Differential effect of protein-bound polysaccharide (PSK) on survival of experimental murine tumors.

The effect of protein-bound polysaccharide (PSK) on the survival of BALB/c and C57BL/6 mice after intravenous injections of syngeneic murine sarcomas (GR9.B9 and Meth-A), LSTRA lymphoma and B16 melanoma cells was studied. Pretreatment of mice with PSK significantly increased survival after the injection of either type of sarcoma cells, although the effect was attenuated when high numbers of cells were injected. Survival was not modified significantly in LSTRA lymphoma or B16 melanoma. Mice pretreated with anti-asialo GMI serum showed significantly decreased survival from all tumors in comparison with untreated mice injected with tumors, regardless of cell dose used. We observed an inverse correlation between H-2 antigen expression and in vitro NK sensitivity of tumor cells from all lines except B16 melanoma cells. These results clearly suggest that pretreatment of mice with PSK prolongs survival and inhibits metastasis formation in mice injected with sarcoma cells, being this effect highly selective, since survival was not improved in mice injected with LSTRA lymphoma or B16 melanoma.     

A suppressive effect of PSK, a protein-bound polysaccharide preparation, on tumor growth: a new effect of PSK on cell motility

We found that PSK has ambidextrous effects on cell motility. PSK enhances macrophage motility and inhibits tumor cell motility, when the capillary tube method was used in vitro. Macrophage motility was enhanced with increasing concentration of PSK. PSK inhibited tumor cell motility, i.e. Ehrlich tumor cells, EL-4 lymphoma cells and human leukemic cells, in dose dependent fashion. Macrophages and tumor cells were incubated with medium containing PSK for varying times at 37 degrees C and then washed with medium to eliminate PSK thoroughly. The motility of macrophages and tumor cells was enhanced and inhibited, respectively, with increasing pre-incubation time. When PSK-treated tumor cells were injected into the abdominal wall of C57BL/6 mice, PSK-treated tumor cells were less invasive than non-treated ones in mice. These phenomena are concern neither with the change of cellular viability nor that of cell proliferation.     

The effect of a protein-bound polysaccharide (PSK) on lymphocyte subsets of peripheral venous blood and thoracic duct lymph

To study the effects of a protein bound polysaccharide (PSK) on the immune system in normal animal, lymphocytes subsets of both peripheral (PBL) and thoracic duct lymphocytes (TDL) were analyzed in 6 week old male SPF Wistar-Imamichi rats before and after free feeding of forage which contained 2% of PSK. PBL and TDL were obtained at the time before, 4 weeks after and 8 weeks after administration of PSK. Age matched rats without administration of PSK were used as a control. Following the peripheral blood cell counts were carried out, lymphocytes subsets such as ratio of total T cells helper/inducer T (Th) cell, suppressor/cytotoxic T (Ts) cell and B cell were assayed using W3/13, W3/25, 0 X 8 and 0 X 4 monoclonal antibodies with laser flow cytometric technique (Orthospectrum III Orthodiagnostics). In terms of the PBL, number of total T cells and Th cells in PSK treated group were significantly lower than that of controls. However no statistical different could be obtained between these two groups in relation to the number of Ts cells and B cells. On the other hand, the number of T cells and Th cells in TDL of PSK treated groups were significantly higher than that of controls while Ts cell and B cell number did not show no apparent differences between these two groups. These findings indicate that oral administration of PSK have a potency to accelerate the transport of T cells from blood stream to lymphatic channel and also regulate their distribution in a normal healthy animals.     

Effectiveness of immunochemotherapy with PSK, a protein-bound polysaccharide, in colorectal cancer and changes of tumor marker

In the present study, curatively resected patients of colorectal cancer at pTNM stages II and III were selected. Patients receiving postoperative combined PSK, a protein-bound polysaccharide, and fluoropyrimidine therapy (PSK + chemotherapy group) were compared with patients receiving postoperative chemotherapy alone (chemotherapy group) during the same period of study. Three-year disease-free survival rates were evaluated and the postoperative changes of serum type IV collagen level were investigated. The results confirmed a significant improvement of the three-year disease-free survival rate in the PSK + chemotherapy group compared to the chemotherapy group, suggesting that PSK is useful as postoperative prognosis control including relapse prevention for colorectal cancers at pTNM stage II and III. Analysis of the postoperative changes of serum type IV collagen level showed significantly higher levels in the chemotherapy group than in the PSK + chemotherapy group, and this tendency was sustained for 12 months after surgery. This observation is speculated to be caused by inhibition of vascular basement membrane destruction by PSK, leading to inhibition of release of type IV collagen into the blood. These results indicated a possibility that combined PSK and chemotherapy inhibited metastasis, thereby reducing the risk of relapse and leading to improvement of the three-year disease-free survival rate.     

PSK,a protein-bound polysaccharide,overcomes defective maturation of dendritic cells exposed to tumor-derived factors in vitro

Dendritic cells (DC) are the most potent antigen-presenting cells that induce specific anti-tumor immunity. To obtain potent efficacy of immunotherapy using infusion of activated DC, it is necessary to overcome defective function of DC in tumor-bearing patients. We examined whether the treatment with PSK, a biological response modifier derived from Basidiomycetes, could allow DC to avoid inhibition of functional maturation by tumor-derived factors in vitro. CD14+ monocyte-derived DC were generated by stimulating with granulocyte-macrophage colony-stimulating factor and interleukin (IL)-4 in the presence or absence of PSK (100 microg/ml), by exposure to a tumor culture supernatant (TSN) of MKN-45P human gastric cancer cells. TSN-exposed DC were not effective in inducing cytotoxic T lymphocyte-mediated growth inhibition of target HT29 human colon cancer cells. In contrast, the presence of PSK significantly resuscitated the defective cytotoxicity. This beneficial outcome was accompanied by an increase in phagocytic activity as measured by fluorescein isothiocyanate-conjugated dextran, expression of CD83 (maturation-specific phenotype), overexpression of a CD86 co-stimulatory molecule, preserved production of IL-12 that plays a key role in the induction of Th1-type immune regulations, and protection against TSN-induced apoptosis of DC. These results demonstrated that PSK overcomes defective maturation of DC exposed to tumor-derived factors in vitro, and suggest the efficacy of PSK in DC-based immunotherapy in cancer patients.     

Antitumor effector mechanism at a distant site in the double grafted tumor system of PSK, a protein-bound polysaccharide preparation

The antitumor effect at a distant site of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received simultaneous intradermal inoculations of Meth-A tumor in the right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with PSK in the right-flank tumor on day 3. PSK inhibited the growth of not only the right but also the left (non-treated) tumor. Immunized spleen cells were taken from mice which had been cured by the intratumoral administration of 5 mg of PSK and were injected into the Meth-A tumor on day 3. Adoptive transfer of PSK immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 monoclonal antibody plus complement. Spleen cells and right and left regional lymph node cells prepared from PSK immunized mice were examined for Thy-1, Lyt-1, Lyt-2 and asialo GM1 phenotypes. The number of Lyt-1-positive lymphocytes increased in the right regional lymph nodes after intratumoral administration of PSK. A massive accumulation of macrophages and polymorphonuclear leukocytes was found in the right tumor and an infiltration of macrophages and Lyt-2-positive lymphocytes was found in the left (non-treated) tumor by immunohistochemical analyses. These results suggest that intratumoral administration of PSK induces Lyt-1-positive cells first in regional lymph nodes, then in the spleen, and subsequently induces macrophages and Lyt-2-positive cells in the left (non-treated) tumor, thus bringing about the regression of metastatic tumors.     

Protective effects of a protein-bound polysaccharide, PSK, against Candida albicans infection in syngeneic tumor-bearing mice via Th1 cell functions

We investigated the effects of a protein-bound polysaccharide, PSK, on the resistance of tumor-bearing mice against Candida albicans infection. In BALB/c mice that had received subcutaneous (sc) transplantation of fibrosarcoma Meth A, viable fungal counts were increased in the kidney and the mean survival period was shortened after challenge with C. albicans, compared with healthy mice. Oral administration of PSK to such mice resulted in a significant decrease of viable fungal counts and a prolongation of the mean survival period. The ratio of CD4-positive T cells in the spleen was decreased in noninfected tumor-bearing mice and the decrease was prevented by PSK, although in vitro anticandida activities of phagocytes were not significantly affected by tumor burden or PSK. Further, intracellular interferon (IFN)-gamma productivity was enhanced and the number of IFN-gamma-producing CD4-positive T cells was enhanced by PSK. PSK enhanced the gene expression of interleukin (IL)-12 and IFN-gamma in the spleen of tumor-bearing mice inoculated with C. albicans. Treatments with anti-IL-12 or anti-IFN-gamma antibody reduced the anti-infectious effects of PSK. These findings suggest that the protective effect of PSK on sublethal inoculation with C. albicans in tumor-bearing mice is possibly mediated by Th1 cell functions.     

Antitumor activity of protein-bound polysaccharide from Cordyceps ophioglossoides in mice

The effects of protein-bound polysaccharide (SN-C) extracted from Cordyceps ophioglossoides on the growth of transplanted allogeneic and syngeneic murine tumors were studied. SN-C given by intraperitoneal administration suppressed the growth of sarcoma-180 transplanted subcutaneously in mice. Intraperitoneal administration of SN-C also caused a significant prolongation of the life span of ICR mice inoculated intraperitoneally with Ehrlich carcinoma, and C3H/He mice inoculated intraperitoneally with a syngeneic tumor (X-5563). SN-C showed a significant cytocidal effect on cultured tumor cells. SN-C did not affect delayed-type hypersensitivity (DTH) in normal mice, but restored the depressed capacity to raise DTH in tumor-bearing mice. These results suggested that SN-C may exert both direct and host-mediated antitumor effects.     

Effects of combined therapies with protein-bound polysaccharide (PSK, Krestin) and fluorinated pyrimidine derivatives on experimental liver metastases and on the immunologic capacities of the hosts.

An experimental model is introduced for the study of liver metastases using intrasplenically injected EL-4 and Lewis lung tumor cells. Fluorinated pyrimidine derivatives, 1-(2-tetrahydrofuryl)-5-fluorouracil and 5-fluorouracil, showed inhibitory effects on the frequencies of liver metastases. Immunosuppressive effects of these drugs were compared at the doses capable of showing 50% inhibition of the development of metastatic nodules. These derivatives strongly suppressed the phagocytic activity and the number of Kupffer cells of the liver and then the humoral response against sheep red blood cells, the delayed hypersensitivity against picryl chloride. On the contrary, combined administration of protein-bound polysaccharide (PSK) and 1-(2-tetrahydrofuryl)-5-fluorouracil showed no inhibitory effect on these activities.     

Krestin (PSK)

A polysaccharide preparation isolated from Coriolus versicolor (Fr.) Quél. of Basidiomycetes (PSK) predominantly consists of glucan and approximately 25% tightly bound protein. PSK was effective against various allogeneic and syngeneic animal tumors and has been given orally to cancer patients. Various suppressed or enhanced immune responses of tumor-bearing animals were restored to normal levels by the administration of PSK in the tumor models tested. The killer T cell activity was augmented in tumor-bearing mice by intraperitoneal or oral administration of PSK, and there was correlation between the PSK associated antitumor effect and the killer T cell activity. It was found that PSK competed with immunosuppressive substances isolated from tumor-bearing mice and that the intestinal immune system appeared to be modulated by oral administration of PSK. After oral administration of 14C- or 35S-labeled PSK to normal rats, it was found that small or large molecular substances appeared in the serum depending on the time elapsed after administration, an indication that large molecular size products were from the digestive tract.     

Usefulness of polysaccharide K (PSK) as postoperative adjuvant immunotherapy in patients with stage IV gastric cancer

Background. We investigated retrospectively the usefulness of polysaccharide K (PSK) administration for prolonging survival after noncurative resection in patients with stage IV gastric cancer who underwent surgery at our department. Methods. 357 patients with gastric cancer evaluated as stage IV according to thegeneral rules for gastric cancer study, 11th edn (1985) of the Japanese Research Society for Gastric Cancer, who were expected to succumb to surgical death or postoperative death within 2 months were studied. The patients were divided into two groups: total PSK dosage greater than or less than 180g. Survival rates were calculated for each group. Results. In these stage-IV gastric cancer patients, the 5-year and 10-year survival rates in the PSK group were both 13.9%. In the non-PSK group, the rates were 8.7% and 7.0%, respectively. The survival rates were significantly higher in the PSK group (p = 0.0001). The survival rate was also calculated with respect to the staging factors. Patients in the PSK group who had H0, P0, se or milder and n3 or n4 showed a significantly higher survival rate than those with these staging factors in the non-PSK group (p = 0.010). Conclusion. PSK administration prolonged survival in stage-IV gastric cancer patients, the effect being marked in patients with severe lymph node metastasis. We suggest that be administered as adjuvant therapy in stage-IV gastric cancer patients with severe lymph node metastasis (n3, n4).     

Neonatal inoculation with the protein-bound polysaccharide PSK increases resistance of adult animals to challenge with syngeneic tumor cells and reduces azoxymethane-induced precancerous lesions in the colon.

We have investigated the results of neonatal inoculation with a protein-bound polysaccharide, PSK, as it affects the defense mechanism of animals against cancer. Male BALB/c mice received a single i.p. injection of 10 mg/kg PSK within 48 h of birth. When the mice were 8 weeks of age, colon adenocarcinoma 26 (C26 tumor) cells were transplanted s.c. Injection of PSK increased the number of tumor-rejecting mice from 10 to 50% compared with the control mice transplanted with 5 x 10(3) tumor cells and prolonged the median survival period to 174% of control mice with tumors. When the number of transplanted tumor cells was increased to 1 x 10(6), PSK injection significantly prolonged the survival period, although tumors grew in all mice. The survival period was also significantly prolonged in male C57BL/6 mice that received an injection neonatally with PSK and were given a s.c. transplant of Lewis lung carcinoma or B16 melanoma at 8 weeks of age. The effect on survival was dependent on the PSK dose and the number of transplanted tumor cells. PSK was as effective for male mice 30 weeks of age as for mice 8 weeks of age treated with PSK during the neonatal period. However, prolongation of the survival period of tumor-bearing mice was not observed in the offspring (F1). Neonatal injection of PSK also significantly reduced the number of metastatic foci in the liver of mice inoculated with 1 x 10(5) C26 tumor cells in the splenic vein after 8 weeks of age. In addition, neonatal injection of PSK significantly reduced the number of aberrant crypts and aberrant crypt foci, the precancerous lesions in the colon of F344 rats that received injections s.c. with azoxymethane after 7 weeks of age, to 47% of that of rats that received an injection with saline at the same age. The effect on precancerous lesions was dependent on the timing of PSK injection and the dose. Regarding the mechanism, when animals thymectomized during the neonatal period or when congenitally athymic animals were used instead of healthy animals, the effect on survival or precancerous lesions did not appear. Neonatal injection of PSK significantly reduced the number of CD4+ CD8+ T cells and significantly increased the number of CD4+ CD8- and CD4- CD8+ T cells in the thymus of healthy mice 10 weeks of age and C26 tumor-bearing mice. Furthermore, neonatal injection of PSK significantly elevated the T-cell differentiation induced by a mouse thymus extract 10 weeks of age. These findings suggest that neonatal injection of PSK induces resistance in adult mice to challenge by syngeneic tumor cells and reduces the azoxymethane-induced precancerous lesions in the colon of adult rats via the thymus functions.     

Mechanism of antimetastatic immunopotentiation by low-dose cyclophosphamide

We have previously reported the antimetastatic effect of a single low-dose of cyclophosphamide (Cy) on L-TACB rat lymphoma. The phenomenon could be adoptively transferred in immunocompetent rats and is abolished in nude mice, facts for which an immunomodulatory explanation was proposed. The aim of this paper was to identify the mechanism(s) by which spleen cells from Cy-treated tumour-bearing rats could exert this antimetastatic activity. Conditioned media obtained by incubation of spleen cells from Cy-treated and non-treated tumour-bearing rats, under specific or non-specific stimulation, were assayed to evaluate their effect on lymphocyte proliferation. The production of transforming growth factor beta (TGF-beta), interleukin-10 (IL-10) and nitric oxide (NO) by conditioned media was also studied. The restoration of spleen lymphoproliferative responses to normal levels when exposed to media conditioned by splenocytes from Cy-treated tumour-bearing rats, together with a decreased production of suppressive cytokines TGF-beta, IL-10 and NO, suggest an enhancement of host antimetastatic immunity triggered by single low-dose Cy treatment.     

Effect of PSK on prostaglandin metabolism (I)

The effect of PSK (Krestin) on the metabolism of prostaglandins was investigated. The effect of PSK on the thromboxane A2 (TXA2) level, which stimulates tumor proliferation and platelet aggregation, was examined using platelets. PSK suppressed platelet aggregation and the production of malondialdehyde (MDA) and TXB2 which is a stable metabolite of TXA2. The effect of PSK on the production of prostacyclin (PGI2), which is an anti-tumor PG, was then examined using rat arterial rings. It was found that PGI2 production was stimulated by PSK. The in vivo inhibition of platelet activation by PSK was then examined using two thrombosis models in which platelet aggregation was mainly involved. PSK exerted its anti-platelet effect by regulation of PG production. It was concluded that not only an immune regulating effect but also PG regulation are involved in the pharmacological action of PSK.     

Experimental study on immunochemotherapy (PSK, CQ) in rat bladder cancer (BC-47)

Cancer of the urinary bladder is a tumor with the highest frequency among urogenital cancers, and more-over, its recurrence rate is high. It is considered important and urgently necessary to conduct studies into the prevention of recurrence of this cancer. We have started a Study Group on Postoperative Maintenance Therapy for Bladder Tumor (Tokyo), and conducted group studies on the prevention of recurrence. Fundamental experiments have been performed, and the following results obtained. Antitumor effects of PSK in combination with CQ for bladder carcinoma were studied using male ACI rats. An established bladder carcinoma cell line, BC47 was transplanted into the backs of rats subcutaneously prior to the administration of PSK and CQ. Inhibitory effect on tumor growth and prolongation of survival period were examined. Although single-agent PSK or CQ both had inhibitory effects on transplanted tumors as well as on metastatic tumors in the lung, more remarkable effects were noticed as a result of combination treatment. Prolongation of lifespan using combination therapy was superior to that using single treatment and the rats of decrease in body weight was also lower. These results do not necessarily clarify the antitumor mechanisms, but the immune system, probably non-specifically, may take part in the mechanism judged from the accumulated results obtained from in vivo systems. We think that the combinational effects of these two drugs on general condition are probably originated from inhibition of tumor growth since the inhibition, prolongation of lifespan, change of body weight and so on were closely correlated with each other. However, the direct effect of these drugs may also be counted. The combination therapy of PSK and CQ seemed to be useful against rat bladder carcinoma, BC47. We therefore intend to proceed with group study trials of Postoperative Maintenance Therapy for Bladder Tumor.     

Induction of macrophage tumoricidal activity, major histocompatibility complex class II antigen (Iak) expression, and interleukin-1 production by swainsonine

Previous studies in our laboratory have shown that the reported antitumor activity of systemically administered swainsonine, an indolizidine alkaloid, is due at least in part to immune modulation involving effector cells (Humphries, M.J.; Matsumoto, K; White, S.L.; Olden, K. Cancer Res. 48:1410-1415; 1988 and White, S. L.; Schweitzer, K.; Humphries, M.J.; Olden, K. Biochem. Biophys. Res. Commun. 150:615-625; 1988). In this report, studies are presented to show that swainsonine was effective in activating peritoneal macrophages to cytotoxicity against tumor cells. Stimulation of tumoricidal activity of macrophages was associated with increased secretion of interleukin-1 (IL-1) and expression of the Iak major histocompatibility complex (MHC) antigen on the cell surface. The 3-fold stimulation of cytotoxicity observed in these in vivo studies was comparable to that obtained with Corynebacterium parvum, a commonly used in vivo activating agent. The in vitro incubation of thioglycollate-elicited peritoneal macrophages with swainsonine consistently resulted in levels of activation (6- to 8-fold) comparable to that obtained by treatment with known in vitro macrophage activating agents such as lipopolysaccharide (LPS) or recombinant gamma-interferon (rIFN-gamma). The stimulation observed by using swainsonine in combination with LPS was additive, suggesting different mechanisms of action. These studies have important implications not only for treatment of cancer, infectious diseases, and immune suppressive disorders, but also for elucidation of the mechanism of macrophage activation.     

Induction of ornithine decarboxylase activity by 4,4'-methylene bis(2-chloroaniline) in the rat.

The effects of the curative extender 4,4'-methylene bis (2-chloraniline) (MOCA), an established experimental carcinogen that exhibits activity in rat liver, on hepatic ornithine decarboxylase (ODC) activity was investigated. Male Sprague-Dawley rats were injected i.p. with 75 mg/kg MOCA and killed 6, 12, 18, 24, 42 and 48 h later. Stimulation with MOCA of liver cytosolic ODC was first evident at 6 h, peaked at 12 h and returned to control levels by 42 h. The liver enzyme was refractory to stimulation by a second treatment of MOCA within the dosing intervals examined. The magnitude of stimulation of the enzyme by this aromatic amine was dependent on dose and route of administration.     

Selective suppression of t-cell activity in tumor-bearing mice and its improvement by lentinan,a potent anti-tumor polysaccharide

The cellular site of immunosuppression in Ehrlich tumor-bearing mice was analysed with particular reference to the T- and B-cell activities. The B-cell activity as measured by the anti-dinitrophenyl (DNP) antibody responses to DNP-thymus-independent carriers (TID) was not impaired in tumor-bearing mice as compared with normal mice, whereas the anti-DNP antibody responses to DNP-thymus-dependent carriers (TD) and the development of helper T-cell activity to TD were markedly suppressed in tumor-bearing animals or mice pretreated with cell-free cancerous ascitic fluid. The selective suppression of T-cell response was not mediated by the generation of suppressor cell activity toward TD, which may depress the manifestation of developed helper T-cell activity. A marked suppression of T-cell response was observed when the animals were inoculated with tumor cells or injected with cancerous ascitic fluid prior to antigenic stimulation, but not when the animals were rendered tumor-bearing by such treatments after the immunization. The suppression of T-cell activity in both sarcoma 180 tumor-bearing mice and cell-free Ehrlich cancerous ascitic fluid-treated mice was prevented by treatment with lentinan, a potent anti-tumor polysaccharide. The applicability of this experimental system to the search for immunopotentiators relevant to tumor immunotherapy is discussed in the light of the preventive effect of lentinan on the suppression of T-cell response in tumor-bearing animals.