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We have investigated the relationship between gene expression of Bcl 2 and Bax and the therapeutic effect in oral cancer patients. A significant correlation between Bcl-2/Bax gene expression ratio in the peripheral blood mononuclear cells (PBMCs) from the patients, and the therapeutic effect of radiation therapy in combination with UFT and OK-432, as well as survival rate, was observed. In addition, the statistically significant correlation was also observed between Bcl-2/Bax ratio and IFN-gamma and NK activity induced with OK-432. These findings suggest that Bcl-2 and Bax gene expression in PBMCs may be useful as a prognostic factor in oral cancer patients.

We investigated the effect of 5-FU and radiation in OK-432-induced cytokine production. Stimulation of peripheral blood mononuclear cells (PBMCs) with OK-432 (1 micro/ml) for 24 h induced Th1-type cytokines (IFN-gamma, TNF-alpha, IL-12, IL-18) as well as IL-10 and TGF-beta. When the PBMCs were stimulated by 5-FU (5 microg/ml) or X-ray (2 Gy) simultaneously with OK-432, production of IL-10 and TGF-beta was significantly inhibited, while no significant change in Th1 cytokine production was observed. Although OK-432 also enhanced the expression of the genes encoding SOCS-1 and SOCS-3, which are negative regulators for cytokine signaling, this was reduced by 5-FU or X-irradiation. Induction of IL-10 and TGF-beta by OK-432 was significantly decreased by adding antisense ODN for SOCS-1 or that for SOCS-3. Radiation and 5-FU induce Th1-dominant state by inhibiting the OK-432-induced production of IL-10 and TGF-beta mediated by regulation of SOCS-1 and SOCS-3 expression, and are suggested to increase anti-cancer immunity.

We have tried to identify the effective components of OK-432, a Streptococcus-derived anti-cancer immunotherapeutic agent. In the current study, we investigated the effect of OK-432-derived DNA (OK-DNA) in augmenting anti-cancer immune response. Analysis of OK-DNA with the restriction enzymes Hpa II and Msp I revealed that OK-DNA contained unmethylated CpG motifs. OK-DNA induced Th1-type cytokines, such as IFN-gamma and IL-12, and augmented killer cell activities in vitro on human peripheral blood mononuclear cells, whereas the methylated OK-DNA did not. Cytokines were also produced by OK-DNA-stimulated splenocytes derived from wild-type mice but not from TLR9-deficient mice. In the in vivo study, a peritumoral administration of OK-DNA resulted in a significant inhibition of tumor growth in syngeneic tumor-bearing wild-type and TLR4-deficient mice but not in TLR9-deficient mice. Anti-tumor effect of OK-432 in TLR9-deficient mice was significantly but partially reduced as compared with that in wild-type mice, while the effect of OK-432 was almost completely eliminated in TLR4-deficient mice. These findings suggest that unmethylated CpG-DNA in OK-432 functions as an active component in OK-432-induced anti-cancer immunity via TLR9, at least in part.

OK-PSA, an active component of OK-432, induces anti-tumor immunity via Toll-like receptor (TLR) 4/MD-2 complex. In the current study, we evaluated the effect of the OK-PSA on human head and neck cancer cell lines. Twelve cancer cell lines including 7 squamous cell carcinoma (SCC) cell lines and 5 salivary gland cancer (SGC) cell lines were examined. The quantitative real-time PCR analysis revealed that TLR4 mRNA was expressed in all 12 cell lines, and that MD-2 mRNA was expressed in 5 cell lines. OK-PSA stimulation resulted in the activation of NF-kappaB in the 4 SCC cell lines which express both TLR4 and MD-2 genes, and in 5 SGC cell lines which express at least TLR4 gene independently of MD-2 expression. In these OK-PSA-responsive cell lines, OK-PSA activated caspase-1, caspase-3 and caspase-8, and induced apoptosis. OK-PSA-induced apoptosis were observed even in a SGC cell line in which p53 is mutated and its function is impaired. These findings strongly suggest that OK-PSA induces apoptosis by the activation of caspases through p53-independent pathway via TLR4 signaling in head and neck cancer cells.

We investigated the in vivo anti-tumor effect of intratumoral administration of dendritic cells (DCs) after chemotherapy using TS-1, and followed by immunopotentiator OK-432. Both in Meth-A-bearing BALB/c and in SCCV II-bearing C3H/HeN mice, one week of oral administration of TS-1 affected a partial eradication of established tumors. TS-1 followed by DCs and OK-432 resulted in a marked inhibition in tumor growth, and also contributed to a greater prolongation of survival. Cytotoxic activities of tumor-infiltrating lymphocytes and draining lymph node cells against inoculated tumor cells were significantly increased by the therapy. Cytotoxic memory T cells were also induced. The same therapy was also applied to SCCV II-bearing C3H/HeJ mice in which the Toll-like receptor (TLR) 4 is mutated; no immunotherapeutic effect was observed in the mice. These findings suggest that local DC therapy in combination with TS-1 and OK-432 may well be a useful strategy for the treatment of solid tumors, and that TLR4 signaling is involved in the success of this therapy.

Although we have reported that Toll-like receptor (TLR) 4 is involved in OK-432-induced anti-cancer immunity, its detailed mechanism remained uncertain. We hypothesized that OK-432 may first be captured, dissolved by phagocytes, and then active components released from the cells may stimulate TLR4. This hypothesis was examined by the current in vitro experiments. We used TS-2 MoAb which recognizes OK-PSA, an active component of OK-432. First, we observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining by using TS-2 clearly demonstrated that OK-432 was captured and dissolved by these cells. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by ELISA with TS-2. The supernatants from OK-432-treated DC culture, but not from untreated DC culture, increased NF-kappaB activity in TLR4-expressing cells. The increased NF-kappaB activity was inhibited by TS-2. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the OK-432 action.

A lipoteichoic acid-related molecule OK-PSA is an active component of OK-432. In the in vitro experiments, OK-PSA enhanced expression of MHC class II, CD80 and CD86, as well as IL-12 production on dendritic cells (DCs) were derived from wild-type mice, but not from TLR4-deficient (TLR4-/-) mice. Next we examined the in vivo anti-cancer effect of intratumoral administration of syngeneic DCs followed by OK-PSA against established tumors in mice. Although OK-PSA augmented anti-tumor effect of DC administration in tumor-bearing wild-type mice, anti-tumor effect of DCs and OK-PSA was not significant in TLR4-/- mice. Interestingly, an administration of wild-type mice-derived DCs followed by OK-PSA exhibited a marked anti-tumor effect even in TLR4-/- mice. These findings suggest that OK-PSA may be a potential adjuvant for local DC therapy, and that DC therapy followed by OK-PSA is able to elicit anti-cancer activity even in TLR4-deficient host when TLR4 is expressed only in DCs injected intratumorally.