Synthesis of model compounds acting as Nitric Oxide (NO)-scavengers and the fabrication of NO-scavenging hydrogels targeting the treatment of triple-negative breast cancer
Abdo, Amir
Abdo, Amir
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Publication Date
2023-07-31
Type
Thesis
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Abstract
Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer, with an early recurrence within two to three years of first diagnosis. Moreover, the heterogeneity of the TNBC tissue creates difficulty in developing specific treatments for different TNBC subtypes. At certain stages of its development, a correlation exists between the tumour progression of TNBC and the overexpression of inducible nitric oxide synthase (iNOS). Hence, the modulation of the intracellular levels of ●NO can be employed to treat this type of tumour by inhibiting the metastatic behaviour of cancer cells and decreasing angiogenesis. Various NOS inhibitors can slow down the migration of TNBC cells. However, these inhibitors have certain drawbacks, such as a lack of specificity and reduced commercial interest in developing related therapeutics. To overcome these limitations, ●NO-scavenging is proposed as an alternative approach to reduce the intracellular levels of ●NO and inhibit cancer cell migration. The goal of this thesis is to target TNBC cell migration and angiogenesis within the tumour tissue through ●NO-scavenging. The goal was to synthesize several hemin-based ●NO-scavenging compounds and develop functional hyaluronic acid (HA)- based ●NO-scavenging hydrogels. The biological effects of these compounds were evaluated in vitro and in vivo. The initial stage of the project involved examining the effectiveness of hemin as a model compound for ●NO-scavenging and its impact on TNBC cell migration, the production of reactive oxygen species, and the expression of glycoproteins associated with metastasis. Additionally, the study examined the expression of epithelial-mesenchymal transition markers and mitochondrial functions in TNBC cells. Next, considering the catalytic processes of hemin for nitrite/H2O2-induced protein nitration, its influence in combination with ●NO on the nitration of cellular proteins was also evaluated. Furthermore, the mechanism of hemin/●NO-catalyzed nitration of BSA as a model protein was studied. This was a mechanistic study for the initial evaluation of the cellular effects of hemin and ●NO, and the influence of ●NO-scavenging in a group of molecular events in the TNBC cells. Modification of the properties of hemin through conjugation to specific aromatic moieties was the next step. The objective of this step was to reduce the aggregation behaviour of hemin molecules, protect them against oxidative degradation, and preserve their ●NO-scavenging efficiency. The properties of hemin and its derivatives were evaluated experimentally and theoretically. Next, their cytocompatibility was evaluated by measuring their effects on the intracellular levels of ●NO in TNBC cells, migration, and the accompanying downstream effects. Furthermore, the effects of different compounds on the ●NOinduced vasodilation in zebrafish embryos were investigated. The study introduced a novel class of iron based ●NO-scavenging compounds, which were synthesized using straightforward procedures and possessed unique chemical properties. Furthermore, the synthesized compounds exhibited diverse abilities to regulate TNBC cell migration and blood vessel dilation, each with distinct molecular effects at the cellular level. Next, the study investigated the interactions between HA, one of the primary components of the hydrogels, and ●NO, as well as how these interactions can lead to HA fragmentation. These interactions inhibited TNBC cell migration and dilation of blood vessels in zebrafish embryos. While ●NO induced the depolymerization of HA chains, with similar effects to other active radicals, hemin and its different derivatives prevented that with different levels. These interactions were examined via several techniques to understand how HA can scavenge ●NO, the involved mechanism, and the further effects on modulation of TNBC cell migration and ●NO-induced vasodilation. Multiple formulations were synthesised, and their properties were adjusted to create biodegradable hydrogels with customized compositions and ●NO-scavenging strength, utilizing surface functional ●NO-scavenging molecules. The hydrogel formulations continuously released active ●NOscavenging molecules, particularly of hemin and its derivative, hemin-tyrosine, which were evaluated in vitro. The ●NO-scavenging properties of certain hydrogels and their products following degradation were accompanied by an ability to decrease the levels of intracellular ●NO and inhibit TNBC cell migration. After collecting these observations, the final step was to develop chemically crosslinked HA-hydrogels with tuneable properties and ●NO-scavenging efficiency. This thesis aims to explore a fresh approach to treating TNBC by targeting one of the main gasotransmitters that play a critical molecular role in cancer metastasis. This study synthesised a new class of functional compounds and hydrogels that can scavenge ●NO and regulate cancer cell migration and angiogenesis. The chemical properties of the synthesized compounds and their ●NO-scavenging efficiencies were evaluated, and the further cellular effects were also assessed in vitro and in vivo. Candidate hydrogel formulations, loaded with candidate ●NO-scavenging compounds, were developed, and their reactivity against ●NO was verified by various assays, including in vitro studies.
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NUI Galway