Joanna Kopecka

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition

BackgroundThe 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.MethodsWe 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.ResultsMDR 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.ConclusionsOur work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Nanoparticle- and liposome-carried drugs: new strategies for active targeting and drug delivery across blood-brain barrier.

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.

iNOS activity is necessary for the cytotoxic and immunogenic effects of doxorubicin in human colon cancer cells

BackgroundDoxorubicin is one of the few chemotherapeutic drugs able to exert both cytotoxic and pro-immunogenic effects against cancer cells. Following the drug administration, the intracellular protein calreticulin is translocated with an unknown mechanism onto the plasma membrane, where it triggers the phagocytosis of tumour cells by dendritic cells. Moreover doxorubicin up-regulates the inducible nitric oxide (NO) synthase (iNOS) gene in cancer cells, leading to huge amounts of NO, which in turn acts as a mediator of the drug toxicity and as a chemosensitizer agent in colon cancer. Indeed by nitrating tyrosine on the multidrug resistance related protein 3, NO decreases the doxorubicin efflux from tumour cells and enhances the drug toxicity. It is not clear if NO, beside playing a role in chemosensitivity, may also play a role in doxorubicin pro-immunogenic effects. To clarify this issue, we compared the doxorubicin-sensitive human colon cancer HT29 cells with the drug-resistant HT29-dx cells and the HT29 cells silenced for iNOS (HT29 iNOS-).ResultsIn both HT29-dx and HT29 iNOS- cells, doxorubicin did not induce NO synthesis, had a lower intracellular accumulation and a lower toxicity. Moreover the drug failed to promote the translocation of calreticulin and the phagocytosis of HT29-dx and HT29 iNOS-cells, which resulted both chemoresistant and immunoresistant. However, if NO levels were exogenously increased by sodium nitroprusside, the chemosensitivity to doxorubicin was restored in HT29 iNOS-cells. In parallel the NO donor per se was sufficient to induce the exposure of calreticulin and to increase the phagocytosis of HT29 iNOS- cells by DCs and their functional maturation, thus mimicking the pro-immunogenic effects exerted by doxorubicin in the parental drug-sensitive HT29 cells.ConclusionOur data suggest that chemo- and immuno-resistance to anthracyclines are associated in colon cancer cells and rely on a common mechanism, that is the inability of doxorubicin to induce iNOS. Therefore NO donors might represent a promising strategy to restore both chemosensitivity and immunosensitivity to doxorubicin in resistant cells.

Mitochondrial-Targeting Nitrooxy-doxorubicin: A New Approach To Overcome Drug Resistance

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.

Modulation of doxorubicin resistance by the glucose-6-phosphate dehydrogenase activity.

How anti-neoplastic agents induce MDR (multidrug resistance) in cancer cells and the role of GSH (glutathione) in the activation of pumps such as the MRPs (MDR-associated proteins) are still open questions. In the present paper we illustrate that a doxorubicin-resistant human colon cancer cell line (HT29-DX), exhibiting decreased doxorubicin accumulation, increased intracellular GSH content, and increased MRP1 and MRP2 expression in comparison with doxorubicin-sensitive HT29 cells, shows increased activity of the PPP (pentose phosphate pathway) and of G6PD (glucose-6-phosphate dehydrogenase). We observed the onset of MDR in HT29 cells overexpressing G6PD which was accompanied by an increase in GSH. The G6PD inhibitors DHEA (dehydroepiandrosterone) and 6-AN (6-aminonicotinamide) reversed the increase of G6PD and GSH and inhibited MDR both in HT29-DX cells and in HT29 cells overexpressing G6PD. In our opinion, these results suggest that the activation of the PPP and an increased activity of G6PD are necessary to some MDR cells to keep the GSH content high, which is in turn necessary to extrude anticancer drugs out of the cell. We think that our data provide a new further mechanism for GSH increase and its effects on MDR acquisition.

Zoledronic Acid Restores Doxorubicin Chemosensitivity and Immunogenic Cell Death in Multidrug-Resistant Human Cancer Cells

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.

Temozolomide downregulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/glycogen synthase-3 kinase/β-catenin pathway

BACKGROUND Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis is poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. METHODS A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, was used. RESULTS Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently noncytotoxic doses of temozolomide, which chemosensitized glioblastoma cells to doxorubicin, vinblastine, and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to an investigation of whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. Temozolomide-treated cells had increased methylation of the cytosine-phosphate-guanine islands in the Wnt3a gene promoter, decreased expression of Wnt3a, disrupted glycogen synthase-3 kinase/β-catenin axis, reduced transcriptional activation of ABCB1, and a lower amount and activity of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary, glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and ABCB1 levels. CONCLUSIONS Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation, and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cell population by downregulating Wnt3a signaling.

Temozolomide down-regulates P-glycoprotein in human blood–brain barrier cells by disrupting Wnt3 signaling

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/β-catenin signaling, and reduces the binding of β-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.

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)/β-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 β-catenin. By contrast, the inhibition of RhoA/RhoAK axis prevented the activation of PTP1B, enhanced the GSK3-induced phosphorylation and ubiquitination of β-catenin, and reduced the β-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.

Sigma-2 receptor and progesterone receptor membrane component 1 (PGRMC1) are two different proteins: Proofs by fluorescent labeling and binding of sigma-2 receptor ligands to PGRMC1

Graphical abstract Figure. No Caption available. Abstract A controversial relationship between sigma‐2 and progesterone receptor membrane component 1 (PGRMC1) proteins, both representing promising targets for the therapy and diagnosis of tumors, exists since 2011, when the sigma‐2 receptor was reported to be identical to PGRMC1. Because a misidentification of these proteins will lead to biased future research hampering the possible diagnostic and therapeutic exploitation of the two targets, there is the need to solve the debate on their identity. With this aim, we have herein investigated uptake and distribution of structurally different fluorescent sigma‐2 receptor ligands by flow cytometry and confocal microscopy in MCF7 cells, where together with intrinsic sigma‐2 receptors, PGRMC1 was constitutively present or alternatively silenced or overexpressed. HCT116 cells, with constitutive or silenced PGRMC1, were also studied. These experiments showed that the fluorescent sigma‐2 ligands bind to their receptor irrespective of PGRMC1 expression. Furthermore, isothermal titration calorimetry was conducted to examine if DTG and PB28, two structurally distinct nanomolar affinity sigma‐2 ligands, bind to purified PGRMC1 proteins that have recently been revealed to form both apo‐monomeric and heme‐mediated dimeric forms. While no binding to apo‐PGRMC1 monomer was detected, a micromolar affinity to heme‐mediated dimerized PGRMC1 was demonstrated in DTG but not in PB28. The current data provide evidence that sigma‐2 receptor and PGRMC1 are not identical, paving the pathway for future unbiased research in which these two attractive targets are treated as different proteins while the identification of the true sigma‐2 protein further needs to be pursued.