Janny A. Villa-Pulgarin

Lipid raft-targeted therapy in multiple myeloma.

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.

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.

Selective Fusion of Azurophilic Granules with Leishmania-containing Phagosomes in Human Neutrophils

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.

In Vitro and In Vivo Efficacy of Ether Lipid Edelfosine against Leishmania spp. and SbV-Resistant Parasites

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.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells.

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-xL 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.

Cytotoxic oxasqualenoids from the red alga Laurencia viridis.

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.

New Polyether Triterpenoids from Laurencia viridis and Their Biological Evaluation

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.

Complete inhibition of extranodal dissemination of lymphoma by edelfosine-loaded lipid nanoparticles

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.

Synthesis and cytotoxic activity of a new potential DNA bisintercalator: 1,4-Bis{3-[N-(4-chlorobenzo[g]phthalazin-1-yl)aminopropyl]}piperazine

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.

C-Geranylated flavonoids from Paulownia tomentosa fruits with antimicrobial potential and synergistic activity with antibiotics

Abstract 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 Leishmania brazilensis, L. donovani, L. infantum, and L. panamensis against methicillin-resistant Staphylococcus aureus (MRSA); and synergistic potential with antibiotics was analyzed. IC50 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 (IC50 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.