| BackgroundBladder cancer is the most common malignancy of the urology tract, and the seventh most common cancer in men and the17th in women. It is estimated annual incidence of330,000new cases. Approximately70%of bladder tumors are classified as superficial, which is associated with a40%to80%risk of recurrence and a10%to27%chance of progressing to muscle-invasive or metastatic disease. Standard treatment of superficial bladder cancer is transurethral resection bladder tumor, subsequently intravesical instillation of chemotherapeutics to prevent recurrence and progression of the disease. OK-432, which is a penicillin-killed and lyophilized preparation of a low-virulence strain of Streptococcus pyogenes (group A), has been successfully used as an immunotherapeutic agent against many types of malignancies, including head and neck cancer, stomach cancer, lung cancer, pancreatic cancer and colorectal cancer. It has been reported that OK-432elicits anti-tumor effects by stimulating immunocompetent cells such as macrophages, T cells, and natural killer (NK) cells, and by inducing helper T-cell1(Th1) type cytokines, such as IFN-y, TNF-a, IL-6, IL-8, IL-10, IL-12, and IL-18. which can augment cytotoxic T Lymphocytes to antitumor.In this study, we investigated whether OK-432exerts its anti-tumor activities through TLR4/NF-κB signal to cascades in tumor cells. Our results demonstrated for the first time that OK-432could inhibit proliferation and suppress metastasis in vitro, and promote apoptosis in vivo. The underlying mechanism of OK-432on inducing apoptosis and inhibiting cell proliferation in bladder cancer should be contributed to the modulation of TLR4/NF-κB Signaling Pathway.Methods1. Different doses of OK-432(0.0.125,0.25.0.5.1,2and4ke/ml) were incubated for the indicated times (24h,48h,72h). At the end of incubation, Colorimetric analysis using a96-well microplate reader was performed at wavelength490nm.2. T24and EJ cells were seeded in6-well plates at1000cells/well in RPMI1640medium containing10%serum. The medium with OK-432was replaced after every2-3. After2weeks of incubation, colonies were manually counted.3. Cells (EJ and T24) were exposed to different doses of OK-432(1,2and4ke/ml) for 24h, then following the protocols provided by the manufacturer. Apoptotic cells were then analyzed by flow cytometry.4. After cells reach to60-80%confluence, cells were treated with different doses of OK-432(1,2and4ke/ml). After24h of incubation, DNA content was then analyzed using the flow cytometry.5. A transwell cell culture chamber supplemented with10%fetal bovine serum was added to each well of the plate to act as a chemoattractant in the lower chamber. Then the cells were suspended in medium with different concentrations of OK-432. Cells were incubated for24h. The cells that invaded through the insert were counted in five random fields and expressed as the average number of cells per field. Differently, the culture chamber was coated with Matrigel in invasion assay, dried and reconstituted at37℃with free fetal bovine culture medium.6. Cells were harvested at24h following OK-432treatment, then extracted the protein to assay by western-blot. Apoptotic proteins, cell cycle proteins. EMT related proteins and TLR4//NF-κB Signaling Pathway proteins were assayed by western-blot.7. N-methyl-N-nitrosourea (MNU) induced rat bladder tumor model after treated for8weeks, then treated with OK-432for8weeks. After that, all all rats were sacrificed and processed for histopathological and immunological evaluation.Results1. Low dose OK-432(ranged0.125ke/ml to0.5ke/ml) inhibited the bladder cancer cells proliferation inconspicuously, while high dose OK-432(ranged1ke/ml to4ke/ml) significantly decreased the T24and EJ cells viability, which showed a time-and dose- dependent manner.2. Colony formation was conducted.0.1ke/ml,0.5ke/ml and1ke/ml OK-432were separately incubated with T24and EJ cells for2weeks, while1ke/ml OK-432inhibited the bladder cancer cells colony formation significantly comparing with blank control.3. EdU assayed show that the proliferation rate of T24cells in the presence of OK-432with different concentrations (0,1,2.4ke/ml) was37.33%,31.33%,11%.1.93%, respectively. In contrast to EJ cells, the proliferation rate was42.33%,38.33%,16.67%and1.67%.4. The apoptosis rate of T24cells which treated with0,1,2, and4ke/ml OK-432was4.04%,13.21%.25.32%, and57.34%respectively, and the apoptosis rate of EJ cells was observed to increase shown dose-dependent.2ke/ml and4ke/ml OK-432increased the apoptosis rate more significantly than control group. The expression of cleavage of caspase-7, caspase-9. caspase-3, PARP, and Bax was upregulated significantly after treated with4ke/ml OK-432, whereas Bcl-2levels were not rEdUced in both cell lines.5. Cell cycle arrest at G0-G1stage by51.31%.51.86%. and67.63%. at1.2. and4ke/mlOK-432treatment, respectively, as in EJ cells while37.43%,49.11%, and65.01%arrest was observed at1,2, and4ke/ml OK-432treatment. Protein expression of the cyclin D1and cyclin D3were decreased in T24and EJ cells upon OK-432treatment. However, the expression of P15and P16up-regulated.6. High dose OK-432(2and4ke/ml) inhibited T24cells migration and invasion significantly, and less effect on low dose group (1ke/ml) comparing with control group. In addition, both results showed dose-dependent with OK-432. EJ cells were conducted with the same results. In present study, we found that the expression of E-cadherin up-regulated in the presence of OK-432, otherwise, N-cadherin, Vimentin, Snail, and MMP9shown less expression with the increasing OK-432concentration in T24and EJ cells.7. Protein of TLR4up-regulated upon OK-432treatment significantly. Phosphorylation of NF-κB and IκBαdown-regulated upon treatment of OK-432comparing to control group significantly, as total NF-κB and IκBα failed to change.8. TUNEL and PCNA protein were examined in IHC. TUNEL down-regulated while PCNA up-regulated treated with OK-432.ConclusionsTaken together, we have shown that OK-432exhibits the suppression of NF-κB and NF-Kb-regulated gene products, which are associated with anti-proliferative, pro-apoptotic and anti-invasive effects. In this regard, our findings that OK-432suppressed NF-Kb activation extend our understanding on the molecular mechanisms underlying the anti-tumor activities of OK-432. |
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