Search

Supplementary Data from In vitro and In vivo Selective Antitumor Activity of Edelfosine against Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Involving Lipid Rafts

Purpose: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL.Experimental Design: Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice.Results: Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine ≫ erucylphosphocholine ≥ miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor.Conclusions: Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine. Clin Cancer Res; 16(7); 2046–54. ©2010 AACR.

Atherosclerosis, a chronic inflammatory condition in which atheroma accumulates within the intima of the arterial wall, is a life-threatening manifestation of cardiovascular disease, due to atheroma rupture, chronic luminal narrowing and thrombosis. Current knowledge of the role of a protective immune response in atherosclerotic lesions has provided promising opportunities to develop new immunotherapeutic strategies. In particular, Tregs exert an atheroprotective role by releasing anti-inflammatory cytokines (IL-10/TGF-β) and suppressing autoreactive T lymphocytes.Lay abstract Atherosclerosis is a chronic inflammatory disease in which the wall of the artery develops abnormalities, and can lead to serious problems, including heart attack, stroke, or even death. Scientific evidence has shown that the immune system is involved in the development and progression of atherosclerosis. Understanding the role of protective immune response in atherosclerosis provided promising opportunities to develop approaches for prevention and treatment.

Background: Cancer is one of the most common diseases in the world, with over 18 million new cases estimated in 2018. Many of the drugs used for cancer can have significant adverse effects and variable effectiveness. Nitroimidazoles are prodrugs that usually have shown antimicrobial activity specifically antiparasitic. However, its antitumor activity in vitro has barely been explored. Objective: The aim of this study is to determine the influence of the length of the substituted N-alkyl chain in the imidazole ring on the antitumor activity in vitro. Methods: Four nitroimidazoles were obtained by chemical synthesis varying the length of the substituted N-alkyl chain from methyl to butyl. The antitumor activity of N-alkyl-nitroimidazoles was evaluated by MTT assay employing two tumor cell lines (MDA-MB231 and A549). Results: In this study, it was reported that N-alkyl nitroimidazoles exhibited an LC50 as low as 16.7 µM in breast tumor cells MDA-MB231 while in normal Vero kidney cells, the LC50 was around 30 µM. It was also reported that the length of the substituted N-Alkyl chain in the imidazole ring affects the antitumoral activity in A549 lung cells. Conclusion: Increasing the length of the substituted N-Alkyl chain in the imidazole ring decreased the antitumor activity against only A549 cancer cells. N-alkyl nitroimidazoles exhibited considerable selectivity towards tumor cell lines.

Production of polysaccharides by white-rot-fungi in submerged cultivation has several advantages due to process control. This work deals with the submerged cultivation, extraction and antitumor activity of polysaccharides from a wild strain of Schizophyllum radiatum isolated from a tropical forest of Colombia. The mushroom was cultivated in laboratory conditions, and classified by classical and molecular taxonomy. Submerged cultivation was performed in a bioreactor of 5 L using a ligninolytic residue as substrate. The fermentation conditions were 30 ± 1 °C, pH 4.5, 300 rpm and 1.5 vvm of air for 4 days. The yields were 16.8 g/L (w/v) of biomass, and after extraction, 0.6 g/L of water-soluble exopolysaccharide (SEPS) and 2.01 % (w/w) of water-soluble intrapolysaccharide (SIPS) were obtained. In each extract total carbohydrate, glucans and protein contents were determined. Also, nuclear magnetic resonance (NMR), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, X-ray diffractometry (XRD), high performance liquid chromatography with refraction index detection (HPLC-RI), high performance gel permeation chromatography (HPGPC) and Nuclear Magnetic Resonance (NMR) analysis were performed. Results indicated that SEPS and SIPS are heteropolysaccharides with amorphous structure and high molecular weights. Antitumor and immunostimulant activity was evaluated in different cancer cell lines. The results suggest these polysaccharides have direct and indirect antitumor activity activating immune cells such as macrophages. These findings enhance our knowledge about new sources of fungal metabolites that serve as adjuvant, cheaper and less harmful alternatives to cancer treatment.

La meningitis es una patología prevalente en pediatría, tiene múltiples causales y alta morbimortalidad. Ante la sospecha se debe iniciar tratamiento empírico de amplio espectro, una rápida identificación del patógeno mejora el pronóstico y disminuye complicaciones. Esta revisión pretende evaluar la utilidad del FilmArray en el diagnóstico de meningitis en pediatría. Se realizó búsquedas en base de todos en Pubmed y Lilacs se encontraron 381, luego de excluir repetidos, por título y resumen; 8 artículos cumplieron los criterios de inclusión y exclusión. Con la estrategia PICO (P: niños entre 2 y 12 años, I: Método del FilmArray ME, C: Métodos cultivo, Gram y PCR específica, D: identificar el impacto sobre un uso racional de antibióticos, disminución del tiempo de hospitalización y otras comorbilidades asociadas). Evidenciando que el FilmArray ME es un examen con igual rendimiento diagnóstico que los métodos convencionales para el diagnóstico de meningitis en población infantil. No se logra evidenciar que mejore tiempo de hospitalización, uso de antibióticos o secuelas; hasta el momento ha igualado y en algunos casos superado el rendimiento del diagnóstico etiológico en la meningitis, aunque faltan estudios para ayudar a dilucidar su real valor en reducción de días de hospitalización y uso de antibióticos.

This work deals with the submerged cultivation, extraction and antitumor activity of polysaccharides from Lentinus crinitus. The fungus was isolated from a tropical forest (Antioquia, Colombia), cultivated in laboratory conditions, and classified by classical and molecular taxonomy. Then, it was cultivated in a bioreactor of 5 L using a ligninolytic residue as substrate. The fermentation conditions were 30 ± 1 °C, pH 4.5, 300 rpm and 1.5 vvm for 4 days. The yields of fermentation were 20 g/L of biomass. After extraction, 0.65 g/L of water-soluble exopolysaccharide (LEPS) and 3.3 mg/100 g of water-soluble intrapolysaccharide (LIPS) were obtained. In each extract total carbohydrate, glucans and protein contents were determined. Also, scanning electron microscopy (SEM), Fourier transform infrared (FTIR), X-ray diffractometry (XRD), high performance liquid chromatography with refraction index detection (HPLC-RI) and high performance gel permeation chromatography (HPGPC) analysis for characterization were performed. The antitumor activity was evaluated and polysaccharides not only showed anti-proliferative activity in breast cancer cells but also they activate J774 macrophages as evidenced by the increase of nitric oxide and tumor necrosis factor-α (inducers of tumor cell apoptosis). Our findings suggest that polysaccharides can activate macrophages to release nitric oxide (NO) and tumor necrosis factor alpha (TNF-α), which directly blocks cancer cell growth. These findings enhance our knowledge about new sources of fungal metabolites that serve as coadjuvant, cheap and less harmful alternatives to cancer treatment.

Consumption of Brassica (Cruciferae) vegetables is associated with a reduced risk of cancer, but identification of the active components and insights into the underlying molecular events are scarce. Here we found that an extract of Lepidium latifolium, a cruciferous plant native to southern Europe, Mediterranean countries and Asia, showed in vitro cytotoxic activity, inducing caspase-dependent apoptosis, in a variety of human tumor cells, and the plant juice showed in vivo antitumor activity in a HT-29 human colon cancer xenograft mouse model. The epithionitrile 1-cyano-2,3-epithiopropane (CETP) was identified as the major active cancer cell-killing principle of L. latifolium. Synthetic and plant-derived CETP displayed similar proapoptotic activities as assessed by biochemical and morphological analyses. Analysis of the antiproliferative capacity of CETP on a wide number of cancer cell lines from the NCI-60 cell line panel followed by COMPARE analysis, showed an activity profile different from known anticancer agents. Flow cytometry and biochemical analyses revealed that CETP-induced apoptosis involved mitochondria, as assessed by loss of mitochondrial transmembrane potential and generation of reactive oxygen species, while overexpression of Bcl-X and Bcl-2 prevented CETP-induced apoptosis. Inhibition of reactive oxygen species by glutathione and N-acetyl cysteine reduced the apoptotic response induced by CETP. FADD dominant negative form, blocking Fas/CD95 signaling, and a specific caspase-8 inhibitor also inhibited CETP-induced killing. Taken together, our data suggest that the cancer cell-killing action of CETP, involving both intrinsic and extrinsic apoptotic signaling pathways, underlies the antitumor activity of L. latifolium plant, which could be of potential interest in cancer treatment., This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2011-30518; SAF2014-59716-R; CTQ2014-55888-C03-01-R), Instituto de Salud Carlos III (RD12/0036/0065 from Red Temática de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund—FEDER), and the European Community's Seventh Framework Programme FP7-2007-2013 (HEALTH-F2-2011-256986, PANACREAS).

Leishmaniasis is a public health disease that requires the development of more effective treatments and the identification of novel molecular targets. Since blocking the PI3K/AKT pathway has been successfully studied as an effective anticancer strategy for decades, we examined whether the same approach would also be feasible in Leishmania due to their high amount and diverse set of annotated proteins. Here, we used a best reciprocal hits protocol to identify potential protein kinase homologues in an annotated human PI3K/AKT pathway. We calculated their ligandibility based on available bioactivity data of the reported homologues and modelled their 3D structures to estimate the druggability of their binding pockets. The models were used to run a virtual screening method with molecular docking. We found and studied five protein kinases in five different Leishmania species, which are AKT, CDK, AMPK, mTOR and GSK3 homologues from the studied pathways. The compounds found for different enzymes and species were analysed and suggested as starting point scaffolds for the design of inhibitors. We studied the kinases’ participation in protein–protein interaction networks, and the potential deleterious effects, if inhibited, were supported with the literature. In the case of Leishmania GSK3, an inhibitor of its human counterpart, prioritized by our method, was validated in vitro to test its anti-Leishmania activity and indirectly infer the presence of the enzyme in the parasite. The analysis contributes to improving the knowledge about the presence of similar signalling pathways in Leishmania, as well as the discovery of compounds acting against any of these kinases as potential molecular targets in the parasite.

The evidence regarding the role of regulatory B cells (Breg) in atherosclerosis are scarce, and there are contradictory data about their atheroprotective properties. Due to the demonstrated protective function of Breg in different inflammatory diseases mainly through interleukin-10 (IL-10) production, the knowledge of their participation in atherosclerosis immunopathology would be very valuable. To further study which B cell subsets participate in IL-10 production and their regulatory role, splenocytes from apolipoprotein-E-deficient mice were evaluated by ex vivo and in vitro cultures. Atherosclerotic mice had increased frequency of IL-10+ B cells, which presented high CD1d, CD19, and IgM, but variable CD5, CD21, and CD23 expression. IL-10+ B cells were not enriched in B cell subsets previously reported as Breg. Increased frequency of IL-10+ B cells with transitional 1-like (T1-like) and follicular (FO) and reduced CD5+ and marginal zone (MZ) phenotypes were observed ex vivo. Increased frequency of IL-10+ B cells with T1-like and MZ, and decreased IL-10+ FO and T2 phenotypes were also observed in vitro. To determine regulatory capacity of B cells in the atherosclerotic model, each subset were co-cultured with CD4+CD25- T cells. CD5+, FO, MZ, and T1-like cells from atherosclerotic mice exhibited regulation in an IL-10-dependent manner. However, only FO cells decreased both frequency of interferon gamma (IFN-γ)+ and tumor necrosis factor alpha (TNF-α)+ and proliferation of T cells. Finally, splenocytes showed increased frequency of IFN-γ+ and TNF-α+ cells only when FO-depleted B cells were evaluated. These results suggest that mainly FO B cells can modulate in some level the inflammatory responses observed in atherosclerosis.

[Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated., [Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast., [Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis., This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.

[Context]: C-6-Geranylated flavonoids possess promising biological activities. These substances could be a source of lead compounds for the development of therapeutics. [Objective]: The study was designed to evaluate their antibacterial and antileishmanial activity. [Materials and methods]: C-6-Geranylated flavanones were tested in micromolar concentrations against promastigote forms of Leishmaniabrazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC values (after 72 h) were calculated and compared with that of miltefosine. Flow cytometry and DNA fragmentation analysis were used the mechanism of the effect. Geranylated flavanones or epigallocatechin gallate were combined with oxacillin, tetracycline, and ciprofloxacin, and the effects of these two-component combinations were evaluated. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were established (after 24 h), the synergy was measured by the checkerboard titration technique, and the sums of the fractional inhibitory concentrations (∑FICs) were computed. [Results]: 3′-O-Methyl-5′-O-methyldiplacone and 3′-O-methyldiplacone showed good antileishmanial activities (IC 8–42 μM). 3′-O-Methyl-5′-hydroxydiplacone activates the apoptotic death at leishmanias, the effect of 3′-O-methyl-5′-O-methyldiplacone has another mechanism. The test of the antibacterial activity showed good effects of 3′-O-methyldiplacol and mimulone against MRSA (MIC 2–16 μg/mL), and in six cases, the results showed synergistic effects when combined with oxacillin. Synergistic effects were also found for the combination of epigallocatechin gallate with tetracycline or oxacillin. [Conclusion]: This work demonstrates anti-MRSA and antileishmanial potential of geranylated flavanones and uncovers their promising synergistic activities with antibiotics. In addition, the mechanism of antileishmanial effect is proposed., The work was supported by 93/2012/FaF, 63/2014/FaF, and 94/2013/FaF of UVPS Brno. Authors also thank the Project CIETUS 2010 for support.

Cardiovascular diseases are the most common cause of death in the world, atherosclerosis being its main underlying disease. Information about the role of B cells during atherosclerotic process is scarce, but both proatherogenic and atheroprotective properties have been described in the immunopathology of this disease. Frequency and phenotype of B cell subpopulations were studied in wild type and apolipoprotein-E-deficient (apoE−/−) mice fed or not with high-fat diet (HFD), by flow cytometry. Here, we provide the information about the materials, methods, analysis and additional information related to our study published in Atherosclerosis (DOI: 10.1016/j.atherosclerosis.2015.12.022, article reference: ATH14410) [1]. The data contained in this article shows and supports that mice with advanced atherosclerosis have a variety of alterations in frequency and phenotype of B cell subsets, most of which associated with dyslipidemia. Keywords: B cells, Atherosclerosis, Apolipoprotein E, Cholesterol, Dyslipidemia

The red seaweed Laurencia viridis is a rich source of secondary metabolites derived from squalene. New polyethers, such as iubol (2), 22-hydroxy-15(28)-dehydrovenustatriol (3), 1,2-dehydropseudodehydrothyrsiferol (4), and secodehydrothyrsiferol (5) have been isolated and characterized from this alga. The structures were determined through the interpretation of NMR spectroscopic data and the relative configuration was proposed on the basis of NOESY spectrum and biogenetic considerations. All new compounds exhibited significant cytotoxic activity against a panel of cancer cell lines.

[Background]: Lipid nanoparticles (LNs) made of synthetic lipids Compritol® 888 ATO and Precirol® ATO 5 were developed with an average size of 110.4 ± 2.1 and 103.1 ± 2.9 nm, and an encapsulation efficiency above 85% for both type of lipids. These LNs decrease the hemolytic toxicity of the drug by 90%. [Materials & methods]: Pharmacokinetic and biodistribution profiles of the drug were studied after intravenous and oral administration of edelfosine-containing LNs. [Results]: This provided an increase in relative oral bioavailability of 1500% after a single oral administration of drug-loaded LNs, maintaining edelfosine plasma levels over 7 days in contrast to a single oral administration of edelfosine solution, which presented a relative oral bioavailability of 10%. Moreover, edelfosine-loaded LNs showed a high accumulation of the drug in lymph nodes and resulted in slower tumor growth than the free drug in a murine lymphoma xenograft model, as well as potent extranodal dissemination inhibition. Original submitted 5 April 2011; Revised submitted 5 July 201. © 2012 Future Medicine Ltd.

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background]: The leishmaniases are a complex of neglected tropical diseases caused by more than 20 Leishmania parasite species, for which available therapeutic arsenal is scarce and unsatisfactory. Pentavalent antimonials (SbV) are currently the first-line pharmacologic therapy for leishmaniasis worldwide, but resistance to these compounds is increasingly reported. Alkyl-lysophospoholipid analogs (ALPs) constitute a family of compounds with antileishmanial activity, and one of its members, miltefosine, has been approved as the first oral treatment for visceral and cutaneous leishmaniasis. However, its clinical use can be challenged by less impressive efficiency in patients infected with some Leishmania species, including L. braziliensis and L. mexicana, and by proneness to develop drug resistance in vitro. [Methodology/Principal Findings]: We found that ALPs ranked edelfosine.perifosine.miltefosine.erucylphosphocholine for their antileishmanial activity and capacity to promote apoptosis-like parasitic cell death in promastigote and amastigote forms of distinct Leishmania spp., as assessed by proliferation and flow cytometry assays. Effective antileishmanial ALP concentrations were dependent on both the parasite species and their development stage. Edelfosine accumulated in and killed intracellular Leishmania parasites within macrophages. In vivo antileishmanial activity was demonstrated following oral treatment with edelfosine of mice and hamsters infected with L. major, L. panamensis or L. braziliensis, without any significant side-effect. Edelfosine also killed SbV-resistant Leishmania parasites in in vitro and in vivo assays, and required longer incubation times than miltefosine to generate drug resistance. [Conclusions/Significance]: Our data reveal that edelfosine is the most potent ALP in killing different Leishmania spp., and it is less prone to lead to drug resistance development than miltefosine. Edelfosine is effective in killing Leishmania in culture and within macrophages, as well as in animal models infected with different Leishmania spp. and SbV-resistant parasites. Our results indicate that edelfosine is a promising orally administered antileishmanial drug for clinical evaluation., This work was supported by the Spanish Ministerio de Ciencia e Innovación (SAF2008-02251; SAF2011-30518; RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union; and TRA2009-0275), European Community’s Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986), Junta de Castilla y León (CSI052A11-2; GR15-Experimental Therapeutics and Translational Oncology Program) and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032).

Two new cytotoxic oxasqualenoids, saiyacenols A and B, were isolated from the red alga Laurencia viridis and their structures elucidated by spectroscopic methods and chemical correlation with the well-known compound dehydrothyrsiferol. The new compounds share an important part of their structure with thyrsiferol and aplysiols B and C. Isolation of the saiyacenols concludes the controversy about the configuration of aplysiols, correcting the structure of aplysiol B. Our results illustrate the current limitations of NMR J-based methods when approaching quaternary carbons and demand a reappraisal of rational biogenetic proposals in structural elucidation., Financial support was provided by grants MAREX [FP 7; KBBE–3-245137 (EU)], SAF2011-28883-03-01 and SAF2008-02251 (MICINN); RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union, and Junta de Castilla y León (CSI052A11-2, and GR15-Experimental Therapeutics and Translational Oncology Program). F.C.P. acknowledges a repatriation fellowship (Consejo Nacional de Ciencia y Tecnología; 168081) and SEGAI-ULL fellowship.

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-x(L) by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine.

Three new polyether squalene derivatives 15-dehydroxythyrsenol A (2), prethyrsenol A (3) and 13-hydroxyprethyrsenol A (4) have been isolated from the red alga Laurencia viridis. Their structures were determined through the interpretation of NMR spectroscopic data and chemical correlations. In addition, four semi-synthetic compounds modulating the solubility of the lead compound dehydrothyrsiferol (1) were prepared without loss of activity. The cytotoxicity of the new compounds exhibited low μM activities. In order to explain their biological properties, docking simulations of the natural and synthetic compounds onto the αvβ3 integrin binding region were carried out. © 2011 Elsevier Masson SAS. All rights reserved., Financial support was provided by the grants CTQ2008-06754- C04-01 and SAF2008-02251 (MEC); C2008000145 and EXMAR (ACISII); RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union, and Junta de Castilla y León (GR15-Experimental Therapeutics and Translational Oncology Program, and Biomedicine Project 2009) and F.C.P. is the recipient of a SEGAI-ULL fellowship.

This work is licensed under the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License.-- et al., Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-xL overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment., This work was supported by the Spanish Ministerio de Ciencia e Innovación (SAF2008-02251, BQU2003-4413 and RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union), Fondo de Investigación Sanitaria and European Commission (FIS-FEDER 06/0813, 08/1434, PS09/01915), European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986), Junta de Castilla y León (CSI052A11-2, GR15-Experimental Therapeutics and Translational Oncology Program, Biomedicine Project 2009 and Biomedicine Project 2010-2011), Fundación para la Investigación Sanitaria en Castilla-La Mancha (FISCAM, PI-2006/10) and Junta de Comunidades de Castilla-La Mancha (I1I09-0163-4002). CG is supported by the Ramón y Cajal Program from the Spanish Ministerio de Ciencia e Innovación. MF is recipient of a postdoctoral fellowship from Fondo de Investigación Sanitaria. VH is recipient of a predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.

The synthesis of new 1,4-bisalkylamino (2-4) and 1-alkylamino-4-chloro (5-6) substituted benzo[g]phthalazines is reported. Compounds 2-4 and 6 were prepared both in the free and heteroaromatic ring protonated forms. Bifunctional 6 contains the 1,4-bisaminopropylpiperazine chain as a linker between the two heteroaromatic units, whereas 5 is its monofunctional analogue. The in vitro antitumour activity of the synthesized compounds has been tested against human colon, breast and lung carcinoma cells, and also against human glioblastoma cells. Results obtained show that all of them are active in all cases, but bifunctional 6·2HCl is remarkably effective against the four cell lines tested, exhibiting IC50 values in the range of 10-7 M, similar to those found for doxorubicin. The bifunctional structure of 6·2HCl enhances activity with respect to the monofunctional related compounds 5 and 7, leading to the highest activity among all the compounds tested. Molecular modelling of 6 suggests that those results could be indicative of DNA bisintercalation, which should be specially favoured in the diprotonated form 6·2HCl, a compound suitable for being studied more in depth in further biological tests. Measure of the DNA thermal melting curves show that the linear rise in Tm for bifunctional 6·2HCl is nearly twice than that one obtained for monofunctional 5, and supports the DNA-binding hypothesis. © 2010 Elsevier Ltd. All rights reserved., The authors thank the Spanish Comision Interministerial de Ciencia y Tecnología for financial support given to this research work. This work was supported by grants from the Spanish Ministry of Science and Innovation (SAF2008-02251, and RD06/0020/ 1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III). We are also grateful to staff of the C.N.Q.O. Manuel Lora-Tamayo (CSIC) for determining the NMR spectra and performing elemental analyses.

[Purpose]: Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL. [Experimental Design]: Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice. [Results]: Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine ≫ erucylphosphocholine ≥ miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor. [Conclusions]: Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine., Ministerio de Ciencia e Innovación grants SAF2005-04293, SAF2006-8850, SAF2007-61261, SAF2007-60964, PCT-090100-2007-27, PS09/01915, and SAF2008-02251; Red Temática de Investigación Cooperativa en Cáncer grants RD06/0020/0014, RD06/0020/0097, and RD06/0020/1037 (Instituto de Salud Carlos III, cofunded by the Fondo Europeo de Desarrollo Regional of the European Union); Fondo de Investigación Sanitaria and European Commission grant FIS-FEDER 06/0813; Junta de Castilla y León (CSI01A08, GR15-Experimental Therapeutics and Translational Oncology Program, and Biomedicine Project 2009); Fundación de Investigación Médica Mutua Madrileña; Fundación “la Caixa” grant BM05-30-0; Caja Navarra Foundation; Department of Health of the Government of Navarra (“Ortiz de Landázuri, 2009” project); and AGAUR-Generalitat de Catalunya grant 2005SGR-00549. C. Gajate is supported by the Ramón y Cajal Program from the Ministerio de Ciencia e Innovación of Spain. A. Estella-Hermoso de Mendoza is supported by Department of Education of the Basque Government research grant BFI06.37. M. de Frias is a recipient of a fellowship from the AGAUR-Generalitat de Catalunya. G. Roué holds a Miguel Servet research contract from Instituto de Salud Carlos III.

Leishmania parasites use polymorphonuclear neutrophils as intermediate hosts before their ultimate delivery to macrophages following engulfment of parasite-infected neutrophils. This leads to a silent and unrecognized entry of Leishmania into the macrophage host cell. Neutrophil function depends on its cytoplasmic granules, but their mobilization and role in how Leishmania parasites evade intracellular killing in neutrophils remain undetermined. Here, we have found by ultrastructural approaches that neutrophils ingested Leishmania major promastigotes, and azurophilic granules fused in a preferential way with parasite-containing phagosomes, without promoting parasite killing. Azurophilic granules, identified by the granule marker myeloperoxidase, also fused with Leishmania donovani-engulfed vacuoles in human neutrophils. In addition, the azurophilic membrane marker CD63 was also detected in the vacuole surrounding the parasite, and in the fusion of azurophilic granules with the parasite-engulfed phagosome. Tertiary and specific granules, involved in vacuole acidification and superoxide anion generation, hardly fused with Leishmania-containing phagosomes. L. major interaction with neutrophils did not elicit production of reactive oxygen species or mobilization of tertiary and specific granules. By using immunogold electron microscopy approaches in the engulfment of L. major and L. donovani by human neutrophils, we did not find a significant contribution of endoplasmic reticulum to the formation of Leishmania-containing vacuoles. Live Leishmania parasites were required to be optimally internalized by neutrophils. Our data suggest that Leishmania promastigotes modulate their uptake by neutrophils, and regulate granule fusion processes in a rather selective way to favor parasite survival in human neutrophils. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported by Spanish Ministry of Science and Innovation (Grant SAF2008-02251, and Grant RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, co-funded by the Fondo Europeo de Desarrollo Regional of the European Union), and Junta de Castilla y León (CSI01A08, GR15-Experimental Therapeutics and Translational Oncology Program, and Biomedicine Project 2009).

Despite recent advances in treatment, multiple myeloma (MM) remains an incurable malignancy. By using in vitro, ex vivo and in vivo approaches, we have identified here that lipid rafts constitute a new target in MM. We have found that the phospholipid ether edelfosine targets and accumulates in MM cell membrane rafts, inducing apoptosis through co-clustering of rafts and death receptors. Raft disruption by cholesterol depletion inhibited drug uptake by tumor cells as well as cell killing. Cholesterol replenishment restored MM cell ability to take up edelfosine and to undergo drug-induced apoptosis. Ceramide addition displaced cholesterol from rafts, and inhibited edelfosine-induced apoptosis. In an MM animal model, edelfosine oral administration showed a potent in vivo antimyeloma activity, and the drug accumulated preferentially and dramatically in the tumor. A decrease in tumor cell cholesterol, a major raft component, inhibited the in vivo antimyeloma action of edelfosine and reduced drug uptake by the tumor. The results reported here provide the proof-of-principle and rationale for further clinical evaluation of edelfosine and for this raft-targeted therapy to improve patient outcome in MM. Our data reveal cholesterol-containing lipid rafts as a novel and efficient therapeutic target in MM, opening a new avenue in cancer treatment., This work was supported by grants from Ministerio de Ciencia e Innovación (SAF2007-61261, SAF2008-02251, PCT-090100-2007-27, RD06/0020/1037 from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III), Fondo de Investigación Sanitaria and European Commission (FIS-FEDER 06/0813, PS09/01915), Junta de Castilla y León (GR15-Experimental Therapeutics and Translational Oncology Program, and Biomedicine Project 2009) and Caja Navarra Foundation, Department of Health of the Navarra Government (‘Ortiz de Landázuri, 2009’ project). CG is supported by the Ramón y Cajal Program from the Ministerio de Ciencia e Innovación of Spain. AEHdM is supported by a research grant (BF106.37) from the Department of Education of the Basque Government.