MiR126-targeted-nanoparticles combined with PI3K/AKT inhibitor as a new strategy to overcome melanoma resistance.

Metastatic melanoma poses significant challenges as a highly lethal disease. Despite the success of molecular targeting using BRAFV600E inhibitors (BRAFis) and immunotherapy, the emergence of early recurrence remains an issue and there is the need for novel therapeutic approaches. This study aimed at creating a targeted delivery system for the oncosuppressor microRNA 126 (miR126) and testing its effectiveness in combination with a phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT) inhibitor for treating metastatic melanoma resistant to BRAFis. To achieve this, we synthesized chitosan nanoparticles containing a chemically modified miR126 sequence. These nanoparticles were further functionalized with an antibody specific to the chondroitin sulfate proteoglycan 4 (CSPG4) melanoma marker. After evaluation in vitro, the efficacy of this treatment was evaluated through an in vivo experiment using mice bearing resistant human melanoma. The co-administration of miR126 and the PI3K/AKT inhibitor in these experiments significantly reduced tumor growth and inhibited the formation of liver and lung metastases. These results provide evidence for a strategy to target an oncosuppressive nucleic acid sequence to tumor cells while simultaneously protecting it from plasma degradation. The system described in this study exhibits encouraging potential for the effective treatment of therapy-resistant metastatic melanoma while also presenting a prospective approach for other forms of cancer.

The Synergistic Combination of Curcumin and Polydatin Improves Temozolomide Efficacy on Glioblastoma Cells.

Glioblastoma (GBL) is one of the more malignant primary brain tumors; it is currently treated by a multimodality strategy including surgery, and radio- and chemotherapy, mainly consisting of temozolomide (TMZ)-based chemotherapy. Tumor relapse often occurs due to the establishment of TMZ resistance, with a patient median survival time of <2 years. The identification of natural molecules with strong anti-tumor activity led to the combination of these compounds with conventional chemotherapeutic agents, developing protocols for integrated anticancer therapies. Curcumin (CUR), resveratrol (RES), and its glucoside polydatin (PLD) are widely employed in the pharmaceutical and nutraceutical fields, and several studies have demonstrated that the combination of these natural products was more cytotoxic than the individual compounds alone against different cancers. Some of us recently demonstrated the synergistic efficacy of the sublingual administration of a new nutraceutical formulation of CUR+PLD in reducing tumor size and improving GBL patient survival. To provide some experimental evidence to reinforce these clinical results, we investigated if pretreatment with a combination of CUR+PLD can improve TMZ cytotoxicity on GBL cells by analyzing the effects on cell cycle, viability, morphology, expression of proteins related to cell proliferation, differentiation, apoptosis or autophagy, and the actin network. Cell viability was assessed using the MTT assay or a CytoSmart cell counter. CalcuSyn software was used to study the CUR+PLD synergism. The morphology was evaluated by optical and scanning electron microscopy, and protein expression was analyzed by Western blot. Flow cytometry was used for the cell cycle, autophagic flux, and apoptosis analyses. The results provide evidence that CUR and PLD, acting in synergy with each other, strongly improve the efficacy of alkylating anti-tumor agents such as TMZ on drug-resistant GBL cells through their ability to affect survival, differentiation, and tumor invasiveness.

Phytocompounds and Nanoformulations for Anticancer Therapy: A Review.

Cancer is a complex disease that affects millions of people and remains a major public health problem worldwide. Conventional cancer treatments, including surgery, chemotherapy, immunotherapy, and radiotherapy, have limited achievements and multiple drawbacks, among which are healthy tissue damage and multidrug-resistant phenotype onset. Increasing evidence shows that many plants' natural products, as well as their bioactive compounds, have promising anticancer activity and exhibit minimal toxicity compared to conventional anticancer drugs. However, their widespread use in cancer therapy is severely restricted by limitations in terms of their water solubility, absorption, lack of stability, bioavailability, and selective targeting. The use of nanoformulations for plants' natural product transportation and delivery could be helpful in overcoming these limitations, thus enhancing their therapeutic efficacy and providing the basis for improved anticancer treatment strategies. The present review is aimed at providing an update on some phytocompounds (curcumin, resveratrol, quercetin, and cannabinoids, among others) and their main nanoformulations showing antitumor activities, both in vitro and in vivo, against such different human cancer types as breast and colorectal cancer, lymphomas, malignant melanoma, glioblastoma multiforme, and osteosarcoma. The intracellular pathways underlying phytocompound anticancer activity and the main advantages of nanoformulation employment are also examined. Finally, this review critically analyzes the research gaps and limitations causing the limited success of phytocompounds' and nanoformulations' clinical translation.

Phytochemical Analysis and In Vitro Antileukemic Activity of Alkaloid-Enriched Extracts from Vinca sardoa (Stearn) Pignatti.

Vinca sardoa (Stearn) Pignatti, known as Sardinian periwinkle, is widely diffused in Sardinia (Italy). This species contains indole alkaloids, which are known to have a great variety of biological activities. This study investigated the antileukemic activity against a B lymphoblast cell line (SUP-B15) of V. sardoa alkaloid-rich extracts obtained from plants grown in Italy, in Iglesias (Sardinia) and Rome (Latium). All the extracts showed a good capacity to induce reductions in cell proliferation of up to 50% at the tested concentrations (1-15 µg/mL). Moreover, none of the extracts showed cytotoxicity on normal cells at all the studied concentrations.

"In vivo" and "in vitro" antimicrobial activity of Origanum vulgare essential oil and its two phenolic compounds on clinical isolates of Candida spp.

A limited therapeutic arsenal is currently available against Candida infections that show high resistance to antifungal agents. For this reason, there is a great need to prioritize testing therapeutic agents for the treatment of candidiasis. The use of essential oils and their phytoconstituents has been emphasized as a new therapeutic approach. The cell surface hydrophobicity (CSH), polysaccharide content, antimicrobial activity of essential oil from Origanum vulgare L. (OVEO), and its two phenolic compounds carvacrol and thymol were evaluated in four different Candida spp. (Candida albicans and emerging non-albicans Candida (NAC) species, such as C. glabrata, C. tropicalis, and C. krusei). The results showed the differences between Candida species; for example, C. tropicalis revealed higher resistance than other strains to different natural molecule treatments. The ultrastructural variabilities in the biomembranes and cell walls of these Candida spp. might explain the different biological effects observed after OVEO, carvacrol and thymol treatments. Therefore, to study the biological effects of these natural compounds on Candida strains, the samples were observed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Moreover, the release of cellular materials and their "in vivo" antimicrobial activity on infected G. mellonella larvae were evaluated. The novelty of this study is the demonstration that exists a close correlation between both structural architecture of cell walls and biomembranes' organization with cell fungal responses to essential oils treatments. Overall, these results suggest practical limits to the predictability.

Peptide-Mediated Targeted Delivery of Aloe-Emodin as Anticancer Drug.

Breast cancer is one of the most diffuse cancers in the world and despite the availability of the different drugs employed against it, the need for new and particularly more specific molecules is ever growing. In this framework, natural products are increasingly assuming an important role as new anticancer drugs. Aloe-emodin (AE) is one of the best characterized molecules in this field. The functionalization of bioactive natural products with selected peptide sequences to enhance their bioavailability and specificity of action is a powerful and promising strategy. In this study, we analyzed the cell specificity, cell viability effects, intracellular distribution, and immune cell response of a new peptide conjugate of Aloe-emodin in SKBR3 and A549 cell lines by means of viability tests, flow cytometry, and confocal microscopy. The conjugate proved to be more effective at reducing cell viability than AE in both cell lines. Furthermore, the results showed that it was mainly internalized within the SKBR3 cells, showing a nuclear localization, while A459 cells displayed mainly a cytoplasmic distribution. A preserving effect of the conjugate on NKs' cell function was also observed. The designed conjugate showed a promising specific activity towards HER2-expressing cells coupled with an enhanced water solubility and a higher cytotoxicity; thus, the resulting proof-of-concept molecule can be further improved as an anticancer compound.

Ultrastructural Damages to H1N1 Influenza Virus Caused by Vapor Essential Oils.

Influenza viruses are transmitted from human to human via airborne droplets and can be transferred through contaminated environmental surfaces. Some works have demonstrated the efficacy of essential oils (EOs) as antimicrobial and antiviral agents, but most of them examined the liquid phases, which are generally toxic for oral applications. In our study, we describe the antiviral activity of Citrus bergamia, Melaleuca alternifolia, Illicium verum and Eucalyptus globulus vapor EOs against influenza virus type A. In the vapor phase, C. bergamia and M. alternifolia strongly reduced viral cytopathic effect without exerting any cytotoxicity. The E. globulus vapor EO reduced viral infection by 78% with no cytotoxicity, while I. verum was not effective. Furthermore, we characterized the EOs and their vapor phase by the head-space gas chromatography-mass spectrometry technique, observing that the major component found in each liquid EO is the same one of the corresponding vapor phases, with the exception of M. alternifolia. To deepen the mechanism of action, the morphological integrity of virus particles was checked by negative staining transmission electron microscopy, showing that they interfere with the lipid bilayer of the viral envelope, leading to the decomposition of membranes. We speculated that the most abundant components of the vapor EOs might directly interfere with influenza virus envelope structures or mask viral structures important for early steps of viral infection.

Drug Delivery Systems of Natural Products in Oncology.

In recent decades, increasing interest in the use of natural products in anticancer therapy field has been observed, mainly due to unsolved drug-resistance problems. The antitumoral effect of natural compounds involving different signaling pathways and cellular mechanisms has been largely demonstrated in in vitro and in vivo studies. The encapsulation of natural products into different delivery systems may lead to a significant enhancement of their anticancer efficacy by increasing in vivo stability and bioavailability, reducing side adverse effects and improving target-specific activity. This review will focus on research studies related to nanostructured systems containing natural compounds for new drug delivery tools in anticancer therapies.

Detection of exosomal prions in blood by immunochemistry techniques.

In most forms of prion diseases, blood is infectious, but detection by immunochemistry techniques of the only available marker of infection (the misfolded prion protein, PrPTSE) in blood remains elusive. We developed a novel method for the detection of PrPTSE in blood of prion-infected rodents based on the finding that PrPTSE is associated with plasma exosomes. However, further purification of the exosomes on a sucrose gradient was necessary to remove plasma immunoglobulins, which interfere with PrPTSE, masking its detection by immunochemistry. Finally, we report that about 20% of plasma infectivity is associated with exosomes.

Immune surveillance properties of human NK cell-derived exosomes.

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56+ and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.

Esculentin(1-21), an amphibian skin membrane-active peptide with potent activity on both planktonic and biofilm cells of the bacterial pathogen Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that forms sessile communities, named biofilms. The non-motile forms are very difficult to eradicate and are often associated with the establishment of persistent infections, especially in patients with cystic fibrosis. The resistance of P. aeruginosa to conventional antibiotics has become a growing health concern worldwide and has prompted the search for new anti-infective agents with new modes of action. Naturally occurring antimicrobial peptides (AMPs) represent promising future template candidates. Here we report on the potent activity and membrane-perturbing effects of the amphibian AMP esculentin(1-21), on both the free-living and sessile forms of P. aeruginosa, as a possible mechanism for biofilm disruption. Furthermore, the findings that esculentin(1-21) is able to prolong survival of animals in models of sepsis and pulmonary infection indicate that this peptide can be a promising template for the generation of new antibiotic formulations to advance care of infections caused by P. aeruginosa.

Fighting Microbial Infections from Escherichia coli O157:H7: The Combined Use of Three Essential Oils of the Cymbopogon Genus and a Derivative of Esculentin-1a Peptide.

The absence of effective therapy against Escherichia coli O157:H7 infections has led to the need to develop new antimicrobial agents. As the use of synergistic combinations of natural antimicrobial compounds is growing as a new weapon in the fight against multidrug-resistant bacteria, here, we have tested new synergistic combinations of natural agents. Notably, we investigated a possible synergistic effect of combinations of essential oils and natural peptides to counteract the formation of biofilm. We chose three essential oils (i.e., Cymbopogon citratus, C. flexuosus and C. martinii) and one peptide already studied in our previous works. We determined the fractional inhibitory concentration (FIC) by analyzing the combination of the peptide derived from esculentin-1a, Esc(1-21), with the three essential oils. We also studied the effects of combinations by time-kill curves, scanning electron microscopy on biofilm and Sytox Green on cell membrane permeability. Finally, we analyzed the expression of different genes implicated in motility, biofilm formation and stress responses. The results showed a different pattern of gene expression in bacteria treated with the mixtures compared to those treated with the peptide or the single C. citratus essential oil. In conclusion, we demonstrated that the three essential oils used in combination with the peptide showed synergy against the E. coli O157:H7, proving attractive as an alternative strategy against E. coli pathogen infections.

The PavA-like fibronectin-binding protein of Enterococcus faecalis, EfbA, is important for virulence in a mouse model of ascending urinary tract infection.

Enterococcus faecalis is an established nosocomial pathogen, yet the pathogenesis of enterococcal infections, particularly of urinary tract infections (UTIs), remains to be fully elucidated. Fibronectin-binding proteins have been identified as potent adhesins in pathogenic Gram-positive cocci. Here, we characterized EfbA, which is encoded by the enterococcal orthologue of Streptococcus pneumoniae pavA. Similar to PavA, the anchorless EfbA protein was localized to the enterococcal cell outer surface and bound to immobilized human fibronectin. In addition to abrogated EfbA expression, deletion of the efbA gene eliminated EfbA from the cell surface and drastically reduced the enterococcal cell binding to immobilized fibronectin. The ΔefbA deletion mutant was highly attenuated vs wild-type in a murine ascending UTI model, consistent with an increased tropism for the kidney relative to the bladder. These results provide the first evidence that EfbA of E. faecalis plays a role in UTIs, probably contributing to the pathogenesis in this site.

Interaction of Drug-Sensitive and -Resistant Human Melanoma Cells with HUVEC Cells: A Label-Free Cell-Based Impedance Study.

Cancer cell extravasation is a crucial step in cancer metastasis. However, many of the mechanisms involved in this process are only now being elucidated. Thus, in the present study we analysed the trans-endothelial invasion of melanoma cells by a high throughput label-free cell impedance assay applied to transwell chamber invasion assay. This technique monitors and quantifies in real-time the invasion of endothelial cells by malignant tumour cells, for a long time, avoiding artefacts due to preparation of the end point measurements. Results obtained by impedance analysis were compared with endpoint measurements. In this study, we used human melanoma M14 wild type (WT) cells and their drug resistant counterparts, M14 multidrug resistant (ADR) melanoma cells, selected by prolonged exposure to doxorubicin (DOX). Tumour cells were co-cultured with monolayers of human umbilical vein endothelial cells (HUVEC). Results herein reported demonstrated that: (i) the trans-endothelial migration of resistant melanoma cells was faster than sensitive ones; (ii) the endothelial cells appeared to be strongly affected by the transmigration of melanoma cells which showed the ability to degrade their cytoplasm; (iii) resistant cells preferentially adopted the transcellular invasion vs. the paracellular one; (iv) the endothelial damage mediated by tumour metalloproteinases seemed to be reversible.

Gold nanorods derivatized with CTAB and hydroquinone or ascorbic acid: spectroscopic investigation of anisotropic nanoparticles of different shapes and sizes.

Gold nanorods stabilized by binary ligand mixtures of cetyltrimethylammonium bromide (CTAB, primary ligand) and ascorbic acid or hydroquinone were investigated by complementary synchrotron radiation-induced spectroscopies and microscopies, with the aim to find evidence of the influence of the secondary ligand molecular and chemical structure on the nanorod shapes and size ratios. Indeed, as it is well known that the CTAB interaction with Ag(i) ions at the NR surface plays a key role in directing the anisotropic growth of nanorods, the possibility to finely control the NR shape and dimension by opportunely selecting the secondary ligands opens new perspectives in the design and synthesis of anisotropic nanoparticles.

Terpinen-4-ol, the Main Bioactive Component of Tea Tree Oil, as an Innovative Antimicrobial Agent against Legionella pneumophila.

Legionella pneumophila (Lp), responsible for a severe pneumonia called Legionnaires' disease, represents an important health burden in Europe. Prevention and control of Lp contamination in warm water systems is still a great challenge often due to the failure in disinfection procedures. The aim of this study was to evaluate the in vitro activity of Terpinen-4-ol (T-4-ol) as potential agent for Lp control, in comparison with the essential oil of Melaleuca alternifolia (tea tree) (TTO. Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of T-4-ol were determined by broth micro-dilution and a micro-atmosphere diffusion method to investigate the anti-Lp effects of T-4-ol and TTO vapors. Scanning Electron Microscopy (SEM) was adopted to highlight the morphological changes and Lp damage following T-4-ol and TTO treatments. The greatest antimicrobial activity against Lp was shown by T-4-ol with a MIC range of 0.06-0.125% v/v and MBC range of 0.25-0.5% v/v. The TTO and T-4-ol MIC and MBC decreased with increasing temperature (36 °C to 45 ± 1 °C), and temperature also significantly influenced the efficacy of TTO and T-4-ol vapors. The time-killing assay showed an exponential trend of T-4-ol bactericidal activity at 0.5% v/v against Lp. SEM observations revealed a concentration- and temperature- dependent effect of T-4-ol and TTO on cell surface morphology with alterations. These findings suggest that T-4-ol is active against Lp and further studies may address the potential effectiveness of T-4-ol for control of water systems.

Design, Synthesis, and In Vitro, In Silico and In Cellulo Evaluation of New Pyrimidine and Pyridine Amide and Carbamate Derivatives as Multi-Functional Cholinesterase Inhibitors.

Alzheimer disease is an age-linked neurodegenerative disorder representing one of the greatest medical care challenges of our century. Several drugs are useful in ameliorating the symptoms, even if none could stop or reverse disease progression. The standard approach is represented by the cholinesterase inhibitors (ChEIs) that restore the levels of acetylcholine (ACh) by inhibiting the acetylcholinesterase (AChE). Still, their limited efficacy has prompted researchers to develop new ChEIs that could also reduce the oxidative stress by exhibiting antioxidant properties and by chelating the main metals involved in the disease. Recently, we developed some derivatives constituted by a 2-amino-pyrimidine or a 2-amino-pyridine moiety connected to various aromatic groups by a flexible amino-alkyl linker as new dual inhibitors of AChE and butyrylcholinesterase (BChE). Following our previous studies, in this work we explored the role of the flexible linker by replacing the amino group with an amide or a carbamic group. The most potent compounds showed higher selectivity against BChE in respect to AChE, proving also to possess a weak anti-aggregating activity toward Aß42 and tau and to be able to chelate Cu2+ and Fe3+ ions. Molecular docking and molecular dynamic studies proposed possible binding modes with the enzymes. It is noteworthy that these compounds were predicted as BBB-permeable and showed low cytotoxicity on the human brain cell line.

High Activity of N-Acetylcysteine in Combination with Beta-Lactams against Carbapenem-Resistant Klebsiella pneumoniae and Acinetobacter baumannii.

AIM: The aim of the study was to evaluate the in vitro activity of N-acetylcysteine (NAC), alone or in combination with beta-lactams, against carbapenem-resistant Klebsiella pneumoniae (CR-Kp) and Acinetobacter baumannii (CR-Ab). METHODS: The antibacterial activity of each compound was tested by broth microdilution and the synergism was evaluated by the checkerboard method. Killing studies of NAC alone and in combination with beta-lactams were performed. Bacterial morphological changes were investigated with scanning electron microscopy (SEM). RESULTS: Overall, 30 strains were included (15 CR-Kp and 15 CR-Ab). The NAC Minimal Inhibitory Concentrations (MIC)50/90 were 5/5 and 2.5/5 mg/mL for CR-Kp and CR-Ab, respectively. For both microorganisms, NAC, in addition to beta-lactams (meropenem for CR-Kp, meropenem and ampicillin/sulbactam for CR-Ab, respectively), was able to enhance their activity. The killing studies showed a rapid and concentration-dependent activity of NAC alone; the addition of NAC to meropenem or ampicillin/sulbactam at subinhibitory concentrations induced a fast and lasting bactericidal activity that persisted over time. The SEM analyses showed evident morphological alterations of the bacterial cells following incubation with NAC, alone and in combination with meropenem. CONCLUSIONS: NAC demonstrated a high in vitro activity against CR-Kp and CR-Ab and was able to enhance beta-lactams' susceptibility in the tested strains. The preliminary data on the SEM analyses confirmed the in vitro results.

The MP65 gene is required for cell wall integrity, adherence to epithelial cells and biofilm formation in Candida albicans.

BACKGROUND: The MP65 gene of Candida albicans (orf19.1779) encodes a putative ß-glucanase mannoprotein of 65 kDa, which plays a main role in a host-fungus relationship, morphogenesis and pathogenicity. In this study, we performed an extensive analysis of a mp65Δ mutant to assess the role of this protein in cell wall integrity, adherence to epithelial cells and biofilm formation. RESULTS: The mp65Δ mutant showed a high sensitivity to a range of cell wall-perturbing and degrading agents, especially Congo red, which induced morphological changes such as swelling, clumping and formation of hyphae. The mp65Δ mutant showed an activation of two MAPKs (Mkc1p and Cek1p), a high level of expression of two stress-related genes (DDR48 and SOD5), and a modulated expression of ß-glucan epitopes, but no gross changes in cell wall polysaccharide composition. Interestingly, the mp65Δ mutant displayed a marked reduction in adhesion to BEC and Caco-2 cells and severe defects in biofilm formation when compared to the wild type. All of the mentioned properties were totally or partially recovered in a revertant strain, demonstrating the specificity of gene deletion. CONCLUSIONS: We demonstrate that the MP65 gene of Candida albicans plays a significant role in maintaining cell wall integrity, as well as in adherence to epithelia and biofilm formation, which are major virulence attributes of this fungus.

Tea tree oil might combat melanoma.

In this study we present new data from experiments focused on the antitumor activity of tea tree oil (TTO), an essential oil distilled from Melaleuca alternifolia. TTO proved to be capable of inhibiting the growth of melanoma cells and of overcoming multidrug resistance (MDR), as we reported in our previous study. Moreover, the survival role of the MDR-marker P-glycoprotein appears to be involved in the mechanism of invasion of melanoma cells. The results reported herein indicate that TTO and its main active component, terpinen-4-ol, can also interfere with the migration and invasion processes of drug-sensitive and drug-resistant melanoma cells.