New forms of induction of apoptosis in aggressive lymphoma using peptides that interrupt the RAS / RAF interaction

Interpretation: RAS-RAF-MEK-ERK is a key pathway for apoptosis regulation in cancer cells. B-Raf-inhibitors such as PLX4032 peptide was developed by Institute Curie-Université Pierre et Marie Curie in order to induce apoptosis in cancer cells. Objectives: To demonstrate pro-apoptotic properties and survival outcome of EP2014/064243 peptide in murine aggressive lymphoma. Material and Methodology: BALBc mice with T-lymphoma were randomized assigned either in Group A (peptide+cyclophosphamide-CFM); Group B (peptides), Group C (CFM-control) or Control D (Cl-Na 0.9%-SF control group). Survival probability was calculated by Kaplan-Meier analysis. Apoptosis was detected using TUNEL technique. The protocol was approved by the Institutional Committee for Animal Care (CICUAL: T04-01-2015) Results: The median survival was 24 days (21.6-26.4) for placebo, 33 days (28.0-35.4) for the CFM monotherapy group, 33 (27.1-35.8) for the peptide group and 34 days (24,4-40) for CFM-peptide combined treatment (p<0.05). In lymph node tissue the mean TUNEL positive cells per field for each treatment group was 2, 12 and 13 and 35 for SF, CFM, peptide and combined therapy (p<0.05). Conclusion: These findings suggest that in murine aggressive lymphoma treated by an experimental peptide in addition with CFM, had an exponentially pro-apoptotic effect than CFM alone, suggesting that the peptide potentiated the anti-tumoural effect of CFM.

Correction to: T cell leukemia control via Ras-Raf pathway inhibition with peptides.

[This corrects the article on p. 172 in vol. 10, PMID: 29075346.].

T cell leukemia control via Ras-Raf pathway inhibition with peptides.

RATIONALE: RAS-RAF-MEK-ERK pathway has been considered a promising target for anticancer therapy. However, tumor cells may develop resistance against such drugs via hyperactivation of N-Ras, which explains why novel therapeut-ic approaches. In this sense, the Institute Curie- Université Pierre et Marie Curie (Paris 6) designed peptides in order to disturb Ras/Raf interaction which showed pro-apoptotic properties. These peptides were patented as WO2015001045 A2 (PCT/EP2014/064243)5. OBJECTIVE: In order to check the anti-tumoral action of WO2015001045 A2 peptides in a very aggressive BALB/c mice spontaneous leukemia called LB, we performed the present study. METHOD & RESULTS: 50 BALB/c mice inoculated with 106 LB tumor cells were randomly assigned either to control (placebo) or treatment group (that daily received 3 mg of peptide per kg of mice) during 30 days. By day 15 only 24% of the control group was alive vs. 100% of the treatment group. The average survival in treated group was 20,27 days while in control group the mean survival was 15,48 days. Either bone marrow, spleen or axillary nodes demonstrated a higher level of malignant T cell presence compare with treated group (89,78% ; 95,64% & 77,68% versus 72,45%, 80,23% & 63.44% respectively for each organ inspected. DISCUSSION: Our study demonstrated an improvement in survival curves in mice model affected by spontaneous T lymphoid leukemia when peptides WO2015001045 A2 were used. These peptides might be a valid option to become part of the therapeutic armory for malignant lymphoproliferative diseases control.

In vitro and in vivo effects of Ranunculus peltatus subsp. baudotii methanol extract on models of eicosanoid production and contact dermatitis.

Ranunculus (Crowfoot) species are numerous and they are all reputed to be counter-irritants and are used in several topical conditions. In order to study the pharmacological mechanisms of action underlying this popular use, a methanol extract of Ranunculus peltatus was tested in vitro in various assays involving eicosanoid and human elastase release by intact cells as well as in vivo, with models of delayed-type hypersensitivity (DTH) contact dermatitis. The extract proved to be a selective inhibitor of the cyclooxygenase-1 pathway, producing the total inhibition of 12-(S)-HHTrE release at 200 microg/mL, while leaving both 5-lipoxygenase and 12-lipoxygenase activities unaffected at the same dose. The n-hexane, chloroform and ethyl acetate fractions of the crude methanol extract inhibited LTB(4) release by intact rat peritoneal neutrophils, but more polar fractions were inactive and did not increase the 5-LOX activity as seen previously for extracts of other Ranunculus species. In the in vivo models, the methanol extract reduced the dinitrofluorobenzene (DNFB)-induced oedema by 40%, but failed to inhibit the oedema brought on by oxazolone. The results agree with the age-old assertion that Water Crowfoot species can be used as a topical antiinflammatory remedy without the prominent irritant action that accompanies the application of non-aquatic Ranunculus species.

Antioxidant activity of Paraguayan plant extracts.

The antioxidant properties of six medical herbs used in the traditional Paraguayan medicine were studied using free radical-generating systems. The methanol extracts from Aristolochia giberti, Cecropia pachystachya, Eugenia uniflora, Piper fulvescens, Schinus weinmannifolia and Schinus terebinthifolia protected against enzymatic and non-enzymatic lipid peroxidation in microsomal membranes of rat. C. pachystachya, E. uniflora, S. weinmannifolia and S. terebinthifolia showed the highest scavenging activity on the superoxide and DPPH radicals.

Inhibition of Trypanosoma cruzi growth by medical plant extracts.

This study describes the screening of extracts obtained from 18 plants and two fungi used in the Chinese and Mediterranean traditional medicines on epimastigote forms of Trypanosoma cruzi. The extracts were tested against epimastigote of T. cruzi Bra C15C2 clone in vitro at 27 degrees C and at a concentration of 250 microg/ml in axenic culture. Angelica dahurica, A. pubescens, A. sinensis, Astragalus membranaceus, Coptis chinensis, Haplophyllum hispanicum, Phellodendron amurense, Poria cocos, Ranunculus sceleratus and Scutellaria baicalensis showed significant effects against the parasite with a percentage of growth inhibition between 20 and 100%. C. chinensis and R. sceleratus showed the greatest activity with IC(50) values of 1.7 microg/ml for C. chinensis and 10.7 microg/ml for R. sceleratus. These activities are greater than that of allopurinol. C. chinesis and R. sceleratus extracts did not show cytotoxic effects on rat polimorphonuclear cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactic dehydrogenase assays. These results allowed us to suggest that R. sceleratus and C. chinensis could be a source of new compounds clinically active against T. cruzi.

Antioxidant activity of anti-inflammatory plant extracts.

The antioxidant properties of twenty medical herbs used in the traditional Mediterranean and Chinese medicine were studied. Extracts from Forsythia suspensa, Helichrysum italicum, Scrophularia auriculata, Inula viscosa, Coptis chinensis, Poria cocos and Scutellaria baicalensis had previously shown anti-inflammatory activity in different experimental models. Using free radical-generating systems H. italicum. I. viscosa and F. suspensa protected against enzymatic and non-enzymatic lipid peroxidation in model membranes and also showed scavenging property on the superoxide radical. All extracts were assayed at a concentration of 100 microg/ml. Most of the extracts were weak scavengers of the hydroxyl radical and C. chinensis and P. cocos exhibited the highest scavenging activity. Although S. baicalensis inhibited the lipid peroxidation in rat liver microsomes and red blood cells, the extract showed inhibitory actions on aminopyrine N-demethylase and xanthine oxidase activities as well as an pro-oxidant effect observed in the Fe3+-EDTA-H2O2 system. The results of the present work suggest that the anti-inflammatory activities of the same extracts could be explained, at least in part, by their antioxidant properties.

Is the protection against ischemia induced by red wine linked to its antioxidant capacity?

OBJECTIVE: To establish whether the total antioxidant capacity of nonalcoholic extracts of three Argentine red wines (RWE) is correlated with their protection against ischemia-reperfusion injury. ANIMALS AND METHODS: The antioxidant properties of three RWE were determined using different free radical-generating systems. To examine the effects of these RWE during a 20 min global ischemic period followed by 30 min of reperfusion, isolated rat hearts received 50 mug/mL of RWE 1 (cabernet-sauvignon), RWE 2 (malbec) or RWE 3 (a commercial mixture of cabernet-sauvignon, malbec and merlot) 10 min before and after ischemia. Left ventricular developed pressure (LVDP), maximal velocity of rise of left ventricular pressure (+dP/dt(max)) and left ventricular end-diastolic pressure (LVEDP) were used to assess contractility and diastolic function. RESULTS: All RWE inhibited lipid peroxidation induced by the Cl(4)C/NADPH system in a similar proportion (42+/-4%, 47+/-9% and 43+/-14% for RWE 1, RWE 2 and RWE 3, respectively). The scavenging activity of superoxide anion and 2,2-diphenyl-1-picryl-hydrazyl radical was about the same with the three RWE. In hearts without RWE treatment, LVDP and +dP/dt(max) were 61+/-4% and 62+/-5%, respectively, at the end of the reperfusion period. Infusion of RWE 1 and RWE 2 significantly improved postischemic recovery (LVDP and +dP/dt(max) were 102+/-4% and 101+/-4% for RWE 1 and 92+/-5% and 91+/-5% for RWE 2, respectively) and attenuated the increase of LVEDP. RWE 3 did not improve either systolic or diastolic dysfunction. CONCLUSION: These data show that although the three non-alcoholic RWE exhibit a similar total antioxidant capacity, only two of them protect the heart against myocardial stunning, suggesting that the protective effect is not primarily linked to the anti-oxidant properties of the extracts.

[Trypanosoma cruzi: obtaining extracellular amastigotes and studying their development under different culture conditions]. / Trypanosoma cruzi: obtención de amastigotes extracelulares y estudio de su crecimiento en diferentes condiciones de cultivo.

This work describes a protocol to obtain pure populations of extracellular amastigotes of Trypanosoma cruzi. The amastigote stage was obtained by means of temperature changes and human plasma added to the culture medium. Epimastigotes (clon BraC15C2) were first grown in F69 medium at 27 degrees C during 96 h and then at 36.5 degrees C. After three subcultures of 96 h each at the latter temperature a subsequent incubation in the presence of 5% human plasma, was needed to obtain a population of amastigotes that could be maintained indefinitely in the F69 or F29 media. This amastigote population was similar morphologically to that obtained through other methods. The kinetic of growth depended on the culture medium used (F29 or Brain-Heart Infusion, BHI). When culture was incubated at 27 degrees C in both media, the pre-exponential and logarithmic phases of growth were observed at 72-96 h and 24-48 h respectively. The change in stage from amastigote to epimastigote dependent whether amastigote were subcultured or not. The growth of amastigotes in BHI medium at 36.5 degrees C did not occurred. The growth of amastigotes was similar to those observed at 27 degrees C when F29 medium was used although the transformation to epimastigotes did not take place at this temperature. A population over 99% of amastigotes were maintained at 36.5 degrees C indefinitely by means of subcultures in F29 medium.

Antioxidant effects of an aqueous extract of Ilex paraguariensis.

In this work we investigate the antioxidant properties of an aqueous extract prepared from an infusion of Ilex paraguariensis (Aquifoliaceae) using free radical-generating systems. The extract inhibited the enzymatic and nonenzymatic lipid peroxidation in rat liver microsomes in a concentration-dependent fashion, with IC(50) values of 18 microg/ml and 28 microg/ml, respectively. The extract also inhibited the H(2)O(2)-induced peroxidation of red blood cell membranes with an IC(50) of 100 microg/ml and exhibited radical scavenging properties toward superoxide anion (IC(50) = 15 microg/ml) and 2,2-diphenyl-1-picrylhydrazyl radical. In the range of concentrations used, the extract was not a scavenger of the hydroxyl radical. Our results suggest that ingestion of extracts of Ilex paraguariensis could contribute to increase the antioxidant defense of an organism against free radicals attack.

Effect of the chloroform extract of Tanacetum vulgare and one of its active principles, parthenolide, on experimental gastric ulcer in rats.

This study examines the anti-ulcerogenic activity of a chloroform extract of Tanacetum vulgare and purified parthenolide, the major sesquiterpene lactone found in the extract. Gastric ulcers induced by oral administration of absolute ethanol to rats were reduced dose-dependently by oral pretreatment of animals with the chloroform extract (2.5-80 mg kg(-1)) or parthenolide (5-40 mg kg(-1)). When administered 30 min before challenge with the alcohol the protection ranged between 34 and 100% for the extract and 27 and 100% for parthenolide. When the products were administered orally 24 h before treatment with ethanol, 40 mg kg(-1) of the extract and of the lactone reduced the mean ulcer index from 4.8+/-0.3 for control animals to 1.4+/-0.2 and 0.5+/-0.1, respectively. The products also prevented alcohol-induced reduction of the number of sulphydryl groups within the gastric mucosa (50.6+/-2.3 microg (mgprotein)(-1) for normal animals compared with 17.7+/-3.0 microg (mg protein)(-1) for alcohol-treated animals). Administration of the extract (80 mg kg(-1)) or parthenolide (40 mg kg(-1)) 24 h before ethanol treatment restored the numbers of mucosal -SH groups to values near those found for normal animals. These results suggest that the products assayed, in particular parthenolide, might find therapeutic application, although further work is required to establish their profit/risk ratio.

Anti-inflammatory effects of South American Tanacetum vulgare.

In recent years the role of phenolic compounds and sesquiterpene lactones, particularly parthenolide, in the anti-migraine and anti-inflammatory effects of Tanacetum parthenium (Asteraceae) has attracted much attention. However, the closely-related cosmopolitan species T. vulgare has remained outside the mainstream of research in this field. After treating the aerial parts of T. vulgare with dichloromethane and methanol, and applying conventional column and thin-layer chromatographic techniques, it was possible to isolate from the moderately lipophilic fractions the principles responsible for the anti-inflammatory activity of this plant against the mouse-ear oedema induced by 12-O-tetradecanoylphorbol 13-acetate. These were identified by ultraviolet and nuclear magnetic resonance spectroscopy as parthenolide (93% oedema inhibition at 0.5 mg/ear, ID50 (dose of drug inhibiting the oedema by 50%) = 0.18 micromol/ear) and the methoxyflavones jaceosidin (80% oedema inhibition at 0.5 mg/ear, ID50 = 0.50 micromol/ear), eupatorin, chrysoeriol and diosmetin. Because in molar terms the potency of parthenolide was nearly three times greater than that of the most active of the flavones and because it is obtained from the plant in considerably larger amounts, the flavonoids must only be partially responsible, and to a minor extent, for the observed in-vivo anti-inflammatory local effect.

Effect of arsenic (V) on the antioxidant defense system: in vitro oxidation of rat plasma lipoprotein.

Adult male rats were treated orally with sodium arsenate (10 mg As/kd/day) for 2 days, and in increase in hepatic glutathione level was seen. Ascorbic acid content increased in both liver and plasma of intoxicated animals. Hepatic activities of superoxide dismutase and glutathione peroxidase did not change with the treatment and there was no increase in the level of lipid peroxidation measured as thiobarbituric acid-reacting substances (TBARS). Arsenic decreased the plasma level of uric acid and increased the plasma triglycerides content without modifying vitamin E levels. Both total lipoproteins and very low density lipoprotein plus low density lipoprotein (VLDL + LDL) fractions demonstrated greater propensity for in vitro oxidation than the corresponding untreated rats. The last finding might be a useful parameter for determining the degree of oxidative stress in the initial steps of intoxication with arsenic.

Effects of lovastatin on the fatty acid desaturation in a human lung mucoepidermoid carcinoma grown in nude mice.

The effect of lovastatin, a hypocholesterolemic drug, on tumor growth and desaturase activity was studied in a human lung mucoepidermoid carcinoma (HLMC) grown in nude mice. After administration of a diet supplemented with 25 mg% (w/w) lovastatin for 30 days the growth of HLMC was not inhibited. Liver and tumor phospholipid/cholesterol ratio was increased in lovastatin group but serum cholesterol was unaffected. Treatment with lovastatin increased delta 5 and delta 9 desaturation in tumor microsomes, whereas delta 6 desaturation did not change in tumors of treated mice. The changes were not reflected in the fatty acid composition of total tumor lipids.

[Trypanosoma cruzi: influence of human plasma on the morphogenesis of blood trypomastigotes in a cell-free culture media]. / Trypanosoma cruzi: influencia del plasma humano en la morfogénesis de tripomastigotes sanguíneos en un medio de cultivo acelular.

Morphogenesis of blood stream trypomastigotes in the cell free culture medium F69 at 37 degrees C for 10 days showed qualitative differences either with or without human plasma. Without human plasma, blood stream trypomastigotes performed only one cycle before disappearing and the culture kept growing as amastigotes and epimastigotes until the end of the experiment. In contrast, human plasma induced multiple cycles of transformation. The sequence was blood stream trypomastigotes, regressive parasites, amastigotes, progressive parasite and again trypomastigotes. Human plasma preserved the trypomastigote stage, produced a blockade of the epimastigote stage and inhibited the division of amastigotes. In this experimental model, human plasma modified the biological cycle of T. cruzi by inducing or inhibiting different stages.

[Effect of dietary supplementation with gamma-linolenic acid on the growth of a human lung carcinoma implanted in nude mice]. / Efecto de la suplementación dietética con acido gama-linolenico sobre el crecimiento de un carcinoma de pulmón humano implantado en ratones atímicos.

We have studied the effect of a gamma-linolenic acid (18:3 n-6, GLA)-supplemented diet on the growth of a human lung mucoepidermoid carcinoma (HLMC) implanted in athymic mice and on its uptake of human low density lipoproteins labeled with 99mTc (99mTc-LDL). Mice bearing the HLMC were divided into two experimental groups. One of them was administered a control diet (C diet) and the other one was given a diet supplemented with 25 mg GLA/g pellet (GLA diet) for three weeks (Table 1). A tumor growth inhibition with the GLA diet was evident at the second week of treatment, and a marked inhibition (56%) was reached at the end of the third week (Fig. 1). The GLA diet produced some changes in the total fatty acid composition of tumor, plasma and liver of host mice: GLA and arachidonic acid (20:4 n-6, AA) induced significant increases, whereas oleic (18:1 n-9, OA) and linoleic acids (18:2 n-6, LA) were decreased (Table 2). Tumors of those animals fed both diets were labeled by 99mTc-LDL, and no difference was observed in the ratio of tumor/liver and tumor/kidney uptake of host animal (Table 3). Results obtained using this experimental model suggest that the inhibitory effect of GLA on tumor growth is not related to the LDL tumor uptake.

Efecto de la suplementación dietética con acido gama-linolenico sobre el crecimiento de un carcinoma de pulmón humano implantado en ratones atímicos. / [Effect of dietary supplementation with gamma-linolenic acid on the growth of a human lung carcinoma implanted in nude mice]

We have studied the effect of a gamma-linolenic acid (18:3 n-6, GLA)-supplemented diet on the growth of a human lung mucoepidermoid carcinoma (HLMC) implanted in athymic mice and on its uptake of human low density lipoproteins labeled with 99mTc (99mTc-LDL). Mice bearing the HLMC were divided into two experimental groups. One of them was administered a control diet (C diet) and the other one was given a diet supplemented with 25 mg GLA/g pellet (GLA diet) for three weeks (Table 1). A tumor growth inhibition with the GLA diet was evident at the second week of treatment, and a marked inhibition (56

) was reached at the end of the third week (Fig. 1). The GLA diet produced some changes in the total fatty acid composition of tumor, plasma and liver of host mice: GLA and arachidonic acid (20:4 n-6, AA) induced significant increases, whereas oleic (18:1 n-9, OA) and linoleic acids (18:2 n-6, LA) were decreased (Table 2). Tumors of those animals fed both diets were labeled by 99mTc-LDL, and no difference was observed in the ratio of tumor/liver and tumor/kidney uptake of host animal (Table 3). Results obtained using this experimental model suggest that the inhibitory effect of GLA on tumor growth is not related to the LDL tumor uptake.