Specific regulation of HSPs in human tumor cell lines by PSK.

Substantial progress has been make in elucidating the biochemical properties of HSPs (Heat Shock Proteins) as molecular chaperones in protein biogenesis and their roles in thermoprotection and cytoprotection against cellular insults. Recently, besides advantageous cellular functions, the detrimental role of HSPs has been implicated in a variety of diseases. HSP47 is assumed to be a collagen specific molecular chaperone and its involvement in the progression of fibrosis was observed. There have been a number of reports that suggest the role of HSP60 as an autoantigen in a variety of autoimmune diseases. PSK, a protein-bound polysaccharide, is a biological response modifier that is clinically used for the treatment of cancer patients in Japan. PSK shows a broad range of biological effects in addition to the antitumor activity. In this study, we evaluated the effect of PSK on the expression of HSPs in human tumor strains at the protein and mRNA levels. PSK was observed to suppress the expression of HSP47 and HSP60 but not HSP72/73 in human tumor cell lines. Thus, the novel pharmacological potential of PSK was suggested in the diseases derived from the aberrant expression of HSPs.

Specific regulation of HSPs in human tumor cell lines by flavonoids.

While the protective role HSPs (Heat Shock Proteins) has been recognized against physiological stress such as heat shock, heavy metals and glucose starvation, recent progress has revealed another aspect of HSPs in various diseases. HSP27 has been shown to be involved in the acquired resistance of tumor cells hyperthermic and chemotherapeutic treatment. In human breast tumors, overexpression of HSP27 is associated with a shorter disease-free survival period. HSP47 is thought to be a collagen specific molecular chaperone. The involvement of HSP47 in the progression of fibrosis has been reported. Aberrant expression of HSP could cause various autoimmune diseases. Manipulation of HSP expression, therefore, could be a therapeutic target to reduce HSP-derived detrimental cellular effects. Flavonoids are a widely distributed group of plant substances, universally present in vascular plants. Although the flavonoids have been known as natural plant products as long as the alkaloids, their pharmacological effects and potential medicinal uses have been little studied by comparison. Today, the picture has changed and the biological and pharmacological activities of plant flavonoids look promising. We investigated the effect of flavonoids on the expression of HSPs in human tumor cell lines. Flavonoids inhibited the expression of HSP27, HSP47, HSP60 and HSP72/73. The results suggested the pharmacological possibilities of flavonoids in diseases derived from abnormal expression of HSPs.

Specific expression of HSP27 in human tumor cell lines in vitro.

The cells of all organisms respond to environmental stress by synthesizing a specific group of proteins called heat shock proteins (HSPs). While the protective role of HSPs has been observed against cellular stress, recent reports have suggested a number of functions of HSPs in various cellular processes in normal and pathological conditions. HSP27 is an important low molecular weight HSP (Mr: 27,000) found in human cells. HSP27 appears to be involved in intracellular signaling and drug resistance in addition to thermotolerance. In human breast tumors, a striking association between HSP27 overexpression and a shorter disease-free survival period was reported. In this paper, we evaluated the expression levels of HSP27 in the cell lines of human breast and other tumors. Western blotting of HSP27 has shown the correlation of HSP27 expression and metastasis and tumorigenicity of mammary and prostate tumors. Thus the biological significance of HSP27 in aggressive mammary and prostate tumors was indicated. HSP27 is suggested to be a prognostic marker for the malignancy of mammary and prostate tumors.

Specific expression of HSP47 in human tumor cell lines in vitro.

HSP47 is a stress-inducible glycoprotein of 47 kd molecular weight, and is assumed to be a collagen specific molecular chaperone. The expression of HSP47 is closely related to that of collagen, which is the main component of the extracellular matrix. We investigated the expression level of HSP47 in human tumor cell lines to elucidate collagen biosynthesis in tumor cells. Western blot analysis showed higher levels of HSP47 in solid tumor cell lines than in leukemia cell lines. Tumor cell lines which were derived from metastatic carcinomas and were still metastatic in animals, synthesized higher levels of HSP47. These results suggested that HSP47 may play an important role in tumor metastasis and can be a prognostic marker for the metastatic activity of human tumor cells.

Decreased expression of HSP47 in highly malignant mouse fibrosarcoma.

HSP47, a heat shock-inducible glycoprotein of M(r) = 47,000, is assumed to be a molecular chaperone specific for collagen. In addition to its stress inducibility, HSP47 has transformation-sensitivity; that is, the synthesis of HSP47 decreases after malignant transformation. The expression of several HSPs in mouse fibrosarcoma, QRsP-1 and QRsP-4 were examined. HSP47 expression was weaker in the highly malignant QRsP-4 than in the weakly malignant QRsP-1, whilst HSC70 expression was similar in both cell lines. These findings suggested an important role of HSP47 as a marker of tumour malignancy of QRsP cell lines.

Inhibition of HSP47 during the transition from solid to ascitic form of Sarcoma 180 in vivo.

HSP47, a collagen-binding stress protein, has transformation-sensitivity; that is, the synthesis of HSP47 decreases after malignant transformation. We examined the expression of HSP47 in transplanted tumours by Western blotting. While HSP47 was not detected in ascites tumours including Ehrlich, P388 and Sarcoma 180, marked expression of HSP47 was observed in solid tumours such as B16, B16-BL6 and Sarcoma 180. The expression of HSP47 in Sarcoma 180 cells disappeared completely after these solid tumour cells were dispersed and inoculated intraperitoneally. These findings suggested an important role of HSP47 as a marker for tumour malignancy.

Marked induction of HSP47, a collagen-binding stress protein, during solid tumor formation of ascitic Sarcoma 180 in vivo.

HSP47, a heat shock-inducible glycoprotein of Mr = 47,000, is assumed to be a molecular chaperone specific for collagen. In addition to its stress inducibility, HSP47 has transformation-sensitivity; that is, the synthesis of HSP47 decreases after malignant transformation. We developed a rat monoclonal antibody against mouse HSP47, which we then used to examine the amounts of HSP47 in transplanted tumors. HSC70 (HSP73) was expressed constitutively in these ascites as well as solid tumors. While HSP47 was not expressed in ascitic form of Sarcoma 180, its expression was markedly induced during solid tumor formation of ascitic Sarcoma 180 cells after subcutaneous transplantation. The average survival period of ascites Sarcoma 180-bearing mice was 20.3 +/- 3.6 days (mean +/- SD), and was much shorter than that of solid Sarcoma 180-bearing mice. These findings suggest an important role of HSP47 in solid tumor formation as well as a possible marker for tumor malignancy.

HSP47 as a possible marker for malignancy of tumors in vivo.

We have developed a rat monoclonal antibody against mouse HSP47 (designated as NK47) and investigated the expression of HSP47 in several mouse ascites and solid tumors. The expression of HSP47 was not detected in ascites tumors such as P388 and Ehrlich ascites tumors while HSP47 was markedly expressed in solid tumors such as Colon-26, B16 and B16-BL6 melanomas. HSP70, HSC70 and HSP90 were expressed in almost the same level among all the tumors examined. These results suggest that the expression of HSP47 decreases as the tumor becomes more malignant.

Enhancement of the antitumor effect by the concurrent use of a monoclonal antibody and the protein-bound polysaccharide PSK in mice bearing a human cancer cell line.

The antitumor effects of a monoclonal antibody against a human cancer cell line and a protein-bound polysaccharide, PSK, obtained from cultured mycelia of Coriolus versicolor in basidiomycetes were examined. The IgG2a monoclonal antibody against the human colon cancer cell line colo 205 induced in vitro antibody-dependent macrophage-mediated cytotoxicity against the cancer cells, but only slightly suppressed the in vivo growth of the cancer cells. Concurrent use of PSK with the antibody enhanced the in vitro antibody-dependent macrophage-mediated cytotoxicity as well as the in vivo antitumor activity. These findings suggest that the combined use of a monoclonal antibody and PSK, which have different modes of action, may be useful in the treatment of cancer.

Effect of a biological response modifier, PSK, on intestinal flora of tumor-bearing mice.

PSK, a protein-bound polysaccharide derived from basidiomycetes, is a biological response modifier that exhibits a variety of activities following oral administration, including prevention of infection. In this study, the effects of oral administration of this drug on microbial flora of tumor-bearing mice were examined. Numbers of Staphylococcus, Streptococcus and Pseudomonas were increased, and those of Bifidobacterium and Lactobacillus were reduced in fresh feces of mice from later than 9 weeks after inoculation of sarcoma 180. However, these changes were prevented by oral administration of PSK. In mice bearing sarcoma 180, the increases in numbers of Staphylococcus and Pseudomonas and the decrease in those of Bifidobacterium were further enhanced by intraperitoneal administration of anticancer agent mitomycin C, but such changes were suppressed by oral administration of PSK. These results suggest that PSK has a preventive effect against abnormal conditions of the intestinal flora induced by tumor inoculation or the administration of anticancer agents.

Effects of biological response modifiers with different modes of action used separately and together on immune responses in mice with syngeneic tumours.

The effect of a protein-bound polysaccharide (PSK) obtained from cultured mycelia of the Basidiomycetes Coriolus versicolor on activities involved in the host defence mechanism of C57BL/6 mice bearing adenocarcinoma 755 was compared with that of live bacille Calmette-Guérin (BCG). Delayed footpad reaction, the activity of splenic natural killer cells and interferon production induced by concanavalin A in splenic cells of healthy mice were little affected by PSK, but in mice bearing tumours PSK prevented the tumour-induced reduction in these activities. Live BCG augmented these activities in healthy mice but had little effect on the reduction of activities induced by a tumour. The immunosuppressive activity of the serum of tumour-bearing mice was reduced by PSK administration; live BCG did not have this effect. The combined use of live BCG and PSK improved these activities in the host, with synergistic increases in the antitumour effect. These results suggest that the combined use of live BCG and PSK, which have different modes of action, may be useful in the treatment of cancer.

Enhancement of effector cell activities in mice bearing syngeneic plasmacytoma X5563 by a biological response modifier, PSK.

We investigated the effect of PSK, a protein-bound polysaccharide obtained from Coriolus versicolor of basidiomycetes, on antitumor immunity in tumor-bearing mice. PSK prolonged significantly the life span of C3H/He mice bearing syngeneic plasmacytoma X5563 in a schedule- and dose-dependent manner. PSK was most effective when administered at 100 mg/kg every other day ten times starting from the day after tumor inoculation. The administration of PSK enhanced significantly the cytostatic activity of peritoneal exudate plastic-adherent cells and the cytolytic activity of spleen cells after in vitro incubation with mitomycin C-treated tumor cells. In addition, PSK restored the cytokine-producing capacity of spleen cells suppressed in tumor-bearing mice after in vitro incubation with mitogen. Sera from tumor-bearing mice suppressed the activity of such effector cells as well as the interleukin 2-producing capacity of spleen cells, but sera from PSK-treated tumor-bearing mice prevented this suppression. These results suggest that PSK enhances antitumor immunity by reducing immunosuppressive activity of serum from tumor-bearing mice.

Competitive action of a biological response modifier, PSK, on a humoral immunosuppressive factor produced in tumor-bearing hosts.

We investigated the effect of PSK, a protein-bound polysaccharide obtained from the basidiomycetes Coriolus versicolor, on an immunosuppressive factor produced in tumor-bearing animals. Oral administration of PSK suppressed the growth of the tumor in C3H/He mice bearing X5563 plasmacytoma or MH134 hepatoma, but affected mice bearing MM102 mammary tumor little. PSK prevented the reduction in splenic lymphocyte blastogenesis caused by phytohemagglutinin that occurs in mice bearing X5563 tumors or MH134 hepatoma. The lymphocyte blastogenesis affected little by tumor or PSK in mice bearing MM102 tumors. The effect of sera on the blastogenesis of lymphocytes caused by phytohemagglutinin was different with different tumors in the C3H/He mice. Serum of mice bearing X5563 tumors inhibited blastogenesis, but serum of mice bearing MH134 hepatoma or MM102 tumors promoted it. The sera of mice bearing MH134 hepatoma contained both inhibitory and promotive factors; those of mice bearing X5563 tumors contained an inhibitory factor, and those of mice bearing MM102 tumors contained a promotive factor. The oral administration of PSK reduced the inhibition caused by the sera of mice bearing X5563 tumors. The promotive activity of sera from mice bearing MH134 hepatoma was augmented by PSK; that of sera in mice bearing MM102 tumors was not affected by PSK. Living Bacillus Calmette-Guérin did not have such effects in any of these mice. Serum immunosuppressive activity was also reduced by PSK in various tumor lines of rodents. These results suggest that PSK acts by reducing the activity of immunosuppressive factors produced in tumor-bearing hosts.

Antitumor effects of 3-[p-(N,N-bis-(2'-chloroethyl)amino)-phenyl]-L- alanine conjugated with human immunoglobulin (K18).

K18 (3-[p-(N,N-bis(2'-chloroethyl)amino)- phenyl]-L-alanine conjugated with human immunoglobulin) is a newly developed antitumor agent. LD50 values of K18 in animals were quite high, suggesting its low acute toxicity. This drug showed anti-tumorigenicity not only on an experimental animal tumor (Walker 256), but also on a human tumor transplantable into nude mice (RCC-13). A distribution study clarified the unique properties of K18 to accumulate and remain in the tumor site with a high rate.

Treatment with Krestin combined with mitomycin C, and effect on immune response.

The combination effects of Krestin (PSK) and mitomycin C (MMC) were examined in experimental tumor models. PSK was administered either orally or intraperitoneally. Delayed-type footpad reaction and antibody formation against sheep erythrocytes were measured in hosts of which immune functions were depressed by tumor burden. Results of the experiment indicated that the simultaneous administration of PSK and MMC significantly increased the survival rate of tumor-bearing mice and restored more effectively their immune functions compared to those of nontreated tumor-bearing controls or tumor-bearing hosts treated with a single agent.

Tumor growth-promoting effect of immunosuppressive substance in mice.

The effect of immunosuppressive (IS) substance obtained from cancerous ascitic fluid on tumor growth and host immunity in plasmacytoma X5563-bearing C3H/He mice is described. IS substance given in three injections, before and after tumor inoculation caused: (a) enhanced tumor growth, (b) marked reduction in survival times, (c) inhibition on Con-A response of spleen cells. Depressed natural killer (NK) activity was observed in normal and tumor-bearing mice treated with IS substance. The data presented here suggest that IS substance suppresses both humoral and cellular immunoresponsiveness and tumor cells evade immune surveillance or immunologically mediated removal.

Tumor and tissue distribution of 125I-labeled natural and antitumor antibodies in murine experimental tumor systems.

The binding and tissue distribution of 125I-labeled rabbit anti-sarcoma 180 (S-180) immunoglobulin G (IgG) (RAS-180G), normal rabbit IgG (NRG) and ICR mouse IgGs from normal (NMG) and S-180-bearing ICR mice (AS-180G) were studied in ICR mice bearing S-180. 125I-labelled IgGs preparted from normal (NC57G) or Adenocarconoma 755 (Ca 755)-bearing C57BL/6 mice (A755G) were also examined in C57BL/6 mice bearing ca755. Not only RAS-180G and AS-180G but also NRG and NMG persisted in the tumor site. This result suggests that allogeneic natural IgG could be used as a carrier protein for antitumor agents.

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.

Comparative study of alpha 1-acid glycoprotein molecular variants in ascitic fluid of cancer and non-cancer patients.

alpha 1-Acid glycoproteins (alpha 1-AG) were collected from the ascites of patients with liver cirrhosis or liver cancer, respectively, and their physical, chemical, and biological properties were compared. The substance obtained from patients with liver cancer showed a 2-3 times higher inhibitory effect on [3H]thymidine uptake by human peripheral lymphocytes stimulated with PHA than that obtained from patients with cirrhosis. The two substances showed differences in their affinity to wheat germ agglutinin (WGA) and concanavalin A (Con A). Of the fractions obtained by lectin affinity chromatography, the Con-A bound fraction showed the greatest lymphocyte proliferation inhibitory activity. The alpha 1-AG levels were elevated in both the patients with cancer and those with infectious disease, but the level of the Con-A bound fraction was elevated only in those with cancer. This study suggests that the molecular variants of alpha 1-AG differ in their carbohydrate structure with the disease, and that the cellular immunity of the host way be partially controlled by changes in the content of these molecular variants.

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 b5 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.