Anti-cancer effect of an intratumoral injection of dendritic cells expressing TLR4 in combination with an active component of OK-432 in TLR4-deficient mice

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.     

Induction of apoptosis in human head and neck cancer cell lines by an active component of OK-432 through p53-independent pathway via toll-like receptor (TLR) 4 signaling

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.     

Immunotherapy, with OK-432 for cases of nasopharyngeal carcinoma

The prognosis of nasopharyngeal carcinoma (NPC) is very poor, due to the extreme failure of the immuno-surveillance mechanism in such cases. As an immunotherapeutic agent, OK-432 was administered to NPC patients. Cases were divided into two groups. One was given OK-432 for less than 6 months (short-term group), while a long-term group, was treated with OK-432 for over 6 months. These two groups were evaluated for immunological activity and prognosis. As immunological parameters, the numbers of white blood cells and lymphocytes were counted, and delayed skin reactivities with Su-PS and PPD were determined. No influence on the number of white blood cells and lymphocytes could be observed in either of the two groups. However, the skin tests showed better improvement of immunity in the long-term group than in the short-term cases. Furthermore, these reactivities correlated well with the clinical status of NPC patients. As to the absolute number of lymphocytes, improvement in the long-term NPC group was delayed in comparison to that of another head and neck carcinoma group given long-term OK-432 treatment. The prognosis of the long-term administered group was apparently better than that of the short-term group. Long-term administration of OK-432 is therefore indispensable for the treatment of NPC cases, because of the extreme decline of their immunity.     

Antitumor activity of a Streptococcus pyogenes preparation (OK-432). II. Analysis of the cytotoxic lymphocytes induced by OK-432 injection into tumor-bearing F344 rats

In the present study, the antigenic phenotype and target cell specificity of the cytotoxic lymphocytes observed in F344 rats following the ip inoculation of a syngeneic MADB106 mammary carcinoma and the single injection of OK-432 were examined. When the cytotoxicity of peritoneal exudate cells (PECs) was measured in a 4-hour 51Cr release assay, appreciable cytotoxicity against MADB106 tumor cells was evident by day 7-14 following OK-432 injection. With the use of an antibody (R1-3B3) and complement depletion of cytotoxic PECs, the MADB106 killer cells appeared to consist of both R1-3B3- (non-T) and R1-3B3+ (T) cells, with most of the anti-MADB106 killing residing in the R1-3B3- cell population. The R1-3B3- killer cells were further defined as: a) phenotypically asialoGM1+, b) present in athymic nude rats, and c) accompanied by some augmentation of YAC-1 killing [the prototype rat natural killer (NK) target], suggesting that some of these R1-3B3- killer cells were typical NK cells. However, it was also observed that most of the R1-3B3- cells that killed MADB106 tumor cells were: 1) phenotypically or functionally different from either cytotoxic T-cells or typical NK cells; 2) observed only in MADB106 tumor-bearing rats challenged with OK-432; 3) not present on day 1-2 following OK-432 injection, the time when YAC-1 killing was maximally augmented; and 4) present in high numbers in a secondary response following reinoculation of the MADB106 tumor cells into cured rats. The in vivo relevance and possible derivation of these various cytotoxic lymphocyte populations in the syngeneic tumor-bearing hosts are discussed.     

In vitro potentiation of human natural killer cell activity by a streptococcal preparation, OK-432: interferon and interleukin-2 participation in the stimulation with OK-432

Inasmuch as human natural killer (NK) cell activity was markedly augmented by a streptococcal immunopotentiator, OK-432, both in vivo and in vitro, the mechanism in which OK-432 augmented human NK cell activity was analyzed. Culture supernatants of nonadherent lymphocytes stimulated with OK-432 significantly augmented NK cell activity. Significant activity of both interferon (IFN) (both alpha- and gamma-types) and interleukin-2 (IL-2) was detected in the culture supernatants from nonadherent lymphocytes. Concomitant treatment of supernatants with anti-IFN-alpha antiserum and pH-2 glycine-HCI buffer or the absorption of supernatants with an IL-2-dependent cell line completely abrogated the NK-augmenting activity, whereas the treatment with either one of these resulted in only partial elimination of the activity. These results indicate that OK-432 stimulates human nonadherent lymphocytes to produce IFN and IL-2 and that both factors are primarily responsible for the NK augmentation by OK-432.     

Induction of mouse peritoneal cytotoxic factor by OK-432. (3). Comparison between LPS and OK-432 as eliciting agents

By injection of OK-432, a cytotoxic factor was induced in peritoneal fluids of mice which had been primed with OK-432. Two-step stimulation (priming and eliciting) was always necessary to induce the cytotoxic factor. OK-432-primed mice did not produce soluble cytotoxic factor spontaneously and no cytotoxic activity was detected in the mice treated by a single injection of OK-432 as an eliciting agent. High doses of OK-432 were required to prime mice for the production of cytotoxic factor, whereas a small amount was enough to elicit it. Pathological studies were also conducted in order to clarify whether the mice were safe under the conditions in which PCF had been induced. Moderate liver damage was observed in the mice injected with OK-432 and LPS, whereas no histological change in the liver or spleen was observed in the mice treated with OK-432 alone. These results suggest that OK-432 is a good candidate as an inducer of cytotoxic factor in the peritoneal cavity.     

Growth inhibition and apoptosis by an active component of OK-432, a streptococcal agent, via Toll-like receptor 4 in human head and neck cancer cell lines

Toll-like receptor 4 (TLR4) plays a significant role in cancer therapy as receptors of bacteria-derived immunotherapeutic agents such as OK-432, a streptococcal immunotherapeutic agent. In addition, recent reports demonstrated that TLRs, including TLR4, are also expressed in cancer cells as well as in immunocompetent cells. It is a problem in cancer therapy that the immunoadjuvant may activate survival signals such as nuclear factor (NF)-κB or mitogen-activated protein kinases (MAPKs) in cancer cells via TLRs. In the current study, we investigated responsiveness of human head and neck cancer cell lines against TLR4 ligands, OK-PSA, an active component of OK-432, and a lipopolysaccharide (LPS). Stimulation with LPS or OK-PSA resulted in the activation of NF-κB in these cell lines expressing TLR4 and MD-2 that is a significant coreceptor for TLR4 signaling. Interestingly, OK-PSA induced cell-growth inhibition, while LPS enhanced the proliferation of the cancer cells. OK-PSA induced NF-κB activation more slowly than that induced by LPS. In addition, phosphorylation of p38 MAPK by OK-PSA was only slight compared with that by LPS. OK-PSA also induced apoptosis of the cancer cells mediated by the activation of caspase 1, 3 and 8 in a p53-independent manner. These findings strongly suggest that active components of OK-432 may elicit anti-cancer effects via enhancing host immunity as well as via directly inducing the growth inhibition and apoptosis of head and neck cancer cells through TLR4 signal.     

The management of malignant ascites with a streptococcal preparation, OK-432: relation between the clinical effect and auto-tumor cell killing activity by OK-432-induced ascites-derived lymphocytes

Twelve patients with malignant ascites caused by gastro-intestinal cancer were treated by intraperitoneal administration of OK-432. Tumor cells from these patients were separated from ascitic fluid and cultured in vitro before OK-432 therapy. OK-432 was given intraperitoneally one to two times a week at doses ranging from 5 to 20 KE suspended in saline. Mononuclear lymphocytes were collected from the fluid at various intervals throughout the therapy. The effect of ascites-derived lymphocytes on ascites-derived autologous tumor cell growth was studied in vitro using microplate assay. Nine (responders) of 12 patients showed complete disappearance or significant reduction of ascitic fluid. Ascites-derived lymphocytes slightly inhibited autologous tumor cell growth only in one case before OK-432 therapy. Lymphocytes collected from ascites after OK-432 injection significantly inhibited auto-tumor cell growth in all of 9 responders. In 3 non-responders, however, auto-tumor cell growth inhibition was found only in one case. Interestingly, lymphocytes from non-responders significantly inhibited the growth of tumor cells taken from responders. Conversely, lymphocytes from responders did not inhibit non-responder-derived tumor cell growth. These findings imply that auto-tumor killing by OK-432-induced lymphocytes may depend more on the condition of the tumor cells than on the condition of the lymphocytes, and that the measurement of auto-tumor killing activity by ascites-derived lymphocytes may be useful as an indicator in OK-432 therapy.     

Intrapericardial OK-432 instillation for the management of malignant pericardial effusion

Ten patients with malignant pericardial effusion were treated with intrapericardial injection of OK-432 (penicillin-treated and heat-treated lyophilized powder of the substrain of Streptococcus pyogenes A3). After intrapericardial insertion of a catheter, a maximal volume of pericardial fluid was withdrawn with cytologic confirmation of malignancy. Five or 10 Klinische Einheit (KE) (KE is a unit used to express the strength of a preparation) of OK-432 diluted in 20 ml of saline was injected into the pericardial space in seven and three patients, respectively. It was repeated in case of reaccumulation. Seven patients were treated only once and the remaining three required a second treatment. Complete control of pericardial effusion was achieved in all patients for an average of 329 days (range, 54 to 790 days). Fever and chest pain were experienced in six and five patients, respectively, but were controlled with antipyretics. Two of three patients who received 10 KE of OK-432 experienced hypotension that was successfully controlled with vasopressor drugs with or without reaspiration of pericardial fluid. Rapid reactive reaccumulation of the pericardial fluid was thought to be a cause of hypotension. A follow-up computed tomography (CT) scan was performed in seven patients and a thickened pericardium was noticed in five; no patients had constrictive pericarditis. These results suggest that intrapericardial administration of 5 KE of OK-432 is an effective and safe treatment for malignant pericardial effusion.     

Synergistic activation of rat alveolar macrophages by cepharanthine and OK-432

We studied the synergistic activation of rat alveolar macrophages (AM) by cepharanthine (Ceph.) and OK-432. Rat AM could be rendered much more tumoricidal against Walker-256 tumors in vitro after the i.v. injection of ceph. (5 mg/Kg) and OK-432 (5 KE/rat) thao of ceph. or OK-432 only. Radioactivity of 14C-acetate-OK-432 trapped in murine lung after the i.v. injection of mixed of ceph. was much higher than of 14C-acetate-OK-432 only. Rat AM could be rendered tumoricidal by incubation in vitro with OK-432, but not with ceph. This finding suggests that if OK-432 is injected intravenously with mixed of with ceph., of OK-432 is trapped much more up by the lung without mixture, therefore AM can be much more tumoricidal. Intravenous administration of OK-432 with ceph. may be useful for reduction of lung metastasis by tumoricidal AM.     

The effect of OK-432 in the treatment of primary lung cancer

Twenty patients with lung cancer were treated with a streptococcal preparation, OK-432 in addition to various other treatments, and we evaluated the effect of OK-432 in comparison with an equivalent number of patients without OK-432. With regard to advanced-stage patients, the median survival time of those treated with OK-432 was longer than that of patients without OK-432. Patients whose PPD or SU-PS skin reactions were positive had a longer survival time than those giving a negative reaction. OK-432 significantly increased the reactions for both PPD and SU-PS. On the other hand, OK-432 did not have any significant effect in increasing the numbers of peripheral lymphocytes and T-cell subsets. Furthermore, there were no effects on tumor markers, such as CEA, beta 2-microglobulin and ferritin. However, OK-432 had a remarkable effect in decreasing immunosuppressive acidic protein.     

Effect of endoscopical administration of OK-432 in 4 patients with advanced esophageal or gastric cancer

OK-432 has been injected endoscopically into 4 patients with an unresectable esophageal or gastric cancer. Three months after the initial injection, a remarkable improvement in the symptoms and a reduction of the tumor size was recognized in all 4 patients. This study indicates that an OK-432 injection provides considerable benefits to patients with an advanced esophageal or gastric cancer.     

Radiation, 5-FU and OK-432: inhibitory effect of IL-10 and TGF-beta

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.     

Experimental and clinical effect of hypertransfusion and OK-432 on granulocyte recovery

The experimental and clinical studies were carried out to alleviate the bone marrow suppression by antineoplastic drugs. Faster recovery of granulopoiesis was observed by pretreating recipients with RBC hypertransfusion and/or OK-432 (Picibanil, biological products of beta-streptococci.) In experimental mice, higher granulocyte counts could be maintained with hypertransfusion in the peripheral blood, and the recovery of granulopoietic series and CFU-S of bone marrow cells were found to be more rapid after cyclophosphamide administration. OK-432 also resulted in higher peripheral granulocyte, whereas the total nucleated cell counts and CFU-S were decreased in the bone marrow, suggesting sparing bone marrow granulocyte reserve and its migration to the peripheral blood. The mechanism of higher granulocyte count after hypertransfusion was not clearly explained but it was considered that erythroid suppression caused colateral flow of multipotential stem cells to granulopoiesis. The effect of both combinations was unexpectedly less significant in the recovery of granulopoiesis, but it was thought that the optimal time interval should be sought between pretreatment and the administration of anti-neoplastic agents. The clinical use of hyper transfusion and OK-432 also proved the alleviation of granulocytopenia, and rapid granulocyte recovery at the time of consolidation therapy among children with AML.