Protein-bound Polysaccharide Research Articles

Isolation of galactosaminoglycan moiety (CO-N) from protein-bound polysaccharide of Cordyceps ophioglossoides and its effects against murine tumors.

A galactosaminoglycan moiety was obtained from an antitumor polysaccharide fraction (SN-C) isolated from Cordyceps ophioglossoides culture. SN-C was subjected to sonication, then a protein-bound galactosaminoglycan (CO-N) was isolated specifically by precipitation with 10% ammonium hydroxide. When given intraperitoneally to mice, CO-N inhibited the proliferation of sarcoma 180 cells inoculated into the peritoneal cavity and exhibited a marked life-prolonging effect against ascitic tumors such as Ehrlich carcinoma and IMC carcinoma. CO-N also showed an inhibitory effect against solid Ehrlich carcinoma when given intratumorally and significantly inhibited the growth of a syngeneic solid tumor (MM46 mammary carcinoma) upon intravenous administration at a low dose. CO-N showed a cytocidal effect against cultured cells of IMC and P388D1 in vitro. Flow cytometric analysis demonstrated that fluorescein isothiocyanate-CO-N binds to the surface of Ehrlich cells.

The correlation between molecular weight and antitumor activity of galactosaminoglycan (CO-N) from Cordyceps ophioglossoides.

A galactosaminoglycan (CO-N) obtained by ultrasonication from a protein-bound polysaccharide SN-C, which was isolated from Cordyceps ophioglossoides culture, has a direct cytotoxicity against tumor cells (Ohmori et al., Chem. Pharm. Bull., 37, 1019 (1989). High performance liquid chromatographic analysis revealed that CO-N shows a broad molecular weight distribution with an average molecular weight of 33000. A potent antitumor activity of CO-N was observed in the higher-molecular-weight fraction on gel filtration, and the low-molecular-weight fraction below 6600 showed a weak activity. However, the depolymerized CO-N (ca. 5500) obtained by further ultrasonication of the original CO-N still retained the antitumor activity of CO-N against Ehrlich ascitic carcinoma or MM46 solid mammary carcinoma.

Effects of the protein-bound polysaccharide preparation, PSK on spontaneous breast cancer in mice.

The protein-bound polysaccharide preparation, PSK was tested for its ability to suppress carcinogenesis in spontaneous tumours in C3H/OuJ mice. They were divided into two groups of 40 individuals each. A normal diet was given to one group, establishing a control population, while the other group had 2% PSK added to their feed. Amount of feed consumed, body weight and tumour size were recorded weekly for 1 year. Within 15 weeks, 90% of the control population had developed tumours. In the test group the incidence of cancer and the number of tumours was significantly suppressed, and survival rates were improved. The amount of feed consumed and body weights of control and test groups were about the same.

Human immunodeficiency virus (HIV)-induced cell fusion: Quantification and its application for the simple and rapid screening of anti-HIV substances in vitro

A coculture system using Molt-4 and its HIV-producing cell, Molt-4/HIV HTLV-IIIB induced syncytia very efficiently. Among the cocultivations with various ratios of two types of cells, multinucleated giant cells were most clearly visible 20 hr after culture in a cell ratio of 5:5. The degree of cell fusion apparently correlated with the decrease in cell number during fusion reaction. The grade of the syncytia formation can be quantitatively expressed by the fusion index (FI). Using this system we evaluated various substances which are known to inhibit HIV replication and virus-induced cytopathogenicity in a cell-free viral infection. Glycyrrhizin sulfate, dextran sulfate, PSK, which is a protein-bound polysaccharide extracted from Basidiomycetes, and human plasma containing anti-HIV antibody gave about 15-fold reduction in F1 when compared to that of control. At the same time no difference was seen between the F1 given by two nucleoside analogs (3′-azido-2′,3′-dideoxythymidine and 2′,3′-didehydro-2′,3′-dideoxythymidine) and that of control.

PSK, a polysaccharide from Coriolus vesicolor, enhances oxygen metabolism of murine peritoneal macrophages and the host resistance to listerial infection.

PSK, a protein-bound polysaccharide isolated from the basidiomycete Coriolus vesicolor (Fr.) Quél. was examined with regard to its effects of macrophage (M phi) oxygen metabolism in mice, a function important for the expression of M phi antimicrobial activity. The O2(-)-producing ability and chemiluminescence (CL) of host peritoneal M phi s in response to phorbol myristate acetate were markedly elevated by preinjection of PSK (1 or 5 mg per mouse intraperitoneally) around 4-7d before M phi-harvest. The enhanced O2(-)-producing ability due to PSK injection persisted much longer than the enhanced CL, indicating a discrepancy in regulation of generation of active oxygen species such as O2-, H2O2, OH, and 1O2. Daily injections of PSK (1 mg per injection) from 10 to 4d before M phi harvest did not increase the efficacy of PSK over that given by a single 1 mg injection. When PSK (5 mg) was given intraperitoneally to mice in a single injection 10, 7 or 4d before the intravenous Listeria monocytogenes inoculation, a similar increase in the host resistance to the bacteria was noted regardless of the timing of the injection. Multiple PSK injections fron 10 to 4d before the infection also enhanced the host resistance, to the same degree. Therefore, PSK is thought to augment the host resistance to certain intracellular parasites including L. monocytogenes at least to some extent by enhancing oxygen metabolism of the host M phi.

Effect of PSK, a protein-bound polysaccharide from Coriolus versicolor, on drug-metabolizing enzymes in sarcoma-180 bearing and normal mice

The effects of PSK and Propionibacterium acnes (anaerobic Corynebacterium) on hepatic drug-metabolizing enzymes were studied using sarcoma-180 bearing and non-tumor bearing mice. PSK had no influence on aminopyrine N-demethylase and aniline hydroxylase activities, cytochrome P-450 concentration in hepatic microsomes, and the reductase activity of cytochrome c in normal mice. The content of cytochrome P-450 was not significantly reduced in S-180 bearing mice. On the other hand, P. acnes administration significantly decreased the amount of cytochromes P-450 and b 5 and aminopyrine N-demethylase activity. When FT-207 (Tegafur) was administered orally to S-180 bearing mice combined with the immunoadjuvants, only P. acnes significantly reduced the 5-FU levels in the serum and some organs.

Component analysis of protein-bound polysaccharide (SN-C) from Cordyceps ophioglossoides and its effects on syngeneic murine tumors.

A protein-bound polysaccharide (SN-C) obtained from the culture filtrate of Cordyceps ophioglossoides, which has been reported to have antitumor activity, was analyzed for sugar and amino acid components.Gas chromatography revealed that the neutral sugar component of SN-C was mainly composed of glucose.By using an amino acid auto analyzer, it was shown that SN-C was composed of galactosamine as the sole aminosugai.An intraperitoneal administration of SN-C into mice inoculated intraperitoneally with syngeneic murine tumors such as MM46 mammary carcinoma or L5178Y leukemia suppressed the tumor growth and resulted in a significant prolongation of the life span.When used in vitro, SN-C significantly inhibited the proliferation of various murine tumor cells including P388.Futhermore, SN-C inhibited the incorporation of glucose into Meth-A tumor cells with a consequent decrease of deoxyribonucleic acid (DNA) synthesis.SN-C may be a new type of antitumor polysaccharide since this material seems to posess both direct antitumor effect and stimulating activity on the host-mediated effect.

Dissociation of a glucan fraction (CO-1) from protein-bound polysaccharide of Cordyceps ophioglossoides and analysis of its antitumor effect.

A glucan moiety (CO-1) was dissociated from the antitumor protein-bound polysaccharide (SN-C) obtained from the culture of Cordyceps ophioglossoides by ultrasonication and heat treatment.CO-1 exhibited antitumor activity against murine sarcoma 180 when given by oral administration.CO-1 was also effective against syngeneic MM46 mammary carcinoma (solid form), but did not show any effect on ascitic tumors such as P388 leukemia.On intraperitoneal administration to normal mice, CO-1 increased the number of peritoneal exudate cells and strongly enhanced the chemiluminescence response of these exudate cells.The activity of CO-1 was higher than that of zymosan-A.

Fate and distribution of an antitumor protein-bound polysaccharide PSK (Krestin ®)

The fate of 14C-labelled PSK in the body was investigated. Although only substances with low mol. wt were observed in blood shortly after the administration, with time, substances with high mol. wt appeared, suggesting absorption of PSK in its original form from the digestive tract. 14C-labelled PSK was distributed in bone marrow, salivary gland, brain, liver, spleen, pancreas and tumor. Approximately 70% of 14C-labelled PSK was excreted by expiratory air after 24 h and approximately 15–20% in urine after 72 h. Only a small amount of 14C-labelled PSK was transferred into lymph and bile. The present study on the in vivo behavior of PSK provides an important basis for further analysis of its pharmacological actions.

Production of antiserum against antitumor protein-bound polysaccharide preparation, PSK (Krestin ®) and its pharmacological application

Antiserum against a protein-bound polysaccharide preparation (PSK) was produced by immunizing New Zealand White rabbits with PSK. The intestinal absorption of PSK in mice was visualized by indirect immunofluorescent staining with anti-PSK serum. The change in blood levels of 14C after oral administration of 14C-PSK and the recovery of 14C by antiserum were determined. The results indicated that antigenic epitopes in PSK are not completely destroyed during the process of digestion, absorption and distribution, but the changes of serum levels of 14C radioactivity differ from those of immunoreactive radioactivity. These results suggest that multiple processes are involved in the fate of PSK administered orally.

Antitumor constituents ofPolyporus giganteus

To investigate antitumor constituents of higher fungi, the carpophores ofPolyporus giganteus Pers. ex. Fr. (81 g, dry weight) which were collected in Indiana, U.S.A. were examined for antitumor activity. Two protein-bound polysaccharide fractions (I and II) were prepared from the hot water extract and one fraction (III) from the 0.1 N NaOH extract of the carpophores. The antitumor effect of each fraction was tested against sarcoma 180 implanted subcutaneously in female ICR mice. Of three fractions, Fraction II showed 85.2% inhibition ratio at the dose of 20 mg/kg/day for 10 days and was named gigantan. Gigantan was found to contain 59% polysaccharide and 27% protein. Its polysaccharide moiety was a heteroglycan that consisted of mainly glucose (89.3%), galactose (7.7%), manose (2.0%) and fructose (1.0%).

Tumor growth promoting activity of an immunosuppressive substance and its modulation by protein-bound polysaccharide PSK

The role of an immunosuppressive substance (IS), which is increased in the serum of tumor-bearing animals, was examined in rats. IS isolated from cancerous ascites fluid of rats was administered to Walker 256 tumor-bearing rats to examine changes in the serum level of IS, tumor growth and survival rate. PSK, an immunomodulator, was also administered. Serum IS increased with tumor growth. The administration of IS to tumor-transplanted rats caused the tumor to grow and shortened the animals' survival time. The administration of PSK, however, inhibited the increase in serum level of IS, resulting in the suppression of tumor growth and a prolongation of survival time. The findings suggested that IS is a useful parameter for predicting not only tumor growth but also the therapeutic effect of immunomodulators such as PSK.

Effect of immunostimulants and antitumor agents on Tumor Necrosis Factor (TNF) production

OK-432, a lyophilized preparation of Streptococcus pyogenes, showed a priming activity for TNF production in mice, associated with an increase of spleen weight. PSK, a protein-bound polysaccharide preparation from coriolus versicolor, did not show such activity. Both OK-432 and PSK potentiated the TNF production in mice primed with Corynebacterium parvum (CP) and challenged with Escherichia coli endotoxin (LPS). Cytotoxic antitumor agents of 5-fluorouracil (5-FU), cyclophosphamide (CY) and bleomycin (BLM) suppressed TNF production in mice primed with CP and challenged with LPS. TNF production suppressed by 5-FU, CY and BLM was partially restored by the combined treatment with OK-432 or PSK. These results suggest that the administration of cytotoxic antitumor agents suppresses the intrinsic TNF production in cancer patients, and the combined use of immunostimulants such as OK-432 and PSK is advantageous in restoring TNF production suppressed by cytotoxic antitumor agents.

Combined therapeutic effects of an immunomodulator, PSK, and chemotherapy with carboquone on rat bladder carcinoma.

Responses of bladder cancer in ACI rats to combination therapy with an immunomodulator, PSK, and an alkylating agent, carboquone, are reported. PSK is a protein-bound polysaccharide isolated from Basidiomycetes, and carboquone is the alkylating agent 2,5-bis(1-aziridinyl)-3-(2-hydroxy-1-methanoxyethyl)-6-methyl-p- benzoquinone carbonate, molecular weight 3,214. The immunomodulator, PSK, was shown to enhance the effectiveness of the chemotherapeutic agent, carboquone. The therapeutic effect of combination treatment was monitored by measuring growth rates of tumors transplanted SC and by measuring decreases in metastatic spread to lungs in tumor-bearing animals. Effects of PSK on host immunity were monitored by measuring serum levels of immunosuppressive substance.

Cloning of sequences induced and suppressed by administration of PSK, antitumor protein-bound polysaccharide

To elucidate the effects of PSK, a protein-bound polysaccharide from Coriolus versicolor, on gene expression in tumor cells, we prepared cDNA clone libraries from PSK-treated and untreated cells of a rat ascites hepatoma line, AH66, which was previously shown to be susceptible to the antitumor action of this compound. Two PSK-induced and one suppressed cDNA clones were selected from these libraries by using a differential colony hybridization and RNA blot hybridization. PSK was thus shown to have a direct effect on the transcription and consequently on the translation of tumor cells.

Therapeutic effects of PS-K and busulfan on the recurrent and metastatic diseases after the surgical removal of 3-methylcholanthrene-induced autochthonous tumors in C57BL/6 mice.

The effectiveness of a protein-bound polysaccharide, PS-K, and an immunoaugmenting antileukemia agent, busulfan (BU), was investigated for the treatment of autochthonous tumors induced by 3-methylcholanthrene in C57BL/6 mice. PS-K and BU were found to be effective in dealing with recurrence and pulmonary metastasis when applied in conjunction with the surgical removal of the primary tumor. The survival time of mice to which PS-K (300 mg/kg/day X 5 or X 2/week for 7 weeks, ip) or BU (50 mg/kg X 3, po) was administered was significantly prolonged when compared with that of mice treated by surgery alone. Moreover, the incidence of the local recurrence of tumors was significantly lower in mice treated with PS-K or BU after the surgical removal of the primary tumor. The effectiveness of the treatment on metastasis was also shown by the prolongation of mean survival time in groups of mice which showed no local recurrence of tumors. The timing-dependency of the treatment with PS-K did not seem to be an important factor and no additive therapeutic effects of PS-K and BU were observed in the present study.

Interaction of protein-bound polysaccharide (PS-K) with microtubule proteins. V. Influence on tubulin-dependent adenosine triphosphatase (ATPase) activity.

In the presence of 5 mM Ca2+ or 2mM Mg2+, a protein-bound polysaccharide (PS-K) isolated from Basidiomycetes inhibited the tubulin-dependent adenosine triphosphatase (ATPase) activity of microtubule-associated proteins (MAPs) in a concentration-dependent manner, though it had little effect on the activity in the absence of tubulin. The extent of inhibition decreased at lower concentrations of Ca2+ or Mg2+ and an excess amount of tubulin largely restored the inhibitory effect. When Mn2+ was added to the reaction mixture instead of these divalent cations, the presence of PS-K conversely stimulated the tubulin-dependent ATPase activity of MAPs at lower Mn2+ concentrations (<1-2 mM) under the conditions tested, though PS-K inhibited the ATPase activity at high Mn2+ concentrations. Maximal stimulation and inhibition were about 160% and 70% of the original level (i.e., in the absence of PS-K), respectively. PS-K could also enhance MAPs ATPase activity under conditions where it enhanced the activity in the presence of tubulin, indicating that PS-K itself interacts with MAPs ATPase as tubulin does. The addition of a large amount of ATP under experimental conditions where PS-K inhibited tubulin-dependent Mn2+-ATPase activity of MAPs, on the other hand, led to stimulation by PS-K. Thus, the influence of PS-K on the ATPase activity was dependent on the molar ratio of metal ions to adenosine triphosphate (ATP).

Interaction of protein-bound polysaccharide (PS-K) with rabbit skeletal muscle actomyosin.

The interaction of protein-bound polysaccharide (PS-K) with actomyosin from rabbit skeletal muscle was investigated by measuring the changes in adenosine triphosphatase (ATPase) activity, turbidity, and viscosity. At low ionic strengths, PS-K reduced the actin-activated Mg2+-ATPase activity of myosin to 20% of the original level, whereas it slightly enhanced the myosin ATPase activity without actin. The inhibition of actin-activated ATPase activity was not reversed by increasing actin concentration and, further, the apparent affinity of myosin for actin was not altered by the addition of PS-K. The extent of inhibition depended on the sequence of addition of myosin, actin, and PS-K, i. e., when PS-K was preincubated first with myosin before adding actin, the most marked inhibition was observed. The changes of actomyosin turbidity induced by PS-K were similar to those of ATPase inhibition. PS-K did not affect the polymerization and depolymerization of actin. Therefore, the inhibitory effects seem to be caused by PS-K bound to myosin filaments.

Inhibition of adenosine-5'-triphosphatase activity in microtubule protein preparation by PS-K.

A protein-bound polysaccharide, PS-K, exhibited an inhibitory action on the ATPase activity of microtubule proteins in a concentration-dependent manner. The extent of inhibition was greater with Mg2+ than with Ca2+. Heat treatment at 100°C and digestion by DNase 1, RNase A, several proteases, and a glycosidase mixture did not affect the inhibitory effect of PS-K.

Effect of protein-bound polysaccharide (PS-K) on microtubule proteins. III. Some properties of PS-K inhibition of microtubule polymerization and the site of its action.

A protein-bound polysaccharide (PS-K) suppressed glycerol-free microtubule polymerization in the same way as it suppressed the polymerization in a reassembly mixture containing glycerol. However, the extent of inhibition was enhanced in the absence of glycerol. Maximal inhibition reached about 85% (the extent of polymerization was 15% of the original level) in the standard assembly medium containing 100mM KCl. The addition of PS-K increased the critical concentration for microtubule polymerization. Electron microscopy indicated that PS-K did not change the ultrastructure of microtubules. During incubation with PS-K, microtubule proteins retained their ability to polymerize, because the addition of taxol largely restored the inhibition by PS-K and the presence of an excess amount of microtubule-associated proteins (MAPs) or tubulin dimers also sequestered the inhibition to some extent. The extent of cancellation was greater by MAPs than by tubulin dimers.