Schinus terebinthifolius Raddi (Brazilian pepper) leaves extract: in vitro and in vivo evidence of anti-inflammatory and antioxidant properties.

The aim of this work was to evaluate the anti-inflammatory and antioxidant effects of ethyl acetate extract obtained from the leaves of Brazilian peppertree Schinus terebinthifolius Raddi (EAELSt). Total phenols and flavonoids, chemical constituents, in vitro antioxidant activity (DPPH and lipoperoxidation assays), and cytotoxicity in L929 fibroblasts were determined. In vivo anti-inflammatory and antioxidant properties were evaluated using TPA-induced ear inflammation model in mice. Phenol and flavonoid contents were 19.2 ± 0.4 and 93.8 ± 5.2 of gallic acid or quercetin equivalents/g, respectively. LC-MS analysis identified 43 compounds, of which myricetin-O-pentoside and quercetin-O-rhamnoside were major peaks of chromatogram. Incubation with EAELSt decreased the amount of DPPH radical (EC50 of 54.5 ± 2.4 µg/mL) and lipoperoxidation at 200-500 µg/mL. The incubation with EAELSt did not change fibroblast viability up to 100 µg/mL. Topical treatment with EAELSt significantly reduced edema and myeloperoxidase activity at 0.3, 1, and 3 mg/ear when compared to the vehicle-treated group. In addition, EAELSt decreased IL-6 and TNF-α levels and increased IL-10 levels. Besides, it modulated markers of oxidative stress (reduced total hydroperoxides and increased sulfhydryl contents and ferrium reduction potential) and increased the activity of catalase and superoxide dismutase, without altering GPx activity.

(+)-Limonene 1,2-Epoxide-Loaded SLNs: Evaluation of Drug Release, Antioxidant Activity, and Cytotoxicity in an HaCaT Cell Line.

In this work, we developed a solid lipid nanoparticle (SLN) formulation with (+)-limonene 1,2-epoxide and glycerol monostearate (Lim-SLNs), stabilized with Poloxamer® 188 in aqueous dispersion to modify the release profile of the loaded monoterpene derivative. We also evaluated the role of SLNs in lipid peroxidation and cytotoxicity in a spontaneously transformed aneuploid immortal keratinocyte cell line from adult human skin (the HaCaT cell line). For the cell viability assay, the colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used. Lim-SLNs with a loading capacity and encapsulation efficiency of 0.39% and 63%, respectively, were produced by high pressure homogenization. A mean particle size of 194 ± 3.4 nm and polydispersity index of 0.244 were recorded for the loaded Lim-SLNs, as compared to 203 ± 1.5 nm (PI 0.213) for the non-loaded (blank) SLNs. The loading of the monoterpene derivative into glycerol monostearate SLNs fitted into the zero-order kinetics, and ameliorated both lipid peroxidation and cytotoxicity in a keratinocyte cell line. A promising formulation for antioxidant and anti-tumoral activities is here proposed.

Cytotoxic, Antitumor and Toxicological Profile of Passiflora alata Leaf Extract.

Passiflora alata or passion fruit is a native flowering plant from Amazon, geographically spread from Peru to Brazil. The plant has long been used in folks medicine for its pharmacological properties and is included in the Brazilian Pharmacopoeia since 1929. The aim of this study was to evaluate the potential cytotoxic and antitumor activities of Passiflora alata leaf extract (PaLE) in S180-tumor bearing mice. The percentage of cell proliferation inhibition (% CPI) and IC50 in relation to 4 tumor cell lines were determined in PC3, K-562, HepG2 and S180 cell lines using the MTT assay. PaLE showed a CPI > 75% and greater potency (IC50 < 30 µg/mL) against PC3 and S180 cell lines. PaLE showed antitumor activity in treatments intraperitoneally (36.75% and 44.99% at doses of 100 and 150 mg/kg/day, respectively). Toxicological changes were shown in the reduced body mass associated with reduced food consumption, increased spleen mass associated with histopathological increase in the white pulp of the spleen and increased number of total leukocytes with changes in the percentage relationship between lymphocytes and neutrophils. Our outcomes corroborate the conclusion that PaLE has antitumor activity in vitro and in vivo with low toxicity.

ß-Cyclodextrin/Isopentyl Caffeate Inclusion Complex: Synthesis, Characterization and Antileishmanial Activity.

Isopentyl caffeate (ICaf) is a bioactive ester widely distributed in nature. Our patented work has shown promising results of this molecule against Leishmania. However, ICaf shows poor solubility, which limits its usage in clinical settings. In this work, we have proposed the development of an inclusion complex of ICaf in ß-cyclodextrin (ß-CD), with the aim to improve the drug solubility, and thus, its bioavailability. The inclusion complex (ICaf:ß-CD) was developed applying three distinct methods, i.e., physical mixture (PM), kneading (KN) or co-evaporation (CO) in different molar proportions (0.25:1, 1:1 and 2:1). Characterization of the complexes was carried out by thermal analysis, Fourier-transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and molecular docking. The ICaf:ß-CD complex in a molar ratio of 1:1 obtained by CO showed the best complexation and, therefore, was selected for further analysis. Solubility assay showed a marked improvement in the ICaf:ß-CD (CO, 1:1) solubility profile when compared to the pure ICaf compound. Cell proliferation assay using ICaf:ß-CD complex showed an IC50 of 3.8 and 2.7 µg/mL against L. amazonesis and L. chagasi promastigotes, respectively. These results demonstrate the great potential of the inclusion complex to improve the treatment options for visceral and cutaneous leishmaniases.

Praziquantel-Solid Lipid Nanoparticles Produced by Supercritical Carbon Dioxide Extraction: Physicochemical Characterization, Release Profile, and Cytotoxicity.

Solid lipid nanoparticles (SLNs) can be produced by various methods, but most of them are difficult to scale up. Supercritical fluid (SCF) is an important tool to produce micro/nanoparticles with a narrow size distribution and high encapsulation efficiency. The aim of this work was to produce cetyl palmitate SLNs using SCF to be loaded with praziquantel (PZQ) as an insoluble model drug. The mean particle size (nm), polydispersity index (PdI), zeta potential, and encapsulation efficiency (EE) were determined on the freshly prepared samples, which were also subject of Differential Scanning Calorimetry (DSC), Fourier-Transform Infrared Spectroscopy (FTIR), drug release profile, and in vitro cytotoxicity analyses. PZQ-SLN exhibited a mean size of ~25 nm, PdI ~ 0.5, zeta potential ~-28 mV, and EE 88.37%. The DSC analysis demonstrated that SCF reduced the crystallinity of cetyl palmitate and favored the loading of PZQ into the lipid matrices. No chemical interaction between the PZQ and cetyl palmitate was revealed by FTIR analysis, while the release or PZQ from SLN followed the Weibull model. PZQ-SLN showed low cytotoxicity against fibroblasts cell lines. This study demonstrates that SCF may be a suitable scale-up procedure for the production of SLN, which have shown to be an appropriate carrier for PZQ.

ATR suppresses apoptosis after UVB irradiation by controlling both translesion synthesis and alternative tolerance pathways.

Ultraviolet (UV) light can stall replication forks owing to the formation of bulky lesions in the DNA. Replication across these blocking lesions occurs through translesion DNA synthesis, and cells activate the ATR damage responses to UV. However, it remains unclear whether lesion bypass requires the replication checkpoint because ATR is not necessary for PCNA ubiquitylation. We observed that ATR knockdown by siRNA increased replication stress and promoted early induction of apoptosis following UVB irradiation in SV40-immortalized human cells, including cells from XP-V and XP-C patients. XP-V cells were further sensitized by the silencing, indicating that DNA polymerase η (Pol η) remains active despite ATR control. However, following UVB irradiation, ATR-depleted cells were unable to achieve mitosis, as would be expected after the loss of a DNA checkpoint control. Thus, ATR also regulates replication arrest recovery following UVB-induced damage, independently of Pol η, in SV40-immortalized cell lines. The ATR-mediated DNA damage response regulates replication and different tolerance pathways, and in these cells, ATR depletion induces replication catastrophe, which contributes to explain the potential of ATR inhibition to protect against UVB-induced carcinogenesis.

Extracellular Vesicle-Packaged miR-195-5p Sensitizes Melanoma to Targeted Therapy with Kinase Inhibitors.

Management of advanced melanoma remains challenging, with most BRAF (B-Raf proto-oncogene, serine/threonine kinase)-mutated metastatic patients relapsing within a few months upon MAPK inhibitors treatment. Modulation of tumor-derived extracellular vesicle (EVs) cargo with enrichment of antitumoral molecules is a promising strategy to impair tumor progression and increase treatment response. Herein, we report that restored expression of miR-195-5p, down-regulated in melanoma favoring drug resistance, increases the release of EVs enriched in the tumor suppressor miRNAs, miR-195-5p, miR-152-3p, and miR-202-3p. Incorporating these EVs by bystander tumor cells resulted in decreased proliferation and viability, accompanied by a reduction in CCND1 and YAP1 mRNA levels. Upon treatment with MAPK inhibitors, miR-195 EVs significantly decreased BCL2-L1 protein levels and increased cell death ratio and treatment efficacy. Additionally, EVs exogenously loaded with miR-195-5p by electroporation reduced tumor volume in vivo and impaired engraftment and growth of xenografts implanted with melanoma cells exposed to MAPK inhibitors. Our study shows that miR-195-5p antitumoral activity can be spread to bystander cells through EVs, improving melanoma response to targeted therapy and revealing a promising EV-based strategy to increase clinical response in patients harboring BRAF mutations.

Antitumor Profile of Combined Matricaria recutita Flower Extract and 5-Fluorouracil Chemotherapy in Sarcoma 180 In Vivo Model.

Medicinal plants have been commonly associated with chemotherapeutic treatments, as an approach to reduce the toxicological risks of classical anticancer drugs. The objective of this study was to evaluate the effects of combining the antineoplastic drug 5-fluorouracil (5-FU) with Matricaria recutita flowers extract (MRFE) to treat mice transplanted with sarcoma 180. Tumor inhibition, body and visceral mass variation, biochemical, hematological, and histopathological parameters were evaluated. The isolated 5-FU, 5-FU+MRFE 100 mg/kg/day, and 5-FU+MRFE 200 mg/kg/day reduced tumor growth; however, 5-FU+MRFE 200 mg/kg/day showed a more significant tumor reduction when compared to 5-FU alone. These results corroborated with the analysis of the tumor histopathological and immunodetection of the Ki67 antigen. In the toxicological analysis of the association 5-FU+MRFE 200 mg/kg/day, an intense loss of body mass was observed, possibly as a result of diarrhea. In addition, spleen atrophy, with a reduction in white pulp, leukopenia and thrombocytopenia, was observed in the 5-FU groups alone and associated with MRFE 200 mg/kg/day; however, there was no statistical difference between these groups. Therefore, the MRFE 200 mg/kg/day did not interfere in myelosuppressive action of 5-FU. In hematological analysis, body and visceral mass variation and biochemical parameters related to renal (urea and creatinine) and cardiac (CK-MB) function, no alteration was observed. In biochemical parameters related to liver function enzymes, there was a reduction in aspartate transaminase (AST) values in the 5-FU groups alone and associated with MRFE 200 mg/kg/day; however, there was no statistical difference between these groups. Therefore, the MRFE 200 mg/kg/day does not appear to influence enzyme reduction. The results of this study suggest that the association between the 5-FU+MRFE 200 can positively interfere with the antitumor activity, promoting the antineoplastic-induced reduction in body mass, while minimizing the toxicity of chemotherapy.

Membrane-derived particles shed by PSMA-positive cells function as pro-angiogenic stimuli in tumors.

Cell membrane-derived particles (Mp) are rounded membrane-enclosed particles that are shed from tumor cells. Mp are formed from tumor membranes and are capable of tumor targeting and immunotherapeutic agents because they share membrane homology with parental cells; thus, they are under consideration as a drug delivery vehicle. Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein with enzymatic functionality, is highly expressed in Mp and extracellular vesicles (EV) from prostate cancer (PCa) with poor clinical prognosis. Although PSMA expression was previously shown in EV and Mp isolated from cell lines and from the blood of patients with high-grade PCa, no pathophysiological effects have been linked to PCa-derived Mp. Here, we compared Mp from PSMA-expressing (PSMA-Mp) and PSMA-non-expressing (WT-Mp) cells side by side in vitro and in vivo. PSMA-Mp can transfer PSMA and new phenotypic characteristics to the tumor microenvironment. The consequence of PSMA transfer to cells and increased secretion of vascular endothelial growth factor-A (VEGF-A), pro-angiogenic and pro-lymphangiogenic mediators, with increased 4E binding protein 1 (4EBP-1) phosphorylation.

Cashew Gum: A Review of Brazilian Patents and Pharmaceutical Applications with a Special Focus on Nanoparticles.

Natural polysaccharides are structures composed of highly diversified biological macromolecules whose properties have been exploited by a diversity of industries. Until 2018, the polysaccharides market raised more than US $ 12 billion worldwide, while an annual growth forecast of 4.8% is expected by 2026. The food industry is largely responsible for the consumption of this plant-source material, produced by microbiological fermentation. Among the used polysaccharides, gums are hydrocolloids obtained from a variety of sources and in different forms, being composed of salts of calcium, potassium, magnesium and sugar monomers. Their non-toxicity, hydrophilicity, viscosity, biodegradability, biocompatibility and sustainable production are among their main advantages. Although Brazil is amongst the largest producers of cashew gum, reaching 50 tons per year, the polysaccharide is not being used to its full potential, in particular, with regard to its uses in pharmaceuticals. Cashew gum (CG), obtained from Anacardium occidentale L., caught the attention of the industry only in 1970; in 1990, its production started to grow. Within the Brazilian academy, the groups from the Federal University of Ceará and Piauí are devoting the most efforts to the study of cashew gum, with a total of 31 articles already published. The number of patents in the country for innovations containing cashew tree gum has reached 14, including the technological process for the purification of cashew tree gum, comparison of physical and chemical methods for physicochemical characterizations, and optimum purification methodology. This scenario opens a range of opportunities for the use of cashew gum, mainly in the development of new pharmaceutical products, with a special interest in nanoparticles.

Analysis of the mechanisms of action of isopentenyl caffeate against Leishmania.

Leishmaniasis is a neglected parasitic disease for which the conventional treatment can be considered inefficient and extremely aggressive, generating several and severe side effects. Therefore, the discovery of new drug candidates is important for the improvement in the quality of life of patients. Previously, we reported the promising results of isopentyl caffeate (ICaf) against Leishmania chagasi (agent of visceral leishmaniasis) and Leishmania amazonensis (agent of cutaneous leishmaniasis) promastigotes, displaying IC50 of 1.56 and 1.71 µM, respectively. Herein, we aimed to decipher the mechanisms of anti-Leishmania action of ICaf. Light and scanning electron microscopy assays showed relevant morphological changes in promastigotes when treated with ICaf, including rounding of the parasite body, shortening of the flagellum, blebs on the plasma membrane and cellular aggregation. The parasite mitochondrion was targeted by ICaf, resulting in a significant reduction in its metabolic activity and electric membrane potential followed by an increase in the production of reactive oxygen species, which culminated in the loss of plasma membrane integrity and parasite death. Relevantly, ICaf also had a potent anti-amastigote action. The IC50 values calculated for intracellular amastigotes of L. amazonensis were 3.27, 1.60 and 1.52 µM, while for L. chagasi the values were 2.48, 1.84 and 1.60 µM, respectively, after treating the infected macrophages with ICaf for 24, 48 and 72 h. ICaf was well tolerated by THP-1 macrophages, which gave rise to excellent selectivity indexes considering both Leishmania species. The current results suggest that ICaf may emerge as a chemotherapeutic alternative for the treatment of leishmaniasis.

Anti-Tumor Efficiency of Perillylalcohol/ß-Cyclodextrin Inclusion Complexes in a Sarcoma S180-Induced Mice Model.

The low solubility and high volatility of perillyl alcohol (POH) compromise its bioavailability and potential use as chemotherapeutic drug. In this work, we have evaluated the anticancer activity of POH complexed with ß-cyclodextrin (ß-CD) using three complexation approaches. Molecular docking suggests the hydrogen-bond between POH and ß-cyclodextrin in molar proportion was 1:1. Thermal analysis and Fourier-transform infrared spectroscopy (FTIR) confirmed that the POH was enclosed in the ß-CD cavity. Also, there was a significant reduction of particle size thereof, indicating a modification of the ß-cyclodextrin crystals. The complexes were tested against human L929 fibroblasts after 24 h of incubation showing no signs of cytotoxicity. Concerning the histopathological results, the treatment with POH/ß-CD at a dose of 50 mg/kg promoted approximately 60% inhibition of tumor growth in a sarcoma S180-induced mice model and the reduction of nuclear immunoexpression of the Ki67 antigen compared to the control group. Obtained data suggest a significant reduction of cycling cells and tumor proliferation. Our results confirm that complexation of POH/ß-CD not only solves the problem related to the volatility of the monoterpene but also increases its efficiency as an antitumor agent.

Platelet-activating factor receptor (PAF-R)-dependent pathways control tumour growth and tumour response to chemotherapy.

BACKGROUND: Phagocytosis of apoptotic cells by macrophages induces a suppressor phenotype. Previous data from our group suggested that this occurs via Platelet-activating factor receptor (PAF-R)-mediated pathways. In the present study, we investigated the impact of apoptotic cell inoculation or induction by a chemotherapeutic agent (dacarbazine, DTIC) on tumour growth, microenvironmental parameters and survival, and the effect of treatment with a PAF-R antagonist (WEB2170). These studies were performed in murine tumours: Ehrlich Ascitis Tumour (EAT) and B16F10 melanoma. METHODS: Tumour growth was assessed by direct counting of EAT cells in the ascitis or by measuring the volume of the solid tumour. Parameters of the tumour microenvironment, such as the frequency of cells expressing cyclo-oxygenase-2 (COX-2), caspase-3 and galectin-3, and microvascular density, were determined by immunohistochemistry. Levels of vascular endothelium growth factor (VEGF) and prostaglandin E2 (PGE2) were determined by ELISA, and levels of nitric oxide (NO) by Griess reaction. PAF-R expression was analysed by immunohistochemistry and flow cytometry. RESULTS: Inoculation of apoptotic cells before EAT implantation stimulated tumour growth. This effect was reversed by in vivo pre-treatment with WEB2170. This treatment also reduced tumour growth and modified the microenvironment by reducing PGE2, VEGF and NO production. In B16F10 melanoma, WEB2170 alone or in association with DTIC significantly reduced tumour volume. Survival of the tumour-bearing mice was not affected by WEB2170 treatment but was significantly improved by the combination of DTIC with WEB2170. Tumour microenvironment elements were among the targets of the combination therapy since the relative frequency of COX-2 and galectin-3 positive cells and the microvascular density within the tumour mass were significantly reduced by treatment with WEB2170 or DTIC alone or in combination. Antibodies to PAF-R stained the cells from inside the tumour, but not the tumour cells grown in vitro. At the tissue level, a few cells (probably macrophages) stained positively with antibodies to PAF-R. CONCLUSIONS: We suggest that PAF-R-dependent pathways are activated during experimental tumour growth, modifying the microenvironment and the phenotype of the tumour macrophages in such a way as to favour tumour growth. Combination therapy with a PAF-R antagonist and a chemotherapeutic drug may represent a new and promising strategy for the treatment of some tumours.

Perillaldehyde 1,2-epoxide Loaded SLN-Tailored mAb: Production, Physicochemical Characterization and In Vitro Cytotoxicity Profile in MCF-7 Cell Lines.

We have developed a new cationic solid lipid nanoparticle (SLN) formulation, composed of Compritol ATO 888, poloxamer 188 and cetyltrimethylammonium bromide (CTAB), to load perillaldehyde 1,2-epoxide, and surface-tailored with a monoclonal antibody for site-specific targeting of human epithelial growth receptor 2 (HER2). Perillaldehyde 1,2-epoxide-loaded cationic SLN (cPa-SLN), with a mean particle size (z-Ave) of 275.31 ± 4.78 nm and polydispersity index (PI) of 0.303 ± 0.081, were produced by high shear homogenization. An encapsulation efficiency of cPa-SLN above 80% was achieved. The release of perillaldehyde 1,2-epoxide from cationic SLN followed the Korsemeyer-Peppas kinetic model, which is typically seen in nanoparticle formulations. The lipid peroxidation of cPa-SLN was assessed by the capacity to produce thiobarbituric acid-reactive substances, while the antioxidant activity was determined by the capacity to scavenge the stable radical DPPH. The surface functionalization of cPa-SLN with the antibody was done via streptavidin-biotin interaction, monitoring z-Ave, PI and ZP of the obtained assembly (cPa-SLN-SAb), as well as its stability in phosphate buffer. The effect of plain cationic SLN (c-SLN, monoterpene free), cPa-SLN and cPa-SLN-SAb onto the MCF-7 cell lines was evaluated in a concentration range from 0.01 to 0.1 mg/mL, confirming that streptavidin adsorption onto cPa-SLN-SAb improved the cell viability in comparison to the cationic cPa-SLN.

In Vitro Characterization, Modelling, and Antioxidant Properties of Polyphenon-60 from Green Tea in Eudragit S100-2 Chitosan Microspheres.

Eudragit S100-coated chitosan microspheres (S100Ch) are proposed as a new oral delivery system for green tea polyphenon-60 (PP60). PP60 is a mixture of polyphenolic compounds, known for its active role in decreasing oxidative stress and metabolic risk factors involved in diabetes and in other chronic diseases. Chitosan-PP60 microspheres prepared by an emulsion cross-linking method were coated with Eudragit S100 to ensure the release of PP60 in the terminal ileum. Different core-coat ratios of Eudragit and chitosan were tested. Optimized chitosan microspheres were obtained with a chitosan:PP60 ratio of 8:1 (Ch-PP608:1), rotation speed of 1500 rpm, and surfactant concentration of 1.0% (m/v) achieving a mean size of 7.16 µm. Their coating with the enteric polymer (S100Ch-PP60) increased the mean size significantly (51.4 µm). The in vitro modified-release of PP60 from S100Ch-PP60 was confirmed in simulated gastrointestinal conditions. Mathematical fitting models were used to characterize the release mechanism showing that both Ch-PP608:1 and S100Ch-PP60 fitted the Korsmeyers-Peppas model. The antioxidant activity of PP60 was kept in glutaraldehyde-crosslinked chitosan microspheres before and after their coating, showing an IC50 of 212.3 µg/mL and 154.4 µg/mL, respectively. The potential of chitosan microspheres for the delivery of catechins was illustrated, with limited risk of cytotoxicity as shown in Caco-2 cell lines using the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The beneficial effects of green tea and its derivatives in the management of metabolic disorders can be exploited using mucoadhesive chitosan microspheres coated with enteric polymers for colonic delivery.

Alginate Nanoparticles for Drug Delivery and Targeting.

Nanotechnology refers to the control, manipulation, study and manufacture of structures and devices at the nanometer size range. The small size, customized surface, improved solubility and multi-functionality of nanoparticles will continue to create new biomedical applications, as nanoparticles allow to dominate stability, solubility and bioavailability, as well controlled release of drugs. The type of a nanoparticle, and its related chemical, physical and morphological properties influence its interaction with living cells, as well as determine the route of clearance and possible toxic effects. This field requires cross-disciplinary research and gives opportunities to design and develop multifunctional devices, which allow the diagnosis and treatment of devastating diseases. Over the past few decades, biodegradable polymers have been studied for the fabrication of drug delivery systems. There was extensive development of biodegradable polymeric nanoparticles for drug delivery and tissue engineering, in view of their applications in controlling the release of drugs, stabilizing labile molecules from degradation and site-specific drug targeting. The primary aim is to reduce dosing frequency and prolong the therapeutic outcomes. For this purpose, inert excipients should be selected, being biopolymers, e.g. sodium alginate, commonly used in controlled drug delivery. Nanoparticles composed of alginate (known as anionic polysaccharide widely distributed in the cell walls of brown algae which, when in contact with water, forms a viscous gum) have emerged as one of the most extensively characterized biomaterials used for drug delivery and targeting a set of administration routes. Their advantages include not only the versatile physicochemical properties, which allow chemical modifications for site-specific targeting but also their biocompatibility and biodegradation profiles, as well as mucoadhesiveness. Furthermore, mechanical strength, gelation, and cell affinity can be modulated by combining alginate nanoparticles with other polymers, surface tailoring using specific targeting moieties and by chemical or physical cross-linking. However, for every physicochemical modification in the macromolecule/ nanoparticles, a new toxicological profile may be obtained. In this paper, the different aspects related to the use of alginate nanoparticles for drug delivery and targeting have been revised, as well as how their toxicological profile will determine the therapeutic outcome of the drug delivery system.

NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients.

The risk of developing metastatic disease in breast cancer patients is traditionally predictable based on the number of positive axillary lymph nodes, complemented with additional clinicopathological factors. However, since lymph node-negative patients have a 20-30% probability of developing metastatic disease, lymph node information alone is insufficient to accurately assess individual risk. Molecular approaches, such as multigene expression panels, analyze a set of cancer-related genes that more accurately predict the early risk of metastasis and the treatment response. Here, we present N-Myc downstream-regulated gene 4 (NDRG4) epigenetic silencing as a mechanistic biomarker of metastasis in ductal invasive breast tumors. While aberrant NDRG4 DNA hypermethylation is significantly associated with the development of metastatic disease, downregulation of NDRG4 transcription and protein expression is functionally associated with enhanced lymph node adhesion and cell mobility. Here, we show that epigenetic silencing of NDRG4 modulates integrin signaling by assembling ß1-integrins into large punctate clusters at the leading edge of tumor cells to promote an "adhesive switch," decreasing cell adhesion to fibronectin and increasing cell adhesion and migration towards vitronectin, an important component of human lymph nodes. Taken together, our functional and clinical observations suggest that NDRG4 is a potential mechanistic biomarker in breast cancer that is functionally associated with metastatic disease.

Structure and spasmolytic activity relationships of monoterpene analogues found in many aromatic plants.

Rotundifolone, a monoterpene isolated from the essential oil of the leaves of Mentha x villosa, is a constituent of several essential oils and known to have spasmolytic activity. The present study aimed to investigate the correlation between structure and spasmolytic activity of rotundifolone and its analogues in ileum isolated from guinea-pig. Five of the seven tested analogues were found to have a spasmolytic effect more potent than rotundifolone itself, except for pulegone and (+)-limonene. The comparison between rotundifolone and limonene oxide showed that the absence of the keto group did not decrease the relaxant effect. Comparison of the spasmolytic activity between rotundifolone and (+)-pulegone showed that the absence of the epoxy group did not decrease the relaxation of the ileum. Carvone epoxide was found to be significantly more potent than rotundifolone. The monoterpene (-)-carvone produced ileum relaxation and was more potent than its enantiomer (+)-carvone. (+)-Limonene and pulegone oxide showed a similar effect. The study showed that the functional groups and their position at the ring of rotundifolone contributed to the relaxation activity of the ileum. The absence of the oxygenated molecular structure is not a critical requirement for the molecule to be bioactive.

Biological applications of a chimeric probe for the assessment of galectin-3 ligands.

Beta1-6 branching of N-linked oligosaccharides has been correlated with the progression of different cancers. The leukoagglutinins of Phaseolus vulgaris (L-PHA) have been used to study this pattern of glycosylation whose biological significance is incompletely understood. The animal lectin, galectin-3, also binds to structures recognized by L-PHA. To develop a functional tool for the in situ identification of this pattern of glycosylation, human galectin-3 was fused to bacterial alkaline phosphatase (gal3/AP). Gal3/AP recognized both A and B blood group saccharides (B>A) and lactosamine derivatives. Gal3/AP recognition depended at least in part on the N-linked oligosaccharides of different glycoproteins. The presence and distribution of galectin-3 ligands were analyzed in both murine and human normal and tumor samples. Loss of apical expression of galectin-3 ligands was commonly found in carcinomas. Endothelial and inflammatory cells were enriched in galectin-3 ligands as compared with tumor cells; thus, gal3/AP is a suitable tool for studying tumor microenvironments. Comparative analysis of both gal3/AP and L-PHA binding patterns indicated that although similar, these patterns are not identical. The probe developed was useful for several immunoenzymatic assays and will allow the physiological and clinical significance of the expression pattern of galectin-3 ligands to be established. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.