Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines.

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YANAE1335_proof ■ 28 August 2014 ■ 1/9

Anaerobe xxx (2014) 1e9

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Clinical microbiology

Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines Q6

Babak Haghshenas a, Norhafizah Abdullah b, **, Yousef Nami a, 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 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 c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 June 2014 Received in revised form 22 July 2014 Accepted 18 August 2014 Available online xxx

Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 mg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics. © 2014 Published by Elsevier Ltd.

Keywords: Apoptosis Anticancer Antibacterial Antibiotic susceptibility

1. Introduction Human life is significantly influenced by bacteria, which are necessary in producing useful biomaterials. Bacteria are the most beneficial prokaryotes; suggesting that their useful metabolites should be characterized and manipulated [1,2]. Fuller (1989) defined probiotics as a bacterial group that balances gastrointestinal microbial population and induces health-promoting effects [3]. Lactic acid bacteria (LAB) include Oenococcus, Lactococcus,

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* Corresponding author. Faculty of Pharmacy, Tabriz University of Medical Sciences, Daneshgah Street, P.O. Box 51664-14766, Tabriz, Iran. Tel.: þ98 411 3372250, þ98 411 3372251; fax: þ98 411 3344798. ** Corresponding author. Tel.: þ603 89466295; fax: þ603 86567120. E-mail addresses: nhafi[email protected] (N. Abdullah), [email protected] (A.Y. Khosroushahi).

Streptococcus, Enterococcus, Leuconostoc, Lactobacillus, and Pediococcus. Among the LAB, Lactobacillus are commonly consumed as probiotics [3e5]. The Lactobacillus genus is rod-shaped, facultatively aerotolerant, gram-positive, catalase-negative, and nonmotile. It has no ability for sporulation but is able to produce lactic acid through glucose fermentation [5e7]. Lactobacillus bacteria have been isolated from a vast range of food products (e.g., fermented milk, cheese, raw milk, vegetables, fruits, and meat) [7]. Lactobacillus plantarum possesses high anti-pathogen activity against Staphylococcus aureus and enterobacteriaceae; it is also used to preserve bio-processed food products [8]. Lactococcus bacteria and Lactobacillus have similar characterizations, except for their morphology. Lactococcus bacteria are cocci-shaped [9,10]. The primary origins of Lactococcus bacteria are plant and animal skin, although a number of these bacteria (e.g., Lactococcus lactis subsp. lactis and Lactococcus garviae) have been isolated from dairy

http://dx.doi.org/10.1016/j.anaerobe.2014.08.009 1075-9964/© 2014 Published by Elsevier Ltd.

Please cite this article in press as: Haghshenas B, et al., Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines, Anaerobe (2014), http://dx.doi.org/10.1016/ j.anaerobe.2014.08.009

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B. Haghshenas et al. / Anaerobe xxx (2014) 1e9

products [11]. L. lactis exhibits anti-pathogen activity through media acidification and bacteriocins production (i.e., nisin) [10,12,13]. By regulating the host immune system, probiotics are able to decrease the risks of irritable bowel syndrome [14,15], diarrhea [16], antibiotic-related diarrhea [17], vaginal infections [18], atopic eczema [19,20], and inflammatory bowel disease [21,22]. The effects of cytotoxic and anti-proliferative probiotics on different cancer cell lines have been previously reported for their healthpromoting effect on human communities [23,24]. This study aims to isolate the LAB of traditional dairy products collected from Iran. A wide range of traditional dairy products, including curd, tarkhineh, shiraz, yogurt, and cheese, is produced in different parts of Iran, because of its diverse climate. Shiraz and tarkhineh are the local names of two traditional dairy products in the west of Iran. These fermented dairy products are consumed in the region or exported to European countries because of increasing demand [25,26]. Isolates were molecularly identified and initially examined to investigate their probiotic capability. The secreted metabolites of probiotic-characterized isolates were screened on different human cancer cell lines (i.e., HT29, MCF-7, AGS, and HeLa) and normal cell lines (i.e., HUVEC) to assay their anti-proliferative or cytotoxic effects. 2. Materials and methods 2.1. Sampling, isolation, and biochemical assessment of bacteria Two hundred samples of traditional dairy products, namely, cheese, yogurt, curd, shiraz, and tarkhineh) were exploited as sources of isolation. A total of 2 g for each sample was completely homogenized in sodium citrate solution (2% w/v) with the use of a circulator. A total of 1 ml of the homogenized samples was added to 9 ml of MRS broth and incubated at 37 C for 24 h. A total of 100 ml of incubating samples was spread on MRS agar media. Single colonies were picked from the agar media and inoculated in 10 ml of MRS broth. Cell suspensions were then incubated for 24 h at 37 C [27,28]. A single colony on the growth agar plate was selected and transferred to a 15 ml media broth culture for the enrichment step. Isolates were screened after 24 h of incubation at 37 C by performing primary morphological and biochemical tests, including cell morphology, gram staining, and catalase tests [27]. 2.2. DNA extraction The total bacterial genomic DNA was extracted based on the method described by Leenhouts et al. (1990) [29]. Each precipitated DNA plate was dissolved in 50 ml DNAase free water after air-drying and then stored in a refrigerator [29].

deposited sequences in GenBank, NCBI site (http://blast.ncbi.nlm. nih.gov/Blast.cgi) to molecularly identify each strain. 2.4. Low pH and high bile salt tolerance test Overnight cultured bacteria in MRS broth were centrifuged at 6000 g for 15 min. Cell plates were then suspended in 0.3% (w/v) oxgall solution or PBS with pH 2.5 for 3 h at 37 C. The resistant rate was calculated on the basis of the pour plate technique on the MRS agar by comparing treated/untreated cell survival. Each examination was performed twice with three repetitions for each time [30,31]. The survival rate was calculated using the following equation:

Survival rateð%Þ ¼ ðlog cfu N1 =log cfu N0 Þ 100 where N1 represents the total viable counts of bacterial isolates in the MRS agar after being treated with extra bile salts or in low acidic conditions; and N0 displays the total viable counts of isolates before incubation in harsh conditions [32]. 2.5. Antimicrobial activity assay of isolates A previously described method was applied to determine the antagonistic activity of isolates against clinically important human pathogens [33]. Table 2 lists the indicator pathogens. A total of 2 ml of overnight cultured bacteria in the MRS broth was filtered using Nalgene syringe filter units (0.2 mm). A total of 50 mL of filtrates was then poured into 7 mm diameter wells created in the MuellereHinton agar plates, which contained the mentioned indicator pathogens. The clear zone diameter for each isolated strain was measured and analyzed after 24 h incubation at 37 C. The areola diameter was an indicator of anti-pathogenic activity. Antagonistic activity was divided into three groups: strong (diameter 20 mm), moderate (20 mm < diameter > 10 mm), and weak (diameter 10 mm) [34]. 2.6. Antibiotic susceptibility assay of isolates Disc diffusion method was exploited against clinically important antibiotics listed in Table 3 to determine the antibiotic susceptibility of isolates. The antibiotic disks were purchased from Padtan Teb Co. (Tehran, Iran). They were manually placed on MuellereHinton agar plates by using sterile forceps, with inoculated isolated strains on each plate. After 24 h incubation at 37 C, clear zones were measured in accordance with disks producer guidelines. Data were analyzed in accordance with recommended standards (Performance Standards for Antimicrobial Susceptibility Testing from the Clinical and Laboratory Standards Institute, Wayne, PA, CLSI, 2007). The isolated strains were classified as sensitive, intermediate, or resistant on the basis of clear zone size [35].

2.3. Amplification and sequencing of 16S rDNA gene 2.7. Metabolite preparation Amplification of the 16S rDNA gene (1500 bp) was performed using specific primers for Lactobacillus and Lactococcus genus (F: 50 AGAGTTTGATCMTGGCTCAG-30 and R: 50 -TACCTTGTTAGGACTTCACC-30 ). The PCR program cycle was set as follows: denaturation for 4 min at 95 C, 32cycles for 5 min (which consisted of 94 C for 1 min, 58 C for 1 min, and 72 C for 95 s), and a final extension at 72 C for 5 min. Amplicons were electrophoresed on 1% agarose gel. The amplified fragment was then purified from the gel using a QIAquick PCR purification kit (QIAGEN, Hilden, Germany). Purified 16S rDNA fragments were sequenced by Macrogene Company (Korean sequencing company). Each sequence was blasted with

Suspension bacterial cell cultures at stationary phase (18 h incubation at 37 C) were centrifuged at 10,000 g for 7 min. The pH adjusted (7.4) supernatant was then sterilized using 0.22 mm filter (Milipore, USA) [36]. Filtrates were applied to anti-proliferative or cytotoxicity assessments. 2.8. Cell line growth Four cancer cell lines [i.e., colon cancer (HT29), cervical cancer (HeLa), gastric cancer (AGS), breast cancer (MCF-7)] and a normal


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

L. plantarum15HN L. lactis subsp. Lactis44Lac

pH 2.5 Final counts (log cfu/ml) after incubation at:

0.3% Bile salt Final counts (log cfu/ml) after incubation at:

0h

1h

2h

3h

SR (%)

0h

1h

2h

3h

SR (%)

8.772 8.401

8.553 8.112

6.618 7.325

6.228 7.14

71 85

8.863 8.783

8.753 8.712

7.818 8.325

7.799 8.256

88 94

SR: Survival Rate.

umbilical vein endothelial cell (i.e., HUVEC) were examined to evaluate the effects of isolated bacteria-secreted metabolites on human cells. Cells were cultured into T-flasks 25 cm2 (TPP, Switzerland) containing 8 ml cell growth media RPMI-1640 (Sigma, USA), 10% (v/v) fetal bovine serum (PAA, Austria), 100 IU/ml penicillin (Sigma, USA), and 100 mg/ml streptomycin (Sigma, USA) at 95% humidity, 5% CO2, 37 C. 2.9. MTT assay Cancer/normal cell lines (with cell density of 1.2 104 cell/well) were seeded into each well of the 96-well microplates (Nunc, Denmark) and incubated under cell growth conditions. According to the adequate number of viable probiotic cells for fresh dairy products (1e10 106 CFU/ml), 10 concentration rates (5 mg/ml to 50 mg/ml) were screened on four cancer cell lines. Different concentrations of sterile bacterial secretions, pronase (Roche Applied Science) treated/untreated secretions, were administrated into each well after post-seeding for 24 h. Treated/untreated cell lines were incubated for 48 h. The medium of each well after incubation was replaced with equally fresh media containing 20 ml of MTT solution (2 mg/ml). Incubation was continued under dark conditions for 4 h. The medium of each well was again replaced with 200 ml DMSO plus 25 ml of Sorenson's glycine buffer (0.1 M glycine, 0.1 M NaCl, pH 10.5) and incubated for 30 min at 37 C. The absorbent of microplates were read using m Quant ELISA Reader (Bio-tek Instruments, USA) at 570 nm. Taxol, as a famous anticancer drug, was used as a positive control in this assessment. 2.10. Fluorescent microscopy Cells were fixed with paraformaldehyde (4%) for 5 min and permeabilized using Triton 100 (0.1%) for 10 min. Permeabilized cells were stained with50 ml DAPI stain for 5 min. Stained cells were washed twice with PBS and assessed morphologically through fluorescent microscopy (BX63, Olympus, Japan) equipped with UMWU2 fluorescence filter (excitation filter BP 330e385, dichromatic mirror DM 400, emission filter LP 420). 2.11. Flow cytometry AGS treated cells with 40 mg/ml L. lactis subsp. Lactis 44Lac secretions for 48 h were exploited for flow cytometry assessments using the BD Bioscience Annexin V-FITC Kit (BD Biosciences, USA). According to the kit instrument, twice-washed cells (1 106 cells/ mL) with cold PBS (pH 7.2) were re-suspended in a 100 mL binding buffer, 10 mL PI (Propidium Iodide) solution, and 5 mL Annexin VFITC for 15 min at room temperature and under dark conditions. A total of 400 mL binding buffer was again added to each tube. Assessments were then conducted using the FACS-Calibur flow cytometer (BD Biosciences, USA), accomplishing analysis on 10,000 cells at a rate of 1000 cells/s. The quadrant setting was performed with an untreated cell line as the negative control. Data

analysis was conducted using CELLQuest Pro software (BD Biosciences, USA). 2.12. Statistical analysis All of the numerical data were analyzed through one-way ANOVA method using SPSS program package (version 18). All of the mean multiple comparisons were conducted using Dunkan tests. P 0.05 was considered a significant difference. Graphs were prepared using Microsoft Office Excel. 3. Results 3.1. Identification of isolates The morphological and biochemical results illustrated that isolates were gram-positive, catalase-negative, rod-shaped, and coccishaped (data are not shown). Molecular identification revealed that isolated bacteria included L. plantarum 15HN and L. lactis subsp. Lactis 44Lac strains, which were isolated from traditional yogurt and cheese, respectively. Among of all isolates, these two strains showed remarkable probiotic properties thus they were selected for continuing anticancer examinations and with detailed presenting their characteristics. 3.2. Low pH and bile salt tolerance Table 1 illustrates the survival rates of L. plantarum 15HN and L. lactis subsp. Lactis 44Lac before and after being incubated for 3 h at pH 2.5 or 0.3% oxgall. L. plantarum 15HN and L. lactis subsp. Lactis 44Lac displayed high survival rates: 71% and 85% after being incubated at pH 2.5 and 88% and 92% after being incubated at 0.3% oxgall, respectively. 3.3. Antimicrobial activity Table 2 presents the antimicrobial activity of L. plantarum 15HN and L. lactis subsp. Lactis 44Lac against examined pathogenic bacteria. L. plantarum 15HN displayed superior antagonistic activity against Staphylococcus typhimurium, Staphylococcus marcesens, Staphylococcus aureus, Lactobacillus monocytogenes, Klebsiella pneumoniae, Staphylococcus flexneri, Pseudomonas aeruginosa, Staphylococcus mutans, Staphylococcus saprophyticus subsp. Saprophyticus, and native Escherichia coli. This strain also showed weak anti-pathogenic activity against Candida albicans, E. coli, and Bacillus cereus subsp. kenyae. Meanwhile, L. lactis subsp. Lactis 44Lac demonstrated moderate anti-bacterial activity against S. marcesens, K. pneumoniae, E. coli, and native E. coli. 3.4. Antibiotic susceptibility The antibiotic susceptibility of L. plantarum 15HN and L. lactis subsp. Lactis 44Lac against high-consumption antibiotics was evaluated. L. plantarum 15HN was sensitive or semi-sensitive to


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Table 2 The inhibitory effect of isolated L. plantarum 15HN and L. lactis subsp. Lactis 44Lac against pathogenic microorganisms. Pathogens

Growth condition

Origin

Diameter of inhibition zone (mm) L. plantarum

MPA, 37 C MHA, 28 C MHA, 37 C MRS, 37 C Blood agar, 37 C MHA, 37 C LB, 37 C MPA, 37 C Blood agar, 37 C LB, 37 C MPA, 37 C BHI, 37 C MPA, 37 C MHA, 37 C

P. aeruginosa C. albicans S. marcesens E. faecalis S. saprophyticus S. mutans E. coli (0157) S. typhimurium S. aureus E. coli (026) B. cereus L. monocytogenes K. pneumoniae S. 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)

11.3 10.0 17.3 0.0 11.3 17.3 10.0 12.3 11.7 12.3 10.0 13.7 12.0 12.0

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.3 0.0 0.0 0.0 0.7 0.6 0.0 1.2 0.3 0.7 0.0 0.9 0.6 0.0

L. lactis subsp. Lactis

M W M

0.0 0.0 12.3 0.0 0.0 0.0 12.0 0.0 0.0 13.7 0.0 0.0 12.7 0.0

M M W M M M M M M

± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.0 0.0 1.2 0.0 0.0 0.0 0.6 0.0 0.0 1.2 0.0 0.0 0.3 0.0

M

M

M

M

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.

Table 3 The antibiotic susceptibility of isolated L. plantarum 15HN and L. lactis subsp. Lactis 44Lac against the high consumption antibiotics.

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Diameter of inhibition zone (mm) Antibiotics

C

Te

E

Am

Gm

CC

Smz

Va

P

Disk content

30 mg

30 mg

15 mg

10 mg

10 mg

2 mg

25 mg

30 mg

10000 units

L. plantarum L. lactis subsp. Lactis

20S 28S

20S 19S

20I 26S

40S 27S

10S 18S

18S 27S

25S 0R

0R 20S

40S 15I

C, chloramphenicol; Te, tetracycline; E, erythromycin; Am, Ampicillin; Gm, gentamycin; CC, clindamycin; Smz, sulfamethoxazol; P, penicillin; and Va, vancomycin. I: intermediate susceptibility (zone diameter 12.5e17.4 mm); R: resistant (zone diameter 12.4 mm); S: susceptible (zone diameter 17.5).

chloramphenicol, tetracycline, erythromycin, ampicillin, gentamycin, clindamycin, sulfamethoxazol, and penicillin and was resistant to vancomycin. L. lactis subsp. Lactis 44Lac was also sensitive or semi-sensitive to chloramphenicol, tetracycline, erythromycin, ampicillin, gentamycin, clindamycin, vancomycin, and penicillin. However, it was resistant to sulfamethoxazol (Table 3). 3.5. Cytotoxicity Different concentrations of bacterial secretion metabolites were applied on four human cancer cell lines by applying 30% (v/v) cellfree total extract with 40 mg/ml dry weight for 12, 24, 48 h (Fig. 1). Screening results indicate that L. lactis subsp. Lactis 44Lac has a significant anti-cancer activity (P 0.05) against all of the examined cell lines. The prescreening results showed that L. plantarum 15HN secretion metabolites can significantly enhanced the growth of human cancer cells with no significant effects on human normal cells at in vitro condition. Based on the screening results, further assessments were continued using L. lactis subsp. Lactis 44Lac 30% (v/v) secretion metabolites as the selected treatment group, Taxoltreated groups as positive control, and untreated cell lines as negative control. Compared with the untreated cancer cell line and isolated L. lactis subsp. Lactis from the intestinal tract as control, cell viability decreased significantly (P 0.05) in all of the human cancer cell lines treated by L. lactis subsp. Lactis 44Lac 30% (v/v) secretion metabolites (Fig. 1, Panels AeD). Moreover, cell viability in all of the treated cancer cells did not show a significant difference (P > 0.05) between Taxol and L. lactis subsp. Lactis 44Lac 30% (v/v) secretion metabolites treated groups, indicating that secretions act

similarly to Taxol with regard to anti-cancer effects (Fig. 2, Panels AeD). The cytotoxicity of secretions on cancer cell lines were the same as that of Taxol; therefore, the cytotoxic effects of secretions were evaluated on human umbilical vein endothelial cells (HUVEC) normal cells by conducting MTT assay. Interestingly, L. lactis subsp. Lactis 44Lac 30% (v/v) secretion metabolites did not show significant cytotoxic effects (P > 0.05) on normal human cells, whereas Taxol displayed high cytotoxic effects on HUVEC (Fig. 2, Panel E). A simple pronase test was conducted to characterize the active material in secretion metabolites. The anti-proliferative effect of pronase-treated/untreated secretions was investigated on the AGS cell line. The results show that the anti-cancer effect was omitted in the pronase treated secretion, suggesting that the bioactive compound is a protein (Fig. 3). 3.6. Apoptosis assessment A comparison of microscopic images of treated cells and untreated control illustrate apoptotic cell increase with condensed and fragmented nucleus, cell shrinkage, and membrane blabbing in treated cells (Fig. 4). Flow cytometry was utilized to quantitatively assay apoptotic cells in AGS treated groups with L. lactis subsp. Lactis 44Lac 30% (v/v) secretion metabolites. The results displayed 75.28% apoptosis (11.05% early and 64.23% late apoptosis) and 1.8% necrosis, indicating approximately 77% cell death in treated groups; untreated cells showed lower than 1% dead cells (Fig. 4, Panel E and F). In consideration of these results, apoptosis is considered the main phenomenon involved with cell death in treated cells.


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Fig. 1. The cytotoxic effects of isolated L. plantarum 15HN and L. lactis subsp. Lactis 44 Lac secretions on different cancer cell lines at three time points 12, 24, and 48 h. Panels represent; a) MCF-7 (human breast cancer cell line), b) AGS (human gastric cancer cell line) c) HeLa (human cervix cancer cell line), and d) HT29 (human colon cancer cell). 0% concentration: MRS is used as control. A series: L. plantarum 15HN. B series: L. lactis subsp. Lactis 44Lac. Error bars represent the standard deviation of the each mean.

4. Discussion Sequencing the 16S-rDNA in accordance with FAO/WHO guidelines is a suitable and accessible technique to identify probiotic bacteria [37]. The results identified isolated strains with 99%e100% homology, which is higher than the threshold for taxonomical studies (97%). Therefore, this method is a valid and accurate technique for phylogenetic clustering of probiotic bacteria [38]. The varieties of Lactobacillus strains isolated from dairy products have proven to be specific to a source and region [39,40]. L. plantarum has a long history as food fermentative [5,7,41]. It is the predominant strain in different traditional dairy products (i.e., Armada cheese [42], Batzos cheese [43], yogurt, Laban zeer, Kulenaoto, M'Bannick, Kwerionik, Koumiss, and Zincica [40]). L. lactis subsp. Lactis can be isolated from traditional dairy products, particularly cheese [11,43,44], despite non-dairy primary origins (plant and animal skin). The molecular identification results reveal that L. plantarum 15HN and L. lactis subsp. lactis 44Lac were isolated from traditional yogurt and cheese, respectively. This is due to the specific preparation process for fermented dairy products in the area, as well as the powerful natural potential for growth of L. lactis subsp. Lactis [11] and L. plantarum [8] in high salt concentrations and low pH environments. Health-improving effects of probiotics include resistance to gastrointestinal acid and bile, susceptibility against antibiotics, and having high anti-microbial activities. Therefore, probiotic characterization must be performed through standard in vitro

experiments [4,37,45,46]. Probiotics must tolerate inverse conditions (i.e., high bile salts [0.3% (w/v)] and low pH [pH 2.0 to pH 3.0]) for a minimum of 90 min [47e51]. The two isolated strains displayed high survival rates under low pH (>71%) and high bile salt conditions (>88%). However, L. lactis subsp. lactis 44Lac showed better tolerance than L. plantarum15HN. This high tolerance capability is related to the bi-layer membrane structure, which enables easy tolerance of inverse conditions [47,50,52e55]. These results are in contrast with other studies that found that Lactobacillus strains (L. plantarum) tolerates inverse conditions better than other genera (Lactococcus) among LAB isolates [47,50,54,56,57]. L. plantarum 15HN displayed superior antagonistic activities against a vast variety of indicator pathogens (e.g., bacteria and fungi); other studies have reported restricted anti-pathogenic activities for this strain [58e62]. L. lactis subsp. Lactis 44Lac showed moderate anti-bacterial activities against S. marcesens, K. pneumoniae, E. coli and native E. coli only. Secretion of bacteriocins (nisin) enables this strain to display anti-pathogen activity and be used as a food preservative [63,64]. Isolated strains display higher antibiotic susceptibility compared with other isolated strains in this genera [40,65e69]. Therefore, they can be safely consumed after antibiotic therapy. By contrast, these antibiotics can be used in selective growth media. Nonetheless, reestablishing bacterial balance in the gastrointestinal tract after antibiotic treatments should be considered. The high rates of antibiotic susceptibility probability were due to the limited


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Fig. 2. The cytotoxic effects of L. lactis subsp. Lactis 44Lac secretions (40 mg/mL) and Taxol on several cancer cell lines and normal cell line for 48 h. Panels represent; A) MCF-7 (human breast cancer cell line), B) AGS (human gastric cancer cell line), C) HeLa (human cervix cancer cell line), D) HT29 (human colon cancer cell), and E) HUVEC (Normal cell). Asterisks illustrate the significant differences at P 0.05. Error bares represent standard deviation of each mean. CON: MRS is used as control. Dla: L. lactis subsp. Lactis isolated from intestinal tract was used as a reference strain for comparison. Taxol: used as a positive control.

use of antibiotics in the isolated area. L. plantarum 15HN was sensitive or semi-sensitive to all of the examined antibiotics except for vancomycin. The strict homo fermentative Lactobacilli are generally sensitive to vancomycin. However, L. plantarum carries vancomycin resistance genes [40,70]. L. lactis subsp. lactis 44Lac was resistant to sulfamethoxazol only. Probiotics are health-promoting agents that promote different therapeutic activities, and being anti-carcinogenic is its most

Fig. 3. Cell viability (%) for 40 mg/mL secreted metabolites of L. lactis subsp. Lactis 44Lac and pronase treated metabolites on AGS (human gastric cancer cell line) cell line after 48 h incubation and untreated and Taxol treated AGS cells as negative and positive controls. Each bar represents the ±SE of each mean. Asterisks denote statistically significant differences at P 0.05.

interesting property. Probiotics display high apoptic and antiproliferative effects on different cancer cell lines (e.g., breast, bladder, colon, liver, and gastric) [71e76]. The majority of the literature has focused on their anti-colorectal properties [76e78]. Our results prove that secreted metabolites of L. lactis subsp. Lactis 44Lac have high anti-cancer activities on different cancer cell lines. Extracted metabolites of this strain showed time-and dosedependent effects, where 30% (v/v) concentration with50 mg/ml dry weight after 48 h incubation displayed better results compared with other concentrations and incubation times. Secreted metabolites of L. lactis subsp. Lactis 44Lac are qualified as safe and cheap anti-cancer agents because they are effective on epithelial origin cancer cell lines, which are less sensitive to anticancer metabolites or drugs compared with other cell lines [79e83]. They did not show any side effects on normal cell lines despite the high cytotoxicity linked to other anticancer agents on sensitive cell lines and tissues [84e88]. Active proteins play a key role in the cytotoxity of extracted metabolites. They suppress the cancer cell lines by binding to procarcinogenic compounds [89,90], carcinogenic fecal enzymes [89,91], or mutagenic substances [89,90]. Moreover, the observed effects for non-protein molecules are linked to short-chain fatty acids (e.g., butyrate and propionate) [89]. Staining treated cancer cell lines and analyzing them through a fluorescent microscope is appropriate for cytotoxic demonstration and assessment of morphological changes in the cell membrane and chromatins [92,93]. The fluorescent microscopy results indicate that apoptosis as the main cytotoxic mechanism, with a vast variety of apoptotic bodies (i.e., number, size, and composition).


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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 Fig. 4. Fluorescent microscopic images of DAPI stained apoptotic cells and the flow cytometry assessment on cancer cell line treated by L. lactis subsp. Lactis 44Lac secretions (40 mg/ 92 mL) for 48 h. Panels (A and B) represent AGS and panels (C and D) illustrate HT29 treated cells. a; membrane blebbing, b; nucleus fragmentation, c; cell shrinkage and d; apoptotic bodies. Panels (E) and (F) represent the flow cytometric analysis of untreated control and treated AGS cells for, respectively. Lower left column: annexin V/PI (viable cells), lower 93 Q7 right column: annexin Vþ/PI (early apoptotic cells), upper right column: annexin Vþ/PIþ (late apoptotic cells) and upper left column annexin V/PIþ (necrotic cells). 94 95 96 Iran also the moral patronages of Mr. Abolfazl Barzegari are grateOther studies have reported these variations of apoptotic bodies 97 fully acknowledged. [94,95]. Q2 98 However, the fluorescent microscopy and DNA fragmentation 99 results are not convincing enough [96].Therefore, the exact death 100 References mode of cells was characterized through flow cytometry. Apoptosis, Q3 101 particularly late apoptosis, was found to be the main cytotoxic 102 [1] Temmerman R, Huys G, Swings J. Identification of lactic acid bacteria: culturemechanism for extracted metabolites of L. lactis subsp. lactis 44Lac. 103 dependent and culture-independent methods. Trends Food Sci Technol In conclusion, L. plantarum 15HN and L. lactis subsp. Lactis 44Lac 104 2004;15:348e59. [2] Pontes DS, Lima-Bittencourt CI, Chartone-Souza E, Amaral Nascimento AM. display desirable tolerance to low pH and high bile salts, as well as 105 Molecular approaches: advantages and artifacts in assessing bacterial difavorable antagonistic activities and acceptable antibiotic suscep106 versity. J Ind Microbiol Biotechnol 2007;34:463e73. tibility. Therefore, they are qualified to be potential probiotics. Anti107 [3] Guarner F, Khan AG, Garisch J, Eliakim R, Gangl A, Thomson A, et al. Probiotics cancer assessments display high cytotoxity for protein nature and prebiotics. World Gastroenterology Organisation practice guideline; 2008. 108 [4] Biradar SS, Bahagvati ST, Shegunshi B. Probiotics and antibiotics: a brief secreted metabolites of L. lactis subsp. Lactis 44Lac on different 109 overview. Internet J Nutr Wellness 2005;2. human cancer cell lines higher than secreted metabolites of 110 [5] Giraffa G, Chanishvili N, Widyastuti Y. Importance of lactobacilli in food and L. plantarum 15HN. The anti-cancer effects for secreted metabolites feed biotechnology. Res Microbiol 2010;161:480e7. 111 € J, De Vos WM. Molecular [6] Satokari RM, Vaughan EE, Smidt H, Saarela M, M€ atto of L. lactis subsp. Lactis 44Lac were interestingly similar to Taxol. 112 approaches for the detection and identification of bifidobacteria and lactoHowever, unlike Taxol, they did not show any cytotoxity on human 113 bacilli in the human gastrointestinal tract. Syst Appl Microbiol 2003;26: normal cells. These bioactive proteins should be investigated 114 572e84. [7] Singh S, Goswami P, Singh R, Heller KJ. Application of molecular identification accurately from a structural, physicochemical, and functional 115 tools for Lactobacillus, with a focus on discrimination between closely related perspective and analyzed through in vitro/in vivo assessments 116 species: a review. LWT e Food Sci Technol 2009;42:448e57. before they are introduced as potential anti-cancer therapeutics. 117 [8] Bengmark S. Immunonutrition: role of biosurfactants, fiber, and probiotic bacteria. Nutrition 1998;14:585e94. 118 [9] Vinh DC, Nichol KA, Rand F, Embil JM. Native-valve bacterial endocarditis Ethical issues 119 caused by Lactococcus garvieae. Diagn Microbiol Infect Dis 2006;56:91e4. 120 [10] Wikipedia TFE. Lactococcus; 2013. [11] Casalta E, Montel M-C. Safety assessment of dairy microorganisms: the LacNo ethical issues to be promulgated. 121 tococcus genus. Int J Food Microbiol 2008;126:271e3. 122 €nlund MM, Isolauri E, Salminen SJ. Adhe[12] Ouwehand AC, Kirjavainen PV, Gro 123 Conflict of interest statement sion of probiotic micro-organisms to intestinal mucus. Int Dairy J 1999;9: 623e30. 124 [13] Davies EA, Delves-Broughton J. BACTERIOCINS j nisin. In: Richard KR, editor. 125 The authors declare that there are no conflicts of interests. Encyclopedia of food microbiology. Oxford: Elsevier; 1999. p. 191e8. 126 [14] Parkes GC. Chapter 30-The role of probiotics in the treatment of irritable bowel syndrome. In: Ronald Ross W, Victor RP, editors. Bioactive foods in 127 Acknowledgment promoting health. Boston: Academic Press; 2010. p. 513e28. 128 [15] Barouei J, Adams MC, Hodgson DM. Prophylactic role of maternal adminis129 The financial support of the University Putra Malaysia and the tration of probiotics in the prevention of irritable bowel syndrome. Med 130 Hypotheses 2009;73:764e7. Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Please cite this article in press as: Haghshenas B, et al., Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines, Anaerobe (2014), http://dx.doi.org/10.1016/ j.anaerobe.2014.08.009

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Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: evaluation of the probiotic potential.

Biosorption of silver cations onto Lactococcus lactis and Lactobacillus casei isolated from dairy products.

Complete Genome Sequence of Lactococcus lactis subsp. lactis KLDS4.0325.

Conjugal mobilization of streptococcal plasmid pMV158 between strains of Lactococcus lactis subsp. lactis.

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

Draft Genome Sequences of 11 Lactococcus lactis subsp. cremoris Strains.

Tryptophan biosynthesis genes in Lactococcus lactis subsp. lactis.

Genome Sequence of Lactococcus lactis subsp. lactis bv. diacetylactis LD61.

Histidine biosynthesis genes in Lactococcus lactis subsp. lactis.

Molecular analyses of the lactococcin A gene cluster from Lactococcus lactis subsp. lactis biovar diacetylactis WM4.

Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363.

Isolation of strong constitutive promoters from Lactococcus lactis subsp. lactis N8.

Complete Genome Sequence of Lactococcus lactis subsp. lactis A12, a Strain Isolated from Wheat Sourdough.

Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin.

Nucleotide sequence of IS904 from Lactococcus lactis subsp. lactis strain NIZO R5.

Construction of a novel inducible expression vector for Lactococcus lactis M4 and Lactobacillus plantarum Pa21.

Physical and genetic map of the chromosome of Lactococcus lactis subsp. lactis IL1403.

Thermosensitive plasmid replication, temperature-sensitive host growth, and chromosomal plasmid integration conferred by Lactococcus lactis subsp. cremoris lactose plasmids in Lactococcus lactis subsp. lactis.

Transfer of Tn916 between Lactococcus lactis subsp. lactis strains is nontranspositional: evidence for a chromosomal fertility function in strain MG1363.

Molecular characterization of a second abortive phage resistance gene present in Lactococcus lactis subsp. lactis ME2.

Branched-chain amino acid biosynthesis genes in Lactococcus lactis subsp. lactis.

IS946-mediated integration of heterologous DNA into the genome of Lactococcus lactis subsp. lactis.

Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3.

Molecular characterization of the integration of the lactose plasmid from Lactococcus lactis subsp. cremoris SK11 into the chromosome of L. lactis subsp. lactis.