Attractive approaches in anticancer therapy: a challenge posed by natural products

Part A. Total synthesis, biological evaluation and SAR studies of Smo and Gli antagonists Hedgehog (Hh) signaling pathway is essential for tissue homeostasis, development and stemness. Since Hh pathway possesses a critical role in cancer initiation, proliferation, metastasis, chemoresistance and in the survival of CSCs, its constituents represent attractive druggable targets for anticancer therapy. With the aim to inhibit the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1, small molecules as emerged as promising anticancer agents. The natural isoflavone Glabrescione B (GlaB) emerged as the first small molecule binding to Gli1 zinc-finger and debilitating Gli1 activity by interfering with its interaction with DNA. Here, taking advantage by the versatile isoflavone scaffold, we have designed, synthesized and tested new Hh inhibitors. The rational introduction of defined substitutions on the isoflavone’s ring B, led us to identify molecules targeting preferentially Gli1 or Smo. Thanks to a multidisciplinary approach, combining chemical, biological and molecular docking fields, it was possible obtain new insights into the mechanism of action of these molecules. The co-administration of two different isoflavones behaving as Smo and Gli antagonists in primary mudulloblastoma (MB) cells, emphasized the synergistic effects of these agents, hence paving the way to additional and innovative strategies for the pharmacological inhibition of Hh signaling. Part B. Toward the total synthesis of madangamine B Marine natural products show increasing interest in biological, ecological, pharmacological and chemical fields1-3. Since their secondary metabolites possess structures not identified in terrestrial organisms, endowed by significant biological activities, they are intriguing candidates as lead compound for drug discovery1-4. Madangamines (A-F), a class of diazapentacyclic alkaloids, were discovered (A-E) by Andersen and co-workers since 1992 from a sponge of the genus Haposclerida, family Petrosiidae, collected off Madang, in Papua New Guinea. The same authors reported in 1994 the isolation of madangamine A, found in the marine sponge Xestospongia ingens, and few years later they described four new related alkaloids, labelled madangamines B-E. It had to wait until 2005 to reach the isolation of madangamine F from Pachychalina alcaloidifera, accomplished by Berlinck and co-workers and reported in 2007. Pure madangamines A-C and F were isolated as optically active compounds3; madangamines D and E were isolated as inseparable mixture2. Until 2014 the absolute configuration of madangamines was only presumed, and it has been hypothesized on the basis of an ingenamine, which is considered a putative precursor of madangamines. The assignment of the absolute configuration of (+)-madangamine D, is based on its first total synthesis by Amat and co-workers11. As had been described for the natural products, synthetic madangamine D has been found dextrorotatory, having unambiguous 2S, 5S, 9R, 12R absolute configuration of this alkaloid family. Madangamines A and F exhibited, in vitro, M IC50 values against a multitude of cancer cell lines, both human and murine’s ones. Thanks to the collaboration with PharmaMar Company it was been possible test the activity of madangamine D, that showed in vitro significant cytotoxic activity against pancreas (GI50 7.4 μg/mL) and colon cancers (GI50 4.4 μg/mL)

Oregonin from Alnus incana bark affects DNA methyltransferases expression and mitochondrial DNA copies in mouse embryonic fibroblasts

Oregonin is an open-chain diarylheptanoid isolated from Alnus incana bark that possesses remarkable antioxidant and anti-inflammatory properties, inhibits adipogenesis, and can be used in the prevention of obesity and related metabolic disorders. Here, we aimed to investigate the effects of oregonin on the epigenetic regulation in cells as well as its ability to modulate DNA methylating enzymes expression and mitochondrial DNA (mtDNA) copies. Our results show that oregonin altered the expression of DNA methyltransferases and mtDNA copy numbers in dependency on concentration and specificity of cells genotype. A close correlation between mtDNA copy numbers and mRNA expression of the mtDnmt1 and Dnmt3b was established. Moreover, molecular modeling suggested that oregonin fits the catalytic site of DNMT1 and partially overlaps with binding of the cofactor. These findings further extend the knowledge on oregonin, and elucidate for the first time its potential to affect the key players of the DNA methylation process, namely DNMTs transcripts and mtDNA

Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold

Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling

Identification of two novel LDLR variants by Next Generation Sequencing

Introduction. Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease characterized by elevated plasma low-density lipoprotein cholesterol (LDL-C). Targeted Next Generation Sequencing (NGS) is a new opportunity to expand the existing pathogenic variants (PVs) spectrum associated to FH. Our aim was to report a diagnostic NGS-based approach to detect variants associated to FH.Methods. We report two patients: a 48-year-old Asian woman, without known history of hypercholesterolemia and a 46-year-old Caucasian man, with childhood hypercholesterolemia.Results. An effective NGS-based pipeline, FH-Devyser kit/Amplicon Suite, beginning from sequencing to data analysis, did not identify known PVs in the LDLR, APOB, APOE, LDLRAP1, STAP1 and PCSK9 genes, but revealed two novel LDLR variants (c.1564A>T, p.Ile522Phe and c.1688C>T, p.Pro563Leu).Discussion and conclusions. This study showed that an effective NGS-based pipeline led to a definitive diagnosis in two FH families, allowing to plan their therapeutic treatment. Although the functional consequence of the two LDLR variants needs to be assessed in vitro, the in silico analysis and high preservation of the two amino acid positions observed in the LDLR protein, across different animal species, suggest that both variants are deleterious

Combined Orthoplastic Approach in Fracture-Related Infections of the Distal Tibia

This series reports on the treatment of distal tibia (DT) fracture-related infections (FRI) with a combined orthoplastic approach. Thirteen patients were included. In eight patients with extensive bone involvement and in those with a non-healed fracture, the DT was resected ("staged approach"). In five cases, the DT was preserved ("single-stage approach"). A wide debridement was performed, and the cavity was filled with antibiotic-loaded PerOssal beads. All patients had a soft-tissue defect covered by a free vascularized flap (anterolateral thigh perforator flap in eight cases, latissimus dorsi flap in five). At the final follow-up (mean 25 months, range, 13-37), no infection recurrence was observed. In one patient, the persistence of infection was observed, and the patient underwent a repeated debridement. In two cases, a voluminous hematoma was observed. However, none of these complications impacted the final outcome. The successful treatment of FRI depends on proper debridement and obliteration of dead spaces with a flap. Therefore, when dealing with DT FRI, debridement of infected bone and soft tissues must be as radical as required, with no fear of the need for massive reconstructions

Assessing the pathogenicity of BRCA1/2 variants of unknown significance: Relevance and challenges for breast cancer precision medicine

IntroductionBreast cancer (BC) is the leading cause of cancer-related death in women worldwide. Pathogenic variants in BRCA1 and BRCA2 genes account for approximately 50% of all hereditary BC, with 60-80% of patients characterized by Triple Negative Breast Cancer (TNBC) at an early stage phenotype. The identification of a pathogenic BRCA1/2 variant has important and expanding roles in risk-reducing surgeries, treatment planning, and familial surveillance. Otherwise, finding unclassified Variants of Unknown Significance (VUS) limits the clinical utility of the molecular test, leading to an “imprecise medicine”.MethodsWe reported the explanatory example of the BRCA1 c.5057A>C, p.(His1686Pro) VUS identified in a patient with TNBC. We integrated data from family history and clinic-pathological evaluations, genetic analyses, and bioinformatics in silico investigations to evaluate the VUS classification.ResultsOur evaluation posed evidences for the pathogenicity significance of the investigated VUS: 1) association of the BRCA1 variant to cancer-affected members of the family; 2) absence of another high-risk mutation; 3) multiple indirect evidences derived from gene and protein structural analysis.DiscussionIn line with the ongoing efforts to uncertain variants classification, we speculated about the relevance of an in-depth assessment of pathogenicity of BRCA1/2 VUS for a personalized management of patients with BC. We underlined that the efficient integration of clinical data with the widest number of supporting molecular evidences should be adopted for the proper management of patients, with the final aim of effectively guide the best prognostic and therapeutic paths

Correction: Lung cancer multi-omics digital human avatars for integrating precision medicine into clinical practice: the LANTERN study

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Introduzione

Introduzione al seminario con contributi vari di dottorandi e dottori di ricerca in studi latin

Epitranscriptomics: A New Layer of microRNA Regulation in Cancer

MicroRNAs are pervasive regulators of gene expression at the post-transcriptional level in metazoan, playing key roles in several physiological and pathological processes. Accordingly, these small non-coding RNAs are also involved in cancer development and progression. Furthermore, miRNAs represent valuable diagnostic and prognostic biomarkers in malignancies. In the last twenty years, the role of RNA modifications in fine-tuning gene expressions at several levels has been unraveled. All RNA species may undergo post-transcriptional modifications, collectively referred to as epitranscriptomic modifications, which, in many instances, affect RNA molecule properties. miRNAs are not an exception, in this respect, and they have been shown to undergo several post-transcriptional modifications. In this review, we will summarize the recent findings concerning miRNA epitranscriptomic modifications, focusing on their potential role in cancer development and progression