Anti-cancer effects of Bifidobacterium species in colon cancer cells and a mouse model of carcinogenesis

Abstract

Introduction: Probiotics are suggested to prevent colorectal cancer (CRC). This study aimed to investigate the anticancer properties of some potential probiotics in vitro and in vivo.

Materials and methods: Anticancer effects of the following potential probiotic groups were investigated in LS174T cancer cells compared to IEC-18 normal cells. 1. a single strain of Bifidobacterium. breve, 2. a single strain of Lactobacillus. reuteri, 3. a cocktail of 5 strains of Lactobacilli (LC), 4. a cocktail of 5 strains of Bifidobacteria (BC), 5. a cocktail of 10 strains from Lactobacillus and Bifidobacterium (L+B). Apoptosis rate, EGFR, HER-2 and PTGS-2 (COX-2 protein) expression levels were assessed as metrics of evaluating anticancer properties. Effect of BC, as the most effective group in vitro, was further assessed in mice models.

Results: BC induced ~21% and only ~3% apoptosis among LS174T and IEC-18 cells respectively. BC decreased the expression of EGFR by 4.4 folds, HER-2 by 6.7 folds, and PTGS-2 by 20 folds among the LS174T cells. In all these cases, BC did not interfere significantly with the expression of the genes in IEC-18 cells. This cocktail has caused only 1.1 folds decrease, 1.8 folds increase and 1.7 folds decrease in EGFR, HER-2 and PTGS-2 expression, respectively. Western blot analysis confirmed these results in the protein level. BC significantly ameliorated the disease activity index, restored colon length, inhibited the increase in incidence and progress of tumors to higher stages and grades.

Conclusions: BC was the most efficient treatment in this study. It had considerable "protective" anti-cancer properties and concomitantly down regulated EGFR, HER-2 and PTGS-2 (COX-2), while having significant anti-CRC effects on CRC mice models. In general, this potential probiotic could be considered as a suitable nutritional supplement to treat and prevent CRC.

Fig 1. Flow cytometry analysis of cancer LS174T and normal IEC-18 cells before and after 120h treatment with bifidobacteria cocktail (100 bacteria/cell), cetuximab and trastuzumab.

Cells considered as viable were Annexin V and PI negative; cells in early apoptosis stage were Annexin V positive and PI negative; and cells in late apoptosis/ necrosis stage were both Annexin V and PI positive. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.

Fig 2. Relative fold change (relative to untreated control cells) of the EGFR gene among LS174T and IEC-18 cells.

Results were expressed as mean; error bars (SD); n = 3. Statistical analysis was performed using one-way ANOVA test. * indicates P-values less than 0.05, ** indicates P-values less than 0.01, and *** indicates P-values less than 0.001. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.

Fig 3. Relative fold change (relative to untreated control cells) of the HER-2 gene among LS174T and IEC-18 cells.

Results were expressed as mean; error bars (SD); n = 3. Statistical analysis was performed using one-way ANOVA test. * indicates P-values less than 0.05, ** indicates P-values less than 0.01, and *** indicates P-values less than 0.001. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.

Fig 4. Relative fold change (relative to untreated control cells) of the PTGS-2 gene among LS174T and IEC-18 cells.

Results were expressed as mean; error bars (SD); n = 3. Statistical analysis was performed using one-way ANOVA test. * indicates P-values less than 0.05, ** indicates P-values less than 0.01, and *** indicates P-values less than 0.001. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.

Fig 5. Western blot analysis demonstrating the expression of EGFR, HER-2 and COX-2 proteins in LS174T and IEC-18 cells.

Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls. β-Actin was used as the loading control.

Fig 6. Macroscopic and histological assessment of colic tumor and inflammation in mice.

A) Average Disease Activity Index (DAI) score, B) Colonic length and macroscopic tumor incidence, C) Semi-quantitative scoring of average histological inflammation, D) Representative H&E-stained images of distal colon tissues from 1) PBS mice group (negative control), AOM/DSS + BC group and 3) AOM/DSS + PBS mice (positive control); scale bars, 200 μm. Data were represented as the mean ± SD (n = 5 mice per group).