Glyphosate-induced glioblastoma cell proliferation: Unraveling the interplay of oxidative, inflammatory, proliferative, and survival signaling pathways.

The aim of the present study was to investigate the impacts of glyphosate herbicide on the survival and proliferation of glioblastoma cells and to explore the molecular mechanisms underlying such effects. For this, cultured human glioblastoma cell line, A172, was exposed to the glyphosate analytical standard, a glyphosate-based herbicide formulation (GBH), or the metabolite aminomethylphosphonic acid (AMPA). The three compounds induced A172 cytotoxicity after 24 h of exposure, with more prominent cytotoxic effects after 48 and 72 h of treatment. Further experiments were performed by treating A172 cells for 6 h with glyphosate, GBH, or AMPA at 0.5 mg/L, which corresponds to the maximum residue limits for glyphosate and AMPA in drinking water in Brazil. Colony forming units (CFU) assay showed that AMPA increased the number of CFU formed, while glyphosate and GBH increased the CFU sizes. The three compounds tested altered the cell cycle and caused DNA damage, as indicated by the increase in γ-H2AX. The mechanisms underlying the pesticide effects involve the activation of Akt and mitogen-activated protein kinases (MAPKs) signaling pathways, oxidative imbalance, and inflammation. Glyphosate led to NLRP3 activation culminating in caspase-1 recruitment, while AMPA decreased NLRP3 immunocontent and GBH did not alter this pathway. Results of the present study suggest that exposure to glyphosate (isolated or in formulation) or to its metabolite AMPA may affect cell signaling pathways resulting in oxidative damage and inflammation, giving glioblastoma cells an advantage by increasing their proliferation and growth.

Estradiol and 1α,25(OH)2 vitamin D3 share plasma membrane downstream signal transduction through calcium influx and genomic activation in immature rat testis.

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

Covalently Bonded N-Acetylcysteine-polyester Loaded in PCL Scaffolds for Enhanced Interactions with Fibroblasts.

Poly(ε-caprolactone) (PCL) is commonly used in devices for tissue reconstruction due to its biocompatibility and suitable mechanical properties. However, its high crystallinity and hydrophobicity do not favor cell adhesion and difficult polymer bioresorption. To improve these characteristics, the development of engineered scaffolds for tissue regeneration, based on poly(globalide-co-ε-caprolactone) (PGlCL) covalently bonded with N-acetylcysteine (PGlCL-NAC) was proposed. The scaffolds were obtained from polymer blends of PCL and PGlCL-NAC, using the electrospinning technique. The use of PGlCL-NAC allowed for the modification of the physical and chemical properties of PCL electrospun scaffolds, including an expressive reduction in the fiber's diameter, hydrophobicity, and crystallinity. All electrospun scaffolds showed no cytotoxicity against fibroblasts (McCoy cells). In vitro biocompatibility assays showed that all tested scaffolds provided high cell viability and proliferation in short-term (NRU, MTT, and nuclear morphology assays) and long-term (clonogenic assay) assays. Nevertheless, PGlCL-NAC based scaffolds have favored the survival and proliferation of the cells in comparison to PCL scaffolds. Cell adhesion on the scaffolds assessed by electronic microscopy images confirmed this behavior. These results suggest that the incorporation of PGlCL-NAC in scaffolds for tissue regeneration could be a promising strategy to improve cell-surface interactions and contribute to the development of more efficiently engineered biomedical devices.

Dietary supplementation with procyanidin-rich Pinus pinaster extract is associated with attenuated Ehrlich tumor development in mice.

Inflammation and oxidative stress are related to cancer initiation and progression. We hypothesized that dietary supplementation with a procyanidin-rich Pinus pinaster extract (Pyc) with known antioxidant and anti-inflammatory effects could induce systemic protection, thereby attenuating tumor development. To test our hypothesis, mice were subjected to long-term supplementation (20 days, every 24 h) with saline, 25 mg/kg resveratrol or 100 mg/kg Pyc. Pyc was administered at a maximum tolerated oral dose, previously determined using toxicity indicators. Ten days after Ehrlich ascites tumor induction, weight gain and abdominal circumference increase were calculated. Ascitic fluid from six mice/group was evaluated by determining total volume; tumor packed cell volume; cell viability; tumor cell death type; inflammatory infiltrate; and levels of tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), carbonyl proteins, lipid peroxidation, cyclooxigenase-2 (COX-2) expression and Akt phosphorylation (p-Akt). Ten mice/group were monitored to evaluate survival. Pyc and resveratrol were associated with reduced weight gain (>30%), abdominal circumference and ascitic volume. Tumor packed cell volume was reduced in Pyc-supplemented mice (26%), which had the largest tumor cell count reduction (>35%), increased ascitic fluid apoptosis rates (20%) and the longest survival (>2-fold). Pyc and resveratrol treatment both reduced inflammatory infiltrate and levels of TNF-α, IL-1ß, carbonyl proteins, lipid peroxidation (~ 30%) and p-Akt (up to 4-fold). Only Pyc significantly inhibited COX-2. Pyc attenuated oxidative and inflammation mediators and impaired tumor development, supporting our hypothesis and suggesting Pyc as a candidate for future studies in multitargeted dietary-based cancer prevention approaches.

Novel selenylated imidazo[1,2-a]pyridines for breast cancer chemotherapy: Inhibition of cell proliferation by Akt-mediated regulation, DNA cleavage and apoptosis.

A novel series of selenylated imidazo[1,2-a]pyridines were designed and synthesized with a view to a promising activity against breast cancer cell. The compounds, 7-methyl-3-(naphthalene-1-ylselanyl)-2-phenylimidazo[1,2-a]pyridine, named IP-Se-05, and 3-((2-methoxyphenyl)selanyl)-7-methyl-2-phenylimidazo[1,2-a]pyridine, named IP-Se-06, showed high cytotoxicity for MCF-7 cells (IC50 = 26.0 µM and 12.5 µM, respectively). Both the compounds inhibited the cell proliferation and caused decrease in the number of cells in the G2/M phase of cell cycle. IP-Se-05 and IP-Se-06 were also evaluated for effects on CT-DNA and DNA of MCF-7 cells. The compounds intercalated into CT-DNA and both treatments caused cleavage of DNA in cells. In addition, the compounds induced cell death by apoptosis. However, the presence of (2-methoxyphenyl) selenyl moiety at the imidazo[1,2-a]pyridine (IP-Se-06) appears to have a better antitumor effect with higher cytotoxicity at a lower concentration and caused less necrosis. Overall, the current study established IP-Se-06 more than IP-Se-05 as a potential prototype compound to be employed as an antiproliferative agent for the treatment of breast cancer.

Novel pyrimidinic selenourea induces DNA damage, cell cycle arrest, and apoptosis in human breast carcinoma.

Novel pyrimidinic selenoureas were synthesized and evaluated against tumour and normal cell lines. Among these, the compound named 3j initially showed relevant cytotoxicity and selectivity for tumour cells. Three analogues of 3j were designed and synthesized keeping in view the structural requirements of this compound. Almost all the tested compounds displayed considerable cytotoxicity. However, 8a, one of the 3j analogues, was shown to be highly selective and cytotoxic, especially for breast carcinoma cells (MCF-7) (IC50 = 3.9 µM). Furthermore, 8a caused DNA damage, inhibited cell proliferation, was able to arrest cell cycle in S phase, and induced cell death by apoptosis in human breast carcinoma cells. Moreover, predictions of pharmacokinetic properties showed that 8a may present good absorption and permeation characteristics for oral administration. Overall, the current study established 8a as a potential drug prototype to be employed as a DNA interactive cytotoxic agent for the treatment of breast cancer.

Ultrasonic-assisted extraction combined with sample preparation and analysis using LC-ESI-MS/MS allowed the identification of 24 new phenolic compounds in pecan nut shell [Carya illinoinensis (Wangenh) C. Koch] extracts.

Ultrasonic-assisted extraction combined with statistical tools (factorial design, response surface methodology and kinetics) were used to evaluate the effects of the experimental conditions of temperature, solid-to-solvent ratio, ethanol concentration and time for the extraction of the total phenolic content from pecan nut shells. The optimal conditions for the aqueous and hydroalcoholic extract (with 20% v/v of ethanol) were 60 and 80 °C; solid to solvent ratio of 30 mL·g-1 (for both) and extraction time of 35 and 25 min, respectively. Using these optimize extraction conditions, 426 and 582 mg GAE·g-1 of phenolic compounds, from the aqueous and hydroalcoholic phases respectively, were obtained. In addition, the analysis of the phenolic compounds using the LC-ESI-MS/MS system allowed the identification of 29 phenolic compounds, 24 of which had not been reported in literature for this raw material yet.

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. AIM OF THE STUDY: Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. MATERIALS AND METHODS: Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. RESULTS: The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). CONCLUSIONS: These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer.

Healing effect of Dillenia indica fruit extracts standardized to betulinic acid on ultraviolet radiation-induced psoriasis-like wounds in rats.

CONTEXT: Dillenia indica Linn. (Dilleniaceae) is traditionally used to treat skin inflammation. OBJECTIVE: This study evaluated the healing effect of Dillenia indica fruit extracts on induced psoriasis-like wounds in Wistar rats. MATERIALS AND METHODS: Extracts were standardized to betulinic acid, including an aqueous ethanolic extract (AEE), ethyl acetate extract (EAE) and petroleum ether extract. Effects against lipid peroxidation were assessed in vitro. Wounds were created at rat tails (n = 12). Topical treatments were applied once daily for 7 days (1 mL of AEE or EAE at 5 or 50 mg/mL). Maximal dose was defined by the extract solubility. A 10-fold lower dose was also tested. Positive and negative controls were treated with clobetasol (0.5 mg/mL) or excipient. Half of each group was euthanized for histology. The remaining animals were observed for 20 days for wound measurements. RESULTS: Yields of AEE and EAE were 4.3 and 0.7%, respectively. Betulinic acid concentrations in AEE and EAE were 4.6 and 107.6 mg/g. Extracts neutralized lipid peroxidation in vitro at 0.02 µg/mL, accelerating healing at 50 mg/mL. Complete healing in mice treated with AEE occurred 16 days after wound induction. This time was 14 and 12 days in mice treated with EAE and clobetasol. Compared to orthokeratosis, parakeratosis was reduced by AEE (25%), EAE (45%) and clobetasol (55%). EAE caused superior protection against biomolecules oxidation of skin compared to AEE. DISCUSSION AND CONCLUSION: EAE exhibited activity closer to that of clobetasol. Betulinic acid may be an active constituent, which should be assessed in future studies.

In vivo inhibition of tumor progression by 5 hydroxy-1,4-naphthoquinone (juglone) and 2-(4-hydroxyanilino)-1,4-naphthoquinone (Q7) in combination with ascorbate.

The purpose of the study was to obtain further in vivo data of antitumor effects and mechanisms triggered by juglone and Q7 in combination with ascorbate. The study was done using Ehrlich ascites tumor-bearing mice. Treatments were intraperitoneal every 24 h for 9 days. Control group was treated with excipient. Previous tests selected the doses of juglone and Q7 plus ascorbate (1 and 100 mg/kg, respectively). Samples of ascitic fluid were collected to evaluate carbonyl proteins, GSH and activity of antioxidant enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase. Hypoxia inducible factor HIF-1α, GLUT1, proteins driving cell cycle (p53, p16 and cyclin A) and apoptosis (poly-ADP-polymerase PARP, Bax and Bcl-xL) were assessed by western blot. Tumor cells were categorized by the phase of cell cycle using flow cytometry and type of cell death using acridine orange/ethidium bromide. A glucose uptake assessment was performed by liquid scintillation using Ehrlich tumor cells cultured with (14)C-deoxyglucose. Treatments caused increased protein carbonylation and activity of antioxidant enzymes and decreased levels of GSH, HIF-1α, GLUT1 and glucose uptake in tumor cells. They also caused increased number of tumor cells in G1, p53 and p16 activation and decreased cyclin A, but only when combined with ascorbate. Apoptosis was induced mostly when treatments were done with ascorbate, causing PARP and Bax cleavage, and increased Bax/Bcl-xL ratio. Juglone and Q7 in combination with ascorbate caused inhibition of tumor progress in vivo by triggering apoptosis and cell cycle arrest associated with oxidative stress, suppression of HIF-1 and uncoupling of glycolytic metabolism.

Piper nigrum ethanolic extract rich in piperamides causes ROS overproduction, oxidative damage in DNA leading to cell cycle arrest and apoptosis in cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Ayurvedic and Chinese traditional medicine and tribal people use herbal preparations containing Piper nigrum fruits for the treatment of many health disorders like inflammation, fever, asthma and cancer. In Brazil, traditional maroon culture associates the spice Piper nigrum to health recovery and inflammation attenuation. AIMS OF THE STUDY: The aim of the current work was to evaluate the relationship between reactive oxygen species (ROS) overproduction, DNA fragmentation, cell cycle arrest and apoptosis induced by Piper nigrum ethanolic extract and its antitumor activity. METHODS: The plant was macerated in ethanol. Extract constitution was assessed by TLC, UV-vis and ESI-IT-MS/MS spectrometry. The cytotoxicity, proliferation and intracellular ROS generation was evaluated in MCF-7 cells. DNA damage effects were evaluated through intercalation into CT-DNA, plasmid DNA cleavage and oxidative damage in CT-DNA. Tumor growth inhibition, survival time increase, apoptosis, cell cycle arrest and oxidative stress were assessed in Ehrlich ascites carcinoma-bearing mice. RESULTS: Extraction yielded 64mg/g (36% piperine and 4.2% piperyline). Treatments caused DNA damage and reduced cell viability (EC50=27.1±2.0 and 80.5±6.6µg/ml in MCF-7 and HT-29 cells, respectively), inhibiting cell proliferation by 57% and increased ROS generation in MCF-7 cells (65%). Ehrlich carcinoma was inhibited by the extract, which caused reduction of tumor growth (60%), elevated survival time (76%), cell cycle arrest and induced apoptosis. The treatment with extract increased Bax and p53 and inhibited Bcl-xL and cyclin A expression. It also induced an oxidative stress in vivo verified as enhanced lipid peroxidation and carbonyl proteins content and increased activities of glutathione reductase, superoxide dismutase and catalase. GSH concentration was decreased in tumor tissue from mice. CONCLUSION: The ethanolic extract has cytotoxic and antiproliferative effect on MCF-7 cells and antitumor effect in vivo probably due to ROS overproduction that induced oxidative stress affecting key proteins involved in cell cycle arrest at G1/S and triggering apoptosis. Finally, the overall data from this study are well in line with the traditional claims for the antitumor effect of Piper nigrum fruits.

Persistence of the benefit of an antioxidant therapy in children and teenagers with Down syndrome.

This study examined the effect of an antioxidant intervention in biomarkers of inflammation and oxidative stress (OS) in the blood of Down syndrome (DS) children and teenagers during four different stages. A control group was composed by healthy children (n=18), assessed once, and a Down group composed by DS patients (n=21) assessed at the basal period (t0), as well as after 6 months of antioxidant supplementation (t1), after 12 months (after interruption of the antioxidant intervention for 6 months) (t2), and again after further 6 months of antioxidant supplementation (t3). Biomarkers of inflammation (myeloperoxidase activity - MPO and levels of IL-1ß and TNF-α) and OS (thiobarbituric acid reactive substances - TBARS, protein carbonyls - PC), reduced glutathione (GSH), uric acid (UA) and vitamin E levels, as well as antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GPx), glutathione reductase (GR), glutathione-S-transferase (GST) and gamma-glutamyltransferase (GGT) activities, were measured after each period. After the antioxidant supplementation, the activities of SOD, CAT, GPx, GR, GGT and MPO were downregulated, while TBARS contents were strongly decreased, the contents of GSH and vitamin E were significantly increased, and no changes in G6PD and GST activity as well as in UA and PC levels were detected. After the interruption of the antioxidant therapy for 6 months, DS patients showed elevated GPx and GGT activities and also elevated UA and TBARS levels. No changes in SOD, CAT, GR, GST, G6PD and MPO activities as well as in GSH, vitamin E, PC, TNF-α and IL-1ß levels were detected. The results showed that the antioxidant intervention persistently attenuated the systemic oxidative damage in DS patients even after a relatively long period of cessation of the antioxidant intervention.

Pheomelanin-coated iron oxide magnetic nanoparticles: a promising candidate for negative T2 contrast enhancement in magnetic resonance imaging.

We describe herein a novel type of monodisperse water-soluble magnetite nanoparticle coated with pheomelanin using an environmentally-friendly approach in aqueous medium. The results indicate superparamagnetic behaviour at room temperature and show improved negative contrast in T2-weighted MRI with a transverse relaxivity of 218 mM(-1) s(-1).

DNA damage and inhibition of akt pathway in mcf-7 cells and ehrlich tumor in mice treated with 1,4-naphthoquinones in combination with ascorbate.

The aim of this study was to enhance the understanding of the antitumor mechanism of 1,4-naphthoquinones and ascorbate. Juglone, phenylaminonaphthoquinone-7, and 9 (Q7/Q9) were evaluated for effects on CT-DNA and DNA of cancer cells. Evaluations in MCF-7 cells are DNA damage, ROS levels, viability, and proliferation. Proteins from MCF-7 lysates were immunoblotted for verifying PARP integrity, γH2AX, and pAkt. Antitumor activity was measured in Ehrlich ascites carcinoma-bearing mice. The same markers of molecular toxicity were assessed in vivo. The naphthoquinones intercalate into CT-DNA and caused oxidative cleavage, which is increased in the presence of ascorbate. Treatments caused DNA damage and reduced viability and proliferation of MCF-7 cells. Effects were potentiated by ascorbate. No PARP cleavage was observed. Naphthoquinones, combined with ascorbate, caused phosphorylation of H2AX and inhibited pAkt. ROS were enhanced in MCF-7 cells, particularly by the juglone and Q7 plus ascorbate. Ehrlich carcinoma was inhibited by juglone, Q7, or Q9, but the potentiating effect of ascorbate was reproduced in vivo only in the cases of juglone and Q7, which caused up to 60% inhibition of tumor and the largest extension of survival. Juglone and Q7 plus ascorbate caused enhanced ROS and DNA damage and inhibited pAkt also in Ehrlich carcinoma cells.

Inhibition of tumor proliferation associated with cell cycle arrest caused by extract and fraction from Casearia sylvestris (Salicaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: Casearia sylvestris is a tree found in tropical America. In Brazil it is known mainly as Guaçatonga. Literature reports suggest that the leaves and other plant parts have been used by indigenous populations from South America in preparations, mainly aqueous or hydroethanolic macerations or decoctions, most times taken orally for the primary treatment of several diseases, including cancer. AIM OF THE STUDY: This article reports the results of an investigation about the antiproliferative effects of Casearia sylvestris on tumor cells in vitro and in vivo. MATERIAL AND METHODS: Aqueous ethanolic maceration and column chromatography were done to obtain a crude aqueous ethanolic extract (CAE) and a chloroform fraction (f-CHCl3). The human breast cancer cell line MCF-7 was used in culture. In vitro, non-cytotoxic concentrations were determined by MTT assay and the antiproliferative effect was assessed by the colony forming unit assay using non-cytotoxic concentrations. Effects on the cell cycle were observed through flow cytometry using a propidium iodide kit. Casearin C was identified in f-CHCl3 by chromatography and H(1) nuclear magnetic resonance. The effect on some key proteins of DNA damage (phosphorylation on the histone H2AX) and cell cycle control (p53, p16, cdk2) was evaluated through immunoblot. Antiproliferative effects in vivo were measured in tumor tissue from Ehrlich ascites-bearing mice through the (3)H-thymidine uptake assay and the trypan blue exclusion method. RESULTS: In vitro, EC50 values found at 24 h on MCF-7 cells were 141 µg/mL for CAE and 66 µg/mL for f-CHCl3. Inhibition on proliferation was recorded at concentrations as low as 4 µg/mL in the case of the f-CHCl3 (up to 40%) and up to 50% when CAE was added at 9 µg/mL. The cell cycle arrest was demonstrated by the reduction in terms of number of cells in phases G2/M and S, up to 38.9% and 51.9% when cells were treated with CAE, and 53.9% and 66.2%, respectively, when cells were treated with f-CHCl3. The number of cells in G1 was increased when the cells were treated with CAE (21.4%) or f-CHCl3 (27.8%). Key proteins of cell cycle control were affected. The treatments caused activation of p53, p16 and DNA damage found by the appearance of bands corresponding to γ-H2AX. The treatments caused inhibition of cdk2. CAE and particularly f-CHCl3 caused significant inhibition on tumor growth in mice (40% and 60%, respectively). Uptake of (3)H-thymidine, thus proliferation was reduced in tumor cells from mice treated with CAE (>30%) or f-CHCl3 (up to 50%) compared to cells from control animals. Data from the trypan blue assay indicating a lower number of tumor cells in treated animals. From the overall, data from this study are in line with the traditional claims for the antitumor effect of Casearia sylvestris. CONCLUSIONS: This investigation suggests that whether the extracts from Casearia sylvestris are cytotoxic at high concentrations, lower concentrations have antiproliferative effect and could be useful to complement conventional cytotoxic chemotherapy, and should be evaluated further.

Substituted 3­acyl­2­phenylamino­1,4­naphthoquinones intercalate into DNA and cause genotoxicity through the increased generation of reactive oxygen species culminating in cell death.

Naphthoquinones interact with biological systems by generating reactive oxygen species (ROS) that can damage cancer cells. The cytotoxicity and the antitumor activity of 3­acyl­2­phenylamino­1,4­naphthoquinones (DPB1­DPB9) were evaluated in the MCF7 human breast cancer cell line and in male Ehrlich tumor­bearing Balb/c mice. DPB4 was the most cytotoxic derivative against MCF7 cells (EC50 15 µM) and DPB6 was the least cytotoxic one (EC50 56 µM). The 1,4­naphthoquinone derivatives were able to cause DNA damage and promote DNA fragmentation as shown by the plasmid DNA cleavage assay (FII form). In addition, 1,4­naphthoquinone derivatives possibly interacted with DNA as intercalating agents, which was demonstrated by the changes caused in the fluorescence of the DNA­ethidium bromide complexes. Cell death of MCF7 cells induced by 3­acyl­2­phenylamino­1,4­naphthoquinones was mostly due to apoptosis. The DNA fragmentation and subsequent apoptosis may be correlated to the redox potential of the 1,4­naphthoquinone derivatives that, once present in the cell nucleus, led to the increased generation of ROS. Finally, certain 1,4­naphthoquinone derivatives and particularly DPB4 significantly inhibited the growth of Ehrlich ascites tumors in mice (73%).

Antioxidant intervention attenuates oxidative stress in children and teenagers with Down syndrome.

We previously demonstrated that systemic oxidative stress is present in Down syndrome (DS) patients. In the present study we investigated the antioxidant status in the peripheral blood of DS children and teenagers comparing such status before and after an antioxidant supplementation. Oxidative stress biomarkers were evaluated in the blood of DS patients (n=21) before and after a daily antioxidant intervention (vitamin E 400mg, C 500 mg) during 6 months. Healthy children (n=18) without DS were recruited as control group. The activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), gamma-glutamyltransferase (GGT), glucose-6-phosphate dehydrogenase (G6PD) and myeloperoxidase (MPO), as well as the contents of reduced glutathione (GSH), uric acid, vitamin E, thiobarbituric acid reactive substances (TBARS), and protein carbonyls (PC) were measured. Before the antioxidant therapy, DS patients presented decreased GST activity and GSH depletion; elevated SOD, CAT, GR, GGT and MPO activities; increased uric acid levels; while GPx and G6PD activities as well as vitamin E and TBARS levels were unaltered. After the antioxidant supplementation, SOD, CAT, GPx, GR, GGT and MPO activities were downregulated, while TBARS contents were strongly decreased in DS. Also, the antioxidant therapy did not change G6PD and GST activities as well as uric acid and PC levels, while it significantly increased GSH and vitamin E levels in DS patients. Our results clearly demonstrate that the antioxidant intervention with vitamins E and C attenuated the systemic oxidative damage present in DS patients.

Anticancer activity of flavonol and flavan-3-ol rich extracts from Croton celtidifolius latex.

CONTEXT: Croton celtidifolius Baill (Euphorbiaceae) is a tree found in the Atlantic Forest in Southern Brazil, where it is commonly known as "Sangue-de-Dragão". Its red latex is used traditionally for treating ulcers, diabetes and cancer. OBJECTIVE: To evaluate antitumor activities of Croton celtififolius latex in vitro and in vivo. MATERIAL AND METHODS: Phytochemical analyses were conducted using HPLC-DAD-MS. Cytotoxic, nuclease and pro-apoptotic properties were determined using the tetrazolium salt assay (MTT), plasmid DNA damage assay and ethidium bromide (EB)/acridine orange methods, respectively, and antitumor activity was determined in the Ehrlich ascites carcinoma (EAC) mouse model. RESULTS: Phytochemical studies indicated a high phenol content of flavonols (45.67 ± 0.24 and 18.01 ± 0.23 mg/mL of myricetin and quercetin, respectively) and flavan-3-ols (114.12 ± 1.84 and 1527.41 ± 16.42 mg/L of epicatechin and epigallocatechin, respectively) in latex. These compounds reduced MCF-7 and EAC cell viability in the MTT assay (IC50 = 169.0 ± 1.8 and 187.0 ± 2.2 µg/mL, respectively). Latex compounds caused significant DNA fragmentation and increased the number of apoptotic cells (negative control (NC), 12%; latex, 41%) as indicated by differential staining in the EB/acridine orange assay. The in vivo latex treatment at 3.12 mg/kg/day reduced the body weight by 7.57 ± 2.04 g and increased median survival time to 17.5 days when compared to the NC group (13.0 days). In addition, the highest latex concentration inhibited tumor growth by 56%. DISCUSSION AND CONCLUSION: These results agree with ethno-pharmacological reports showing cytotoxicity and antitumor activity of C. celtidifolius latex. The mechanism of antitumor action may be related to direct DNA fragmentation that reduces survival and induces apoptosis.

Sodium orthovanadate associated with pharmacological doses of ascorbate causes an increased generation of ROS in tumor cells that inhibits proliferation and triggers apoptosis.

Pharmacological doses of ascorbate were evaluated for its ability to potentiate the toxicity of sodium orthovanadate (Na(3)VO(4)) in tumor cells. Cytotoxicity, inhibition of cell proliferation, generation of ROS and DNA fragmentation were assessed in T24 cells. Na(3)VO(4) was cytotoxic against T24 cells (EC(50)=5.8 µM at 24 h), but in the presence of ascorbate (100 µM) the EC(50) fell to 3.3 µM. Na(3)VO(4) plus ascorbate caused a strong inhibition of cell proliferation (up to 20%) and increased the generation of ROS (4-fold). Na(3)VO(4) did not directly cleave plasmid DNA, at this aspect no synergism was found occurring between Na(3)VO(4) and ascorbate once the resulting action of the combination was no greater than that of both substances administered separately. Cells from Ehrlich ascites carcinoma-bearing mice were used to determine the activity of antioxidant enzymes, the extent of the oxidative damage and the type of cell death. Na(3)VO(4) alone, or combined with ascorbate, increased catalase activity, but only Na(3)VO(4) plus ascorbate increased superoxide dismutase activity (up to 4-fold). Oxidative damage on proteins and lipids was higher due to the treatment done with Na(3)VO(4) plus ascorbate (2-3-fold). Ascorbate potentiated apoptosis in tumor cells from mice treated with Na(3)VO(4). The results indicate that pharmacological doses of ascorbate enhance the generation of ROS induced by Na(3)VO(4) in tumor cells causing inhibition of proliferation and apoptosis. Apoptosis induced by orthovanadate and ascorbate is closer related to inhibition on Bcl-xL and activation of Bax. Our data apparently rule out a mechanism of cell demise p53-dependent or related to Cdk2 impairment.