Fermentation supernatants of Lactobacillus delbrueckii inhibit growth of human colon cancer cells and induce apoptosis through a caspase 3-dependent pathway

Abstract

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Therefore, the present study explored the effect of the supernatants obtained from Lactobacillus delbrueckii fermentation (LBF) on colon cancer. The results indicated that the proliferation of LBF solution-treated colon cancer SW620 cells was arrested and accumulated in the G1 phase in a concentration-dependent manner. The LBF solution efficiently induced apoptosis through the intrinsic caspase 3-depedent pathway, with a corresponding decreased expression of Bcl-2. The activity of matrix metalloproteinase 9, which is associated with the invasion of colon cancer cells, was also decreased in the LBF-treated cells. In conclusion, the results demonstrate the antitumor effect of LBF in vitro and may contribute to the development of novel therapies for the treatment of colon cancer.

Keywords: Lactobacillus delbrueckii; apoptosis; caspase 3; colon cancer; matrix metalloproteinase-9.

Figure 1

Effect of LBF solution on the proliferation of colon cancer SW620 cells. The SW620 cells were treated with various concentrations of LBF solution (0, 0.025, 0.038, 0.05, 0.063, 0.076, 0.1, 0.2, 0.25, 0.4, 0.6, 0.625 and 0.75 mg/ml) for 24 h. Growth is expressed as relative to untreated control cells. Data are presented as the mean ± SEM, from three independent experiments. One-way analysis of variance with Bonferroni’s multiple comparison test was used for statistical analysis.^**P<0.01 and ^***P<0.001, vs. untreated control cells. LBF, Lactobacillus delbrueckii fermentation.

Figure 2

Annexin V/propidium iodide double staining assay. (A and B) Results of flow cytometry analysis of the control and 0.25 mg/ml LBF solution-treated groups; lower left quadrant indicates surviving cells and the lower and upper right quadrants indicate the early and late apoptotic cells, respectively. Upper left quadrant indicates necrotic cells, lower left quadrant indicates surviving cells and the lower and upper right quadrants indicate the early and late apoptotic cells, respectively. Images are representative of the results from two independent experiments. (C) Bar graph demonstrating the results of the flow cytometry analysis evaluating the apoptotic rate of cells. Data are presented as the mean ± SEM, from three independent experiments. One way analysis of variance with Bonferroni’s multiple comparison test was used for statistical analysis. ^***P<0.001, vs. untreated control. LBF, Lactobacillus delbrueckii fermentation.

Figure 3

LBF solution induces the activation of the intrinsic apoptotic pathway. Immunohistochemistry images were captured under a microscope (magnification, ×40), of the control and 0.25 mg/ml LBF solution-treated cells stained for (A and B) caspase 3 and (C and D) Bcl-2. (E) Western blot analysis of the SW620 cell lysates treated with and without 0.25 mg/ml LBF solution for caspase 3 and Bcl-2. Uniform loading was confirmed by β-actin and the images are representative of the results from three independent experiments. LBF, Lactobacillus delbrueckii fermentation.

Figure 4

Effects of the LBF solution on the gelatinolytic activity of MMP-9. Gelatin zymography was performed on the untreated and LBF-treated (0.25 mg/ml for 24 h) cells to detect MMP-9 activity. Images are representative of the results from three independent experiments. LBF, Lactobacillus delbrueckii fermentation; MMP-9, matrix metalloproteinase-9.