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Abstract
The striking increase in colorectal cancer (CRC) has shown the great fatality in Korea for more than 15 years. The leading edge of this rising incidence rate is mainly due to the people's dietary changes in Korea. Some studies have reported that the dietary fiber does not have significant cytotoxic effects on CRC cells, which contrasts to the effects of probiotics. It gives a positive evaluation that the nonpathogenic spore-forming Bacillus species among the probiotics including fermented bacteria might have optimistic effects on CRC incidence rate. Recently, we isolated Bacillus lentus (BL) from Korean soybean fermented food. BL showed the cytotoxic effect on human colon carcinoma cell lines HCT116 and SW480. Interestingly, BL did not have effect on human dermal fibroblast cells and human hepatoma cell line HepG2. It suggested that BL has the target cell-specific cytotoxicity toward human colon carcinoma cells. To clarify the death signaling pathway underlying the BL-induced apoptosis in cancer cells, we analyzed the expression of caspases, Bax and Bcl-2 by western blotting. The apoptotic effects by cytotoxic elements were executed by direct BL contact or membrane-derived vesicles isolated from BL. Treatment of HCT116 with BL activated caspase-9, -3 and increased cleavage form of poly (ADP-ribose) polymerase (PARP). However, caspase-8 activity was not increased by BL. BL-activated intrinsic pathway increased the pro-apoptotic Bax, decreased the anti-apoptotic Bcl-2 proteins on mitochondria, disrupted the mitochondrial membrane potential, and then released the cytochrome c from mitochondria. The membrane-derived vesicles (MVs) from BL induced apoptosis of the HCT116. Here, we propose that BL as a strong candidate for the development of apoptosis-specific antitumor agent will give great contribution to the understandings of the tumor-microbe interdisciplinary areas.
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Figure 1.
B. lentus (BL)-induced cytotoxic effects on human dermal fibroblabst (HDF) cells and human cancer cell lines HCT116, HepG2, SW480E, SW480R. Cells (5 × 104 cells per well in 96-well plate) were treated with a various MOI (0.1, 1, or 10) of BL for 72 hours. Cell survival was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Figure 2.
B. lentus (BL)-induced apoptosis in cancer cells. (A) After treatment with BL (MOI=1) for the indicated times, HCT116 cells were fixed, stained with fluorescein isothiocyanate-conjugated annexin V and PI, and analyzed by Flow cytometry. Data are the mean ± SD of five experiments. The result demonstrated the per-centage of the ratio of cells in apoptosis and necrosis. (B) HCT116 cells were pretreated with 50 μM z-VAD-fmk (a pan-caspase in-hibitor) for 45 min and then treated with BL (MOI=1) for 3 days. Cell survival was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The mean levels of cell viability following medium were set to 100, and the relative loss of cell viability in presence of BL or inhibitor is shown.
Figure 3.
B. lentus (BL)-induced mitochondria-dependent apoptosis in HCT116. (A) HCT116 cells were treated with BL (MOI=1) for the indicated times. The cells were harvested and subjected to Western blot analysis for cleaved caspase-8, -9, -3, and PARP. (B) Caspase-3, -8, and -9 activities were determined using the caspase-3, -8, or -9 fluorogenic peptide substrates DEVD-AFC, IETD-AFC, and LETD-AFC, respectively.
Figure 4.
The effect of B. lentus (BL) on Bcl-2 family members and on cytochrome c release. (A) At the indicated time points after treatment with BL (MOI=1), the cells were harvested and subjected to Western blot analysis for phosphorylated Bcl-2, Bcl-2, BCL-XL, and Bax. Subcellular fractions were prepared as described in Materials and Methods. Cytochrome c release from mitochondria to cytosol was analyzed using anti-cytochrome c antibody. (B) The effect of BL on mitochondrial transmembrane potentials (Δψm) in HCT116 cells. The Cells were treated with and without BL (MOI=1). After 4 days, cells were incubated with 10 μM rhodamine123 for 20 min and scored immediately by flow cytometry. Mean fluorescent intensity in HCT 116 cells was determined by flow cytometry (Bottom).
Figure 5.
Cytotoxic effects of membrane vesicles (MVs) from B. lentus (BL) on human colorectal cancer cell line HCT116. Cells were treated with various concentrations of BL OMV (5, 10,20 μg/ml) for 48 h. Cell survival was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were conducted on HCT116 cells as described in Materials and Methods.
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