"Nitric Oxide Donors for Biomedical Applications: A Themed Issue Dedicated to Professor Alberto Gasco": Special Issue Editorial Overview.

The Guest Editors Federica Sodano, Elena Gazzano, and Roberta Fruttero are pleased to present this editorial overview of the Special Issue entitled "Nitric Oxide Donors for Biomedical Applications: A Themed Issue Dedicated to Professor Alberto Gasco" [...].

Cytotoxicity of fibrous antigorite from New Caledonia.

Exposure to asbestos and asbestos-like minerals has been related to the development of severe lung diseases, including cancer and malignant mesothelioma (MM). A high incidence of non-occupational MM was observed in New Caledonia (France) in people living in proximity of serpentinite outcrops, containing chrysotile and fibrous antigorite. Antigorite is a magnesium silicate, which shares with chrysotile asbestos the chemical formula. To achieve information on antigorite toxicity, we investigated the physico-minero-chemical features relevant for toxicity and cellular effects elicited on murine macrophages (MH-S) and alveolar epithelial cells (A549) of three fibrous antigorites (f-Atg) collected in a Caledonian nickel lateritic ore and subjected to supergene alteration. Field Atg were milled to obtain samples suitable for toxicological studies with a similar particle size distribution. UICC chrysotile (Ctl) and a non-fibrous antigorite (nf-Atg) were used as reference minerals. A high variability in toxicity was observed depending on shape, chemical alteration, and surface reactivity. The antigorites shared with Ctl a similar surface area (16.3, 12.1, 20.3, 13.4, and 15.6 m2/g for f-Atg1, 2, 3, nf-Atg, and Ctl). f-Atg showed different level of pedogenetic weathering (Ni depletion f-Atg1 ≪ f-Atg2 and 3) and contained about 50% of elongated mineral particles, some of which exhibited high aspect ratios (AR > 10 µm, 20%, 26%, 31% for f-Atg1, 2, and 3, respectively). The minerals differed in bio-accessible iron at pH 4.5 (f-Atg1 ≪ f-Atg3, < f-Atg2, nf-Atg < Ctl), and surface reactivity (ROS release in solution, f-Atg1 ≪ f-Atg2, 3, nf-Atg, and Ctl). f-Atg2 and f-Atg3 induced oxidative stress and pro-inflammatory responses, while the less altered, poorly reactive sample (f-Atg1) induced negligible effects, as well nf-Atg. The slow dissolution kinetics observed in simulated body fluids may signal a high biopersistence. Overall, our work revealed a significative cellular toxicity of f-Atg that correlates with fibrous habit and surface reactivity.

Changes of physico-chemical properties of nano-biomaterials by digestion fluids affect the physiological properties of epithelial intestinal cells and barrier models.

BACKGROUND: The widespread use of nano-biomaterials (NBMs) has increased the chance of human exposure. Although ingestion is one of the major routes of exposure to NBMs, it is not thoroughly studied to date. NBMs are expected to be dramatically modified following the transit into the oral-gastric-intestinal (OGI) tract. How these transformations affect their interaction with intestinal cells is still poorly understood. NBMs of different chemical nature-lipid-surfactant nanoparticles (LSNPs), carbon nanoparticles (CNPs), surface modified Fe3O4 nanoparticles (FNPs) and hydroxyapatite nanoparticles (HNPs)-were treated in a simulated human digestive system (SHDS) and then characterised. The biological effects of SHDS-treated and untreated NBMs were evaluated on primary (HCoEpiC) and immortalised (Caco-2, HCT116) epithelial intestinal cells and on an intestinal barrier model. RESULTS: The application of the in vitro SDHS modified the biocompatibility of NBMs on gastrointestinal cells. The differences between SHDS-treated and untreated NBMs could be attributed to the irreversible modification of the NBMs in the SHDS. Aggregation was detected for all NBMs regardless of their chemical nature, while pH- or enzyme-mediated partial degradation was detected for hydroxyapatite or polymer-coated iron oxide nanoparticles and lipid nanoparticles, respectively. The formation of a bio-corona, which contains proteases, was also demonstrated on all the analysed NBMs. In viability assays, undifferentiated primary cells were more sensitive than immortalised cells to digested NBMs, but neither pristine nor treated NBMs affected the intestinal barrier viability and permeability. SHDS-treated NBMs up-regulated the tight junction genes (claudin 3 and 5, occludin, zonula occludens 1) in intestinal barrier, with different patterns between each NBM, and increase the expression of both pro- and anti-inflammatory cytokines (IL-1ß, TNF-α, IL-22, IL-10). Notably, none of these NBMs showed any significant genotoxic effect. CONCLUSIONS: Overall, the results add a piece of evidence on the importance of applying validated in vitro SHDS models for the assessment of NBM intestinal toxicity/biocompatibility. We propose the association of chemical and microscopic characterization, SHDS and in vitro tests on both immortalised and primary cells as a robust screening pipeline useful to monitor the changes in the physico-chemical properties of ingested NBMs and their effects on intestinal cells.

NO in Viral Infections: Role and Development of Antiviral Therapies.

Nitric oxide is a ubiquitous signaling radical that influences critical body functions. Its importance in the cardiovascular system and the innate immune response to bacterial and viral infections has been extensively investigated. The overproduction of NO is an early component of viral infections, including those affecting the respiratory tract. The production of high levels of NO is due to the overexpression of NO biosynthesis by inducible NO synthase (iNOS), which is involved in viral clearance. The development of NO-based antiviral therapies, particularly gaseous NO inhalation and NO-donors, has proven to be an excellent antiviral therapeutic strategy. The aim of this review is to systematically examine the multiple research studies that have been carried out to elucidate the role of NO in viral infections and to comprehensively describe the NO-based antiviral strategies that have been developed thus far. Particular attention has been paid to the potential mechanisms of NO and its clinical use in the prevention and therapy of COVID-19.

SKP2 drives the sensitivity to neddylation inhibitors and cisplatin in malignant pleural mesothelioma.

BACKGROUND: The combination of pemetrexed and cisplatin remains the reference first-line systemic therapy for malignant pleural mesothelioma (MPM). Its activity is moderate because of tumor aggressiveness, immune-suppressive environment and resistance to chemotherapy-induced immunogenic cell death (ICD). Preliminary and limited findings suggest that MPM cells have deregulated ubiquitination and proteasome activities, although proteasome inhibitors achieved disappointing clinical results. METHODS: Here, we investigated the role of the E3-ubiquitin ligase SKP/Cullin/F-box (SCF) complex in cell cycle progression, endoplasmic reticulum (ER)/proteostatic stress and ICD in MPM, and the therapeutic potential of the neddylation/SCF complex inhibitor MLN4924/Pevonedistat. RESULTS: In patient-derived MPM cultures and syngenic murine models, MLN4924 and cisplatin showed anti-tumor effects, regardless of MPM histotype and BAP1 mutational status, increasing DNA damage, inducing S- and G2/M-cell cycle arrest, and apoptosis. Mechanistically, by interfering with the neddylation of cullin-1 and ubiquitin-conjugating enzyme UBE2M, MLN4924 blocks the SCF complex activity and triggers an ER stress-dependent ICD, which activated anti-MPM CD8+T-lymphocytes. The SKP2 component of SCF complex was identified as the main driver of sensitivity to MLN4924 and resistance to cisplatin. These findings were confirmed in a retrospective MPM patient series, where SKP2 high levels were associated with a worse response to platinum-based therapy and inferior survival. CONCLUSIONS: We suggest that the combination of neddylation inhibitors and cisplatin could be worth of further investigation in the clinical setting for MPM unresponsive to cisplatin. We also propose SKP2 as a new stratification marker to determine the sensitivity to cisplatin and drugs interfering with ubiquitination/proteasome systems in MPM.

Glabratephrin reverses doxorubicin resistance in triple negative breast cancer by inhibiting P-glycoprotein.

Triple-negative breast cancer is one of the most aggressive breast cancer. The first therapeutic option is chemotherapy, often based on anthracycline as doxorubicin. However, chemotherapy efficacy is limited in by the presence of P-glycoprotein (Pgp), a membrane transporter protein that effluxes doxorubicin, reducing its cellular accumulation and toxicity. Inhibiting Pgp activity with effective and non-toxic products is still an open challenge. In this work, we demonstrated that the natural product Glabratephrin (Glab), a prenylated flavonoid from Tephrosia purpurea with a unique chemical structure, increased doxorubicin accumulation and cytotoxicity in triple negative breast cancer cells with high levels of Pgp, characterized by both acquired or intrinsic resistance to doxorubicin. Glab also reduced the growth of Pgp-expressing tumors, without adding significant extra-toxicities to doxorubicin treatment. Interestingly, Glab did not change the expression of Pgp, but it reduced the affinity for Pgp and the efflux of doxorubicin, as suggested by the increased Km and the reduced Vmax. In silico molecular docking predicted that Glab binds two residues (phenylalanine 322, glutamine 721) localized in the transmembrane domains of Pgp, facing the extracellular environment. Moreover, site-directed mutagenesis identified glycine 185 as a critical residue mediating the reduced catalytic efficacy of Pgp elicited by Glab. We propose Glab as an effective and safe compound able to reverse doxorubicin resistance mediated by Pgp in triple negative breast cancers, opening the way to a new combinatorial approach that may improve chemotherapy efficacy in the most refractory and aggressive breast cancer.

Ketogal Safety Profile in Human Primary Colonic Epithelial Cells and in Mice.

In our previous studies, a ketorolac-galactose conjugate (ketogal) showed prolonged anti-inflammatory and analgesic activity, causing less gastric ulcerogenic effect and renal toxicity than its parent drug ketorolac. In order to demonstrate the safer profile of ketogal compared to ketorolac, histopathological changes in the small intestine and liver using three staining techniques before and after repeated oral administration in mice with ketorolac or an equimolecular dose of its galactosylated prodrug ketogal were assessed. Cytotoxicity and oxidative stress parameters were evaluated and compared in ketorolac- and ketogal-treated Human Primary Colonic Epithelial cells at different concentrations and incubation times. Evidence of mitochondrial oxidative stress was found after ketorolac treatment; this was attributable to altered mitochondrial membrane depolarization and oxidative stress parameters. No mitochondrial damage was observed after ketogal treatment. In ketorolac-treated mice, severe subepithelial vacuolation and erosion with inflammatory infiltrates and edematous area in the intestinal tissues were noted, as well as alterations in sinusoidal spaces and hepatocytes with foamy cytoplasm. In contrast, treatment with ketogal provided a significant improvement in the morphology of both organs. The prodrug clearly demonstrated a safer profile than its parent drug both in vitro and ex vivo, confirming that ketogal is a strategic alternative to ketorolac.

The heme synthesis-export system regulates the tricarboxylic acid cycle flux and oxidative phosphorylation.

Heme is an iron-containing porphyrin of vital importance for cell energetic metabolism. High rates of heme synthesis are commonly observed in proliferating cells. Moreover, the cell-surface heme exporter feline leukemia virus subgroup C receptor 1a (FLVCR1a) is overexpressed in several tumor types. However, the reasons why heme synthesis and export are enhanced in highly proliferating cells remain unknown. Here, we illustrate a functional axis between heme synthesis and heme export: heme efflux through the plasma membrane sustains heme synthesis, and implementation of the two processes down-modulates the tricarboxylic acid (TCA) cycle flux and oxidative phosphorylation. Conversely, inhibition of heme export reduces heme synthesis and promotes the TCA cycle fueling and flux as well as oxidative phosphorylation. These data indicate that the heme synthesis-export system modulates the TCA cycle and oxidative metabolism and provide a mechanistic basis for the observation that both processes are enhanced in cells with high-energy demand.

Identification of Redox-Sensitive Transcription Factors as Markers of Malignant Pleural Mesothelioma.

Although asbestos has been banned in most countries around the world, malignant pleural mesothelioma (MPM) is a current problem. MPM is an aggressive tumor with a poor prognosis, so it is crucial to identify new markers in the preventive field. Asbestos exposure induces oxidative stress and its carcinogenesis has been linked to a strong oxidative damage, event counteracted by antioxidant systems at the pulmonary level. The present study has been focused on some redox-sensitive transcription factors that regulate cellular antioxidant defense and are overexpressed in many tumors, such as Nrf2 (Nuclear factor erythroid 2-related factor 2), Ref-1 (Redox effector factor 1), and FOXM1 (Forkhead box protein M1). The research was performed in human mesothelial and MPM cells. Our results have clearly demonstrated an overexpression of Nrf2, Ref-1, and FOXM1 in mesothelioma towards mesothelium, and a consequent activation of downstream genes controlled by these factors, which in turn regulates antioxidant defense. This event is mediated by oxidative free radicals produced when mesothelial cells are exposed to asbestos fibers. We observed an increased expression of Nrf2, Ref-1, and FOXM1 towards untreated cells, confirming asbestos as the mediator of oxidative stress evoked at the mesothelium level. These factors can therefore be considered predictive biomarkers of MPM and potential pharmacological targets in the treatment of this aggressive cancer.

Efficacy, biocompatibility and degradability of carbon nanoparticles for photothermal therapy of lung cancer.

Aim: To investigate near infrared-induced phototoxicity toward lung cancer cells, and the biodegradability and effect on immune cells of glucose-derived carbon nanoparticles (CNPs). Methods: The human A549 lung adenocarcinoma cell line was used as a model to study the phototoxicity of CNPs. The biodegradability and the effect on immune cells was demonstrated in primary human neutrophils and macrophages. Results: Near infrared-activated CNPs elicited rapid cell death, characterized by the elevation of heat shock proteins and the induction of DNA damage. CNPs were found to be noncytotoxic toward primary human macrophages and were susceptible to biodegradation when cocultured with human neutrophils. Conclusions: Our results identify CNPs as promising platforms for photothermal therapy of lung cancer.

Tuning NO release of organelle-targeted furoxan derivatives and their cytotoxicity against lung cancer cells.

We herein report a study on a set of hybrid compounds in which 3-R-substituted furoxan moieties (R = CH3, CONH2, CN, SO2C6H5), endowed with varying NO-releasing capacities, are joined to a mitochondrial probe, rhodamine B. Each product has been investigated for its ability to release NO both in physiological solution, in the presence of cysteine, and in A549 lung adenocarcinoma cancer cells. The cytotoxicity of all the products against the aforementioned cancer cells has been assessed, including the structurally related compounds with no mitochondrial targeting, which were taken as a reference. In the case of the models bearing the -CH3 and -CONH2 groups at the 3-position on the furoxan, only the targeted models showed a significant cytotoxic activity, and only at the highest concentrations, in accordance with their weak NO-releasing properties. On the contrary, the presence of the strong electron-withdrawing groups, as -CN and -SO2C6H5, at the 3-position gave rise to anticancer agents, likely because of the high NO-releasing and of their capability of inhibiting cellular proteins by covalent binding. In detail, the rhodamine hybrid containing the 3-SO2C6H5 substituted furoxan moiety emerged as the most interesting product as it showed high cytotoxicity over the entire concentration range tested. This substructure was also linked to a phenothiazine scaffold that is able to accumulate in lysosomes. Nevertheless, mitochondrial targeting for these NO-donor furoxan substructures was found to be the most efficient.

Synthesis of defined oligohyaluronates-decorated liposomes and interaction with lung cancer cells.

In this work hyaluronic acid (HA) oligosaccharides with degree of polymerization (DP) 4, 6 and 8, obtained by enzymatic depolymerization of HA, were conjugated to a PEG-phospholipid moiety. The products (HA-DP4, HA-DP6 and HA-DP8) were used to prepare decorated liposomes. The cellular uptake of HA-DP4, HA-DP6 and HA-DP8-decorated fluorescently labelled liposomes was significantly higher (12 to 14-fold) in lung cancer cell lines with high CD44 expression than in those with low CD44 expression, suggesting a receptor-mediated entry of HA-conjugated formulations. Competition assays showed that the uptake followed this rank order: HA-DP8>HA-DP6>HA-DP4 liposomes. Moreover, they are capable of a faster interaction with CD44, followed by phagocytosis, than HA liposomes obtained from HA of higher molecular weight (4800 and 14800 Da). HA-DP4, HA-DP6 and HA-DP8-liposomes did not show cytotoxicity or inflammatory effects. Overall, we propose our new HA-DP oligosaccharides as biocompatible and effective tools for a potential drug delivery to CD44-positive cells.

MRP5 nitration by NO-releasing gemcitabine encapsulated in liposomes confers sensitivity in chemoresistant pancreatic adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is a therapy recalcitrant disease characterized by the aberrations in multiple genes that drive pathogenesis and drug chemoresistance. In this study, we synthesize a library of seven novel nitric oxide-releasing gemcitabine pro-drugs (NO-GEMs) in order to improve the effectiveness of GEM by exploiting the therapeutic effects of NO. Among these NO-GEM pro-drugs we select 5b as the most effective compound in GEM-resistant PDAC cells. After its encapsulation in liposomes for drug delivery the intracellular NO level increases and nitration associated to activity inhibition of the multidrug resistance associated protein 5 (MRP5; ABCC5) occurs. This results in GEM intracellular accumulation and enhanced apoptotic cell death in GEM-resistant PDAC cells, which express MRP5 at higher levels than GEM-sensitive cells. Our results support the development of a new anti-tumoral strategy to efficiently affect GEM-resistant PDAC cells based on the usage of NO-GEM pro-drugs.

Overcoming Doxorubicin Resistance with Lipid-Polymer Hybrid Nanoparticles Photoreleasing Nitric Oxide.

We report on tailored lipid-polymer hybrid nanoparticles (NPs) delivering nitric oxide (NO) under the control of visible light as a tool for overcoming doxorubicin (DOX) resistance. The NPs consist of a polymeric core and a coating. They are appropriately designed to entrap DOX in the poly(lactide-co-glycolide) core and a NO photodonor (NOPD) in the phospholipid shell to avoid their mutual interaction both in the ground and excited states. The characteristic red fluorescence of DOX, useful for its tracking in cells, is well preserved upon incorporation within the NPs, even in the copresence of NOPD. The NP scaffold enhances the NO photoreleasing efficiency of the entrapped NOPD when compared with that of the free compound, and the copresence of DOX does not significantly affect such enhanced photochemical performance. Besides, the delivery of DOX and NOPD from NPs is also not mutually influenced. Experiments carried out in M14 DOX-resistant melanoma cells demonstrate that NO release from the multicargo NPs can be finely regulated by excitation with visible light, at a concentration level below the cytotoxic doses but sufficient enough to inhibit the efflux transporters mostly responsible for DOX cellular extrusion. This results in increased cellular retention of DOX with consequent enhancement of its antitumor activity. This approach, in principle, is not dependent on the type of chemotherapeutic used and may pave the way for new treatment modalities based on the photoregulated release of NO to overcome the multidrug resistance phenomenon and improve cancer chemotherapies.

Coencapsulation of disulfiram and doxorubicin in liposomes strongly reverses multidrug resistance in breast cancer cells.

Disulfiram (DSF) is an inhibitor of P-glycoprotein (Pgp), the main obstacle limiting the success of doxorubicin (DOX), but it has poor solubility and stability. With the aim to overcome these limitations we prepared liposomes coencapsulating DSF and DOX (LipoDSF-DOX). Liposome stability, drugs release profile, effects on DOX cytotoxicity, Pgp activity and expression in breast cancer cells were evaluated. We observed that LipoDSF-DOX with a 1:3 weight ratio, with DSF in lipid bilayer and DOX in aqueous core, released DSF faster than DOX. LipoDSF-DOX increased DOX intracellular accumulation and cytotoxicity in Pgp-expressing breast cancer cells, with an efficacy superior to the mixture of free DSF and DOX, thanks to a differential kinetics of release of DSF and DOX when carried by liposomes. The mechanism of the increased DOX retention relied on the DSF-induced sulfhydraton of Pgp and followed by its ubiquitination. These events reduced Pgp expression and catalytic activity in LipoDSF-DOX-treated cells. Our results show that LipoDSF-DOX effectively reversed DOX resistance in Pgp-expressing breast cancer cells, exploiting the temporally different kinetics of release of DSF and DOX, optimized to decrease expression and activity of Pgp.

Improvement of conventional anti-cancer drugs as new tools against multidrug resistant tumors.

Multidrug resistance (MDR) is the dominant cause of the failure of cancer chemotherapy. The design of antitumor drugs that are able to evade MDR is rapidly evolving, showing that this area of biomedical research attracts great interest in the scientific community. The current review explores promising recent approaches that have been developed with the aim of circumventing or overcoming MDR. Encouraging results have been obtained in the investigation of the MDR-modulating properties of various classes of natural compounds and their analogues. Inhibition of P-gp or downregulation of its expression have proven to be the main mechanisms by which MDR can be surmounted. The use of hybrid molecules that are able to simultaneously interact with two or more cancer cell targets is currently being explored as a means to circumvent drug resistance. This strategy is based on the design of hybrid compounds that are obtained either by merging the structural features of separate drugs, or by conjugating two drugs or pharmacophores via cleavable/non-cleavable linkers. The approach is highly promising due to the pharmacokinetic and pharmacodynamic advantages that can be achieved over the independent administration of the two individual components. However, it should be stressed that the task of obtaining successful multivalent drugs is a very challenging one. The conjugation of anticancer agents with nitric oxide (NO) donors has recently been developed, creating a particular class of hybrid that can combat tumor drug resistance. Appropriate NO donors have been shown to reverse drug resistance via nitration of ABC transporters and by interfering with a number of metabolic enzymes and signaling pathways. In fact, hybrid compounds that are produced by covalently attaching NO-donors and antitumor drugs have been shown to elicit a synergistic cytotoxic effect in a variety of drug resistant cancer cell lines. Another strategy to circumvent MDR is based on nanocarrier-mediated transport and the controlled release of chemotherapeutic drugs and P-gp inhibitors. Their pharmacokinetics are governed by the nanoparticle or polymer carrier and make use of the enhanced permeation and retention (EPR) effect, which can increase selective delivery to cancer cells. These systems are usually internalized by cancer cells via endocytosis and accumulate in endosomes and lysosomes, thus preventing rapid efflux. Other modalities to combat MDR are described in this review, including the pharmaco-modulation of acridine, which is a well-known scaffold in the development of bioactive compounds, the use of natural compounds as means to reverse MDR, and the conjugation of anticancer drugs with carriers that target specific tumor-cell components. Finally, the outstanding potential of in silico structure-based methods as a means to evaluate the ability of antitumor drugs to interact with drug transporters is also highlighted in this review. Structure-based design methods, which utilize 3D structural data of proteins and their complexes with ligands, are the most effective of the in silico methods available, as they provide a prediction regarding the interaction between transport proteins and their substrates and inhibitors. The recently resolved X-ray structure of human P-gp can help predict the interaction sites of designed compounds, providing insight into their binding mode and directing possible rational modifications to prevent them from becoming P-gp drug substrates. In summary, although major efforts were invested in the search for new tools to combat drug resistant tumors, they all require further implementation and methodological development. Further investigation and progress in the abovementioned strategies will provide significant advances in the rational combat against cancer MDR.

Curcumin-Loaded Solid Lipid Nanoparticles Bypass P-Glycoprotein Mediated Doxorubicin Resistance in Triple Negative Breast Cancer Cells.

Multidrug resistance (MDR) is a critical hindrance to the success of cancer chemotherapy. The main thing responsible for MDR phenotypes are plasma-membranes associated with adenosine triphosphate (ATP) Binding Cassette (ABC) drug efflux transporters, such as the P-glycoprotein (Pgp) transporter that has the broadest spectrum of substrates. Curcumin (CURC) is a Pgp inhibitor, but it is poorly soluble and bioavailable. To overcome these limitations, we validated the efficacy and safety of CURC, loaded in biocompatible solid lipid nanoparticles (SLNs), with or without chitosan coating, with the goal of increasing the stability, homogeneous water dispersibility, and cellular uptake. Both CURC-loaded SLNs were 5-10-fold more effective than free CURC in increasing the intracellular retention and toxicity of doxorubicin in Pgp-expressing triple negative breast cancer (TNBC). The effect was due to the decrease of intracellular reactive oxygen species, consequent inhibition of the Akt/IKKα-ß/NF-kB axis, and reduced transcriptional activation of the Pgp promoter by p65/p50 NF-kB. CURC-loaded SLNs also effectively rescued the sensitivity to doxorubicin against drug-resistant TNBC tumors, without signs of systemic toxicity. These results suggest that the combination therapy, based on CURC-loaded SLNs and doxorubicin, is an effective and safe approach to overcome the Pgp-mediated chemoresistance in TNBC.

Mitochondrial metabolism: Inducer or therapeutic target in tumor immune-resistance?

Mitochondria have been considered for a long time only as the principal source of building blocks and energy upon aerobic conditions. Recently they emerged as key players in cell proliferation, invasion and resistance to therapy. The most aggressive tumors are able to evade the immune-surveillance. Alterations in the mitochondria metabolism either in cancer cells or in host immune system cells are involved in such tumor-induced immune-suppression. This review will focus on the main mitochondrial dysfunctions in tumor and immune cell populations determining immune-resistance, and on the therapies that may target mitochondrial metabolism and restore a powerful anti-tumor immune-activity.

Phospholipids and cholesterol: Inducers of cancer multidrug resistance and therapeutic targets.

Lipids, phospholipids and cholesterol in particular, are the predominant components of the plasma membrane, wherein multidrug efflux transporters of the ATP-binding cassette (ABC) superfamily reside as integral pump proteins. In the current review, we discuss how lipids potently modulate the expression and activity of these multidrug efflux pumps, contributing to the development of the multidrug resistance (MDR) phenotype in cancer. The molecular mechanisms underlying this modulation of the MDR phenotype are pleiotropic. First, notwithstanding the high intra-and inter-tumor variability, MDR cells display an altered composition of plasma membrane phospholipids and glycosphingolipids, and are enriched with very long saturated fatty acid chains. This feature, along with the increased levels of cholesterol, decrease membrane fluidity, alter the spatial organization of membrane nano- and micro-domains, interact with transmembrane helices of ABC transporters, hence favoring drug binding and release. Second, MDR cells exhibit a peculiar membrane lipid composition of intracellular organelles including mitochondria and endoplasmic reticulum (ER). In this respect, they contain a lower amount of oxidizable fatty acids, hence being more resistant to oxidative stress and chemotherapy-induced apoptosis. Third, drug resistant cancer cells have a higher ratio of monosatured/polyunsatured fatty acids: this lipid signature reduces the production of reactive aldehydes with cytotoxic and pro-inflammatory activity and, together with the increased activity of anti-oxidant enzymes, limits the cellular damage induced by lipid peroxidation. Finally, specific precursors of phospholipids and cholesterol including ceramides and isoprenoids, are highly produced in MDR cells; by acting as second messengers, they trigger multiple signaling cascades that induce the transcription of drug efflux transporter genes and/or promote a metabolic reprogramming which supports the MDR phenotype. High-throughput lipidomics and computational biology technologies are a great tool in analyzing the tumor lipid signature in a personalized manner and in identifying novel biomarkers of drug resistance. Moreover, beyond the induction of MDR, lipid metabolism offers a remarkable opportunity to reverse MDR by using lipid analogues and repurposing lipid-targeting drugs (e.g. statins and aminobisphosphonates) that reprogram the lipid composition of drug resistant cells, hence rendering them drug sensitive.