Enhanced Therapeutic Efficacy of Immunostimulatory CpG-ODN by Silencing SOCS-1 with Polysaccharide/miR-155 Complexes.

For the induction of antigen-specific immune responses, adjuvants as well as antigens are essential. CpG-ODN is a potent agonist of toll-like receptor 9 (TLR9) and is known as an adjuvant to induce cellular immune responses. We previously developed a therapeutic oligonucleotide delivery system based on the formation of a complex between schizophyllan (SPG), a kind of ß-1,3-glucan, and poly(dA), which actively delivered CpG-ODN to antigen-presenting cells (APCs) in the draining lymph nodes and induced antigen-specific immune responses. However, unfortunately, the signaling pathway of TLR9 is negatively regulated by an intracellular protein called suppressor of cytokine signaling-1 (SOCS-1), which suppresses the adjuvant effect of CpG-ODN. To solve this, we focused on microRNA-155 (miR-155), which regulates innate and autoimmune processes by targeting SOCS-1. In this study, we proposed a strategy of combining miR-155 and CpG-ODN, each complexed with SPG (denoted as SPG/miR-155 and SPG/CpG, respectively), to induce a more potent immune response. As a result, we showed that the efficient delivery of miR-155 to APCs by a complex form could induce much more potent cellular immune responses than SPG/CpG alone. Furthermore, the mice treated with the combination of SPG/miR-155 and SPG/CpG showed a long delay in tumor growth occurrence and improved survival after tumor inoculation. These results indicate the possibility of therapeutic strategies for cancer.

The targeted delivery of the c-Src peptide complexed with schizophyllan to macrophages inhibits polymicrobial sepsis and ulcerative colitis in mice.

Hyper-inflammatory responses triggered by intracellular reactive oxygen species (ROS) can lead to a variety of diseases, including sepsis and colitis. However, the regulators of this process remain poorly defined. In this study, we demonstrate that c-Src is a negative regulator of cellular ROS generation through its binding to p47phox. This molecule also competitively inhibits the NADPH oxidase complex (NOX) assembly. Furthermore, we developed the schizophyllan (SPG)-c-Src SH3 peptide, which is a ß-1,3-glucan conjugated c-Src SH3-derived peptide composed of amino acids 91-108 and 121-140 of c-Src. The SPG-SH3 peptide has a significant therapeutic effect on mouse ROS-mediated inflammatory disease models, cecal-ligation-puncture-induced sepsis, and dextran sodium sulfate-induced colitis. It does so by inhibiting the NOX subunit assembly and proinflammatory mediator production. Therefore, the SPG-SH3 peptide is a potential therapeutic agent for ROS-associated lethal inflammatory diseases. Our findings provide clues for the development of new peptide-base drugs that will target p47phox.

Molecular function of macrophage migration inhibitory factor and a novel therapy for inflammatory bowel disease.

Macrophage migration inhibitory factor (MIF) is a unique protein that participates in inflammation, immune responses, and cell growth. An array of in vitro and in vivo experiments has demonstrated that MIF is profoundly involved in the pathogenesis of acute and chronic inflammatory disorders, such as inflammatory bowel disease (IBD). Blockade of MIF bioactivities by either neutralizing anti-MIF antibodies or antagonists prevents inflammatory cytokine cascade, which strongly suggests that an anti-MIF therapeutic strategy is feasible for treatment of IBD. Recently, we developed a new therapeutic approach for IBD by administration of antisense MIF oligonucleotides in conjugation with schizophyllan (SPG), a member of the glucan family. SPG specifically binds Dectin-1 expressed in antigen-presenting cells (APCs), and the antisense MIF/SPG complex is incorporated into the cells. In in vivo experiments of colitis models in mice, we found that intraperitoneal administration of the complex ameliorated the clinical signs of colitis and improved the histological scores. This novel therapy designed to knock down the MIF production in APCs is expected to be clinically applicable for the treatment of IBD.

Medicinal mushrooms and cancer therapy: translating a traditional practice into Western medicine.

Modern medical practice relies heavily on the use of highly purified pharmaceutical compounds whose purity can be easily assessed and whose pharmaceutical activity and toxicity show clear structure-function relationships. In contrast, many herbal medicines contain mixtures of natural compounds that have not undergone detailed chemical analyses and whose mechanism of action is not known. Traditional folk medicine and ethno-pharmacology coupled to bioprospecting have been an important source of many anticancer agents as well as other medicines. With the current decline in the number of new molecular entities from the pharmaceutical industry, novel anticancer agents are being sought from traditional medicine. As the example of medicinal mushrooms demonstrates, however, translating traditional Eastern practices into acceptable evidence-based Western therapies is difficult. Different manufacturing standards, criteria of purity, and under-powered clinical trials make assessment of efficacy and toxicity by Western standards of clinical evidence difficult. Purified bioactive compounds derived from medicinal mushrooms are a potentially important new source of anticancer agents; their assimilation into Western drug discovery programs and clinical trials also provides a framework for the study and use of other traditional medicines.

Effects of sizofiran on endotoxin-enhanced cold ischemia-reperfusion injury of the rat liver.

Kupffer cells (KC), resident macrophages of the liver, have been strongly implicated in lipopolysaccharide (LPS)-induced liver graft injury. However, our recent study showed that sizofiran (schizophyllan glucan) (SPG), which activates KC, did not influence cold ischemia-reperfusion liver injury of LPS-exposed rats. Here we investigated some mechanisms by which SPG does not aggravate LPS-enhanced cold ischemia-reperfusion rat liver injury. Control and SPG-treated rats were exposed to LPS for 2 h prior to hepatectomy. The livers were cold-preserved in University of Wisconsin solution followed by reperfusion with Krebs-Henseleit buffer. We found that SPG dramatically inhibited LPS-induced increases of tumor necrosis factor-alpha (TNF-alpha) in the plasma and bile in vivo. Moreover, LPS-induced TNF- release into the washout solution after cold ischemia was also abrogated by SPG pretreatment. However, SPG increased TNF- release into the perfusate after reperfusion. On the other hand, SPG completely abolished expression of c-myc protooncogene, which is known to sensitize cells to TNF-alpha cytotoxicity. In conclusion, inhibition of both TNF- release after LPS challenge and c-myc expression may explain why activation of KC with SPG does not aggravate endotoxin-enhanced cold ischemia-reperfusion liver injury.

[Phase III study of gynecologic cancer in Japan].

Gynecologic Oncologists in Japan has increasingly much interest in the phase III study to define the standard treatment modality. It is very difficult, however, to conduct a phase III trial because a very few medical staff including doctors have experience and understanding of such studies and also poor understanding of patients to be entered. According to the literature and papers in the meetings, several phase III studies were conducted unexpectedly. I reviewed these data and pointed out the problems included in the studies. The results of these trials and related problems prompted Japanese Gynecologic Oncologists to conduct a high quality phase III study to clarify the standard treatment modality for gynecologic cancer.

Prognostic value of thymidine phosphorylase immunostaining in patients with uterine cervical cancer treated concurrently with doxifluridine, radiotherapy and immunotherapy.

Thymidine phosphorylase (dThdPase) is reportedly identical to platelet-derived endothelial cell growth factor (PD-ECGF). We conducted immunohistochemical staining of dThdPase to assess correlation between its expression in cancer tissue and efficacy of a combination therapy with 5'-DFUR, radiotherapy and sizofilan (SPG) in uterine cervical cancer patients. No difference in response rates was observed between dThdPase positive and negative tumor and stromal cells. Survival curves significantly differed between stromal dThdPase positive and negative groups (p=0.032). Results showed that dThdPase immunostaining is possibly prognostic and predictive in determining success of the combination therapy.

Effects of concomitant use of doxifluridine, radiotherapy and immunotherapy in patients with advanced cervical cancer.

Clinical effects of doxifluridine (group A, 600 mg/body/day; group B, 800 mg/body/day) combined with radiotherapy and immunotherapy were evaluated in patients with advanced cancer of the uterine cervix. Response rates were 84.2% (16/19 patients) in group A and 100% (18/18 patients) in group B, respectively (p=0.230). There was no significant difference in adverse reaction incidence between the methods but significantly higher grade adverse reaction were observed in group B than in group A (p=0.048). Time to progression (TTP) was longer in group B than in group A (p=0.081). The optimal 5'-DFUR dose was 800 mg/body (group B), by which higher grade adverse reactions were fully controlled and TTP was prolonged.

Mushrooms, tumors, and immunity.

Medicinal properties have been attributed to mushrooms for thousands of years. Mushroom extracts are widely sold as nutritional supplements and touted as beneficial for health. Yet, there has not been a critical review attempting to integrate their nutraceutical potential with basic science. Relatively few studies are available on the biologic effects of mushroom consumption, and those have been performed exclusively in murine models. In this paper, we review existing data on the mechanism of whole mushrooms and isolated mushroom compounds, in particular (1-->3)-beta-D-glucans, and the means by which they modulate the immune system and potentially exert tumor-inhibitory effects. We believe that the antitumor mechanisms of several species of whole mushrooms as well as of polysaccharides isolated from Lentinus edodes, Schizophyllum commune, Grifola frondosa, and Sclerotinia sclerotiorum are mediated largely by T cells and macrophages. Despite the structural and functional similarities of these glucans, they differ in their effectiveness against specific tumors and in their ability to elicit various cellular responses, particularly cytokine expression and production. Unfortunately, our data base on the involvement of these important mediators is still rather limited, as are studies concerning the molecular mechanisms of the interactions of glucans with their target cells. As long as it remains unclear what receptors are involved in, and what downstream events are triggered by, the binding of these glucans to their target cells, it will be difficult to make further progress in understanding not only their antitumor mechanisms but also their other biological activities.

Preparation and antitumor activities of beta-(1-->6) branched (1-->3)-beta-D-glucan derivatives.

The formylmethylated and aminoethylated derivatives of schizophyllan (SPG), a beta-(1-->6)-branched (1-->3)-beta-D-glucan from Schizophyllum commune Fries, were prepared through dimethoxyethylated SPG which was synthesized by the reaction of SPG with dimethylchloracetal under an alkaline condition. The degree of the substitution of formylmethyl groups in the formylmethylated derivative of SPG was estimated as approximately 0.19, and the locations of formylmethyl groups in the derivative were predominantly located at O-6 and/or O-4 positions in glucose residues. The molecular weights of these derivatives were similar to that of SPG, and the helical structure of the derivatives did not seem to be different. The antitumor activities of the formylmethylated and aminoethylated derivatives of SPG against subcutaneously implanted sarcoma 180 solid tumor in mice by intraperitoneal (i.p.) administration were increased more effectively than that of SPG at a dose of 10 mg/kg/d for 7 d starting from 7 d after transplantation of sarcoma 180. The activities inducing tumor regressing factor of the formylmethylated and aminoethylated derivatives were 1.5 to 2 times stronger than that of SPG at a dose of 100 mg/kg. Formylmethylated and aminoethylated derivatives of SPG as well as SPG itself retained the potentiating activity for the reticuloendothelial system. The productions of soluble cytotoxic factors secreted from murine macrophages by the administration of the formylmethylated and aminoethylated derivatives of SPG, which were probably superoxide anion and tumor necrosis factor as measured by the potency of the nitro blue tetrazolium reduction and the cytotoxic activity against L929 cells, respectively, appeared to be more efficient than that of SPG.

Combination treatment with cisplatin and schizophyllan for 7,12-dimethylbenz(a)anthracene-induced rat ovarian adenocarcinoma.

OBJECTIVE: Experimental chemotherapy was conducted to investigate the combined effect of Schizophyllan (SPG) and Cisplatin (CDDP). METHODS: Rats with 7,12-dimethylbenz(a)anthracene (DMBA)-induced ovarian adenocarcinoma received SPG and/or CDDP, were observed for the anti-tumor effect of the drugs and were subjected to immunohistochemical study. RESULTS: 1) Tumor reduction was enhanced by the combined use of SPG and CDDP; 2) The survival rate of rats given SPG alone was significantly higher than that of the control rats, and treatment with SPG combined with CDDP tended to improve the survival rate; 3) Immunohistochemically, an infiltrative increase in cytotoxic T-lymphocytes (CTL) was induced, although the development of helper T-cells and macrophages was immunohistochemically weak at the tumor site. CONCLUSION: The administration of SPG enhanced the anti-tumor effect of CDDP.

Activated (HLA-DR+) T-lymphocyte subsets in cervical carcinoma and effects of radiotherapy and immunotherapy with sizofiran on cell-mediated immunity and survival.

A prospective randomized trial on 312 patients with locally advanced cervical carcinoma (FIGO stages IB-IV) was carried out. The 5-year survival in 90 patients treated with radiotherapy and antitumor polysaccharide sizofiran, an extract from the culture broth of Schizophyllum commune Fries, in combination was significantly (P = 0.045) better than that in 82 patients treated with radiotherapy alone. Treatment with sizofiran and 5-fluorouracil in combination improved (P = 0.003) the 5-year survival in 60 patients treated with radiotherapy. In 244 cervical carcinoma patients, the percentage of activated CD8+ (CD8+HLA-DR+) T cells in the CD8+ T-cell subsets in peripheral lymphocytes increased significantly as the disease progressed. A similar tendency was observed in the percentage of activated CD4+ (CD4+HLA-DR+) T cells in the CD4+ T-cell subsets. These immunologic parameters were significantly increased by radiotherapy, but not by surgery. Sizofiran accelerated a recovery in the activated CD8+ T cells in the CD8+ T-cell subsets compared with that of sizofiran nontreated patients after radiotherapy. Our data show that possible immune impairment in cervical carcinoma may be caused by disturbances in cell-mediated immunity, and that sizofiran is an effective immunotherapeutic agent for cervical carcinoma because it stimulates a rapid recovery of the immunologic parameters impaired by radiotherapy.

A case report of recurrent cervical cancer which responded to a combination of biological therapies.

Adoptive immunotherapy with tumor-infiltrating lymphocytes (TIL) has been challenged to human cancer patients during these years. However, as the efficacy of the therapy alone is still limited, another approach, a combination with other therapies may be required to obtain more favorable antitumor effects. Tumor necrosis factor alpha (TNF) is worth considering for combination, because at least its various potential roles in immune response suggest that it might increase the killing activity of T cells against autologous tumor cells. We attempted to treat the cancer patients with TIL combined with TNF therapy. A patient with recurrent cervical cancer was treated with multiple biological therapies including TIL and TNF therapy, and irradiation. Biological therapies consisted of endogenous and exogenous TNF (EET) therapy, TIL, administration of rTNF-SAM2 combined with hyperthermia and several biological response modifiers such as schizophyllan and lentinan. After 50 days of therapy, the recurrent tumor had remarkably decreased in size, and cystic change of the tumor was observed by CT scanning. By further continuation of these therapies, the disease condition was much improved. As these antitumor effects cannot well be explained by irradiation therapy alone, multiple biological therapies are considered to be potentially effective against the highly malignant advanced tumors.

Clinical evaluation of sizofilan as assistant immunotherapy in treatment of head and neck cancer.

Sizofilan (SPG), a simple glucan produced in a culture medium by Schizophyllum commune Feries, was used as an assistant immunotherapy in 15 patients with head and neck cancer. The cumulative 5-year survival rate was 86.7% in the SPG-treated group and 73.4% in the control group. Immunological parameters showed that the SPG group quickly recovered the cellular immunity damaged by radiation, chemotherapy and surgical procedure. SPG was found to be effective as an assistant immunotherapeutic agent in the treatment of head and neck.

Antitumor activity of Langerhans cells in radiation therapy for cervical cancer and its modulation with SPG administration.

Correlations between infiltration of Langerhans cells (ILC) in tumor tissues and radiation curability were investigated in 449 patients with cervical cancer treated with radiation alone, including 390 squamous cell carcinomas and 59 adenocarcinomas. No significant difference in prognosis was noted in stage I, II, and IV squamous cell carcinomas between positive and negative ILC. However, in the patients with stage III squamous cell carcinoma, a significantly better survival was observed for patients with ILC than for those without, 10 year survival rates being 78% vs. 54%, P < 0.01. In adenocarcinoma, the patients with ILC also showed significantly better survival than those without ILC, the 10 years survival rates being 45% vs. 25%, P < 0.025. An analysis of failure patterns following radiation treatment demonstrated that the favorable prognosis in patients with ILC in squamous cell carcinoma was due to improvement of local control rates and somewhat lower metastatic rates, whereas in adenocarcinoma it was only due to better local control rate. The ILC was significantly associated with T-cell infiltration in tumor tissues. The immunological stimulation with Sizofiran in 20 patients led to an augmentation of ILC in tumor tissues. The present study suggests that the ILC in cancer tissues improves local response to radiation treatment partly by T-cell mediated anti-tumor activity.

Improvement of long-term prognosis in patients with ovarian cancers by adjuvant sizofiran immunotherapy: a prospective randomized controlled study.

The effect of immunotherapy using sizofiran (SPG) on the prognosis of patients with ovarian cancers was prospectively studied in a total of 68 patients, who were randomly assigned to either a cisplatin, adriamycin and cyclophosphamide (PAC) therapy group or a PAC plus SPG combination therapy group. The survival rate was significantly higher in patients with stage Ic, II or III cancers treated with the PAC plus SPG combination, compared with the patients treated with PAC alone. In the SPG-receiving patients with stage Ic or more advanced cancers who were treated with four cycles or more of PAC, the outcome was improved (Cox-Mantel, p = 0.074; generalized Kruskal-Wallis, p = 0.032). Similar improvement was also observed in the patients with non-serous adenocarcinomas (Cox-Mantel, p-0.076; generalized Kruskal-Wallis, p = 0.045). No side effects attributable to SPG were recorded. The present results suggest that the use of SPG in combination with long-term chemotherapy improves the postoperative prognosis in ovarian cancer patients.

Augmenting effect of sizofiran on the immunofunction of regional lymph nodes in cervical cancer.

The immunomodulating effect of sizofiran on regional lymph nodes (RLN) was studied in cervical cancer and benign gynecologic tumors comparatively between treated patients (sizofiran intramuscularly (1) 1 day (20 mg) before and (2) 8 days (20 mg) and 1 day (20 mg) before surgery; n = 34) and untreated patients (n = 21). The RLN (internal iliac lymph nodes) were dissected surgically, and freshly obtained mononuclear (MN) cells were studied to observe interleukin-2 (IL-2) production and lymphokine-activated killer cell and natural-killer cell activities. The infiltration of surface phenotype of MN cells into RLN was determined semiquantitatively by immunohistochemistry. Sizofiran augmented IL-2 production of RLN, which was accompanied by a marked increase in the number of cells stained with anti-Leu-3a and IL-2 receptor. This effect was found more often in Stage Ib disease than in Stage 0, and it was not observed in benign tumors. These results suggest that stimulation with some antigens (e.g., cancer antigen in the current study) is necessary to induce immunoaugmentation by sizofiran which has no antigenicity. The augmenting effect was seen even in lymph nodes involved by advanced cervical cancer. Therefore, sizofiran was found to be a potent biologic response modifier in patients with cervical cancer.

Clinical effect of sizofiran combined with irradiation in cervical cancer patients: a randomized controlled study. Cooperative Study Group on SPG for Gynecological Cancer.

To evaluate the clinical effect of a biological response modifier (BRM), sizofiran (SPG), combined with irradiation, a randomized controlled study was performed in patients with stage II or III cervical cancer involving the collaboration of 52 institutes throughout Japan. Patients were randomly allocated to the control group (radiotherapy only) and the SPG group (radiotherapy + SPG). SPG was given intramuscularly, 40 mg once and 20 mg twice, a week concomitantly with radiotherapy. A total 315 patients were enrolled for the study but 23 were excluded from analysis. Of the remaining 292 patients, 121 were of stage II (43 controls and 78 SPG) and 171 of stage III (49 controls and 122 SPG). The results were as follows. (1) The complete response (CR) rate among stage II patients was higher in the SPG group (91.0%) than in the control group (79.1%); also the CR rate among stage III patients was significantly higher in the SPG group (77.9%) than in the control group (61.2%). (2) The SPG group showed a significantly rapid recovery from the decreased lymphocyte counts due to radiotherapy (P less than 0.05). (3) Side effects, probably associated with SPG administration, were observed in 11 cases (5.2%).

Combination therapy of radiation and Sizofiran (SPG) on the tumor growth and metastasis on squamous-cell carcinoma NR-S1 in syngeneic C3H/He mice.

The efficacy of Sizofiran(SPG), a highly purified beta-1,3-D-glucan from the culture broth of basidiomycetes Schizophyllum commune Fries, in combination with local irradiation was investigated using squamous-cell carcinoma NR-S1 and syngeneic hosts of C3H/He mice. NR-S1 tumor was implanted sc in the thigh of C3H/He mice. When tumor grew to 4 mm in diameter, the local irradiation of 55 Gy was delivered. SPG was injected im at a dose of 5 mg/kg. When SPG was administered after irradiation, remarkable inhibition of tumor growth was observed in comparison with the radiation alone group. Furthermore, the combination effect of radiation and active immunotherapy using mitomycin C-treated NR-S1 cells as vaccine was examined. When radiotherapy and active immunotherapy were combined with SPG, suppression of tumor growth was observed from an early stage in comparison with the group which was not administered SPG. SPG also inhibited the pulmonary metastasis of NR-S1 tumor after radiotherapy.

Maintenance of the activation of peritoneal macrophages in patients with ovarian cancer by sizofiran and recombinant interferon-gamma.

Four patients with ovarian cancer received 20 mg of sizofiran, a beta-1,3-glucan (molecular weight: 450,000), intramuscularly one day before and 4, 7, 11, 14, 18 and 21 days after second look laparotomy and recombinant interferon-gamma (rIFN-gamma) intraperitoneally on the day of second look laparotomy and 4, 7, 11, 14, 18 and 21 days thereafter. The peritoneal cavity was washed with physiological saline and peritoneal macrophages (M phi) were isolated. The number of M phi increased 30-1600 times during the treatment period. The concentrations of interleukin-1, interferon-gamma, tumor necrosis factor and prostaglandin E2 were also found increased in the supernatant fluid of M phi cultured for 24 hours with 10 micrograms/ml of lipopolysaccharide. The present study demonstrated that the activation of peritoneal M phi could be maintained and its number was increased by repeated dosing of sizofiran and rIFN-gamma in combination every three or four days in patients with ovarian cancer. Peritoneal M phi thus activated may exert an antitumor effect on ovarian cancer.