Neuroprotective effects of niclosamide on disease progression via inflammatory pathways modulation in SOD1-G93A and FUS-associated amyotrophic lateral sclerosis models.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease influenced by genetic, epigenetic, and environmental factors, resulting in dysfunction in cellular and molecular pathways. The limited efficacy of current treatments highlights the need for combination therapies targeting multiple aspects of the disease. Niclosamide, an anthelminthic drug listed as an essential medicine, has been repurposed in clinical trials for different diseases due to its anti-inflammatory and anti-fibrotic properties. Niclosamide can inhibit various molecular pathways (e.g., STAT3, mTOR) that are dysregulated in ALS, suggesting its potential to disrupt these altered mechanisms associated with the pathology. We administered niclosamide intraperitoneally to two transgenic murine models, SOD1-G93A and FUS mice, mimicking key pathological processes of ALS. The treatment was initiated at the onset of symptoms, and we assessed disease progression by neurological scores, rotarod and wire tests, and monitored survival. Furthermore, we investigated cellular and molecular mechanisms affected by niclosamide in the spinal cord and muscle of ALS mice. In both models, the administration of niclosamide resulted in a slowdown of disease progression, an increase in survival rates, and an improvement in tissue pathology. This was characterised by reduced gliosis, motor neuron loss, muscle atrophy, and inflammatory pathways. Based on these results, our findings demonstrate that niclosamide can impact multiple pathways involved in ALS. This multi-targeted approach leads to a slowdown in the progression of the disease, positioning niclosamide as a promising candidate for repurposing in the treatment of ALS.

Lipid-Based Self-Microemulsion of Niclosamide Achieved Enhanced Oral Delivery and Anti-Tumor Efficacy in Orthotopic Patient-Derived Xenograft of Hepatocellular Carcinoma in Mice.

Introduction: We previously identified niclosamide as a promising repurposed drug candidate for hepatocellular carcinoma (HCC) treatment. However, it is poorly water soluble, limiting its tissue bioavailability and clinical application. To overcome these challenges, we developed an orally bioavailable self-microemulsifying drug delivery system encapsulating niclosamide (Nic-SMEDDS). Methods: Nic-SMEDDS was synthesized and characterized for its physicochemical properties, in vivo pharmacokinetics and absorption mechanisms, and in vivo therapeutic efficacy in an orthotopic patient-derived xenograft (PDX)-HCC mouse model. Niclosamide ethanolamine salt (NEN), with superior water solubility, was used as a positive control. Results: Nic-SMEDDS (5.6% drug load) displayed favorable physicochemical properties and drug release profiles in vitro. In vivo, Nic-SMEDDS displayed prolonged retention time and plasma release profile compared to niclosamide or NEN. Oral administration of Nic-SMEDDS to non-tumor bearing mice improved niclosamide bioavailability and Cmax by 4.1- and 1.8-fold, respectively, compared to oral niclosamide. Cycloheximide pre-treatment blocked niclosamide absorption from orally administered Nic-SMEDDS, suggesting that its absorption was facilitated through the chylomicron pathway. Nic-SMEDDS (100 mg/kg, bid) showed greater anti-tumor efficacy compared to NEN (200 mg/kg, qd); this correlated with higher levels (p < 0.01) of niclosamide, increased caspase-3, and decreased Ki-67 in the harvested PDX tissues when Nic-SMEDDS was given. Biochemical analysis at the treatment end-point indicated that Nic-SMEDDS elevated lipid levels in treated mice. Conclusion: We successfully developed an orally bioavailable formulation of niclosamide, which significantly enhanced oral bioavailability and anti-tumor efficacy in an HCC PDX mouse model. Our data support its clinical translation for the treatment of solid tumors.

Pharmacokinetic considerations for enhancing drug repurposing opportunities of anthelmintics: Niclosamide as a case study.

Recently, anthelmintics have showcased versatile therapeutic potential in addressing various diseases, positioning them as promising candidates for drug repurposing. However, challenges such as low bioavailability and a lack of a solid pharmacokinetic basis impede successful repurposing. To overcome these flaws, we aimed to investigate the key pharmacokinetic factors of anthelmintics mainly focusing on the absorption, distribution, and metabolism profiles by employing niclosamide (NIC) as a model drug. The intestinal permeability of NIC is significantly influenced by solubility and doesn't function as a substrate for efflux transporters. It showed high plasma protein binding. Also, the metabolism study indicated that NIC would have low metabolic stability by extensively undergoing the intestinal glucuronidation. Additionally, we investigated the CYP-mediated drug-drug interaction potential of NIC in both direct and time-dependent ways. NIC showed strong inhibitory effects on CYP1A2 and CYP2C8 and is not likely to become a time-dependent inhibitor. Our findings could contribute to the identification of essential factors in the pharmacokinetics of anthelmintics, potentially facilitating their repositioning.

ROS-Suppression Nanoplatform Combined Activation of STAT3/Bcl-2 Pathway for Preventing Myocardial Infarction in Mice.

Myocardial infarction (MI) is the leading cause of death worldwide. The most effective way to treat myocardial infarction is to rescue ischemic cardiomyocytes. After an ischemic event, the overproduction of reactive oxygen species (ROS) is a key driver of myocardial injury. The produced ROS affects mitochondrial function and induces apoptosis in cardiomyocytes. This was accomplished by constructing platelet-membrane-encapsulated ROS-responsive drug-releasing nanoparticles (PMN@NIC-MalNPs) to deliver malonate and niclosamide (NIC). The results revealed that PMN@NIC-MalNPs degraded and released malonate and niclosamide in a high-level ROS microenvironment, effectively reducing the oxidative stress and apoptosis rate. By enhancing basal mitochondrial oxygen consumption rate (OCR), adenosine triphosphate (ATP) production, and spare respiratory capacity (SRC) in vitro, reduced the oxidative stress levels and restored mitochondrial function. In vivo studies revealed that the PMN@NIC-MalNPs improved cardiac dysfunction, inhibited succinate dehydrogenase (SDH) activity, increased ATP production, and reduced the myocardial infarct size in myocardial infarction model mice. Further, transcriptome analysis and Western blot revealed that PMN@NIC-MalNPs prevented apoptosis by activating the expressions of the signal transducer and activator of transcription 3 (STAT3) and Bcl-2, and inhibiting the expression of Bax. Thus, this study provides a novel therapeutic solution for treating myocardial infarction and predicting the viability of an antioxidant and antiapoptotic therapeutic solution in the treatment of myocardial injury.

Repurposing Niclosamide as a plausible neurotherapeutic in autism spectrum disorders, targeting mitochondrial dysfunction: a strong hypothesis.

Autism Spectrum Disorders (ASD) are a complex set of neurodevelopmental manifestations which present in the form of social and communication deficits. Affecting a growing proportion of children worldwide, the exact pathogenesis of this disorder is not very well understood, and multiple signaling pathways have been implicated. Among them, the ERK/MAPK pathway is critical in a number of cellular processes, and the normal functioning of neuronal cells also depends on this cascade. As such, recent studies have increasingly focused on the impact this pathway has on the development of autistic symptoms. Improper ERK signaling is suspected to be involved in neurotoxicity, and the same might be implicated in autism spectrum disorders (ASD), through a variety of effects including mitochondrial dysfunction and oxidative stress. Niclosamide, an antihelminthic and anti-inflammatory agent, has shown potential in inhibiting this pathway, and countering the effects shown by its overactivity in inflammation. While it has previously been evaluated in other neurological disorders like Alzheimer's Disease and Parkinson's Disease, as well as various cancers by targeting ERK/MAPK, it's efficacy in autism has not yet been evaluated. In this article, we attempt to discuss the potential role of the ERK/MAPK pathway in the pathogenesis of ASD, specifically through mitochondrial damage, before moving to the therapeutic potential of niclosamide in the disorder, mediated by the inhibition of this pathway and its detrimental effects of neuronal development.

Niclosamide, but not ivermectin, inhibits anoctamin 1 and 6 and attenuates inflammation of the respiratory tract.

Inflammatory airway diseases like cystic fibrosis, asthma and COVID-19 are characterized by high levels of pulmonary cytokines. Two well-established antiparasitic drugs, niclosamide and ivermectin, are intensively discussed for the treatment of viral inflammatory airway infections. Here, we examined these repurposed drugs with respect to their anti-inflammatory effects in airways in vivo and in vitro. Niclosamide reduced mucus content, eosinophilic infiltration and cell death in asthmatic mouse lungs in vivo and inhibited release of interleukins in the two differentiated airway epithelial cell lines CFBE and BCi-NS1.1 in vitro. Cytokine release was also inhibited by the knockdown of the Ca2+-activated Cl- channel anoctamin 1 (ANO1, TMEM16A) and the phospholipid scramblase anoctamin 6 (ANO6, TMEM16F), which have previously been shown to affect intracellular Ca2+ levels near the plasma membrane and to facilitate exocytosis. At concentrations around 200 nM, niclosamide inhibited inflammation, lowered intracellular Ca2+, acidified cytosolic pH and blocked activation of ANO1 and ANO6. It is suggested that niclosamide brings about its anti-inflammatory effects at least in part by inhibiting ANO1 and ANO6, and by lowering intracellular Ca2+ levels. In contrast to niclosamide, 1 µM ivermectin did not exert any of the effects described for niclosamide. The present data suggest niclosamide as an effective anti-inflammatory treatment in CF, asthma, and COVID-19, in addition to its previously reported antiviral effects. It has an advantageous concentration-response relationship and is known to be well tolerated.

Head-to-head comparison of BAM15, semaglutide, rosiglitazone, NEN, and calorie restriction on metabolic physiology in female db/db mice.

Metabolic disorders such as type 2 diabetes, fatty liver disease, hyperlipidemia, and obesity commonly co-occur but clinical treatment options do not effectively target all disorders. Calorie restriction, semaglutide, rosiglitazone, and mitochondrial uncouplers have all demonstrated efficacy against one or more obesity-related metabolic disorders, but it currently remains unclear which therapeutic strategy best targets the combination of hyperglycaemia, liver fat, hypertriglyceridemia, and adiposity. Herein we performed a head-to-head comparison of 5 treatment interventions in the female db/db mouse model of severe metabolic disease. Treatments included ∼60 % calorie restriction (CR), semaglutide, rosiglitazone, BAM15, and niclosamide ethanolamine (NEN). Results showed that BAM15 and CR improved body weight and liver steatosis to levels superior to semaglutide, NEN, and rosiglitazone, while BAM15, semaglutide, and rosiglitazone improved glucose tolerance better than CR and NEN. BAM15, CR, semaglutide, and rosiglitazone all had efficacy against hypertriglyceridaemia. These data provide a comprehensive head-to-head comparison of several key treatment strategies for metabolic disease and highlight the efficacy of mitochondrial uncoupling to correct multiple facets of the metabolic disease milieu in female db/db mice.

Combination therapy of niclosamide with gemcitabine inhibited cell proliferation and apoptosis via Wnt/ß-catenin/c-Myc signaling pathway by inducing ß-catenin ubiquitination in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a type of cancer with high morbidity and mortality rates worldwide. Owing to a lack of therapeutic options, the overall survival rate of patients with pancreatic cancer is low. Gemcitabine has been mainly used to treat patients with pancreatic cancer, but its efficacy is limited by chemoresistance. Therefore, a novel therapeutic agent for PDAC therapy is urgently needed. An anthelminthic drug, niclosamide, has already been researched in breast, lung, colon, and pancreatic cancer as an anti-cancer purpose by re-positioning its original purpose. However, combination therapy of gemcitabine and niclosamide was not informed yet. Here, we found that niclosamide co-administered with gemcitabine significantly inhibited tumorigenesis of pancreatic cancer compared to gemcitabine alone. Further, combining niclosamide and gemcitabine inhibited cell proliferation and induced apoptosis. Niclosamide induced cell cycle arrest at the G1 phase, and the levels of CDK4/6 and cyclin D1 were lowered after gemcitabine treatment. In addition, the combination of these chemical compounds more effectively increased the binding level of activated ß-catenin destruction complex and ß-catenin to enable phosphorylation, compared to gemcitabine alone. After phosphorylation, niclosamide - gemcitabine upregulated the ubiquitin level, which caused phosphorylated ß-catenin to undergo proteasomal degradation; the combination was more potent than gemcitabine alone. Finally, the combination more effectively suppressed tumor growth in vivo, compared to gemcitabine alone. Altogether, our results indicate that niclosamide synergistically enhances the antitumor effect of gemcitabine in pancreatic cancer, by inducing the degradation of ß-catenin with ubiquitination. Therefore, this drug combination can potentially be used in PDAC therapy.

Targeting the epithelial-mesenchymal transition (EMT) pathway with combination of Wnt inhibitor and chalcone complexes in lung cancer cells.

Non-small cell lung cancer (NSCLC) is the most common type of the lung cancer. Despite development in treatment options in NSCLC, the overall survival ratios is still poor due to epithelial and mesenchymal transition (EMT) feature and associated metastasis event. Thereby there is a need to develop strategy to increase antitumor response against the NSCLC cells by targeting EMT pathway with combination drugs. Niclosamide and chalcone complexes are both affect cancer cell signaling pathways and therefore inhibit the EMT pathway. In this study, it was aimed to increase antitumor response and suppress EMT pathway in NSCLC cells by combining niclosamide and chalcone complexes. SRB cell viability assay was performed to investigate the anticancer activity of drugs. The drugs were tested on both NSCLC cells (A549 and H1299) and normal lung bronchial cells (BEAS-2B). Then the two drugs were combined and their effects on cancer cells were evaluated. Fluorescence imaging and enzyme-linked immunosorbent assay were performed on treated cells to observe the cell death manner. Wound healing assay, real-time quantitative polymerase chain reaction, and western blot analysis were performed to measure EMT pathway activity. Our results showed that niclosamide and chalcone complexes combination kill cancer cells more than normal lung bronchial cells. Compared to single drug administration, the combination of both drugs killed NSCLC cells more effectively by increasing apoptotic activity. In addition, the combination of niclosamide and chalcone complexes decreased multidrug resistance and EMT activity by lowering their gene expressions and protein levels. These results showed that niclosamide and chalcone complexes combination could be a new drug combination for the treatment of NSCLC.

Combination of niclosamide and quinacrine inactivates Akt/HK2/Cyclin D1 axis mediated by glucose deprivation towards the inhibition of melanoma cell proliferation.

Malignant melanoma is one of the most aggressive and lethal skin cancer. At present, the treatment methods for melanoma have shortcomings. Glucose is the primary energy source of cancer cells. However, it is unclear whether glucose deprivation can be used to treat melanoma. Herein, we first found glucose played an essential role in melanoma proliferation. We then further found a drug combination of niclosamide and quinacrine could inhibit melanoma proliferation and glucose intake. Thirdly, we revealed the mechanism of anti-melanoma effect of the drug combination, which suppressed the Akt pathway. In addition, the first-rate limiting enzyme HK2 of glucose metabolism was inhibited. This work also disclosed that the decrease of HK2 inhibited cyclin D1 by reducing the activity of transcription factor E2F3, which further suppressed the proliferation of melanoma cells. The drug combination treatment also resulted in significant tumor regression in the absence of obvious morphologic changes in primary organ in vivo. In summary, our study demonstrated that the drug combination treatment created glucose deprivation to inactive the Akt/HK2/cyclin D1 axis, thereby inhibited the proliferation of melanoma cells, providing a potential anti-melanoma strategy.

A dual-action niclosamide-based prodrug that targets cancer stem cells and inhibits TNBC metastasis.

Chemotherapy typically destroys the tumor mass but rarely eradicates the cancer stem cells (CSCs) that can drive metastatic recurrence. A key current challenge is finding ways to eradicate CSCs and suppress their characteristics. Here, we report a prodrug, Nic-A, created by combining a carbonic anhydrase IX (CAIX) inhibitor, acetazolamide, with a signal transducer and transcriptional activator 3 (STAT3) inhibitor, niclosamide. Nic-A was designed to target triple-negative breast cancer (TNBC) CSCs and was found to inhibit both proliferating TNBC cells and CSCs via STAT3 dysregulation and suppression of CSC-like properties. Its use leads to a decrease in aldehyde dehydrogenase 1 activity, CD44high/CD24low stem-like subpopulations, and tumor spheroid-forming ability. TNBC xenograft tumors treated with Nic-A exhibited decreased angiogenesis and tumor growth, as well as decreased Ki-67 expression and increased apoptosis. In addition, distant metastases were suppressed in TNBC allografts derived from a CSC-enriched population. This study thus highlights a potential strategy for addressing CSC-based cancer recurrence.

Niclosamide modulates cyclosporin A-induced hepatotoxicity in a mouse model: PPAR-γ and Wnt/ß-catenin crosstalk.

OBJECTIVES: The aim of this study was to evaluate whether: 1) Wnt/ß-catenin signaling is involved in cyclosporin A (CsA)-induced hepatotoxicity, and 2) knockdown of this pathway by niclosamide (NCL) attenuate CsA-induced hepatotoxicity. METHODS: The experiment was accomplished in 21 days. Adult male mice were randomly distributed into five groups: control group, CsA (25 mg/kg/day) group, CsA + NCL (2.5 mg/kg/day) group, CsA + NCL (5 mg/kg/day) group, and NCL (5 mg/kg/day) group. RESULTS: NCL showed marked hepatoprotection by significantly decreasing liver enzymes activities and ameliorating the histopathological alterations induced by CsA. Besides, NCL alleviated oxidative stress and inflammation. NCL-treated groups (2.5 and 5 mg/kg) displayed rise in the expression of hepatic peroxisome proliferator-activated receptor-γ (PPAR-γ) by 2.1- and 2.5-fold, respectively. Notably, NCL (2.5 and 5 mg/kg) significantly inhibited Wnt/ß-catenin signaling, evidenced by a marked decrease in the hepatic expression of Wnt3a by 54 % and 50 %, frizzled-7 receptor by 50 % and 50 %, ß-catenin by 22 % and 49 %, and c-myc by 50 % and 50 %, respectively. CONCLUSIONS: NCL can be regarded as a potential agent to mitigate CsA-induced hepatotoxicity.

Niclosamide causes lysosome-dependent cell death in endometrial cancer cells and tumors.

Endometrial cancer is the most common female cancer showing continuous rise in its incidence and mortality rate. Despite the extensive research efforts in cancer therapeutics, still there is a lack of effective treatment options and the outcome is poor for patients with advanced and recurrent endometrial cancers. In this study, we aimed to evaluate the efficacy of niclosamide (NIC) against endometrial cancer. NIC is an FDA-approved anti-helminthic drug, which has been recently extensively studied as a potent anti-cancerous agent in several cancers. The anti-cancerous activity of NIC was analyzed in-vitro (ANC3A, Hec1B, and Ishikawa endometrial cancer cell lines) by cell viability-, soft agar-, invasion- and migration- assay. The action mechanism of NIC was demonstrated by western blot analysis and immune-fluorescence imaging and validated by specific inhibitors. The in-vivo efficacy of NIC was studied in the Ishikawa xenograft animal model. NIC effectively suppressed the viability (IC50<1 µM), colony formation ability, migration, and invasion of all endometrial cancer cells tested. We demonstrated that NIC inhibited AKT/mTOR signaling pathway and induced apoptosis and autophagy in endometrial cancer cells. Further study demonstrated that although NIC induced autophagosome formation, it inhibits autolysosome formation. In addition, we observed that NIC induced BAX co-localization with lysosome and inhibited Cathepsin B maturation from pro-cathepsin B, thereby inducing the lysosomal membrane permeability and release of hydrolytic enzymes from the lysosome to cytosol, which eventually contributed cell death. NIC also inhibited tumor weight and volume in the Ishikawa xenograft animal model without having any evidence of toxicity. Due to its potent anti-cancerous activity and safety profile, NIC seems to be a promising agent for human endometrial cancer therapeutics.

Identification of niclosamide as a novel antiviral agent against porcine epidemic diarrhea virus infection by targeting viral internalization.

Porcine epidemic diarrhea virus (PEDV), an enteropathogenic coronavirus, has catastrophic impacts on the global pig industry. However, there remain no effective drugs against PEDV infection. In this study, we utilized a recombinant PEDV expressing renilla luciferase (PEDV-Rluc) to screen potential anti-PEDV agents from an FDA-approved drug library in Vero cells. Four compounds were identified that significantly decreased luciferase activity of PEDV-Rluc. Among them, niclosamide was further characterized because it exhibited the most potent antiviral activity with the highest selectivity index. It can efficiently inhibit viral RNA synthesis, protein expression and viral progeny production of classical and variant PEDV strains in a dose-dependent manner. Time of addition assay showed that niclosamide exhibited potent anti-PEDV activity when added simultaneously with or after virus infection. Furthermore, niclosamide significantly inhibited the entry stage of PEDV infection by affecting viral internalization rather than viral attachment to cells. In addition, a combination with other small molecule inhibitors of endosomal acidification enhanced the anti-PEDV effect of niclosamide in vitro. Taken together, these findings suggested that niclosamide is a novel antiviral agent that might provide a basis for the development of novel drug therapies against PEDV and other related pathogenic coronavirus infections.

Niclosamide in prostate cancer: An inhibitor of AR-V7, a mitochondrial uncoupler, or more?

A recent phase Ib study investigating the use of reformulated niclosamide in combination with abiraterone and prednisone in patients with castration-resistant prostate cancer (CRPC) demonstrated encouraging preliminary efficacy with low toxicity. Preclinical studies have reported that niclosamide at clinically relevant concentrations inhibits androgen receptor splice variant 7 (AR-V7), a known tumor driver in CRPC. However, the magnitude of anti-tumor effects of niclosamide either used alone or in combination with abiraterone in these experimental models, far exceeded what could have been explained as a simple AR-V7 inhibition. Niclosamide at clinically relevant concentrations also acts as an oxidative phosphorylation (OxPhos) uncoupler in mitochondria. This raises the question whether the observed effects of niclosamide were partly mediated by OxPhos inhibition. Most OxPhos inhibitors did not demonstrate selectivity towards cancer cells and failed to enter clinical practice due to unacceptable toxicity. However, some mitochondrial uncouplers have greater cytotoxicity against cancerous cells compared to non-cancerous. Hyperpolarization of cancer cell mitochondria, or the more alkaline mitochondrial matrix of cancer cells could be potential reasons for this. Niclosamide can also alter Wnt/ß-catenin, mTOR, Notch, NF-kB and STAT3 signaling pathways. Hence, the mechanism of action of reformulated niclosamide in CRPC patients requires further investigation. This will potentially lead to new opportunities to develop and investigate even more selective and effective treatments against prostate cancer.

Niclosamide as a Promising Therapeutic Player in Human Cancer and Other Diseases.

Niclosamide is an FDA-approved anthelmintic drug for the treatment of parasitic infections. However, over the past few years, increasing evidence has shown that niclosamide could treat diseases beyond parasitic diseases, which include metabolic diseases, immune system diseases, bacterial and viral infections, asthma, arterial constriction, myopia, and cancer. Therefore, we systematically reviewed the pharmacological activities and therapeutic prospects of niclosamide in human disease and cancer and summarized the related molecular mechanisms and signaling pathways, indicating that niclosamide is a promising therapeutic player in various human diseases, including cancer.

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model.

Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a promising therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of a perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophages including three intermediate subtypes with sharing characteristics of traditional "small" or "large" peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased the ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide restored intercellular communications between macrophages and B cells. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages. Validation of similar macrophage pathogenesis in patients will further promote the clinical usage of niclosamide for endometriosis treatment.

Selectively Targeting Breast Cancer Stem Cells by 8-Quinolinol and Niclosamide.

Cancer maintenance, metastatic dissemination and drug resistance are sustained by cancer stem cells (CSCs). Triple negative breast cancer (TNBC) is the breast cancer subtype with the highest number of CSCs and the poorest prognosis. Here, we aimed to identify potential drugs targeting CSCs to be further employed in combination with standard chemotherapy in TNBC treatment. The anti-CSC efficacy of up to 17 small drugs was tested in TNBC cell lines using cell viability assays on differentiated cancer cells and CSCs. Then, the effect of 2 selected drugs (8-quinolinol -8Q- and niclosamide -NCS-) in the cancer stemness features were evaluated using mammosphere growth, cell invasion, migration and anchorage-independent growth assays. Changes in the expression of stemness genes after 8Q or NCS treatment were also evaluated. Moreover, the potential synergism of 8Q and NCS with PTX on CSC proliferation and stemness-related signaling pathways was evaluated using TNBC cell lines, CSC-reporter sublines, and CSC-enriched mammospheres. Finally, the efficacy of NCS in combination with PTX was analyzed in vivo using an orthotopic mouse model of MDA-MB-231 cells. Among all tested drug candidates, 8Q and NCS showed remarkable specific anti-CSC activity in terms of CSC viability, migration, invasion and anchorage independent growth reduction in vitro. Moreover, specific 8Q/PTX and NCS/PTX ratios at which both drugs displayed a synergistic effect in different TNBC cell lines were identified. The sole use of PTX increased the relative presence of CSCs in TNBC cells, whereas the combination of 8Q and NCS counteracted this pro-CSC activity of PTX while significantly reducing cell viability. In vivo, the combination of NCS with PTX reduced tumor growth and limited the dissemination of the disease by reducing circulating tumor cells and the incidence of lung metastasis. The combination of 8Q and NCS with PTX at established ratios inhibits both the proliferation of differentiated cancer cells and the viability of CSCs, paving the way for more efficacious TNBC treatments.

Multiple approaches to repurposing drugs for neuroblastoma.

Neuroblastoma (NB) is the second leading extracranial solid tumor of early childhood with about two-thirds of cases presenting before the age of 5, and accounts for roughly 15 percent of all pediatric cancer fatalities in the United States. Treatments against NB are lacking, resulting in a low survival rate in high-risk patients. A repurposing approach using already approved or clinical stage compounds can be used for diseases for which the patient population is small, and the commercial market limited. We have used Bayesian machine learning, in vitro cell assays, and combination analysis to identify molecules with potential use for NB. We demonstrated that pyronaridine (SH-SY5Y IC50 1.70 µM, SK-N-AS IC50 3.45 µM), BAY 11-7082 (SH-SY5Y IC50 0.85 µM, SK-N-AS IC50 1.23 µM), niclosamide (SH-SY5Y IC50 0.87 µM, SK-N-AS IC50 2.33 µM) and fingolimod (SH-SY5Y IC50 4.71 µM, SK-N-AS IC50 6.11 µM) showed cytotoxicity against NB. As several of the molecules are approved drugs in the US or elsewhere, they may be repurposed more readily for NB treatment. Pyronaridine was also tested in combinations in SH-SY5Y cells and demonstrated an antagonistic effect with either etoposide or crizotinib. Whereas when crizotinib and etoposide were combined with each other they had a synergistic effect in these cells. We have also described several analogs of pyronaridine to explore the structure-activity relationship against cell lines. We describe multiple molecules demonstrating cytotoxicity against NB and the further evaluation of these molecules and combinations using other NB cells lines and in vivo models will be important in the future to assess translational potential.

Combination of niclosamide and current therapies to overcome resistance for cancer: New frontiers for an old drug.

Niclosamide is a drug used to treat parasitic infections. Recent studies have shown that niclosamide may have a wide range of clinical applications and can be used to treat cancer and other diseases. However, its application is also limited by its water solubility and safety, and drug resistance to cancer. To solve these problems, some studies have shown that niclosamide can be used in combination with chemotherapeutic drugs, targeted drugs, radiotherapy, and immunotherapy to enhance the anti-tumor effect. This review summarizes the drug combination strategies and therapeutic effect of niclosamide, to provide a reference for the combination therapy of niclosamide and wider application of antitumor drugs.