Tropane alkaloids from the stem bark of Erythroxylum bezerrae.

Six previously undescribed tropane alkaloids, designated as erythrobezerrines A-F, were isolated from the EtOH extract from the stem bark of Erythroxylum bezerrae Plowman. Their structures were elucidated based on the interpretation of the NMR and MS data and in some instances, confirmed by X-ray diffraction analysis. The cytotoxicity of the isolated compounds was evaluated against the cancer cell lines L929, PC-3, HCT-116, SNB-19 and NCI-H460, but only erythrobezerrine C showed moderate activity with IC50 values of 3.38 and 5.43 µM for HCT-116 and NCI-H460, respectively.

Effect of EGF on expression and localization of maturation-promoting factor, mitogen-activated protein kinase, p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes.

This study aimed to investigate the localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus-oocyte complexes. Cumulus-oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.

New flavone and other compounds from Tephrosia egregia: assessing the cytotoxic effect on human tumor cell lines

ABSTRACT The organic extracts from stems, roots and leaves of Tephrosia egregia Sandwith, Fabaceae, provided a new flavone, 5-hydroxy-8-(1",2"-epoxy-3"-hydroxy-3"-methylbutyl)-7-methoxyflavone (1), in addition to eleven known compounds: pongaflavone (2), praecansone B (3), 12a-hydroxyrotenone (4), praecansone A, 2',6'-dimethoxy-4',5'-(2",2"-dimethyl)-pyranochalcone, pongachalcone, maackiain, β-sistosterol and its glucoside, p-cumaric acid and cinnamic acid. The structures of all compounds were established on the basis of spectroscopic methods, mainly 1D and 2D NMR and HRESIMS, involving comparison with literature data. Cytotoxicity of compounds 1-4 was evaluated against AGP-01 (cancerous ascitic fluid), HCT-116 (colon adenocarcinoma), HL-60 (leukemia), PC-3 (prostate carcinoma), SF-295 (glioblastoma) and SKMEL 28 (melanoma) cell lines.

Ritterostatin GN 1N , a Cephalostatin-Ritterazine Bis-steroidal Pyrazine Hybrid, Selectively Targets GRP78.

Natural products discovered by using agnostic approaches, unlike rationally designed leads or those obtained through high-throughput screening, offer the ability to reveal new biological pathways and, hence, serve as an important vehicle to unveil new avenues in drug discovery. The ritterazine-cephalostatin family of natural products displays robust and potent antitumor activities, with sub-nanomolar growth inhibition against multiple cell lines and potent activity in xenograft models. Herein, we used comparative cellular and molecular biological methods to uncover the ritterazine-cephalostatin cytotoxic mode of action (MOA) in human tumor cells. Our findings indicated that, whereas ritterostatin GN 1N , a cephalostatin-ritterazine hybrid, binds to multiple HSP70s, its cellular trafficking confines activity to the endoplasmic reticulum (ER)-based HSP70 isoform, GRP78. This targeting results in activation of the unfolding protein response (UPR) and subsequent apoptotic cell death.

Withanolides from leaves of cultivated Acnistus arborescens.

Seven withanolides, including four previously unknown, were isolated from the acetone and ethanol extracts of cultivated specimens of Acnistus arborescens. These four compounds were identified as rel-(18R,22R)-5ß,6ß:18ß,20-diepoxy-3ß,18α-dimethoxy-4ß-hydroxy-1-oxowith-24-enolide, rel-(20R,22R)-5ß,6ß-epoxy-4ß,16α,20-trihydroxy-1-oxowitha-2,24dienolide, rel-(20R,22R)-16α-acetoxy-6α-chloro-4ß,5ß,20-trihydroxy-1-oxowitha-2,24-dienolide and rel-(20R,22R)-16α-acetoxy-20-hydroxy-1-oxowitha-2,5,24-trienolide. Their structures were elucidated by interpretation of spectroscopic data (1D and 2D NMR), HRESIMS experiments and comparison with published data for similar compounds. Cytotoxicity of the isolated compounds was evaluated against a panel of four tumor cell lines (HL-60, HCT-116, SF-268 and PANC-1). Withanolide D was the most active, with an IC50 value in the range of 0.3-1.7 µM, rel-(18R,22R)-5ß,6ß:18ß,20-diepoxy-3ß,18α-dimethoxy-4ß-hydroxy-1-oxowith-24-enolide and rel-(20R,22R)-5ß,6ß-epoxy-4ß,16α,20-trihydroxy-1-oxowitha-2,24dienolide were moderately active, while all the others were non-cytotoxic.

The Hybrid Pyrroloisoindolone-Dehydropyrrolizine Alkaloid (-)-Chlorizidine†A Targets Proteins within the Glycolytic Pathway.

The cytotoxic activity of (-)-chlorizidine A, a marine alkaloid containing a unique fusion between a pyrroloisoindolone and dehydropyrrolizine, was explored by using a combination of cellular and molecular methods. Our studies began by applying preliminary SAR evidence gathered from semisynthetic bioactivity evaluations to prepare an active immunoaffinity fluorescent (IAF) probe. This probe was then used to identify two cytosolic proteins, GAPDH and hENO1, as the targets of (-)-chlorizidine A.

Fluorescent kapakahines serve as non-toxic probes for live cell Golgi imaging.

AIMS: There is an ongoing need for fluorescent probes that specifically-target select organelles within mammalian cells. This study describes the development of probes for the selective labeling of the Golgi apparatus and offers applications for live cell and fixed cell imaging. MAIN METHODS: The kapakahines, characterized by a common C(3)-N(1') dimeric tryptophan linkage, comprise a unique family of bioactive marine depsipeptide natural products. We describe the uptake and subcellular localization of fluorescently-labeled analogs of kapakahine E. Using confocal microscopy, we identify a rapid and selective localization within the Golgi apparatus. Comparison with commercial Golgi stains indicates a unique localization pattern, which differs from currently available materials, therein offering a new tool to monitor the Golgi in live cells without toxic side effects. KEY FINDINGS: This study identifies a fluorescent analog of kapakahine E that is rapidly uptaken in cells and localizes within the Golgi apparatus. SIGNIFICANCE: The advance of microscopic methods is reliant on the parallel discovery of next generation molecular probes. This study describes the advance of stable and viable probe for staining the Golgi apparatus.

Cytotoxic Plakortides from the Brazilian Marine Sponge Plakortis angulospiculatus.

Three new plakortides, 7,8-dihydroplakortide E (1), 2, and 10, along with known natural products 3, 4, spongosoritin A (5), 6-8, and plakortide P (9), were isolated from Brazilian specimens of Plakortis angulospiculatus. Compounds 2, 3, 5, and 7-9 displayed cytotoxic activities with IC50 values ranging from 0.2 to 10 µM. Compounds that contained a dihydrofuran ring were generally less active and displayed time dependence in their activity. The activities of compounds 2 and 7-9, carboxylic acids bearing a common six-membered endoperoxide, were higher overall than for compounds 3 and 5. The modes underlying the cytotoxic actions of plakortides 2, 3, 5, 7, and 9 were further investigated using HCT-116 cells. While dihydrofurans 3 and 5 induce a G0/G1 arrest, six-membered peroxides 2, 7, and 9 delivered a G2/M arrest and an accumulation of mitotic figures, indicating a distinctly different antimitotic response. Confocal analysis indicated that microtubules were not altered after treatment with 2, 7, or 9, therein suggesting that the mitotic arrest may be unrelated to cytoskeletal targets. Overall, we find that two related classes of natural products obtained from the same extract offer cytostatic activity, yet they do so through discrete pathways.

Structure-activity relationships for withanolides as inducers of the cellular heat-shock response.

To understand the relationship between the structure and the remarkably diverse bioactivities reported for withanolides, we obtained withaferin A (WA; 1) and 36 analogues (2-37) and compared their cytotoxicity to cytoprotective heat-shock-inducing activity (HSA). By analyzing structure-activity relationships for the series, we found that the ring A enone is essential for both bioactivities. Acetylation of 27-OH of 4-epi-WA (28) to 33 enhanced both activities, whereas introduction of ß-OH to WA at C-12 (29) and C-15 (30) decreased both activities. Introduction of ß-OAc to 4,27-diacetyl-WA (16) at C-15 (37) decreased HSA without affecting cytotoxicity, but at C-12 (36), it had minimal effect. Importantly, acetylation of 27-OH, yielding 15 from 1, 16 from 14, and 35 from 34, enhanced HSA without increasing cytotoxicity. Our findings demonstrate that the withanolide scaffold can be modified to enhance HSA selectively, thereby assisting development of natural product-inspired drugs to combat protein aggregation-associated diseases by stimulating cellular defense mechanisms.

Identification of pyrroloformamide as a cytokinesis modulator.

Discovered in the late 1940s, the pyrrolinonodithioles represent a family of potent disulfide-containing natural products. Although they are understood in a synthetic and biosynthetic context, the biological role of these materials remains unresolved. To date, their activity has been suggested to arise through regulating RNA metabolism, and more recently they have been suggested to function as backup thiols for detoxification. Using materials identified through a natural products program, we now identify the biological function of one member of this family, pyrroloformamide, as an antimitotic agent acting, in part, by disrupting cytokinesis.

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H(2)O(2)-induced DNA damage in cultured human leukocytes.

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50-300 µg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 µg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H(2)O(2) toxicity was observed only when cells were treated with EO during and after exposure to H(2)O(2). With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 µg/mL EO. In conclusion, EO at concentrations up to 300 µg/mL or doses up to 400mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H(2)O(2).

Geopyxins A-E, ent-kaurane diterpenoids from endolichenic fungal strains Geopyxis aff. majalis and Geopyxis sp. AZ0066: structure-activity relationships of geopyxins and their analogues.

Four new ent-kaurane diterpenoids, geopyxins A-D (1-4), were isolated from Geopyxis aff. majalis, a fungus occurring in the lichen Pseudevernia intensa, whereas Geopyxis sp. AZ0066 inhabiting the same host afforded two new ent-kaurane diterpenoids, geopyxins E and F (5 and 6), together with 1 and 3. The structures of 1-6 were established on the basis of their spectroscopic data, while the absolute configurations were assigned using modified Mosher's ester method. Methylation of 1-3, 5, and 6 gave their corresponding methyl esters 7-11. On acetylation, 1 and 7 yielded their corresponding monoacetates 12 and 14 and diacetates 13 and 15. All compounds were evaluated for their cytotoxic and heat-shock induction activities. Compounds 2, 7-10, 12, 14, and 15 showed cytotoxic activity in the low micromolar range against all five cancer cell lines tested, but only compounds 7-9, 14, and 15 were found to activate the heat-shock response at similar concentrations. From a preliminary structure-activity perspective, the electrophilic α,ß-unsaturated ketone carbonyl motif present in all compounds except 6 and 11 was found to be necessary but not sufficient for both cytotoxicity and heat-shock activation.

Cell cycle arrest through inhibition of tubulin polymerization by withaphysalin F, a bioactive compound isolated from Acnistus arborescens.

Many compounds used in the treatment of cancer possess tubulin-interacting properties that lead to mitotic arrest. Withaphysalins are potent cytotoxic compounds that are commonly found in plants belonging to the Solanaceae family, such as Acnistus arborescens; however, the cytotoxic mechanisms or molecular targets of these compounds remain unknown. Thus, the aim of this study was to evaluate the effects of whitaphysalins on cancer cell cycle progression and tubulin interaction. In this report, we show the antiproliferative activity of withaphysalin F and its effect in arresting cells in the G(2)/M phase of the cell cycle. These two effects are the result of the interference of withaphysalin F in the polymerization of microtubules. Withaphysalin F also induced DNA fragmentation, which can be related to an increase in mitochondrial membrane depolarization. These results suggest that interference of withaphysalin F in microtubule polymerization may induce cell cycle arrest in the G(2)/M phase and therefore contribute to growth inhibition of tumor cells in vitro. Taken together, these studies indicate that withaphysalin F could potentially be used as an anticancer drug.

Studies on the cytotoxicity of miscellaneous compounds from Eupatorium betonicaeforme (D.C.) Baker (Asteraceae).

A detailed study on the cytotoxic effects of five known constituents isolated from the flowers and roots of Eupatorium betonicaeforme is reported, including 2,2-dimethyl-6-vinylchroman-4-one (1), 2-senecioyl-4-vinylphenol (2), 6-acetyl-2,2-dimethylchroman-4-one (3), (4E)-8beta-angeloyloxy-9beta,10beta-dihydroxy-1-oxogermacra-4,11(13)-dien-12,6alpha-olide (4), and 3beta-hydroxyicosan-1,5beta-olide (5). The sesquiterpene lactone 4 exhibited the highest cytotoxicity, with IC50 values ranging from 3.9 to 9.9 microM, showing some degree of cell selectivity. The antiproliferative activity of 4 was examined towards HL-60 cells, and found to diminish cell viability in a dose-dependent manner. Moreover, at all concentrations tested, there was a decrease in the number of cells capable of incorporating 5-bromo-2'-deoxyuridine (BrdU), indicating disruption of DNA synthesis. The morphological changes induced by 4 were compatible with apoptotic cell death. This work, thus, corroborates the anticancer potential of Eupatorium secondary metabolites.