New pharmacological strategies for cutaneous malignant melanoma

Human cutaneous melanoma is an aggressive and chemotherapy resistant type of cancer. Although the development of new targeted therapies and immunologic agents has completely changed the treatment guidelines, one of the most important tasks for the future will be to overcome acquired resistance. In this thesis we investigated different pharmacological strategies against human melanoma cells. Particularly, we demonstrated the theranostic properties (i.e., the ability of imaging and pharmacological silencing activity) of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. This may represent an innovative approach for cancer diagnosis and treatment in melanoma patients because survivin is an inhibitor of apoptosis overexpressed in tumor cells and almost undetectable in human melanocytes. We also provide evidence of the pro-apoptotic effect and cell cycle arrest ability of AM251, a cannabinoid type 1 receptor antagonist/inverse agonist with an anticancer potency comparable to that observed for cisplatin. This compound may be a potential prototype for the development of promising diarylpyrazole derivatives to be evaluated in human cutaneous melanoma. Finally, we demonstrated that the cannabinoid type 1 receptor is markedly expressed in stem-like cells and not expressed in the BRAF-wild type parental cells. Otherwise, both primary BRAF-mutated melanoma cultures and their correspondent melanoma-initiating cells expressed high levels of this receptor subtype. These findings suggest a possible role of the endocannabinoid system in determining the phenotype of melanoma cells and their potential to cause central nervous system metastases

Circulating MicroRNAs in Cutaneous Melanoma Diagnosis and Prognosis

Cutaneous melanoma represents a challenge for pharmacologists and clinicians due to their high degree of genetic and phenotypic heterogeneity. The identification of new non-invasive and informative biomarkers would therefore represent a substantial step to adequately treat melanoma patients. MicroRNAs (miRNAs) are a class of small non-coding RNAs that play an important role as negative regulators of gene expression. Several studies have demonstrated their correlation with disease status in different types of cancer including melanoma. Extracellular miRNAs are released from both tumor cells and/or normal cells. MiRNAs do not circulate freely in biological fluids but they are incorporated into extracellular vesicles or form complexes with lipids and proteins. Circulating miRNAs may represent potential biomarkers of cutaneous melanoma diagnosis and patient prognosis. Longitudinal monitoring of cell-free miRNAs in biological fluids of melanoma patients could help clinicians to predict disease progression before the tumor becomes resistant to a given drug. However, to confirm their clinical utility it will be necessary to validate the best available technique for their detection and quantification and to test selected miRNAs in prospective clinical trials

Intake of Natural Compounds and Circulating microRNA Expression Levels: Their Relationship Investigated in Healthy Subjects With Different Dietary Habits

Diet has a strong influence on many physiological processes, which in turn have important implications on a variety of pathological conditions. In this respect, microRNAs (miRNAs), a class of small non-coding RNAs playing a relevant epigenetic role in controlling gene expression, may represent mediators between the dietary intake and the healthy status. Despite great advances in the field of nutri-epigenomics, it remains unclear how miRNA expression is modulated by the diet and, specifically, the intake of specific nutrients. We investigated the whole circulating miRNome by small RNA-sequencing performed on plasma samples of 120 healthy volunteers with different dietary habits (vegans, vegetarians, and omnivores). Dietary intakes of specific nutrients were estimated for each subject from the information reported in the food-frequency questionnaire previously validated in the EPIC study. We focused hereby on the intake of 23 natural compounds (NCs) of the classes of lipids, micro-elements, and vitamins. We identified 78 significant correlations (rho > 0.300, p-value < 0.05) among the estimated daily intake of 13 NCs and the expression levels of 58 plasma miRNAs. Overall, vitamin D, sodium, and vitamin E correlated with the largest number of miRNAs. All the identified correlations were consistent among the three dietary groups and 22 of them were confirmed as significant (p-value < 0.05) by age-, gender-, and body-mass index-adjusted Generalized Linear regression Model analysis. miR-23a-3p expression levels were related with different NCs including a significant positive correlation with sodium (rho = 0.377) and significant negative correlations with lipid-related NCs and vitamin E. Conversely, the estimated intake of vitamin D was negatively correlated with the expression of the highest number of circulating miRNAs, particularly miR-1277-5p (rho = −0.393) and miR-144-3p (rho = −0.393). Functional analysis of the targets of sodium intake-correlated miRNAs highlighted terms related to cardiac development. A similar approach on targets of those miRNAs correlated with vitamin D intake showed an enrichment in genes involved in hormone metabolisms, while the response to chronic inflammation was among the top enriched processes involving targets of miRNAs negatively related with vitamin E intake. Our findings show that nutrients through the habitual diet influence circulating miRNA profiles and highlight that this aspect must be considered in the nutri-epigenomic research

Cholinesterase-like organocatalysis by imidazole and imidazole-bearing molecules

Organocatalysis, which is mostly explored for its new potential industrial applications, also represents a chemical event involved in endogenous processes. In the present study, we provide the first evidence that imidazole and imidazole derivatives have cholinesterase-like properties since they can accelerate the hydrolysis of acetylthiocholine and propionylthiocholine in a concentration-dependent manner. The natural imidazole-containing molecules as L-histidine and histamine show a catalytic activity, comparable to that of imidazole itself, whereas synthetic molecules, as cimetidine and clonidine, were less active. In the experimental conditions used, the reaction progress curves were sigmoidal and the rational of such unexpected behavior as well as the mechanism of catalysis is discussed. Although indirectly, findings of the present study suggests that imidazolic compounds may interfere with the homeostasis of the cholinergic system in vivo

Tumor Suppressor Role of hsa-miR-193a-3p and -5p in Cutaneous Melanoma

Background: Remarkable deregulation of several microRNAs (miRNAs) is demonstrated in cutaneous melanoma. hsa-miR-193a-3p is reported to be under-expressed in tissues and in plasma of melanoma patients, but the role of both miR-193a arms in melanoma is not known yet. Methods: After observing the reduced levels of miR-193a arms in plasma exosomes of melanoma patients, the effects of hsa-miR-193a-3p and –5p transfection in cutaneous melanoma cell lines are investigated. Results: In melanoma cell lines A375, 501Mel, and MeWo, the ectopic over-expression of miR-193a arms significantly reduced cell viability as well as the expression of genes involved in proliferation (ERBB2, KRAS, PIK3R3, and MTOR) and apoptosis (MCL1 and NUSAP1). These functional features were accompanied by a significant downregulation of Akt and Erk pathways and a strong increase in the apoptotic process. Since in silico databases revealed TROY, an orphan member of the tumor necrosis receptor family, as a potential direct target of miR-193a-5p, this possibility was investigated using the luciferase assay and excluded by our results. Conclusions: Our results underline a relevant role of miR-193a, both -3p and -5p, as tumor suppressors clarifying the intracellular mechanisms involved and suggesting that their ectopic over-expression could represent a novel treatment for cutaneous melanoma patients

Harnessing therapeutic viruses as a delivery vehicle for RNA-based therapy

Messenger RNA (mRNA) and microRNA (miRNA)-based therapeutics have become attractive alternatives to DNA-based therapeutics due to recent advances in manufacture, scalability and cost. Also, RNA-based therapeutics are considered safe since there are no risk of inducing genomic changes as well as the potential adverse effects would be only temporary due to the transient nature of RNA-based therapeutics. However, efficient in vivo delivery of RNA-based therapeutics remains a challenge. We have developed a delivery platform for RNA-based therapeutics by exploiting the physicochemical properties of enveloped viruses. By physically attaching cationic liposome/RNA complexes onto the viral envelope of vaccinia virus, we were able to deliver mRNA, self-replicating RNA as well as miRNA inside target cells. Also, we showed that this platform, called viRNA platform, can efficiently deliver functional miRNA mimics into B16.OVA tumour in vivo.Peer reviewe

miRNA Modulation and Antitumor Activity by the Extra-Virgin Olive Oil Polyphenol Oleacein in Human Melanoma Cells

Extra-virgin olive oil (EVOO) polyphenols contribute to Mediterranean diet health- promoting properties. One of the most abundant secoiridoid present in EVOO, Oleacein (OA), demonstrated anticancer activity against several tumors. Nevertheless, its role against melanoma has not still investigated. This study aimed at determining in vitro the antimelanoma activity of OA and the relative mechanism of action. OA induced cell growth inhibition in 501Mel melanoma cells with an IC50 in the low micromolar range of concentrations. Moreover, an OA concentration approximating the IC50 induced G1/S phase arrest, DNA fragmentation, and downregulation of genes encoding antiapoptotic (BCL2 and MCL1) and proproliferative (c-KIT, K-RAS, PIK3R3, mTOR) proteins, while increased transcription levels of the proapoptotic protein BAX. Concordantly, OA increased the levels of miR-193a-3p (targeting MCL1, c-KIT and K-RAS), miR-193a-5p (targeting PIK3R3 and mTOR), miR-34a-5p (targeting BCL2 and c-KIT) and miR-16-5p (miR-16-5p targeting BCL2, K-RAS and mTOR), while decreased miR-214-3p (targeting BAX). These modulatory effects might contribute to the inhibition of 501Mel melanoma cell growth observed after treatment with an olive leaves-derived formulation rich in OA, with potential application against in situ cutaneous melanoma. Altogether, these results demonstrate the ability of OA to contrast the proliferation of cutaneous melanoma cells through the transcriptional modulation of relevant genes and microRNAs, confirming the anticancer potential of EVOO and suggesting OA as a chemopreventive agent for cancer disease therapy

Molecular beacon-decorated polymethylmethacrylate core-shell fluorescent nanoparticles for the detection of survivin mRNA in human cancer cells.

One of the main goals of nanomedicine in cancer is the development of effective drug delivery systems, primarily nanoparticles. Survivin, an overexpressed anti-apoptotic protein in cancer, represents a pharmacological target for therapy and a Molecular Beacon (MB) specific for survivin mRNA is available. In this study, the ability of polymethylmethacrylate nanoparticles (PMMA-NPs) to promote survivin MB uptake in human A549 cells was investigated. Fluorescent and positively charged core PMMA-NPs of nearly 60nm, obtained through an emulsion co-polymerization reaction, and the MB alone were evaluated in solution, for their analytical characterization; then, the MB specificity and functionality were verified after adsorption onto the PMMA-NPs. The carrier ability of PMMA-NPs in A549 was examined by confocal microscopy. With the optimized protocol, a hardly detectable fluorescent signal was obtained after incubation of the cells with the MB alone (fluorescent spots per cell of 1.90±0.40 with a mean area of 1.04±0.20µm2), while bright fluorescent spots inside the cells were evident by using the MB loaded onto the PMMA-NPs. (27.50±2.30 fluorescent spots per cell with a mean area of 2.35±0.16µm2). These results demonstrate the ability of the PMMA-NPs to promote the survivin-MB internalization, suggesting that this complex might represent a promising strategy for intracellular sensing and for the reduction of cancer cell proliferation

Topical application of silymarin enhances cutaneous wound healing in rats

Abstract Wound healing in a short period with minimum side effects is one of the major goals of medical sciences. Silymarin, an extract from Silybum marianum, has been shown to have antioxidant and anti-inflammatory properties. This study investigates the wound healing activity of silymarin topical formulation in an in experimental model. A 875 mm2 (25 × 35 mm) full-thickness excision was made on the abdominal region of each rat by a surgical blade and the day on which the wound was made considered as day 0. Each rat was treated two times each day. On days 1,4, 8 and 12, the wound area was measured using precise caliber and camera imaging. On day 12, blood samples were collected for the analysis of antioxidant, malondialdehyde and estradiol levels. After 12 days of treatment, rats were sacrificed and abdominal region tissues used for histological analyses. The study showed that topical application of silymarin on wound in rats improved wound healing correlating with less redness, exudates and swelling. Furthermore, in serum of rats treated with silymarin ointment improved antioxidant and estradiol levels, while decreased malondialdehyde levels, a marker of oxidative stress. Histological analyses showed also an improve of novel blood vessels. This effect on angiogenesis correlated with improve nitric oxide synthase expression and epithelial cells after treatment with silymarin. Silymarin ointment represents a promising therapeutic agent for the treatment of wounds through its antioxidant and anti-inflammatory properties

Tailoring of silica-based nanoporous pod by spermidine multi-activity

Ubiquitous in nature, polyamines (PAs) are a class of low-molecular aliphatic amines critically involved in cell growth, survival and differentiation. The polycation behavior is validated as a successful strategy in delivery systems to enhance oligonucleotide loading and cellular uptake. In this study, the chemical features and the functional roles of the PA spermidine are synergistically exploited in the synthesis and bioactive functionalization of SiO2-based structures. Inspired by biosilicification, the role of spermidine is assessed both as catalyst and template in a biomimetic one-pot synthesis of dense silica-based particles (SPs) and as a competitive agent in an interfacial reassembly strategy, to empty out SPs and generate spermidine-decorated hollow silica nanoporous pods (spd-SNPs). Spermidine bioactivity is then employed for targeting tumor cell over-expressed polyamine transport system (PTS) and for effective delivery of functional miRNA into melanoma cells. Spermidine decoration promotes spd-SNP cell internalization mediated by PTS and along with hollow structure enhances oligonucleotide loading. Accordingly, the functional delivery of the tumor suppressor miR-34a 3p resulted in intracellular accumulation of histone-complexed DNA fragments associated with apoptosis. Overall, the results highlight the potential of spd-SNP as a multi-agent anticancer therapy