Xylodiol from Xylopia langsdorfiana induces apoptosis in HL60 cells

An atisane diterpene was isolated from Xylopia langsdorfiana St. Hilaire & Tulasne, Annonaceae, leaves, ent-atisane-7α,16α-diol (xylodiol). Preliminary study showed that xylodiol was cytotoxic and induced differentiation on human leukemia cell lines. However, the molecular mechanisms of xylodiol-mediated cytotoxicity have not been fully defined. Thus, we investigated the anti-tumor effect of xylodiol in human leukemia HL60 cell line. Xylodiol induced apoptosis and necrosis. HL60 cells treated with xylodiol showed biochemical changes characteristic of apoptosis, including caspases-8, -9 and -3 activation and loss of mitochondrial transmembrane potential (∆ Ψm). However, there was a condensation rather than swelling of mitochondria. Moreover, the formation of condensed mitochondria and the loss of ∆ Ψm occurred downstream of caspase activation. Cyclosporine A did not protect HL60 cells from the cytotoxic effects of xylodiol, suggesting that the loss of ∆ Ψm is a late event in xylodiol-induced apoptosis. Oxidative stress was involved in xylodiol-induced apoptosis. Thus, we conclude that activated caspases cleave cellular proteins resulting in mitochondrial damage leading to mitochondrial condensation, loss of ∆ Ψm and ROS release from the mitochondria. ROS can further induce and maintain a collapse of ∆ Ψm leading to cellular damage through oxidation of lipids and proteins resulting in apoptotic cell death.

Antitumoral activity of L-ascorbic acid-poly- D,L-(lactide-co-glycolide) nanoparticles containing violacein.

It has been demonstrated that tumoral cells have a higher uptake of ascorbic acid compared to normal cells. This differential characteristic can be used as a way to improve the specificity of antitumoral compounds if combined with polymeric drug delivery systems. The aim of this study was to prepare, characterize and evaluate the antitumoral activity of poly- D,L-(lactide-co-glycolide) 50:50 loading the antitumoral compound violacein and capped with L-ascorbic acid. Nanoparticles were prepared using the nanoprecipitation method and morphologically characterized by scanning electron microscopy (SEM). The average diameter and Zeta potential were determined by photon correlation spectroscopy method (PCS), and assays were carried out to determine the content of ascorbic acid and in vitro drug release kinetics. The antitumoral activity of this system was also evaluated against HL-60 cells by tetrazolium reduction assay. Nanoparticles with size distribution between 300-400 nm and strong negative outer surface (-40 mV) were obtained by this method. Analysis of ascorbic acid content showed that this compound was mainly localized on the external surface of nanoparticles. Violacein loading efficiency was determined as 32% +/- 1% and this drug was gradually released from nanoparticles at different rates depending on the composition of the release media. In addition, this system was observed to be 2 x more efficient as an antitumoral compared with free violacein.

Diterpenes from Xylopia langsdorffiana inhibit cell growth and induce differentiation in human leukemia cells.

Two new diterpenes were isolated from stems and leaves of Xylopia langsdorffiana, ent-atisane-7alpha,16alpha-diol (xylodiol) and ent-7alpha-acetoxytrachyloban-18-oic acid (trachylobane), along with the known 8(17),12E,14-labdatrien-18-oic acid (labdane). We investigated their antitumour effects on HL60, U937 and K562 human leukemia cell lines. We found that xylodiol was the most potent diterpene in inhibiting cell proliferation of HL60, U937 and K562 cells, with mean IC50 values of 90, 80 and 50 microM, respectively. Based on the nitroblue tetrazolium (NBT) reduction assay, all the diterpenes were found to induce terminal differentiation in HL60 and K562 cells, with xylodiol being the most effective. NBT reduction was increased by almost 120% after 12 h exposure of HL60 cells to xylodiol at a concentration lower than the IC50 (50 microM). Thus, xylodiol inhibited human leukemia cell growth in vitro partly by inducing cell differentiation, and merits further studies to examine its mechanism of action as a potential antitumoural agent.

Comparação da oscilação postural estática na posição sentada entre jovens e idosos saudáveis / Comparing static sitting postural sway of healthy young and older adults

OBJETIVOS: Descrever uma nova metodologia de análise da oscilação postural estática sentada e comparar os resultados de jovens e idosos saudáveis. MÉTODOS: Participaram do estudo 38 indivíduos saudáveis, 17 jovens (idade média 23±2,38 anos) e 21 idosos (idade média 67±2,42 anos). A oscilação postural foi mensurada por meio do sistema eletromagnético Polhemus® 3Space Isotrack II. As avaliações foram feitas nas condições olhos abertos (OA) e fechados (OF), com os voluntários sentados sem apoio plantar e sem encosto em suportes de madeira (superfície estável-SE) e de espuma (superfície instável-SI). Cada condição sensorial foi avaliada durante 90 segundos. Os parâmetros analisados foram: deslocamento máximo (Dmáx), trajetória total (Traj) e velocidade média (Vel) nos planos sagital (X) e frontal (Y). RESULTADOS: Nas condições OA e OF em SE, foram encontradas oscilações nos planos X e Y dos parâmetros Traj e Vel maiores em jovens que em idosos. Em SI, foram observadas maiores Traj Y e Vel Y nos jovens, sem diferença significativa entre os grupos quanto a Traj X e Vel X com olhos fechados. Em relação ao Dmáx, tanto no plano X quanto no Y, em todas as condições sensoriais, só houve diferença significativa na condição OASE no plano sagital, sendo maior nos jovens. CONCLUSÕES: Jovens saudáveis oscilam mais que os idosos saudáveis na posição sentada. Além disso, a ferramenta utilizada mostrou ser útil para análise da oscilação postural estática na posição sentada, possibilitando o surgimento de estudos que a associem com o efeito de diversas tarefas motoras.

OBJECTIVES: To describe a new method to analyze the static sitting postural sway and to compare the results of healthy young and older adult subjects. METHODS: Thirty-eight healthy subjects took part in the study, including 17 young adults (mean age 23±2.38 years old) and 21 older adults (mean age 67±2.42 years old). The device used to quantify trunk sway was the magnetic field sensor Polhemus® 3Space Isotrack II. The measurements were taken in the eyes-opened (EO) and eyes-closed (EC) condition with the subjects seated first on a wooden stable surface (SS) then on a foam unstable surface (US) without back or foot support. Each sensory condition was assessed for 90 seconds. The analyzed parameters were: maximum amplitude (Amp), total trajectory (Traj) and mean velocity (Vel) in the sagittal (X) and frontal (Y) planes. RESULTS: In the EO and EC conditions on SS, young adults presented greater postural sway in the X and Y planes on the Traj and Vel parameters. In the US, young adults showed greater Y Traj and Y Vel in the EO and EC conditions, and there was no significant difference between the groups with regard to X Traj and X Vel in the EC condition. The young adults presented greater Amp only in the EOSS condition in the X plane. CONCLUSIONS: The young adult subjects presented greater sway in the sitting position than the older adult subjects. In addition, the Polhemus® device was a useful tool to analyze static sitting postural sway and can be used in future studies that associate static sitting postural sway with the effect of various motor tasks.

Nanocytotoxicity: violacein and violacein-loaded poly (D, L-lactide-co-glycolide) nanoparticles acting on human leukemic cells.

Violacein is a compound obtained from Chromobacterium violaceum, a bacterium found in the Amazonian region. Violacein-loaded poly (D, L-lactide-co-glycolide) nanoparticles has a similar inhibitory effect evaluated by trypan blue assay on leukemic HL60 cells than the free form. However, the cytotoxic effects evaluated by phosphatase activity and MTT reduction assays were lower for the encapsulated form than for free violacein. Based on morphological changes, violacein and violacein entrapped in nanoparticles were found to induce terminal differentiation (assessed by nitro blue tetrazolium reduction) in HL60 cells. Thus, both formulations inhibit HL60 cell growth in vitro, partly by inducing cytotoxic effects and cell differentiation. Flow cytometric analysis of HL60 cells after treatment for 12 h showed that violacein-loaded PLGA induced apoptosis, with maximum cell death at a concentration of 2 microM. Violacein and violacein/PLGA induced opposite effects on the mitochondrial swelling which indicates altered mitochondrial function. The mitochondrial activity was also checked by flow cytometry studies. Labelled cells with the probe JC1 displayed a basal hypopolarized status of the mitochondria in treated cells. Based on morphological changes, alterations in phospholipid asymmetry and changes in mitochondrial polarization, violacein and nanoparticles containing violacein were found to trigger cell death by apoptosis. These methodologies are promising as diagnostic and mechanistic effects of nanoparticles in cell cultures.

Violacein: properties and biological activities.

The violet pigment violacein is an indole derivative, isolated mainly from bacteria of the genus Chromobacterium, which exhibits important antitumoral, antimicrobial and antiparasitary properties. Furthermore, the formulation of violacein in different polymeric carriers developed so far offers alternative approaches to overcoming physiological barriers and undesirable physicochemical properties in vivo, thus improving its efficacy.

ent-Trachylobane diterpenoids from Xylopia langsdorffiana.

Two new diterpenes of the ent-trachylobane type were isolated from the stems of Xylopia langsdorffiana, ent-7alpha-acetoxytrachyloban-18-oic acid (1) and ent-7alpha-hydroxytrachyloban-18-oic acid (2). The structures of these isolates were deduced by spectroscopic data interpretation. X-ray crystallography of 1 was used to confirm its structure. The cytotoxic activity of 1 against V79 fibroblasts and rat hepatocytes was investigated.

Dehydrocrotonin and its beta-cyclodextrin complex: cytotoxicity in V79 fibroblasts and rat cultured hepatocytes.

Trans-dehydrocrotonin has antiulcerogenic and antitumor activities. A complex of beta-cyclodextrin with dehydrocrotonin was developed to improve the delivery of dehydrocrotonin. Complex in solid state was evaluated using X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA) and scanning electron microscopy (SEM). X-ray diffraction and scanning electron microscopy studies showed that dehydrocrotonin exists in a semicrystalline state in the complexed form with beta-cyclodextrin. Differential scanning calorimetry studies showed the existence of a complex of dehydrocrotonin with beta-cyclodextrin. The thermal gravimetric analysis studies confirmed the differential scanning calorimetry results of the complex. Free dehydrocrotonin and the dehydrocrotonin/beta-cyclodextrin inclusion complex were assayed in freshly isolated rat hepatocytes and in V79 cells. Cytotoxicity was determined using nucleic acid content, methylthiazoletetrazolium (MTT) reduction and neutral red uptake assays. In all assays, there was a large reduction (3.5-16.1-fold) in the cytotoxicity of dehydrocrotonin in hepatocytes when complexed with beta-cyclodextrin, whereas for V79 cells the decrease in cytotoxicity was 1.7- and 1.87-fold for MTT reduction and nucleic acid content assays, respectively. The lower cytotoxicity of the dehydrocrotonin/beta-cyclodextrin complex compared to free dehydrocrotonin in rat hepatocytes and V79 cells suggests that such a complex may be useful for the administration of dehydrocrotonin in vivo.

Dehydrocrotonin and its derivative, dimethylamide-crotonin induce apoptosis with lipid peroxidation and activation of caspases-2, -6 and -9 in human leukemic cells HL60.

A variety of stimuli can induce cells to undergo apoptosis, with one of the most reproducible inducers being mild oxidative stress following exposure to anticancer agents. Apoptosis involves events mediated by cysteine proteases (caspases) that are classified as initiators (-8, -9 and -12) or executors (-2, -3, -6 and -7). In this study, we examined the mechanisms of apoptosis induced by dehydrocrotonin (DHC), a diterpene lactone isolated from the Amazonian plant Croton cajucara, and its synthetic derivative, dimethylamide-crotonin (DCR), in human HL60 promyelocytic leukemia cells. Flow cytometric analysis of HL60 cells after treatment for 72 h showed that DCR- and DHC-induced apoptosis, with maximum cell death at a concentration of 250 microM for both compounds. DCR and DHC were effective in triggering the activation of caspases-2, -6 and -9. The level of reduced glutathione (GSH) decreased, whereas there was an increase in thiobarbituric acid-reactive substance (TBARS) production and in mitochondrial swelling. These effects on mitochondrial swelling, GSH content and lipid peroxidation were abolished by cyclosporine A, an inhibitor of the membrane permeability transition. The cytotoxicity of DHC and DCR was prevented by a high concentration of GSH (15 mM) in the culture medium. These results indicate that DCR and DHC produced apoptosis partly by oxidative stress-induced lipid peroxidation, which triggered the caspase cascade, that lead to apoptotic cell death in HL60 cells. Based on the pattern of caspase activation, on the increase in mitochondrial swelling and on the inhibitory action of cyclosporine A, we conclude that DCR and DHC triggered apoptosis in HL60 cells probably through cytochrome c release and apoptosome formation.

Natural killer cell activity and anti-tumour effects of dehydrocrotonin and its synthetic derivatives.

In this work, the anti-tumour properties of dehydrocrotonin and its derivatives were investigated in vitro and in vivo using the Ehrlich ascites tumour model. Treatment of Ehrlich ascites tumour-bearing mice with 20 mg/kg dehydrocrotonin for 4 days significantly increased survival, whereas administration of dehydrocrotonin derivatives was ineffective in affording protection. Compound IV exhibited little activity against Ehrlich tumour cells in vitro. Investigation of the effects of dehydrocrotonin treatment on total natural killer (NK) cell activity of tumour-bearing mice as a possible mechanism of dehydrocrotonin action in vivo revealed that this sesquiterpene lactone significantly improved NK cytotoxicity against YAC-1, a Moloney virus-induced mouse T-cell lymphoma of A/SN origin. As expected, tumour growth in non-treated mice markedly suppressed NK cell cytolysis. No effects on NK functional activity were observed in normal mice receiving dehydrocrotonin. In summary, only the natural compound exhibits anti-tumour efficacy and immunomodulatory actions in vivo, which may be related to its chemical structure.

Antiulcerogenic effect and cytotoxic activity of semi-synthetic crotonin obtained from Croton cajucara Benth.

Trans-dehydrocrotonin, the major diterpene isolated from the bark of Croton cajucara, has good antiulcerogenic activity which, however, is accompanied by toxic effects. On the basis of these results, a semi-synthetic crotonin, named 4SRC, was prepared to determine whether this substance has similar antiulcerogenic activity with lower or no toxicity. The natural crotonin was also isolated from the bark of C. cajucara but was not used due to the small amount obtained. The cytotoxic effect of semi-synthetic crotonin, expressed as cell viability, was assessed in (a) lung fibroblast cell line (V79) derived from Chinese hamsters, a system commonly used for cytotoxicity studies, and (b) rat hepatocytes isolated from male Wistar rats. After treatment, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (MTT reduction), total acid content and neutral red uptake assays. To evaluate V79 cell viability, different concentrations of semi-synthetic crotonin were incubated with the cells. To evaluate the antiulcerogenic effects of semi-synthetic crotonin (50, 100 and 200 mg/kg), we used the models of gastric ulcer induced by ethanol/HCl, stress, indomethacin/bethanechol, and ethanol in male Swiss mice and male Wistar rats. The substance had an IC(50)=500 microM in the neutral red uptake and MTT reduction tests and an IC(50)=200 microM in the nucleic acid content test. With regard to hepatocyte viability after treatment with semi-synthetic crotonin at different concentrations, semi-synthetic crotonin had an IC(50)=10-500 microM in the nucleic acid content and MTT reduction tests and an IC(50)=120 microM in the neutral red uptake test. In another experiment, V79 cells were incubated with the metabolites produced by hepatocytes treated with different concentrations of semi-synthetic crotonin. After a 4-h incubation, semi-synthetic crotonin had an IC(50)=500 microM in the MTT reduction and neutral red uptake tests and an IC(50)=370 microM in nucleic acid content test. The substance had significant antiulcerogenic activity in all models studied, suggesting the presence of a possible antisecretory effect combined with a cytoprotective effect. For this reason, the effect of semi-synthetic crotonin was also evaluated on biochemical parameters of gastric juice and gastric wall mucus, both obtained from pylorus-ligated mice. No significant differences were observed in these parameters between semi-synthetic crotonin-treated and control animals. The results obtained with semi-synthetic crotonin are promising, with a significant preventive effect against gastric ulcer induced by different agents. Our data also show that semi-synthetic crotonin was less toxic than dehydrocrotonin and that the cytotoxic effects decreases with the time that isolated hepatocytes were in culture.

Evaluation of the antiulcerogenic activity of violacein and its modulation by the inclusion complexation with beta-cyclodextrin.

The effects of beta-cyclodextrin (betaCD) inclusion complexation on the ability of violacein to prevent gastric ulceration in mice were studied. Violacein-betaCD inclusion complexes were prepared in 1:1 and 1:2 molar ratios and analysed by differential scanning calorimetry and powder X-ray diffractometry. Violacein previously administered orally at 10 mg/kg significantly reduced indomethacin-induced gastric lesions, as well as 100 mg/kg of cimetidine (positive control). However, betaCD complexation in both molar ratios significantly potentiated the protective action of violacein. In the HCl--ethanol-induced gastric ulcer model, violacein and the 1:2 inclusion complex (10 mg/kg, p.o.) inhibited gastric damage by almost 85%, whereas a 63% reduction was observed for the positive control, lansoprazole, at 30 mg/kg. In contrast, treatment with the 1:1 inclusion complex resulted in almost total disappearance of the antiulcer activity in this model. No significant changes in stress-induced gastric injury were found. In addition, the 1:2 inclusion complex improved the antilipoperoxidant activity of violacein in rat liver cells exposed to t-butyl hydroperoxide, whereas the 1:1 complex was less active than violacein. In summary, the 1:2 betaCD inclusion complex has gastroprotective properties similar to or higher than that of violacein. An increase in mucosal defensive mechanisms and protection against peroxidative damage might be involved.

Violacein and its beta-cyclodextrin complexes induce apoptosis and differentiation in HL60 cells.

Violacein, a pigment isolated from Chromobacterium violaceum, has been reported to have multiple biological activities including in vitro antitumor effects. Certain anticancer agents are known to induce apoptosis in human tumor cell lines. In this work, our aim was to investigate the effectiveness of violacein/beta-cyclodextrin (beta-CD)-containing systems to produce lethal effects in the human promyelocytic leukemia cell line HL60. Using the MTT tetrazolium reduction test, IC(50) for the inclusion complexes (1:1 and 1:2 violacein:beta-CD molar ratios) and violacein alone were less than 1 microM. Violacein and violacein/beta-CD complexes were able to induce NBT reduction. Moreover, by using the Feulgen reaction, all the compounds were found to trigger apoptosis in HL60 cells, inducing around 35% of DNA fragmentation, as analyzed through the diphenylamine assay. In addition, caspases seem to play an important role in the activation of the executioner phase of apoptosis induced by violacein and its derivatives.

3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N, N-dimethyl-3-(2-thienyl)-2-propen-1-amine: synthesis, cytotoxicity, and leishmanicidal, trypanocidal and antimycobacterial activities.

Current therapies for Chagas' disease, leishmaniasis and tuberculosis are unsatisfactory because of the failure rates, significant toxicity and/or drug resistance. In this study, the compound 3-[4'-bromo-(1,1'-biphenyl)-4-yl]-N,N-dimethyl-3-(2-thienyl)-2-propen-1-amine (IV) was synthesized and its trypanocidal, leishmanicidal and antimycobacterial activities were investigated. The cytotoxicity was determined on V79 cells with three endpoints: nucleic acid content, 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide reduction and Neutral Red uptake. This compound was active against different species of mycobacteria and different life cycle stages of Trypanosoma cruzi. In experiments with trypomastigotes performed at 4 degrees C in the presence of blood, the activity was 8.8-fold more active than the standard drug, Crystal Violet. Higher activity was achieved against Leishmania amazonensis, with an ED(50)/24 h of 3.0 +/- 0.3 micro mol/L. The effect against trypanosomatids, which suggests high activity of compound IV against promastigotes of L. amazonensis and amastigotes of T. cruzi, stimulated further studies in vitro with amastigotes interiorized in macrophages and with in vivo models. Our results indicate that mammalian V79 cells are less susceptible to the action of compound IV than promastigotes of L. amazonensis (8.0-13.3-fold) and axenic amastigotes of T. cruzi (3.5-5.9-fold).