Evaluation of Probiotic Properties and Toxic Effects of Lactobacilli Cell Extract Isolated from Dairy Products of Guilan Province on HT29 Cancer Cells and HUVEC Normal Cells

Due to the negative effects of cancer, various methods such as surgery, chemotherapy and radiotherapy are used to treat cancer and are used as primary methods in the treatment of colorectal cancer. However, the success rate of this treatment is not enough and the death rate due to it increases every year, and a series of necessary strategies are needed to control this deadly disease (5). In general, the main characteristic of cancer cells is uncontrollable cell proliferation and resistance to programmed death, so an agent that causes apoptosis in cancer cells can be known as an anticancer substance (8). Among various cases, probiotics control the digestive enzymes of animals and humans, inhibit cancer agents in the body and in laboratory conditions, and also play an important role in suppressing compounds and tumors caused by cancer in laboratory animals (9, 10). HT29 is not only used to study the biology of human colon cancers, but is of particular interest in studies focused on digestion and accessibility due to its ability to express the characteristics of colon cells (11). HUVEC are human umbilical vein endothelial cells used to study endothelial cell function and pathology (eg, angiogenesis). Primary isolated HUVECs are probably the most popular used in research, as human umbilical vessels are more readily available than other blood vessel types (8). Today, due to the increasing prevalence of gastrointestinal cancer, this issue is considered as an important global health challenge, and in the past few decades, it has been shown that the imbalance in the intestinal microbiota is related to the rate of various chronic disorders, including cancer. Oral administration of different strains of probiotics can prevent the occurrence of cancer or reduce the incidence of inflammation after surgery (12). Therefore, the researcher is trying to answer the question whether probiotic properties and toxic effects of Lactobacillus cell extract isolated from dairy products of Gilan province have an effect on HT-29 cancer cell line and normal HUVEC cell or not.

In order to carry out this research, lactobacilli were first isolated from dairy samples, then morphological, biochemical, genetic and phylogenetic identification was performed on them. Using sequencing and then analyzing the determined sequences on the NCBI site, Lactobacillus fermentum species (L. Fermentum) was detected with 88.61% homology and its phylogenetic tree was drawn and registered as Limosilactobacillus fermentum GL strain. The isolated lactobacillus extract was affected on the HT-29 cancer cell line and normal HUVEC cells, and the survival of the cells under the effect of lactobacillus was determined using the MTT test (3-(4,5 dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide) was investigated. Finally, considering that all the tests were performed in triplicate, Minitab ver16 software and GraphPad Prism ver9 software were used to determine the mean (One-Way Unstacked ANOVA) and standard deviation, respectively (p < 0.05).

The results showed that the bacteria were gram positive, catalase negative, oxidase negative, negative movement, no growth at 15°C and growth at 37°C and 45°C. Also, molecular analysis showed the amplification of S rRNA16 gene in two of the 4 samples. Further, the blast results showed that the desired sequence of one of the samples with 61.88% similarity belongs to Lactobacillus fermentum. Then, the phylogenetic tree resulting from the analysis of S rRNA 16 sequence was performed by NCBI genome database and MEGA 7 software, and its relationship with other bacteria is shown. Finally, gene registration was done from the sequence obtained in the NCBI database and the desired bacterium was named Limosilactobacillus fermentum GL. Also, the results of cell viability were evaluated using the MTT colorimetric method. Based on the results, cytoplasmic extract in concentrations of 0.5, 0.75 mg/ml did not significantly change the cell viability of HT-29 cells, and concentrations of 1, 1.5, 2 mg/ml increased cell viability by 4.87±50%, respectively, decreased 8.61±40%, 5.80±25%. The IC50 value of cytoplasmic extract for HT-29 cells was 1.43 mg/ml. Also, the results of the cytotoxicity study showed that the concentrations used of the cytoplasmic extract had no inhibitory effect on the normal HUVEC cell line for 24 hours (P<0.05).

Despite recent advances in cancer treatment strategies, cancers are one of the most important causes of death worldwide. Although some anticancer treatments such as surgery, chemotherapy, and radiotherapy have been used with varying degrees of success in many types of cancer patients, these treatments are expensive and have harmful side effects including cardiotoxicity, diarrhea, intestinal strictures, and Inability to effectively absorb nutrients. Probiotics have been suggested as supplements to increase the effectiveness of anti-cancer treatments (24). The results showed that the concentrations of 1, 1.5 and 2 mg/ml of lactobacillus extract can reduce the cell viability by 50±4.87, 40±8.61 and 25±5.80% respectively during 24 hours. . Based on the MTT test, a concentration of 1 mg/ml of the cytoplasmic extract inhibited 50% of HT29 cells and had no toxic effect on normal HUVEC cells. In a study after 24-hour treatment of HT29 cells with 109 CFU/mL of Lactobacillus casei ATCC 393, cell survival decreased by 78% (13). Probably, in the future, these species or their metabolites can be used as food supplements or additives with anti-cancer activity, and for the production of such products, the selection of Lactobacillus strain is of high value (4). As shown in the results, the cell extract of Limosilactobacillus fermentum GL had important effects on growth inhibition in HT-29 cells by suppressing cell proliferation. However, the exact underlying mechanisms of the effects of probiotics on cancer cells have not been fully defined (4). It should be noted that the bacteria used in this study (Limosilactobacillus fermentum GL) were isolated from traditional dairy samples of Gilan and their anti-proliferative and anti-cancer effects were investigated for the first time.