Antipsychotic Drug Thioridazine Research Articles

P10.02.B THE PSYCHOTROPIC AGENT THIORIDAZINE IMPEDES MELANOMA BRAIN METASTASIS CELL ACTIVITY THROUGH INHIBITION OF AUTOPHAGY

Abstract BACKGROUND Neural tissue and melanocytes share the same germ layer in early embryonic development. It is then perhaps not by coincidence that melanoma is one of the most common cancer types to metastasize to the brain. With aggressive treatment the median survival of melanoma brain metastases (MBM) is now estimated to be approximately 13 months. Targeted therapies and immunotherapy have armed us with new systemic weapons and shown great responses, but unfortunately most of these patient relapse. Drug repurposing, using old drugs for new purposes, has had several successes for other diseases. The anti-psychotic drug thioridazine (THD), a dopamine receptor 2 antagonist, with properties to cross an intact blood brain barrier, has earlier shown promising effects on melanoma cells that escaped prior treatment and on cancer stem cells. However, the therapeutic effects on MBM have not been previously investigated. MATERIAL AND METHODS We used three BRAF V600E (H1, H2 and H10) and one L577F mutated patient derived MBM cell lines (H3) developed in our lab in our experiments. A monolayer viability assay (WST-1) was utilized to determine IC50 doses. Migrational and proliferational effects of THD was studied by IncuCyte live cell imaging. Colony forming properties and the ability of cells to grow in an anchorage independent environment after THD treatment was investigated using a 2D clonogenic assay and a 3D soft agar model. A potential therapeutic window was assessed through normal human astrocytes (NHA) and fetal rat brain organoids (FRBO). Apoptosis and necrosis were studied by Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis after 72 hours of treatment. Western blots after THD treatment was done to elucidate underlying molecular mechanisms. RESULTS Cell viability studies showed that THD exerted a dose-dependent inhibition of tumor cell viability (IC50 doses between 9-12 µM), while NHA tolerated similar drug concentrations. Tumor cell migration and proliferation were almost completely inhibited with concentrations below the IC50 doses. An inhibition of clonogenic formation and 3D growth was also observed. Bright field microscopy showed changes in MBM morphology in monolayers, but FRBOs were intact after drug treatment. Apoptosis in MBM cells was induced after treatment with THD through PARP/cleaved caspase 3/Bcl-2. Expression levels of LC3B-II and p62 were significant increase in treated cells, indicating inhibition of autophagic flux at a late stage. CONCLUSION Our data shows that THD has a strong selective cytotoxicity in MBM cells through inhibition of autophagic flux, with a minimal effect on NHA and FBRO.

Screen-printed gold electrode for ultrasensitive voltammetric determination of the antipsychotic drug thioridazine

A highly sensitive, fast and simple analytical procedure for analysis of the antypsychotic drug thioridazine (TDZ) using an original screen-printed gold electrode (SPAuE) was developed. The cyclic voltammetry, scanning electron microscopy, electrochemical impedance and energy-dispersive X-ray spectroscopy were used for the SPAuE characterization. The results showed the advantage of the commercially available SPAuE in terms of working electrode active surface and the efficiency of kinetics of electron transfer compare to unmodified and ex situ modified gold film screen-printed carbon electrode. Moreover, the results displayed that an irreversible, adsorption-controlled oxidation process of TDZ in phosphate buffered saline of pH 2.6 is connected with the strong affinity of sulfur atoms for gold. The SPAuE allows for a much lower detection limit (2.9 × 10−12 M) than in the case of other electrochemical sensors used for TDZ analysis. The measurements results were validated by official and standard addition methods.

Thioridazine protects against disturbed flow-induced atherosclerosis by inhibiting RhoA/YAP-mediated endothelial inflammation.

Atherosclerotic diseases remain the leading cause of adult mortality and impose heavy burdens on health systems globally. Our previous study found that disturbed flow enhanced YAP activity to provoke endothelial activation and atherosclerosis, and targeting YAP alleviated endothelial inflammation and atherogenesis. Therefore, we established a luciferase reporter assay-based drug screening platform to seek out new YAP inhibitors for anti-atherosclerotic treatment. By screening the FDA-approved drug library, we identified that an anti-psychotic drug thioridazine markedly suppressed YAP activity in human endothelial cells. Thioridazine inhibited disturbed flow-induced endothelial inflammatory response in vivo and in vitro. We verified that the anti-inflammatory effects of thioridazine were mediated by inhibition of YAP. Thioridazine regulated YAP activity via restraining RhoA. Moreover, administration of thioridazine attenuated partial carotid ligation- and western diet-induced atherosclerosis in two mouse models. Overall, this work opens up the possibility of repurposing thioridazine for intervention of atherosclerotic diseases. This study also shed light on the underlying mechanisms that thioridazine inhibited endothelial activation and atherogenesis via repression of RhoA-YAP axis. As a new YAP inhibitor, thioridazine might need further investigation and development for the treatment of atherosclerotic diseases in clinical practice.

Abstract 12359: Thioridazine Suppresses Endothelial Inflammation and Atherogenesis Through Inhibition of Yap-jnk Signaling Axis

Introduction: Atherosclerosis is a critical public health issue as cardiovascular disease has been on the rise globally for decades. Our previous study found that YAP plays a critical role in endothelial inflammation and atherosclerosis, indicating that YAP is a promising therapeutic target against atherosclerotic vascular diseases. Therefore, we established the drug screening platform, aiming to seek new YAP inhibitors through repurposing FDA-approved drugs. Methods and Results: Drug screening was initially performed by luciferase reporter gene assay. We found that the anti-psychotic drug thioridazine can significantly inhibit the YAP/TAZ activity in human endothelial cells. Thioridazine downregulated the mRNA levels of YAP/TAZ target genes (ANKRD1, CTGF, CYR61) in endothelial cells. Thioridazine also increased the phosphorylation of YAP and inhibited the nuclear translocation of YAP in endothelial cells. Thioridazine suppressed endothelial inflammation, as demonstrated by downregulation of JNK phosphorylation, activity of JNK effector AP-1 as well as mRNA expression of pro-inflammatory markers including JNK target genes. Moreover, we observed that oral administration of thioridazine alleviated the formation of atherosclerotic plaques in ApoE -/- mice on a western diet. Conclusion: Overall, the FDA-approved clinical drug, thioridazine suppresses endothelial inflammation and atherogenesis through inhibition of YAP-JNK signaling pathway. As a novel YAP inhibitor, thioridazine might need further investigation and development for potential use of treating atherosclerotic vascular disease.

P10.16.A Repurposing thioridazine as a novel treatment of melanoma brain metastasis

Abstract Background Melanoma shows increasing incidence and has the highest propensity to metastasize to the brain of all primary malignant tumors. Untreated, the median survival time is 2-3 months after diagnosis, while aggressive treatment extends survival to only 4-12 months. Even though targeted therapies and immunotherapy has changed the therapeutic landscape, many of these patient relapse. In this respect, drug repurposing, using old drugs for new purposes, has had several successes for other diseases. The anti-psychotic drug thioridazine (THD) is a dopamine receptor 2 antagonist, with properties to cross an intact blood brain barrier, has shown promising effect on melanoma cells that has escaped prior treatment and on cancer stem cells. However, the therapeutic effects on melanoma brain metastasis (MBM) have not been previously investigated. Material and Methods All experiments were done on MBM cell lines (H1, H2, H3 and H10), which were developed in our lab from patient biopsies obtained after surgery. A monolayer viability assay (WST-1) was used to determine IC50 doses. Migration and proliferation after treatment was studied by IncuCyte live cell imaging. Colony forming properties and the ability of cells to grow in an anchorage free environment after THD treatment was investigated using a 2D clonogenic assay and a 3D soft agar model. Potential drug effects were also studied on normal human astrocytes (NHA) and on fetal rat brain organoids (FRBO), using a Live/Dead viability kit and confocal imaging. Apoptosis and necrosis was studied by Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis after 72 hours of treatment. Results THD shows a strong cytotoxic effect on MBM cell lines. Cell viability studies showed that THD exerted a dose-dependent inhibition of tumor cell viability (IC50 doses 9-12 µM), while NHA tolerated similar drug concentrations. Tumor cell migration and proliferation were almost completely inhibited with concentrations below the IC50 doses. An inhibition of clonogenic formation and 3D growth was also observed. Bright field microscopy showed changes in MBM morphology in monolayers, but FRBOs were intact after drug treatment. Further, apoptosis in MBM cells was induced after treatment with THD. Conclusion Our preliminary data shows that THD has a strong selective cytotoxicity in MBM cells with minimal effects on NHA and FBRO.

EXTH-32. NOVEL THIORIDAZINE DERIVATES: ANTIPROLIFERATIVE AND APOPTOSIS-INDUCING ACTIVITY ON GLIOBLASTOMA CELLS IN VITRO

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we showed that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of this study was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay and real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p< 0,0001, U251 p< 0,0001, LN18 p= 0,0004) as well as on glioma stem cells (GSC) (P3 p< 0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

Highly selective voltammetric detection of antipsychotic drug thioridazine hydrochloride based on NiO@Gd2O3 modified screen printed carbon electrode

• NiO@Gd 2 O 3 was synthesized via the facile co-precipitation method. • Modified sensor were studied by CV and DPV techniques for TRH detection. • NiO@Gd 2 O 3 electrode displayed WLR, low LOD, and excellent sensitivity. • The fabricated sensor shows good selectivity in MT, DA, UA, and CPZ towards TRH. The construction of mixed metal oxides matrix acts as highly active electrode materials in the field of an electrochemical sensor, owing to high conductivity, a large number of active sites, fast kinetics, and superior sensing activity. In this study, the spherical-like NiO@Gd 2 O 3 (NGO) microstructures were successfully synthesized by a simple co-precipitation method and employed for the selective electrochemical detection of thioridazine hydrochloride (TRH). The as-prepared NGO-500 material was confirmed with suitable spectrometric techniques such as X-ray diffraction (XRD), Fourier transform infrared (FT-IR), Raman spectroscopy, Brunauer–Emmett–Teller (BET) method, field emission scanning electron microscope (FE-SEM), transmission electron microscopy (TEM), and X-ray photoelectron spectrometry (XPS) analyses. The electrocatalytic performance of TRH on modified screen-printed carbon electrode (NGO-500/SPCE) was investigated through cyclic voltammetric (CV), and differential pulse voltammetric (DPV) methods. As a result, the proposed electrode demonstrated a wide linear response of 0.04–151.6 µM, a low detection limit of 0.004 µM with an excellent sensitivity of 3.09 µA µM −1 cm −2 . Besides, the modified electrode establishes outstanding selectivity in the presence of the various interfering compounds towards TRH detection. The fabricated electrode displayed notable repeatability, reproducibility, acceptable cyclic and storage stability. The feasibility of the NGO-500 electrode was used to sense TRH in urine and serum samples with satisfactory results.

Hydrothermal synthesis of iron vanadate nanoparticles for voltammetric detection of antipsychotic drug thioridazine

Antipsychotic medications play an important role in treating psychotic disorders such as delusions, chaotic thoughts, and hallucinations. However, the excessive usage of antipsychotic drugs can cause numerous undesired effects which results in strong demand for drug detection. In this report, iron vanadate nanoparticles (FeV NPs) were synthesized using a mild hydrothermal process for the electrooxidation of thioridazine hydrochloride (TDZ). In detail, FeV NPs with 50–100 nm size were obtained from the starting materials of Fe(NO3)3·9H2O/NH4VO3/(C6H9NO)n at 120 °C for 6 h. Systematic investigations such as XRD, FT–IR, Raman, FE-SEM, TEM, and XPS analyses were performed to establish the structural, morphological, and compositional profile of the prepared FeV NPs. The electrochemical impedance spectroscopy (EIS) analysis exhibited fast electron transfer and the electrochemical experiments showed an excellent catalytic activity of FeV NPs modified electrode towards the electrooxidation of TDZ. As a result, the proposed electrode revealed a broad linear concentration range of 0.02–122.1 µM and the detection limit of 0.008 µM with exceptional selectivity in the existence of organic and inorganic compounds. As a proof of concept, the electrode was tested for the recognition of TDZ in human blood serum, with a high recovery range between 93% and 102%. The electrochemical sensing results put forward that the FeV NPs and their related composites will be a favorable candidate for sensing a variety of analytes in the future.

Autophagy in Osteosarcoma Cancer Stem Cells Is Critical Process which Can Be Targeted by the Antipsychotic Drug Thioridazine.

Simple SummaryCancer stem cells (CSCs) represent a minor population of cancer cells with stem cell-like properties and appear as a crucial target in oncology as they are the origin of relapses and resistance to current treatments. Autophagy, which allows the degradation and recycling of cellular components for survival purposes, has been shown to be upregulated in some CSCs, participating in the resistance of these cells. The aim of our study was to analyze the autophagy level and the consequences of targeting this process in osteosarcoma CSCs. Our results indicate that autophagy is a critical process in osteosarcoma CSCs and that targeting this pathway allows to switch their fate from survival to death.Cancer stem cells (CSCs) represent a minor population of cancer cells with stem cell-like properties which are able to fuel tumor growth and resist conventional treatments. Autophagy has been described to be upregulated in some CSCs and to play a crucial role by maintaining stem features and promoting resistance to both hostile microenvironments and treatments. Osteosarcoma (OS) is an aggressive bone cancer which mainly affects children and adolescents and autophagy in OS CSCs has been poorly studied. However, this is a very interesting case because autophagy is often deregulated in this cancer. In the present work, we used two OS cell lines showing different autophagy capacities to isolate CSC-enriched populations and to analyze the autophagy in basal and nutrient-deprived conditions. Our results indicate that autophagy is more efficient in CSCs populations compared to the parental cell lines, suggesting that autophagy is a critical process in OS CSCs. We also showed that the antipsychotic drug thioridazine is able to stimulate, and then impair autophagy in both CSC-enriched populations, leading to autosis, a cell death mediated by the Na+/K+ ATPase pump and triggered by dysregulated accumulation of autophagosomes. Taken together, our results indicate that autophagy is very active in OS CSCs and that targeting this pathway to switch their fate from survival to death could provide a novel strategy to eradicate these cells in osteosarcoma.

Abstract 3803: Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells

Abstract In the past decades, chemotherapy has shown encouraging but limited efficacy treating triple negative breast cancer (TNBC). Targeting cancer stem cells (CSCs) has been recognized as a promising method to inhibit tumor growth and dissemination. This study aimed to determine whether an anti-psychotic drug thioridazine (THZ) could sensitize metastatic TNBC cells to anti-cancer drug carboplatin (CBP). To this end, metastatic TNBC (4T1)-bearing animals were treated with THZ, CBP and their combination respectively. MTT, flow cytometry, electron microscope and western blot were performed to evaluate the treated 4T1 cells. Tumor size and lung metastasis were monitored. The percentage of residual 4T1 cancer stem cells and the tumorigenecity of residual tumor cells from each treated group were compared. We found that both mono- and combination treatments suppressed 4T1 cells and induced apoptosis in a dose-dependent manner. These effects involved mitochondrial dysfunction and mitochondria-mediated apoptosis. The structure of mitochondrial membrane was impaired as observed by electron microscope, and the mitochondrial membrane stability was interrupted as evident by down-regulated Bcl-2 & Bcl-xL expression, and up-regulated BAX expression. Both extrinsic and intrinsic apoptosis were demonstrated by enrichment of activated caspase 3, 8, 9. Tumor growth and lung metastasis in 4T1-bearing animals were markedly inhibited by CBP combined with THZ. We also found suppression of PI3k/AKT/mTOR pathway and activation of ER stress by CSP + THZ both in vitro and in vivo. Moreover, angiogenesis was inhibited, demonstrating an important mechanism for tumor metastasis inhibition. More importantly, THZ demonstrated the capability of destroying CSCs and this effect was amplified when combined with CBP. The tumorigenecity of CBP + THZ combination-treated residual tumor cells was significantly impaired. These results show that THZ could inhibit 4T1 tumor cell proliferation and induce apoptosis and significantly destroy the stability of mitochondrial membrane, causing extrinsic and intrinsic apoptosis when combined with CBP. A signature of dysangiogenesis indicated that angiogenesis might be involved in this process. These findings suggest that the anti-psychotic drug THZ is a promising chemosensitizer in TNBC tumor and TNBC patients may benefit from THZ + CBP administration treatment by targeting cancer stem cells. Keywords: Triple-negative breast cancer (TNBC); cancer stem cells (CSCs); thioridazine (THZ); carboplatin (CBP). Citation Format: Yi Wang, Leiming Xia, Li Gong, Yang Xia, Liu Liu, Yuanyuan Liu, Wicha Max, Alfred E. Chang, Qiao Li, Yangyi Bao. Synergistic inhibition of thioridazine combined with carboplatin on triple negative breast cancer by targeting cancer stem cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3803.

One-pot sonochemical synthesis of marigold flower-like structured ruthenium doped bismuth sulfide for the highly sensitive detection of antipsychotic drug thioridazine in the human serum sample

We demonstrate the synthesis and application of marigold flower-like structured ruthenium doped bismuth sulfide (Ru-Bi2S3). The Ru-Bi2S3 was synthesized by a simple one-pot sonochemical technique at different experimental conditions. The structural behavior, morphology, and catalytic activity of the resultant material were probed via suitable spectrophotometry and voltammetric methods. The fabricated Ru-Bi2S3 modified electrode applied to investigate the electrochemical oxidation of thioridazine (TZ). Captivatingly, the modified electrode has dynamic linear ranges from 0.005 to 7.415 µM and 7.415 to 1432 µM. Further, the lowest detection limit (0.001 µM) and higher sensitivity (8.639 µA µM−1 cm−2) was attained towards the electrochemical sensing of TZ. Besides, the practicability of the TZ sensor was evaluated in a human serum sample using the standard addition method.

Structures of Ebola Virus Glycoprotein Complexes with Tricyclic Antidepressant and Antipsychotic Drugs.

A large number of Food and Drug Administration (FDA)-approved drugs have been found to inhibit the cell entry of Ebola virus (EBOV). However, since these drugs have various primary pharmacological targets, their mechanisms of action against EBOV remain largely unknown. We have previously shown that six FDA-approved drugs inhibit EBOV infection by interacting with and destabilizing the viral glycoprotein (GP). Here we show that antidepressants imipramine and clomipramine and antipsychotic drug thioridazine also directly interact with EBOV GP and determine the mode of interaction by crystallographic analysis of the complexes. The compounds bind within the same pocket as observed for other, chemically divergent complexes but with different binding modes. These details should be of value for the development of potent EBOV inhibitors.

Environmental fate and effect assessment of thioridazine and its transformation products formed by photodegradation

An experimental and in silico quantitative structure-activity relationship (QSAR) approach was applied to assess the environmental fate and effects of the antipsychotic drug Thioridazine (THI). The sunlight-driven attenuation of THI was simulated using a Xenon arc lamp. The photodegradation reached the complete primary elimination, whereas 97% of primary elimination and 11% of mineralization was achieved after 256 min of irradiation for the initial concentrations of 500 μg L−1 and 50 mg L−1, respectively. A non-target approach for the identification and monitoring of transformation products (TPs) was adopted. The structure of the TPs was further elucidated using liquid chromatography–high resolution mass spectrometry (LC–HRMS). The proposed photodegradation pathway included sulfoxidation, hydroxylation, dehydroxylation, and S- and N-dealkylation, taking into account direct and indirect photolysis through a self-sensitizing process in the higher concentration studied. The biodegradability of THI and photolytic samples of THI was tested according to OECD 301D and 301F, showing that THI and the mixture of TPs were not readily biodegradable. Furthermore, THI was shown to be highly toxic to environmental bacteria using a modified luminescent bacteria test with Vibrio fischeri. This bacteriotoxic activity of THI was significantly reduced by phototransformation and individual concentration-response analysis confirmed a lowered bacterial toxicity for the sulfoxidation products Thioridazine-2-sulfoxide and Thioridazine-5-sulfoxide. Additionally, the applied QSAR models predicted statistical and rule-based positive alerts of mutagenic activities for carbazole derivative TPs (TP 355 and TP 339) formed through sulfoxide elimination, which would require further confirmatory in vitro validation tests.

Channeling brain cancer

Drug development based on the antipsychotic drug thioridazine may lead to treatments for metastatic medulloblastoma without compromising quality of life.

EAG2 potassium channel with evolutionarily conserved function as a brain tumor target

Over 20% of the drugs for treating human diseases target ion channels, but no cancer drug approved by the US Food and Drug Administration (FDA) is intended to target an ion channel. We found that the EAG2 (Ether-a-go-go 2) potassium channel has an evolutionarily conserved function for promoting brain tumor growth and metastasis, delineate downstream pathways, and uncover a mechanism for different potassium channels to functionally cooperate and regulate mitotic cell volume and tumor progression. EAG2 potassium channel was enriched at the trailing edge of migrating medulloblastoma (MB) cells to regulate local cell volume dynamics, thereby facilitating cell motility. We identified the FDA-approved antipsychotic drug thioridazine as an EAG2 channel blocker that reduces xenografted MB growth and metastasis, and present a case report of repurposing thioridazine for treating a human patient. Our findings illustrate the potential of targeting ion channels in cancer treatment.

Identification of thioridazine, an antipsychotic drug, as an antiglioblastoma and anticancer stem cell agent using public gene expression data.

Glioblastoma (GBM) is a common and malignant tumor with a poor prognosis. Glioblastoma stem cells (GSCs) have been reported to be involved in tumorigenesis, tumor maintenance and therapeutic resistance. Thus, to discover novel candidate therapeutic drugs for anti-GBM and anti-GSCs is an urgent need. We hypothesized that if treatment with a drug could reverse, at least in part, the gene expression signature of GBM and GSCs, this drug may have the potential to inhibit pathways essential in the formation of GBM and thereby treat GBM. Here, we collected 356 GBM gene signatures from public databases and queried the Connectivity Map. We systematically evaluated the in vitro antitumor effects of 79 drugs in GBM cell lines. Of the drugs screened, thioridazine was selected for further characterization because it has potent anti-GBM and anti-GSCs properties. When investigating the mechanisms underlying the cytocidal effects of thioridazine, we found that thioridazine induces autophagy in GBM cell lines, and upregulates AMPK activity. Moreover, LC3-II was upregulated in U87MG sphere cells treated with thioridazine. In addition, thioridazine suppressed GBM tumorigenesis and induced autophagy in vivo. We not only repurposed the antipsychotic drug thioridazine as a potent anti-GBM and anti-GSCs agent, but also provided a new strategy to search for drugs with anticancer and anticancer stem cell properties.

Differential effects of thioridazine enantiomers on action potential duration in rabbit papillary muscle

The antipsychotic drug thioridazine has potential for treatment of multidrug-resistant microbes including tuberculosis but also causes cardiotoxic QT interval prolongation. Both thioridazine enantiomers have potent antimicrobial effects, but the neuroleptic effect primarily resides with (+)-thioridazine. In this study we for the first time investigate the cardiotoxicity of the isolated thioridazine enantiomers and show their effects on ventricular repolarization. The effects of (+)-thioridazine, (−)-thioridazine, and racemate on the rabbit ventricular action potential duration (APD) were investigated in a randomized controlled blinded experiment. Action potentials were measured in papillary muscles isolated from 21 female rabbits, and the drug effect on 90% APD in comparison with control (ΔΔ-APD90) was evaluated. Increasing concentrations of (+)-thioridazine and the racemate caused significant dose-dependent ΔΔ-APD90 prolongation, while (−)-thioridazine did not. At 0.5 and 2Hz pacing, (+)-thioridazine caused 19.5% and 20.1% ΔΔ-APD90 prolongation, the racemate caused 8.0% and 12.9%, and (−)-thioridazine caused 1.5% and 1.1%. The effect of (−)-thioridazine on APD90 was significantly less than that of the other drugs at both pacing rates (P<0.01 in all cases), and there was no significant difference between (−)-thioridazine and control. The results of this study indicate that the APD prolonging effect of thioridazine is primarily due to the (+)-thioridazine enantiomer. If these results are valid in humans, (−)-thioridazine may be a safer drug for treatment of multidrug-resistant tuberculosis and other microbes.

Reduced Emergence of Isoniazid Resistance with Concurrent Use of Thioridazine against Acute Murine Tuberculosis

The repurposing of existing drugs is being pursued as a means by which to accelerate the development of novel regimens for the treatment of drug-susceptible and drug-resistant tuberculosis (TB). In the current study, we assessed the activity of the antipsychotic drug thioridazine (TRZ) in combination with the standard regimen in a well-validated murine TB model. Single-dose and steady-state pharmacokinetic studies were performed in BALB/c mice to establish human-equivalent doses of TRZ. To determine the bactericidal activity of TRZ against TB in BALB/c mice, three separate studies were performed, including a dose-ranging study of TRZ monotherapy and efficacy studies of human-equivalent doses of TRZ with and without isoniazid (INH) or rifampin (RIF). Therapeutic efficacy was assessed by the change in mycobacterial load in the lung. The human-equivalent dose of thioridazine was determined to be 25 mg/kg of body weight, which was well tolerated in mice. TRZ was found to accumulate at high concentrations in lung tissue relative to serum levels. We observed modest synergy during coadministration of TRZ with INH, and the addition of TRZ reduced the emergence of INH-resistant mutants in mouse lungs. In conclusion, this study further illustrates the opportunity to reevaluate the contribution of TRZ to the sterilizing activity of combination regimens to prevent the emergence of drug-resistant M. tuberculosis.

Thioridazine, an antipsychotic drug, elicits potent antitumor effects in gastric cancer

Thioridazine, an antipsychotic drug, has been reported to induce apoptosis in various types of cancer cells, with specificity on targeting cancer stem cells (CSCs). However, whether it elicits anticancer effects in gastric cancer has never been reported. In the present study, we examined the ability of thioridazine to induce cell death in the gastric cancer cell lines NCI-N87 and AGS, and detected its in vivo tumor inhibition capacity. Thioridazine elicited cytotoxic effects on NCI-N87 and AGS cells in a dose-dependent manner, and inhibited the colony formation abilitiy of the NCI-N87 and AGS cells. Thioridazine treatment induced nuclear fragmentation, increased the proportion of sub-G1 phase cells, and elevated the percentage of Annexin V-positive cells, suggesting the occurrence of apoptosis. Moreover, thioridazine induced gastric cancer cell apoptosis in a caspase-dependent manner, as shown by a decrease in the precursors of casapse-9, caspase-8 and caspase-3, and by the ability of the caspase inhibitor Z-VAD-FMK to reverse the cytotoxic effect of thioridazine. JC-1 staining further revealed that thioridazine induced gastric cancer cell apoptosis via the mitochondrial pathway. In addition, thioridazine pretreatment inhibited the growth of NCI-N87 cell-derived tumors. The present study demonstrated that the antipsychotic drug thioridazine possesses anti-gastric cancer ability through in vitro and in vivo experiments, suggesting thioridazine as a potential drug in gastric cancer therapy.