Antonella Capuozzo

The lysosomal calcium channel TRPML1, whose mutations cause the lysosomal storage disorder (LSD) mucolipidosis type IV (MLIV), contributes to upregulate autophagic genes by inducing the nuclear translocation of the transcription factor EB (TFEB). Here we show that TRPML1 activation also induces autophagic vesicle (AV) biogenesis through the generation of phosphatidylinositol 3-phosphate (PI3P) and the recruitment of essential PI3P-binding proteins to the nascent phagophore in a TFEB-independent manner. Thus, TRPML1 activation of phagophore formation requires the calcium-dependent kinase CaMKKβ and AMPK, which increase the activation of ULK1 and VPS34 autophagic protein complexes. Consistently, cells from MLIV patients show a reduced recruitment of PI3P-binding proteins to the phagophore during autophagy induction, suggesting that altered AV biogenesis is part of the pathological features of this disease. Together, we show that TRPML1 is a multistep regulator of autophagy that may be targeted for therapeutic purposes to treat LSDs and other autophagic disorders.

According to WHO, breast cancer incidence is increasing so that the search for novel chemotherapeutic options is nowadays an essential requirement to fight neoplasm subtypes. By exploring new effective metal-based chemotherapeutic strategies, many ruthenium complexes have been recently proposed as antitumour drugs, showing ability to impact on diverse cellular targets. In the framework of different molecular pathways leading to cell death in human models of breast cancer, here we demonstrate autophagy involvement behind the antiproliferative action of a ruthenium(III)-complex incorporated into a cationic nanosystem (HoThyRu/DOTAP), proved to be hitherto one of the most effective within the suite of nucleolipidic formulations we have developed for the in vivo transport of anticancer ruthenium(III)-based drugs. Indeed, evidences are implicating autophagy in both cancer development and therapy, and anticancer interventions endowed with the ability to trigger this biological response are currently considered attractive oncotherapeutic approaches. Moreover, crosstalk between apoptosis and autophagy, regulated by finely tuned metallo-chemotherapeutics, may provide novel opportunities for future improvement of cancer treatment. Following this line, our in vitro and in vivo preclinical investigations suggest that an original strategy based on suitable formulations of ruthenium(III)-complexes, inducing sustained cell death, could open new opportunities for breast cancer treatment, including the highly aggressive triple-negative subtype.

In the search for more efficient anticancer treatments, Ru(III) complexes have attracted much interest among metal-based candidate drugs, showing marked antitumor and antimetastatic activity associated with lower systemic toxicity. Remarkable examples are the Ru(III) complexes NAMI-A and KP1019, which have reached advanced clinical evaluation. In order to improve the in vivo stability of Ru(III)-based drugs, as well as their cellular uptake and effectiveness, a new approach has been proposed by our research group, based on the incorporation of the active, NAMI-A-like Ru(III) complex into highly functionalized nucleolipidic structures, i.e., hybrid molecules containing a nucleoside or nucleotide central core derivatized with a lipid chain, ensuring both efficient protection against extracellular degradation and high cellular internalization of the metal. Aiming at expanding the chemical diversity of available amphiphilic Ru(III) complexes, we here selected a trifunctional α-amino acid to replace the nucleosidic core of previously prepared nucleolipid-based Ru(III) complexes. The amino acidic scaffold, linked to the Ru(III) complex, is decorated with both hydrophilic and lipophilic moieties, conferring high propensity to form stable aggregates in water, which is required to obtain a suitable nanocarrier for the drug delivery. Following this approach, a novel compound, indicated here as compound I, was successfully prepared and characterized, then studied in coformulation with the biocompatible cationic lipid 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) by dynamic light scattering (DLS), small angle neutron scattering (SANS), and UV–vis analysis. Evaluated in vitro on a panel of human and nonhuman cell lines, it showed good antiproliferative activity on cancer cells, with IC₅₀ values in the μM range, and no relevant cytotoxicity on the healthy cells used as control.

Looking for new metal-based anticancer treatments, in recent years many ruthenium complexes have been proposed as effective and safe potential drugs. In this context we have recently developed a novel approach for the in vivo delivery of Ru(III) complexes, preparing stable ruthenium-based nucleolipidic nanoaggregates endowed with significant antiproliferative activity. Herein we describe the cellular response to our ruthenium-containing formulations in selected models of human breast cancer. By in vitro bioscreens in the context of preclinical studies, we have focused on their ability to inhibit breast cancer cell proliferation by the activation of the intrinsic apoptotic pathway, possibly via mitochondrial perturbations involving Bcl-2 family members and predisposing to programmed cell death. In addition, the most efficient ruthenium-containing cationic nanoaggregates we have hitherto developed are able to elicit both extrinsic and intrinsic apoptosis, as well as autophagy. To limit chemoresistance and counteract uncontrolled proliferation, multiple cell death pathways activation by metal-based chemotherapeutics is a challenging, yet very promising strategy for targeted therapy development in aggressive cancer diseases, such as triple-negative breast cancer with limited treatment options. These outcomes provide valuable, original knowledge on ruthenium-based candidate drugs and new insights for future optimized cancer treatment protocols.

A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin A ruthenium anticancer compound interacts with histones and impacts differently on epigenetic and death pathways compared to cisplatin

Liposomes are complex aggregates, often including polyethylene glycol (PEG) to expand their life span in vivo, although their full biocompatibility is still questioned. With the aim to suitably replace PEG within liposomal formulations, here we propose a new liposomes formulation, which includes an amphiphilic molecule of natural origin: the lipooligosaccharide (LOS) from the Gram‐negative bacterium Rhizobium rubi . LOS architecture is bifunctional: the lipid moiety at one terminus promotes its insertion into the liposome, the other terminus is hydrophilic and in this case presents an oligosaccharide motif similar to the human Lewis B antigen. Liposomes were prepared by co‐formulating de‐ O ‐acylated LOS ( de ‐LOS) with the lipid 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and the anticancer nucleolipid‐based Ru(III) complex, ToThyRu. In‐depth microstructural characterization shows that de ‐LOS containing liposomes are stable aggregates. In vitro preliminary bioscreens have disclosed their negligible toxic profile and a good uptake in MCF‐7 and HaCaT cells. The results validate the use of lipooligosaccharides in formulating liposomes and pave the way to their use in drug delivery applications.

L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia. J. Cell. Biochem. 117: 402–412, 2016.

Aiming for novel tools for anticancer therapies, a ruthenium complex, covalently linked to a cholesterol-containing nucleolipid and stabilized by co-aggregation with a biocompatible lipid, is here presented. The amphiphilic ruthenium complex, named ToThyCholRu, is intrinsically negatively charged and has been inserted into liposomes formed by the cationic 1,2-dioleyl-3-trimethylammoniumpropane chloride (DOTAP) to hinder the degradation kinetics typically observed for known ruthenium-based antineoplastic agents. The here described nanovectors contain up to 30% in moles of the ruthenium complex and are stable for several weeks. This drug delivery system has been characterized using dynamic light scattering (DLS), small angle neutron scattering (SANS), neutron reflectivity (NR) and electron paramagnetic resonance (EPR) techniques. Fluorescence microscopy, following the incorporation of rhodamine-B within the ruthenium-loaded liposomes, showed fast cellular uptake in human carcinoma cells, with a strong fluorescence accumulation within the cells. The in vitro bioactivity profile revealed an important antiproliferative activity and, most remarkably, the highest ability in ruthenium vectorization measured so far. Cellular morphological changes and DNA fragmentation provided evidence of an apoptosis-inducing activity, in line with several in vitro studies supporting apoptotic events as the main cause for the anticancer properties of ruthenium derivatives. Overall, these data highlighted the crucial role played by the cellular uptake properties in determining the anticancer efficacy of ruthenium-based drugs, showing DOTAP as a very efficient nanocarrier for their stabilization in aqueous media and transport in cells. In vitro bioscreens have shown the high antiproliferative activity of ToThyCholRu–DOTAP liposomes against specific human adenocarcinoma cell types. Furthermore, these formulations have proved to be over 20-fold more effective against MCF-7 and WiDr adenocarcinoma cells with respect to the nude ruthenium complex AziRu we have previously described.