Lactobacillus casei Exerts Anti-Proliferative Effects Accompanied by Apoptotic Cell Death and Up-Regulation of TRAIL in Colon Carcinoma Cells

Abstract

Probiotic microorganisms such as lactic acid bacteria (LAB) exert a number of strain-specific health-promoting activities attributed to their immunomodulatory, anti-inflammatory and anti-carcinogenic properties. Despite recent attention, our understanding of the biological processes involved in the beneficial effects of LAB strains is still limited. To this end, the present study investigated the growth-inhibitory effects of Lactobacillus casei ATCC 393 against experimental colon cancer. Administration of live Lactobacillus casei (as well as bacterial components thereof) on murine (CT26) and human (HT29) colon carcinoma cell lines raised a significant concentration- and time-dependent anti-proliferative effect, determined by cell viability assays. Specifically, a dramatic decrease in viability of colon cancer cells co-incubated with 10(9) CFU/mL L. casei for 24 hours was detected (78% for HT29 and 52% for CT26 cells). In addition, live L. casei induced apoptotic cell death in both cell lines as revealed by annexin V and propidium iodide staining. The significance of the in vitro anti-proliferative effects was further confirmed in an experimental tumor model. Oral daily administration of 10(9) CFU live L. casei for 13 days significantly inhibited in vivo growth of colon carcinoma cells, resulting in approximately 80% reduction in tumor volume of treated mice. Tumor growth inhibition was accompanied by L. casei-driven up-regulation of the TNF-related apoptosis-inducing ligand TRAIL and down-regulation of Survivin. Taken together, these findings provide evidence for beneficial tumor-inhibitory, anti-proliferative and pro-apoptotic effects driven by this probiotic LAB strain.

Fig 1. Lactobacillus casei inhibits proliferation of colon cancer cells.

Anti-proliferative effect of increasing concentrations of selected preparations of L. casei cell-free-supernatant (CFS), heat-killed sonicated (HK-SON) and live L. casei (LC) at different time points on murine CT26 (A) and human HT29 (B) colon cancer cells, determined by the sulforhodamine B assay. Percentages of growth inhibition of L. casei-treated to control PBS-treated cells and CFS/HK-SON-treated to MRS-treated cells are presented as mean values ± s.d. from eight replicates. All data shown are representative of at least 4 independent experiments. Data were analyzed with the statistical software SPSS using Student’s t-test. Differences between control and treated groups are considered statistically significant when p < 0.05 (*).

Fig 2. Acidic pH in culture medium is only partly involved in the Lactobacillus casei-induced anti-proliferative effect.

(A) pH values measured in culture supernatants (see Materials and Methods) after co-incubation of CT26 cells with increasing concentrations of live L. casei for 24 hours. Control cells (indicated as 0) were cultured for the same time period in the absence of L. casei. (B) Lactic acid production in the supernatant of L. casei-treated CT26 cells after a 24 hour co-incubation period. Note the consumption of glucose. (C) Effect of acidic pH on the proliferation of CT26 cells. The percentage growth inhibition refers to control CT26 cells cultured in standard DMEM medium having a pH of 7.66 (D) Comparison of the growth-inhibitory effect of different concentrations of live L. casei on CT26 cells versus the effect of pH adjusted medium. Values of pH are comparable (see 2A) to those induced by co-incubation with higher concentrations of live bacteria. Results were reproduced in 3 independent experiments.

Fig 3. Fluorescently-labeled live Lactobacillus casei adheres to human and murine colon cancer cells.

(A) Live L. casei or E. coli were stained with 20 μM CFSE. Live CT26 or HT29 cells were stained with the nuclear dye Hoechst 33342 and the cytoplasmic membrane dye CellBrite Red dye, and then treated with 10⁹ CFU/mL L. casei or E. coli for 5 h and observed by confocal fluorescence microscopy (Stack of 2D images, Magnification: 1000x). (B) Flow cytometric analysis of adhesion. The adhesion of CFSE-labeled L. casei on murine CT26 or human HT29 cells was detected by the shift in FL1 intensity. Cells were co-incubated with 10⁹ CFU/ml CFSE-stained L. casei for 2 hours or with 10⁸ CFU/mL for 24 hours. At least 50.000 cells per sample were analyzed. CFSE-labeled bacteria in suspension were also analyzed as a background control but due to the size difference were not detected in the flow cytometric settings used. (C) Comparison of fluorescence intensity profiles of CT26 and HT29 cells treated with 10⁸ CFU/mL L. casei for 24 hours.

Fig 4. Lactobacillus casei induces apoptotic cell death in colon cancer cells detected by confocal microscopy.

(A) CT26 or HT29 cells were co-incubated with 10⁹ CFU/mL L. casei for 24 hours and stained with propidium iodide (PI). Following treatment, live PI-positive CT26 (A) or HT29 (B) cells were visualized (red) as compared to control non-treated cells. Nuclei were counter-stained with Hoechst 33342 (blue) (Magnification: 400x). (B) A representative late apoptotic HT29 cell following L. casei treatment (Magnification: 800x). HT29 cells were co-incubated with 10⁹ CFU/mL L. casei for 24 h and stained with Hoechst, PI and Annexin V-FITC. (C) Detection of apoptotic cell death by chromatin condensation and nucleus segmentation. Fluorescence confocal images (black/white and colour) from successive focal planes of apoptotic nuclei of HT29 (a) or CT26 (b) cells following treatment of cells with 10⁹ CFU/mL L. casei for 24 h. DNA was stained with Hoechst 33342. Apoptotic nuclei with condensed chromatin (top nucleus in (a) and nuclei in (bii), (biv), (biii)) and fragmented nuclei (a) down and both nuclei in (bi)) are visualized (Magnification: 400x).

Fig 5. Lactobacillus casei induces apoptotic cell death in colon cancer cells detected by flow cytometry.

CT26 (upper row) or HT29 cells (lower row) were co-incubated with 10⁸ or 10⁹ CFU/mL L. casei for 24 hours. Apoptotic cell death of treated cells was detected by dual staining with Annexin V-FITC and PI followed by flow cytometric analysis (see Material and Methods). The percentage of the following cell populations is indicated: Annexin V-FITC and PI negative stained, indicating viable cells (lower left quadrant), Annexin V-positive and PI-negative stained, indicating early apoptotic cells (lower right quadrant), and Annexin V/PI double-stained cells showing late apoptosis (upper right quadrant).

Fig 6. Lactobacillus casei upregulates the expression of the apoptosis-inducing ligand TRAIL and downregulates transcriptional expression of cyclin D1 and BIRC5a in colon cancer cells.

(A) Relative gene expression (mean fold change) of TRAIL (Ai), BIRC5a (Aii) and cyclin D1 (Aiii) in L. casei-treated versus non-treated CT26 cells. The values of mean fold change in gene expression are presented in Aiv). CT26 cells were co-incubated with increasing concentrations of L. casei at indicated timepoints, and then RT-PCR was carried out with specific primers. For analysis, beta-actin was used as the internal reference and non-treated CT26 cells were used as the calibrator. mRNA relative expression for all genes was calculated by the comparative quantification Ct method (ΔΔCt). Results are representative of three independent experiments. Asterisks in (Ai), (Aii) and (Aiii) and numbers in bold in (Aiv) represent statistically significant differences. (B) TRAIL expression is detected by confocal fluorescence microscopy in CT26 or HT29 colon cancer cells after incubation with 10⁸ CFU/mL L. casei for 12 hours or 10⁹ CFU/ml L. casei for 5 hours. Cell nuclei were stained with DAPI and TRAIL was detected using a specific antibody and visualized by an AlexaFluor647-conjugated secondary antibody (Stack of 2D images, Magnification: 1000x). (C) TRAIL expression was examined by Western blot analysis in CT26 and HT29 cells. Cells were treated with 10⁸ CFU/mL L. casei for different time points. Membrane protein extract was used to detect TRAIL protein with a specific antibody against TRAIL and the respective soluble protein fraction was used to determine β-tubulin protein expression with a specific anti-β-tubulin antibody. Left gel, control non-treated CT26 cells (lane 1), CT26 cells treated with 10⁸ CFU/mL L. casei for: 5 hours (lane 2); 12 hours (lane 3); 24 hours (lane 4). Right gel, non-treated HT29 cells (lane 1); HT29 cells treated with 10⁸ CFU/mL L. casei for 12 hours (lane 2).

Fig 7. Oral administration of live Lactobacillus casei inhibits in vivo growth of colon carcinoma tumors in mice.

(A) Schematic representation of the in vivo tumor model. Live L. casei was administered per os daily to BALB/c mice for 13 days. At day 10, 5x10⁶ CT26 cells were inoculated subcutaneously. Mice in the control group received PBS. Tumors were harvested from euthanized animals 7 days after administration of CT26 cells. (B) Mean tumor volume of tumors excised from mice that received L. casei (LC) or PBS (control). A statistically significant (p < 0.001) reduction of ≈80% in tumor volume was observed in L. casei-treated mice as compared to control. (C) Photographic observation of tumors harvested from control (PBS)- or L. casei (LC)-treated mice. (D) Immunohistochemical detection of TRAIL (Di, Diii) and Survivin (Dii, Div) in CT26 tumors in BALB/c mice. Detection of TRAIL in tumor tissue from L. casei-treated (Diii) compared to control (Di) mice, and Survivin in tumor tissue from L. casei-treated (Div) or control (Dii) mice. At least ten sections per tumor and seven tumors per group from two independent experiments were analyzed. The difference in the expression of TRAIL in tumor tissue from treated (Diii) versus control (Di) mice is statistically significant (p = 0.007), as calculated using the SigmaPlot 11.0 statistical software.