Terpenic subfraction of Pterodon pubescens induces apoptosis of K562 leukemic cells by modulating gene expression

International audienceDeregulation of cell proliferation and apoptosis is linked to malignant cell development. Leukemia is the most frequent cancer in children, and plants are important sources for new potential anti-cancer agents. Although anti-tumoral effects have been shown for Pterodon pubescens extracts, the mechanisms are still obscure. This study describes in Pterodon pubescens a furane diterpene only reported in Pterodon polygalaeflorus, the methyl-6 alpha-acetoxy-7 beta-hydroxyvouacapan-17 beta-oate, indicated by HRMS and (13)C-NMR analysis, and demonstrates some mechanisms of the anti-leukemia action of its terpene subfraction SF5. SF5 induced cytotoxic and anti-proliferative effects on K562 cells. Increased sub-G1 nuclei and Annexin V(+)-FITC cells confirmed apoptosis of leukemic cells by treatment of these cells with SF5. Down-regulation of DNMT1 gene transcription and over-expression of Apaf-1 mRNA suggested that SF5 may be inducing apoptosis of K562 cells by epigenetic up-regulation of pro-apoptotic proteins involved in the mitochondrial intrinsic pathway

Heme-Induced ROS in Trypanosoma Cruzi Activates CaMKII-Like That Triggers Epimastigote Proliferation. One Helpful Effect of ROS

Heme is a ubiquitous molecule that has a number of physiological roles. The toxic effects of this molecule have been demonstrated in various models, based on both its pro-oxidant nature and through a detergent mechanism. It is estimated that about 10 mM of heme is released during blood digestion in the blood-sucking bug's midgut. The parasite Trypanosoma cruzi, the agent of Chagas' disease, proliferates in the midgut of the insect vector; however, heme metabolism in trypanosomatids remains to be elucidated. Here we provide a mechanistic explanation for the proliferative effects of heme on trypanosomatids. Heme, but not other porphyrins, induced T. cruzi proliferation, and this phenomenon was accompanied by a marked increase in reactive oxygen species (ROS) formation in epimastigotes when monitored by ROS-sensitive fluorescent probes. Heme-induced ROS production was time-and concentration-dependent. In addition, lipid peroxidation and the formation of 4-hydroxy-2-nonenal (4-HNE) adducts with parasite proteins were increased in epimastigotes in the presence of heme. Conversely, the antioxidants urate and GSH reversed the heme-induced ROS. Urate also decreased parasite proliferation. Among several protein kinase inhibitors tested only specific inhibitors of CaMKII, KN93 and Myr-AIP, were able to abolish heme-induced ROS formation in epimastigotes leading to parasite growth impairment. Taken together, these data provide new insight into T. cruzi- insect vector interactions: heme, a molecule from the blood digestion, triggers epimastigote proliferation through a redox-sensitive signalling mechanism

Morphological aspects of fruits, seeds, seedlings and in vivo and in vitro germination of species of the genus Cleome

The genus Cleome is widely distributed in drier areas of the tropics and subtropics. Cleome dendroides and C. rosea are Brazilian native species that occur mainly in Atlantic Forest and sandy coastal plains, respectively ecosystems negatively affected by human impacts. Cleome spinosa is frequently found in urban areas. Many Cleome species have been used in traditional medicine, as C. spinosa. In the present work, was investigated C. dendroides, C. rosea and C. spinosa germinative behavior under in vivo conditions, as well as was established suitable conditions to in vitro germination and seedling development. The in vivo germination was performed evaluating the influence of temperature, substrate and light. It was observed that only C. spinosa seeds presents physiological dormancy, which was overcome by using alternate temperatures. The substrate influenced significantly the germination of C. rosea and the seeds of C. dendroides showed the highest germination percentages in the different conditions evaluated. The post-seminal development stages under in vivo and in vitro conditions were defined. It was observed that the development was faster under in vitro than in vivo conditions. An effective methodology for in vitro germination, enabling the providing of material to experiment on plant tissue culture was established to C. dendroides and C. spinosa

The heme uptake process in Trypanosoma cruzi epimastigotes is inhibited by heme analogues and by inhibitors of ABC transporters

Heme (iron protoporphyrin IX) is an important molecule involved in many biological reactions, including oxygen transport, respiration, photosynthesis and drug detoxification. Trypanosoma cruzi parasites, the etiological agent of Chagas’ disease, take up heme from the environment to supply their nutritional needs because they do not synthesize this cofactor. However, the mechanisms involved in heme transport across biological membranes are poorly understood. Indeed, in T. cruzi, no heme transporter has yet been characterized. In the present work, we evaluate the heme uptake processes by T. cruzi epimastigotes using fluorescent heme-analogues. Heme uptake decreased significantly when cells were pretreated with different concentrations of SnPPIX, PdMPIX or ZnMPIX, this observed competition suggests that they are taken up by the same transport system. We studied the growth behavior of epimastigotes using the same heme-analogues and the treatments with SnPPIX or PdMPIX impaired cell growth but when heme was added to the culture medium the observed inhibition was partially reversed. In addition, we tested how the heme uptake processes are affected by the presence of different transporter inhibitors. When the cells were treated with inhibitors and then incubated with heme, heme uptake decreased significantly for all treatments. These results constitute a strong indication for the existence of a protein associated with porphyrin transport in T. cruzi, possibly ATP-binding cassette transporters (ABC-transporter).Fil: Cupello Peixoto, Mauricio. Universidade do Estado do Rio de Janeiro. Instituto de Biologia Roberto Alcântara Gomes (IBRAG). Departamento de Bioquímica. Laboratório de Interação Tripanossomatídeos e Vetores; Brazil.Fil: Fernandes de Souza, Cíntia. Universidade do Estado do Rio de Janeiro. Instituto de Biologia Roberto Alcântara Gomes (IBRAG). Departamento de Bioquímica. Laboratório de Interação Tripanossomatídeos e Vetores; Brazil.Fil: Buchensky, Celeste. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Rocha Corrêa Soares, Juliana Baptista. Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica. Programa de Biologia Molecular e Biotecnologia. Laboratório de Bioquímica Redox; BrazilFil: Travassos Laranja, Gustavo Augusto. Universidade do Estado do Rio de Janeiro. Instituto de Biologia Roberto Alcântara Gomes (IBRAG). Departamento de Bioquímica. Laboratório de Interação Tripanossomatídeos e Vetores; Brazil.Fil: Garcia Pinto Coelho, Marsen. Universidade do Estado do Rio de Janeiro. Instituto de Biologia Roberto Alcântara Gomes (IBRAG). Departamento de Bioquímica. Laboratório de Interação Tripanossomatídeos e Vetores; Brazil.Fil: Cricco, Julia Alejandra. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Química Biológica. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Paes, Marcia Cristina. Universidade do Estado do Rio de Janeiro. Instituto de Biologia Roberto Alcântara Gomes (IBRAG). Departamento de Bioquímica. Laboratório de Interação Tripanossomatídeos e Vetores; Brazil.Fil: Paes, Marcia Cristina. Instituto Nacional de Ciência e Tecnologia. Entomologia Molecular (INCT-EM); Brazi

Avaliação da proteína amilóide A sérica na atividade clínica da artrite reumatóide

A artrite reumatóide (AR) é uma doença auto-imune, crônica, caracterizada pelo comprometimento inflamatório das articulações sinoviais periféricas. A proteína amilóide A sérica (SAA) é uma das principais proteínas de fase aguda (PFA), porém seu uso na rotina do laboratório clínico ainda é pouco difundido. OBJETIVO: O objetivo deste trabalho foi analisar a utilidade da SAA na avaliação da atividade clínica da AR. MÉTODOS: Foram estudados 113 pacientes com AR, diagnosticados segundo os critérios do Colégio Americano de Reumatologia. Para a caracterização da atividade de doença, foi utilizado o Índice de Atividade de Doença (IAD), proposto pela Liga Européia Contra o Reumatismo. RESULTADOS: A SAA apresentou correlação positiva, estatisticamente significativa, com a proteína C-Reativa (PCR), tanto como a α-1-glicoproteína ácida (AGP), quanto com o IAD. Nossos resultados demonstraram que a SAA apresentou, particularmente, uma maior sensibilidade na determinação da atividade inflamatória da AR, em comparação às outras PFA. Apresentou, também, uma boa capacidade de discriminar os grupos de atividade moderada e alta do IAD. Como o IAD não mede unicamente o componente inflamatório da AR, a dosagem de uma PFA é de grande utilidade para a caracterização da atividade dessa enfermidade. CONCLUSÕES: Os resultados deste estudo sugerem que a SAA pode ser de grande valor na determinação da atividade inflamatória da AR

Serum cartilage biomarkers in late rheumatoid arthritis

Objectives: To evaluate the clinical value of two new cartilageserum markers in patients with rheumatoid arthritis of longduration. Methods: One hundred and forty patients followed for anaverage period of twelve years were evaluated for the presence ofhuman cartilage glycoprotein-39 and cartilage serum oligomericmatrix protein and the respective coefficient correlation betweenmarkers and disease activity score. Results: The mean values ofcartilage serum oligomeric matrix protein and human cartilageglycoprotein-39 were respectively 9 ± 6 ug/ml and 36 ± 16 mg/ml (p > 0,001) when compared to controls. A positive correlationwas observed between disease activity score and the cartilagebiomarkers (r = 0,67 for human cartilage glycoprotein-39 and r =0,83 for cartilage serum oligomeric matrix protein. Conclusion:This study shows in a inequivocal form that patientswith rheumatoid arthritis of long duration show higher values ofserum biochemical markers of cartilage metabolism and a positivecorrelation with disease activity score

Terpenic fraction of <it>Pterodon pubescens</it> inhibits nuclear factor kappa B and extracellular signal-regulated protein Kinase 1/2 activation and deregulates gene expression in leukemia cells

Abstract Background Plant derived compounds have been shown to be important sources of several anti-cancer agents. As cell cycle deregulation and tumor growth are intimately linked, the discovery of new substances targeting events in this biochemical pathway would be of great value. The anti-leukemic effect of an ethanolic extract of Pterodon pubescens seeds (EEPp) has been previously demonstrated and now we show that a terpenic subfraction (SF5) of EEPp containing farnesol, geranylgeraniol and vouacapan derivatives induces apoptosis in the human chronic myelogenous leukemia cell line K562. This work addresses SF5’s antiproliferative mechanisms in these cells since they are still unclear. Methods DNA synthesis in K562 cells was assessed by [3H]-methyl-thymidine incorporation and cell cycle status by flow cytometry. The expression of cyclins D1 and E2, of the cell cycle inhibitor p21 and of the proto-oncogene c-myc was evaluated by semi-quantitative RT-PCR. Extracellular-signal-regulated kinases (ERK) 1/2 and nuclear factor kappa B (NF-κB) activation was evaluated by western blotting. Results In K562 cells, SF5 treatment induced a higher inhibition of DNA synthesis and cell growth than the original EEPp hexanic fraction from which SF5 originated, and also arrested the cell cycle in G1. Exposure of these cells to SF5 led to a decrease in cyclin E2 and c-myc expression while p21 mRNA levels were increased. Furthermore, SF5 inhibited the activation of mitogen-activated protein kinase (MAPK) ERK 1/2 and NF-κB. Conclusions This work suggests that the anti-leukemic action of SF5 is linked to the inhibition of ERKs, NF-κB and c-myc signaling pathways resulting in reduced cyclin E2 mRNA expression and cell cycle arrest in the G1 phase.</p