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International audience; The adverse outcome pathway (AOP) is a conceptual construct that facilitates organisation and interpretation of mechanistic data representing multiple biological levels and deriving from a range of methodological approaches including in silico, in vitro and in vivo assays. AOPs are playing an increasingly important role in the chemical safety assessment paradigm and quantification of AOPs is an important step towards a more reliable prediction of chemically induced adverse effects. Modelling methodologies require the identification, extraction and use of reliable data and information to support the inclusion of quantitative considerations in AOP development. An extensive and growing range of digital resources are available to support the modelling of quantitative AOPs, providing a wide range of information, but also requiring guidance for their practical application. A framework for qAOP development is proposed based on feedback from a group of experts and three qAOP case studies. The proposed framework provides a harmonised approach for both regulators and scientists working in this area.

In a century where toxicology and chemical risk assessment are embracing alternative methods to animal testing, there is an opportunity to understand the causal factors of neurodevelopmental disorders such as learning and memory disabilities in children, as a foundation to predict adverse effects. New testing paradigms, along with the advances in probabilistic modelling, can help with the formulation of mechanistically-driven hypotheses on how exposure to environmental chemicals could potentially lead to developmental neurotoxicity (DNT). This investigation aimed to develop a Bayesian hierarchical model of a simplified AOP network for DNT. The model predicted the probability that a compound induces each of three selected common key events (CKEs) of the simplified AOP network and the adverse outcome (AO) of DNT, taking into account correlations and causal relations informed by the key event relationships (KERs). A dataset of 88 compounds representing pharmaceuticals, industrial chemicals and pesticides was compiled including physicochemical properties as well as in silico and in vitro information. The Bayesian model was able to predict DNT with an accuracy of 76%, and classify the compounds into low, medium or high probability classes for their potential to induce DNT. The modelling workflow achieved three further goals: it dealt with missing values; accommodated unbalanced and correlated data; and followed the structure of a directed acyclic graph (DAG) to simulate the simplified AOP network. Overall, the model demonstrated the utility of Bayesian hierarchical modelling for the development of quantitative AOP (qAOP) models and for informing the use of new approach methodologies (NAMs) in chemical risk assessment.

The Adverse Outcome Pathway (AOP) framework is increasingly used to integrate data based on traditional and emerging toxicity testing paradigms. As the number of AOP descriptions has increased, so has the need to define the AOP in computable terms. Herein, we present a comprehensive annotation of 172 AOPs housed in the AOP-Wiki as of December 4, 2016, using terms from existing biological ontologies. AOP Key Events (KEs) were assigned ontology terms using a concept called the Event Component, which consists of a Process, an Object, and an Action term, with each term originating from ontologies and other controlled vocabularies. Annotation of KEs with ontology classes from 14 ontologies and controlled vocabularies resulted in a total of 685 KEs being annotated with a total of 809 Event Components. A set of seven conventions resulted, defining the annotation of KEs via Event Components. This expanded annotation of AOPs allows computational reasoners to aid in both AOP development and applications. I...

The immunoglobulin heavy-chain variable region (IGHV) mutational status is a strong determinant of remission duration in chronic lymphocytic leukemia (CLL). The aim of this work was to compare the multidrug resistance (MDR) signature of IGHV mutated and unmutated CLL cells, identifying biochemical and molecular targets potentially amenable to therapeutic intervention. We found that the mevalonate pathway-dependent Ras/ERK1–2 and RhoA/RhoA kinase signaling cascades, and the downstream HIF-1α/P-glycoprotein axis were more active in IGHV unmutated than in mutated cells, leading to a constitutive protection from doxorubicin-induced cytotoxicity. The constitutive MDR phenotype of IGHV unmutated cells was partially dependent on B cell receptor signaling, as shown by the inhibitory effect exerted by ibrutinib. Stromal cells further protected IGHV unmutated cells from doxorubicin by upregulating Ras/ERK1–2, RhoA/RhoA kinase, Akt, HIF-1α and P-glycoprotein activities. Mevalonate pathway inhibition with simvastatin abrogated these signaling pathways and reversed the resistance of IGHV unmutated cells to doxorubicin, also counteracting the protective effect exerted by stromal cells. Similar results were obtained via the targeted inhibition of the downstream molecules ERK1–2, RhoA kinase and HIF-1α. Therefore, targeting the mevalonate pathway and its downstream signaling cascades is a promising strategy to circumvent the MDR signature of IGHV unmutated CLL cells.

In this work we investigated how the surface charge and the presence of polyethylene glycol (PEG) on liposome carriers affect the delivery of the encapsulated doxorubicin in P-glycoprotein (Pgp)-overexpressing cells. We found that neutral net charge was critical to favour the liposome uptake and decrease the Vmax of doxorubicin efflux. PEG-coating was necessary to increase the Km of doxorubicin for Pgp. In particular the PEGylated phospholipid present in neutral liposomes, i.e. PEGylated distearoyl-phosphatidylethanolamine (DSPE-PEG), was a Pgp allosteric inhibitor, increased doxorubicin Km and inhibited Pgp ATPase activity. Site-directed mutagenesis experiments suggested that the domain centred around glycine 185 of Pgp was necessary for these inhibitory properties of DSPE-PEG and PEGylated neutral liposomes. We conclude that both surface charge and PEGylation must be considered to optimize the doxorubicin delivery within chemoresistant cells. DSPE-PEG-enriched particles may represent promising tools for therapeutic and diagnostic applications in tissues with high levels of Pgp.These authors investigated how surface charge and PEGylation of liposome carriers affect the delivery of encapsulated doxorubicin to Pgp-overexpressing cells, concluding that both factors need to be considered in order to optimize doxorubicin delivery to chemoresistant cells.

Glioblastoma multiforme (GBM) is the most malignant and frequent intra-axial tumor of the central nervous system. Despite improvements in neurosurgery, radiotherapy, and chemotherapy, no local control of the disease is today possible.1,2 The failure of chemotherapy is due to the tumor's polyclonality, to its modalities of invasion, and to resistance to therapies. The presence of so-called cancer stem cells (CSCs) within GBM has been suggested as another possible reason for chemoresistance.3–5 GBM CSCs are believed to originate from the transformation of neural stem cells of the embryonic matrix and the neurogenic zones of the adult brain6 or from the dedifferentiation of hemispheric astrocytes or tumor cells. The latter reacquire stemness through embryonic regression upon the progressive accumulation of mutations.7–10 GBM CSCs show self-renewal, clonogenicity, and tumorigenicity when transplanted into mice.11 In culture systems without a coated matrix, the occurrence of CSCs in a tumor is denoted by the generation of neurospheres (NSs) beside adherent cells (ACs) after the addition of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF).12 GBM CSCs share with neural stem cells stemness antigens, such as CD133, Musashi1, nestin, and Sox2,12,13 along with genetic alterations with primary tumors.10,14 They often show high activity of transforming growth factor/bone morphogenetic protein, Notch, Wnt, and sonic hedgehog homolog pathways, which are involved in self-renewal, proliferation, and invasion.15 The molecular basis of chemoresistance in GBM CSCs is under intensive investigation.3,16–18 The main drugs used in the therapy of GBM are DNA-damaging agents, such as the alkylating agents temozolomide (TMZ) and carmustine (BCNU), the topoisomerase II inhibitor etoposide, the topoisomerase I inhibitor irinotecan, and the DNA-intercalating drug doxorubicin.2 TMZ methylates the O6 position on guanine that mismatches with thymine in genomic DNA. The subsequent DNA aberration blocks replication and induces cell death. It is generally recognized that the overexpression of O6-methylguanine-DNA methyltransferase (MGMT), which removes the alkyl group from guanine, mediates the resistance to TMZ in GBM.3,19 However, MGMT alone is not always correlated with resistance to TMZ in GBM CSCs. A plethora of additional intrinsic factors (eg, the activity of DNA-repairing systems or the activity of specific signaling pathways involved in cell proliferation and apoptosis protection) or extrinsic factors (eg, the hypoxic environment or the presence of CSC niches rich in protective growth factors) may contribute to the chemoresistant phenotype of GBM CSCs.3,20 The role of membrane ATP-binding cassette (ABC) transporters—such as P-glycoprotein (Pgp/ABCB1), multidrug-resistance related protein 1 (MRP1/ABCC1), and breast cancer resistance protein (BCRP/ABCG2)—as inducers of the chemoresistant phenotype in GBM CSCs is still a matter of debate.3 Pgp,21,22 MRP1/3/5,21 and BCRP23,24 have been found highly expressed in GBM CSCs, either derived from primary tumors21,23 or obtained in vitro by growing ACs in the NS-generating medium.22 The overexpression of Pgp in NS U87MG cells is predictive of resistance to doxorubicin, etoposide, carboplatin, BCNU,22 and vincristine.25 TMZ has been reported to be a substrate of Pgp,26 although this observation is still controversial. To our knowledge, the regulation of ABC transporters in GBM CSCs has not yet been investigated. In this work we found that Pgp levels in GBM CSCs are controlled by Wnt3a oncogene and that clinically achievable doses of TMZ interfere with Wnt3a-dependent signaling and decrease the expression of Pgp in this tumor population.

The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.

Products 4 and 5, obtained by conjugation of doxorubicin with nitric oxide (NO) donor nitrooxy and phenylsulfonyl furoxan moieties, respectively, accumulate in doxorubicin-resistant human colon cancer cells (HT29-dx), inducing high cytotoxicity. This behavior parallels the ability of the compounds to generate NO, detected as nitrite, in these cells. Preliminary immunoblotting studies suggest that the mechanism that underlies the cytotoxic effect could involve inhibition of cellular drug efflux due to nitration of tyrosine residues of the MRP3 protein pump.

Background and purpose: Statins, inhibitors of hydroxymethylglutaryl-CoA reductase, reduce the intracellular synthesis of cholesterol and prevent the onset of atherosclerosis. They also decrease the synthesis of isoprenoid molecules, such as the side chain of ubiquinone and geranylgeranyl pyrophosphate. As a consequence, statins impair mitochondrial metabolism and the activation of small monomeric GTPases (such as Rho and Ras), causing toxic effects. To date, a successful strategy to prevent statin toxicity is lacking. Experimental approach: In human monocytic THP-1 cells, we measured the synthesis of cholesterol and isoprenoids, mitochondrial electron flow, the activity of RhoA and Rac, cell death and proliferation. Key results: Mevastatin reduced the synthesis of cholesterol, geranylgeranyl pyrophosphate and ubiquinone, mitochondrial electron transport, activity of RhoA and Rac, and cell proliferation, accompanied by increased cell death. Geranylgeraniol, a cell-permeable analogue of geranylgeranyl pyrophosphate, reversed all these effects of mevastatin, without affecting its ability to reduce cholesterol synthesis. Notably, geranylgeraniol was more effective than the addition of exogenous ubiquinone, which rescued mitochondrial respiratory activity and reversed mevastatin cytotoxicity, but did not alter the decrease in cell proliferation. The same results were obtained in human liver HepG2 cells. Conclusions and implications: Geranylgeraniol had a broader protective effect against the cytotoxicity of statins than exogenous ubiquinone. Therefore, geranylgeraniol may be a more useful and practical means of limiting the toxicities of statins, without reducing their efficacy as cholesterol lowering agents.

// Iris Chiara Salaroglio 1, * , Ivana Campia 1, * , Joanna Kopecka 1 , Elena Gazzano 1 , Sara Orecchia 2 , Dario Ghigo 1 , Chiara Riganti 1 1 Department of Oncology, University of Torino, Italy 2 S.C. Anatomia Patologica, Azienda Ospedaliera S.S. Antonio e Biagio, Alessandria, Italy * These authors have contributed equally to this work Correspondence to: Dario Ghigo, e-mail: dario.ghigo@unito.it Keywords: malignant mesothelioma, chemoresistance, immunosuppression, indoleamine 1,2 dioxygenase, zoledronic acid Received: August 28, 2014 Accepted: November 11, 2014 Published: December 31, 2014 ABSTRACT 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.

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., Oncotarget, 6 (9), ISSN:1949-2553

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-1β, 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.

BACKGROUND: Treatment of fludarabine-resistant chronic lymphocytic leukemia (CLL) patients is an unmet clinical need. Fludarabine resistance in CLL depends on intrinsic molecular features of the tumor cells, and on bidirectional interactions occurring between CLL cells and stromal cells (SC) of the tumor microenvironment. One of the main players of SC-induced fludarabine resistance is the CXCL12/CXCR4 axis. CXCR4 is a G protein-coupled receptor constitutively expressed on CLL cells. The binding of CXCR4 with CXCL12 activates the Ras/ERK1-2/Akt and the RhoA-dependent signalling pathways. To be active transducers Ras and RhoA need to undergo a post-translational modification (i.e. isoprenylation) by means of small molecules produced by the mevalonate (Mev) pathway. We have recently demonstrated that the Mev pathway is more active in IGHV unmutated than in mutated CLL cells, and is amenable to pharmacological manipulation by statins (i.e. simvastatin [Sim]). It is currently unknown whether the Mev pathway and its pharmacological targeting are implicated in the modulation of the CXCL12/CXCR4 axis and in the SC-induced fludarabine resistance of CLL cells. AIM: The aim of this study was to investigate the reciprocal interactions between the Mev pathway and the CXCL12/CXCR4 axis, in order to identify potential targets to counteract the constitutive and SC-induced fludarabine resistance of CLL cells. METHODS: Immuno-magnetically purified patient-derived CLL cells were cultured alone or with murine SC (M2-10B4 cell line). In selected experiments, cell cultures were exposed to human recombinant CXCL12 (100 μg/ml), CXCR4 inhibitor AMD3100 (5 μg/ml), fludarabine (F-ara-A, 10 μM), Sim (1 μM), ERK1-2 kinase inhibitor PD98059 (10 μM), RhoA kinase inhibitor Y276 (10 μM), HIF-1α inhibitor YC-1 (10 μM). The activity of the Mev pathway was measured by the quantification of metabolites [i.e. cholesterol and farnesyl pyrophosphate (FPP)] produced by CLL cells after 24 h incubation with 1 μCi of [3H]acetate. Ras and RhoA activities were evaluated measuring their GTP-bound fraction, taken as an index of the G-protein activation, respectively by pull-down assay and by an ELISA based assay. ERK1-2 and HIF-1α phosphorylation were evaluated by Western Blot. RhoA kinase, Akt and HIF-1α activities were measured with specific immunoassay kit. The amount of CXCL12 in culture supernatants was assessed by ELISA assay. Cell viability was determined by Annexin-V/propidium Iodide immunostaining and flow cytometry analysis. RESULTS: Co-culture with SC upregulated the Mev pathway activity of CLL cells, as shown by the increased production of cholesterol and FPP. This SC-induced increase in the Mev pathway activity was followed by the activation of the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling, the upregulation of the pro-survival factor Akt, and an increase in the transcriptional activity of HIF-1α. These biological and molecular effects were identically observed when CLL cells were exposed to recombinant CXCL12, and were completely abrogated by the CXCR4 antagonist AMD3100, thus showing the key role of the CXCL12/CXCR4 axis in the SC-induced modulation of the Mev pathway and the downstream Ras/ERK1-2 and RhoA/RhoA kinase signalling. On the other hand, blocking the Mev pathway by Sim and targeting ERK1-2 kinases, RhoA kinase and HIF-1α by specific small-molecule inhibitors significantly reduced the constitutive activity and the SC-induced upregulation of the Ras/ERK1-2 and RhoA/RhoA kinase signal transduction. A regulatory role of the Mev pathway on the CXCL12/CXCR4 axis was observed not only in CLL cells but also in SC, as shown by the significant reduction in CXCL12 secretion by SC exposed to Sim. In the last set of experiments, we found that the inhibition of the Mev pathway by Sim potentiated the direct cytotoxic effect of fludarabine against CLL cells. Even more importantly, both Sim and the downstream HIF-1α inhibitor YC-1 were capable of counteracting the SC-mediated protection of CLL cells from fludarabine-induced cytotoxicity. CONCLUSIONS: Our data demonstrate that the Mev pathway has a regulatory role on the CXCL12/CXCR4 axis and on the SC-mediated protective effects toward spontaneous and fludarabine-induced CLL cell death. The upstream inhibition of the Mev pathway and the downstream targeting of HIF-1α are promising strategies to circumvent fludarabine resistance in CLL. Disclosures Boccadoro: Janssen-Cilag: Consultancy, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Rho GTPases, which control processes such as cell proliferation and cytoskeleton remodeling, are often hyperexpressed in tumors. Several members, such as RhoA/B/C, must be isoprenylated to interact with their effectors. Statins, by inhibiting the synthesis of prenyl groups, may affect RhoA/B/C activity and represent a promising tool in anticancer therapy.

Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/?-catenin signaling, and reduces the binding of ?-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma. © 2013 Springer Basel.

The blood-brain barrier (BBB), the unusual microvascular endothelial interface between the central nervous system (CNS) and the circulatory system, is a major hindrance to drug delivery in the brain parenchyma. Besides the absence of fenestrations and the abundance of tight junctions, ATP-binding cassette (ABC) transporters critically reduce drug entry within the CNS, as they carry many drugs back into the bloodstream. Nanoparticle-and liposome-carried drugs, because of their increased cellular uptake and reduced efflux through ABC transporters, have been developed in recent times to circumvent the low drug permeability of the BBB. This review discusses the role of ABC transporters in controlling drug penetration into the brain parenchyma, the rationale for using nanoparticle-and liposome-based strategies to increase drug delivery across the BBB and new therapeutic strategies for using nanoparticle-and liposome-carried drugs in different conditions, ranging from CNS tumors and neurodegenerative diseases to viral infections and epilepsy. © 2013 Bentham Science Publishers.

In previous studies, we showed that nitric oxide (NO) donors and synthetic doxorubicins (DOXs) modified with moieties containing NO-releasing groups--such as nitrooxy-DOX (NitDOX) or 3-phenylsulfonylfuroxan-DOX (FurDOX)--overcome drug resistance by decreasing the activity of ATP-binding cassette (ABC) transporters that can extrude the drug. Here, we have investigated the biochemical mechanisms by which NitDOX and FurDOX exert antitumor effects. Both NitDOX and FurDOX were more cytotoxic than DOX against drug-resistant cells. Interestingly, NitDOX exhibited a faster uptake and an extranuclear distribution. NitDOX was preferentially localized in the mitochondria, where it nitrated and inhibited the mitochondria-associated ABC transporters, decreased the flux through the tricarboxylic acid cycle, slowed down the activity of complex I, lowered the synthesis of ATP, induced oxidative and nitrosative stress, and elicited the release of cytochrome c and the activation of caspase-9 and -3 in DOX-resistant cells. We suggest that NitDOX may represent the prototype of a new class of multifunctional anthracyclines, which have cellular targets different from conventional anthracyclines and greater efficacy against drug-resistant tumors.

Durable tumor cell eradication by chemotherapy is challenged by the development of multidrug-resistance (MDR) and the failure to induce immunogenic cell death. The aim of this work was to investigate whether MDR and immunogenic cell death share a common biochemical pathway eventually amenable to therapeutic intervention. We found that mevalonate pathway activity, Ras and RhoA protein isoprenylation, Ras- and RhoA-downstream signalling pathway activities, Hypoxia Inducible Factor-1alpha activation were significantly higher in MDR+ compared with MDR- human cancer cells, leading to increased P-glycoprotein expression, and protection from doxorubicin-induced cytotoxicity and immunogenic cell death. Zoledronic acid, a potent aminobisphosphonate targeting the mevalonate pathway, interrupted Ras- and RhoA-dependent downstream signalling pathways, abrogated the Hypoxia Inducible Factor-1alpha-driven P-glycoprotein expression, and restored doxorubicin-induced cytotoxicity and immunogenic cell death in MDR+ cells. Immunogenic cell death recovery was documented by the ability of dendritic cells to phagocytise MDR+ cells treated with zoledronic acid plus doxorubicin, and to recruit anti-tumor cytotoxic CD8+ T lymphocytes. These data indicate that MDR+ cells have an hyper-active mevalonate pathway which is targetable with zoledronic acid to antagonize their ability to withstand chemotherapy-induced cytotoxicity and escape immunogenic cell death.

Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition of Na+/K+-ATPase. We showed previously that in human hepatoma cells, digoxin and ouabain increase the rate of the mevalonate cascade and therefore have Na+/K+-ATPase-independent effects. In the present study we found that they increase the expression and activity of 3-hydroxy-3 methylglutaryl-CoA reductase and the synthesis of cholesterol in cardiomyocytes, their main target cells. Surprisingly this did not promote intracellular cholesterol accumulation. The glycosides activated the liver X receptor transcription factor and increased the expression of ABCA1 (ATP-binding cassette protein A1) transporter, which mediates the efflux of cholesterol and its delivery to apolipoprotein A-I. By increasing the synthesis of ubiquinone, another derivative of the mevalonate cascade, digoxin and ouabain simultaneously enhanced the rate of electron transport in the mitochondrial respiratory chain and the synthesis of ATP. Mice treated with digoxin showed lower cholesterol and higher ubiquinone content in their hearts, and a small increase in their serum HDL (high-density lipoprotein) cholesterol. The results of the present study suggest that cardioactive glycosides may have a role in the reverse transport of cholesterol and in the energy metabolism of cardiomyocytes.

Abstract 3881 Background: The mutational status of tumor immunoglobulin heavy chain variable region (IGHV) is a reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. The tumor microenvironment actively supports the survival of CLL cells and confers a multidrug resistance (MDR) phenotype to CLL cells. MDR is due to the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. Pgp is under the positive control of the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α) which is activated by isoprenylated Ras/Rho-dependent downstream signaling pathways. Ras and Rho isoprenylation are regulated by the mevalonate (Mev) pathway activity suggesting that this pathway can be exploited as a metabolic checkpoint to regulate chemresistance. Aim: The aim of this study was twofold: 1) to investigate the correlation between chemoresistance and the activity of the Mev pathway and Ras/Rho-A downstream signaling pathways in purified M and UM CLL cells under basal conditions and after incubation with stromal cells; 2) to evaluate the chemosensitizing effects of agents specifically targeting the Mev pathway and downstream signaling pathways under the same culture conditions. Methods: M and UM CLL cells were cultured in the presence and in the absence of murine stromal cells (M210B4) and exposed to Zoledronic acid (ZA) (1 μmol/L), Simvastatine (Sim) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L), HIF-1α inhibitor YC-1 (10 μmol/L) and Doxorubicine (Doxo) (1 μmol/L). The Mev pathway activity was measured by cells radiolabelling with [14C]-mevalonic acid and thin layer chromatography. Ras, ERK1/2 and Akt activity were detected by Western blot. Rho, Rho Kinase and HIF-1α activity were assessed by ELISA. Mdr1 expression was measured by Real Time-PCR. PgP activity was evaluated by measuring Doxo intracellular accumulation. Doxo cytotoxicity was assessed by annexin V and propidium iodide staining. Results: The Mev pathway is significantly more active in UM than in M CLL cells. This hypermetabolic activity translates into a higher activation of Ras/Akt and Rho/Rho kinase signaling pathways and higher expression of the phosphorylated active form of HIF-1α. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to a more effective Doxo extrusion and therefore better survival upon Doxo exposure. M210B4 stromal cells further protect UM CLL cells from Doxo induced cell death by upregulating Mev pathway activity, HIF-1α/mdr1/PgP axis activation, and Doxo extrusion. Targeting the Mev pathway of UM cells with ZA and Mev reduces the basal activity of HIF-1α/mdr1/PgP axis and significantly increases Doxo retention and cytotoxicity. Similar effects are obtained with PD85 and YC1–10 which are specific inhibitors of the downstream molecules ERK-1/2 and HIF-1α, respectively. All these agents are able to overcome the protective effect exerted by stromal cells by significantly increasing PgP activity and Doxo-induced cell death. Conclusions: Our data demonstrate that the Ras- and Rho-dependent HIF-1α/mdr1/PgP axis is more active and associated with higher levels of MDR in UM compared with M CLL cells. Targeting the Mev pathway and/or downstream signalling pathways is a promising strategy to circumvent basal and stroma-mediated chemoresistance especially in UM CLL cells. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding.

Cardioactive glycosides, like digoxin, ouabain and related compounds, are drugs that inhibit Na(+)/K(+)-ATPase and have a strong inotropic effect on heart: they cause the Na(+)/Ca(2+) exchanger to extrude Na+ in exchange with Ca(2+) and therefore increase the [Ca(2+)](i) concentration. For this reason, some of these drugs are currently used in the treatment of congestive heart failure and cardiac arrhythmias. Recently it has been discovered that cardiac glycosides exert pleiotropic effects on many aspects of cell metabolism. Na(+)/K(+)-ATPase is not the exclusive target, as they affect the cell response to hypoxia, modulate several signaling pathways involved in cell death and proliferation, regulate the transcription of different genes and modify the pharmacokinetics of other drugs, by altering the expression and activity of drug-metabolizing enzymes. Some of these effects are related to the steroid structure of glycosides, a property which also makes them fine modulators of the synthesis of cholesterol and steroid hormones. Moreover, new endogenously synthesized glycosides have been discovered in the last years: these molecules are involved in the balance of salt and in the control of blood pressure. This review will focus on the recent studies which have demonstrated that exogenous and endogenous glycosides, besides playing a role as inotropic agents, are also important in the pathogenesis and therapy of different human diseases, such as stroke, diabetes, neurological diseases and cancer.

Doxorubicin is one of the most employed anticancer drugs, but its efficacy is limited by the onset of adverse effects such as drug resistance, due to the drug efflux via P-glycoprotein (Pgp). Several factors are associated to a high Pgp activity, including the amount of cholesterol in plasma membrane, which is essential to maintain the pump function. In this work we started from the following observations: 1) the drug-resistant colon cancer HT29-dx cells had a higher content of cholesterol in plasma membrane than drug-sensitive HT29 cells and a higher activity of Pgp, which was decreased by the cholesterol-lowering agent β-methyl-cyclodextrin; 2) HT29-dx cells showed a higher synthesis of endogenous cholesterol and a higher expression of the low-density lipoprotein receptor (LDLR); 3) the anti-cholesterolemic drug simvastatin reduced the cholesterol synthesis, increased the synthesis of LDLR and lowered the Pgp activity in resistant cells. In order to circumvent drug resistance we designed a new liposomal doxorubicin, conjugated with a recombinant LDLR-binding peptide from human apoB100: this LDL-masked doxorubicin ("apo-Lipodox") was efficiently internalized by a LDLR-driven endocytosis and induced cytotoxic effects in HT29-dx cells, reversing their drug resistance. Its efficacy was further increased by simvastatin, which up-regulates the LDLR levels and contemporarily reduces the Pgp activity, thus increasing the liposomes uptake and limiting the drug efflux. We propose that the association of liposomal doxorubicin and statins may be a future promising strategy to reverse drug-resistance in human cancer cells.

Digoxin and ouabain are cardioactive glycosides, which inhibit the Na+/K+-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca2+](i)). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca2+](i) and this event was dependent on the calcium influx via the Na+/Ca2+ exchanger. The increased [Ca2+](i) enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na+/Ca2+ exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.

Digoxin and ouabain are steroid drugs that inhibit the Na(+)/K(+)-ATPase, and are widely used in the treatment of heart diseases. They may also have additional effects, such as on metabolism of steroid hormones, although until now no evidence has been provided about the effects of these cardioactive glycosides on the synthesis of cholesterol. Here we report that digoxin and ouabain increased the synthesis of cholesterol in human liver HepG2 cells, enhancing the activity and the expression of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), the rate-limiting enzyme of the cholesterol synthesis. This effect was mediated by the binding of the sterol regulatory element binding protein-2 (SREBP-2) to the HMGCR promoter, and was lost in cells silenced for SREBP-2 or loaded with increasing amounts of cholesterol. Digoxin and ouabain competed with cholesterol for binding to the SREBP-cleavage-activating protein, and are critical regulators of cholesterol synthesis in human liver cells.

NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive nitrogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism's homeostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabetes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopathological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two compounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role of NOXs.

Abstract 735 The mutational status of the tumor immunoglobulin heavy chain variable region (IGHV) is a very reliable prognosticator in chronic lymphocytic leukemia (CLL): patients with unmutated (UM) IGHV have a worse prognosis than patients with mutated (M) IGHV. We have recently shown that UM CLL cells are more dependent on microenvironment-mediated signals for survival (Coscia M. et al, Leukemia, 2011), but it is currently unknown whether this also translates into an enhanced chemotherapy resistance. Multi drug resistance (MDR) is mediated by the over-expression of membrane transporters, like P-glycoprotein (Pgp), which actively extrudes several anticancer drugs. The Pgp protein is the product of the mdr1 gene whose regulation is under the positive control of the mevalonate (Mev) metabolic pathway, the downstream Ras/RhoA dependent signaling pathways and the transcription factor Hypoxia-Inducible-Factor-1-alfa (HIF-1α). The aim of this study was to investigate the MDR phenotype and signaling cascade in M and UM CLL cells under basal conditions and after exposure to prosurvival microenvironment-mediated signals. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from untreated CLL patients. More than 90% pure CLL cells were co-cultured with the murine stromal cell line M2-10B4. Culture conditions included: medium alone, Zoledronic acid (ZA) (1 μmol/L), ERK1/2 kinase inhibitor PD98059 (10 μmol/L) and the HIF-1α inhibitor YC-1 (10 μmol/L). The level of activity of the Mev pathway was evaluated by measuring the intermediate metabolite farnesyl pyrophosphate (FPP) and the final product cholesterol (Chol) as previously described (Coscia M. et al, J Cell Mol Med, 2010). The Ras and RhoA activity, was evaluated by measuring the GTP binding. Rho kinase activity was measured using the CycLex Rho Kinase Assay Kit (CycLex Co., Nagano). The following antibodies were used for Western Blot (WB) analyses: anti phospho-(Thr202/Tyr204, Thr185/Tyr187)-ERK1/2; anti-ERK 1/2; anti-HIF-1α; anti-Pgp; anti-GAPDH. The activity of HIF-1 in the nuclear extracts was assessed by the TransAM™ HIF-1 Transcription Factor Assay Kit (Active Motif). Mdr1 gene expression was evaluated by RT-PCR. Intracellular Doxorubicine (Doxo) accumulation was evaluated by cytofluorimetric analyses. We have found that the Mev pathway is significantly accelerated in freshly isolated UM vs M CLL cells. This hypermetabolic activity translates into a higher activation of the Ras/ERK-1/2 and RhoA/Rho kinase signaling pathways, and to a significantly more active HIF-1α in UM compared to M CLL cells. HIF-1α activation positively regulates mdr1 gene expression in UM CLL cells leading to Pgp expression and more effective Doxo extrusion in these cells. The co-culture of CLL cells with the murine stromal cell line M2-10B4 further accelerated the Mev pathway and activated the HIF-1α/mdr1/PgP axis leading to even lower intracellular Doxo retention. This stroma-mediated effect was particularly evident in UM CLL cells. Targeting of the Mev pathway with ZA and statins, and targeting of ERK-1/2 and HIF-1α with specific inhibitors (PD85 and YC1-10, respectively) significantly reduced basal and stroma-induced activity of HIF-1α/mdr1/PgP axis, and significantly increased the intracellular concentrations of Doxo in UM CLL cells. These data indicate that the pharmacological inhibition of the Mev pathway and of the downstream HIF-1α/mdr1/PgP axis can be regarded as a novel potential strategy to circumvent basal and environment-mediated chemoresistance of CLL cells. Disclosures: Massaia: Novartis Farma S.p.A: Honoraria, Research Funding, scientific adviser board.

Background The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. Methods We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. Results MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells. Conclusions Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.