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.

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.

Aiming for novel tools for anticancer therapies, a ruthenium complex, covalently linked to a cholesterol-​contg. nucleolipid and stabilized by co-​aggregation with a biocompatible lipid, is here presented. The amphiphilic ruthenium complex, named ToThyCholRu, is intrinsically neg. charged and has been inserted into liposomes formed by the cationic 1,​2-​dioleyl-​3-​trimethylammoniumpropane chloride (DOTAP) to hinder the degrdn. kinetics typically obsd. 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 ESR 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 morphol. 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 derivs. Overall, these data highlighted the crucial role played by the cellular uptake properties in detg. the anticancer efficacy of ruthenium-​based drugs, showing DOTAP as a very efficient nanocarrier for their stabilization in aq. 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

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.