Study of molecular mechanisms in cardio- and neuroprotection and possibility of modulation by nutraceutical phytocomponents

Ischemic preconditioning is a complex cardioprotective phenomenon that involves adaptive changes in cells and molecules. This adaptation occurs in a biphasic pattern: an early phase which develops after 1-2 h, and a late phase that develops after 12-24 h. While it is widely accepted that reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. Methylglyoxal, a metabolic compound formed mainly from the glycolytic intermediate glyceraldehyde-3-phosphate., is a precursor of advanced glycation end product (AGEs) .It is more reactive than glucose and shows a stronger ability to cross-link with protein amino groups to form AGEs. Methylglyoxal induced cytotoxicity may be at least partially responsible for cardiovascular and Alzheimer diseases. Methylglyoxal omeostasis is controlled by the glyoxalase system that consists of two enzyme, glyoxalase 1 (GLO1) and glyoxalase 2. In a recent study it was demonstrated that the transcriptional levels of GLO1 are controlled by NF-E2-related factor 2 (Nrf2). The isothiocyanate sulforaphane, derived from the hydrolysis of glucoraphanin abundantly present in broccoli, represents one of the most potent inducers of phase II enzymes through the Keap1–Nrf2 pathway. The aim of this thesis was evaluated molecular mechanisms in cardio- and neuroprotection and the possibility of modulation by nutraceutical phytocomponents This thesis show to one side that the protection induced by H2O2 is mediated by detoxifying and antioxidant phase II enzymes induction, regulated, not only by transcriptional factor Nrf2, but also by Nrf1; on the other side our data represent an innovative result because for the first time it was demonstrated the possibility of inducing GLO1 by SF supplementation.

Exploring novel targeted therapeutic strategies against Acute Myeloid Leukemia cells based on inhibition of bromodomain proteins and CDC20 activity

Acute myeloid leukemia (AML) is a haematological malignancies arising from the accumulation of undifferentiated myeloid progenitors with an uncontrolled proliferation. The genomic landscape of AML revealed that the disease is characterized by high level of heterogeneity and is subjected to clonal evolution driven by selective pressure of chemotherapy. In this study, we investigated the therapeutic effects of the inhibition of BRD4 and CDC20 in vitro and ex vivo. We demonstrated that inhibition of BRD4 with GSK1215101A in AML cell lines was effective under hypoxia. It induced the activation of antioxidant response both, at transcriptomic and metabolomic levels, driven by enrichment of NRF2 pathway under normoxic and hypoxic condition. Moreover, the combined treatment with Omaveloxolone, a drug inducing NRF2 activation and NF-κB inhibition, potentiated the effects on apoptosis and colony forming capacity of stem progenitor cells. Lastly, gene expression profiling data revealed that combination treatment induced major changes in genes related to cell cycle, together with enrichment of cell differentiation pathways and negative regulation of WNT, in normoxia and hypoxia. Regarding CDC20, we observed its up-regulation in AML patients. Treatment with two different inhibitors, Apcin and proTAME, was effective in primary AML cells and in AML cell lines, through induction of apoptosis and mitotic arrest. The lack of correlation between proliferation markers and CDC20 levels in AML cell subpopulations supports the idea of alternative CDC20 functions, independent from its essential role during mitosis. CDC20-KD experiments conducted in AML cell lines revealed a mild effect on apoptosis induction, but no significant change in cell cycle progression. In summary, these results allowed the identification of a new strategy combination to improve the effects of BRD4 inhibition on LSC residing in the BM hypoxic niche, and provide some new evidence regarding the potential role of CDC20 as a new target for AML treatment.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.