In Silico Analysis of the Cytotoxic Influence of Nitric Oxide Induced by Lactic Acid Bacteria Cell-Free Supernatant on Colorectal Cancer Cells (HCT 116)

Abstract

Colorectal cancer (CRC) treatments not only cause immunogenic cell death of tumors but also targets normal healthy cells. Thus, novel treatments which target cancer cells only are being discovered to reduce colorectal incidence through chemoprevention. This research aims to determine the influence of nitric oxide production on the cytotoxicity of HCT 116 cancer cells induced by two lactic acid bacteria (LAB) strains (Lactobacillus plantarum BS25 and Pediococcus acidilactici S3) cell free supernatants (CFS) grown in deMan-Rogosa- Sharpe (MRS) broth and McCoy’s 5A basal medium. Viable plate count shows MRS broth has a higher yield of LAB culture compared to McCoy’s 5A medium thus being able to produce more metabolites as demonstrated by spectrophotometric reading. Moreover, there is no significant difference in the cytotoxic effect of LpBS25 and PaS3 CFS in terms of time collection (at 24th hour and 72nd hour) and media used (MRS and McCoy’s 5A medium). Hence, MRS and McCoy’s 5A media can be both used in collecting LAB CFS at 24th and 72nd hour since it has the same cytotoxic effect. However, among all the concentrations used in this study, LAB CFS at 25% in MRS and McCoy’s 5A medium collected in both 24th hour and 72nd hour exhibited higher cytotoxicity against HCT 116 showing membrane blebbing. Based on the whole genome analysis of L. plantarum, NOS genes are not present in this bacterium but nitrite reductase (nir)-like protein homologues are present instead, as determined by the nir conserved sequence. This suggests that nitric oxide in L. plantarum is produced through the nitrite reductase substrate which is recommended to be validated through RT-PCR or DNA microarray. Since LAB CFS has cytotoxic effect to colorectal cancer cells and L. plantarum is capable of producing nitric oxide, in silico analysis of HCT 116 treated with nitric oxide was done to determine if the form of cell death induced by LAB CFS is apoptosis. Gene expression profiling has shown ten genes are responsible for apoptosis, wherein the HMGB1 and HMGB2 genes function on DNA change and fragmentation while the genes AURKA, CCNB1, GTSE1, PLK2, and PLK3 involved in p53 network. Downregulation of the genes THBS1, TRIAP1, BIRC3, CDKN1A, and FHL2 that inhibit apoptosis and the downregulation of H2AFX, HMGB1, HMGB2, and PIF1 genes which functions for DNA repair were also triggered. Thus, this in silico study shows that L. plantarum is capable of producing nitric oxide from nitrite and NO-treated HCT 116 triggers the upregulation and downregulation of apoptotic genes. It is recommended to do nitric oxide assay and apoptotic assay to further confirm these findings.