Sulforaphane was purchased from LKT Laboratories (St. Paul, MN, USA) and dissolved in dimethyl sulfoxide (DMSO). Lactobacillus pentosus strain S-PT84 was kindly gifted from the Suntory Global Innovation Center (Kyoto, Japan) (37). Heat-killed L. pentosus S-PT84 was freeze-dried and used for the following experiments: Propidium iodide was purchased from Sigma (St. Louis, MO, USA). 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) was purchased from FUJIFILM Wako pure chemical corporation (Osaka, Japan). Annexin V-FITC apoptosis detection kit was purchased from Nacalai Tesque (Kyoto, Japan). Antibodies against cIAP-1, cIAP-2, Bcl-xL, acetylated-Histone H4, and Histone H4 were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against TNFR1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Bcl-2 and Bim were purchased from Abcam (Cambridge, MA, USA). Antibody against Bax was purchased from BD Biosciences (San Jose, CA, USA). Antibody against GAPDH was purchased from HyTest (Turku, Finland). Antibodies against XIAP, human recombinant TNFα/Fc chimera, human recombinant DR5/Fc chimera, human recombinant Fas/Fc chimera, and zVAD-fmk were obtained from R&D Systems (Minneapolis, MN, USA). Granzyme B inhibitor I was obtained from Calbiochem (San Diego, CA, USA). HRP-conjugated anti-mouse IgG and HRP-conjugated anti-rabbit IgG were purchased from GE Healthcare (Piscataway, NJ, USA).

Human colon cancer HCT116 and SW480 cells and normal human colon epithelial CCD 841 CoN cells were obtained from the American Type Culture Collection (Rockville, MD, USA). HCT116 and SW480 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 4 mmol/l glutamine, 50 U/ml of penicillin G, and 100 µg/ml of streptomycin. CCD 841 CoN cells were maintained in Eagle's minimum essential medium supplemented with 10% (v/v) fetal bovine serum, 4 mmol/l glutamine, 50 U/ml of penicillin G, and 100 µg/ml of streptomycin. Normal human PBMCs were isolated as previously described (25). All cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂.

For co-culture, HCT116 or SW480 cells were seeded onto 12-well plates (BD Falcon, no. 353503). The next day, PBMCs were seeded on cell culture inserts (0.4 µm pores; BD Falcon no. 353494) in a 12-well plate.

The number of viable cells was determined using the Cell Counting Kit-8 according to the manufacturer's instructions (Dojindo Molecular Technology, Kumamoto, Japan).

Cells were stained with 10 µg/ml of propidium iodide. Flow cytometry was performed with FACSCalibur (BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest software (BD Biosciences).

Cells were stained with 2 µmol/l JC-1 and incubated for 15 min. Cells were washed twice and resuspended with PBS. Flow cytometry was performed with FACSCalibur and CellQuest software.

Annexin V-FITC apoptosis detection kit was used according to the manufacturer's protocol. Briefly, cells were resuspended with Annexin V binding buffer and incubated with Annexin V and propidium iodide for 15 min. Flow cytometry was performed with FACSCalibur and CellQuest software.

Cells were centrifuged and the supernatant was collected. A human TNFα ELISA kit (Abcam, Cambridge, UK) was used according to the manufacturer's instructions.

Cells were lysed in buffer containing 50 mmol/l Tris-HCl (pH 7.5), 1% SDS, 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 1 mmol/l dithiothreitol (DTT). The lysate was sonicated and centrifuged at 14,000 g for 20 min at 4°C, and the supernatant was collected. Equal amounts of lysate were analyzed by SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA, USA). The blots were blocked in blocking buffer for 1 h at room temperature and incubated with the appropriate primary antibody in blocking buffer for 1 h at room temperature. The blots were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h, and signals were detected using a Chemilumi-one chemiluminescent kit (Nacalai Tesque, Kyoto, Japan) or Immobilon Western Chemiluminescent HRP Substrate (Millipore). The band intensities were quantified by ImageJ software (NIH).

Data are the means ± standard deviation of three experiments. For simple group means analysis, data were analyzed using a Student's t-test and differences were considered significant at P<0.05. For between-group comparisons, one-way ANOVA and Tukey's post hoc test were performed using Microsoft Excel 2007 (Microsoft Corporation). P<0.05 was considered to indicate a statistically significant difference.

Sulforaphane, a well-known phytochemical, induces apoptosis in cancer cells (10–14). We investigated the effect of sulforaphane on cell growth in human colon cancer HCT116 and SW480 cells and found that sulforaphane suppressed the growth of both cells in a dose-dependent manner (Fig. 1A).

Previously, we showed that Lactobacillus strains facilitated natural killer activity against human cancer cells in vitro (25). Here, we evaluated the effect of L. pentosus S-PT84 on cellular immunity by inducing apoptosis in human colon cancer HCT116 and SW480 cells in co-culture with PBMCs treated by L. pentosus S-PT84. First, isolated PBMCs from a healthy volunteer were pre-treated by L. pentosus S-PT84 for 24 h. Second, PBMCs were co-cultured with human colon cancer cells. After 48 h, the sub-G1 population was examined by flow cytometry. Although non-treated PBMCs showed basal levels of cytotoxicity, Lactobacillus-treated PBMCs induced apoptosis against HCT116 and SW480 cells (Fig. 1). Similarly, the sub-G1 population was significantly increased by sulforaphane in both human colon cancer cell lines tested (Fig. 1B).

We evaluated the effect of the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs in human colon cancer cells. As shown in Fig. 1B, the addition of sulforaphane increased the sub-G1 population from 33 to 57% in HCT116 cells and from 19 to 49% in SW480 cells under co-culture with Lactobacillus-treated PBMCs. Next, Annexin V/propidium iodide staining was used to analyze apoptosis by treatment in human colon cancer HCT116 and SW480 cells or in normal human colon CCD 841 CoN cells. Sulforaphane markedly enhanced apoptosis induced by co-culture with Lactobacillus-treated PBMCs in human colon cancer cells more than in normal human colon cells (Fig. 2A). In addition, we used PBMCs from another volunteer to assess the combination of sulforaphane and L. pentosus S-PT84-treated PBMCs (Fig. S1). As a result, the combination also induced apoptosis in HCT116 cells markedly. Furthermore, we assessed the mitochondrial transmembrane potential in human colon cancer HCT116 and SW480 cells by JC-1 staining (Fig. 2B). To investigate the mitochondria membrane potential (ΔΨm) involved in apoptotic induction, we analyzed apoptosis using a mitochondria-specific dye JC-1. As shown in Fig. 2B, the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs markedly decreased ΔΨm in human colon cancer HCT116 and SW480 cells, which indicates an increase in apoptosis.

Three death factors (TNFα, FasL, and TRAIL) and their receptors have been identified (38). Furthermore, cytotoxic lymphocytes induce apoptosis in target cells by these death factors and/or perforin and granzyme B (39,40). To investigate the apoptosis pathway induced by sulforaphane and co-culture with Lactobacillus-treated PBMCs, we used a TNFR/Fc chimera, DR5/Fc chimera, Fas/Fc chimera, Granzyme B inhibitor I, and pan-caspase inhibitor. Apoptosis was markedly blocked by the pan-caspase inhibitor zVAD-fmk, which indicates that apoptosis was caspase-dependent in HCT116 and SW480 cells (Fig. 3). Furthermore, the TNFR/Fc chimera partially inhibited apoptosis in both cells (Fig. 3). On the other hand, the DR5/Fc chimera, Fas/Fc chimera, and Granzyme B inhibitor I did not affect apoptosis. These results suggest that the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs induced caspase-dependent apoptosis by the TNFα-TNFR pathway.

We found that TNFα was involved in apoptosis by a combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs. Microbial stimuli increases the secretion of various cytokines, including TNFα, from PBMCs (26). Therefore, we evaluated the amount of TNFα secretion from the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs using culture supernatant by ELISA. As shown in Fig. 4, TNFα concentration markedly increased by co-culture with Lactobacillus-treated PBMCs, regardless of sulforaphane treatment, in both cells.

To investigate the extrinsic and intrinsic pathways of apoptosis induced by the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs, we examined the expression of apoptosis-related proteins using western blotting (Fig. 5), and normalized histograms were shown in Fig. S2. As shown in these data, cIAP-1 and cIAP-2, anti-apoptotic IAP family proteins, were significantly upregulated by co-culture with Lactobacillus-treated PBMCs in both cancer cells. However, sulforaphane treatment suppressed the induction of cIAP-1 and cIAP-2 (Figs. 5A, B and S2). Co-culture with Lactobacillus-treated PBMCs slightly induced the expression of TNFR1, a TNFα receptor, in HCT116 cells (Figs. 5A and S2). Additionally, sulforaphane further increased the induction of TNFR1 in HCT116 cells (Figs. 5A, B and S2). XIAP was markedly downregulated by sulforaphane in SW480 cells with or without co-culture with Lactobacillus-treated PBMCs (Figs. 5B and S2). The expression of Bcl-2 was slightly induced by sulforaphane with Lactobacillus-treated PBMCs in HCT116 cells (Figs. 5A and S2). Co-culture with Lactobacillus-treated PBMCs increased Bax in SW480 cells with or without sulforaphane treatment (Figs. 5B and S2). The expression of Bim, Bcl-xL and Bcl-2 did not significantly change in SW480 cells (Figs. 5B and S2). These results raise the possibility that TNFR1 in the extrinsic pathway and cIAP-1, cIAP-2 and Bax in the intrinsic pathway were involved in apoptosis by the combination of sulforaphane and co-culture with Lactobacillus-treated PBMCs in human colon cancer cells.