Compared antioxidant activity among corticosteroids on cultured retinal pigment epithelial cells.

PURPOSE: The aim of this study is to determine if dexamethasone, prednisolone and triamcinolone acetonide (TA), three anti-inflammatory drugs commonly used for ocular treatments, could affect the oxidative status of cultured human cells of the retinal pigment epithelium (RPE) and protect them against oxidative injury. METHODS: ARPE-19 cells were used as an in vitro model of RPE. Glutathione (GSH) levels were assessed to evaluate the effects of dexamethasone, prednisolone and triamcinolone on cellular antioxidant status. Oxidative stress was induced in ARPE-19 cells by treatment with the oxidizing agent menadione, and the effects of dexamethasone, prednisolone and triamcinolone were evaluated. Release of lactate dehydrogenase (LDH) in the culture medium was used to measure cytotoxicity. RESULTS: Incubation with triamcinolone or prednisolone was not able to revert menadione-induced cytotoxicity and GSH depletion; furthermore, it significantly decreased GSH levels in ARPE-19 cells (nmol of GSH/mg cellular protein: 99.7 ± 0.1 in untreated controls vs. 52.6 ± 5.2 with triamcinolone vs. 77.6 ± 5.2 with prednisolone; p < 0.001). Treatment with dexamethasone protected ARPE-19 cells from cytotoxicity and oxidative damage: lactate dehydrogenase release and GSH depletion were significantly decreased after incubation with this compound (LDHout/LDHtot: 0.221 ± 0.038 with menadione vs. 0.041 ± 0.007 with menadione + dexamethasone; p < 0.001; nmol of GSH/mg cellular protein: 5.7 ± 4.2 with menadione vs. 53.2 ± 6.1 with menadione + dexamethasone, respectively; p < 0.001) and did not induce GSH depletion (nmol of GSH/mg cellular protein: 99.7 ± 0.1 vs. 86.5 ± 8.1 nmol/min/mg prot with dexamethasone; p > 0.05). CONCLUSIONS: Dexamethasone, besides suppressing intraocular inflammation, may protect human RPE cells from oxidative stress and decrease the oxidation rate of GSH. Triamcinolone and prednisolone, inducing GSH depletion, may contribute to reduce antioxidant capacity of ARPE-19 cells.

Editor's Highlight: Abrasion of Artificial Stones as a New Cause of an Ancient Disease. Physicochemical Features and Cellular Responses.

New outbursts of silicosis were recently reported among workers manufacturing an engineered material known as "artificial stone," composed by high percentages of quartz (up to 98%) agglomerated with pigments and polymeric resins. Dusts released by abrasion during artificial stone polishing were characterized for particle size, morphology, and elemental composition and studied for (1) ability to catalyze free radical generation in acellular tests, (2) membranolytic potential on human erythrocytes, (3) cytotoxic activity (lactate dehydrogenase release) on murine alveolar macrophages (MH-S) and human bronchial epithelial (BEAS-2B) cell lines, (4) induction of epithelial-mesenchymal transition (EMT) in BEAS-2B cells. Min-U-Sil 5 was used as reference quartz. Artificial stone dusts exhibited morphological features close to quartz, but contained larger amount of metal transition ions (mainly, Fe, Cu, and Ti), potentially responsible for the high reactivity in free radical generation observed. Opposite to Min-U-Sil 5, they were neither hemolytic nor cytotoxic on MH-S cells, a low cytotoxicity only being observed with BEAS-2B cells. The presence on the particle surface of residues of the resin accounts for this attenuated behavior, as hemolysis appeared and cytotoxicity increased after thermal degradation of the resin, when the free quartz surface was exposed. All dusts induced EMT with loss of E-cadherin expression and increased the expression of mesenchymal proteins (α-smooth muscle actin and vimentin). This may contribute to explain the development of fibrosis on workers exposed to artificial stone dusts.

Multi-walled carbon nanotubes directly induce epithelial-mesenchymal transition in human bronchial epithelial cells via the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 signalling pathway.

BACKGROUND: Multi-walled carbon nanotubes (MWCNT) are currently under intense toxicological investigation due to concern on their potential health effects. Current in vitro and in vivo data indicate that MWCNT exposure is strongly associated with lung toxicity (inflammation, fibrosis, granuloma, cancer and airway injury) and their effects might be comparable to asbestos-induced carcinogenesis. Although fibrosis is a multi-origin disease, epithelial-mesenchymal transition (EMT) is recently recognized as an important pathway in cell transformation. It is known that MWCNT exposure induces EMT through the activation of the TGF-ß/Smad signalling pathway thus promoting pulmonary fibrosis, but the molecular mechanisms involved are not fully understood. In the present work we propose a new mechanism involving a TGF-ß-mediated signalling pathway. METHODS: Human bronchial epithelial cells were incubated with two different MWCNT samples at various concentrations for up to 96 h and several markers of EMT were investigated. Quantitative real time PCR, western blot, immunofluorescent staining and gelatin zymographies were performed to detect the marker protein alterations. ELISA was performed to evaluate TGF-ß production. Experiments with neutralizing anti-TGF-ß antibody, specific inhibitors of GSK-3ß and Akt and siRNA were carried out in order to confirm their involvement in MWCNT-induced EMT. In vivo experiments of pharyngeal aspiration in C57BL/6 mice were also performed. Data were analyzed by a one-way ANOVA with Tukey's post-hoc test. RESULTS: Fully characterized MWCNT (mean length < 5 µm) are able to induce EMT in an in vitro human model (BEAS-2B cells) after long-term incubation at sub-cytotoxic concentrations. MWCNT stimulate TGF-ß secretion, Akt activation and GSK-3ß inhibition, which induces nuclear accumulation of SNAIL-1 and its transcriptional activity, thus contributing to switch on the EMT program. Moreover, a significant increment of nuclear ß-catenin - due to E-cadherin repression and following translocation to nucleus - likely reinforces signalling for EMT promotion. In vivo results supported the occurrence of pulmonary fibrosis following MWCNT exposure. CONCLUSIONS: We demonstrate a new molecular mechanism of MWCNT-mediated EMT, which is Smad-independent and involves TGF-ß and its intracellular effectors Akt/GSK-3ß that activate the SNAIL-1 signalling pathway. This finding suggests potential novel targets in the development of therapeutic and preventive approaches.

H2S-Donating Doxorubicins May Overcome Cardiotoxicity and Multidrug Resistance.

Doxorubicin (DOXO) is one of the most effective antineoplastic agents in clinical practice. Its use is limited by acute and chronic side effects, in particular by its cardiotoxicity and by the rapid development of resistance to it. As part of a program aimed at developing new DOXO derivatives endowed with reduced cardiotoxicity, and active against DOXO-resistant tumor cells, a series of H2S-releasing DOXOs (H2S-DOXOs) were obtained by combining DOXO with appropriate H2S donor substructures. The resulting compounds were studied on H9c2 cardiomyocytes and in DOXO-sensitive U-2OS osteosarcoma cells, as well as in related cell variants with increasing degrees of DOXO-resistance. Differently from DOXO, most of the products were not toxic at 5 µM concentration on H9c2 cells. A few of them triggered high activity on the cancer cells. H2S-DOXOs 10 and 11 emerged as the most interesting members of the series. The capacity of 10 to impair Pgp transporter is also discussed.

Physico-chemical properties of quartz from industrial manufacturing and its cytotoxic effects on alveolar macrophages: The case of green sand mould casting for iron production.

Industrial processing of materials containing quartz induces physico-chemical modifications that contribute to the variability of quartz hazard in different plants. Here, modifications affecting a quartz-rich sand during cast iron production, have been investigated. Composition, morphology, presence of radicals associated to quartz and reactivity in free radical generation were studied on a raw sand and on a dust recovered after mould dismantling. Additionally, cytotoxicity of the processed dust and ROS and NO generation were evaluated on MH-S macrophages. Particle morphology and size were marginally affected by casting processing, which caused only a slight increase of the amount of respirable fraction. The raw sand was able to catalyze OH and CO2(-) generation in cell-free test, even if in a lesser extent than the reference quartz (Min-U-Sil), and shows hAl radicals, conventionally found in any quartz-bearing raw materials. Enrichment in iron and extensive coverage with amorphous carbon were observed during processing. They likely contributed, respectively, to increasing the ability of processed dust to release CO2- and to suppressing OH generation respect to the raw sand. Carbon coverage and repeated thermal treatments during industrial processing also caused annealing of radiogenic hAl defects. Finally, no cellular responses were observed with the respirable fraction of the processed powder.

Zoledronic acid-encapsulating self-assembling nanoparticles and doxorubicin: a combinatorial approach to overcome simultaneously chemoresistance and immunoresistance in breast tumors.

The resistance to chemotherapy and the tumor escape from host immunosurveillance are the main causes of the failure of anthracycline-based regimens in breast cancer, where an effective chemo-immunosensitizing strategy is lacking.The clinically used aminobisphosphonate zoledronic acid (ZA) reverses chemoresistance and immunoresistance in vitro. Previously we developed a nanoparticle-based zoledronic acid-containing formulation (NZ) that allowed a higher intratumor delivery of the drug compared with free ZA in vivo. We tested its efficacy in combination with doxorubicin in breast tumors refractory to chemotherapy and immune system recognition as a new combinatorial approach to produce chemo- and immunosensitization.NZ reduced the IC50 of doxorubicin in human and murine chemoresistant breast cancer cells and restored the doxorubicin efficacy against chemo-immunoresistant tumors implanted in immunocompetent mice. By reducing the metabolic flux through the mevalonate pathway, NZ lowered the activity of Ras/ERK1/2/HIF-1α axis and the expression of P-glycoprotein, decreased the glycolysis and the mitochondrial respiratory chain, induced a cytochrome c/caspase 9/caspase 3-dependent apoptosis, thus restoring the direct cytotoxic effects of doxorubicin on tumor cell. Moreover, NZ restored the doxorubicin-induced immunogenic cell death and reversed the tumor-induced immunosuppression due to the production of kynurenine, by inhibiting the STAT3/indoleamine 2,3 dioxygenase axis. These events increased the number of dendritic cells and decreased the number of immunosuppressive T-regulatory cells infiltrating the tumors.Our work proposes the use of nanoparticle encapsulating zoledronic acid as an effective tool overcoming at the same time chemoresistance and immunoresistance in breast tumors, thanks to the effects exerted on tumor cell and tumor-infiltrating immune cells.

Overcoming multidrug resistance by targeting mitochondria with NO-donating doxorubicins.

A library of nitric oxide-donor doxorubicins (NO-DOXOs) was synthesized by linking appropriate NO-donor moieties at C-14 position through an ester bridge. Their hydrolytic stability was evaluated. The intracellular accumulation and cytotoxicity of these novel NO-DOXOs were studied in DOXO-sensitive (HT29) and DOXO-resistant (HT29/dx) tumor-cells. Hydrolytically-stable compounds accumulated in HT29 and HT29/dx cells, thanks to the nitration of plasma-membrane efflux transporters. Surprisingly, no close correlation was found between intracellular accumulation and cytotoxicity. Only compounds with high mitochondria retention (due to nitration of mitochondrial efflux transporter) exert high cytotoxicity, through the activation of a mitochondrial-dependent apoptosis.

Effects of Chrysotile Exposure in Human Bronchial Epithelial Cells: Insights into the Pathogenic Mechanisms of Asbestos-Related Diseases.

BACKGROUND: Chrysotile asbestos accounts for > 90% of the asbestos used worldwide, and exposure is associated with asbestosis (asbestos-related fibrosis) and other malignancies; however, the molecular mechanisms involved are not fully understood. A common pathogenic mechanism for these malignancies is represented by epithelial-mesenchymal transition (EMT), through which epithelial cells undergo a morphological transformation to assume a mesenchymal phenotype. In the present work, we propose that chrysotile asbestos induces EMT through a mechanism involving a signaling pathway mediated by tranforming growth factor beta (TGF-ß). OBJECTIVES: We investigated the role of chrysotile asbestos in inducing EMT in order to elucidate the molecular mechanisms involved in this event. METHODS: Human bronchial epithelial cells (BEAS-2B) were incubated with 1 µg/cm2 chrysotile asbestos for ≤ 72 hr, and several markers of EMT were investigated. Experiments with specific inhibitors for TGF-ß, glycogen synthase kinase-3ß (GSK-3ß), and Akt were performed to confirm their involvement in asbestos-induced EMT. Real-time polymerase chain reaction (PCR), Western blotting, and gelatin zymography were performed to detect mRNA and protein level changes for these markers. RESULTS: Chrysotile asbestos activated a TGF-ß-mediated signaling pathway, implicating the contributions of Akt, GSK-3ß, and SNAIL-1. The activation of this pathway in BEAS-2B cells was associated with a decrease in epithelial markers (E-cadherin and ß-catenin) and an increase in mesenchymal markers (α-smooth muscle actin, vimentin, metalloproteinases, and fibronectin). CONCLUSIONS: Our findings suggest that chrysotile asbestos induces EMT, a common event in asbestos-related diseases, at least in part by eliciting the TGF-ß-mediated Akt/GSK-3ß/SNAIL-1 pathway. CITATION: Gulino GR, Polimeni M, Prato M, Gazzano E, Kopecka J, Colombatto S, Ghigo D, Aldieri E. 2016. Effects of chrysotile exposure in human bronchial epithelial cells: insights into the pathogenic mechanisms of asbestos-related diseases. Environ Health Perspect 124:776-784; http://dx.doi.org/10.1289/ehp.1409627.

Development of sigma-1 (σ1) receptor fluorescent ligands as versatile tools to study σ1 receptors.

Despite their controversial physiology, sigma-1 (σ1) receptors are intriguing targets for the development of therapeutic agents for central nervous system diseases. With the aim of providing versatile pharmacological tools to study σ1 receptors, we developed three σ1 fluorescent tracers by functionalizing three well characterized σ1 ligands with a fluorescent tag. A good compromise between σ1 binding affinity and fluorescent properties was reached, and the σ1 specific targeting of the novel tracers was demonstrated by confocal microscopy and flow cytometry. These novel ligands were also successfully used in competition binding studies by flow cytometry, showing their utility in nonradioactive binding assays as an alternative strategy to the more classical radioligand binding assays. To the best of our knowledge these are the first σ1 fluorescent ligands to be developed and successfully employed in living cells, representing promising tools to strengthen σ1 receptors related studies.

Fibrinogen enhances the inflammatory response of alveolar macrophages to TiO2, SiO2 and carbon nanomaterials.

Many studies have shown that the composition of the protein corona dramatically affects the response of cells to nanomaterials (NMs). However, the role of each single protein is still largely unknown. Fibrinogen (FG), one of the most abundant plasma proteins, is believed to mediate foreign-body reactions. Since this protein is absent in cell media used in in vitro toxicological tests the possible FG-mediated effects have not yet been assessed. Here, the effect of FG on the toxicity of three different kinds of inorganic NMs (carbon, SiO2 and TiO2) on alveolar macrophages has been investigated. A set of integrated techniques (UV-vis spectroscopy, dynamic light scattering and sodium dodecyl sulphate-polyacrylamide gel electrophoresis) have been used to study the strength and the kinetics of interaction of FG with the NMs. The inflammatory response of alveolar macrophages (MH-S) exposed to the three NMs associated with FG has also been investigated. We found that FG significantly enhances the cytotoxicity (lactate dehydrogenase leakage) and the inflammatory response (increase in nitric oxide (NO) concentration and NO synthase activation) induced by SiO2, carbon and TiO2 NMs on alveolar macrophages. This effect appears related to the amount of FG interacting with the NMs. In the case of carbon NMs, the activation of fibrinolysis, likely related to the exposure of cryptic sites of FG, was also observed after 24 h. These findings underline the critical role played by FG in the toxic response to NMs.

Influence of posterior vitreous detachment and type of intraocular lens on lipid peroxidation in the human vitreous.

PURPOSE: The aim of this study was to evaluate the relationship between oxidative stress and human vitreous degeneration, using the presence of an evident posterior vitreous detachment (PVD) as a clinical sign and thiobarbituric acid-reactive substances (TBARS) and nitrite as oxidative biomarkers. METHODS: We collected 42 vitreous samples from patients undergoing pars plana vitrectomy for two groups of vitreoretinal diseases (macular holes and epimacular membranes). TBARS and nitrite were assessed spectrophotometrically and compared to the presence of an evident PVD, considering other clinical features potentially related to the oxidative stress in the vitreous: diabetes, plasma fibrinogen, type of intraocular lens (IOL), and the vitreoretinal disease requiring the surgery. RESULTS: Vitreous TBARS levels were significantly higher in patients with artificial IOLs compared to those with natural lenses and cataracts (1.39±0.64 versus 0.75±0.45; p=0.003). Furthermore, patients with PVD had a significant increase in vitreous TBARS compared to those without PVD (1.45±0.54 versus 0.53±0.38; p=0.001). The plasma fibrinogen levels explained 17% of the TBARS variance, with a significant correlation between these two markers (p=0.011). No significant differences were observed when nitrites were used as biomarkers. CONCLUSIONS: Current IOLs, even with ultraviolet (UV) absorber, do not ensure the same photoprotection offered by natural lenses affected by corticonuclear cataracts. Furthermore, we observed a relevant correlation between the increased presence of peroxidation products in the vitreous and an evident PVD, but the nature of this relationship requires further study.

Self-assembling nanoparticles encapsulating zoledronic acid revert multidrug resistance in cancer cells.

The overexpression of ATP binding cassette (ABC) transporters makes tumor cells simultaneously resistant to several cytotoxic drugs. Impairing the energy metabolism of multidrug resistant (MDR) cells is a promising chemosensitizing strategy, but many metabolic modifiers are too toxic in vivo. We previously observed that the aminobisphosphonate zoledronic acid inhibits the activity of hypoxia inducible factor-1a (HIF-1a), a master regulator of cancer cell metabolism. Free zoledronic acid, however, reaches low intratumor concentration. We synthesized nanoparticle formulations of the aminobisphosphonate that allow a higher intratumor delivery of the drug. We investigated whether they are effective metabolic modifiers and chemosensitizing agents against human MDR cancer cells in vitro and in vivo. At not toxic dosage, nanoparticles carrying zoledronic acid chemosensitized MDR cells to a broad spectrum of cytotoxic drugs, independently of the type of ABC transporters expressed. The nanoparticles inhibited the isoprenoid synthesis and the Ras/ERK1/2-driven activation of HIF-1α, decreased the transcription and activity of glycolytic enzymes, the glucose flux through the glycolysis and tricarboxylic acid cycle, the electron flux through the mitochondrial respiratory chain, the synthesis of ATP. So doing, they lowered the ATP-dependent activity of ABC transporters, increasing the chemotherapy efficacy in vitro and in vivo. These effects were more pronounced in MDR cells than in chemosensitive ones and were due to the inhibition of farnesyl pyrophosphate synthase (FPPS), as demonstrated in FPPS-silenced tumors. Our work proposes nanoparticle formulations of zoledronic acid as the first not toxic metabolic modifiers, effective against MDR tumors.

An Autocrine Cytokine/JAK/STAT-Signaling Induces Kynurenine Synthesis in Multidrug Resistant Human Cancer Cells.

BACKGROUND: Multidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells. RESULTS: We found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1ß, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells. CONCLUSIONS: Our work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors.

Novel and Selective Fluorescent σ2 -Receptor Ligand with a 3,4-Dihydroisoquinolin-1-one Scaffold: A Tool to Study σ2 Receptors in Living Cells.

Although sigma-2 (σ2 ) receptors are still enigmatic proteins, they are promising targets for tumor treatment and diagnosis. With the aim of clarifying their role in oncology, we developed a σ2 -selective fluorescent tracer (compound 5) as a specific tool to study σ2 receptors. By using flow cytometry with 5, we performed competition binding studies on three different cell lines where we also detected the content of the σ2 receptors, avoiding the inconvenient use of radioligands. Comparison with a previously developed mixed σ1 /σ2 fluorescent tracer (1) also allowed for the detection of σ1 receptors within these cells. Results obtained by flow cytometry with tracers 1 and 5 were confirmed by standard methods (western blot for σ1 , and Scatchard analysis for σ2 receptors). Thus, we have produced powerful new tools for research on the σ whose reliability and adaptability to a number of fluorescence techniques will be useful to elucidate the roles of σ receptors in oncology.

Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells.

Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer.We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

Two repeated low doses of doxorubicin are more effective than a single high dose against tumors overexpressing P-glycoprotein.

Standard chemotherapeutic protocols, based on maximum tolerated doses, do not prevent nor overcome chemoresistance caused by the efflux transporter P-glycoprotein (Pgp). We compared the effects of two consecutive low doses versus a single high dose of doxorubicin in drug-sensitive Pgp-negative and drug-resistant Pgp-positive human and murine cancer cells. Two consecutive low doses were significantly more cytotoxic in vitro and in vivo against drug-resistant tumors, while a single high dose failed to do so. The greater efficacy of two consecutive low doses of doxorubicin could be linked to increased levels of intracellular reactive oxygen species. These levels were produced by high electron flux from complex I to complex III of the mitochondrial respiratory chain, unrelated to the synthesis of ATP. This process induced mitochondrial oxidative damage, loss of mitochondrial potential and activation of the cytochrome c/caspase 9/caspase 3 pro-apoptotic axis in drug-resistant cells. Our work shows that the "apparent" ineffectiveness of doxorubicin against drug-resistant tumors overexpressing Pgp can be overcome by changing the timing of its administration and its doses.

Zoledronic acid overcomes chemoresistance and immunosuppression of malignant mesothelioma.

The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.

Liposomal nitrooxy-doxorubicin: one step over caelyx in drug-resistant human cancer cells.

In this work we prepared and characterized two liposomal formulations of a semisynthetic nitric oxide (NO)-releasing doxorubicin (Dox), called nitrooxy-Dox (NitDox), which we previously demonstrated to be cytotoxic in Dox-resistant human colon cancer cells. Liposomes with 38.2% (Lip A) and 19.1% (Lip B) cholesterol were synthesized: both formulations had similar size and zeta potential values and caused the same intracellular distribution of free NitDox, but Lip B accumulated and released NitDox more efficiently. In Dox-resistant human colon cancer cells, Lip A and Lip B exhibited a more favorable kinetics of drug uptake and NO release, and a stronger cytotoxicity than Dox and free NitDox. While Caelyx, one of the liposomal Dox formulations approved for breast and ovary tumors treatment, was ineffective in Dox-resistant breast/ovary cancer cells, Lip B, and to a lesser extent Lip A, still exerted a significant cytotoxicity in these cells. This event was accompanied in parallel by a higher release of NO, which caused nitration of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two transporters involved in Dox efflux, and impaired their pump activity. By doing so, the efflux kinetics of Dox after treatment with Lip B was markedly slowed down and the intracellular accumulation of Dox was increased in breast and ovary drug-resistant cells. We propose these liposomal formulations of NitDox as new tools with a specific indication for tumors overexpressing Pgp and MRP1.

The cross-talk between canonical and non-canonical Wnt-dependent pathways regulates P-glycoprotein expression in human blood-brain barrier cells.

In this work, we investigate if and how transducers of the 'canonical' Wnt pathway, i.e., Wnt/glycogen synthase kinase 3 (GSK3)/ß-catenin, and transducers of the 'non-canonical' Wnt pathway, i.e., Wnt/RhoA/RhoA kinase (RhoAK), cooperate to control the expression of P-glycoprotein (Pgp) in blood-brain barrier (BBB) cells. By analyzing human primary brain microvascular endothelial cells constitutively activated for RhoA, silenced for RhoA or treated with the RhoAK inhibitor Y27632, we found that RhoAK phosphorylated and activated the protein tyrosine phosphatase 1B (PTP1B), which dephosphorylated tyrosine 216 of GSK3, decreasing the GSK3-mediated inhibition of ß-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of ß-catenin, and reduced the ß-catenin-driven transcription of Pgp. The RhoAK inhibition increased the delivery of Pgp substrates like doxorubicin across the BBB and improved the doxorubicin efficacy against glioblastoma cells co-cultured under a BBB monolayer. Our data demonstrate that in human BBB cells the expression of Pgp is controlled by a cross-talk between canonical and non-canonical Wnt pathways. The disruption of this cross-talk, e.g., by inhibiting RhoAK, downregulates Pgp and increases the delivery of Pgp substrates across the BBB.