Male BALB/c nude mice, aged 6–8 weeks (20±2 g), were purchased from Belda Biomedical Science and Technology Co., Ltd. (Suzhou, China). The mice were maintained and used in strict accordance with international standards of animal care guidelines. All experimental procedures were carried out in accordance with the regulations of the Zhengzhou University Committee on Ethics in the Care and Use of Laboratory Animals and were approved by the University Committee for Animal Experiments.

Human prostate cancer cell line PC3 was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). PC3 cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Gibco, Rockville, MD, USA), 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Rockville, MD, USA), in a humidified atmosphere of 5% CO₂–95% air at 37°C. Sesamin was dissolved initially in dimethyl sulfoxide (DMSO), stored as small aliquots at −20°C, then thawed and diluted in a cell culture medium as required to a final concentration of 0, 10, 50 or 100 μg/ml.

PC3 cells were seeded into 96-well plates (Corning Inc., Corning, NY, USA) at a density of 5×10³ cells/well in RPMI-1640 medium with 10% FBS. After 24 h, the cells were pre-incubated with 0, 10 (S10), 50 (S50) or 100 (S100) μg/ml of sesamin, for 1 h before treatment with 1 μg/ml of LPS (both from Sigma-Aldrich, St Louis, MO, USA) at 37°C in 5% of CO₂. Next, 24 or 48 h later, 20 μl of modified tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5-dipheny-2H-tetrazolium-bromide (MTT, 5 mg/ml; Sigma-Aldrich) was added to each well and samples were incubated at 37°C for 4 h. Then the supernatant was carefully removed and 100 μl of DMSO (Sigma-Aldrich) was added to lyse the cells. After the dark-blue MTT crystals dissolved, the absorbance was measured at 490 nm using a Benchmark microplate reader (Bio-Rad, Hercules, CA, USA).

In regards to the Transwell system, Matrigel-coated 24-well Transwell chambers with 8.0-μm polycarbonated filters (Corning Inc.) were used for the in vitro invasion assay. Here, 1×10⁵ cells/300 μl were suspended in serum-free RPMI-1640 medium in the upper chamber of each well, and the lower well of each chamber was filled with 750 μl RPMI-1640 medium supplemented with 15% of FBS. LPS (1 μg/ml) and sesamin (0, 10, 50 and 100 μg/ml) were added to both the upper and the lower chambers as described above. After 24 h of treatment, the filters were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (both from Sigma-Aldrich). Then, the number of invasive cells in at least five randomly selected microscope fields was counted and analyzed statistically.

PC3 cells were cultured in 6-well plates (Corning Inc.) at a density of 4×10⁵ cells/well in RPMI-1640 medium. LPS (1 μg/ml) and sesamin (0, 10, 50 and 100 μg/ml) were added as described previously. Conditioned medium was collected over 48 h. Cytokines, including TNF-α and IL-6, contained in the conditioned medium were analyzed in triplicate using a sandwich-type enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. The absorbance at 450 nm was determined using a microplate reader (Bio-Rad).

For siRNA transfection, PC3 cells were seeded in 6-well plates at a density of 4×10⁵ cells/well and transfected on the following day with p38-siRNA (25 nM; Cell Signaling Technology, Beverly, MA, USA) or the non-silencing control siRNA (si-NS, 25 nM), as indicated using Lipofectamine 2000 (both from Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The transfection efficiency of p38-siRNA was determined by western blotting.

Cultured PC3 cells were extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) after transfection and stimulation of LPS (1 μg/ml) and sesamin (0, 10, 50 and 100 μg/ml) or SB203580 (10 nM; Calbiochem-Novabiochem Corp., San Diego, CA, USA) as described above for 48 h. Protein concentrations of the cell lysates were determined by the bicinchoninic acid assay (BCA; Thermo Scientific, Rockford, IL, USA) and equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), before being transferred to PVDF membranes (Millipore Corp., Bedford, MA, USA), and then processed according to the standard instructions. The antibodies used were rabbit anti-human phospho-p38 mitogen-activated protein kinase (p38-MAPK) (Thr180/Tyr182) monoclonal antibody (#4631) and rabbit anti-p38-MAPK polyclonal antibody (#9212) (both from Cell Signaling Technology); mouse anti-human cyclin D1 monoclonal antibody (sc20044), goat anti-COX-2 polyclonal antibody (sc23983), mouse anti-human Bcl-2 monoclonal antibody (sc509), rabbit anti-survivin polyclonal antibody (sc10811), mouse anti-human matrix metalloproteinase 9 (MMP-9) monoclonal antibody (sc21733), mouse anti-human intercellular adhesion molecule-1 (ICAM-1) monoclonal antibody (sc107), mouse anti-human vascular endothelial growth factor (VEGF) monoclonal antibody (sc7269) and rabbit anti-β-actin polyclonal antibody (sc130657) (dilution, 1:1,000) (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). The secondary antibodies were anti-rabbit or anti-mouse immunoglobulin G conjugated to horseradish peroxidase (dilution, 1:5,000; Beyotime). Signals were detected by enhanced chemiluminescence (ECL) reagent (Beyotime). The absorbance values of target proteins and data analysis were performed using Gel-Pro Analyzer version 4.0 software (Media Cybernetics, Silver Spring, MD, USA).

Cultured PC3 cells were seeded in 6-well plates at a density of 4×10⁵ cells/well as indicated, respectively. Stimulation of LPS (1 μg/ml), sesamin (100 μg/ml) or SB203580 (10 nM) was performed as described above after p38-siRNA/p38-siNS transfection or not. To prepare nuclear extracts for the NF-κB assay, the cells were washed with cold PBS, collected by centrifugation, resuspended in hypotonic lysis buffer, incubated on ice for 15 min and then mixed with 0.75% Nonidet P-40 (NP-40) solution. The nuclear extract was collected and centrifuged at 4,000 × g for 2 min, and stored at −80°C. Protein concentration was determined by the BCA assay. Then, NF-κB activity in nuclear extracts was analyzed by a NF-κB p65 ActivELISA kit (Imgenex, San Diego, CA, USA) according to the manufacturer’s instructions. The absorbance was determined using a microplate reader set at 405 nm.

Cultured PC3 cells were washed 3 times with PBS. For this, 1×10⁷ cells/100 μl PBS were inoculated subcutaneously at the right armpit of BALB/c nude mice. After 7 days, the mice were administered PBS (control) only, LPS (2 mg/kg) only or sesamin (10 mg/kg) and SB203580 (10 mg/kg), respectively before injection with LPS (n=4 per group). The sesamin and SB203580 were injected every 3 days, respectively. Twenty-one days later, all mice from the different groups were sacrificed. Caliper measurement of tumor volume was conducted every other day, and the tumor volume (V) was calculated according to the following formula: V = (largest diameter) x (smallest diameter)²/2.

Results are expressed as the mean ± SD representative of three individual experiments performed in triplicate. Statistical analysis was performed by the Student’s t-test and ANOVA test. P<0.05 was considered to be statistically significant compared to the respective control.

To examine whether sesamin modulates prostate cancer cell proliferation stimulated by LPS, we used the MTT assay and further investigated the effect of sesamin (0, 10, 50 and 100 μg/ml) on the expression of proliferative-related proteins, cyclin D1 and COX-2, in the LPS-stimulated PC3 cells. We found that the proliferation rate of PC3 cells was distinctly promoted by LPS, but was distinctly suppressed by sesamin pretreatment in a dose- and time-dependent manner (Fig. 1A). Moreover, sesamin pretreatment dose-dependently downregulated the expression of cyclin D1 and COX-2 gene products induced by LPS (Fig. 1B and C). Therefore, sesamin had a negative effect on the cell proliferation and the expression of tumor cell-proliferative proteins induced by LPS in the PC3 cells.

To evaluate the effect of LPS and sesamin on prostate cancer cell apoptosis, we determined the expression of Bcl-2 and survivin proteins related to tumor cell survival with sesamin pretreatment under LPS stimulation. The results indicated that sesamin pretreatment dose-dependently inhibited the expression of Bcl-2 and survivin proteins induced by LPS (Fig. 2), thus implying that the possible mechanism for the anti-apoptotic effect of sesamin may be through the downregulation of cell survival proteins, including Bcl-2 and survivin.

To determine the effect of LPS and sesamin on prostate cancer cell invasion, we used the Matrigel invasion assay and western blotting of invasion-related proteins, which are known to be involved in cancer cell invasion, adhesion and angiogenesis. The results revealed that the invasive ability of the PC3 cells was markedly promoted by LPS and further markedly and dose-dependently inhibited by sesamin pretreatment (Fig. 3A). More importantly, sesamin pretreatment dose-dependently suppressed the increased expression of MMP-9, ICAM-1 and VEGF proteins induced by LPS (Fig. 3B and C). Thus, these data confirmed the inhibitory role of sesamin in the invasiveness of prostate cancer and on the expression of tumor cell metastatic proteins.

To investigate the effect of LPS and sesamin on the secretion of TGF-α and IL-6 in PC3 cells, we detected the concentration of TGF-α and IL-6 production in the culture supernatant of PC3 cells under LPS stimulation with sesamin pretreatment. The MTT assay revealed that there was no toxic impact on cells following these processes for 48 h. The data revealed that the accumulation of TGF-α and IL-6 production induced by LPS in conditioned medium was decreased dose-dependently following sesamin pretreatment in PC3 cells (Fig. 4).

To examine whether the p38-MAPK signaling pathway is involved in the enhanced cell proliferation and invasion in response to TNF-α and IL-6 accumulation, we detected the p-p38 and p38 proteins following either sesamin (100 μg/ml) or SB203580 (10 nM) pretreatment or p38-siRNA/p38-siNS transfection in the LPS-stimulated PC3 cells. The results revealed that sesamin pretreatment inhibited the phosphorylation of the p38 protein increased by LPS stimulation, and pretreatment with SB203580, a specific inhibitor of p38-MAPK, showed the same degree of inhibition (Fig. 5A and B). In addition, compared to the control and p38-siNS transfection group, pretreatment with p38-siRNA transfection obviously decreased both p-p38 and p38 proteins under LPS stimulation.

Importantly, to assess the effect of sesamin on NF-κB activity induced by LPS, we used SB203580 pretreatment or p38-siRNA/p38-siNS transfection to investigate the action mechanism of sesamin on NF-κB activity in the LPS-stimulated PC3 cells. Similar to the inhibitory effect of SB203580 and p38-siRNA, sesamin pretreatment clearly suppressed the constitutive and inducible NF-κB activity at a concentration of 100 μg/ml (Fig. 6). Therefore, it can be concluded that LPS-induced translocation of the NF-κB p65 subunit to the nucleus could be inhibited by sesamin to some extent.

To evaluate the effect of sesamin on tumor growth in vivo, we used the SB203580 pretreatment to determine the LPS-stimulated tumor progression derived from PC3 cells in BALB/c nude mice. Compared to the control group, the tumor volume in the LPS pretreatment group was distinctly larger, and the tumor volume in the LPS plus sesamin group was relatively smaller after 14 days post-incubation (Fig. 7). The results illustrated that either sesamin or SB203580 treatment could obviously inhibit PC3 cell-derived tumor growth induced by LPS in vivo.