The Phosphodiesterase-5 Inhibitor Vardenafil Is a Potent Inhibitor of ABCB1/P-Glycoprotein Transporter

One of the major causes of chemotherapy failure in cancer treatment is multidrug resistance (MDR) which is mediated by the ABCB1/P-glycoprotein. Previously, through the use of an extensive screening process, we found that vardenafil, a phosphodiesterase 5 (PDE-5) inhibitor significantly reverses MDR in ABCB1 overexpressing cancer cells, and its efficacy was greater than that of tadalafil, another PDE-5 inhibitor. The present study was designed to determine the reversal mechanisms of vardenafil and tadalafil on ABC transporters-mediated MDR. Vardenafil or tadalafil alone, at concentrations up to 20 µM, had no significant toxic effects on any of the cell lines used in this study, regardless of their membrane transporter status. However, vardenafil when used in combination with anticancer substrates of ABCB1, significantly potentiated their cytotoxicity in ABCB1 overexpressing cells in a concentration-dependent manner, and this effect was greater than that of tadalafil. The sensitivity of the parenteral cell lines to cytotoxic anticancer drugs was not significantly altered by vardenafil. The differential effects of vardenafil and tadalafil appear to be specific for the ABCB1 transporter as both vardenafil and tadalafil had no significant effect on the reversal of drug resistance conferred by ABCC1 (MRP1) and ABCG2 (BCRP) transporters. Vardenafil significantly increased the intracellular accumulation of [3H]-paclitaxel in the ABCB1 overexpressing KB-C2 cells. In addition, vardenafil significantly stimulated the ATPase activity of ABCB1 and inhibited the photolabeling of ABCB1 with [125I]-IAAP. Furthermore, Western blot analysis indicated the incubation of cells with either vardenafil or tadalafil for 72 h did not alter ABCB1 protein expression. Overall, our results suggest that vardenafil reverses ABCB1-mediated MDR by directly blocking the drug efflux function of ABCB1

Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE

TTFields alone and in combination with chemotherapeutic agents effectively reduce the viability of MDR cell sub-lines that over-express ABC transporters

<p>Abstract</p> <p>Background</p> <p>Exposure of cancer cells to chemotherapeutic agents may result in reduced sensitivity to structurally unrelated agents, a phenomenon known as multidrug resistance, MDR. The purpose of this study is to investigate cell growth inhibition of wild type and the corresponding MDR cells by Tumor Treating Fields - TTFields, a new cancer treatment modality that is free of systemic toxicity. The TTFields were applied alone and in combination with paclitaxel and doxorubicin.</p> <p>Methods</p> <p>Three pairs of wild type/MDR cell lines, having resistivity resulting from over-expression of ABC transporters, were studied: a clonal derivative (C11) of parental Chinese hamster ovary AA8 cells and their emetine-resistant sub-line Emt^R1; human breast cancer cells MCF-7 and their mitoxantrone-resistant sub lines MCF-7/Mx and human breast cancer cells MDA-MB-231 and their doxorubicin resistant MDA-MB-231/Dox cells. TTFields were applied for 72 hours with and without the chemotherapeutic agents. The numbers of viable cells in the treated cultures and the untreated control groups were determined using the XTT assay. Student t-test was applied to asses the significance of the differences between results obtained for each of the three cell pairs.</p> <p>Results</p> <p>TTFields caused a similar reduction in the number of viable cells of wild type and MDR cells. Treatments by TTFields/drug combinations resulted in a similar increased reduction in cell survival of wild type and MDR cells. TTFields had no effect on intracellular doxorubicin accumulation in both wild type and MDR cells.</p> <p>Conclusions</p> <p>The results indicate that TTFields alone and in combination with paclitaxel and doxorubicin effectively reduce the viability of both wild type and MDR cell sub-lines and thus can potentially be used as an effective treatment of drug resistant tumors.</p

Towards the clinical implementation of pharmacogenetics in bipolar disorder.

BackgroundBipolar disorder (BD) is a psychiatric illness defined by pathological alterations between the mood states of mania and depression, causing disability, imposing healthcare costs and elevating the risk of suicide. Although effective treatments for BD exist, variability in outcomes leads to a large number of treatment failures, typically followed by a trial and error process of medication switches that can take years. Pharmacogenetic testing (PGT), by tailoring drug choice to an individual, may personalize and expedite treatment so as to identify more rapidly medications well suited to individual BD patients.DiscussionA number of associations have been made in BD between medication response phenotypes and specific genetic markers. However, to date clinical adoption of PGT has been limited, often citing questions that must be answered before it can be widely utilized. These include: What are the requirements of supporting evidence? How large is a clinically relevant effect? What degree of specificity and sensitivity are required? Does a given marker influence decision making and have clinical utility? In many cases, the answers to these questions remain unknown, and ultimately, the question of whether PGT is valid and useful must be determined empirically. Towards this aim, we have reviewed the literature and selected drug-genotype associations with the strongest evidence for utility in BD.SummaryBased upon these findings, we propose a preliminary panel for use in PGT, and a method by which the results of a PGT panel can be integrated for clinical interpretation. Finally, we argue that based on the sufficiency of accumulated evidence, PGT implementation studies are now warranted. We propose and discuss the design for a randomized clinical trial to test the use of PGT in the treatment of BD

The boron-oxygen core of borinate esters is responsible for the store-operated calcium entry potentiation ability

International audienceBACKGROUND: Store-Operated Calcium Entry (SOCE) is the major Ca2+ ion entry pathway in lymphocytes and is responsible of a severe combined immunodeficiency (SCID) when deficient. It has recently been observed or highlighted in other cell types such as myoblasts and neurons, suggesting a wider physiological role of this pathway. Whereas Orai1 protein is considered to be the channel allowing the SOCE in T cells, it is hypothesized that other proteins like TRPC could associate with Orai1 to form SOCE with different pharmacology and kinetics in other cell types. Unraveling SOCE cell functions requires specific effectors to be identified, just as dihydropyridines were crucial for the study of Ca2+ voltage-gated channels, or spider/snake toxins for other ion channel classes. To identify novel SOCE effectors, we analyzed the effects of 2-aminoethyl diphenylborinate (2-APB) and its analogues. 2-APB is a molecule known to both potentiate and inhibit T cell SOCE, but it is also an effector of TRP channels and endoplasmic reticulum Ca2+-ATPase. RESULTS: A structure-function analysis allowed to discover that the boron-oxygen core present in 2-APB and in the borinate ester analogues is absolutely required for the dual effects on SOCE. Indeed, a 2-APB analogue where the boron-oxygen core is replaced by a carbon-phosphorus core is devoid of potentiating capacity (while retaining inhibition capacity), highlighting the key role of the boron-oxygen core present in borinate esters for the potentiation function. However, dimesityl borinate ester, a 2-APB analogue with a terminal B-OH group showed an efficient inhibitory ability, without any potentiating capacity. The removal or addition of phenyl groups respectively decrease or increase the efficiency of the borinate esters to potentiate and inhibit the SOCE. mRNA expression revealed that Jurkat T cells mainly expressed Orai1, and were the more sensitive to 2-APB modulation of SOCE. CONCLUSIONS: This study allows the discovery of new boron-oxygen core containing compounds with the same ability as 2-APB to both potentiate and inhibit the SOCE of different leukocyte cell lines. These compounds could represent new tools to characterize the different types of SOCE and the first step in the development of new immunomodulators

Single-step doxorubicin-selected cancer cells overexpress the ABCG2 drug transporter through epigenetic changes

Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines

The art of cellular communication: tunneling nanotubes bridge the divide

The ability of cells to receive, process, and respond to information is essential for a variety of biological processes. This is true for the simplest single cell entity as it is for the highly specialized cells of multicellular organisms. In the latter, most cells do not exist as independent units, but are organized into specialized tissues. Within these functional assemblies, cells communicate with each other in different ways to coordinate physiological processes. Recently, a new type of cell-to-cell communication was discovered, based on de novo formation of membranous nanotubes between cells. These F-actin-rich structures, referred to as tunneling nanotubes (TNT), were shown to mediate membrane continuity between connected cells and facilitate the intercellular transport of various cellular components. The subsequent identification of TNT-like structures in numerous cell types revealed some structural diversity. At the same time it emerged that the direct transfer of cargo between cells is a common functional property, suggesting a general role of TNT-like structures in selective, long-range cell-to-cell communication. Due to the growing number of documented thin and long cell protrusions in tissue implicated in cell-to-cell signaling, it is intriguing to speculate that TNT-like structures also exist in vivo and participate in important physiological processes

The Drosophila TRPP Cation Channel, PKD2 and Dmel/Ced-12 Act in Genetically Distinct Pathways during Apoptotic Cell Clearance

Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway

BCRP expression does not result in resistance to STX140 in vivo, despite the increased expression of BCRP in A2780 cells in vitro after long-term STX140 exposure

The anti-proliferative and anti-angiogenic properties of the endogenous oestrogen metabolite, 2-methoxyoestradiol (2-MeOE2), are enhanced in a series of sulphamoylated derivatives of 2-MeOE2. To investigate possible mechanisms of resistance to these compounds, a cell line, A2780.140, eightfold less sensitive to the 3,17-O,O-bis-sulphamoylated derivative, STX140, was derived from the A2780 ovarian cancer cell line by dose escalation. Other cell lines tested did not develop STX140 resistance. RT–PCR and immunoblot analysis demonstrated that breast cancer resistance protein (BCRP) expression is dramatically increased in A2780.140 cells. The cells are cross-resistant to the most structurally similar bis-sulphamates, and to BCRP substrates, mitoxantrone and doxorubicin; but they remain sensitive to taxol, an MDR1 substrate, and to all other sulphamates tested. Sensitivity can be restored using a BCRP inhibitor, and this pattern of resistance is also seen in a BCRP-expressing MCF-7-derived cell line, MCF-7.MR. In mice bearing wild-type (wt) and BCRP-expressing tumours on either flank, both STX140 and mitoxantrone inhibited the growth of the MCF-7wt xenografts, but only STX140 inhibited growth of the MCF-7.MR tumours. In conclusion, STX140, a promising orally bioavailable anti-cancer agent in pre-clinical development, is highly efficacious in BCRP-expressing xenografts. This is despite an increase in BCRP expression in A2780 cells in vitro after chronic dosing with STX140