Pyrvinium Pamoate Induces Cell Apoptosis and Autophagy in Colorectal Cancer.

BACKGROUND/AIM: In earlier research, we demonstrated that pyrvinium pamoate (PP) can effectively inhibit the proliferation and migration of colorectal cancer (CRC) cells. In the current study, we further explore the possibility of PP, as a potential therapeutic drug in the treatment of CRC. MATERIALS AND METHODS: Hoechst 33258 staining, immunofluorescence, and western blotting were used to further investigate the connection between PP and CRC cell apoptosis and autophagy. RESULTS: We found that PP promoted apoptosis and autophagy of CRC cells. At the protein level, the expression of proteins related to the PI3K/mTOR signaling pathway exhibited a negative correlation with the dosage of PP. PP may therefore induce apoptosis and autophagy by inhibiting the PI3K/mTOR signaling pathway. CONCLUSION: Our in vitro experiments demonstrated that PP could inhibit the progression of colorectal cancer cells by inducing apoptosis and autophagy. The detailed mechanism needs further investigation.

Pyrvinium doubles against WNT-driven cancer.

The WNT-ß-catenin signaling pathway has a major role in regulating cell proliferation and differentiation. Aberrant activation of the pathway contributes to various human cancer types. Because casein kinase CK1α-initiated phosphorylation of ß-catenin is a key first step to restrain WNT signaling, effective restoration of CK1α activity represents an innovative strategy to combat WNT-driven cancer. A recent study in JBC reveals the anthelmintic pyrvinium directly binds to CK1α as an activator and also stabilizes CK1α protein, doubling against WNT-driven cancer activity.

Chemical genomics with pyrvinium identifies C1orf115 as a regulator of drug efflux.

Pyrvinium is a quinoline-derived cyanine dye and an approved anti-helminthic drug reported to inhibit WNT signaling and have anti-proliferative effects in various cancer cell lines. To further understand the mechanism by which pyrvinium is cytotoxic, we conducted a pooled genome-wide CRISPR loss-of-function screen in the human HAP1 cell model. The top drug-gene sensitizer interactions implicated the malate-aspartate and glycerol-3-phosphate shuttles as mediators of cytotoxicity to mitochondrial complex I inhibition including pyrvinium. By contrast, perturbation of the poorly characterized gene C1orf115/RDD1 resulted in strong resistance to the cytotoxic effects of pyrvinium through dysregulation of the major drug efflux pump ABCB1/MDR1. Interestingly, C1orf115/RDD1 was found to physically associate with ABCB1/MDR1 through proximity-labeling experiments and perturbation of C1orf115 led to mis-localization of ABCB1/MDR1. Our results are consistent with a model whereby C1orf115 modulates drug efflux through regulation of the major drug exporter ABCB1/MDR1.

The Casein kinase 1α agonist pyrvinium attenuates Wnt-mediated CK1α degradation via interaction with the E3 ubiquitin ligase component Cereblon.

The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.

Biochemical interaction of pyrvinium in gentamicin-induced acute kidney injury by modulating calcium dyshomeostasis and mitochondrial dysfunction.

Acute kidney injury (AKI) has a poor clinical prognosis and increases the risk of chronic kidney failure (CKD). It is a common complication of organ failure in hospitalised patients (10-15% of all hospitalizations) and in intensive care unit (ICU) patients, with an incidence of up to 50%. Concerning ICU, AKI has a mortality rate ranging from 27% to 35%, rising to 60%-65% when dialysis is needed, with roughly 5%-20% of survivors requiring dialysis on discharge. AKI is believed to cause over 7 million deaths per year worldwide. Currently, there is no treatment for AKI or its progression to CKD. When activated by AKI, numerous pathways have been suggested as possible contributors to CKD progression. Wnt/ß-catenin is a crucial regulator of kidney development that increases following the injury. Despite the overwhelming evidence that Wnt/ß-catenin promotes AKI, tubulointerstitial fibrosis, a hallmark of CKD progression, is also promoted by this pathway. The therapeutic potential of Wnt/ß-catenin in the treatment of AKI and the progression from AKI to CKD is being studied. This hypothesis aims to determine whether the Wnt/ß-catenin inhibitor pyrvinium has a beneficial effect on the renal dysfunction and damage caused by Gentamicin.

Wnt/ß-catenin antagonist pyrvinium rescues high dose isoproterenol induced cardiotoxicity in rats: Biochemical and immunohistological evidences.

The up-regulation of Wnt/ß-catenin pathway induces cardiac function abnormalities, hypertrophy, and fibrosis in diabetic hypertensive and pressure overload models. The present study investigates the cardioprotective effects of Wnt/ß-catenin inhibition on isoproterenol (ISO) induced cardiotoxicity in rats. ISO was administered at a dose of 85 mg/kg (s.c) for 2 days. Wnt/ß-catenin inhibitor pyrvinium (60 µg/kg, p.o) was given 2h prior and glibenclamide at a dose of 5 mg/kg; p.o, 2 h after ISO injection. Cardiac function parameters were assessed on isolated hearts by using automated Biopac apparatus. The ß-catenin transcription and expression was detected by RT-PCR technique and immunohistochemical method. Serum and cardiac tissue biochemical changes including cardiac troponin-I, CK-MB, LDH, anti-oxidant enzyme levels, inflammatory cytokines, and membrane associated Na+/K + ATPase and Ca2+ATPase and caspase-3 activity, collagen content, fibronectin protein levels were evaluated in various study groups. Histological studies were also carried out to analyze the cardiomyocyte damage, hypertrophy, fibrosis, and necrosis, while α-SMA, TGF-ß expression was checked by immunostaining. ISO administration enhanced ß-catenin gene expression and transcription which promoted oxidative and nitrosative stress, inflammatory cytokine release, reduced ATP levels, induced over-expression of fibrotic proteins resulting in cardiac hypertrophy, myocardial necrosis, functional and histological changes. However, antagonism of Wnt/ß-catenin pathway attenuated these ISO induced pathological manifestations. Notably, the co-treatment with ATP-sensitive K+ channel inhibitor partially, reduced the cardioprotective effects of Wnt/ß-catenin blocker pyrvinium in ISO rats. Thus Wnt/ß-catenin inhibition exhibits cardioprotective in ISO model by anti-oxidant, anti-inflammatory, anti-fibrotic properties and by possible involvement of ATP-sensitive potassium channel activation.

Pyrvinium pamoate ameliorates cyclosporin A- induced hepatotoxicity via the modulation of Wnt/ß-catenin signaling and upregulation of PPAR-γ.

BACKGROUND: Cyclosporin A (CsA) is an immunosuppressive agent that can be used to treat autoimmune diseases. Despite its hepatotoxicity, CsA is a backbone in organ transplantation. Pyrvinium pamoate (PP) is an inhibitor of Wnt signaling approved by the U.S. Food and Drug Administration for its anthelmintic properties. AIM: The goal of this investigation was to determine whether PP could protect against CsA-induced hepatotoxicity. METHOD: Five groups of 50 albino male mice were selected and divided into five groups; group 1 was the control, groups 2 to 4 were subjected to daily CsA (25 mg/kg, i.p), in which groups 3 and 4 were treated with graded dose of PP (0.25, 0.5 mg/kg), and group 5 was treated with PP (0.5 mg/ kg) for 21 days. The mice were sacrificed under anesthesia, and their livers were removed for histological and biochemical assessment. RESULTS: CsA was found to cause a striking increase in liver enzymes, total bilirubin, and malondialdehyde levels while significantly decreasing the levels of albumin, glutathione, and antioxidant enzymes in the treated groups. The tissue levels of tumor necrosis factor-α, interleukin-1ß, and NFКB were also significantly higher with CsA treatment. Moreover, CsA triggered a notable increase in the levels of apoptotic marker P53. CsA activated the Wnt/ß-catenin pathway by increasing WNT3a expression, frizzled receptor-7, ß-catenin, and c-myc. On the other hand, the levels of PPAR-γ decreased significantly with CsA. CsA-induced alterations in the previously stated parameters were greatly reduced by PP, indicating its antioxidant, anti-inflammatory, and antiapoptotic properties. CONCLUSIONS: PP may be considered as a promising agent to prevent CsA hepatotoxicity.

Cancer as an infectious disease: A different treatment alternative using a combination of tigecycline and pyrvinium pamoate - An example of breast cancer.

BACKGROUND: Tigecycline is an antibiotic that well tolerated for treating complicated infections. It has received attention as an anti-cancer agent and expected to solve two major obstacles, sides effects that accompany chemotherapy and drug resistance, in the breast cancer treatment. However, previous studies reported that the levels in the blood are typically low of tigecycline, so higher doses are needed to treat cancer, that may increase the risk of side effects. To achieve better anti-cancer effects for tigecycline, we need to find a novel adjunct agent. METHODS: In this study, we used different concentration of pyrvinium pamoate combined with tigecycline to treat cell. And assess the effect of two drugs in inhibit cell proliferation, induce cell autophagy, or increase cell apoptosis to evaluate the consequent of combined therapy. RESULTS: We observed that after the combined therapy, the cell cycle arrest at G1/s phase, the level of p21 increased, but decreased the levels of CDK2. Others, two drugs via different mechanisms to inhibit cancer cell proliferation and with selective cytotoxic to different cell lines. That could enhance the effect of breast cancer treatment. CONCLUSION: Combining low dose of tigecycline use with pyrvinium pamoate is a novel approach for breast cancer treatment. Appropriate combined therapy in breast cancer is recommended to improve outcomes. Other problems like drug resistance occur in patients or the microbes surrounding breast tissues would confer susceptibility to cancers then influence the effectiveness of treatment, which could be improved through combined therapy.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Lipidomic Analyses Uncover Apoptotic and Inhibitory Effects of Pyrvinium Pamoate on Cholangiocarcinoma Cells via Mitochondrial Membrane Potential Dysfunction.

Pyrvinium pamoate (PP), an FDA-approved anthelmintic drug, has been validated as a highly potent anti-cancer agent and patented recently as a potential chemotherapeutic drug for various cancers. The aims of this study were, therefore, to investigate the ability of PP in anti-proliferative activity and focused on the lipid profiles revealing the alteration of specific lipid species in the liver fluke Opisthorchis viverrini (Ov)-associated cholangiocarcinoma (CCA) cells. PP inhibited CCA cell viability through suppressing mitochondrial membrane potential (MMP) and ATP productions, leading to apoptotic cell death. Liquid chromatography-mass spectrometry combined with chemometrics was performed to investigate lipid alteration during PP-induced apoptosis. The lipidomic analyses showed the altered lipid signatures of CCA cell types including S-acetyldihydrolipoamide, methylselenopyruvate, and triglycerides that were increased in PP-treated CCA cells. In contrast, the levels of sphinganine and phosphatidylinositol were lower in the PP-treated group compared with its counterpart. The orthogonal partial-least squares regression analysis revealed that PP-induced MMP dysfunction, leading to remarkably reduced ATP level, was significantly associated with triglyceride (TG) accumulation observed in PP-treated CCA cells. Our findings indicate that PP could suppress the MMP function, which causes inhibition of CCA cell viability through lipid production, resulting in apoptotic induction in CCA cells. These findings provide an anti-cancer mechanism of PP under apoptotic induction ability that may serve as the alternative approach for CCA treatment.

The FDA-Approved Anthelmintic Pyrvinium Pamoate Inhibits Pancreatic Cancer Cells in Nutrient-Depleted Conditions by Targeting the Mitochondria.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal aggressive cancer, in part due to elements of the microenvironment (hypoxia, hypoglycemia) that cause metabolic network alterations. The FDA-approved antihelminthic pyrvinium pamoate (PP) has previously been shown to cause PDAC cell death, although the mechanism has not been fully determined. We demonstrated that PP effectively inhibited PDAC cell viability with nanomolar IC50 values (9-93 nmol/L) against a panel of PDAC, patient-derived, and murine organoid cell lines. In vivo, we demonstrated that PP inhibited PDAC xenograft tumor growth with both intraperitoneal (IP; P < 0.0001) and oral administration (PO; P = 0.0023) of human-grade drug. Metabolomic and phosphoproteomic data identified that PP potently inhibited PDAC mitochondrial pathways including oxidative phosphorylation and fatty acid metabolism. As PP treatment reduced oxidative phosphorylation (P < 0.001), leading to an increase in glycolysis (P < 0.001), PP was 16.2-fold more effective in hypoglycemic conditions similar to those seen in PDAC tumors. RNA sequencing demonstrated that PP caused a decrease in mitochondrial RNA expression, an effect that was not observed with established mitochondrial inhibitors rotenone and oligomycin. Mechanistically, we determined that PP selectively bound mitochondrial G-quadruplexes and inhibited mitochondrial RNA transcription in a G-quadruplex-dependent manner. This subsequently led to a 90% reduction in mitochondrial encoded gene expression. We are preparing to evaluate the efficacy of PP in PDAC in an IRB-approved window-of-opportunity trial (IND:144822).

Identification of the FDA-Approved Drug Pyrvinium as a Small-Molecule Inhibitor of the PD-1/PD-L1 Interaction.

Immune checkpoint blockade involving inhibition of the PD-1/PD-L1 interaction has provided unprecedented clinical benefits in treating a variety of tumors. To date, a total of six antibodies that bind to either PD-1 or PD-L1 protein and in turn inhibit the PD-1/PD-L1 interaction have received clinical approvals. Despite being highly effective, these expensive large biotherapeutics possess several inherent pharmacokinetic limitations that can be successfully overcome through the use of low-molecular-weight inhibitors. One such promising approach involves small-molecule induced dimerization and sequestration of PD-L1, leading to effective PD-1/PD-L1 inhibition. Herein, we present the discovery of such potential bioactive PD-L1 dimerizers through a structure- and ligand-based screening of a focused library of approved and investigational drugs worldwide. Pyrvinium, an FDA-approved anthelmintic drug, showed the highest activity in our study with IC50 value of ∼29.66 µM. It is noteworthy that Pyrvinium, being an approved drug, may prove especially suitable as a good starting point for further medicinal chemistry efforts, leading to design and development of even more potent structural analogs as selective PD-1/PD-L1 inhibitors. Furthermore, the adopted integrated virtual screening protocol may prove useful in screening other larger databases of lead- and drug-like molecules for hit identification in the domain of small-molecule PD-1/PD-L1 inhibitors.

Pyrvinium pamoate can overcome artemisinin's resistance in anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid carcinoma is a highly lethal subtype of thyroid cancer without effective therapies. Drug resistance in anaplastic thyroid carcinoma poses a significant problem. Although artemisinin exerts antitumor effects, but its efficacy in anaplastic thyroid carcinoma is unknown. METHODS: We used RNA sequencing to identify differentially expressed genes. Next, we determined the cause of ART resistance by testing the expression and activity of ß-catenin, and enhanced ART activity with a WNT signaling inhibitor. RESULTS: Artemisinin suppressed the growth of BHT-101 but not human thyroid anaplastic carcinoma (CAL-62) cells. The mechanism of artemisinin resistance in CAL-62 was associated with the aberrant activation of WNT signaling. Pyrvinium pamoate, an inhibitor of WNT signaling, was used to overcome ART resistance in CAL-62 cells. The combination of artemisinin and pyrvinium pamoate suppressed the growth of CAL-62 cells and induced the apoptosis. CONCLUSIONS: Our study is the first to prove the efficacy of ART as monotherapy or in combination with PP in the management of anaplastic thyroid cancer, and that the inhibition of WNT signaling may overcome ART resistance.

Wnt/ß-Catenin Antagonist Pyrvinium Exerts Cardioprotective Effects in Polymicrobial Sepsis Model by Attenuating Calcium Dyshomeostasis and Mitochondrial Dysfunction.

Calcium dysregulation and mitochondrial dysfunction are key elements in the development of sepsis-induced cardiac dysfunction. Evidences have suggested that inhibition of Wnt/ß-Catenin signalling prevents cardiac dysfunction and remodelling in surgical, hypertension and pressure overload models. The present study investigated the effects of Wnt/ß-Catenin inhibitor on calcium overload and mitochondrial dysfunction in rat sepsis model of cardiomyopathy. Induction of sepsis by cecal ligation puncture (CLP) resulted in the up-regulation of cardiac ß-catenin transcriptional levels and cardiac dysfunction depicted by increased serum lactate dehydrogenase, CK-MB levels reduced maximum (dp/dt max.) and minimum developed pressure (dp/dt min.), increased LVEsDP and relaxation constant tau values. Moreover, oxidative and inflammatory stress, immune cell infiltration, increased myeloperoxidase activity, enhanced caspase-3 activity and fibronectin protein levels were observed in septic rat's heart. Also, septic rat's heart displayed mitochondrial dysfunction due to mPTP opening, increased calcium up-regulation in left ventricular apex tissues and whole heart, increased collagen staining, necrosis and structural damage. Pre-treatment with Wnt/ß-Catenin antagonist attenuated sepsis-induced serum and tissue biochemical changes, cardiac dysfunction and structural alterations by inhibiting mitochondrial mPTP opening and restricting calcium overloading in cardiac tissue.

Anti-Tumor Efficacy of Pyrvinium Pamoate Nanoliposomes in an Experimental Model of Melanoma.

BACKGROUND: Pyrvinium Pamoate (PP) is an old drug approved by the FDA for the treatment of pinworm infections. Recently, it has been introduced as an anti-tumor agent, however, low aqueous solubility severely limits its potential effects. In this study, we developed a liposomal formulation of pyrvinium pamoate to investigate its in vitro cytotoxicity and in vivo efficacy against melanoma cells. MATERIALS & METHODS: As drug carriers, liposomes were fabricated using the thin-film method. PP was encapsulated within the liposomes using a remote loading method. We evaluated the morphology, particle size, and Zeta potential of the liposomes. Additionally, High-Performance Liquid Chromatography (HPLC) was employed for qualitative and quantitative analysis. Then we investigated our liposomal PP for its in vitro cytotoxicity as well as the tumor growth inhibition in C57BL/6 mice bearing B16F0 melanoma tumors. RESULTS: Based on the analytical result, the liposomal drug delivery system is a homogeneous and stable colloidal suspension of PP particles. The images of Atomic force microscopy and particle size data showed that all the prepared nanocarriers were spherical with a diameter of approximately 101 nm. According to both in vitro and in vivo studies, nanoliposomal PP exhibited an improved anti-proliferative potential against B16F10 melanoma tumor compared to free PP. CONCLUSION: Liposomal encapsulation improves the water solubility of PP and enhances its anti-cancer activity.

Pyrvinium pamoate inhibits cell proliferation through ROS-mediated AKT-dependent signaling pathway in colorectal cancer.

The use of the anthelmintic drug pyrvinium pamoate (PP) in cancer therapy has been extensively investigated in the last decade. PP has been shown to have an inhibitory effect in colorectal cancer (CRC), but the underlying mechanism remains elusive. We aimed to investigate the antitumor activity and mechanisms of PP in CRC. In the present study, we used CCK-8 assays, colony formation assays, and western blotting to reveal that PP effectively suppressed CRC cell proliferation and the AKT-dependent signaling pathway in a concentration-dependent and time-dependent manner. Flow cytometric analysis and fluorescence microscopy demonstrated that PP increased intracellular reactive oxygen species (ROS) accumulation. We found that the inhibitory effect of PP on cell proliferation and AKT protein expression induced by PP could be partially reversed by N-acetyl-L-cysteine (NAC), an ROS scavenger. In addition, the results also demonstrated that PP inhibited cell migration by modulating epithelial-to-mesenchymal transition (EMT)-related proteins, including E-cadherin and vimentin. In conclusion, our data suggested that PP effectively inhibited cell proliferation through the ROS-mediated AKT-dependent signaling pathway in CRC, further providing evidence for the use of PP as an antitumor agent.

Targeting of canonical WNT signaling ameliorates experimental sclerodermatous chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) is a major life-threatening complication of allogeneic hematopoietic stem cell transplantation. The molecular mechanisms underlying cGVHD remain poorly understood, and targeted therapies for clinical use are not well established. Here, we examined the role of the canonical WNT pathway in sclerodermatous cGVHD (sclGVHD). WNT signaling was activated in human sclGVHD with increased nuclear accumulation of the transcription factor ß-catenin and a WNT-biased gene expression signature in lesional skin. Treatment with the highly selective tankryase inhibitor G007-LK, the CK1α agonist pyrvinium, or the LRP6 inhibitor salinomycin abrogated the activation of WNT signaling and protected against experimental cGVHD, without a significant impact on graft-versus-leukemia effect (GVL). Treatment with G007-LK, pyrvinium, or salinomycin almost completely prevented the development of clinical and histological features in the B10.D2 (H-2d) → BALB/c (H-2d) and LP/J (H-2b) → C57BL/6 (H-2b) models of sclGVHD. Inhibition of canonical WNT signaling reduced the release of extracellular matrix from fibroblasts and reduced leukocyte influx, suggesting that WNT signaling stimulates fibrotic tissue remodeling by direct effects on fibroblasts and by indirect inflammation-dependent effects in sclGVHD. Our findings may have direct translational potential, because pyrvinium is in clinical use, and tankyrase inhibitors are in clinical trials for other indications.

Pyrvinium pamoate induces in-vitro suppression of IL-6 and IL-8 produced by human endometriotic stromal cells.

Endometriosis, a chronic inflammatory disease, is identified by the presence of endometrial tissue outside the uterus. The prevalence of this disease among reproductive-age women is almost 10-15%. High levels of IL-6 and IL-8 have been found in the peritoneal fluid (PF) of women with endometriosis and are involved in its pathogenesis. Isolated stromal cells from 12 ectopic and eutopic endometrial biopsies of women with ovarian endometrioma and also 12 endometrial biopsies of nonendometriotic controls were treated with 1.1 µM pyrvinium pamoate, a Wnt/ß-catenin signaling pathway inhibitor, for 72 hrs. Before treatment, mRNA gene expression and secretion of IL-6 and IL-8 were significantly higher in ectopic (EESCs) than eutopic (EuESCs) and control (CESCs) endometrial stromal cells. After treatment, mRNA gene expression and also secretion of IL-6 and IL-8 were significantly reduced. Our Findings showed that pyrvinium pamoate suppresses the mRNA gene expression and secretion of IL-6 and IL-8 in human endometriotic stromal cells. Additional investigations on this compound are required before clinical application.

In vitro and in vivo Study of Antifungal Effect of Pyrvinium Pamoate Alone and in Combination With Azoles Against Exophiala dermatitidis.

Infections of Exophiala dermatitidis are often chronic and recalcitrant. Combination therapies with novel compounds and azoles could be an effective solution. Previously, we have demonstrated that pyrvinium pamoate exerted antifungal activity alone and favorable synergy with azoles against planktonic E. dermatitidis. Herein, the underlying antifungal mode of action were investigated. Pyrvinium alone showed sessile MIC50 (SMIC50) of 8->16 µg/ml against E. dermatitidis biofilms. However, synergism of PP with itraconazole, voriconazole, and posaconazole were observed against 16 (88.9%), 9 (50%), and 13 (72.2%) strains of E. dermatitidis biofilms. In accordance with in vitro susceptibilities, pyrvinium alone at concentration of 2 µg/ml resulted in significant growth restriction of planktonic E. dermatitidis. Pyrvinium alone resulted in reduction of biofilm formation. Higher concentration of pyrvinium was associate with more progressive reduction of biofilm mass. The in vivo activity of pyrvinium alone and combined with azoles was evaluated using Galleria mellonella model. Pyrvinium alone significantly improved the survival rate of larvae (P < 0.0001). The combination of pyrvinium and voriconazole or posaconazole acted synergistically in vivo (P < 0.05). Fungal burden determination revealed significant reduction of numbers of colony forming unit (CFU) in larvae treated with pyrvinium-itraconazole and pyrvinium-posaconazole compared to itraconazole or posaconazole alone group, respectively. The effect of pyrvinium on apoptosis, expression of TOR and HSP90, and drug efflux reversal were evaluated by PI/Annexin V staining, Real-Time Quantitative PCR and Rhodamine 6G assay, respectively. Pyrvinium alone or combined with azoles significantly (P < 0.05) increased late apoptosis or necrosis of E. dermatitidis cells. Pyrvinium combined with posaconazole significantly decreased the expression of TOR and Hsp90 compared to posaconazole alone group (P < 0.05). Pyrvinium resulted in significant (P < 0.05) decrease of the efflux of Rhodamine 6G. These findings suggested pyrvinium could be a promising synergist with azoles. The underlying mechanisms could be explained by inducing apoptosis/necrosis, inhibition of drug efflux pumps, and signaling pathways related with stress response and growth control.

Targeting Mitochondria in Melanoma.

Drastically elevated glycolytic activity is a prominent metabolic feature of cancer cells. Until recently it was thought that tumor cells shift their entire energy production from oxidative phosphorylation (OXPHOS) to glycolysis. However, new evidence indicates that many cancer cells still have functional OXPHOS, despite their increased reliance on glycolysis. Growing pre-clinical and clinical evidence suggests that targeting mitochondrial metabolism has anti-cancer effects. Here, we analyzed mitochondrial respiration and the amount and activity of OXPHOS complexes in four melanoma cell lines and normal human dermal fibroblasts (HDFs) by Seahorse real-time cell metabolic analysis, immunoblotting, and spectrophotometry. We also tested three clinically approved antibiotics, one anti-parasitic drug (pyrvinium pamoate), and a novel anti-cancer agent (ONC212) for effects on mitochondrial respiration and proliferation of melanoma cells and HDFs. We found that three of the four melanoma cell lines have elevated glycolysis as well as OXPHOS, but contain dysfunctional mitochondria. The antibiotics produced different effects on the melanoma cells and HDFs. The anti-parasitic drug strongly inhibited respiration and proliferation of both the melanoma cells and HDFs. ONC212 reduced respiration in melanoma cells and HDFs, and inhibited the proliferation of melanoma cells. Our findings highlight ONC212 as a promising drug for targeting mitochondrial respiration in cancer.