Anti-proliferative effects of Enterococcus strains isolated from fermented dairy products on different cancer cell lines

journal of functional foods 11 (2014) 363–374

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Anti-proliferative effects of Enterococcus strains isolated from fermented dairy products on different cancer cell lines Babak Haghshenas a, Yousef Nami a, Norhafizah Abdullah b,*, Dayang Radiah b, Rozita Rosli a, Ahmad Yari Khosroushahi c,d,** a

Institute of Biosciences, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Chemical and Environmental Engineering Department, Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia c Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran d Department of Pharmacognosy, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran b

A R T I C L E

I N F O

A B S T R A C T

Article history:

Among the lactic acid bacteria, Enterococcus strains can be utilized as probiotic and preser-

Received 15 March 2014

vative agents. Enterococcus strains from traditional Iranian dairy products were isolated,

Received in revised form 27

identified by sequencing 16s rDNA gene, and biologically characterized in this study. Primary

September 2014

assessments, including low pH and high bile salt tolerance tests and experiments on an-

Accepted 2 October 2014

tagonistic activity against pathogens and antibiotic susceptibility, verified the probiotic property

Available online

of strains. The secreted metabolites from the strains and Taxol, as an anti-cancer drug, were analyzed through MTT assay to investigate their cytotoxicity in different cancer (MCF-7, HeLa,

Keywords:

HT29, and AGS) and normal human (HUVEC) cell lines. Results revealed the anticancer char-

Apoptosis

acteristics of the secreted metabolites of E. durans 39C against all cancer cell lines, similar

Anticancer

to Taxol. The metabolites did not exhibit cell toxicity in the normal cell line. Fluorescent

Bioactive protein

microscopy and flow cytometry confirmed that apoptosis is the main cytotoxic mecha-

Probiotics

nism of E. durans 39C secretion metabolites. © 2014 Elsevier Ltd. All rights reserved.

1.

Introduction

Probiotics as live microorganisms can equilibrate the microbial population in the gastrointestinal tract and thereby maintain the health of the host. Hence, an increasing number of doctors and nutritionists worldwide prescribe probiotic products (Guarner et al., 2008). Recent studies have revealed that

the majority of probiotic bacteria belong to the lactic acid bacteria (LAB) group. LAB allows for fermentation and has been utilized in food preservation for thousands of years. The LAB group includes the genera Oenococcus, Lactococcus, Streptococcus, Enterococcus, Leuconostoc, Lactobacillus, and Pediococcus (Guarner et al., 2008). Following Lactobacillus and Streptococcus genera, the third largest genus in the LAB group is Enterococcus (Franz, Huch,

* Corresponding author. Chemical and Environmental Engineering Department, Faculty of Engineering, University Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. Tel.: +60 3 89466295; fax: +60 3 86567120. E-mail address: [email protected] (N. Abdullah). ** Corresponding author. Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah Street, P.O.Box 51664-14766, Tabriz, Iran. Tel.: +98 41 33372250-1; fax: +98 41 33344798. E-mail address: [email protected] (A.Y. Khosroushahi). http://dx.doi.org/10.1016/j.jff.2014.10.002 1756-4646/© 2014 Elsevier Ltd. All rights reserved.

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Abriouel, Holzapfel, & Gálvez, 2011). Enterococcus species are catalase negative, Gram-positive, and have low guanine/ cytosine content in their genome. They do not sporulate and can produce a large amount of lactic acid as the end product after sugar fermentation (Cebrián et al., 2012). A probiotic must have a number of characteristics to be effective in improving its host’s health; such characteristics include resistance to gastrointestinal conditions, antipathogenicity, and high susceptibility to antibiotics (Biradar, Bahagvati, & Shegunshi, 2005). Enterococcus species are resistant to undesirable conditions and can thus be isolated from different environments, such as oceans, sewages, soil, plants, animals, food, and dairy products (Franz et al., 2011). Moreover, the use of Enterococcus species in probiotic products has recently increased because of their positive effects on human health (Rubio, Bover-Cid, Martin, Garriga, & Aymerich, 2013). Several Enterococcus species, such as E. faecium, show high healthpromoting capacities and have been utilized in commercial probiotic products, such as Fargo 688s, Cylactins, ECOFLOR, and Symbioflor. These products are prescribed for diseases, such as irritable bowel syndrome, recurrent chronic sinusitis, and bronchitis (Cebrián et al., 2012; Foulquié Moreno, Sarantinopoulos, Tsakalidou, & De Vuyst, 2006; Hadji-Sfaxi et al., 2011). Probiotics as health-promoting microorganisms exhibit different therapeutic activities. Among these activities, anticarcinogenic activity is the most interesting and has been linked to probiotics (Rafter, 2002). The most common mechanisms for the anti-proliferative effects of probiotics are reduction in carcinogenic enzymes/compounds, production of fatty acids/antimutagenic substances, and modulation of the immune system (Rafter, 2003). Traditional dairy products from the western Iran are prepared according to traditional methods employed in this region. No antibiotics are used in animal food, and the acidity of products is higher than those of commercial dairy products. Meanwhile, cancer epidemiology has a lower number of recorded cases in the rural areas of this region (Kermanshah Province) compared with other areas in Iran (Mirmomeni, Mohammadi, Sisakhtnezhad, Hashemi, & Nazari, 2009; Najafi et al., 2011). This difference could be related to the daily dietary intake of people. In this study, new Enterococcus strains with high probiotic capability were identified and isolated from traditional dairy products originally collected from Kermanshah Province in western Iran. The effects of the secretion metabolites of the isolated strains were evaluated by screening the total secretions in various human and normal cancer cell lines from the viewpoint of anti-proliferative and possible cytotoxicity efficacies.

2.

Materials and methods

2.1.

Sampling and isolation of acid/bile resistant bacteria

A total of 150 samples of traditional dairy products, including cheese, yogurt, curd, shiraz (a kind of dairy product in west of Iran), and tarkhineh were prepared. Two grams of each dairy sample was suspended in a 10 mL sodium citrate solution

(pH 7.0) and homogenized with a Stomacher 400 Circulator (Seward Laboratory Systems Inc, Bohemia, NY, USA) for 10 min. Afterward, 1 mL of the samples was added to 9 mL of de Man Rogosa Sharpe (MRS) broth (Merck, Germany) containing 5% CO2. After 24 h of anaerobic growth (37 °C), the harsh conditiontolerant strains were isolated. For this purpose, 0.05 mL of their growth solutions were homogenized in 1 mL of PBS supplemented with 0.3% (w/v) oxgall salt (pH 3) and incubated for 3 h at 37 °C. The treated samples were centrifuged at 4000 × g for 10 min after incubation. The precipitated bacteria were transferred into 10 mL MRS broth and then incubated at 37 °C for 24 h. Finally, 0.05 mL of the diluted solution was spread for 48 h on MRS agar-based media (Merck, Germany) (Gilliland, Staley, & Bush, 1984; Mirzaei & Barzgari, 2012). The single colonies on the growth agar plate were selected and transferred to 10 mL of broth culture medium for 24 h at 37 °C. The isolates were stored in 25% (w/v) glycerol at −70 °C for further assessment.

2.2.

DNA extraction

The single colonies of isolated bacteria were cultured in 10 mL of broth (MRS) at 37 °C overnight. Afterward, 1 mL of each bacterial culture was centrifuged at 10,000 × g for 5 min. The supernatant was outpoured, and the cell plate was washed with dH2O. The bacterial cell plate was re-suspended in 750 µL of GTE (glucose–Tris–EDTA) and incubated for 1 h at 37 °C. Approximately 50 µL of sodium dodecyl sulphate (SDS) solution (10% (w/v)) and 50 µL of proteinase K were added. The mixture was incubated again for 1 h at 65 °C. An equal volume of chloroform–isoamylalcohol (24:1, v/v) was added to each mixture. The mixtures were then centrifuged at 10,000 × g for 30 min, and the two phases were separated from each other. The upper layer was transferred into clean tubes, and an equal volume of each isopropanol solution containing sodium acetate (3 M) was added to each tube. The resulting mixture was then incubated at −20 °C for 20 min. The genomic DNA was precipitated by centrifugation at 10,000 × g for 30 min at 4 °C. The DNA pellets were air-dried and then dissolved in 50 µL of DNase free water. The dissolved DNA were treated by 10 µL of RNase and kept in a fridge for future assessment (Leenhouts, Kok, & Venema, 1990).

2.3.

Amplification and sequencing of 16S rDNA gene

The amplification of 16S rDNA fragments was conducted in a thermal cycler PTC 200 (MJC research, Waltham, USA) with a pair of Enterococcus-specific universal primers (Hal6F/Hal6R) (F: 5′-AGAGTTTGATCMTGGCTCAG-3′ and R: 5′-TACCTTGTTAG GACTTCACC-3′) that have been previously described in (Mirzaei & Barzgari, 2012). The PCR program cycles were set as follows: denaturation at 95 °C for 4 min; 32 cycles at 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 95 s; and final extension at 72 °C for 5 min. The amplified products were electrophoresed at a constant voltage of 60 V for 1 h and purified with QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The amplified 16S rDNA fragments (1500 bp) were sequenced with Macrogen (Korea). The sequences were then analyzed with the BLAST program of the National Center for Biotechnology Information

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(http://blast.ncbi.nlm.nih.gov/Blast.cgi), in which the isolated bacteria were identified at the strain level.

2.4.

Survival in acidic and bile salt conditions

A total of 100 µL of respective stock cultures of the probiotic candidates was incubated in a 10 mL growth medium (MRS broth) at 37 °C for 24 h. Each cell culture medium was centrifuged at 4000 × g for 10 min; the supernatants were removed, and the cell plates were re-suspended for 3 h in 1 mL of PBS (pH 2.5) at 37 °C (Conway, Gorbach, & Goldin, 1987). To determine the survival rates of the isolates in high bile concentrations, 100 µL of stock cultures of the isolated bacteria was incubated in 10 mL of MRS growth medium at 37 °C for 24 h. The respective bacterial cultures (100 µL) were similarly re-suspended in 10 mL of MRS-THIO broth containing MRS supplemented with 0.3% (w/v) oxgall (Sigma-Aldrich, St. Louis, MO, USA, pH 7.0) and 0.2% (w/v) sodium thioglycolate (SigmaAldrich, USA) for 3 h at 37 °C (Walker & Gilliland, 1993). The treated cells were separated in a related agar medium, and bacteria maintenance was evaluated through pour plate technique on MRS agar at time points of 0 and 3 h of incubation. The survival rates for low pH and bile resistance were calculated with the following equation: survival rate (%) = (log cfu N1/log cfu N0) × 100%. Total clones were displayed by N1 after treatment with the acid and bile salt, whereas total clones were displayed by N 0 before incubation in low pH and bile salt conditions.

2.5.

Antimicrobial activity assay of isolates

Modified agar diffusion well method, which was previously described by Bauer, Kirby, Sherris, and Turck (1966), was employed to determine the antimicrobial activities of the isolated bacteria against 14 clinically important human pathogens (Table 2). The isolated strains cultured overnight in MRS broth medium were centrifuged at 12,000 × g for 10 min. Then, 2 mL of the supernatant was filtered with Nalgene® syringe filter units (0.2 µm). A total of 50 µL of each filtrate was added to 7 mm diameter wells on Mueller–Hinton agar plates (Sigma-Aldrich, USA), which were incubated overnight before hand using indicator pathogens at 37 °C. The isolated active supernatants had low pH in certain cases. Thus, the pH of the isolated active supernatants was adjusted by adding NaOH to the physiological solution (pH 7.2) in the experiment. After overnight incubation of the plates at 37 °C, the clear zones around each well were measured and regarded as positive antibacterial activity. According to the diameter of the inhibition zone, the anti-pathogen activity was divided into strong (diameter ≥20 mm), moderate (20 mm ≤ diameter ≥ 10 mm), and weak (diameter ≤10 mm) (Bauer et al., 1966; Nami et al., 2014a, 2014b).

2.6.

Antibiotic susceptibility assays of isolates

Modified disc diffusion method was applied with nine clinically important antibiotics to determine the antibiotic susceptibility of each isolated strain (Table 3). The isolated strains in the anaerobic condition were incubated in MRS broth medium at 37 °C for 24 h. Then, 50 µL of the diluted cultures (approximately 106–107 viable cells) was

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diffused onto Mueller–Hinton agar. After spreading each strain on the Mueller–Hinton agar plates, antibiotic discs purchased from Padtan Teb Co. (Tehran, Iran) were manually placed on the plates by using sterilized forceps. The plates were subjected to incubation for 24 h at 37 °C, and the clear zones around each disc were measured after incubation (Domig et al., 2007). Based on the areola diameters, the guidelines of the producer of the antibiotic discs, and recommended standards (Performance Standards for Antimicrobial Susceptibility Testing from Clinical and Laboratory Standards Institute, Wayne, PA, CLSI, 2007), the strains were grouped into sensitive, intermediate, and resistant from the viewpoint of antibiotic susceptibility (CLSI, 2007).

2.7.

Bacterial secreted metabolite preparation

By measuring the bacterial OD, 1–10 × 106 CFU/mL (standard number of viable probiotic cells) was selected as the appropriate and ready-to-use cell culture for the MTT assay. The active bacterial supernatants were separated via the centrifugation of ready-to-use cell cultures at 10,000 × g for 10 min at 4 °C. The isolated active supernatants have low pH. Thus, the pH of the isolated active supernatants was adjusted by adding NaOH to the physiological solution (pH 7.2) in the cytotoxicity assay experiments. The pH-adjusted bacterial supernatants were carefully filtered with Nalgene syringe filter units (0.22 µm), and 10 concentration rates (5–50 µg/mL) were screened in various cancer and normal cell lines (Foo et al., 2003).

2.8.

Cell line growth

Human cervix cancer (HeLa), human gastric cancer (AGS), human breast cancer (MCF-7), human colon cancer (HT29), and human normal (human umbilical vein endothelial cells or HUVEC) cell lines were obtained from Pastor Institute, Tehran, Iran. All cancerous/normal cells were cultured in RPMI-1640 medium. The media were supplemented with 100 IU/mL penicillin, 10 µg/mL streptomycin, and 10% (v/v) fetal bovine serum at similar incubation conditions for all cells (37 °C, 95% humidity, and 5% CO2).

2.9.

MTT assay

For the MTT assay, the cancer and normal cell lines were plated on a 96-well microplate (Nunc, Roskilde, Denmark). Each well was seeded with 1.2 × 104 cells in 150 µL of growth medium. After 24 h of post seeding (40–60% confluency), different concentrations (5–50 µg/mL) of the filtered probiotic supernatants (using 0.22 µm Nalgene syringe filter units) were administered to each well to achieve a total volume of 200 µL. All treated cells were then incubated for 12, 24, and 48 h. After incubation, 50 µL of the MTT solution (5 mg/mL in PBS) was administered to each microplate well. The plates were again incubated for 4 h in 5% CO 2 at 37 °C in dark conditions. Formazan crystals created with MTT-exposed live cells were dissolved by adding 200 µL of dimethyl sulphoxide (DMSO) and 25 µL of Sorenson’s glycine buffer (0.1 M glycine and 0.1 M NaCl, pH 10.5) into each well. The wells were then incubated at 37 °C while being gently shaken for 20 min. The adsorption of each

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well was measured with a µQuant ELISA Reader (Biotek, ELx 800, Winooski, VT, USA) at 570 nm.

2.10.

pronase by heat denaturation, the treated supernatants were assessed by MTT assay, similar to the untreated supernatants.

Morphological apoptosis assessment 2.13.

All treated and untreated cells were cultured on sterile cover slip sets at the bottom of each 96-well cell culture plate (cell density 120 × 104/each well). The cells were fixed with 4% paraformaldehyde (PFA) for 5 min at the end of each time point (24 and 48 h). The fixed cells were permeabilized with 0.1% Triton X-100 for 5 min. Then, 50 µL of the diluted 4′,6-diamidino2-phenylindole (DAPI; 1:2000) and its buffer (FOXP3 Perm Buffer, BioLegend, San Diego, USA) were added to each well. The cells were washed with PBS (pH 7.2) after 5 min of incubation at room temperature. The stained cells on the cover slips were reversely placed on slides and analyzed under a fluorescent microscope (Olympus BX61, Center Valley, PA, USA) equipped with a U-MWU2 fluorescence filter (excitation filter BP 330– 385, dichromatic mirror DM 400, and emission filter LP 420).

2.11.

Flow cytometry

The flow cytometry assessments were conducted on AGS cells treated with bacterial secreted metabolites (40 µg/mL) for periods of 24 and 48 h using the BD Biosciences Annexin V-FITC Kit (San Jose, CA, USA). After 24 and 48 h of incubation and after being washed twice with cold PBS (pH 7.2), the cells detached by trypsin were separated from their supernatant by centrifugation at 2000 × g for 5 min at 28 °C. The cell pellets were resuspended in one time binding buffer at a concentration of 1 × 106 cells/mL and transferred to new tubes (5 mL). Then, 100 µL of the cell suspension was mixed with 10 µL of propidium iodide (PI) solution and 5 µL of Annexin V-FITC. Thereafter, the cells were placed in 5 mL culture tubes for 15 min at room temperature in dark conditions. A total of 400 µL binding buffer (one time) was added again to each tube. The assessments were conducted with a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA). The analysis of 100,000 cells was accomplished at a rate of 1000 cells/s. Quadrant setting was conducted with the untreated cell line as the negative control. Data analysis was performed with CellQuest Pro software (BD Biosciences, San Jose, CA, USA).

2.12. Pronase test to identify the nature of the supernatant

All experiments were based on a completely random design, and all data were analyzed through one-way ANOVA with an appropriate multiple-comparison test using SPSS 19.0 software. All graphs were prepared in Microsoft Office Excel. A P value ≤0.01(**P ≤ 0.01) was considered statistically significant. Data were presented as the mean ± standard deviation of three measurements.

3.

Results

3.1.

Amplification and sequencing of 16S rDNA

The sequence of the 16S rDNA gene fragments (1500 bp) of three isolated bacteria was blasted with deposited sequences in the GenBank of the NCBI site. Based on the blasting results, the three isolated bacteria were molecularly identified as E. mundtii 50H, E. durans 39C, and E. faecalis 13C isolated from shiraz, yogurt, and curd, respectively.

3.2.

Low pH and bile salt tolerance test

The survival rates for E. mundtii 50H, E. durans 39C, and E. faecalis 13C before and after incubation in low pH/high bile salt conditions are presented in Table 1. All the selected strains retained their viability even after 3 h of exposure to pH 2.5 and 0.3% (w/ v) oxgall. Notably, a broad variation in the survival rates was observed at these conditions. E. mundtii 50H, E. durans 39C, and E. faecalis 13C displayed 78, 82, and 73% survival rates, respectively, after incubation at a low pH condition. Meanwhile, all three isolated strains displayed high tolerance to bile salt conditions, which is 14–25% higher than their low pH tolerance. The survival rates of E. mundtii 50H, E. durans 39C, and E. faecalis 13C strains in bile salt conditions were 98, 96, and 98%, respectively (Table 1).

3.3.

To evaluate the nature of the secreted metabolites (active proteins), pronase (Roche Applied Science, Penzberg, Germany) was added to the supernatants at a concentration of approximately 1 mg/mL, and the mixture was incubated at 37 °C for 30 min for protein content digestion. After the deactivation of

Statistical analysis

Antimicrobial activity of isolates

E. mundtii 50H, E. durans 39C, and E. faecalis 13C displayed significant antagonistic activities against indicator pathogens (Table 2). E. mundtii 50H showed the most efficient antimicrobial activity and inhibited the growth of 11 indicator pathogens, including P. aeruginosa, C. albicans, S. marcesens, E. faecalis, E. coli (0157), E. coli (026), S. typhimurium, S. aureus, L. monocytogenes,

Table 1 – The survival rates of isolated strains after 3 h incubation at pH 2.5 and 0.3% bile salt. Bacteria

E. mundtii 50H E. durans 39C E. faecalis 13C

pH 2.5

0.3% Bile salt

0 h (log cfu/mL)

3 h (log cfu/mL)

Survival rate (%)

0 h (log cfu/mL)

3 h (log cfu/mL)

Survival rate (%)

8.683 8.291 8.900

6.772 6.798 6.497

78 82 73

8.434 8.394 8.687

8.265 8.058 8.513

98 96 98

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Table 2 – The inhibitory effect of isolated strains against pathogenic microorganisms. Pathogens

Origin

Diameter of inhibition zone (mm)

Pseudomonas aeruginosa Candida albicans Serratia marcesens Enterococcus faecalis Staphylococcus saprophyticus Streptococcus mutans Escherichia coli (0157) Salmonella typhimurium Staphylococcus aureus Escherichia coli (026) Bacillus cereus Listeria monocytogenes Klebsiella pneumoniae Shigella flexneri

PTCC 1181 PTCC 5027 (ATCC 10231) PTCC 1187 (Native strain) PTCC 1394 PTCC 1440 (CIP 76.125) PTCC 1683 (ATCC 35668) PTCC 1276 ATCC 14028 ATCC 25923 Native strain PTCC 1539 (ATCC 11778) PTCC 1163 PTCC 1053 (ATCC 10031) PTCC 1234 (NCTC 8516)

E. mundtii 50H

E. durans 39C

E. faecalis 13C

16.7 ± 0.3M 13.0 ± 0.0M 14.0 ± 1.0M 12.3 ± 1.2M 0.0 ± 0.0W 0.0 ± 0.0W 13.3 ± 0.3M 13.3 ± 0.3M 13.0 ± 0.0M 15.7 ± 0.3M 0.0 ± 0.0W 17.0 ± 0.0M 13.3 ± 1.2M 14.0 ± 0.6M

15.0 ± 0.6M 0.0 ± 0.0W 11.0 ± 0.0M 0.0 ± 0.0W 0.0 ± 0.0W 12.3 ± 1.2M 12.0 ± 0.0M 0.0 ± 0.0W 13.7 ± 1.2M 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 13.0 ± 1.0M 11.3 ± 0.7M

15.3 ± 0.7M 0.0 ± 0.0W 13.0 ± 0.6M 0.0 ± 0.0W 14.0 ± 0.6M 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 0.0 ± 0.0W 12.7 ± 1.2M 11.0 ± 0.0M

Values are mean ± standard error. S (strong r ≥ 20 mm), M (moderate r < 20 mm and >10 mm), and W (weak ≤10 mm). CIP: Collection of Bacteries de l’Institute Pasteur, Paris, France; ATCC: American Type Culture Collection, Virginia, USA; NCTC: National Collection of Type Cultures, London, UK. PTCC: Persian Type Culture Collection, Tehran, Iran.

K. pneumonia, and S. flexneri. Meanwhile, E. durans 39C exhibited an overall good antagonistic activity and inhibited the growth of P. aeruginosa, S. marcesens, S. mutans, E. coli (0157), S. aureus, K. pneumonia, and S. flexneri. E. faecalis 13C showed a moderate antimicrobial activity and inhibited the growth of P. aeruginosa, S. marcesens, S. saprophyticus, K. pneumonia, and S. flexneri (Table 2).

3.4.

Antibiotic susceptibility of isolates

The antibiotic susceptibility patterns were species specific. E. durans 39C displayed desirable susceptibility and was sensitive or semi-sensitive to all nine selected antibiotics, including vancomycin, chloramphenicol, erythromycin, tetracycline, gentamycin, clindamycin, penicillin, sulfamethoxazol, and ampicillin. Meanwhile, E. mundtii 50H displayed high susceptibility to eight out of nine antibiotics and was resistant to erythromycin only. E. faecalis 13C was sensitive to tetracycline, gentamycin, clindamycin, and sulfamethoxazol and resistant to chloramphenicol, erythromycin, ampicillin, penicillin, and vancomycin (Table 3).

3.5. lines

Anti-proliferative effect on cancer and normal cell

The time- and dose-dependent anti-proliferative effects of E. durans 39C on various cancer cells revealed that the cell viability of all treated cancer cell lines decreased gradually when incubation time and applied dose increased. The most significant anti-proliferative effect was observed in the last three highest doses (40–50 µg/mL) after 48 h (Fig. 1). Meanwhile, the anti-proliferative activities of E. faecalis 13C secretions were only detected in the AGS cancer cell line. E. mundtii 50H did not exhibit any strong anti-proliferative effect on various cancer cell lines. The variables showed significant differences (P ≤ 0.01) in anticancer activity at 48 h (treatment period) at 40 µg/mL concentration. Thus, these variables were selected for further assessment of E. durans 39C metabolites in various cancer and normal cell lines (Fig. 2). The cytotoxic assessments were continued with Taxol-treated groups as the positive control and untreated cell lines as negative control. The results were compared with those for the secreted metabolites of the reference

Table 3 – Antibiotic susceptibility of isolated strains against the high consumption antibiotics. Antibiotics

Diameter of inhibition zone (mm) C

TE

ER

AM

GE

CC

SLX

P

V

E. mundtii 50H E. durans 39C E. faecalis 13C

18S 22S 0R

20S 30S 28S

0R 20I 0R

22S 25S 0R

12S 13S 15S

30S 20S 21S

15I 22S 18S

25S 26S 0R

30S 17S 0R

C, chloramphenicol; TE, tetracycline; ER, erythromycin; AM, ampicillin; GE, gentamycin; CC, clindamycin; SLX, sulfamethoxazol; P, penicillin; V, vancomycin. Erythromycin results based on R ≤ 13 mm; I: 13–23 mm; S ≥ 23 mm. Gentamycin results based on R ≤ 6 mm; I: 7–9 mm; S ≥ 10 mm. Vancomycin results based on R ≤ 12 mm; I: 12–13 mm; S ≥ 13 mm. I: intermediate (zone diameter, 12.5–17.4 mm); R: resistant (zone diameter, ≤12.4 mm); S: susceptible (zone diameter, ≥17.5).

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Fig. 1 – The cytotoxic effects of isolated E. durans 39C secretions on different cancer cell lines at three time points 12, 24, and 48 h. Panels represent (A) MCF-7, (B) AGS, (C) HeLa and (D) HT29. 0% concentration: RPMI and MRS were used as control. Error bars represent the standard deviation of each mean.

E. durans strain (E. durans LMG 10746T). The results revealed that all cancer cells lines were significantly (P > 0.01) inhibited by E. durans 39C secretions compared with the untreated cancer cell line and reference E. durans strain as control. The cell proliferation percentages of cancerous cells treated with E. durans 39C metabolites during incubation time were found to be 25, 33, 26, and 37% for HeLa, AGS, HT29, and MCF-7 cells, respectively. No significant difference (P > 0.01) was detected between the cytotoxic effects of E. durans 39C metabolites and Taxoltreated groups (Fig. 2). The cell proliferation percentage of HUVEC cells treated with E. durans 39C metabolites during incubation time was found to be 81%. Cell viability in the treated HUVEC cells did not show a significant difference (P ≤ 0.01) between the isolates and secretion metabolites of treated control groups (E. durans LMG 10746T) and untreated cells. However, the cell proliferation percentage of HUVEC cells treated with Taxol metabolites during incubation time was 33% (Fig. 2).

3.6.

Morphological apoptosis assessment

The AGS cell lines treated with metabolites secreted by E. durans 39C (40 µg/mL) for 24 and 48 h showed apoptosis symptoms, such as cell shrinkage, membrane blebbing, nucleus fragmentation, and apoptotic body formation; necrotic bodies were rarely observed. Hence, apoptosis was induced more often than necrosis. The number of apoptotic cells with fragmented and

condensed nucleus was significantly higher than that of normal cells (P ≤ 0.01). However, none of the distinctive apoptotic features were observed in the untreated AGS cell lines (Fig. 3A). Cell shrinkage (early apoptosis) and apoptotic bodies (late apoptosis) were the predominant apoptosis signals in the AGS cells treated with E. durans 39C secretions after 24 and 48 h, respectively (Fig. 3B, C).

3.7.

Quantitative apoptosis assessment

E. durans 39C secretions (40 µg/mL) after 24 h showed 39.83% early apoptosis, 19.74% late apoptosis, and 2.54% necrosis (Fig. 4B). By contrast, the untreated cells showed <1% cell death (Fig. 4A). A total of 0.29% of the untreated cells showed early apoptosis, 0.36% exhibited late apoptosis, and 0.28% displayed necrosis at 24 h; 99.07% of the cells were viable. In consideration of these results, early apoptosis is considered the main phenomenon involved with cell death in the treated samples (24 h). The untreated cell lines showed 0.34% early apoptosis, 4.05% late apoptosis, and 2.73% necrosis at 48 h, whereas 92.88% of cells were viable (Fig. 4C). The AGS cells treated with E. durans 39C secretions after 48 h displayed 28.14% early apoptosis, 48.24% late apoptosis, and 1.87% necrosis (Fig. 4D). The flow cytometry results revealed that no significant differences (P < 0.01) exist in necrosis induction by E. durans 39Csecreted metabolites between 24 and 48 h in the AGS cell line.

journal of functional foods 11 (2014) 363–374

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Fig. 2 – The cytotoxic effects of E. durans 39C secretions (40 µg/mL) and Taxol on several cancer and normal cell lines for 48 h. Asterisks illustrate the significant differences (**P ≤ 0.01). Error bars represent standard deviation of each mean. CON: untreated cancer cell line. MRS: MRS treated cancer cell line. Dla: E. durans LMG 10746T was used as a reference strain for comparison. Taxol: used as a positive control. LMG: Laboratoriumvoor Microbiologie, Universiteit Gent, Gent, Belgium.

Fig. 3 – Fluorescent photomicrographs after incubation of E. durans 39C secreted metabolite (40 µg/mL) on AGS cell line. Panels represent (A) untreated AGS cancer cell line, (B) treated AGS cancer cell line after 24 h incubation, (C) treated AGS cancer cell line after 48 h incubation. A, membrane blebbing; b, nucleus fragmentation; c, cell shrinkage; and d, apoptotic bodies.

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Fig. 4 – Flow cytometry assessment of AGS cancer cell line after 24 and 48 h incubation with the extracted metabolites (40 µg/mL) of E. durans 39C. (A) Untreated control after 24h, (B) treated with E. durans 39C secretions after 24∙h, (C) untreated control after 48 h, (D) treated with E. durans 39C secretions after 48h. Lower left column: annexin V−/PI− (viable cells), lower right column: annexin V+/PI− (early apoptotic cells), upper right column: annexin V+/PI+ (late apoptotic cells) and upper left column: (necrotic cells).

Apoptosis induction by bacterial secretions in the AGS cancer cell lines was exhibited in a time-dependent manner, where the significant predominance of late apoptotic cells at 48 h and early apoptotic cells at 24 h (P < 0.01) was observed. Moreover, the percentage of viable cells decreased steadily by increasing the incubation time from 24 h to 48 h (Fig. 4). These results indicate that apoptosis is the main phenomenon for cell death and not necrosis.

3.8.

homology was performed as a valid and accurate identification technique (Deng, Xi, Mao, & Wanapat, 2008). According to FAO/WHO guidelines and our results, sequencing 16S rDNA is an accessible and suitable technique for the identification of

Pronase test

Similar to Taxol, the pronase-untreated secretions showed significant cytotoxic effects (P ≤ 0.01) on the treated AGS cell line. The cell proliferation percentages of AGS cells treated with E. durans 39C and Taxol metabolites during incubation time (48 h) were 29% and 28%, respectively. The cytotoxic effects of the secretions on the cancer cells after pronase treatment decreased significantly (P ≤ 0.01) compared with those on the untreated cells. This finding verifies the crucial function of secreted proteins in cytotoxic effects (Fig. 5).

4.

Discussion

The threshold value for taxonomical studies is approximately 97%. Hence, 16S rDNA sequencing with 99–100%

Fig. 5 – The cytotoxic effects of E. durans 39C and its pronase treated secretions (40 µg/mL) on the AGS cell line for 48 h. Asterisks illustrate the significant differences (**P ≤ 0.01). Error bars represent standard deviation of each mean. CON: untreated cancer cell line. P: pronase treated metabolites. Taxol: used as a positive control.

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Enterococcus strains (FAO/WHO, 2002). The biodiversity of Enterococcus species in fermented dairy products is variable and region specific (Foulquié Moreno et al., 2006; Manolopoulou et al., 2003). The microbiota of traditional dairy products is supposed to be dominated by E. durans and E. faecium strains (Malek et al., 2012; McAuley, Gobius, Britz, & Craven, 2012). However, the predominant isolated Enterococci in Iranian dairy products belong to E. mundtii, E. durans, and E. faecalis species according to our results. This condition shows that our results are different from the results of other studies in terms of species and prevalence. This difference could be due to the diverse climate in Kermanshah Province and the different preparation processes of fermented dairy products. Probiotics must have several characteristics to be effective in improving the host’s health. Such characteristics include resistance to gastrointestinal acid and bile, susceptibility against antibiotics, and high anti-microbial activity. As agreed upon by scientists, the characterization and assessment of a probiotic’s properties should therefore be performed through standard in vitro experiments (Morelli, 2007). Probiotic bacteria are normally delivered through food. Hence, they must survive for a minimum 90 min in low pH (pH 2.0–3.0) and high bile salt (0.3% (w/v)) conditions of the digestive system before colonizing in the gastrointestinal tract and displaying health promoting effects (Del Piano et al., 2006; Liong & Shah, 2005). The isolated strains displayed good acidic tolerance (73– 82%). The same results were observed in E. faecium and E. faecium strains after incubation at pH 3.0 (Bhardwaj et al., 2010; Strompfová, Lauková, & Ouwehand, 2004). The Enterococcus strains can maintain difference between environmental and cytoplasmic pH via a unique homeostasis mechanism with a bilayer membrane structure; consequently, these strains can tolerate a wide range of pH (Musikasang, Tani, H-kittikun, & Maneerat, 2009). All isolated strains displayed high tolerance in bile salt conditions, which was 14–25% higher than their tolerance in the low pH condition. This high bile tolerance capability has also been detected in other Enterococcus species, such as E. faecium (Saavedra, Taranto, Sesma, & de Valdez, 2003). The effects of bile salts on bacterial cells can be separated from acidic effects, but some combined results maybe observed. Stress adaptation mechanisms triggered by acidic environments can result in bile salt resistance (as shown in our findings), which can be unpredictable and higher than the resistance to acidic conditions (Begley, Gahan, & Hill, 2005). Among the isolated strains, E. mundtii 50H displayed a good antagonistic activity as verified by its capability to inhibit most of the indicator pathogens. Similar results have been reported by other researchers (Settanni et al., 2014; Vera Pingitore, Todorov, Sesma, & Gombossy de Melo Franco, 2012). Meanwhile, all three examined strains showed high inhibition activities for Gram-negative pathogens, such as K. pneumoniae and S. flexneri. This sensitivity may be related to the thin cell walls and the susceptibility of these Gram-negative bacteria to acidic metabolites. Gram-negative pathogenic bacteria are involved in different hospital-acquired infections, such as wound and urinary tract infections; hospital outbreaks caused by their antibiotic-resistant strains have also been reported (Raja, Murali, & Devaraj, 2008; Shoma, Kamruzzaman, Ginn,

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Iredell, & Partridge, 2014). Therefore, these Enterococcus strains may be prescribed against antibiotic-resistant strains of Gramnegative bacteria, particularly for the treatment of individuals with a weak immune system. Antibiotic-resistant genes can spread across a region and transfer to other microorganism societies because of antibiotic overuse. Therefore, sensitivity to conventional antibiotics is a fundamental health-promoting characteristic of probiotics (Temmerman, Pot, Huys, & Swings, 2003). The frequency of antibiotic resistance among the isolated strains was remarkably low. Similar results have been reported for Enterococci in other food products (Abriouel et al., 2008). Resistance to tetracycline, gentamycin, clindamycin, and sulfamethoxazol is the most common resistance among food products’ Enterococci; however, E. mundtii 50H, E. durans 39C, and E. faecalis 13C are sensitive or semi-sensitive to these four antibiotics (Jahan, Krause, & Holley, 2013; Werner et al., 2013). Sensitivity to these antibiotics was probably due to the limited usage of antibiotics in the rural area of Iran (Kermanshah Province). Hence, isolated Enterococci can be safely consumed after antibiotic (tetracycline, gentamycin, clindamycin, and sulfamethoxazol) therapy. Moreover, these four antibiotics can be utilized in their selective growth media. However, the reestablishment of bacterial balance in the gut tract after antibiotic treatment must be considered. Meanwhile, maximum resistance was observed for erythromycin. According to various reports, different strains of the Enterococcus genus carry erythromycin-resistant genes, which further support our results (Jamet et al., 2012; Klibi et al., 2013). Most of the beneficial properties attributed to probiotic bacteria are the results of preclinical studies. The use of in vitro assessments in different cancer and normal cell lines is a necessary step to study the safety of probiotics and understand their possible anticancer mechanisms before evaluation in humans (Hirayama & Rafter, 2000). Most studies on probiotics have focused on the anti-colorectal properties of probiotics (Rafter, 2003). However, according to our results, the secretion metabolites of E. durans 39C display high anticancer activity (similar to Taxol) in various cancer cell lines, such as HeLa, AGS, MCF-7, and HT29. The cytotoxic effects of the extracted metabolites of E. durans 39C in all treated cancer cell lines were dose dependent, where the concentration of 40–50 µg/mL displayed a significantly lower cell viability (P ≤ 0.01) compared with other concentrations (5–35 µg/mL). Similar results have been reported by Nami et al. (2014a, 2014b). The treated cancer cell lines in this study were clustered as epithelial origin cancer cell lines, which are less sensitive to cytotoxic agents compared with other cancer cell lines (hematological origin cancer cell lines). Thus, the effective extracted metabolites on them are appropriate for further anti-cancer assessments (Ali et al., 2000; Su, Wang, Liang, & Zha, 2005). Taxol, a conventional anticancer drug, is relatively expensive, and its source (Pacific yew tree) is limited. Hence, finding safe and cheaper anti-cancer replacements, such as extracted metabolites of E. durans 39C, is critical (Glück, 2014; Hoffman & Shahidi, 2009; Strobel, Stierle, & Hess, 1993). Anticancer drugs, such as Taxol, also display cytotoxic effects on sensitive cell lines and tissues, such as bone marrow, stem cells, and embryo cells (Akagi & Lesser, 2000; Aquino Esperanza et al., 2008). However, the extracted metabolites of E. durans 39C,

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despite their strong cytotoxic effects on various cancer cell lines, did not generate any side effects on rapidly dividing normal cell lines, such as HUVEC, and can thus be introduced safely. Fluorescent dye staining with fluorescent microscopy is one of the approprite techniques to assess cell nucleous morphology and evaluatie morphological changes in treated cancer cell lines. In addition to cytotoxic demonstration in cancerous cells, this technique allows for the assessment of several morphological changes in the cell membrane and chromatins (Savitskiy, Shman, & Potapnev, 2003). Apoptotic cells can be distinguished from necrotic and viable cells based on nuclear morphology, such as nucleus fragmentation and chromatin condensation (Baskic´, Popovic´, Ristic´, & Arsenijevic´, 2006). Our results prove that apoptosis is the main cytotoxic mechanism for the extracted metabolites of E. durans 39C. Similar to our results, the size, composition, and number of apoptotic bodies in treated cell lines in other studies can vary (Wyllie, Kerr, & Currie, 1980). The results of fluorescent microscopy alone were not convincing. Therefore, the exact death mode was characterized by performing flow cytometry assessments and quantifying the entire cell population. However, this method is very sensitive, and differentiation between late apoptosis and necrosis can sometimes become complicated (Li oon, 2010). According to the cytometry results, apoptosis is the main cytotoxic mechanism for E. durans 39C-secreted metabolites. The exact mechanisms for the inhibition of cancer cell lines by probiotics are unclear. Probiotics reduce pro-carcinogenic compounds and carcinogenic fecal enzymes through binding via their active proteins (de LeBlanc, Bibas Bonet, LeBlanc, Sesma, & Perdigón, 2010; Tuohy, Probert, Smejkal, & Gibson, 2003). They also produce anti-mutagenic substances, such as brush border enzymes and dipeptidyl peptidase IV (de LeBlanc et al., 2010; Rafter, 2003). Our results support the finding of other studies that active proteins have a key role in the cytotoxity of E. durans 39C-secreted metabolites.

5.

Conclusion

E. mundtii 50H, E. durans 39C, and E. faecalis 13C were isolated from shiraz, curd, and yogurt, respectively. They displayed desirable tolerance for low pH and high bile salts, favorable antipathogen activity, and acceptable antibiotic susceptibility. Thus, they can be introduced as potential probiotics. The anticancer effect of E. durans 39C secretion metabolites was higher than that of E. mundtii 50H and E. faecalis 13C secretions in all treated cancer cell lines. The cytotoxic effect of E. durans 39C secretions was similar to that of Taxol, which is a conventional anticancer drug. Taxol possesses high cytotoxicity in normal cell lines, but the extracted metabolites of E. durans 39C did not show any significant cytotoxic effects on rapidly dividing normal cell lines, such as HUVEC. These secreted proteins should be investigated properly in terms of physicochemical, structural, and functional properties before they are introduced as anticancer therapeutics.

Ethical issues No ethical issues to be promulgated.

Conflict of interest statement The authors declare that there are no conflicts of interests.

Acknowledgment The financial support of the University Putra Malaysia and the Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran also the moral patronages of Mr. Abolfazl Barzegari are gratefully acknowledged.

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