Introduction: Colorectal cancer is one of the most common and most diagnosed cancers in the world.
There are many predisposing factors, for example, genetic predisposition, smoking, or a diet rich in
red, processed meat and poor in vegetables and fruits. Probiotics may be helpful in the prevention of
cancer and may provide support during treatment. The main aim of this study is to characterize the
potential mechanisms of action of probiotics, in particular the prevention and treatment of colorectal
cancer. Probiotics’ potential mechanisms of action are, for example, modification of intestinal
microbiota, improvement of colonic physicochemical conditions, production of anticancerogenic
and antioxidant metabolites against carcinogenesis, a decrease in intestinal inflammation, and the
production of harmful enzymes. The prevention of colorectal cancer is associated with favorable
quantitative and qualitative changes in the intestinal microbiota, as well as changes in metabolic
activity and in the physicochemical conditions of the intestine. In addition, it is worth noting that the
effect depends on the bacterial strain, as well as on the dose administered.
Age, genetic and environmental factors play an important role in the development of colorectal cancer. Hereditary colorectal cancer syndromes include Lynch syndrome (hereditary non-polyposis colon cancer), familial adenomatous polyposis (FAP), MUTYH-associated polyposis (MAP).
Methods: Gram Staining
The Gram staining of the isolate was determined by light microscopy using Gram staining reagents. It is known that LABs are gram-positive. This means that these cultures will produce blue-violet color for Grampositive bacteria and vice-versa. The cultures were grown in MRS media at 37 °C for 24 h under micro-aerophilic conditions. Fresh cultures were used for gram staining. After incubation, the cultures were aseptically transferred into 1.5 ml of eppendorf tubes and centrifuged for 3 min at 9000 rpm. The cells were resuspended in sterile water by removing the supernatant.
Catalase test
The catalase test was carried out on the isolates to see their reactions to catalase. To do this, two methods can be performed. 18 h incubated cultures of isolates were grown on MRS agar at room temperature. Furthermore, for the catalase test fresh liquid cultures of LAB were used in which 3% hydrogen peroxide solution was added to 1 ml of cultures.
Arginine hydrolysis test
The arginine MRS modified medium and the Nessler reagent was used to view ammonia release from arginine. The freshly prepared 1% culture of the isolates was added into the MRS of 5 ml tubes containing 0.3% of L-arginine hydrochloride. The tubes were further incubated for 18 hours at 37 °C. After incubation, 50 μl of cultures were observed against the white background. 50 μl of the Nessler reagent was pipetted into the cultures and the change in the color was observed. The positive reaction was indicated by a bright orange color, while the yellow color determines the negative reaction. For the negative control, arginine free MRS was used
Hemolysin production test
Hemolysin production was detected using Columbia agar plates supplemented with 5% of sheep blood. The presence of α or β-hemolysis was assessed by the formation of clear or greenish zones around the colonies, respectively.
Low pH and high bile salt concentration tolerance test
The isolate was incubated in 5 mL MRP broth. The 24-h incubated cells were centrifuged at 2000 g for 10 min. The cell plates were suspended in PBS (pH 2.5), followed by incubation for 2 h at 37 C. The cells resistant to low pH were calculated by using the pour plate technique on MRS agar and compared with the isolates in normal PBS for 0 and 3 h. Each examination was conducted twice with three repetitions for each time.
MRS broth medium supplemented with 0.3% Oxgall (Sigma, UK) was inoculated with active cultures (1% v/v) and incubated at 37˚C. Control consisted of MRS broth with the respective bile salt concentrations. Growth was monitored at 0 and 8 hours by recording absorbance at 650 nm. Calculation of coefficient of inhibition (Cinh) was performed via this formula:
Formula
Cinh = ∆T8-T0 Control – ∆T8 - T0 Treatment / ∆T8 - T0 Control
Where, ∆T8 - T0 represents the difference in absorbance at time zero (T0) and after 8 hours (T8). Cinh of less than 0.4 is desirable.
Heat-killed cells (HK cell)
Overnight cultures of the strain was centrifuged (9000 g, 10 min, 4º C) to harvest bacterial cells mass. Next, the cells were washed twice with PBS buffer and resuspended in the buffer and heated at 95º C for 1 h. After that, the mixtures were lyophilized, and the 50mg of lyophilized cells mass were suspended in 1 mL cell culture medium. The prepared stocks were stored at 20º C until they were used in further experiments
Cell-free supernatant (CFS)
The CFS samples were provided by centrifuging overnight bacterial cultures (9000 g, 10 min, 4º C), lyophilizing the supernatant, and solving 50mg of them in 1 mL RPMI media. The final suspension was sterilized using 0.22mm Millipore filters. In a parallel path, to minimize the probable effects of organic acids, the pH of the CFS was adjusted to 7.4 by adding NaOH (1 M), which resulted in neutralized cell-free supernatant (NCFS).
Assessment of cell viability using MTT assay
CLORECTALcancer cell lines HT29 were seeded in a 96-well plate (1.5 × 104 cells/well) and incubated for 24 hours in standard conditions. The cells were treated with different concentrations of supernatant (25, 50, and 100 µg/ml) and heat killed cells (25, 50, and 100 µg/ml) of of isolated strain for 24, 48, 72, and 96 hours. The cancer cell viability was evaluated using the MTT assay (Gibco, United States America).
Results: Conventional lab techniques
The isolated strain was subjected to Gram staining and examined under a light microscope (100X magnification). The strain showed blue-purple color staining. Hence the isolated strain was found Gram-positive bacterium.
According to our result the strain was recorded as catalase negative. Absence of hemolytic activity was also observed. The result of Arginine hydrolysis test showed that the isolated stain did not produce ammonia from arginine.
Identification of Lactobacillus stain
The result showed that the isolated strain belonged to genus Lactobacillus. The 16S rDNA gene sequence result showed that isolate had 99% homology with L.Pediococus
MTT assay results
The MTT assay result showed that the CFS of isolated strain in concentration of 100 µg/ml after 72 hours decreased the viability of HT29 cell line.
The heat killed cells in concentration of 25 µg/ml after 72 hours decreased the viability of HT29 cell lines.
Conclusion: In the present study, the probiotic characteristics and anti-tumor activity of a human breast milk isolated Lactobacillus was investigated. Our results demonstrated that Lactobacillus strain exhibited many typical probiotic characteristics such as Gram staining (Gram-Positive Bacilli), higher survival rate under gastric conditions (lower pH), catalase-negative, L-Arginine test, Lack of hemolytic activity, Lack of Arginase activity.
Despite the fact that this strain had most of the properties of probiotic bacteria, it did not show resistance to bile salts, and among the 6 antibiotics that were examined in this study, this strain was only resistant to gentamicin and sensitive to 5 other antibiotics.
In the investigation of the effect of cell-free supernatant and killed cells of the isolated strain on the viability of two breast cancer cell lines, it was found that the cell-free supernatant of this strain had no effect on the viability of HT29 cell line but decreased the viability of HT29 cell line in concentration of 100 µg/ml after 72 hours.
The heat killed cells of this strain (in concentration of 25 100 µg/ml after 2 h and 100 µg/ml after 48 hours) significantly reduced the viability of HT29cell line.
In investigating the effect of killed cells on HT29cell line in all three concentrations (25, 50, and 100 µg/ml) after 2h the viability) significantly decreased.
Keywords: cancer- colorectal cancer - probiotic - Lactobacillus pediococcus-anti cancer