Pyrvinium Research Articles

DiskImageR: quantification of resistance and tolerance to antimicrobial drugs using disk diffusion assays.

Microbial pathogens represent an increasing threat to human health. Although many infections can be successfully treated and cleared, drug resistance is a widespread problem. The existence of subpopulations of 'tolerant' cells (where a fraction of the population is able to grow above the population resistance level) may increase the rate of treatment failure; yet, existing methods to measure subpopulation effects are cumbersome. Here we describe diskImageR, a computational pipeline that analyses photographs of disk diffusion assays to determine the degree of drug susceptibility [the radius of inhibition, (RAD)], and two aspects of subpopulation growth [the fraction of growth (FoG) within the zone of inhibition, (ZOI), and the rate of change in growth from no drug to inhibitory drug concentrations, (SLOPE)]. diskImageR was used to examine the response of the human fungal pathogen Candida albicans to the antifungal drug fluconazole across different strain backgrounds and growth conditions. Disk diffusion assays performed under Clinical and Laboratory Standards Institute (CLSI) conditions led to more susceptibility and less tolerance than assays performed using rich medium conditions. We also used diskImageR to quantify the effects of three drugs in combination with fluconazole, finding that all three combinations affected tolerance, with the effect of one drug (doxycycline) being very strain dependent. The three drugs had different effects on susceptibility, with doxycycline generally having no effect, chloroquine generally increasing susceptibility and pyrvinium pamoate generally reducing susceptibility. The ability to simultaneously quantitate different aspects of microbial drug responses will facilitate the study of mechanisms of subpopulation responses in the presence of antimicrobial drugs.

WNT pathway inhibitor pyrvinium pamoate inhibits the self-renewal and metastasis of breast cancer stem cells.

Acquisition of chemoresistance and metastatic phenotype are the major causes of breast cancer treatment failure and cancer-related mortality. Recently, a plethora of experimental and clinical studies points toward a central role of cancer stem cells (CSCs) in the chemoresistance and metastasis. In the present study, we demonstrated that pyrvinium pamoate (PP), an anthelmintic drug, inhibited proliferation of different subtypes of breast cancer cells (luminal: MCF-7, claudin-low: MDA-MB‑231, basal-like: MDA-MB‑468 and Her-2 enriched: SkBr-3) as a novel WNT pathway inhibitor. Additionally, PP was also shown to inhibit self-renewal of breast cancer stem cells (BCSCs) and decrease both CD44+CD24-/low and ALDH-positive BCSCs content in a panel of breast cancer cell lines. Besides, the metastatic potential and expression of EMT markers (such as N-cadherin, vimentin, Snail) were also found suppressed by PP. By using a xenograft model, we next tested the efficacy of PP on tumorigenicity of MDA-MB‑231, one of the most aggressive breast cancer cell lines, and we observed PP significantly delayed tumor growth in vivo. Moreover, in-depth analysis revealed that PP caused inhibition of WNT pathway activity and stemness regulator expression including NANOG, SOX2 and OCT4, which were inherently upregulated in the BCSCs as compared with the bulk of cells within the tumor. Collectively, our findings provide direct evidence for PP serving as a promising high-yield agent targeting BCSCs and cancer heterogeneity. Therefore, strategies combining PP with standard chemotherapy drugs which fail to eliminate the BCSCs hold promise to overcome BCSCs associated treatment resistance and achieve a better therapeutic outcome.

AR DNA binding domain inhibitors to treat AR splice variant-driven prostate cancer.

293 Background: Nearly all prostate cancer (PC) cells depend on androgen receptor (AR) signaling for growth. Resistance to novel PC drugs like abiraterone and enzalutamide appears to occur through ligand-independent AR signaling, often by expression of AR splice variants (ARv’s) that lack the ligand binding domain (LBD). Thus, drugs that inhibit AR activity via domains other than the LBD may provide a way to inhibit the growth of drug-resistant PC. We discovered the first non-competitive, ligand-independent AR inhibitor, pyrvinium pamoate (PP), which functions by interacting with the DNA-binding domain (DBD) and preventing the recruitment of RNA polymerase II to transcription start sites. PP inhibits AR activity with low nanomolar potency and functions synergistically with competitive antagonists. Here, we undertook experiments to learn more about PP’s mechanism of action and developed an orally available derivative for translation to human studies. Methods: AR immunoprecipitation (IP) followed by mass spec sequencing was used to identify proteins that differentially bind to AR in the presence of PP. Additionally, RNA-seq was used to identify pathways affected by PP treatment. Functional studies of hits were conducted using over-expression and knock down in relevant PC cell lines. PP derivatives were developed through a medicinal chemistry approach. Lead compounds were tested in a battery of assays to determine potency in cultured cells and in vivo, as well as their solubility and pharmicokinetics. Results: RNA-seq results suggest that AR and hormone pathways are the primary target of PP in PC cells but that the splicing machinery is also be affected. Mass spec results also demonstrate that PP affects the interaction between AR and splicing machinery, as well as many transcription and translation-related proteins. Medicinal chemistry efforts led to a molecule with superior aqueous solubility, oral availability, and a half-life > 6hrs. This compound inhibited the activity of AR and ARv’s in cultured cells and in xenograft models. Conclusions: PP was the first identified AR DBD inhibitor and the development of an orally available derivative holds great promise for the treatment of ARv-driven PCs.

Synergistic Targeting of Protein Translation and Inhibition of NOTCH Signaling in T-ALL

Oncogenic NOTCH signaling is a major driver of T-cell transformation in T-cell acute lymphoblastic leukemia (T-ALL). However, clinical studies testing the efficacy of NOTCH1 inactivation with γ-secretase inhibitors (GSIs) have shown limited antileukemic activity for these drugs as single agents. Here we used an expression-based virtual screening approach and network perturbation analyses to identify and functionally characterize new highly active antileukemic drugs synergistic with NOTCH1 inhibition in T-ALL. Gene expression profiling studies have shown a prominent gene expression signature dominated by genes involved in growth and metabolism downstream of NOTCH1 in T-ALL. Notably, loss of the PTEN tumor suppressor gene confers resistance to GSI therapy and effectively rescues the gene expression signature induced by NOTCH1 inhibition in T-ALL. We hypothesized that drugs inducing transcriptional programs overlapping with those driven by NOTCH1 inhibition and antagonizing those resulting from PTEN loss could have synergistic antileukemic effects with GSIs in PTEN wild type and PTEN null leukemia cells. To address this question we generated gene expression signatures from Pten conditional-inducible knockout NOTCH1-driven leukemias in basal condition, upon NOTCH1 inhibition by GSI treatment and upon deletion of Pten. Connectivity Map (cMAP) analysis in this series identified 17 high scoring compounds as candidate antileukemic drugs (p<0.01). Reassuringly these included two inhibitors of the mTOR/PI3K/AKT pathway (rapamycin, wortmannin), but also histone deacetylase inhibitors (vorinostat, trichostatin A and valproic acid), phenothiazine antipsychotic drugs (trifluoperazine and thioridazine), antimalarial agents (astemizole, mefloquine) and compounds with less characterized activities such as withaferin A, parthenolide and pyrvinium pamoate. Transcriptional profiling followed by pairwise gene set enrichment analysis of these compounds identified groups of drugs with highly interconnected transcriptional programs suggestive of an overlapping mechanism of action (e.g. mTOR/PI3K inhibitors, HDAC inhibitors and phenothiazines), as well as compounds with more unique expression signatures suggestive of a more distinct mode of action (e.g. withaferin A, astemizole and mefloquine). Detailed characterization of the antileukemic effects of these 17 cMAP hits alone and in combination with the GSI DBZ in a broad panel of human NOTCH1-mutated T-ALL cell lines, identified withaferin A, rapamycin, wortmannin, parthenolide and vorinostat as the most active (lethal dose 50 <0.5 µM) and GSI-synergistic (combination index <0.4) drugs in this series. Among these, withaferin A, stood out as the most cytotoxic and GSI-synergistic compound against both PTEN positive and PTEN null T-ALL cell lines. Moreover, withaferin A treatment of primary mouse NOTCH1-induced T-ALLs and primary human T-ALL xenografts demonstrated strong and GSI-synergistic antileukemic activity in vivo.To address the mechanisms mediating the antileukemic effects of withaferin A we performed a detailed analysis of the gene expression signatures induced by this drug in T-ALL lymphoblasts. These studies revealed a strong enrichment of downregulated genes involved in translation regulation in T-ALL cells upon treatment with withaferin A (p<0.001). Mechanistically, transcriptional network perturbation analysis identified the eIF2A translation initiation complex as a potential effector of the antileukemic effects of withaferin A, and withaferin A treatment induced strong dose dependent phosphorylation of eIF2S1 in position S51, a modification responsible for blocking the activity of the eIF2A complex. Consistently, polysome profiling and nascent-protein assays revealed decreased translation in T-ALL cells treated with withaferin A. In this context, expression a phosphomimetic mutant form of eIF2S1 (S51D) impaired leukemia cell viability. Moreover, expression of a non-phosphorylatable form of eIF2S1 (eIF2S1 S51A) in T-ALL cells abrogated the antileukemic effects of withaferin A.These results support a direct role of eIF2S1 phosphorylation and the inhibition of eIF2A-dependent translation as a critical mediators of the antileukemic effects of withaferin A in T-ALL and a role for the combination of GSIs and inhibitors of protein translation for the treatment of high risk T-ALL. DisclosuresCalifano:Therasis Inc: Employment; Cancer Genetics Inc: Consultancy; Ipsen pharmaceuticals: Consultancy; Thermo Fischer Scientific: Consultancy.

Three-Dimensional Cell Culture-Based Screening Identifies the Anthelmintic Drug Nitazoxanide as a Candidate for Treatment of Colorectal Cancer.

Because dormant cancer cells in hypoxic and nutrient-deprived regions of solid tumors provide a major obstacle to treatment, compounds targeting those cells might have clinical benefits. Here, we describe a high-throughput drug screening approach, using glucose-deprived multicellular tumor spheroids (MCTS) with inner hypoxia, to identify compounds that specifically target this cell population. We used a concept of drug repositioning-using known molecules for new indications. This is a promising strategy to identify molecules for rapid clinical advancement. By screening 1,600 compounds with documented clinical history, we aimed to identify candidates with unforeseen potential for repositioning as anticancer drugs. Our screen identified five molecules with pronounced MCTS-selective activity: nitazoxanide, niclosamide, closantel, pyrvinium pamoate, and salinomycin. Herein, we show that all five compounds inhibit mitochondrial respiration. This suggests that cancer cells in low glucose concentrations depend on oxidative phosphorylation rather than solely glycolysis. Importantly, continuous exposure to the compounds was required to achieve effective treatment. Nitazoxanide, an FDA-approved antiprotozoal drug with excellent pharmacokinetic and safety profile, is the only molecule among the screening hits that reaches high plasma concentrations persisting for up to a few hours after single oral dose. Nitazoxanide activated the AMPK pathway and downregulated c-Myc, mTOR, and Wnt signaling at clinically achievable concentrations. Nitazoxanide combined with the cytotoxic drug irinotecan showed anticancer activity in vivo. We here report that the FDA-approved anthelmintic drug nitazoxanide could be a potential candidate for advancement into cancer clinical trials.

A novel function for Wnt signaling modulating neuronal firing activity and the temporal structure of spontaneous oscillation in the entorhinal–hippocampal circuit

During early and late postnatal developments, the establishment of functional neuronal connectivity depends on molecules like Wnt that help the recently formed synapses to establish and consolidate their new cellular interactions. However, unlike other molecules, whether Wnt can modulate the firing properties of cells is unknown. Here, for the first time we explore the physiological effect of the canonical and non-canonical Wnt pathways on a circuit that is currently generating oscillatory activity, the entorhinal cortex–hippocampal circuit. Our results indicate that Wnt pathways have strong influence in the circuital and cellular properties depending on the Wnt protein isoforms, concentration, and type of neuronal circuit. Antibodies against canonical and non-canonical ligands, as well as WASP-1 and sFRP-2, demonstrate that constitutive release of Wnts contributes to the maintenance of the network and intrinsic properties of the circuit. Furthermore, we found that the excess of Wnt3a or the permanent intracellular activation of the pathway with BIO-6 accelerates the period of the oscillation by disrupting the oscillatory units (Up states) in short units, presumably by affecting the synaptic mechanisms that couples neurons into the oscillatory cycle, but without affecting the spike generation. Instead, low doses of Wnt5a increase the period of the oscillation in EC by incorporating new cells into the network activity, probably modifying firing activity in other places of the circuit. Moreover, we found that Wnt signaling operates under different principles in the hippocampus. Using pyrvinium pamoate, a Wnt/β-catenin dependent pathway inhibitor, we demonstrated that this pathway is essential to keep the firing activity in the circuit CA3, and in less degree of CA1 circuit. However, CA1 circuit possesses homeostatic mechanisms to up-regulate the firing activity when it has been suppressed in CA3, and to down-modulate the cellular excitability when exacerbated circuital activity has dominated. In summary, the amount of Wnt that is being released can exert a fine tuning of the physiological output, modulating firing activity, improving reliability of communication between neurons, and maintaining a continuous self-regulatory cycle of synaptic structure–function that can be present during all postnatal life.

Wnt blockers inhibit the proliferation of lung cancer stem cells

BackgroundPrevious study has confirmed that the occurrence of Wnt pathway activation is associated with risk of non-small-cell lung cancer recurrence. However, whether the pharmacologic blocking of the Wnt signaling pathway could provide therapeutic possibility remains unknown. The aim of the present study was to evaluate the therapeutic functions of the Wnt signaling pathway inhibitor pyrvinium pamoate (PP) on lung cancer stem cells (LCSCs) in vitro.MethodsColony formation and sphere culture were performed to enrich LCSCs from three lung cancer cell lines: PC9, SPC-A1, and A549. After confirming stemness by immunofluorescence, PP was employed for cell viability assay by comparison with three other kinds of Wnt signaling inhibitor: salinomycin, ICG-001, and silibinin. The effect of PP on LCSCs was further verified by colony formation assay and gene expression analysis.ResultsLCSCs were successfully generated by sphere culture from SPC-A1 and PC9 cells, but not A549 cells. Immunofluorescence assay showed that LCSCs could express pluripotent stem cell markers, including NANOG, Oct4, KLF5, and SOX2, and Wnt signaling pathway molecules β-catenin and MYC. Half-maximal inhibitory concentrations of PP on SPC-A1, PC9, and A549 were 10 nM, 0.44 nM, and 0.21 nM, respectively, which are much lower than those of salinomycin, ICG-001, and silibinin. Moreover, significantly decreased colony formation and downregulation of pluripotent stem cell signaling pathway were observed in lung cancer cells after treatment with PP.ConclusionWnt signaling inhibitor PP can inhibit proliferation of LCSCs, and the Wnt signaling pathway could be considered a promising therapeutic or interventional target in lung adenocarcinoma.

The activation of the(Pro)renin receptor (PRR) stimulates fibrotic factors expression independent of b‐catenin signaling pathway in collecting duct cells

Binding of the PRR to renin and prorenin activates intracellular signals independent of angiotensin II. The PRR is a component of the Wnt signaling pathway working as an adaptor between Wnt receptor and the vacuolar H+‐ATPase. The expression of PRR is increased in the heart and kidney of hypertensive and diabetic animals; however its role in renal damage remains unclear. In this study we tested the hypothesis that the PRR activation induces the expression of fibronectin and collagen I via the b‐catenin pathway in mouse collecting duct M‐1 cells. Human recombinant prorenin (hrPR) for 16 h in M‐1 cells increased the mRNA and protein levels of fibronectin (1.48±0.1 fold change), collagen I (1.53±0.1 fold‐change) as well as the b‐catenin target genes, cyclin D1 (1.9 ± 0.1 fold change) and c‐myc (2.1 ± 0.2 fold change). Inhibition of b‐catenin degradation with pyrvinium pamoate (10‐9 M) did not prevent the hrPR‐induced expression of all these genes. However, when the cells were transfected with a siRNA‐PRR, the hrPR‐dependent increases in fibronectin, collagen, cyclin D1 and c‐myc were prevented. Furthermore, hrPR treatment did not change the stationary protein level of β‐catenin or phosphorylation of GSK‐3β until 24h, but the level of pERK1/2 displayed a significant increase after 8h of treatment (2.9 ± 0.21 fold change, P&lt;0.05).These results indicate that in collecting duct cells, PRR activation stimulates fibrotic genes independent of β‐catenin pathway.

Antibiotics that target mitochondria effectively eradicate cancer stem cells, across multiple tumor types: treating cancer like an infectious disease.

Here, we propose a new strategy for the treatment of early cancerous lesions and advanced metastatic disease, via the selective targeting of cancer stem cells (CSCs), a.k.a., tumor-initiating cells (TICs). We searched for a global phenotypic characteristic that was highly conserved among cancer stem cells, across multiple tumor types, to provide a mutation-independent approach to cancer therapy. This would allow us to target cancer stem cells, effectively treating cancer as a single disease of "stemness", independently of the tumor tissue type. Using this approach, we identified a conserved phenotypic weak point - a strict dependence on mitochondrial biogenesis for the clonal expansion and survival of cancer stem cells. Interestingly, several classes of FDA-approved antibiotics inhibit mitochondrial biogenesis as a known "side-effect", which could be harnessed instead as a "therapeutic effect". Based on this analysis, we now show that 4-to-5 different classes of FDA-approved drugs can be used to eradicate cancer stem cells, in 12 different cancer cell lines, across 8 different tumor types (breast, DCIS, ovarian, prostate, lung, pancreatic, melanoma, and glioblastoma (brain)). These five classes of mitochondrially-targeted antibiotics include: the erythromycins, the tetracyclines, the glycylcyclines, an anti-parasitic drug, and chloramphenicol. Functional data are presented for one antibiotic in each drug class: azithromycin, doxycycline, tigecycline, pyrvinium pamoate, as well as chloramphenicol, as proof-of-concept. Importantly, many of these drugs are non-toxic for normal cells, likely reducing the side effects of anti-cancer therapy. Thus, we now propose to treat cancer like an infectious disease, by repurposing FDA-approved antibiotics for anti-cancer therapy, across multiple tumor types. These drug classes should also be considered for prevention studies, specifically focused on the prevention of tumor recurrence and distant metastasis. Finally, recent clinical trials with doxycycline and azithromycin (intended to target cancer-associated infections, but not cancer cells) have already shown positive therapeutic effects in cancer patients, although their ability to eradicate cancer stem cells was not yet appreciated.

β-Catenin-Dependent Signaling Pathway Contributes to Renal Fibrosis in Hypertensive Rats.

The mechanism of hypertension-induced renal fibrosis is not well understood, although it is established that high levels of angiotensin II contribute to the effect. Since β-catenin signal transduction participates in fibrotic processes, we evaluated the contribution of β-catenin-dependent signaling pathway in hypertension-induced renal fibrosis. Two-kidney one-clip (2K1C) hypertensive rats were treated with lisinopril (10 mg/kg/day for four weeks) or with pyrvinium pamoate (Wnt signaling inhibitor, single dose of 60 ug/kg, every 3 days for 2 weeks). The treatment with lisinopril reduced the systolic blood pressure from 220 ± 4 in 2K1C rats to 112 ± 5 mmHg (P < 0.05), whereas the reduction in blood pressure with pyrvinium pamoate was not significant (212 ± 6 in 2K1C rats to 170 ± 3 mmHg, P > 0.05). The levels of collagen types I and III, osteopontin, and fibronectin decreased in the unclipped kidney in both treatments compared with 2K1C rats. The expressions of β-catenin, p-Ser9-GSK-3beta, and the β-catenin target genes cyclin D1, c-myc, and bcl-2 significantly decreased in unclipped kidney in both treatments (P < 0.05). In this study we provided evidence that β-catenin-dependent signaling pathway participates in the renal fibrosis induced in 2K1C rats.

Mitochondrial Proteins (e.g., VDAC, Bcl-2, HK, ANT) as Major Control Points in Oncology.

Mitochondria are fascinating organelles, at least in part, because two membranes with very different permeability properties and functions surround them. These membranes contribute to the intracellular compartmentation between mitochondria and the cytosol in eukaryotic cells. Thus, lipids (e.g., phospholipids, cholesterol, cardiolipin), membrane proteins [e.g., voltage-dependent anion channel (VDAC), ion exchangers, mitochondrial carriers such as adenine nucleotide translocase (ANT)], as well as soluble intermembrane space proteins cooperate to allow the channeling of metabolites (e.g., ATP, ADP), ions (e.g., calcium, potassium, sodium) and water across mitochondrial membranes. An increasing body of literature suggests that mitochondrial proteins behave also as control point in oncology (1, 2). For example, they can be involved (i) in the metabolic shift of cancer cells from oxidative phosphorylation to glycolysis via its binding to cytosolic proteins such as hexokinase II (i.e., Warburg effect), (ii) in the control of calcium fluxes between the endoplasmic reticulum (ER) and the mitochondrion via interaction with the inositol-3-phosphate receptor (IP3R), (iii) in the control of inner mitochondrial membrane potential (ΔΨm) via interaction with tubulin and finally, (iv) in the regulation of mitochondrial membrane permeability via interaction with Bax/Bcl-2 family members and/or the mitochondrial permeability transition pore complex (PTPC). Moreover, some proteins, via a shift in their expression, appear to be critical for the cancer cell response to chemotherapy (e.g., cisplatin, oxaliplatin, carboplatin). However, how these diverse functions are modulated and coordinated in cancer cells to control the balance between life and death is still largely unknown and requires extensive review and discussion to broaden our view of the role of mitochondrial proteins in oncology, the role of upstream signaling pathways controlling mitochondrial function and how these proteins and/or signaling pathways might be selective target for anti-cancer therapy. Thus, this research topic presents seven manuscripts, which reviewed the current knowledge of mitochondrial proteins and polyprotein complexes as druggable target for anti-cancer therapy and also explored new putative targets. Thus, Suh et al. reviewed the literature on the anti-cancer potential of the modulation of the permeability transition pore, a megachannel in the inner membrane (3). Sutendra and Michelakis proposed to reverse mitochondrial suppression, which is an hallmark of many cancer cells, with metabolic-modulating drugs, like pyruvate dehydrogenase kinase (PDK) inhibitors or M2 isoenzyme of pyruvate kinase (PKM2) activators as a novel anti-cancer strategy (4). In a similar perspective, Rasola and Chiara discussed the mechanistic interactions among oncogenic kinase pathways, glycogen synthase kinase (GSK-3) activity and subsequent modulation of mitochondrial functions that shape the pro-survival phenotype of cancer cells, such as control of redox homeostasis and inhibition of the mitochondrial permeability transition pore (5). In addition, Shoshan-Barmatz et al. review current evidence pointing to the function of VDAC1 in cell life and death, and highlight these functions in relation to cancer (6). Alternatively, Fulda proposed to shift the balance of mitochondrial apoptosis to by pass evasion of apoptosis and treatment resistance in human cancers (7). Ishii et al. reviewed the accumulating knowledge on pyrvinium pamoate, an FDA-approved anthelmintic, as an anti-cancer drug targeting mitochondrial respiration (8). Interestingly, this drug acts synergistically with dexamethasone to block the proliferation if human myeloma cells and might be used in combination. In a more exploratory approach, Kang and Pervaiz explored the crosstalk between Bcl-2 family and Ras family small GTPases (9). They analyzed their work and the recent literature to show how this molecular crosstalk can regulate the cell fate. Previously, they showed a converging role of Rac1 and Bcl-2 to promote a pro-oxidant state, frequently observed in cancer cells. Despite the complexity of GTPases family, they argued that the modulation of the physical interactions between guanine exchange factors (GEF) and GTPases could open new avenues for future redox-based therapeutic designs. We hope that this research topic provides new insights into the role of mitochondrial proteins as major control points in oncology and help the reader to elaborate new strategies for cancer therapy.

Angiotensin II increases fibronectin and collagen I through the β-catenin-dependent signaling in mouse collecting duct cells.

The contribution of angiotensin II (ANG II) to renal and tubular fibrosis has been widely reported. Recent studies have shown that collecting duct cells can undergo mesenchymal transition suggesting that collecting duct cells are involved in interstitial fibrosis. The Wnt/β-catenin signaling pathway plays an essential role in development, organogenesis, and tissue homeostasis; however, the dysregulation of this pathway has been linked to fibrosis. In this study, we investigated whether AT1 receptor activation induces the expression of fibronectin and collagen I via the β-catenin pathway in mouse collecting duct cell line M-1. ANG II (10(-7) M) treatment in M-1 cells increased mRNA, protein levels of fibronectin and collagen I, the β-catenin target genes (cyclin D1 and c-myc), and the myofibroblast phenotype. These effects were prevented by candesartan, an AT1 receptor blocker. Inhibition of the β-catenin degradation with pyrvinium pamoate (pyr; 10(-9) M) prevented the ANG II-induced expression of fibronectin, collagen I, and β-catenin target genes. ANG II treatment promoted the accumulation of β-catenin protein in a time-dependent manner. Because phosphorylation of glycogen synthase kinase-3β (GSK-3β) inhibits β-catenin degradation, we further evaluated the effects of ANG II and ANG II plus pyr on p-ser9-GSK-3β levels. ANG II-dependent upregulation of β-catenin protein levels was correlated with GSK-3β phosphorylation. These effects were prevented by pyr. Our data indicate that in M-1 collecting duct cells, the β-catenin pathway mediates the stimulation of fibronectin and collagen I in response to AT1 receptor activation.

Artemisinins, new miconazole potentiators resulting in increased activity against Candida albicans biofilms.

Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.

Abstract CT213: Arctigenin, an antiausterity agent shows high safety and promising clinical response in phase I clinical trial in patient with gemcitabine-refractory pancreatic cancer

Abstract Cancer microenvironment is characterized by severe hypoxia and nutrient deprivation, especially glucose. Cancer depends largely on glycolysis for energy production (Warburg effect), therefore tumor hypoxia is mainly caused by poor blood supply despite vigorous angiogenesis. Severe tumor hypoxia and glucose deprivation promote tumor progression and affects tumor behavior especially resistance to anticancer drugs. We have focused on the invention of agents that cancel cancer cells' ability to tolerate poor oxygen and glucose supply (antiausterity) and found many agents including kigamicin, pyrvinium pamoate, and arctigenin. Because antitumor activity and safety profile were excellent in preclinical test, arctigenin was chosen for the first compound for clinical trial. Arctigenin is rich in fruit of Arctinum lappa which is filed in Japanese Pharmacopoeia, we have invented an extraction method to enrich arctigenin (GBS-01, Kracie Pharma, Ltd.) and used in clinical trial. GBS-01 contained arctigenin about 10% in weight. Pancreatic cancer is characterized for its hypovascularity and severe hypoxia in cancer tissue has been reported and, in addition, only limited benefit of chemotherapy has been achieved so far. Patients, material and design of trial: Pancreatic cancer patients refractory to gemcitabine were selected for the phase I clinical trial of GBS-01. Primary endpoint of the trial was determination of recommended dose by assessing DLT in dose escalation study from 1 g/day to 4 g/day, corresponding to 100 mg to 400 mg arctigenin/day. GBS-01 administration was continued until either unacceptable adverse effcts or evident progression of the cancer appeared. Pharmacokinetic study was also carried out as a secondary endpoint. Results:Fifteen patients were enrolled and three patients were not evaluated because of early stopping due to progression of the disease. Results: Among 12 patients evaluated, no DLT was observed. No serious adverse effects on bone marrow, liver and kidney function was observed. Pharmacokinetic study revealed that arctigenin is efficiently absorbed and rapidly conjugated with glucuronic acid. Serum concentration of arctgenin glucuronide was about 100 to several hundreds higher than that of arctigenin, indicating glucuronidation in the first pass. Urinary secretion of arctigenin and its glucuronide was more than half of administered dose in more than half of patients, indicating high bioavailability. Regarding clinical tumor response, partial response was observed in one patient at 2.5 g/day (level 2) for 2 months followed by 2four months stable disease (SD) and SD was observed in other four patients. Median progression free survival was 1.05 months and median overall survival was 5.68 months.Conclusion: Arctigenin showed high bioavailability, high safety profile, and promising clinical responses. Citation Format: Hiroyasu Esumi, Satoshi Owada, Rumi Fujioka, Katsuya Tsuchihara, Atsushi Ohtsu, Aihiro Sato, Masafumi Ikeda, Nobuo Mochizuki, Satoshi Kishino, Takanori Kawashima, Satoshi Yomoda. Arctigenin, an antiausterity agent shows high safety and promising clinical response in phase I clinical trial in patient with gemcitabine-refractory pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT213. doi:10.1158/1538-7445.AM2014-CT213

The impact of pyrvinium pamoate on colon cancer cell viability.

The in vitro and in vivo effects of pyrvinium pamoate (PP), a newly identified WNT signaling inhibitor, were evaluated against colon cancer cell lines and primary colon cancer samples. Antiproliferative activity of PP and its effects on protein and RNA levels of WNT targets were evaluated on adenomatous polyposis coli (APC (mut)) and β-catenin(mut) cell lines, one WNT(wt) colon cancer cell line, as well as six primary colon cancer samples with mutant APC in vitro. In addition, the effect of PP on the growth of liver metastasis was examined. PP blocked colon cancer cell growth in vitro in a dose-dependent manner with great differences in the inhibitory concentration (IC(50)), ranging from 0.6 × 10(-6) to 65 × 10(-6) mol/L for colon cancer cells with mutations in WNT signaling. In addition, PP demonstrated a cytotoxic effect on primary colon cancer samples. A combined cytotoxic effect of PP with 5-fluorouracil (5-FU) was observed for two cell lines. PP decreased messenger RNA (mRNA) and protein levels of known WNT target genes as c-MYC and thereby led to the induction of p21. PP inhibited the migration of HCT116 colon cancer cells in vitro and decreased tumor growth in vivo after intraportal injection of HCT116 cells in nude mice. PP displays promising anticancer activity against a broad panel of human colon cancer cell lines, as well as primary colon cancer samples. However, our findings do not demonstrate a predominant cytotoxic effect of PP on colon cancer cells with mutations in WNT signaling.

Casein kinase 1 suppresses activation of REST in insulted hippocampal neurons and halts ischemia-induced neuronal death.

Repressor Element-1 (RE1) Silencing Transcription Factor/Neuron-Restrictive Silencer Factor (REST/NRSF) is a gene-silencing factor that is widely expressed during embryogenesis and plays a strategic role in neuronal differentiation. Recent studies indicate that REST can be activated in differentiated neurons during a critical window of time in postnatal development and in adult neurons in response to neuronal insults such as seizures and ischemia. However, the mechanism by which REST is regulated in neurons is as yet unknown. Here, we show that REST is controlled at the level of protein stability via β-TrCP-dependent, ubiquitin-based proteasomal degradation in differentiated neurons under physiological conditions and identify Casein Kinase 1 (CK1) as an upstream effector that bidirectionally regulates REST cellular abundance. CK1 associates with and phosphorylates REST at two neighboring, but distinct, motifs within the C terminus of REST critical for binding of β-TrCP and targeting of REST for proteasomal degradation. We further show that global ischemia in rats in vivo triggers a decrease in CK1 and an increase in REST in selectively vulnerable hippocampal CA1 neurons. Administration of the CK1 activator pyrvinium pamoate by in vivo injection immediately after ischemia restores CK1 activity, suppresses REST expression, and rescues neurons destined to die. Our results identify a novel and previously unappreciated role for CK1 as a brake on REST stability and abundance in adult neurons and reveal that loss of CK1 is causally related to ischemia-induced neuronal death. These findings point to CK1 as a potential therapeutic target for the amelioration of hippocampal injury and cognitive deficits associated with global ischemia.

Ligand-independent and tissue-selective androgen receptor inhibition by pyrvinium.

Pyrvinium pamoate (PP) is a potent noncompetitive inhibitor of the androgen receptor (AR). Using a novel method of target identification, we demonstrate that AR is a direct target of PP in prostate cancer cells. We demonstrate that PP inhibits AR activity via the highly conserved DNA binding domain (DBD), the only AR inhibitor that functions via this domain. Furthermore, computational modeling predicts that pyrvinium binds at the interface of the DBD dimer and the minor groove of the AR response element. Because PP acts through the DBD, PP is able to inhibit the constitutive activity of AR splice variants, which are thought to contribute to the growth of castration resistant prostate cancer (CRPC). PP also inhibits androgen-independent AR activation by HER2 kinase. The antiandrogen activity of pyrvinium manifests in the ability to inhibit the in vivo growth of CRPC xenografts that express AR splice variants. Interestingly, PP was most potent in cells with endogenous AR expression derived from prostate or bone. PP was able to inhibit several other hormone nuclear receptors (NRs) but not structurally unrelated transcription factors. PP inhibition of other NRs was similarly cell-type selective. Using dual-energy X-ray absorptiometry, we demonstrate that the cell-type specificity of PP manifests in tissue-selective inhibition of AR activity in mice, as PP decreases prostate weight and bone mineral density but does not affect lean body mass. Our results suggest that the noncompetitive AR inhibitor pyrvinium has significant potential to treat CRPC, including cancers driven by ligand-independent AR signaling.

An Anthelmintic Drug, Pyrvinium Pamoate, Thwarts Fibrosis and Ameliorates Myocardial Contractile Dysfunction in a Mouse Model of Myocardial Infarction

Metabolic adaptation to limited supplies of oxygen and nutrients plays a pivotal role in health and disease. Heart attack results from insufficient delivery of oxygen and nutrients to the heart, where cardiomyocytes die and cardiac fibroblasts proliferate – the latter causing scar formation, which impedes regeneration and impairs contractility of the heart. We postulated that cardiac fibroblasts survive metabolic stress by adapting their intracellular metabolism to low oxygen and nutrients, and impeding this metabolic adaptation would thwart their survival and facilitate the repair of scarred heart. Herein, we show that an anthelmintic drug, Pyrvinium pamoate, which has been previously shown to compromise cancer cell survival under glucose starvation condition, also disables cardiac fibroblast survival specifically under glucose deficient condition. Furthermore, Pyrvinium pamoate reduces scar formation and improves cardiac contractility in a mouse model of myocardial infarction. As Pyrvinium pamoate is an FDA-approved drug, our results suggest a therapeutic use of this or other related drugs to repair scarred heart and possibly other organs.

The Antihelmintic Drug Pyrvinium Pamoate Targets Aggressive Breast Cancer

WNT signaling plays a key role in the self-renewal of tumor initiation cells (TICs). In this study, we used pyrvinium pamoate (PP), an FDA-approved antihelmintic drug that inhibits WNT signaling, to test whether pharmacologic inhibition of WNT signaling can specifically target TICs of aggressive breast cancer cells. SUM-149, an inflammatory breast cancer cell line, and SUM-159, a metaplastic basal-type breast cancer cell line, were used in these studies. We found that PP inhibited primary and secondary mammosphere formation of cancer cells at nanomolar concentrations, at least 10 times less than the dose needed to have a toxic effect on cancer cells. A comparable mammosphere formation IC50 dose to that observed in cancer cell lines was obtained using malignant pleural effusion samples from patients with IBC. A decrease in activity of the TIC surrogate aldehyde dehydrogenase was observed in PP-treated cells, and inhibition of WNT signaling by PP was associated with down-regulation of a panel of markers associated with epithelial-mesenchymal transition. In vivo, intratumoral injection was associated with tumor necrosis, and intraperitoneal injection into mice with tumor xenografts caused significant tumor growth delay and a trend toward decreased lung metastasis. In in vitro mammosphere-based and monolayer-based clonogenic assays, we found that PP radiosensitized cells in monolayer culture but not mammosphere culture. These findings suggest WNT signaling inhibition may be a feasible strategy for targeting aggressive breast cancer. Investigation and modification of the bioavailability and toxicity profile of systemic PP are warranted.

Novel Conjugated Quinoline–Indoles Compromise Plasmodium falciparum Mitochondrial Function and Show Promising Antimalarial Activity

A novel class of antimalarial compounds, based on an indol-3-yl linked to the 2-position of a 4-aminoquinoline moiety, shows promising activity against the malaria parasite, Plasmodium falciparum . Compounds with a quaternary nitrogen on the quinoline show improved activity against the chloroquine-resistant K1 strain. Nonquaternerized 4-aminoquinolines retain significant potency but are relatively less active against the K1 strain. Alkylation of the 4-amino group preferentially improves the activity against the chloroquine-sensitive 3D7 strain. The quinoline-indoles show only weak activity as inhibitors of β-hematin formation, and their activities are only weakly antagonized by a hemoglobinase inhibitor. The compounds appear to dissipate mitochondrial potential as an early event in their antimalarial action and therefore may exert their activity by compromising Plasmodium mitochondrial function. Interestingly, we observed a structural relationship between our compounds and the anticancer and anthelminthic compound, pyrvinium pamoate, which has also been proposed to exert its action via compromising mitochondrial function.