Anti-Proliferative Effects of Two New Lactobacillus Strains of Human Origin on Caco-2 Cell Line


Simin Arian 1 , Hami Kaboosi ORCID 1 , * , Zaheir Heshmatipour 2 , Zeinab Khazaei Koohpar 3 , Fatemeh Pyravii-Ghadikolaii ORCID 4

1 Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran

2 Department of Microbiology, Faculty of Biological Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran

3 Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran

4 Department of Microbiology, Ghaemshahr Branch, Islamic Azad University, Ghaemshahr, Iran

How to Cite: Arian S, Kaboosi H, Heshmatipour Z , Khazaei Koohpar Z, Pyravii-Ghadikolaii F. Anti-Proliferative Effects of Two New Lactobacillus Strains of Human Origin on Caco-2 Cell Line, Iran Red Crescent Med J. 2019 ; 21(3):e84683. doi: 10.5812/ircmj.84683.


Iranian Red Crescent Medical Journal: 21 (3); e84683
Published Online: March 18, 2019
Article Type: Research Article
Received: September 27, 2018
Revised: March 3, 2019
Accepted: March 5, 2019




Background: Anti-proliferative effects of probiotics are considerable in the treatment of various cancers, including colon cancer. In the present study, two new Lactobacillus strains as probiotics were isolated from stool samples at a clinical lab.

Objectives: The aim of this study was to evaluate the effects of the cell-free lyophilized filtrate of two new strains of Lactobacillus, isolated on viability on Caco-2 cells.

Methods: Two new strains of Lactobacillus were isolated from 1 gr of each infant stool specimens from a total of fifty volunteers, according to the principles of a scientific questionnaire. The anti-proliferative effects of the strains were investigated using the MTT assay with Caco-2 cell lines.

Results: Out of 50 samples, seven isolates were lactic acid bacteria, two strains of which were probiotics related to L. fermentum (E) and L. rhamnosus (G). The results showed that the two Lactobacillus strains had good anti-proliferative effects against the cancer cell lines tested. These strains were resistant to low pH and 0.3% bile salt. Cytotoxicity assay revealed that the most effective concentration of strains E (~55% to ~72%) and G (~60% to ~80%) on Caco-2 cells was 10000 µg/mL after 24 to72 hours.

Conclusions: Cytotoxicity effect of the cell-free lyophilized filtrate of bacteria on Caco-2 cells in a dose- and time-dependent manner suggested that these strains might be used in colon cancer therapy.


Bacteria Caco-2 Cells Cancer Cell Line Colon Lactic Acid Lactobacillales L. fermentum L. rhamnosus MTT Assay

Copyright © 2019, Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License ( which permits copy and redistribute the material just in noncommercial usages, provided the original work is properly cited

1. Background

Probiotics are known as live microorganisms (bacteria or yeasts) and when administered in sufficient numbers, confer a health benefit on the host (1, 2). The colonic microflora is dominated by anaerobic bacteria, such as Lactobacillus spp., Bacteroides spp., Fusobacterium spp., and many others. Consumption of probiotics in humans has shown that they are effective in medical problems, such as lactose intolerance, antibiotic-induced diarrhea, gastroenteritis, constipation, and genitourinary tract infections (3). Different studies have shown the anti-proliferative and anti-cancerous properties of probiotics (4). Anti-cancer effects of probiotic bacteria are mediated by inhibition of carcinogens products (5), DNA protection from oxidation, immune system regulation (6), and deregulation of genes implicated in apoptosis, invasion, metastasis, stem cell maintenance, and cell cycle control (5). Furthermore, it appears that probiotic therapy is an effective strategy to improve and overcome gastrointestinal cancers and inflammations. The aim of this study was to evaluate the antiproliferative efficacy of two isolated strains of L. fermentum (E) and L. rhamnosus (G) from healthy volunteers’ stools in Tonekabon city (west of Mazandaran province, Iran) that were cultured on colorectal cancer cell lines (Caco-2). Colorectal Cancer (CRC) is one of the important causes of morbidity and mortality, worldwide, through cancer (7). Genetic background and environmental factors, such as lifestyle and diet, play critical roles (8). The intestinal microbiota is composed of bacteria, viruses, archaea, and fungal species (9), which is essential to protect the local homeostasis and epithelial defense against pathogens (10). Probiotics can regulate unbalanced intestinal microbiota, and by decreasing carcinogenic-stimulating occurrence in the colorectal area, can be used as a therapeutic and preventive strategy (8).

2. Objectives

Novel strains with more functional probiotic properties than the existing ones are the object of new research due to the growing demand for “healthy” foods in the food industry. In the present study, two isolated and identified Lactobacillus strains, which were first isolated from human stools, were used to investigate their anti-cancer activity.

3. Methods

3.1. Sampling and Isolation of Bacteria

The isolated samples were human stool samples obtained from 50 healthy children, according to the principles of a scientific questionnaire at Tonekabon Hospital of Mazandaran province of Iran. Infants were selected at the age of two months to two years, and those with digestive problems were excluded. Once samples were delivered to the laboratory, they were taken to the procedure for isolation and were stored at 4°C. The isolates were grown on MRS agar (de Man, Rogosa, and Sharpe medium; Quelab, Canada) and incubated at 37°C for 24, 48, and 72 hours under anaerobic conditions. All isolates were sub-cultured at least three times before being used. Standard diagnostic and biochemical tests were used to isolate probiotic strains, such as carbohydrate fermentations, acid tolerance assay, and tolerance against bile (4, 11).

3.2. Carbohydrate Fermentations

Positive isolates were characterized according to their fermentation profiles of ability to ferment 15 different carbohydrates. Overall, 2% final sugar concentration was obtained. After overnight incubation at 37°C, the turbidity and the color changed from purple to yellow, which was recorded as positive fermentation results. Also, positive and negative controls were used to indicate any contamination (Table 1) (12).

Table 1. Biochemical Test Results of Isolates
TestsStrain Code
Gram test++
Bile salt tolerance < 2 h24 h
pH tolerancea+++++

a ++, Good; +++, Excellent.

3.3. Tolerance to Acidic pH Values

After isolation of seven lactobacilli, to define gastric acidic tolerance conditions, isolates were grown in MRS broth at 37°C for 18 hours. Then, 0.1 mL aliquots of each active culture was adjusted to pH 2.5 with 5M HCl and cultured in MRS agar and incubated at 37°C under anaerobic conditions for 24 to 48 hours. All the experiments were replicated twice (13).

3.4. Bile Tolerance

The isolates were cultured in MRS broth at 37°C overnight; saturated bile solution was prepared separately by dissolving the powdered bile extract (Oxoid). Bile solution was added to two of the cultures to achieve a final concentration of 0.3% and the second culture with 0% bile served as the control sample. The cultures were incubated at 37°C for 30 minutes, two, four, and 24 hours. The MRS medium containing 0.3% bile was inoculated with active cultures (incubated for 16 to 18 hours). During the incubation for four hours, viable colonies were enumerated for every hour with pour plate technique and also bacterial growth was monitored by measuring absorbance with a spectrophotometer (Analytik Jena, Germany) at 600 nm. All the experiments were replicated twice (11).

3.5. Lactobacillus Identification by Molecular Analysis

Genomic DNA was an extract from isolated lactic acid bacteria by the High Pure PCR Template Preparation Kit (Roche Molecular Systems, Switzerland). Amplification of the 16S rDNA gene was carried out by the master mix kit (Amplification, Denmark ) and in ABI2720 Thermal Cycler (Thermo Fisher Scientific, USA), according to the following program: An initial denaturation at 95°C for five minutes, 35 cycles at 95°C for one minute, 60°C for one minute, 72°C for 75 seconds, and a final extension step at 72°C for 10 minutes. After the purification of PCR products from agarose gel, DNA sequencing was performed by Macrogen Inc. (Korea). Bacteria-specific primer pairs were 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') (14). The sequencing results were blasted with the deposited sequences in the National Center for Biotechnology Information (NCBI) and GenBank site ( to identify the isolated bacteria cells. Based on the sequencing results, a phylogenic tree was drawn for probiotic strains (Figure 1).

The phylogeny tree plotted for two probiotic s, The G and E strains determined as a new strain as Lactobacillus rhamnosus (61%) and Lactobacillus fermentum (86%) respectively.
Figure 1. The phylogeny tree plotted for two probiotic s, The G and E strains determined as a new strain as Lactobacillus rhamnosus (61%) and Lactobacillus fermentum (86%) respectively.

3.6. Cell Viability Test (MTT Assay)

The MTT assay (3- (4, 5-dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) is an essential test to determine the percentage of cell viability. Briefly, 15 × 103 Caco-2 cells/well were seeded in 0.2-mL 96-well flat-bottomed tissue culture-untreated plates and cultured for 24 hours. The culture medium was removed and different concentrations (0, 0.1, 1, 10, 100, 1000, and 10000 µg/mL) of cell-free lyophilized filtrate (for L. fermentum and L. rhamnosus separately) were added to the culture medium for eight, 12, 24, 48 and 72 hours at 37°C. Then, MTT dye (0.5 mg/mL; Sigma Aldrich) was added to each well and incubated at 37°C for two hours. To dissolve the formazan crystals, 100 μL of Dimethyl-Sulfoxide (DMSO) was added. The optical density was measured at 570 nm using an Enzyme-Linked Immunosorbent Assay (ELISA) microplate reader. Each experiment was performed a minimum of three times (15).

3.7. Statistical Analysis

One-way Analysis of Variance (ANOVA) with post-hoc Tukey and Kruskal-Wallis tests were used to compare the differences between bacteria strains. The results are presented as mean ± SD.

4. Results

4.1. Chemical Molecular Identification of the Isolates

Out of 50 isolates, seven isolates were gram-positive, catalase negative, and rods shape. These isolates gave positive results with the carbohydrates, such as glucose, xylose, ribose, arabinose, mannose, raffinose, galactose, fructose, sucrose, fructose, and lactose. The microbial isolates were taxonomically characterized by 16S rDNA sequence analysis. The analysis of the 16S rDNA sequence of strain E indicated that this new strain is closely related (86%) to Lactobacillus fermentum. Also, strain G was determined as a new strain that is closely related (61%) to Lactobacillus rhamnosus, which is observed in the phylogenetic tree pattern, as shown in the Figure 1. Other lactic acid bacteria strains, including strains K, O, N3, and N5 were identified as closely related strains to Lactobacillus fermentum, Enterococcus faecium, Enterococcus durans, and Enterococcus faecium, respectively.

4.2. Tolerance to Acid of Lactobacilli

According to this experiment, G and E isolates were resistant to low pH. Bacteria growth at different pH values was measured in three grades (weak = 1, good = 2, and perfect = 3). Strain G had good growth at pH 2.5 and pH 3.0, while at pH 3.5 to 5.0, revealed perfect growth for this strain. Strain E had perfect growth at all low pH values. Thus, strain E was more tolerated to low pH ( < 3.0) than strain G. According to the current results based on Kruskal Wallis test, there was no significant difference to resistance level between isolates (P > 0.05), as shown in Table 2.

Table 2. Results of the Kruskal Wallis Test. Comparison of Isolate Resistance in Terms of PH (Median, Mode)
StrainpHTest StatisticP Value

4.3. Bile Salt Tolerance Assay

The strains, resistant to low pH, were evaluated for tolerance to the bile salt. All seven lactic acid bacteria isolates were able to grow in 0.3% bile salt (P < 0.05). Best growth ability (OD = 0.163 ± 0.004) of strain G was exhibited after two hours in 0.3% bile (P < 0.05). However, strain E was most tolerated to 0.3% bile (OD = 0.249 ± 0.003) after 24 hours (P < 0.05) compared to other time points. The results showed that strain E was further tolerated to 0.3% bile than strain G as shown in the Table 3.

Table 3. Results of Analysis of Variance Analysis of the Strain Isolations Rate in Terms of Time
Strain030 min2 h4 h24 hTest StatisticP Value
SG0.003 ± 0.0550.004 ± 0.0930.004 ± 0.1630.060 ± 0.0450.003 ± 0.0639.2370.002
SE0.003 ± 0.0860.003 ± 0.0760.003 ± 0.0820.003 ± 0.0700.003 ± 0.2492201.9830.001
SK0.004 ± 0.1210.004 ± 0.1120.003 ± 0.1580.003 ± 0.1540.003 ± 0.255931.3650.001
SL0.004 ± 0.1240.004 ± 0.0730.004 ± 0.1250.397 ± 0.2410.035 ± 1.07316.7560.001
SO0.153 ± 0.2330.300 ± 0.4000.035 ± 0.8470.004 ± 0.7970.003 ± 0.99113.5140.001
SN30.003 ± 0.0850.003 ± 0.0780.003 ± 0.0610.005 ± 0.4870.003 ± 0.44412140.1320.001
SN50.004 ± 0.2040.003 ± 0.0240.004 ± 0.1070.035 ± 0.1530.003 ± 0.9921830.1870.001

4.4. Cytotoxic Effects of Lactobacillus fermentum and Lactobacillus rhamnosus on Caco-2 cells

Inhibition of Caco-2 cell proliferation by the cell-free lyophilized filtrate from L. fermentum (strain E) and L. rhamnosus (strain G) is shown in Tables 4 - 9 respectively, based on different concentrations and times. At concentrations of 0.1 µg/mL, 1 µg/mL, 10 µg/mL, and 100 µg/mL of strain G cell-free lyophilized filtrate, a weak inhibition of cell proliferation (~20% to ~30%) occurred at 24 and 48 hours of incubation. However, 72 hours after treatment with same concentrations, a few different patterns of cell cytotoxicity were observed (~5% to ~30%). The cytotoxicity rates were ~40% for the 1000 µg/mL lyophilized filtrate at 24, 48, and 72 hours of incubation. Nevertheless, 10000 µg/mL lyophilized filtrate of strain G was defined as effective concentration of lyophilized filtrate of these bacteria by cytotoxicity amount of ~60% at 24 hours, ~70% at 48 hours, and ~80% at 72 hours. At concentrations of 0.1 µg/mL, 1µg/mL, 10µg/mL, and 100 µg/mL of strain E cell-free lyophilized filtrate, a weak inhibition of cell proliferation (~4% to ~30%) occurred at 24, 48, and 72 hours of incubation. At concentration of 1000 µg/mL, inhibition of Caco-2 cells viability was ~43% to ~45% after one, two, and three days. Effective concentration of strain E cell-free lyophilized filtrate was determined as 10000 µg/mL by cytotoxicity rates of ~55% to ~72% after 24 to 72 hours.

Table 4. Anti-Proliferative Effect of Lactobacillus (E Strain) Extracts by MTT Assay (24 h)a
Doses, µg/mLViabilityP Value
10000 E45.6 ± 18.410.0001
1000 E55.7 ± 22.510.0001
100 E66.8 ± 27.030.0001
10 E77.1 ± 31.210.0001
1 E85.7 ± 34.670.0001
0.1 E90.3 ± 36.570.011
Positive control E24.1 ± 9.700.0001

a Values are expressed as mean ± SD.

Table 5. Anti-Proliferative Effect of Lactobacillus (G Strain) Extracts by MTT Assay (24 h)a
Doses, µg/mLViabilityP Value
10000 E39.3 ± 15.850.0001
1000 E59.3 ± 23.970.0001
100 E70.8 ±28.620.0001
10 E74.5 ± 30.100.0001
1 E83.2 ± 33.640.0001
0.1 E88.1 ± 35.610.0001
Positive control E17.7 ± 7.090.0001

a Values are expressed as mean ± SD.

Table 6. Anti-Proliferative Effect of Lactobacillus (G Strain) Extracts by MTT Assay (48 h)a
Doses, µg/mLViabilityP Value
10000 G31.4 ± 12.680.0001
1000 G63.3 ± 25.590.0001
100 G71.6 ± 28.980.0001
10 G76.8 ± 31.100.0001
1 G84.6 ± 34.260.0001
0.1G89.9 ± 36.390.0001
Positive control G13.6 ± 5.450.0001

a Values are expressed as mean ± SD.

Table 7. Anti-Proliferative Effect of Lactobacillus (E Strain) Extracts by MTT Assay (48 h)a
Doses, µg/mLViabilityP Value
10000 E37.2 ± 15.000.0001
1000 E56.4 ± 22.780.0001
100 E68.0 ± 27.510.0001
10 E78.6 ± 31.810.0001
1 E86.7 ± 35.080.0001
0.1 E92.0 ± 37.25.011
Positive control E16.9 ± 6.780.0001

a Values are expressed as mean ± SD.

Table 8. Anti-Proliferative Effect of Lactobacillus (G Strain) Extracts by MTT Assay (72 h)a
Doses, µg/mLViabilityP Value
10000 G21.9 ± 8.820.0001
1000 G64.2 ± 25.960.0001
100 G72.1 ± 29.180.0001
10 G82.4 ± 33.350.0001
1 G87.6 ± 35.460.0001
0.1G95.3 ± 38.560.002
Positive control G12.8 ± 5.140.0001

a Values are expressed as mean ± SD.

Table 9. Anti-Proliferative Effect of Lactobacillus (E Strain) Extracts by MTT Assay (72 h)a
Doses, µg/mLViabilityP Value
10000 E27.9 ± 11.260.0001
1000 E57.2 ± 23.140.0001
100 E68.6 ± 27.760.0001
10 E80.0 ± 32.390.0001
1 E89.0 ± 36.030.0001
0.1 E96.5 ± 39.052370.048
Positive control E13.3 ± 5.3490770.0001d

a Values are expressed as mean ± SD.

5. Discussion

According to past studies, probiotics can inhibit colorectal cancer initiation or progression through change of intestinal microbial compounds, protection against pathogens, production of biological components, such as short-chain fatty acid, inactivation of carcinogenic compounds, regulation of immune responses, apoptosis induction, anti-proliferative activity, and antioxidant properties (16-19). The study of Lee et al. demonstrated that LAB plays an important role in the host immune system to produce anti-tumor effects (20, 21). They showed that the butanol extract of B. adolescentis SPM0212 dose-dependently inhibited the growth of Caco-2, HT-29, and SW480 cells by 70%, 30%, and 40%, at 200 μg/mL, and induced macrophage activation and significantly increased the production of TNF-α and NO, which regulated immune modulation and was cytotoxic to tumor cells (21).

Kumar et al. observed in Caco-2 cells that VK1 inhibited proliferation significantly (P < 0.05) at the highest concentrations (100 and 200 μM). The effect began after 24 hours of infusion and persisted up to 72 hours (by 43.4% and 44.3% at 100 and 200 μM, respectively) (18). All of these prove lactobacillus anti-cancer effects. In this study, the researchers assessed the effect of cell-free lyophilized filtrate from L. fermentum and L. rhamnosus on Caco-2 cells. The results showed that cell-free lyophilized filtrate of these bacteria diminished proliferation and increased Caco2 cell death in a dose-dependent manner. In Choi et al. study, the inhibitory effect of the soluble polysaccharide derived from Lactobacillus acidophilus 606 was evaluated on the growth of colon cancer cells and hEF cells using MTT. Similar results were found on other cancer cell lines of the present study (22).

Ewaschuk et al. showed that Lactobacillus acidophilus, L. bulgaricus, L. casei, L. plantarum, Bifidobacterium breve, B. infantis, B. longum, and Streptococcus thermophiles decreased the viability of HT-29 and Caco-2 cells and induced cell death (23). Chen et al. found that oral inoculation of probiotics L. acidophilus on CT-26 murine colon adenocarcinoma cells in mice had a cytotoxicity effect and increased apoptosis (24). Gamallat et al. reported that L. rhamnosus had a protection effect against colon carcinogenesis and induction of apoptosis in a rat model (25). Gayathri and Asha showed that Lactobacillus fermentum and Lactobacillus plantarum had synergistic effects with vincristine on 1,2-dimethylhydrazine-induced colorectal carcinogenesis in mice (26). In this study, probiotics increased anti-cancerous activity of vincristine. The present study showed that cell-free lyophilized filtrate of L. fermentum and L. rhamnosus had anti-proliferative properties on Caco-2 cells.

In a study about anti-cancer effects of probiotics in animal models, Tiptiri-Kourpeti et al. demonstrated that tumor growth inhibited by 109 CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Their findings provided evidence for beneficial tumor-inhibitory, anti-proliferative, and proapoptotic effects driven by this probiotic LAB strain (4). Kahouli and Malhotra reported that L. fermentum NCIMB 5221 significantly inhibited more proliferation of cancer cells than L. fermentum NCIMB 2797 after 48 hours (~46%) and 72 hours (~58%). However, the probiotic treatment was efficient after 72 hours. Also, their study revealed that from three L. fermentum strains, only L. fermentum NCIMB 5221 had low anti-cancerous activity (~6%) after 24 hours (27). In Er et al.’s study 1 × 106 Caco-2 cells/well was cultured on a 96-well plate. The effect of L. plantarum was examined by the MTT assay for viability of cultured Caco-2 cells. They reported that incubation time may influence anti-proliferative activity (28). However in the current study, the researchers found that L. fermentum (strain E) had high anti-cancerous activity after 24 hours with concentration of 10000 µg/mL. Also, other concentrations were more effective than L. fermentum NCIMB 5221 after 24 hours. On the other side, after 48 hours and 72 of the L. fermentum (strain E) at concentration of 10000 µg/mL was more effective than L. fermentum NCIMB 5221. Furthermore, Sadeghi-Aliabadi et al. showed that cell-free filtrate of a commercial strain of L. rhamnosus GG had anti-proliferative effect on Caco-2 at 2500 to 10000 µg/mL after 48 hours (~21% to ~66%) (29). However, in the current study L. rhamnosus (strain G) at concentrations of 1000 µg/mL and 10000 µg/mL had anti-proliferative properties with a rate of ~40% and ~70% at 48 hours, respectively. Thus, L. rhamnosus (strain G) was more effective than L. rhamnosus GG. There was a correlation between percentages of anti-proliferation and adhesion to cancer cells from probiotic bacteria (30). Thirabunyanon and Hongwittayakorn showed that ability of the L. fermentum RM28 strain to adhere to Caco-2 cells was 7%. Anti-proliferative rate of this strain was determined as ~23% by the MTT assay (30). Verdenelli et al. reported that L. rhamnosus IMC501 had ~15% adhesion activity to HT-29 colorectal cancer cell line (31). According to these studies, it seems that L. fermentum (strain E) and L. rhamnosus (strain G), through rate of adhesion to Caco-2 cells, can induce anti-proliferative components on these cells. Er et al. investigated the effect of the cell-free lyophilized filtrate of L. plantarum isolated from meat on Caco-2 cell line and observed weak inhibition of Caco-2 cell proliferation (~5% to 14%) at concentrations of 0.1 to 1000 μg/mL during 24 hours. The cytotoxicity effect of probiotic extract at concentration of 10 000 μg/mL was 33% at 24 hours of incubation (28). However, extracts of both L. fermentum (strain E) and L. rhamnosus (strain G) in the current study were more effective than L. plantarum in the above-mentioned study at different concentrations after 24 hours. Thus, the use of individual Lactobacillus, such as L. fermentum (strain E) and L. rhamnosus (strain G) or combination of different Lactobacillus strains, to eliminate colorectal cancer cells can be a useful strategy in prevention and treatment. However, further evaluations are required to uncover the usefulness of probiotics in treatment of colorectal cancer in clinical stages. One of the limitations of this study was the lack of time required during the dissertation process, which the researchers hope will continue the project in the future.

5.1. Conclusions

In conclusion, both the L. fermentum and L. rhamnosus strains were used in this study, and exhibited 50% to 80% killing of the Caco-2 cells. Hence, the two Lactobacillus strains could be considered as common probiotic for human consumption, due to their beneficial anticancer effects.



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