Assessment of the Anti-proliferative Capabilities of Probiotic Bacteria on some Malignant Cell Lines

Abstract

The incidence of colorectal and breast cancer increases worldwide, breast cancer is the most common cancer among women worldwide, Breast cancer pervasion is rising worldwide with an increase in attacker neoplasia in young women. 375, 000 deaths worldwide were caused in 1 million new cases per year. Colon and rectal cancers are often grouped together because they have many features in common. Colorectal cancer is the third most common type of cancer in oncologic pathology. Currently, it is the most common malignant cancer in the gastrointestinal tract, representing 13% of all malignant tumors. In this context we assess the efficiency of Lactic acid bacteria as anti-tumor. The present study aimed to maximize the anti-tumor activity of probiotics. The obtained results can be summarized as the following: Four probiotic bacterial strains were used in the study; Lactobacillus casei ss. casei (LC 1093), (isolated from cheese), Lactobacillus delbreuckii ss. bulgaricus (Isolated from yoghurt), (LD 1102), Bifidobacterium bifidium (isolated from the intestine of adult) (BB 1334) and Lactobacillus acidophilus (ATCC 4356) isolated from human. Two different types of cancer were tested, thus human breast cancer (MCF7) and human colon cancer (Caco) cell lines. To evaluate the effect of probiotics strains on cancer cell lines, Trypan blue test was performed to determine the viable cancer cells as affected by the treatment with supernatant of probiotic culture for breast cancer, results indicated that percentages of dead cells produced by the treatment with supernatants of L. casei, L. delbrueckii, B. bifidium L. acidophilus, were 9,11,14,15 %, respectively, For colon cancer, results indicated that percentages of dead cells produced by the treatment with supernatants of L. casei, L. delbrueckii, B. bifidium L. acidophilus, were 4.79,5.32,6.38, 7.98 %, respectively) MTT assay was used to measure cancer cells viability after application of probiotic strain, Results of MTT assay for breast cancer, indicated that percentages of dead cells produced by the treatment with supernatants of L. acidophilus, B. bifidium, L. delbrueckii and L. casei 10, 2, 3and 9 % respectively, Results of MTT assay for colon cancer, indicated that percentages of dead cells produced by the treatment with supernatants of L. acidophilus, B. bifidium, L. delbrueckii and L. casei 12, 4, 4 and 10 % respectively Bisulfite-treated DNA of MCF7 cells after been treated with probiotic supernatant was subjected to methylation-specific PCR (MSP) to amplify two tumor-suppressor genes (RASSF1A and RB1). Our results indicated that probiotic supernatant of L. casei, L. delbrueckii, B. bifidium, L. acidophilus had a significant effect on the methylation status of RASSF1A and RB1promotor region in breast cancer cells. The unmethylation-specific primers of both genes were able to generate a defined band. The methylation patterns were reshaped when compared to the untreated MCF7 cell. Probiotic strain which showed the highest anti-proliferative activities (Lactobacillus acidophilus,) determined by the MTT assay, was chosen for this part of the study Lactobacillus acidophilus was secerned for it is antimicrobial activity using well diffusion method against Staphylococcus aureus, Bacillus subtilis, Listeria monocytogenes, (Gram positive), E. coli, Salmonella typhi, Pseudomonas aeruginosa, and Shigella shiga (Gram negative). All these Gram positive and negative bacteria were identified by Microbiology Department, Faculty of Agriculture, Ain Shams University. To optimize the anti-proliferative activities of the chosen strain, Lactobacillus acidophilus the following growth conditions parameters were examined Plackett-Burman (PB) to determine their significant effects on the growth and anti-proliferative activities of the chosen strain: MRS, Molasses, Treated Molasses, Whey, Temperature, pH, Inoculum size, Agitation, Incubation time. With the help of Statistical software package Design-Expert software 11.0.0 (Stat-Ease, Inc., Minneapolis, MN 55413, USA 2018). In the experimental design, the independent variables were screened at two levels, low (-) and high (+) in 12 trials Once critical factors were identified via screening using PBD, the central composite design (CCD) was examined to obtain a quadratic model and to determine the optimal levels of the two significant factors, (agitation and treated molasses) to estimate the pure process variability with antitumor production as response Based on these results the compositions of the optimized medium that produce the maximum anti-cancerous effect was Treated molasses =32.5gm/L, Agitation = 25 RPM, Inoculum size =3%, Temperature=30℃, pH=7, whey =55gm/L, MRS =0, Molasses 55gm/L and incubation time= 2 days.