Ferraro, Maria Grazia (2023) Mechanisms regulating cell death/cell survival behind biological responses to novel ruthenium–based chemotherapeutics in human preclinical models of cancer. [Tesi di dottorato]

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Abstract

Based on the wide use of cisplatin and congeners in anticancer treatments, but also taking benefit from limitations of conventional chemotherapy detected throughout application in clinic, the design and development of novel non-platinum metal-based agents is nowadays believed as a challenging therapeutic option. The shared strategy behind the development of a next-generation of metal-based chemotherapeutics is thereby to overcome the current limits of Pt-based clinical drugs, including toxicity and chemoresistance. Unconventional candidate compounds can be fine-tuned to access interactions with druggable biological targets for the onset of distinctive anticancer activities. Indeed, the variety of metal centers to be explored, as well as chemical diversity of the ligands to be selected, give life to molecular platforms endowed with unique chemical properties, which can be exploited to accomplish specific biological features, as well as to interact with distinct biomolecular targets. Moving in this direction, over the last years platinum family metals, e.g., ruthenium and palladium, have been largely proposed. In this frame, our research group has recently focused on a bioactive Ru(III) complex – named AziRu – nanostructured into a suite of ad hoc designed nucleolipid formulations to enhance its stability and delivery in the context of new anticancer strategies. Specifically, by profitably blending amphiphilic nanomaterials as nucleolipids and the AziRu complex, we have developed variously decorated anticancer nanosystems proved to be very effective against cancer cells. One of our most promising Ru-based nanosystems (named HoThyRu), once co-aggregated with the cationic lipid DOTAP (HoThyRu/DOTAP formulation), has shown superior anticancer activity against breast cancer (BC), one of the most widespread human malignancies. In order to further develop HoThyRu/DOTAP for a final preclinical validation, we first confirmed its bioactivity by exploiting two mammary malignant cellular models, currently considered among the most reliable in vitro models of BCs, i.e., the endocrine-responsive (ER) breast adenocarcinoma MCF-7 and the triple-negative breast adenocarcinoma (TNBC) MDA-MB-231 cell models. According to WHO, TNBC is more aggressive and with fewer available treatment options than other BC, which makes it a complex metastatic disease with a very poor prognosis. By targeted experiments, we have demonstrated the ability of HoThyRu/DOTAP to trigger and sustain multiple mechanisms of programmed cell death (PCD) pathways, such as apoptosis and autophagy. Activation of different cell death pathways in the context of a multimodal action accounts for enhanced antiproliferative effects against BC cells, as well as for reduced chemoresistance to treatments. Through functional assays, we have also demonstrated in vitro HoThyRu/DOTAP ability to reduce MDA-MB-231 migration and invasion, a distinctive behavior of the triple negative metastatic phenotype. Moreover, we have next explored possible interference between “ruthenotherapy” and regulation of iron homeostasis in cancer cells. Indeed, misregulation of iron metabolism is now considered as a valuable cancer hallmark and may have an overwhelming impact on tumor growth and progression. Consequently, cancer cells are more susceptible to iron depletion and oxidative stress with respect to healthy counterparts. In this framework, we showed that the association between HoThyRu/DOTAP and an iron depletion therapy by iron chelators can be therapeutically beneficial ensuing in a more effective antiproliferative action. Brand new investigations are underway to clarify biomolecular targets of AziRu for a further advance of this perspective curative strategy. Following comprehensive in vitro research by targeted bioscreens, we have finally explored HoThyRu/DOTAP behavior in vivo by means of preclinical animal models. Xenograft mice models of estrogen receptor positive (ER-positive) BC and TNBC were set up to investigate HoThyRu/DOTAP safety and efficacy. In the up-to-date panorama of Ru-based candidate drugs, we have in this way showcased that AziRu, inserted in a nucleolipid structure and ad hoc nano-delivered by the positively charged lipid DOTAP, can effectively counteract BC cell proliferation in vivo being concurrently a well-tolerated agent, which is a crucial property for anticancer drug candidates in preclinical studies to progress in clinical stage. Ex vivo investigations are currently ongoing to deepen knowledge on the multi-target action of AziRu on cancer cells and give additional insights into its mode of action in vivo. In the meantime, looking for increasingly effective metal-based anticancer agents and as a further evolution of our ruthenium-containing nanosystems, the last part of this Ph.D. program was committed to the progress of bioengineered lipophilic Ru(III) complexes. In particular, lipid-conjugated Ru(III) complexes - designed to obtain specific lipophilic analogues of AziRu, have been synthesized and fully characterized. As discussed, preliminary biological investigations by means of a selected panel of human cancer cells have been already performed. Considering that to date only a few ruthenium-based agents have advanced in clinical trials compared to their potential and to the number of the investigated derivatives, we can reasonably assume the HoThyRu/DOTAP biocompatible nanosystem as a potential future candidate drug for clinical trials. Upcoming developments mainly aimed at additional nanosystem decorations to ensure selective targeting towards human BC cells could further improve efficacy and safety of this nano-formulation, while in-depth SAR studies could shed light on its biomolecular targets and mode of action.

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