Synthesis and Biological Evaluation of Cyclic Analogues from Nitrone LQB-278: A New Potential Antileukemia Compound.

BACKGROUND/AIM: A new set of LQB-nitrones and analogues was synthesized to evaluate anticancer activity based on the substitution of the terpenyl moiety of the antileukemic compound LQB-278 by the conformationally restricted cinnamyl ether. MATERIALS AND METHODS: A structure-activity relationship study was performed in vitro on Jurkat cells to screen the antileukemic activity of LQB-nitrones and analogues and elucidate the mechanisms of action of the most active derivatives. RESULTS: The cynamyl ramification and its ortho position aldehyde substitution improved the antileukemic activity. Three compounds showed an in vitro antiproliferative action, but only 5b induced apoptosis. Analysis of the molecular mechanisms showed increased expression of the cell cycle inhibitor p21CIP1/WAF1/Sdi1, caspase 3, Fas receptor, and Bax/Bcl-2 ratio. CONCLUSION: The cinnamyl derivative 5b (LQB-461) presented higher antileukemic effects than the prototype terpenyl nitrone, inducing Jurkat cell death by activating both extrinsic and intrinsic pathways of apoptosis. Therefore, this compound is a new promising candidate drug against leukemia.

Anti-arthritic properties of crude extract from Chenopodium ambrosioides L. leaves.

OBJECTIVES: To evaluate the effect of hydroalcoholic crude extract (HCE) from Chenopodium ambrosioides leaves on the development of type II collagen-induced arthritis (CIA) and on pro-inflammatory cytokine balance. METHODS: Collagen-induced arthritis was induced in DBA1/J mice. On the 21st day, the mice were treated orally with HCE or methotrexate, daily. Six weeks after beginning the treatment, the following measures were determined: lymphoid organs cell numbers, percentage of blood cells, IL-6, IFN-γ, TNF-α and IL-17 serum concentrations, activity of hepatic and kidney glutathione S-transferase, hepatic 7-ethoxyresorufin-O-deethylase activity, bone density and histopathology. KEY FINDINGS: Treatment of CIA mice with HCE 5 mg/kg (HCE5) reduced the percentage of neutrophils and macrophages and the number of bone marrow cells and increased the lymphocyte numbers and the inguinal lymph node cellularity. This treatment inhibited the serum concentration of IL-6 and TNF-α, which may be related to the preservation of bone density and to the slight thickening of periarticular tissues, with minimal fibrosis and fibroblast proliferation in the joints. The CIA group presented advanced articular erosion and synovial hyperplasia. Phytochemical analysis showed mainly flavonols. CONCLUSIONS: HCE5 presented anti-arthritic potential and reduced IL-6 and TNF-α, which participate directly in the development and maintenance of the inflammatory process in rheumatoid arthritis.

Anti-inflammatory effect of diterpenes-enriched fractions from Pterodon polygalaeflorus through inhibition of macrophage migration and cytokine production.

OBJECTIVES: To evaluate the anti-inflammatory potential of Pterodon polygalaeflorus hexane extract (HE) and its fractions on macrophage migration in vitro and in vivo. METHODS: Hexane extract from P. polygalaeflorus fruits was fractionated and yielded four fractions. RAW 264.7 cells were treated with samples to evaluate cell viability (MTT assay), cell migration (wound healing and transwell assays), CD14 expression (flow cytometry), iNOS and cytokine mRNA expression (RT-qPCR), NO (Griess reaction) and cytokine (ELISA) production. In vivo migration was evaluated on the thioglycollate-induced peritonitis model. Qualitative analysis was performed by GC-MS. KEY FINDINGS: All fractions inhibited the NO production by LPS-stimulated RAW 264.7 cells. Fr3 and Fr4 presented the lowest IC50 values. The expressions of iNOS and IL-1ß, TNF-α and IL-10 cytokines were inhibited by Fr3 and Fr4, whereas the CD14 expression was only inhibited by Fr3. All the samples inhibited RAW 264.7 migration in the wound healing and transwell assays. Fr3 and Fr4 reduced the migration of Mac-1+ Gr-1- cells to the peritoneum and presented in their compositions: 6α-hydroxy-7ß-acetoxyvouacapan-17ß-oate, methyl 6α,7ß-dihydroxyvouacapan-17ß-oate, methyl 6α-acetoxy-7ß-hydroxyvouacapan-17ß-oate, geranylgeraniol and 14,15-epoxy-geranylgeraniol. CONCLUSIONS: The anti-inflammatory effects of Fr3 and Fr4 involve inhibition of cell migration, iNOS expression and NO production, cytokine expression (mRNA and proteins) and CD14 expression (Fr3).

The orally active pterocarpanquinone LQB-118 exhibits cytotoxicity in prostate cancer cell and tumor models through cellular redox stress.

BACKGROUND: The targeted induction of reactive oxygen species (ROS) is a developing mechanism for cancer therapy. LQB-118 is a pterocarpanquinone and ROS-inducing agent with proven antineoplastic activity. Here, LQB-118 efficacy and mechanism of activity, were examined in Prostate Cancer (PCa) cell and tumor models. METHODS: PC3, LNCaP, and LAPC4 PCa cells were applied. Dicoumarol treatment was used to inhibit quinone reductase activity. N-acetylcysteine (NAC) was applied as a ROS scavenger. ROS production was quantified by H2 DCFDA flow cytometry. LQB-118 treated cells were evaluated for changes in lipid peroxidation, viability, and apoptosis. Treatment-induced gene expression was measured by RT-qPCR and Western Blot. SOD1 knockdown was achieved with siRNA or miRNA mimic transfection. MicroRNA specificity was determined by 3'UTR reporter assay. Oral LQB-118 treatment (10 mg/kg/day) efficacy was determined in athymic male nude mice bearing subcutaneous PC3 xenograft tumors. RESULTS: LQB-118 treatment triggered PCa cell death and apoptosis. Therapeutic activity was at least partially dependent upon quinone reduction and ROS generation. LQB-118 treatment caused an increase in cellular ROS and lipid peroxidation. Treated cells exhibited elevated levels of NQO1, Nrf2, and SOD1. The miRNAs miR-206, miR-1, and miR-101 targeted and reduced SOD1 expression. The knockdown of SOD1, by siRNA or miRNA, enhanced LQB-118 cytotoxicity. Orally administered LQB-118 treatment significantly reduced the growth of established PCa xenograft tumors. CONCLUSION: LQB-118 is a developing and orally active pterocarpanquinone agent that effectively kills PCa cells through quinone reduction and ROS generation. The inhibition SOD1 expression enhances LQB-118 activity, presumably by impairing the cellular antioxidant response.

In Vitro Antileukemic Activity of Xanthosoma sagittifolium (Taioba) Leaf Extract.

Xanthosoma sagittifolium Schott is a herb of the Araceae family, popularly known as taioba, which is consumed as food in some regions of Brazil, Africa, and Asia. This species has already been evaluated for the antifungal activities. However, based on its potential antitumor activity, the present study further aimed to examine the antitumor, as well as chelation, activity of X. sagittifolium leaf extract. Results showed that hydroethanolic extract of X. sagittifolium leaves (HEXs-L) exhibits cytotoxic effects against the immortalized line of human T-lymphocytic (Jurkat) and myelogenous (K562) leukemia cells, but not nontumor RAW 264.7 macrophages or NIH/3T3 fibroblasts. HEXs-L inhibited 50.3% of Jurkat cell proliferation, reducing by 20% cells in G2/M phase, but increasing cells in sub-G1 phase, thereby inducing apoptosis by 54%. In addition, HEXs-L inhibited NO production by 59%, as determined by Griess reaction, and chelated 93.8% of free Fe(II), as demonstrated by ferrozine assay. Phytochemical studies were carried out by ESI-MS, identifying apigenin di-C-glycosides as major compounds. Overall, this work revealed that leaf extract of Xanthosoma sagittifolium presented chelating activity and in vitro antitumor activity, arresting cell cycle and inducing apoptosis of leukemia cells, thus providing evidence that taioba leaves may have practical application in cancer therapy.

Synthesis of N-methylarylnitrones derived from alkyloxybenzaldehydes and antineoplastic effect on human cancer cell lines.

New O-isoprenylated-N-methylarylnitrones derived from isomeric o, m and p-hydroxybenzaldehydes have been prepared and the antineoplastic effects on human cancer cell lines were evaluated. The O-geranylated nitrone LQB-278 (1b) and its isomers 2b and 3b inhibited the NO production, but the anti-leukemic activity was drastically dependent on nitrone isomer, with the 1b being the most effective one (IC50 of 6.7 µM) on Jurkat leukemia cell, by MTT assay. In addition, 1b up-regulated p21CIP1/WAF1/Sdi1 protein expression (flow cytometry), a cell cycle inhibitor, reduced cell growth, and induced DNA fragmentation (increased sub-G1 phase cells) and phosphatidylserine externalization in plasmatic membrane (increased annexin V positive cells). Finally, the 1b up-regulation of p21 expression and apoptosis induction seem to be the mechanisms by which it promotes its anti-leukemic effects, making this new molecular architecture a promising prototype for leukemia intervention.

Proliferation and differentiation of Trypanosoma cruzi inside its vector have a new trigger: redox status.

Trypanosoma cruzi proliferate and differentiate inside different compartments of triatomines gut that is the first environment encountered by T. cruzi. Due to its complex life cycle, the parasite is constantly exposed to reactive oxygen species (ROS). We tested the influence of the pro-oxidant molecules H2O2 and the superoxide generator, Paraquat, as well as, metabolism products of the vector, with distinct redox status, in the proliferation and metacyclogenesis. These molecules are heme, hemozoin and urate. We also tested the antioxidants NAC and GSH. Heme induced the proliferation of epimastigotes and impaired the metacyclogenesis. ß-hematin, did not affect epimastigote proliferation but decreased parasite differentiation. Conversely, we show that urate, GSH and NAC dramatically impaired epimastigote proliferation and during metacyclogenesis, NAC and urate induced a significant increment of trypomastigotes and decreased the percentage of epimastigotes. We also quantified the parasite loads in the anterior and posterior midguts and in the rectum of the vector by qPCR. The treatment with the antioxidants increased the parasite loads in all midgut sections analyzed. In vivo, the group of vectors fed with reduced molecules showed an increment of trypomastigotes and decreased epimastigotes when analyzed by differential counting. Heme stimulated proliferation by increasing the cell number in the S and G2/M phases, whereas NAC arrested epimastigotes in G1 phase. NAC greatly increased the percentage of trypomastigotes. Taken together, these data show a shift in the triatomine gut microenvironment caused by the redox status may also influence T. cruzi biology inside the vector. In this scenario, oxidants act to turn on epimastigote proliferation while antioxidants seem to switch the cycle towards metacyclogenesis. This is a new insight that defines a key role for redox metabolism in governing the parasitic life cycle.

Antitumor screening of Pterodon pubescens terpenic fraction indicates high sensitivity for lymphocytic leukemia cells.

Cancer is the second leading cause of human mortality worldwide. Therefore, the search for new drugs or alternative therapy strategies has been required. Anticancer agents have been developed from plants since the 1950s and natural products still represent an important source of new and promising bioactive molecules. This work describes the cytotoxic effects of SF5 on tumor cells of high prevalence in the world and investigated some mechanisms of its antitumor action. The antitumor screening was performed with human lung carcinoma (A549), human breast (MCF-7) and prostate (PC-3) adenocarcinoma and chronic myeloid and acute lymphocytic leukemia cell lines. The acute lymphocytic leukemia Jurkat cells presented high sensitivity to the cytotoxic effects of SF5 (inhibition of 85-90%), compared with either the chronic myeloid leukemia K562 or solid tumor cell lines (lung, breast and prostate). SF5 arrested the cell cycle in G1 phase, which may be related with the observed downregulation of mRNA expression of c-Myc transcription factor at 24 h and 36 h. SF5 treatment induced cytochrome c release from mitochondria to cytosol, leading the Jurkat cells into apoptosis, which was evidenced by the internucleosomal fragmented DNA and increased number of annexin V-FITC positive cells. The SF5 showed high cytotoxicity for lymphocytic leukemia cells and low or none for solid tumor cells, without toxicity for peripheral mononuclear cells of healthy humans. SF5 altered gene expression, arrested the cell cycle and induced apoptosis via the mitochondrial pathway, similar to traditional antineoplastic chemotherapeutic drugs.

The pterocarpanquinone LQB-118 inhibits tumor cell proliferation by downregulation of c-Myc and cyclins D1 and B1 mRNA and upregulation of p21 cell cycle inhibitor expression.

The incidence of cancer grows annually worldwide and in Brazil it is the second cause of death. The search for anti-cancer drugs has then become urgent. It depends on the studies of natural and chemical synthesis products. The antitumor action of LQB-118, a pterocarpanquinone structurally related to lapachol, has been demonstrated to induce mechanisms linked to leukemia cell apoptosis. This work investigated some mechanisms of the in vitro antitumor action of LQB-118 on prostate cancer cells. LQB-118 reduced the expression of the c-Myc transcription factor, downregulated the cyclin D1 and cyclin B1 mRNA levels and upregulated the p21 cell cycle inhibitor. These effects resulted in cell cycle arrest in the S and G2/M phases and inhibition of tumor cell proliferation. LQB-118 also induced programmed cell death of the prostate cancer cells, as evidenced by internucleosomal DNA fragmentation and annexin-V positive cells. Except the cell cycle arrest in the S phase and enhanced c-Myc expression, all the mechanisms observed here for the in vitro antitumor action of LQB-118 were also found for Paclitaxel, a traditional antineoplastic drug. These findings suggest new molecular mechanisms for the LQB-118 in vitro antitumor action.

Pterodon polygalaeflorus essential oil modulates acute inflammation and B and T lymphocyte activation.

The increased life expectancy of the population has led to increasing incidences of cancer, chronic inflammatory and autoimmune diseases. Thus the continuous search for new drugs is necessary because ineffectiveness and adverse effects have been described for standard drugs. Essential oils are important sources of bioactive metabolites and several clinical trials have been developed using them. The Pterodon genus has been used in traditional medicine to treat rheumatic disorders, thus this work investigated the properties of essential oil from Pterodon polygalaeflorus fruits (EsOPpg) on acute inflammation and lymphocyte activation. The essential oil was obtained by hydrodistillation and its components were identified by GC/MS. The anti-inflammatory response was assessed using the air pouch model. Antinociceptive potential was evaluated using the writhing model. Lymphocyte phenotyping, cell cycle and apoptosis were analyzed by flow cytometry. EsOPpg promoted a reduction in leukocyte counts and protein concentration in the exudate, and reduced vasodilatation and inflammatory cell infiltrate in air pouch tissue. No antinociceptive effect was demonstrated for the doses tested. EsOPpg inhibited lymphocyte proliferation, arresting the cell cycle in G1 phase, and induced apoptosis in these cells. EsOPpg downregulated both the total number of CD8(+) T cells and the activated subpopulation (CD8(+)CD69(+)), while promoting upregulation of the total number of CD19(+) and CD19(+)CD69(+) B cells. In conclusion, Pterodon polygalaeflorus essential oil diminished the acute inflammatory response and inhibited lymphocyte proliferation, reducing neutrophil recruitment into the cavity and air pouch tissue and promoting distinct modulations of the activation level of each lymphocyte subpopulation.

Hypoglycemic effect of Bumelia sartorum polyphenolic rich extracts.

Bumelia sartorum (Sapotaceae) is used ethnomedicinally for treatment of several diseases, including diabetes mellitus. The aim of this work was to investigate the hypoglycemic effect of B. sartorum extracts, rich in polyphenolic compounds, and the possible mechanisms of action. Assessment of B. sartorum hypoglycemic activity was performed from the blood glucose level in normoglycemic mice after administration of the extract by oral gavage. The hypothesis that sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibition could prolong the increase in cytoplasmic Ca2+ concentration, thus leading to an increase of insulin release was evaluated. The enzyme inhibition was measured by ATP hydrolysis using SERCA1 isolated from rabbit skeletal muscle. The total content of phenolic compounds was determined by the Folin-Ciocalteau method. The ethyl acetate (EtOAc) partition and F5 fraction obtained from B. sartorum, both of them rich in polyphenolics, were shown to have a hypoglycemic effect on normoglycemic mice, more significant than that of the known antidiabetic drug, glibenclamide used as a standard comparable compound. Both samples significantly inhibited SERCA activity. Different extracts of B. sartorum, rich in polyphenolic compounds, were able to reduce blood glucose in normoglycemic mice and inhibit SERCA activity. SERCA inhibition may be one of the possible mechanisms involved in glucose decrease.

Innate profiles of cytokines implicated on oral tolerance correlate with low- or high-suppression of humoral response.

SUMMARY: Oral tolerance (OT) is being studied with great interest because of its therapeutic potential in allergy and autoimmunity. In the present study, two mouse strains with extreme phenotypes of OT susceptibility (TS) or resistance (TR) to ovalbumin (OVA) were used to demonstrate whether the tr and ts genes, cumulated during 18 generations of bi-directional genetic selection, influence expression of immunobiological traits in naive or antigen-gavaged TR/TS mice. The difference in anti-OVA titres was 2048-fold between OVA-gavaged TS and TR mice. Tolerance susceptibility to OVA gavage in individuals from a (TS x TR)F(2) population was 24% high-susceptibility, 62% low-susceptibility and 14% non-tolerant. Different antigens, unrelated to OVA, were tested by gavage and TS mice were generally susceptible while TR mice were resistant. The stability of TS and TR phenotypes was not affected by the use of strict protocols of intraperitoneal immunization or feeding over 30 consecutive days. The levels of interleukin-2 (IL-2), IL-4, interferon-gamma and IL-10 cytokines evaluated in concanavalin A-stimulated spleen cells from naive mice and in OVA-stimulated spleen cells from OVA-gavaged mice were higher in TS mice. Interleukin-10 was up-regulated in OVA-gavaged TS mice and down-regulated in TR mice. In naive mice, the percentage of CD4(+) CD25(+) and CD4(+) Foxp3(+) spleen cells and IL-10 expression by CD4(+) cells was significantly higher in TS mice. These results indicate that regulation of IL-10 expression could be an important factor contributing to the mechanisms controlling OT susceptibility, and that the OT responses of TR and TS individuals strongly correlate with their innate potential to secrete this cytokine.

Medicinal potential from in vivo and acclimatized plants of Cleome rosea.

Methanolic extracts obtained from different organs of Cleome rosea, collected from its natural habitat and from in vitro-propagated plants, were submitted to in vitro biological assays. Inhibition of nitric oxide (NO) production by J774 macrophages and antioxidant effects by protecting the plasmid DNA from the SnCl(2)-induced damage were evaluated. Extracts from the stem of both origins and leaf of natural plants inhibited NO production. The plasmid DNA strand breaks induced by SnCl(2) were reduced by extracts from either leaf or stem of both sources. On the other hand, root extracts did not show any kind of effects on plasmid DNA, and presented significant toxic effects to J774 cells. The results showed that C. rosea presents medicinal potential and that the acclimatization process reduces the plant toxicity both to plasmid DNA and to J774 cells, suggesting the use of biotechnology tools to obtain elite plants as source of botanical material for pharmacological and phytochemical studies.