Bibliothek

Notizen: Abstract Purpose: The overall purpose of this study was to determine the potential efficacy of epoxide-containing piperazines as a new class of anti-cancer agents. Two representative compounds, specifically NCO-700, a 4-trimethoxyphenyl-substituted epoxide-piperazine, and TOP-008, a 4-phenylpropenyl-substituted epoxide-piperazine were tested in cytotoxic assays with human breast and prostate cancer cell lines. A second objective was to determine if these two compounds had anti-cancer activity in vivo when tested against xenograft tumors in nude mice or human tumors grown under the kidney capsule in mice. A final objective of this study was to establish if NCO-700 and TOP-008 achieved cancer cell killing through an apoptotic mechanism. Methods: The anti-proliferative activity of NCO-700 and TOP-008 were tested in a 7 day cell-survival assay utilizing a number of well characterized breast (HS-578T, T47D, MCF-7) and prostate (DU-145, PC-3, LNCaP) cancer cell lines. In vivo studies with the two compounds were performed, in nude mice bearing DU-145 xenograft tumors, and in normal mice in which DU-145 prostate cancer cells and HS-578T breast cancer cells were grown as solid tumors in the subrenal capsules of the animals. Apoptotic cell death of cancer cells was determined by a number of established techniques that detect apoptosis, including the confocal laser microscopy of treated cells and mitochondrial leakage assays utilizing the cationic dye, JC-1. Finally, the activation of the caspase cascade, enzymes that carry out apoptosis in mammalian cells, was examined in treated cells by immunoblot assays. Results: NCO-700 and TOP-008 displayed cytotoxicity to HS-578T human breast cancer cells, with ED50 values in the 3–6 μM range. Cytotoxicity to androgen receptor-negative human prostate cancer cells (PC-3 and DU-145 cells) occurred with ED50 values in the 5–20 μM range. Cytotoxicity to hormone receptor-positive breast and prostate cancer cell lines occurred at 10 to 20-fold higher concentrations of the two compounds. When human prostate (DU-145) or breast cancer (HS-578T) cells were grown as solid tumors in the subrenal capsules of mice, significant anti-tumor activity of NCO-700 was observed at 20 mg/kg and 50 mg/kg body weight respectively, for prostate and breast tumors. In nude mice bearing DU-145 prostate tumor xenografts, 50 mg/kg doses of the two compounds either stopped (TOP-008) tumor growth or slowed (NCO-700) growth. The mechanism of cytotoxicity was shown to be through apoptosis, (a) by confocal microscopy studies revealing nuclear fragmentation, (b) by mitochondrial studies revealing disruption of the mitochondrial membrane and release of the cationic dye, JC-1, into the cytoplasm and (c) by protein immunoblot assays indicating that over a 6 h period, TOP-008 induced a significant accumulation of the pro-apoptotic protein, bak, in the mitochondrial fraction of HS-578T human breast cancer cells, accompanied by activation, at 2.5 h, of caspase-3. Conclusions: These studies indicated that the epoxide-containing piperazines, as exemplified by NCO-700 and TOP-008, were effective anti-cancer agents when tested in vitro and in vivo against human breast and prostate tumors. Our studies also indicated that TOP-008 induced the initiation of the caspase cascade leading to apoptosis. Previous toxicology studies in rodents and dogs, as well as a Phase I study in humans, showed NCO-700 to be a well-tolerated, non-toxic compound. Taken together with our current findings, these results suggest that this class of compounds has the potential to be relatively safe, new chemotherapeutic agents for refractory breast and prostate cancers.

Notizen: Abstract.  Three non-allelic radial swelling mutants (rsw1, rsw2 and rsw3) of Arabidopsisthaliana L. Heynh. were shown to be specifically impaired in cellulose production. Fractionation methods that identify, characterise and quantify some of the major cell wall polysaccharides in small quantities of seedlings demonstrated that changes in the production of cellulose are much more pronounced than changes in the production of non-cellulosic polysaccharides. A crude cell wall pellet was sequentially extracted with chloroform methanol (to recover lipids), dimethyl sulphoxide (starch), ammonium oxalate (pectins) and alkali (hemicelluloses). Crystalline cellulose remained insoluble through subsequent treatments with an acetic/nitric acid mixture and with trifluoroacetic acid. Cetyltrimethylammonium bromide precipitation resolved neutral and acidic polymers in the fractions, and precipitation behaviour, monosaccharide composition and glycosidic linkage patterns identified the major polysaccharides. The deduced composition of the walls of wild-type seedlings and the structure and solubility properties of the major polymers were broadly typical of other dicots. The three temperature-sensitive, radial swelling mutants produced less cellulose in their roots than the wild type when grown at their restrictive temperature (31 °C). There were no significant differences at 21 °C where no radial swelling occurs. The limited changes seen in the monosaccharide compositions, glycosidic linkage patterns and quantities of non-cellulosic polysaccharides support the view that the RSW1, RSW2 and RSW3 genes are specifically involved in cellulose synthesis. Reduced deposition of cellulose was accompanied by increased accumulation of starch.