Structure and antitumor activity of the less-branched derivatives of an alkali-soluble glucan isolated from Omphalia lapidescens. (Studies on fungal polysaccharide. XXXVIII).

The structure and antitumor activity of Smith-type degradation products (OL-2-I, OL-2-II and OL-2-III) of an alkali-soluble glucan, OL-2, isolated from a crude fungal drug "Leiwan" (Omphalia lapidescens) were investigated. Methylation analysis suggested that OL-2-I was a (1----3)-beta-D-glucan with approximately one branch at every three main chain glucosyl units at each C-6 position; OL-2-II was a (1----3)-beta-D-glucan with approximately one branch at twenty four main chain glucosyl units at each C-6 position (number of all main chain glucosyl units is on average). OL-2-I, OL-2-II and OL-2-III which were Smith-type degradation products of OL-2, showed potent antitumor activity against the solid form of sarcoma 180 in ICR mice. These results indicated that the degree of beta-linked branching at position 6 was remarkably related to the antitumor activity.     

Polysaccharides in fungi. XXVI. Two branched (1----3)-beta-D-glucans from hot water extract of Yu ?r

Two water-insoluble glucans, U-3-N ([alpha]D +1.0 degree, 0.5 M sodium hydroxide) and U-3-AP1 ([alpha]D +2.5 degrees, 1 M sodium hydroxide) were isolated from hot-water extract of the fruiting bodies of Y? ?r (Chinese name) (Auricularia sp.). U-3-N and U-3-AP1 were investigated by a combination of chemical and spectroscopic methods. The results indicated that U-3-N (molecular weight, 6.1 x 10(5)) was similar to beta-(1----6)-branched (1----3)-beta-D-glucan (N-5P: molecular weight, 5.6 x 10(5)) isolated from the alkaline extract of the fruiting bodies, and U-3-AP1 (molecular weight, 6.3 x 10(4)) was beta-(1----6)-branched (1----3)-beta-D-glucan containing beta-(1----6)-linked D-glucopyranosyl residues. U-3-N showed potent anti-tumor activity against the solid form of sarcoma 180, although U-3-AP1 had little effect on the tumor.     

Physicochemical characteristics and antitumor activities of a highly branched fungal (1----3)-beta-D-glucan, OL-2, isolated from Omphalia lapidescens.

Physicochemical properties and antitumor activities of a fungal (1----3)-beta-D-glucan, OL-2, isolated from Leiwan (Omphalia lapidescens) were examined. OL-2 showed sharp signals on carbon-13 nuclear magnetic resonance spectrum in dimethylsulfoxide-d6 as a solvent, and these signals were significantly reduced by the addition of distilled water to the concentration of 20%. This phenomenon is consistent with the general property of the gel forming (1----3)-beta-D-glucan. Binding of OL-2 to Congo red induced a significant change of lambda max to a longer wavelength, and the concentration to induce gel to sol transition was about 0.7 N; in contrast, the concentration was about 0.2 N in the cases of SPG and curdlan. These observations suggested that the gel structure would be significantly stabilized in the case of OL-2. OL-2 showed no or low antitumor activity against the solid form of Sarcoma 180 by intraperitoneal and intralesional administrations; however, it was effective on the ascites form of Sarcoma 180. Of interest, OL-2 also showed significant antitumor activity against the ascites form of MH-134 when administered with 5-fluorouracil. These results indicated that OL-2 showed characteristic features regarding its physicochemical properties and antitumor activity.     

Separation of the hot water extracts of sclerotia of Sclerotinia sclerotiorum IFO 9395 into mitogenic and antitumor-active subfractions

The hot water extract of sclerotia of Sclerotinia sclerotiorum IFO 9395 (TSHW) was divided into representative fractions by ammonium sulfate and ethanol precipitations, and (1----3)-beta-D-glucanase treatment. The ammonium sulfate and ethanol precipitations gave a (1----3)-beta-D-glucan fraction (TSG) and a mannan fraction (TSM). After the degradation of (1----3)-beta-D-glucan in TSHW by (1----3)-beta-D-glucanase treatment, a water-insoluble protein fraction (EDP) and supernatant (EDS) were obtained. Among these fractions, the mitogenic and antitumor activities were mainly observed in EDP and TSG, respectively. On the other hand, the stimulatory effect on the reticuloendothelial system was mainly found in EDP and EDS, and a weak effect was observed in TSG. These findings suggest that the mitogenic and antitumor activities of TSHW were mainly due to the protein and (1----3)-beta-D-glucan, respectively, and that the mitogenic substance (EDP) is tightly bound to (1----3)-beta-D-glucan (TSG) in TSHW, accounting for its solubility in aqueous solution.     

Antitumor activity of Hypsizigus marmoreus. I. Antitumor activity of extracts and polysaccharides.

Antitumor activity of Hypsizigus marmoreus, one of the most popular Japanese edible mushrooms, was investigated. The aqueous and methanol extracts were tested against allogeneic tumor, solid sarcoma 180 and syngeneic tumor, Meth A fibrosarcoma. The aqueous extract was highly active in inhibiting growth of solid sarcoma 180, but not as much for Meth A fibrosarcoma. Fractionation of antitumor substances of the aqueous extract isolated four polysaccharides. Chemical analysis revealed one of them to be beta-(1-3)-glucan with a remarkable inhibitory effect against tumor-growth of sarcoma 180.     

Conformation-dependent change in antitumor activity of linear and branched (1----3)-beta-D-glucans on the basis of conformational elucidation by carbon-13 nuclear magnetic resonance spectroscopy.

The antitumor activity of (1----3)-beta-D-glucans was tested in order to clarify its conformation-dependent response together with conformational elucidation by carbon-13 nuclear magnetic resonance (13C-NMR) spectroscopy. It was shown that the following three conformations, single chain, single helix and triple helix, are readily distinguished by the high-resolution solid-state 13C-NMR method. It turned out that preparations of linear (1----3)-beta-D-glucans of a triple helical conformation were ineffective in the inhibition of tumor growth. These linear (1----3)-beta-D-glucans were converted to an effective form in the inhibition of tumor growth when they were lyophilized from dimethyl sulfoxide (DMSO) solutions as a result of a conformational change from the triple helical to the single chain forms. They were not effective, however, when assayed in DMSO solution. In contrast, it was found that a branched (1----3)-beta-D-glucan is effective not only in either saline solutions of the triple helical sample or the lyophilized sample from DMSO, but also in DMSO solution. The aforementioned drastic change in antitumor activity was interpreted in terms of resulting conformational changes as analyzed by the 13C-NMR method.     

Synthesis and antitumor activity of a novel water soluble mitomycin analog; 7-N-[2-[[2-(gamma-L-glutamylamino)ethyl]dithio]ethyl]mitomycin C

Introducing the mercaptoethyl group at the 7-N position of mitomycin C 1 has led to the isolation of 7-N,7'-N'-dithiodiethylenedimitomycin C 2. The compound 2 showed excellent antitumor activity against sarcoma 180 (sc-ip) and leukemia P388 (ip-ip) in mice. As an extension of this study, we synthesized mitomycin dimers with symmetrical disulfide and mitomycin derivatives with unsymmetrical disulfide at the 7-N side chain. Among these compounds, the water soluble conjugate 3 with ethyl gamma-L-glutamyl-L-cysteinylglycinate was far more effective against sarcoma 180 and leukemia P388 than 1. During the subsequent stage of inquiry for the potent congeners of 3, the compound 4 (water solubility: greater than 500 mg/ml), designated as KW2149, with the gamma-L-glutamylcystamino group at the 7th position was finally selected for further evaluation.     

Studies on the biological activity of tocotrienols

Tocotrienols were evaluated for activity against transplantable murine tumors inoculated i.p. into mouse, and the activities of two tocotrienols and alpha-tocopherols were compared. When the compounds were injected i.p., alpha- and gamma-tocotrienols were effective against sarcoma 180, Ehrlich carcinoma, and IMC carcinoma, and gamma-tocotrienol showed a slight life-prolonging effect in mice with Meth A fibrosarcoma, but the tocotrienols had no antitumor activity against P388 leukemia at doses of 5-40 mg/kg/d. On the other hand alpha-tocopherol had only a slight effect against sarcoma 180 and IMC carcinoma. The antitumor activity of gamma-tocotrienol was higher than that of alpha-tocotrienol. Tocotrienols showed growth inhibition of human and mouse tumor cells when the cells were exposed to these agents for 72 h in vitro, whereas tocopherol did not show any marked cytotoxic activity. Alpha- and gamma-tocotrienols had inhibitory effects on lipid peroxidation of murine microsomes by adriamycin.     

Discrimination between endotoxin and (1----3)-beta-D-glucan using turbidimetric kinetic assay with Limulus amebocyte lysate

A procedure for the Limulus amebocyte lysate (LAL) test discriminating between endotoxin and (1----3)-beta-D-glucan based on the turbidimetric kinetic method was proposed. Endotoxin and (1----3)-beta-D-glucan, which are elicitors of the activation of LAL, showed different reaction courses with this lysate. To analyze the difference in the reactions, two parameters, the maximum differential coefficient of the reaction (Dmax) and the reaction time required to obtain Dmax (Tp) were defined. The logarithmic plottings of Tp versus Dmax (Tp-Dmax plot) discriminated between endotoxin and (1----3)-beta-D-glucan. Endotoxin was measured with a standard curve plotting logarithmic endotoxin concentration versus Dmax (ET-Dmax plot). The endotoxin calculated from Dmax was less influenced by (1----3)-beta-D-glucan than that calculated from the usual gelation time. A small amount of endotoxin in a sample could be concealed by the addition of polymyxin B, which inhibited the activation of LAL by endotoxin. (1----3)-beta-D-glucan was measured without being affected by the presence of a small amount of endotoxin using LAL with polymyxin B. The following procedure is proposed as a LAL test to discriminate between endotoxin and (1----3)-beta-D-glucan. (1) Identify the main substance (endotoxin or (1----3)-beta-D-glucan) triggering the activation of LAL using the Tp-Dmax plot. (2) Use the appropriate method to measure the main substance: the ET-Dmax plot for endotoxin or the LAL with polymyxin B for (1----3)-beta-D-glucan.     

Synthesis and antitumor activity of new amphiphilic alkylglycerolipids substituted with a polar head group, 2-(2-trimethylammonioethoxy)ethyl or a congeneric oligo(ethyleneoxy)ethyl group

A new series of amphiphilic 1-octadecyl glycerolipids (eleven compounds, 1a-k) were designed and synthesized, in which the 3-phosphocholine portion of platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) was replaced by the 2-(2-trimethylammonioethoxy)ethyl group and congeneric groups having oligo(ethyleneoxy)ethyl bridges of various lengths at position 3, together with modification at position 2 (lower alkyl, acetonyl, acetoacetyl, carboxymethyl and pyrimidin-2-yl groups). These ether lipids, characterized by a nonphosphorus lysoglycerolipid structure, showed potent antitumor activity in vitro (human promyelocytic leukemia cells, HL-60, and human epidermoid carcinoma cells, KB) and in vivo (mouse sarcoma S180 and mouse mammary carcinoma MM46). Maximal in vitro potency was obtained with 1-O-octadecyl-2-O-(2-pyrimidinyl)-3-O-[2-(2-trimethylammonioethoxy )ethyl] glycerol (1g; IC50 values for both HL-60 and KB were 0.32 microgram/ml, indicating a higher activity than alkyl-lysophospholipid, ET18-OMe). Several appropriately 2-substituted 1-octadecylglycerolipids with the 3-[2-(2-trimethylammonioethoxy)ethyl] group (e.g., methyl, 1b; butyl, 1f; 2,2,2-trifluoroethyl, 1j; and acetonyl, 1k) showed a potent life-span-prolonging effect on mice with ascites sarcoma S180 and on those with mammary carcinoma MM46, when administered intraperitoneally at 16.5 and 12.5 mg/kg/d, respectively. Compounds 1b and 1k showed definite tumor growth inhibition against solid sarcoma S180 in mice, whether given p.o. or i.v. at 16.5 mg/kg/d. Studies on the structure-activity relationships indicate that the metabolic stability to phospholipase C or related enzymes is at least partly responsible for the potent antitumor activity of this series of ether lipids.     

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.     

Preparation and antitumor activity of hydroxyethylated derivatives of 6-branched (1----3)-beta-D-glucan, SSG, obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395

SSG is an antitumor branched (1----3)-beta-D-glucan obtained from the culture filtrate of Sclerotinia sclerotiorum IFO 9395. Hydroxyethylation of SSG higher than MS 0.45 (MS value represents molar ratio of hydroxyethyl group vs. glucosyl group) by ethyleneoxide in aqueous sodium hydroxide lose the antitumor activity. Degradation of branching point of hydroxyethylated SSG (HE-SSG) by the sequential treatments of periodate oxidation, borohydride reduction, and mild acid hydrolysis of these derivatives regenerated the antitumor activity. These results directly demonstrated that the branching point covered, at least a part of, the dormant active site of SSG.     

Tissue distribution and in vivo photosensitizing activity of 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester

Photosensitizers 13,15-[N-(3-hydroxypropyl)]cycloimide chlorin p6 (HPC) and 13,15-(N-methoxy)cycloimide chlorin p6 methyl ester (MMC) absorb at 711 nm and possess high photoinduced cytotoxicity in vitro. Here we report, that photodynamic therapy with HPC and MMC provide considerable antitumor effect in mice bearing subcutaneous P338 lymphoma. The highest antitumor effect was achieved at a dose of 4 micromol/kg when 1.5 h delay between dye injection and light irradiation (drug-light interval) was used. According to the confocal spectral imaging studies of tissue sections this drug-light interval corresponds to a maximum of tumor accumulation of MMC and HPC (tumor to skin accumulation ratio is 8-10). Short (15 min) drug-light interval can be used for efficient vasculature-targeted photodynamic therapy with HPC at a dose of 1 micromol/kg, whereas MMC is ineffective at the short drug-light interval. Relationships between the features of tissue distribution and efficacy of photodynamic therapy at different drug-light intervals are discussed for HPC and MMC.     

Synthesis and antitumor activity of 6-O-carboxymethyl chitin fixing 5-fluorouracils through pentamethylene, monomethylene spacer groups via amide, ester bonds.

In order to provide the water-soluble and biodegradable macromolecular prodrug of 5-fluorouracil (5FU), the fixation of 5FUs to 6-O-carboxymethyl chitin(CM-chitin) through pentamethylene, monomethylene spacer groups via amide, ester bonds was carried out. The obtained CM-chitin/5FU conjugate showed the slow release of 5FU and exhibited remarkable antitumor activity against P388 lymphocytic leukemia in mice by intraperitoneal(i.p.) implantation/i.p. injection.     

枸橼酸锗抗肿瘤作用的实验研究

观察了有机锗化合物枸橼酸锗对小鼠移植性肿瘤Ｓ１８０、Ｈ２２和Ｐ３３８的抗肿瘤作用。结果表明，枸橼酸锗０．２、０．４ｇ／ｋｇ体重灌胃给药，对小鼠Ｓ１８０的平均抑瘤率为４７．６２％和５２．３８％；对小鼠Ｈ２２的平均抑瘤率在４６．１５％和５２．２０％；对Ｐ３８８小鼠生命延长率为２４．４２％和３０．１８％，均优于对照组。     

Synthesis and antitumor activity of new alkylphospholipids containing modifications of the phosphocholine moiety

New antitumor alkylglycerophospholipids, in which primarily the phosphocholine moiety of the platelet activating factor (PAF) molecule was modified, were synthesized from 1-alkyl-2-substituted glycerols by introducing polar head phosphoryl groups having methylene bridges of various lengths (from 2 to 14 carbons). They were tested for PAF agonistic activity and antitumor properties. In a series of 1-octadecyl-2-acetoacetylglycerophospholipids (1a-f), an increase in the length of the methylene bridge separating the phosphate and trimethylammonio group in the polar head side chain at position 3 of the glycerol backbone resulted in a progressive decrease in PAF agonistic activity and a characteristic change in antitumor activity against human promyelocytic leukemia cells (HL-60). Maximal potency was obtained with the compound having a decamethylene bridge (1e, IC50 value = 1.5 microgram/ml). Thus, alkylphospholipids possessing a decamethylene bridge and a variety of substituents at position 2 (1g-n) were synthesized. They showed potent inhibitory activity with IC50 values ranging from 0.4 to 1.9 micrograms/ml, depending on the nature of the 2-substituent in the phospholipid molecule. In in vivo tests of the present series of alkylglycerophospholipids (1a--n), using mice bearing sarcoma 180 and mice with mammary carcinoma MM46 (both cells and compounds were given i.p.), 1-octadecyl-2-acetoacetyl-3-glyceryl omega-trimethylammoniodecyl phosphate (1e) showed the most potent life-prolonging effect. The structure-activity relationships are discussed.     

Solubilization of limulus test reactive material(s) from Candida cells by murine phagocytes.

Solubilization of limulus test reactive materials from Candida was examined in the presence or absence of phagocytic cells. Solubilized limulus test reactive materials (LTRM) were detected in culture supernatant, and hot water and sodium hydroxide extracts of the acetone dried cells of Candida parapsilosis. Suspensions of Candida cells also reacted with limulus test, and LTRM were released from the acetone dried cells by serum treatment. After treatment of the acetone dried cells with polymorphonuclear leucocytes (PMN) or macrophages (M phi), a significant amount of LTRM was solubilized. Significant amounts of LTRM were also released by PMN during treatment of live and growing C. parapsilosis. The reactivity of LTRM was completely inhibited by the addition of excess amount of purified (1----3)-beta-D-glucan, suggesting LTRM from Candida cells as described above would contain (1----3)-beta-D-glucan. These results suggested that LTRM during fungal infection would come from the extracellular water soluble polysaccharide fraction as well as the insoluble cell wall fraction solubilized by the action of phagocytes.     

Inhibition of experimental pulmonary metastasis of Lewis lung carcinoma by orally administered beta-glucan in mice

The inhibitory effect on experimental pulmonary metastasis of Lewis lung carcinoma (3LL) of SSG, a (1----3)-beta-D-glucan obtained from the fungus Sclerotinia sclerotiorum IFO 9395, administered orally was examined in mice. Oral administration of SSG for 10 consecutive days just after the intravenous implantation of tumor cells significantly inhibited the experimental pulmonary metastasis of 3LL at a dose of 2000 micrograms. However, SSG administered orally involving other timings was less effective. In comparison with oral administration, SSG was effective when administered intraperitoneally for 10 consecutive days at a dose of 200 micrograms. These results suggest that SSG given by both parenteral and nonparenteral routes is effective in the inhibition of experimental pulmonary metastasis of tumors.     

Polysaccharides in fungi. XXX. Antitumor and immunomodulating activities of two polysaccharides from the fruiting bodies of Armillariella tabescens.

The effects of two polysaccharides, AT-HW and AT-AL obtained from the fruiting bodies of Armillariella tabescens on murine sarcoma 180 tumor and peritoneal macrophages were examined at intraperitoneal administration. AT-HW from the hot-water extract and AT-AL from the alkaline extract significantly inhibited the tumor, and the results of different administration schedule and phagocytic system blockade suggested that the mechanism of AT-AL differed from that of AT-HW and branched (1----3)-beta-D-glucans. AT-HW and AT-AL showed reticuloendothelial system-potentiating activity, increased the number of peritoneal exudate cells, activated on macrophages (acid phosphatase activity, glucose consumption, superoxide anion production), and enhanced mitogenic reaction, although AT-HW did not produce superoxide anion in vitro.     

Antitumour Activity of Chitosan Hydrogen Selenites

Selenium is a biologically important element and essential for life1. Selenium deficiency causes severe degenerative disease, such as Kesan cardiomyopathy, and has been implicated in increasing the incidence of human cancer and some chronic ailment as well as ageing. Administration of selenium compounds such as sodium selenite protects animals against carcinogens and markedly reduces the incidence of various neoplasms in human2. Chitosan is one of the most abundant glycans in nature and its…