Antiamoebic effects of sesquiterpene lactones isolated from the zoanthid Palythoa aff. clavata.

Opportunistic parasitic protozoa of genus Acanthamoeba are responsible to cause severe infections in humans such as Acanthamoeba Keratitis or Amoebic Granulomatous Encephalitis. Current treatments are usually toxic and inefficient and there is a need to access new therapeutic agents. The antiamoebic effects of nephthediol (1) and fourteen germacranolide and eudesmanolide sesquiterpene lactones (2-5, 7-12) isolated from the indigenous zoanthid Palythoa aff. clavata collected at the coast of Lanzarote, Canary Islands were studied against Acanthamoeba castellanii Neff, and the clinical strains A. polyphaga and A. griffini. 4-epi-arbusculin A (11) presented the lowest IC50 value (26,47 ± 1,69 µM) against A. castellanii Neff and low cytotoxicity against murine macrophages, followed by isobadgerin (2), which also showed to be active against A. castellanii Neff cysts. The studies on the mode of action of compounds 2 and 11 revealed these sesquiterpene lactones induce mechanisms of PDC on A. castellanii Neff.

Evaluation of Oxasqualenoids from the Red Alga Laurencia viridis against Acanthamoeba.

Acanthamoeba genus is a widely distributed and opportunistic parasite with increasing importance worldwide as an emerging pathogen in the past decades. This protozoan has an active trophozoite stage, a cyst stage, and is dormant and very resistant. It can cause Acanthamoeba keratitis, an ocular sight-threatening disease, and granulomatous amoebic encephalitis, a chronic, very fatal brain pathology. In this study, the amoebicidal activity of sixteen Laurencia oxasqualenoid metabolites and semisynthetic derivatives were tested against Acanthamoeba castellanii Neff. The results obtained point out that iubol (3) and dehydrothyrsiferol (1) possess potent activities, with IC50 values of 5.30 and 12.83 µM, respectively. The hydroxylated congeners thyrsiferol (2) and 22-hydroxydehydrothyrsiferol (4), active in the same value range at IC50 13.97 and 17.00 µM, are not toxic against murine macrophages; thus, they are solid candidates for the development of new amoebicidal therapies.

Pinnatifidenyne-Derived Ethynyl Oxirane Acetogenins from Laurencia viridis.

Red algae of Laurencia continue to provide wide structural diversity and complexity of halogenated C15 acetogenin medium-ring ethers. Here, we described the isolation of three new C15 acetogenins (3-5), and one truncated derivative (6) from Laurencia viridis collected on the Canary Islands. These compounds are interesting variations on the pinnatifidenyne structure that included the first examples of ethynyl oxirane derivatives (3-4). The structures were elucidated by extensive study of NMR (Nuclear Magnetic Resonance) data, J-based configuration analysis and DFT (Density Functional Theory) calculations. Their antiproliferative activity against six human solid tumor cell lines was evaluated.

Additional Insights into the Obtusallene Family: Components of Laurencia marilzae.

The obtusallenes are a significant subset of C15-halogenated acetogenins that incorporate 12-membered cyclic ethers. We have recently reported the isolation from Laurencia marilzae of 12-epoxyobtusallene IV (1) and its related α,ß-unsaturated carboxylate ester (2), both of special biogenetic relevance. Here we describe the final step of our study, the isolation of three new analogues (3-5), among these, the first bromopropargylic derivative (3) of this class of macrocyclic C15-acetogenins. The structures were elucidated by analysis of NMR and X-ray data. 12-Epoxyobtusallene IV (1), its new isomer 4, and known obtusallene IV (6) were evaluated for their apoptosis-inducing activities in a human hepatocarcinoma cell line.

Stereochemical determination of five-membered cyclic ether acetogenins using a spin-spin coupling constant approach and DFT calculations.

Five-membered rings are of particular interest, due to their presence in some of the most common molecules in chemistry and biology. Despite their apparent simplicity, the structural resolution of these rings is complex, due to their inherent conformational flexibility. Here, we describe an application of a recently reported simple and efficient NMR protocol based on the measurement of spin-spin coupling constants to achieve the challenging relative configurations of five new halogenated C15 tetrahydrofuranyl-acetogenins, marilzafurollenes A-D (1-4) and 12-acetoxy-marilzafurenyne (5), isolated from the red alga, Laurencia marilzae. Although DFT chemical shift calculations were used to connect remote stereocenters, the NMR-based approach seems advantageous over computational techniques in this context, as the presence of halogens may interfere with reliable calculations.

New bicyclotridecane C15 nonterpenoid bromoallenes from Laurencia marilzae.

Nonterpenoid bromoallenes possessing a novel skeleton that incorporates an unprecedented [5.5.1]bicyclotridecane ring system, marilzabicycloallenes A-D (1-4), were isolated from specimens of Laurencia marilzae collected on the Canary Islands. The framework of these metabolites strongly reinforces Braddock's hypothesis concerning the biosynthesis via electrophilic bromination of the obtusallene family.

Nonterpenoid C15 acetogenins from Laurencia marilzae.

Eight new halogenated C(15) acetogenins, 1-8, were isolated from the organic extract of the red alga Laurencia marilzae. The structure elucidation and the assignments of the relative configurations were established by extensive use of spectroscopic studies, particularly 1D and 2D NMR data, while the absolute configurations of compounds 1 and 5 were determined by single-crystal X-ray diffraction analysis. Compounds 1, 2, 4, 5, and 7, along with the previously reported related cyclic ether obtusallene IV (9), were evaluated against six human solid tumor cell lines. All compounds were found to be essentially inactive (GI(50) > 10 µg/mL).

Micromelones A and B, noncontiguous polypropionates from Micromelo undata.

In this paper we report on the isolation and structural elucidation of two new noncontiguous polypropionates, micromelones A ( 10) and B ( 11), that have been isolated from the marine gastropod Micromelo undata. Their structures were determined through the interpretation of their spectroscopic data, and a biosynthetic pathway from a common linear precursor with other polypropionate groups has been proposed.

Chemical modulation of VLA integrin affinity in human breast cancer cells.

The fact that disruption of integrin-extracellular matrix contacts leads to cell death, has converted cell adhesion into a potential target for the control of invasive cancer. In this work, we studied the functional consequences of the interference with the activity of the very late activation antigen (VLA) family of integrins in human breast cancer cell lines of distinct malignancy. The alpha2beta1-mediated adhesion reduced the entry of highly malignant, hormone-independent breast cancer cells into apoptosis. Adhesion of breast cancer cells through the VLA integrins alpha2beta1 and alpha5beta1 was significantly reduced by an apoptosis-inducing natural triterpenoid, dehydrothyrsiferol (DT), when studied on low amounts of extracellular matrix. This effect was dose-dependent, not related to cell toxicity and not shared with apoptosis-inducing standard chemotherapeutics, such as doxorubicin and taxol. The compound did not affect either the cell surface expression level of VLA integrins or cell distribution of vinculin and actin during cell spreading. In addition, neither phosphorylation of the focal adhesion kinase pp125FAK on Tyr397 nor the protein kinase B (Akt/PKB) on Ser473 was significantly altered by DT. The integrin activation level, assessed by binding of soluble collagen to the alpha2beta1 integrin, was reduced upon cell treatment with DT. Importantly, the TS2/16, an anti-beta1 activating monoclonal antibody was able to rescue DT-treated cells from apoptosis. Since the activation state of integrins is increasingly recognized as an essential factor in metastasis formation, findings presented herein reveal that the chemical regulation of integrin affinity may be a potential therapeutic strategy in cancer therapy.

Induction of apoptosis in estrogen dependent and independent breast cancer cells by the marine terpenoid dehydrothyrsiferol.

Breast cancer (BCA) represents the highest incidence of death in 35- to 60-year-old women. Above all, hormone unresponsive BCA is still associated with poorer prognosis than hormone receptor expressing malign, mammary tumors. There is a consistent need for effective compounds to treat especially the first variant of this disease. Therefore, we investigated the cytotoxic effects of the marine polyether triterpenoid dehydrothyrsiferol (DT) in four BCA cell lines. Annexin V labeling revealed higher rates of DT-induced apoptosis in hormone insensitive than in estrogen receptor expressing cells. Flow cytometric analysis of combined DNA fragmentation and total DNA labeling allowed us to ascribe apoptotic cells to their cell cycle stage. Although, high cell mortality was detected in mitogen dependent G(1)-phase, time, concentration, and cell line dependent populations of apoptotic cells were also found to be of S-phase and G(2)/M-phase origin. These results suggest that the induction of apoptosis by DT might be transduced through more than one effector pathway. Cell cycle distributions and 5-bromo-2'-deoxyuridine incorporation varied in a treatment dependent manner and differed from control experiments with colchicine and doxorubicin which exclude that DT functions as a mitosis inhibitor. In summary, we propose that DT might be an interesting candidate for an antitumor drug development regimen.

Dehydrothyrsiferol does not modulate multidrug resistance-associated protein 1 resistance: a functional screening system for MRP1 substrates.

We had shown previously that the novel, marine, anticancer compound dehydrothyrsiferol (DHT) does not modulate P-glycoprotein (P-gp) dependent drug efflux. Many chemotherapeutics with clinical impact are substrates for the structurally distant related membrane transport protein MRP1 (multidrug resistance-associated protein 1). Thus, we were interested in analysing the behaviour of DHT and control compounds in specific drug transport of MRP1 overexpressing cells. We established a fluorescence based drug efflux system for specific, functional detection of interference of a test compound in MRP1 mediated drug extrusion. Briefly, MRP1 overexpressing HL60/Adr cells were incubated to uptake and then efflux fluorescent 5(6)-carboxyfluorescein diacetate (CFDA), rhodamine 123 (Rh123), or 3,3-diethylocarbocyanine iodide (DiOC2), respectively. Changes in cell fluorescence intensity after coincubation with the compound of interest were determined by flow cytometry. MRP1 mediated efflux of CFDA was analysed in the presence of DHT, the known substrates genistein, probenecid, and the specific inhibitor MK-571. To exclude unknown P-gp related interference in drug transport, efflux of the fluorescent P-gp substrate DiOC2 and specific inhibition by cyclosporin A (CsA) were analysed. Cytotoxicity of DHT in resistant HL60/Adr cells was found to be even superior to that in the parental HL60 leukaemia cell line. Consequently, DHT did not interfere in MRP1 mediated drug transport. In contrast to DiOC2, rhodamine 123 was not specifically effluxed by P-gp but also by MRP1. Therefore, we propose the MRP1 specific CFDA efflux model as a screening and/or excluding system for MRP1 substrates. Together with previous data our results suggest DHT to be an interesting candidate for further investigation directed towards a drug development regimen.