Pro-apoptotic activity of ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione in human prostate cancer cells.

With the aim of identifying novel agents with antigrowth and pro-apoptotic activity on prostate cancer cells, we assayed the effect of ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione, a semisynthetic compound, against androgen-sensitive (LNCaP) and androgen-insensitive (DU-145) human prostate cancer cells. Our results indicate that after 72h of incubation, ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione at micromolar concentrations exhibited an inhibitory effect on LNCaP and DU-145 cell growth (MTT assay), but the semisynthetic compound was the most active. In addition, our results indicate that apoptotic cell demise is induced in LNCaP and DU-145 cells. In fact, a significant increase of caspase-3 activity, not correlated to LDH release, marker of membrane breakdown, was observed in both cell lines treated with ergosterol peroxide and the semisynthetic compound. With respect to genomic DNA damage, determined by COMET and TUNEL assays, the results obtained show a significant increase in DNA fragmentation when compared with the untreated control. In conclusion, the results obtained in this study, demonstrating that ergosterol peroxide and (22E)-ergosta-7,22-dien-5alpha-hydroxy-3,6-dione attenuate the growth of prostate cells, at least in part, triggering an apoptotic process, permit to confirm the use of mushrooms as origin of compounds to be used as novel therapeutic agents for prostate cancer treatment, or as models for molecules more active and selective.

Demalonyl thyrsiflorin A, a semisynthetic labdane-derived diterpenoid, induces apoptosis and necrosis in human epithelial cancer cells.

In a previous study, we isolated thyrsiflorin A, a new diterpene with the scopadulane skeleton, from Calceolaria thyrsiflora (Scrophulariaceae family). Experimental evidences on the semisynthetic analogues of scopadulane diterpenes have permitted to hypothesize that a polar substituent is important for the antitumor activity of this class of compounds. Therefore, the present study was undertaken to investigate the effect of the semisynthetic compound, demalonyl thyrsiflorin A, on cell growth and death in two human epithelial cell lines, DU-145 cells (androgen-insensitive prostate cancer cells) and KB cells (oral squamous carcinoma cells). The results obtained, show that our compound, exhibited comparable degrees of antigrowth effect on cancer cells examined as judged by IC(50) values, 9.77 microM (2.73 microg/ml) and 10.86 microM (3.04 microg/ml) in DU-145 and KB cells, respectively, and support the hypothesis that also for diterpenoid compounds an available hydroxyl group is important for decreased cancer cell viability. In addition, we demonstrated an apoptotic response after treatment of DU-145 and KB cells with this semisynthetic compound at 6-12 microM concentrations, together with a necrosis process at higher doses (25-50 microM). Both apoptotic and necrotic pathway implicated in demalonyl thyrsiflorin A-treated cells are correlated with the elevation of ROS generation.

Diterpenoids from Calceolaria alba.

Chemical examination of the petrol ether (60-80) extract of the aerial parts of Calceolaria alba R. et Pav., collected in Santa Juana, VIII Region, Chile, resulted in the isolation of 3 new diterpenoids. Their structures have been elucidated by a study of their physical and spectral data; in particular using 2 NMR spectroscopy (DEPT, 1H-1H, COSY, NOESY, HMQC and HMBC).

Putrescine-1,4-dicinnamide from Pholiota spumosa (Basidiomycetes) inhibits cell growth of human prostate cancer cells.

Previously, it was isolated from the fruiting bodies of the gilled mushroom Pholiota spumosa (Basidiomycetes, Strophariaceae), putrescine-1,4-dicinnamide, a phenylpropanoid derivative conjugated with polyamine putrescine never isolated before as a natural compound. Recently, polyamine analogs that are similar in structure to the natural polyamines but that cannot mimic their functions that are essential for cellular growth and differentiation, have shown antitumor activity in several types of human cancer cells. Therefore, we have now investigated the response of DU-145 cells, a well characterized androgen-independent human prostate cancer (PCA) cell line, to this phenylpropanoid derivative. The results presented here demonstrate that putrescine-1,4-dicinnamide, as suggested for polyamine analogs synthesized artificially, inhibits the cell growth of cancer cells inducing apoptosis cell death, mediated, at least in part, by the activation of caspase cascades, that at higher doses shift to necrosis, through the increase of reactive oxygen species (ROS) generation.

Propolis protects human spermatozoa from DNA damage caused by benzo[a]pyrene and exogenous reactive oxygen species.

Many environmental, physiological and genetic factors have been implicated in defective sperm function, the most common cause of infertility. In addition, sperm preparation techniques such as centrifugation, used prior to in vitro fertilization, are associated with the generation of reactive oxygen species (ROS) and an increase in the level of DNA damage. Factors that can offer spermatozoa protection are, therefore, of great importance. This study was designed to examine in vitro the effect of a Chilean propolis ethanolic extract on human spermatozoa treated with benzo[a]pyrene and exogenous reactive oxygen species. Our experimental evidence demonstrated that the natural drug under investigation is able to protect genomic DNA by damage induced by benzo[a]pyrene, hydrogen peroxide (H2O2) and hydrogen peroxide in combination with adenosine 5'-diphosphate (ADP) and ferrous sulfate (FeSO4), determining a significant reduction of the intracellular oxidants. An increase in membrane damage, measured by monitoring the formation of thiobarbituric acid-reactive substances (TBARS) and lactic dehydrogenase (LDH) release, was observed only in sperm treated with H2O2, ADP and FeSO4. The propolis extract was shown to possess the capacity to protect sperm membrane from the deleterious action of oxidative attack, reducing TBARS formation and LDH release. In summary, our results evidence that the protective effect exhibited by this natural compound in human spermatozoa is correlated, at least in part, to the antioxidant capacity of its active components, and suggest that propolis may have a role in protection against male infertility.

Chilean propolis: antioxidant activity and antiproliferative action in human tumor cell lines.

Propolis, a natural product derived from plant resins collected by honeybees, has been used for thousands of years in traditional medicine all over the world. The composition of the propolis depends upon the vegetation of the area from where it was collected and on the bee species. In this study, we investigated the antioxidant activity of a propolis sample, provided by NATURANDES-CHILE, collected in a temperate region of central Chile. In addition, this natural compound was tested for its antiproliferative capacity on KB (human mouth epidermoid carcinoma cells), Caco-2 (colon adenocarcinoma cells) and DU-145 (androgen-insensitive prostate cancer cells) human tumor cell lines. Results showed that this Chilean propolis sample exhibits interesting biological properties, correlated with its chemical composition and expressed by its capacity to scavenge free radicals and to inhibit tumor cell growth.

Cytotoxic activity of the root extract from Myoschilos oblongum.

The crude methanolic root extract of Myoschilos oblongum exhibited a significant cytotoxic activity against PZ-HPV-7 human prostate cells. Furthermore, two esters of docosanol were isolated from the CH(2)Cl(2) extract.

The biotransformation of 18-hydroxy-9-epi-ent-pimara-7,15-diene by Gibberella fujikuroi.

Incubation of 18-hydroxy-9-epi-ent-pimara-7,15-diene with the fungus Gibberella fujikuroi gave the compounds 18-hydroxy-7 alpha,8 alpha-epoxy-9-epi-ent-pimara-15-ene, 18-hydroxy-7-oxo-ent-pimara-15-ene, 6 beta, 18-dihydroxy-7 alpha, 8 alpha-epoxy-9-epi-ent-pimara-15-ene, 9 beta,18-dihydroxy-7 alpha, 8 alpha-epoxy-ent-pimara-15-ene and 6 beta, 14 alpha, 18-trihydroxy-9-epi-ent-pimara-7,15-diene. Oxidation of C-19, which is characteristic of the biosynthesis pathway of the gibberellins is not produced.

CD8+ T cells are the effectors of the contact dermatitis induced by urushiol in mice and are regulated by CD4+ T cells.

BACKGROUND: The exposure of human skin to leaves and branches of litre (Lithraea caustica), a Chilean endemic tree, induces a severe contact dermatitis characterized by swelling and pruritus in susceptible individuals. The allergenic priniciple of litre is 3-pentadecyl (10-enyl) catechol (litreol), which is structurally similar to the allergens isolated from poison oak and poison ivy. All of them belong to a family of compounds named urushiols. As a proelectrophilic allergen, litreol must be intracellularly activated before modifying proteins of individuals exposed to it. As a result, self-peptides derived from litreol-modified intracellular proteins would be presented in the context of class I MHC molecules. We hypothesized that CD8+ T lymphocytes would play a major role during the effector phase of the immune response induced by those modified peptides. In order to test this hypothesis, we investigated the cellular immune response to litreol in Balb/cJ mice. The role of the different lymphocyte subpopulations in this response was assessed by immunodepleting mice of CD4+ or CD8+ T lymphocytes using specific monoclonal antibodies (mAbs). We report the observation that the contact dermatitis induced by litreol has two components: a primary response which does not require TCRalpha beta+ T cells, and a secondary response mediated mainly by CD8+ T cells and regulated by CD4+ T cells. Our results show that CD8+ lymphocytes play a central role as effectors of the secondary response to litreol. Furthermore, our data suggest that two functionally different CD4+ T subpopulations serve as regulators of the CD8+ T cell function: a CD4+ T helper population sensitive to a low dose of the depleting mAb, and CD4+ T suppressor population which is eliminated only with a high dose of depleting mAb.

Modulation of fatty acid oxidation alters contact hypersensitivity to urushiols: role of aliphatic chain beta-oxidation in processing and activation of urushiols.

Lithraea caustica, or litre, a tree of the Anacardiaceae family that is endemic to the central region of Chile, induces a severe contact dermatitis in susceptible human beings. The allergen was previously isolated and characterized as a 3-(pentadecyl-10-enyl) catechol, a molecule belonging to the urushiol group of allergens isolated from poison ivy and poison oak plants. Because urushiols are pro-electrophilic haptens, it is believed that the reactive species are generated intracellularly by skin keratinocytes and Langerhans cells. The active species are presumed to modify self proteins which, after proteolytic processing, would generate immunogenic peptides carrying the hapten. The presence of a 15-carbon-length hydrophobic chain should impair antigen presentation of self-modified peptides by class I MHC molecules, either by steric hindrance or by limiting their sorting to the ER lumen. We have proposed that the shortening of the aliphatic chain by beta-oxidation within peroxisomes and/or mitochondria should be a requirement for the antigen presentation process. To test this hypothesis we investigated the effect of drugs that modify the fatty acid metabolism on urushiol-induced contact dermatitis in mice. Clofibrate, a peroxisomal proliferator in mice, increased the immune response to the urushiols from litre by 50%. Conversely, tetradecyl glycidic acid, an inhibitor of the uptake of fatty acids by mitochondria, decreased the hypersensitivity to the hapten. An increase in the level in glutathione by treatment of the animals with 2-oxotiazolidin-4-carboxilic acid lowered the response. Those findings strongly support a role for the fatty acid oxidative metabolism in the processing and activation of urushiols in vivo.

Effects and mode of action of 1,4-naphthoquinones isolated from Calceolaria sessilis on tumoral cells and Trypanosoma parasites.

The naphthoquinones 2-hydroxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-1), (-)-2,3,3-trimethyl-2-3-dihydronaphtho[2,3-b]furan-4,9-quinone (CS-3), and 2-acetoxy-3-(1,1-dimethylallyl)-1,4-naphthoquinone (CS-5) isolated from Calceolaria sessilis were tested against Trypanosoma cruzi epimastigotes, the TA3 tumor cell line and the methotrexate-resistant subline TA3-MTX-R. Naphthoquinone CS-3 was the most active; the 50% culture growth inhibition (I50) on T. cruzi (Tulahuén and LQ strain and DM28c clone) was at concentrations ranging from 2.1 to 5.2 mumolar. Also CS-3 inhibited TA3 and TA3-MTX-R culture growth with an I50 of 2.1 and 3.8 mumolar, respectively. Naphthoquinone CS-3 inhibited the respiration of the tumor cells by interfering with the electron transport at some point between NADH and ubiquinone. The respiration of T. cruzi was not inhibited by naphthoquinone CS-3. Naphthoquinone CS-3 produced a temporary increase of oxygen consumption in T. cruzi and tumor cells, suggesting the generation and participation of free radicals.

A New Non-Glycosidic Iridoid and a New Bisiridoid from Argylia radiata1.

From ARGYLIA RADIATA a non-glycosidic iridoid, the (4a S, 7 S, 7a S)-7-hydroxy-7-methyl-1,4a,5,6,7,7a-heptahydrocyclopenta[ C]pyran-3(4 H)-one, and a bisiridoid, derived from the junction of catalpol and 7-deoxyloganic acid, were isolated.

Polar Constituents of Calceolaria ascendens1.

A new phenylpropanoid glucoside, 1'- O-beta- D-(3,4-dihydroxy-beta-phenyl)-ethyl-4'- O-caffeoyl-beta- D-apiosyl-(1'''-->3')-glucopyranoside, named calceolarioside E, was isolated from CALCEOLARIA ASCENDENS Lind., together with two other phenylpropanoid glucosides, verbascoside and forsythoside A, and cyclohexanols rengyol, isorengyol, and 4-hydroxy-4-(2'-hydroxyethyl)-cyclohexanone.

Radiatoside B and C, Two New Bisiridoid Glucosides from Argylia radiata1.

Two new bisiridoid glucosides, radiatoside B and C, together with the known compounds mussaenosidic acid and geniposidic acid, were isolated from the aerial parts of ARGYLIA RADIATA. The structures of the new compounds were established by spectroscopic methods.