Effects of Lactobacillus casei Culture Supernatant on Differentiation of K562 Cell Line

Abstract

Background and objective: Considering the toxic side effects of chemotherapy in treatment of cancer, anticancer drugs of natural origin including probiotic Lactobacillus strains have recently attracted a lot of attention. Methods: After culturing chronic myeloid leukemia cell line K562 in 96-well plates, effects of different concentrations of culture supernatant from Lactobacillus casei on differentiation of the cells were investigated after 48 and 72 hours under an inverted microscope. Number of live cells and percentage of viable cells were determined by trypan blue exclusion test of cell viability. Cytotoxicity was assessed by MTT assay. Data analysis was performed by SPSS (version) 22 using one-way analysis of variance and Tukey's test at significance level of 0.05. Results: Secondary metabolites from the probiotic bacteria L. casei induced cellular differentiation, exerted anti-cancer effects and inhibited growth in K562 cells. Apoptotic cell death was confirmed by MTT and DNA fragmentation assays in a way that increasing the dilution from 1.2 to 1.32 significantly increased the viability of cells (P=0.001). In addition, increasing the dilution significantly increased the number of live cells in the first 48 hours (P=0.001). Conclusion: Culture supernatant of L. casei reduces the number of live cells, and induces apoptosis and monocytic differentiation in K562 cells in a dose-and time-dependent manner. Therefore, combined chemotherapy and differentiation therapy using such supernatants could be useful for treatment of cancer. This paper should be cited as: Maftuni K, Zare P[Effects of Lactobacillus casei Culture Supernatant on Differentiation of K562 Cell Line]. mljgoums. 2017; 11(5):16-21 the bacterium. These supernatants are not involved in the growth, proliferation and evolution of bacteria, and are usually produced in the stationary phase of growth (4). In this study, we evaluate the anticancer effects of culture supernatant of this bacterium on K562 cells. The K562 cell line is composed of undifferentiated blast cells that are rich in glycophorin, and derived from chronic myeloid leukemia (CML) patients. The cells were obtained from the Pasteur Institute of Iran. Given that the supernatant derived from Lactobacilli such as L. casei has strong antibacterial properties, and the compounds derived from these bacteria have anti-cancer properties (5), we aimed to evaluate the anticancer effects of culture supernatant from L. casei. MATERIAL AND METHODS For bacterial culture and supernatant extraction, lyophilized L. casei (PTCC 1608) were purchased from the industrial fungal and bacterial collection of Iran. To prepare the bacteria, external surface of the ampules was first disinfected with 70% ethanol, scratched with a diamond scratch pen, and then broken by applying pressure on the scratched area. After addition of 400 μl of MRS broth to each ampule, a uniform suspension was achieved by thorough mixing. The suspension was then added to 20 ml of MRS broth. Remaining drops of the suspension were grown on MRS agar medium to ensure the purity of the bacteria. The media containing L. casei were incubated at 37 °C for 48 hours until the end of the stationary phase. Finally, bacteria-free supernatant was used for testing. Batch culture was performed in order to obtain the supernatant. In this technique, no compound is added or removed during fermentation.