Marine-Inspired Drugs and Biomaterials in the Perspective of Pancreatic Cancer Therapies.

Despite its low prevalence, pancreatic cancer (PC) is one of the deadliest, typically characterised as silent in early stages and with a dramatically poor prognosis when in its advanced stages, commonly associated with a high degree of metastasis. Many efforts have been made in pursuing innovative therapeutical approaches, from the search for new cytotoxic drugs and other bioactive compounds, to the development of more targeted approaches, including improved drug delivery devices. Marine biotechnology has been contributing to this quest by providing new chemical leads and materials originating from different organisms. In this review, marine biodiscovery for PC is addressed, particularly regarding marine invertebrates (namely sponges, molluscs, and bryozoans), seaweeds, fungi, and bacteria. In addition, the development of biomaterials based on marine-originating compounds, particularly chitosan, fucoidan, and alginate, for the production of advanced cancer therapies, is also discussed. The key role that drug delivery can play in new cancer treatments is highlighted, as therapeutical outcomes need to be improved to give further hope to patients.

Fluorometric Liposome Screen for Inhibitors of a Physiologically Important Bacterial Ion Channel.

The bacterial K+ homeostasis machinery is widely conserved across bacterial species, and different from that in animals. Dysfunction in components of the machinery has an impact on intracellular turgor, membrane potential, adaptation to changes in both extracellular pH and osmolarity, and in virulence. Using a fluorescence-based liposome flux assay, we have performed a high-throughput screen to identify novel inhibitors of the KtrAB ion channel complex from Bacillus subtilis, a component of the K+ homeostasis machinery that is also present in many bacterial pathogens. The screen identified 41 compounds that inhibited K+ flux and that clustered into eight chemical groups. Many of the identified inhibitors were found to target KtrAB with an in vitro potency in the low µM range. We investigated the mechanisms of inhibition and found that most molecules affected either the membrane component of the channel, KtrB alone or the full KtrAB complex without a preference for the functional conformation of the channel, thus broadening their inhibitory action. A urea derivative molecule that inhibited the membrane component of KtrAB affected cell viability in conditions in which KtrAB activity is essential. With this proof-of-concept study, we demonstrate that targeting components of the K+ homeostasis machinery has the potential as a new antibacterial strategy and that the fluorescence-based flux assay is a robust tool for screening chemical libraries.

PLGA nanoparticles optimized by Box-Behnken for efficient encapsulation of therapeutic Cymbopogon citratus essential oil.

This study aimed to optimize Cymbopogon citratus essential oil loaded into PLGA-nanoparticles by investigating the effect of processing variables (sonication time, ultrasound power, and essential oil/polymer ratio) on encapsulation efficiency and particle mean hydrodynamic diameter using Box-Behnken design. Nanoparticles were prepared by an emulsification/solvent diffusion method and physicochemically characterized by FTIR, DSC and TGA/DTA. Cytotoxicity was evaluated in human HaCat keratinocytes by WST-1 and LDH assays. The optimized formulation had a hydrodynamic mean diameter of 277 nm, a polydispersity index of 0.18, a Zeta potential of -16 mV and an encapsulation efficiency of 73%. Nanoparticle characterization showed that only citral was incorporated in nanocarriers, with some amount adsorbed on their surface, and highlighted the potential in increasing the oil thermal stability. The drug release profile demonstrated a biphasic pattern with a substantial sustained release depending on diffusion from the polymeric matrix. Toxicity effects on cell viability of pure essential oil at low concentrations were significantly eliminated when encapsulated. Results revealed the ability of PLGA-nanoparticles to improve essential oil physicochemical characteristics, by controlling release and reducing toxicity, suggesting their potential use in pharmaceutical preparations.

Changes in the Biophysical Properties of the Cell Membrane Are Involved in the Response of Neurospora crassa to Staurosporine.

Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasma membrane of conidia. This leads to higher fluidity in other membrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15 min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa.

Antioxidant Activity and Genotoxic Assessment of Crabwood (Andiroba, Carapa guianensis Aublet) Seed Oils.

The seed oil of Carapa guianensis (Aublet), a tree from the Meliaceae family commonly known as andiroba, is widely used in Brazilian traditional medicine because of its multiple curative properties against fever and rheumatism and as an anti-inflammatory agent, antibacterial agent, and insect repellant. Since there is no consensus on the best way to obtain the C. guianensis oil and due to its ethnomedicinal properties, the aim of the present research was to evaluate the chemical composition, free-radical scavenging activity, and mutagenic and genotoxicity properties of three C. guianensis oils obtained by different extraction methods. The phenolic contents were evaluated by spectrophotometry. Oil 1 was obtained by pressing the dried seeds at room temperature; oil 2 was obtained by autoclaving, drying, and pressing; oil 3 was obtained by Soxhlet extraction at 30-60°C using petroleum ether. The oil from each process presented differential yields, physicochemical properties, and phenolic contents. Oil 1 showed a higher scavenging activity against the DPPH radical when compared to oils 2 and 3, suggesting a significant antioxidant activity. All oils were shown to be cytotoxic to bacteria and to CHO-K1 and RAW264.7 cells. At noncytotoxic concentrations, oil 2 presented mutagenicity to Salmonella enterica serovar Typhimurium and induced micronuclei in both cell types. Under the same conditions, oil 3 also induced micronucleus formation. However, the present data demonstrated that oil 1, extracted without using high temperatures, was the safest for use as compared to the other two oils, not showing mutagenicity or micronucleus induction.

Reorganization of plasma membrane lipid domains during conidial germination.

Neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (SL)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. Several changes in lipid metabolism and in the membrane composition of N. crassa occur during spore germination. However, the biophysical impact of those changes is unknown. Thus, a biophysical study of N. crassa plasma membrane, particularly SL-enriched domains, and their dynamics along conidial germination is prompted. Two N. crassa strains, wild-type (WT) and slime, which is devoid of cell wall, were studied. Conidial growth of N. crassa WT from a dormancy state to an exponential phase was accompanied by membrane reorganization, namely an increase of membrane fluidity, occurring faster in a supplemented medium than in Vogel's minimal medium. Gel-like domains, likely enriched in SLs, were found in both N. crassa strains, but were particularly compact, rigid and abundant in the case of slime cells, even more than in budding yeast Saccharomyces cerevisiae. In N. crassa, our results suggest that the melting of SL-enriched domains occurs near growth temperature (30°C) for WT, but at higher temperatures for slime. Regarding biophysical properties strongly affected by ergosterol, the plasma membrane of slime conidia lays in between those of N. crassa WT and S. cerevisiae cells. The differences in biophysical properties found in this work, and the relationships established between membrane lipid composition and dynamics, give new insights about the plasma membrane organization and structure of N. crassa strains during conidial growth.

Molecular Insights into the Mechanism of Calmodulin Inhibition of the EAG1 Potassium Channel.

The human EAG1 potassium channel belongs to the superfamily of KCNH voltage-gated potassium channels that have roles in cardiac repolarization and neuronal excitability. EAG1 is strongly inhibited by Ca2+/calmodulin (CaM) through a mechanism that is not understood. We determined the binding properties of CaM with each one of three previously identified binding sites (BDN, BDC1, and BDC2), analyzed binding to protein stretches that include more than one site, and determined the effect of neighboring globular domains on the binding properties. The determination of the crystal structure of CaM bound to BDC2 shows the channel fragment interacting with only the C lobe of calmodulin and adopting an unusual bent conformation. Based on this structure and on a functional and biochemical analysis of mutants, we propose a model for the mechanism of inhibition whereby the local conformational change induced by CaM binding at BDC2 lies at the basis of channel modulation.

Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain.

The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

Differential toxicity of an organic PM2.5 extract to human lung cells cultured in three dimensions (3D) and monolayers.

Several epidemiological studies have associated PM2.5 (particulate matter, aerodynamic diameter 2.5 µm) exposure with an increase in morbidity and mortality attributed to cardiopulmonary diseases. Based upon these observations and the growing effort to replace the use of animals in research, in vitro A549 cells cultured in three dimensions (3D), an alternative method to the use of animals, as well as monolayers were investigated to examine whether organic PM2.5 extract induced equivalent cytotoxic changes in vitro as compared to in vivo. PM2.5 was collected on Brazil Avenue, Rio de Janeiro, Brazil, from November 2010 to May 2011, except March, and analyzed for the ability to induce cytotoxicity in A549 cells using various established assays. Samples collected in all months significantly decreased viability of A549 cells using both types of cell death assays, and those collected in November showed lower cytotoxicity. It is worthwhile noting that for samples collected in all months except for April, PM2.5 induced greater toxicity in cells grown in monolayers than in 3D. Data demonstrated that cell behavior varied based upon type of culture system employed. Since the 3D cell culture mimics the architecture of in vivo tissue to a greater extent than monolayers, it is suggested that data from 3D studies resemble more closely human exposure conditions and thus may provide more reliable findings to be utilized in risk assessment following PM exposure than results obtained in traditional culture system.

Screening for Non-Pore-Binding Modulators of EAG K+ Channels.

Members of the ether-à-go-go (EAG) family of voltage-gated K(+) channels are involved in several pathophysiological diseases, and there has been a great interest in screening for drugs that modulate the activity of these channels. Many drugs have been shown to bind in the pore of these channels, blocking ion flux and causing disease pathology. In this report, we present two independent screening campaigns in which we wanted to identify small molecules that bind to either the intracellular cytoplasmic amino terminal Per-Arnt-Sim (PAS) domain from the human EAG-related gene (ERG) channel or the amino or carboxy terminal globular domains from the mouse EAG1 channel, affecting their interaction. We report that in both cases, compounds were identified that showed weak, nonspecific binding. We suggest alternative routes should be pursued in future efforts to identify specific, high-affinity binders to these cytoplasmic domains.

Reduced glutathione export during programmed cell death of Neurospora crassa.

In a previous study, we demonstrated that staurosporine (STS) induces programmed cell death (PCD) in the fungus Neurospora crassa and that glutathione has the capability of inhibiting both STS-induced reactive oxygen species (ROS) formation and cell death. Here, we further investigated the role of glutathione in STS-induced PCD in N. crassa and observed an efflux of reduced glutathione (GSH) together with a change in the cell internal redox state to a more oxidative environment. This event was also observed with another PCD inducer, phytosphingosine (PHS), although externally added GSH did not prevent PHS-induced PCD. The nature of ROS, detected under the experimental conditions at which GSH export occurred, is also different in the two systems, predominantly superoxide in the case of STS and hydrogen peroxide in the case of PHS. In both cases, GSH export preceded the alterations in the plasma membrane that lead to selective dye permeation. We conclude that glutathione export in the context of PCD is not exclusive of certain mammalian cells and can be extended to Fungi, being an early PCD event in N. crassa. In addition, STS and PHS induce different PCD pathways in this fungus and the role of GSH export in each of them is likely different.

Modulation of fungal sensitivity to staurosporine by targeting proteins identified by transcriptional profiling.

An analysis of the time-dependent genetic response to the death-inducer staurosporine was performed in Neurospora crassa by transcriptional profiling. Staurosporine induced two major genes encoding an ABC transporter and a protein with similarity to regulatory subunits of potassium channels. The transcriptional response is dependent on the activity of a novel transcription factor. Deletion mutants in differentially expressed genes displayed altered sensitivity to staurosporine, underscoring significant proteins involved in the response to the drug. A null-mutant of the ABC transporter (abc3) is extremely sensitive to staurosporine, accumulates more staurosporine than the wild type strain and is defective in energy-dependent export of the drug, indicating that the ABC3 protein is the first described staurosporine transporter. It was located in the plasma membrane by immunofluorescence microscopy. The combination of inhibitors of ABC transporters or of potassium channels with staurosporine leads to an enhanced activity against N. crassa and pathogenic fungi paving the way to the development of more potent and specific antifungals. Our results highlight the general use of transcriptional profiling for the identification of novel proteins involved in cell death and their potential use as drug targets.

Rotenone enhances the antifungal properties of staurosporine.

We studied staurosporine-induced cell death in the filamentous fungus Neurospora crassa. The generation of reactive oxygen species during the process appears to be an important signaling event, since addition of the antioxidant glutathione prevents the effects of staurosporine on fungal growth. Selected mutants with mutations in respiratory chain complex I are extremely sensitive to the drug, stressing the involvement of complex I in programmed cell death. Following this finding, we determined that the complex I-specific inhibitor rotenone used in combination with staurosporine results in a synergistic and specific antifungal activity, likely through a concerted action on intracellular glutathione depletion. Paradoxically, the synergistic antifungal activity of rotenone and staurosporine is observed in N. crassa complex I mutants and in Saccharomyces cerevisiae, which lacks complex I. In addition, it is not observed when other complex I inhibitors are used instead of rotenone. These results indicate that the rotenone effect is independent of complex I inhibition. The combination of rotenone and staurosporine is effective against N. crassa as well as against the common pathogens Aspergillus fumigatus and Candida albicans, pointing to its usefulness as an antifungal agent.

Energy conservation by Rhodothermus marinus respiratory complex I.

A sodium ion efflux, together with a proton influx and an inside-positive DeltaPsi, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by (23)Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.

Electron paramagnetic resonance studies of the iron-sulfur centers from complex I of Rhodothermus marinus.

Rhodothermus marinus, a thermohalophilic gram negative bacterium, contains a type I NADH/quinone oxidoreductase (complex I). Its purification was optimized, yielding large amounts of pure and active protein. Furthermore, the stoichiometry of NADH oxidation and quinone reduction was shown to be 1:1. The large amounts of protein enabled a thorough characterization by electron paramagnetic resonance (EPR) spectroscopy at different temperatures and microwave powers, using NADH, NADPH, and dithionite as reducing agents. A minimum of two [2Fe-2S](2+/1+) and four [4Fe-4S](2+/1+) centers were observed in the purified complex. Redox titrations monitored by EPR spectroscopy made possible the determination of the reduction potentials of the iron-sulfur centers; with the exception of one of the [4Fe-4S](2+/1+) centers, which has a lower reduction potential, all the other centers have reduction potentials of -240 +/- 20 mV, pH 7.5.

A nhaD Na+/H+ antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes.

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo1 to nqo7 (nqoA) and nqo10 to nqo14 (nqoB)] and two independent genes (nqo8 and nqo9). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4alpha-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo13 and nqo14. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.

Quinone reduction by Rhodothermus marinus succinate:menaquinone oxidoreductase is not stimulated by the membrane potential.

Succinate:quinone oxidoreductase (SQR), a di-haem enzyme purified from Rhodothermus marinus, reveals an HQNO-sensitive succinate:quinone oxidoreductase activity with several menaquinone analogues as electron acceptors that decreases with lowering the redox midpoint potential of the quinones. A turnover with the low-potential 2,3-dimethyl-1,4-naphthoquinone that is the closest analogue of menaquinone, although low, can be detected in liposome-reconstituted SQR. Reduction of the quinone is not stimulated by an imposed K+-diffusion membrane potential of a physiological sign (positive inside the vesicles). Nor does the imposed membrane potential increase the reduction level of the haems in R. marinus SQR poised with the succinate/fumarate redox couple. The data do not support a widely discussed hypothesis on the electrogenic transmembrane electron transfer from succinate to menaquinone catalysed by di-haem SQRs. The role of the membrane potential in regulation of the SQR activity is discussed.

Respiratory chains from aerobic thermophilic prokaryotes.

Thermophiles are organisms that grow optimally above 50 degrees C and up to approximately 120 degrees C. These extreme conditions must have led to specific characteristics of the cellular components. In this paper we extensively analyze the types of respiratory complexes from thermophilic aerobic prokaryotes. The different membrane-bound complexes so far characterized are described, and the genomic data available for thermophilic archaea and bacteria are analyzed. It is observed that no specific characteristics can be associated to thermophilicity as the different types of complexes I-IV are present randomly in thermophilic aerobic organisms, as well as in mesophiles. Rather, the extensive genomic analyses indicate that the differences concerning the several complexes are related to the organism phylogeny, i.e., to evolution and lateral gene transfer events.

Quinol:fumarate oxidoreductases and succinate:quinone oxidoreductases: phylogenetic relationships, metal centres and membrane attachment.

A comprehensive phylogenetic analysis of the core subunits of succinate:quinone oxidoreductases and quinol:fumarate oxidoreductases is performed, showing that the classification of the enzymes as type A to E based on the type of the membrane anchor fully correlates with the specific characteristics of the two core subunits. A special emphasis is given to the type E enzymes, which have an atypical association to the membrane, possibly involving anchor subunits with amphipathic helices. Furthermore, the redox properties of the SQR/QFR proteins are also reviewed, stressing out the recent observation of redox-Bohr effect upon haem reduction, observed for the Desulfovibrio gigas and Rhodothermus marinus enzymes, which indicates a direct protonation event at the haems or at a nearby residue. Finally, the possible contribution of these enzymes to the formation/dissipation of a transmembrane proton gradient is discussed, considering recent experimental and structural data.

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus.

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.