howarth School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, Adelaide, Australia and Microbiology Department, SA Pathology at Women’s and Children’s Hospital, Adelaide, Australia
We evaluated supernatants (SNs) from Escherichia coli Nissle 1917 (EcN) grown in commonly used growth media for their capacity to affect the viability of Caco-2 colon cancer cells in the presence and absence of 5-Fluorouracil (5-FU) chemotherapy. EcN was grown in Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17). Human Caco-2 colon cancer cells were treated with DMEM (control), growth media alone (LB, TSB, MRS, and M17) or EcN SNs derived from these 4 media, in the presence and absence of 5-FU. Cell viability, reactive oxygen species (ROS), and cell monolayer permeability were determined. EcN SN in LB medium reduced Caco-2 cell viability significantly, to 51% at 48 h. The combination of this EcN SN and 5-FU further reduced cell viability to 37% at 48 h, compared to 5-FU control. MRS broth and EcN SN in MRS, together with 5-FU, generated significantly lower levels of ROS compared to 5-FU control. However, all 5-FU treatments significantly disrupted the Caco-2 cell barrier compared to control; with no significant differences observed among any of the 5-FU treatments. EcN SNs (LBC) was most effective at decreasing the viability of Caco-2 cells. This could indicate a potential role for this EcN SN in chemoprevention for colon cancer. Received 4 April 2014; accepted in final form 29 October 2014. Address correspondence to Gordon S. Howarth, School of Animal and Veterinary Sciences, University of Adelaide, Roseworthy Campus, South Australia, 5371, Australia. Tel: C61 8 8313 7885. Fax: C61 8 8313 7972. E-mail: [email protected]
INTRODUCTION Colorectal cancer is the fourth most frequently diagnosed and the second most deadly type of cancer worldwide, with approximately 100,000 new cases diagnosed and 50,000 estimated deaths each year in the United States (1). To date, colon cancer cannot be treated effectively. Clinical trials have shown 5-fluorouracil (5-FU) to be effective as adjuvant chemotherapy for high-risk Stage II and III colon cancer (2). Nevertheless, chemotherapy is currently used as an effective method to treat colon cancer, although it commonly results in debilitating side-effects (e.g., mucositis). The severity of mucositis varies from mild to severe, characterized by pain, ulceration, and bleeding along the entire digestive tract. Indeed, mucositis may be so severe that death can result (3). Chemotherapy induces the generation of reactive oxygen species (ROS), which cause tissue damage (4). Chemotherapy also damages the DNA of epithelial basal cells leading to cell death (4). Combined, these biological reactions are responsible for the pathogenesis of mucositis (4). The development of new agents, which would not compromise the efficacy of existing chemotherapy regimens but would 1
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attenuate the intestinal damage induced by chemotherapy, could hold great potential for the future treatment of colon cancer. Investigations into the clinical application of probiotics such as lactobacillus and bifidobacterium for the potential prevention or treatment of intestinal disorders have been growing steadily (5,6). Mechanisms of probiotic action described to date include adhesion to the intestinal–lumen interface; enhancement of mucosal barrier function; competition with pathogens for receptor binding, nutrients and colonization; promotion of immune responses; elaboration of bacteriocins; and modulation of cell kinetics (6,7). Escherichia coli Nissle 1917 (EcN) was the first bacterium to be identified as a probiotic (8). EcN has been reported to exert therapeutic activity against several intestinal disorders such as inflammatory bowel disease (9), particularly the ulcerative colitis variant (9) and other forms of gut inflammation in humans (10). Moreover, EcN has been reported to improve mucosal integrity by modulating the tight junction molecule, zonula occludens-1 (ZO-1), thereby reducing intestinal permeability in the colon in mice following dextran sodium sulfate (DSS)-induced damage (11). In recent years, factors released from probiotics have created a new frontier of disease therapy as a result of their capacity to combat intestinal disorders in the absence of living probiotic bacteria (12,13). Probiotic-derived factors encompass proteins and other molecules released from living probiotics into their respective culture media (supernatants [SN]) and have been shown to exert beneficial biological and physiological properties in certain circumstances; although studies have been limited (6). For example, bacteriocins and reuterin released from probiotics have been shown to inhibit the adhesion and viability of known enteric pathogens such as grampositive and gram-negative bacteria as well as yeasts, fungi, and protozoa (7,14). Probiotic factors released from Lactobacillus rhamnosus GG (LGG) were reported to prevent alcoholinduced Caco-2 cell monolayer barrier dysfunction (15). To this end, 2 new proteins, p75 and p40, from LGG supernatants have been shown to play pivotal roles in reducing tumor necrosis factor alpha (TNF-a)-induced epithelial cell apoptosis and promoting the growth of epithelial cells (16). In addition, EcN SN partially protected the intestine from 5-FU-induced damage in rats and in IEC-6 cells (12,13). EcN SN has also revealed promise in the treatment of human gastrointestinal motility disorders (17). The potential utility of probiotic-derived factors in cancer chemotherapy is revealing some promise. In human studies, it has been reported that EcN SNs, as well as other metabolic factors derived from the probiotic formulation VSL#3 (Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus delbrueckii subsp. bulgaricus), significantly decreased cyclooxygenase-2 (COX-2) gene expression
induced by gastrin or the proinflammatory cytokine TNF-a (18). The inhibition of COX-2 expression is reported to be an important therapeutic approach in the prevention of colon carcinogenesis (18,19). Moreover, Propionibacterium freudenreichii ITG P9 strain fermented milk SN induced apoptosis of HGT-1 cancer cells (20). Short chain fatty acids (SCFAs) produced from certain bacterial strains such as Clostridium butyricum, have also been shown to exert anti-carcinogenic properties (6,21). The SCFA butyrate, has also been demonstrated to impose cell cycle arrest, differentiation and apoptosis in many tumor cell lines (21). In addition, butyrate has been found to inhibit the action of histone deacetylases, involved in tumorigenesis (21). Importantly, however, the composition and bioactivities of factors from the same bacterial strain could vary, depending on the composition of the growth medium. The resultant factors could, therefore, have differential and possibly disparate efficacies in the context of cancer treatment. For example, Streptococcus thermophilus grown in milk produced high levels of enzymes such as BCAA aminotransferase, ketol-acid reductoisomerase, and pyruvate formate-lyase (22). However, these enzymes were barely detectable when the same cells were grown in M17 medium. The primary aim of the current study was therefore to investigate the potential for EcN when grown in different growth media to produce SN derived factors able to differentially kill Caco-2 cells. Specifically, effects on Caco-2 cell viability, ROS generation, and cell monolayer permeability were sought. The second aim was to identify a mechanism of action for EcN SNs by determining effects on these end-points in the presence and absence of the antimetabolite chemotherapy drug, 5-FU.
METHODS EcN SN Preparation The method for preparing bacterial SNs has previously been described by Prisciandaro et al. (12,23). Briefly, E. coli Nissle 1917 (EcN) was purchased from Ardeypharm (Herdecke, Germany). EcN [25 £ 107 colony forming units (CFU)] was grown separately in 4 different broth growth media; Luria-Bertani (LB) (GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia), tryptone soya (TSB), de Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (the last 3 all from Oxoid, Adelaide, South Australia, Australia). EcN (80 mL) was incubated at 37 C for 24 h and reached a concentration of approximately 109 CFU/mL. EcN mixtures were then centrifuged at 6000 rpm for 20 min (HeraeusÒ MegafugeÒ 1.0, Thermo Fisher Scientific, Waltham, MApH). SN were then collected and buffered with HEPE to a final concentration of 20 mM. The pH was adjusted to 7.2 using HCl (3 M)/NaOH (3 M). SNs were then filtered through a 0.22-mm low protein-binding
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syringe driven filter (MillexÒ Syringe Filter Units, Merck KGaA, Darmstadt, Germany) and stored at -20 C. Each SN (0.5 mL) was transferred to a 1.5-mL Eppendorf tube and the total moisture content was removed by using a Savant SpeedVacÒ Concentrator (Savant SpeedVac SC110Ò , Thermo Fisher Scientific, Waltham, MA,) for at least 24 h. Dried SNs were then weighed and suspended in serum free Dulbecco’s Modified Eagle Medium (DMEM) to reach a final concentration of 100 mg/mL. All SNs were refiltered through a 0.22-mm syringe driven filter (MillexÒ Syringe Filter Units, Merck KGaA, Darmstadt, Germany) prior to use. All control growth media SNs were prepared the same way as EcN SNs without previously growing EcN. The resultant treatment groups were as follows: DMEM (control), LB, TSB, MRS, and M17; EcN SNs were LBC, TSBC, MRSC, and M17C. The “C” symbol indicates that each broth had been used to grow EcN.
Cell Culture Caco-2 human colorectal adenocarcinoma cells (passage 25-35) were cultured in medium containing DMEM supplemented with 10% (v/v) fetal calf serum (FCS) and 1% (v/v) antibiotics (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia). All cells were maintained at 37 C in a humidified incubator with 5% CO2 and grown in 75 cm2 vented tissue culture flasks (CELLSTARÒ , Greiner Bio-One, Frankenhausen, Germany). Culture medium was changed twice each week and passaged when 80% confluence had been reached.
Cell Viability Caco-2 cell viability was examined by MTT assay (24,25). Cells (5 £ 103 cells/mL; 100 uL) were placed in each well of a 96-well tissue culture plate for 48 h for attachment to the bottom of the well. After 48 h, the culture medium was replaced with serum-free DMEM (control) and media (100 mg/mL), EcN SNs (100 mg/mL) and/or 5-FU (0.5 mM). The cells were then cultured for either 24 or 48 h. MTT powder [3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide; Molecular ProbesÒ , Life Technologies, Mulgrave, Victoria, Australia] was dissolved in Dulbecco’s Phosphate Buffered Saline (DPBS) (GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) to reach a final concentration of 1 mg/mL. The MTT solution (50 mL) was then sterilized by 0.22-mm filtration and added to each well. After the cells were incubated at 37 C for 4 h, the medium was then replaced with 100 mL of dimethyl sulfoxide (Sigma-Aldrich, Sydney, Australia) for 15 min with minor shaking. Absorbance was read at 570 nm by a spectrophotometer (Tecan infinite 200Ò PRO, M€annedorf, Switzerland). Data were expressed as number of viable cells compared to the percentage of control cells treated with serum-free DMEM (25). Treatments were performed in
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triplicate, whereas the entire experiment was conducted 3 times (n D 9). Detection of ROS Through Flow Cytometry A modified assay using a CellROXÒ Oxidative Stress Reagent was performed to detect ROS by flow cytometry (C10444, Life Technologies, Mulgrave, Victoria, Australia). The cell-permeable CellROXÒ reagent is nonfluorescent while in a reduced state but exhibits a strong fluorescent signal upon oxidation, which can be detected with excitation and emission values of 508 and 525 nm (C10444 manual, Life Technologies, Mulgrave, Victoria, Australia). Caco-2 cells were grown in 12-well culture plates at a concentration of 7 £ 104 cells/mL for 48 h to allow attachment. Broths (LB, TSB, MRS, and M17) and EcN SNs including LBC, TSBC, MRSC, and M17C at a final concentration of 100 mg/mL and/or 5-FU at a final concentration of 2 mM were used to replace DMEM for a further 24 or 48 h. Cell preparation followed the method recommended by the manufacturer (C10444, Life Technologies, Mulgrave, Victoria, Australia) with minor adjustments based on other relevant publications (26). Briefly, after 24 or 48 h, all media were aspirated and cells harvested by trypsinization. Cells were then washed with 3 mL of DPBS and centrifuged at 1500 rpm for 2 min at room temperature (Eppendorf Centrifuge 5810, Hamburg, Germany). Supernatants were then discarded and cell pellets were resuspended with 1 mL of DPBS containing CellROXÒ reagent (1 mM) for 30 min at 37 C in the dark. Next, each cell suspension was gently mixed before analysis by flow cytometry (FACSCalibur, Becton Dickinson Biosciences, San Jose, CA), using a 488-nm laser with a 530/30 BP filter. Ten thousand events were collected for each treatment. Results were presented by histogram and data were analyzed by BD CellQuest ProTM software Ó 2002 (Becton Dickinson Biosciences, San Jose, CA).
Transepithelial Electrical Resistance Assay Caco-2 cells were harvested from a 75 cm2 vented tissue culture flask. Cell suspensions (200 mL) were added to the upper layer of 0.33 cm2 Transwell clear polyester permeable membranes (Corning Costar, Tewksbury, MA) at a density of 1 £ 105 cells/Transwell. Next, 600 mL of DMEM supplemented with 10% FCS and 1% antibodies (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) were added to the bottom well. Cells were incubated at 37 C in a humidified incubator with 5% CO2 for the duration of the study. Cells were cultured with DMEM supplemented with 10% FCS and 1% antibodies (penicillin, gentamicin, and streptomycin, GibcoÒ , Life Technologies, Mulgrave, Victoria, Australia) for the first 23 days to allow sufficient time to form a healthy monolayer barrier. Media were changed every 2 to 3 days prior to the beginning of the
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experiment. On the 23rd day, for control treatment groups, media were replaced with DMEM control, broth controls (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) at a concentration of 100 mg/mL on the upper layer of the Transwell and DMEM at the lower layer for all control groups. For 5-FU treatment groups, all media were replaced with DMEM control, broth controls (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) at a concentration of 100 mg/mL on the upper layer of the Transwell and 5-FU (2 mM) at the lower layer for all 5-FU treated groups. Transepithelial electrical resistance (TER) measurements were recorded prior to changes of media for the first 23 days and 24, 48, 72, and 96 h after adding treatments. Treatments were carried out in duplicate and repeated 3 times for the entire experiment (n D 6). Monolayer resistance was measured by a millicell-ERS volt-ohm meter (Millipore, Billerica, MA) with electrodes. Values were expressed as ohms per square centimeter (V/cm2) (12).
Ferric Reducing Antioxidant Power Assay The antioxidant activity of all treatments including DMEM control, broths (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) was measured by ferric reducing antioxidant power (FRAP) assay (27,28). Total moisture content in each treatment was removed by using a Speedvac (Servant Speedvac SC110). Dried samples were then weighed and suspended in Milli-Q water to reach a final concentration of 50 mg/mL. Ascorbic acid (0.1–1.0 mg/mL) was dissolved in Milli-Q water as a positive control. Ferrous sulphate prepared in Milli-Q water (0.1–1.0 mM) was used to generate a standard curve. FRAP reagent was prepared with 300 mM acetate buffer (pH 3.6), 10 mM TPTZ (2,4,6-Tripyridyl-s-Triazine) in 40 mM HCL, and 20 mM ferrous chloride at a ratio of 10:1:1 and kept in the dark. All chemicals were purchased from Sigma Aldrich (Sydney, Australia). Each sample (15 mL) was added in triplicate into each well of a nonsterile 96-well plate (Grenier Bio-one, Basel, Switzerland). FRAP reagent (150 mL) was immediately added into each sample. The plate was then kept in the dark with minor shaking for 4 min and read at 593 nm using a spectrophotometer (Tecan infinite 200Ò PRO, M€annedorf, Switzerland). A linear equation was generated based on the ferrous sulphate standard curve and Fe2C concentration (mM) in a sample was calculated based on this equation. FRAP values of test samples were expressed as mmol Fe2C per gram of sample weight (mmol/g). Treatments were performed in three replicates while the entire experiment was conducted 3 times (n D 9). Statistical Analysis Results are expressed as mean § SEM. IBM SPSS Statistics for Windows, version 20.0 (SPSS, Armonk, NY) was used for all statistical analyses. One-way analysis of variance with a
Tukey’s post hoc test was performed to determine the difference between means, with statistical significance set at P < 0.05.
RESULTS Effects of Different Broths and EcN SNs on Cell Viability of Caco-2 Cells in the Presence and Absence of 5-FU The cell viability of Caco-2 cells cultured for 24 h in broth LB and EcN SNs M17C was not significantly different compared to DMEM controls (100%) (Fig. 1A). However, the number of viable cells was significantly reduced to approximately 60–87% when Caco-2 cells were cultured in TSB, MRS, and M17 broths and EcN SNs (LBC, TSBC, and MRSC) (Fig. 1A; P < 0.05). Surprisingly, EcN SNs (LBC and MRSC) and broth (TSB and MRS) reduced the cell viability to approximately 60% compared to DMEM controls, with LBC producing the greatest decrease in cell viability (61.1 § 5.7%) among all treatments (Fig. 1A; P < 0.001). The cytotoxic effects were also observed in broths and EcN SNs at 48 h (Fig. 1B). Broths (LB, TSB, MRS and M17) and EcN SNs (LBC, MRSC and M17C), excluding EcN SN TSBC, significantly reduced cell viability to 51–78%, compared to DMEM controls (100%) (Fig. 1B; P < 0.05); whereas only TSBC did not significantly reduce cell viability, compared to DMEM controls (P > 0.05). More importantly, EcN SNs (LBC and MRSC) and MRS broth decreased cell viability to almost 50% compared to DMEM controls (P < 0.001) at 48 h (Fig. 1B). 5-FU treatment reduced viable cell numbers in all groups compared to DMEM treated controls. The cell viability of 5FU treated Caco-2 cells (5-FU control) was significantly reduced, to 79.3 § 1.0% at 24 h and 63.8 § 5.2% at 48 h, compared to DMEM control (100%; Fig. 1A and B; P < 0.05). All broths (LB, TSB, MRS, and M17) and EcN SNs (LBC, TSBC, MRSC, and M17C) together with 5-FU significantly decreased Caco-2 cell viability to approximately 55– 78% at 24 h and 38–60% at 48 h compared to DMEM controls (Fig. 1A and B; P < 0.05). TSB and MRS broths, excluding LB and M17, in combination with 5-FU further reduced cell viability to approximately 38–50%, compared to 5-FU controls (79.3 § 1.0%) at 24 h (Fig 1A; P < 0.05). Meanwhile, EcN SNs (LBC and MRSC), excluding TSBC and M17C, together with 5-FU further decreased the proportion of viable cells to approximately 37–60% compared to 5-FU controls (79.3 § 1.0%) at 24 h (Fig 1A; P < 0.001). At 48 h, LB, TSB, and M17 broths and EcN SNs (TSBC and M17C), together with 5-FU, tended to decrease Caco-2 cell viability to approximately 50–63%, consistent with 5-FU control (63.8 § 5.2%), although statistical significance was not attained (Fig. 1B; p D 1.00). However, EcN SNs (LBC and MRSC) and MRS broth, together with 5-FU, further reduced cell viability to approximately 37–43% compared
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FIG. 1. Combined effects of media, Escherichia coli Nissle 1917 (EcN) supernatants (SNs), and/or 5-fluorouracil (5-FU; mM) on the viability of Caco-2 cells for 24 h (Fig. 1A) or 48 h (Fig. 1B). Cells were treated with serum free Dulbecco’s Modified Eagle Medium (control), bacterial media alone [Luria-Bertani (LB), tryptone soya (TSB), Man Rogosa Sharpe (MRS), and M17 broth supplemented with 10% (v/v) lactose solution (M17)] and EcN SNs (LBC, TSBC, MRSC, and M17C) at a final concentration of 100 mg/mL, either alone or in combination with 5-FU (500 mM). Data are expressed as percentage of viable cells relative to untreated cell controls. Data are presented as means § SEM of three independent experiments (n D 9). Bar data not sharing the same letter are significantly different (P < 0.05). # indicates a significant difference compared to DMEM control in the groups without 5-FU. *indicates a significant difference compared to 5-FU control in all 5-FU treatment groups (P < 0.05).
with 5-FU control (63.8 § 5.2%) at 48 h (P < 0.001). LBC together with 5-FU produced the lowest proportion of viable cells (37.4 § 3.4%) among all the treatments tested at 48 h (P < 0.001). Effects of Different Broths and EcN SNs on the Generation of ROS from Caco-2 Cells in the Presence and Absence of 5-FU The intensity histogram of ROS generation under treatments of DMEM (control) and 5-FU (5-FU control) for 24 h and 48 h is shown in Fig. 2A and 2B, respectively. The histogram was gated as M1. The percentage of ROS generation was
calculated within the area of gate M1 by BD CellQuest ProTM software Ó 2002 (Becton Dickinson Biosciences, San Jose, CA). The percentage difference (PD) in ROS generation from Caco-2 cells cultured in different broths and EcN SNs and/or 5-FU as compared to Caco-2 cells cultured in DMEM, is shown in Fig. 3A (24 h) and B (48 h). ROS generation from Caco-2 cells cultured in broths (LB, TSB, MRS, and M17) and EcN SNs (TSBC and MRSC) generated similar levels of ROS (approximately minus 5%) compared to DMEM controls at 24 h (Fig. 3A). However, EcN SNs (LBC and M17C) reduced ROS generation (approximately minus 9%) compared to the DMEM control at 24 h (Fig. 3A; P < 0.05) There were no significant difference in
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DMEM, broths, EcN SNs, and/or 5-FU. TER values 24, 48, 72, and 96 h after adding treatments are shown in Fig. 5. The TER value of the DMEM treatment, broths and EcN SNs decreased gradually over time from 9.8 § 0.1 £ 103 V/cm2 at 24 h to 4.9 § 0.1 £ 103 V/cm2 at 96 h (Fig. 5A). The resistance value of all broths and EcN SNs did not significantly change, compared to DMEM controls at all time-points measured (Fig. 5A; P > 0.05). 5-FU control, broths C 5-FU and EcN SNs C 5-FU did not change the resistance value at 24 h compared to DMEM control (Fig. 5B; P > 0.05). However, the TER values of 5-FU control significantly decreased over time to 5.5 § 0.1 £ 103 V/cm2 at 48 h, 3.4 § 0.1 £ 103 V/cm2 at 72 h, and 2.0 § 0.1 £ 103 V/cm2 at 96 h compared to DMEM controls (Fig. 5B; P < 0.05). All broths and EcN SNs together with 5-FU did not significantly change the resistance value compared to 5-FU control at all time-points measured (P > 0.05). FIG. 2. Representative histogram of reactive oxygen species (ROS) density demonstrating the gating of Dulbecco’s Modified Eagle Medium and 5-FU (2 mM) treated Caco-2 cells for 24 h (A) and 48 h (B) assessed by flow cytometry.
ROS generation between broths or EcN SNs and DMEM control at 48 h (Fig. 3B; P > 0.05). At 24 h the PD in ROS generation from Caco-2 cells treated with 5-FU increased significantly (12.3 § 1.7%) compared to the DMEM control (Fig. 3A; P < 0.05). This became more apparent at 48 h with 22.5 § 2.9% of PD in ROS generation resulting from 5-FU treatment (Fig. 3B; P < 0.05). All broths and EcN SNs (LBC, TSBC, MRSC, and M17C) in combination with 5-FU produced similar values of PD for ROS generation compared to 5-FU control with no significant difference among treatments at 24 h (Fig. 3B; P > 0.05). Broths (LB, TSB, and M17) and EcN SNs (LBC, TSBC, and M17C) cultured Caco-2 cells together with 5-FU increased PD in ROS generation compared to DMEM control to approximately 20%, which was consistent with 5-FU control (22.5 § 2.9%) (Fig. 3B). Moreover, broth MRS and EcN SNs MRSC together with 5-FU significantly reduced PD in ROS generation to approximately 10% compared to 5-FU control (Fig. 3B; P < 0.05).
Effects of Different Broths and EcN SNs on the Cell Barrier of Caco-2 Cells in the Presence and Absence of 5-FU Caco-2 cells were cultured in Transwells for 23 days prior to treatment to allow cells to form a tight cell barrier (Fig. 4). On day 23, TER values (9.5 § 0.3 £ 103 V/cm2) were significantly increased compared to day 1 (3.8 § 0.2 £ 103 V/cm2) (Fig. 4; P < 0.05). On Day 23, cells were treated with either
Antioxidant Capacity of Broths and EcN SNs The FRAP values of broths and EcN SNs are shown in Fig. 6. The ascorbic acid, FRAP value of 13.2 § 1.1, was used as a positive control. All media (DMEM, LB, TSB, MRS and M17) and all EcN SNs (LBC, TSBC, MRSC and M17C) recorded extremely low FRAP values (
