ABSTRACT
Chronic myeloid leukemia (CML) is a clonal myeloproliferative hematological disease characterized by the chimeric breakpoint-cluster region/Abelson kinase1 (BCR::ABL1) oncoprotein; playing a pivotal role in CML molecular pathology, diagnosis, treatment, and possible resistance arising from the success and tolerance of tyrosine kinase inhibitor (TKI)-based therapy. The transcription factor STAT5 constitutive signaling, which is influenced by the cytokine signaling network, triggers BCR::ABL1-based CML pathogenesis and is also relevant to acquired TKI resistance. The unsuccessful therapeutic approaches targeting BCR::ABL1, in particular third-line therapy with ponatinib, still need to be further developed with alternative combination strategies to overcome drug resistance. As treatment with the STAT5 inhibitor pimozide in combination with ponatinib resulted in an efficient and synergistic therapeutic approach in TKI-resistant CML cells, this study focused on identifying the underlying amplification of ponatinib response mechanisms by determining different cytokine expression profiles in parental and ponatinib-resistant CML cells, in vitro. The results showed that expression of interleukin (IL) 1B, IL9, and IL12A-B was increased by 2-fold, while IL18 was downregulated by 2-fold in the ponatinib-resistant cells compared to sensitive ones. Importantly, ponatinib treatment upregulated the expression of 21 of the 23 interferon and IL genes in the ponatinib-resistant cells, while treatment with pimozide or a combination dose resulted in a reduction in the expression of 19 different cytokine genes, such as for example, inflammatory cytokines, IL1A-B and IL6 or cytokine genes associated with supporting tumor progression, leukemia stem cell growth or poor survival, such as IL3, IL8, IL9, IL10, IL12, or IL15. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis results showed that the genes were mainly enriched in the regulation of receptor signaling through the Janus kinase/signal transducer and activator of transcription pathway, cytokine-cytokine receptor interaction, and hematopoietic cell lineage. Protein-protein interaction analysis showed that IL2, IL6, IL15, IFNG, and others appeared in the top lists of pathways, indicating their high centrality and importance in the network. Therefore, pimozide could be a promising agent to support TKI therapies in ponatinib resistance. This research would help to clarify the role of cytokines in ponatinib resistance and advance the development of new therapeutics to utilize the STAT5 inhibitor pimozide in combination with TKIs.
Subject(s)
Imidazoles , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Pimozide , Pyridazines , Humans , Pimozide/pharmacology , Pimozide/therapeutic use , Cytokines/metabolism , Drug Resistance, Neoplasm/genetics , Fusion Proteins, bcr-abl/genetics , Fusion Proteins, bcr-abl/metabolism , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , Interleukin-15/metabolism , Interleukin-15/therapeutic use , Interleukin-6/metabolism , Interleukin-9/metabolism , Interleukin-9/therapeutic use , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathologyABSTRACT
The antipsychotic drug pimozide has been demonstrated to inhibit cancer. However, the precise anti-cancer mechanism of pimozide remains unclear. The purpose of this study was to investigate the effects of pimozide on human MCF-7 and MDA-MB-231 breast cancer cell lines, and the potential involvement in the RAF/ERK signaling. The effects of pimozide on cells were examined by 4,5-dimethylthiazol-2-yl-3,5-diphenylformazan, wound healing, colony formation, transwell assays, and caspase activity assay. Flow cytometry and acridine orange and ethidium bromide staining were performed to assess changes in cells. Transmission electron microscopy and monodansylcadaverine staining were used to observe autophagosomes. The cyclic adenosine monophosphate was evaluated using the FRET system. Immunohistochemistry, immunofluorescence, RNA interference, and western blot investigated the expression of proteins. Mechanistically, we focus on the RAF1/ERK signaling. We detected pimozide was docked to RAF1 by Schrodinger software. Pimozide down-regulated the phosphorylation of RAF1, ERK 1/2, Bcl-2, and Bcl-xl, up-regulated Bax, and cleaved caspase-9 to induce apoptosis. Pimozide might promote autophagy by up-regulating cAMP. The enhancement of autophagy increased the conversion of LC3-I to LC3-II and down-regulated p62 expression. But mTOR signaling was not involved in promoting autophagy. The knockdown of RAF1 expression induced autophagy and apoptosis in breast cancer cells, consistent with the results of pimozide or sorafenib alone. Blocked autophagy by chloroquine resulted in the impairment of pimozide-induced apoptosis. These data showed that pimozide inhibits breast cancer by regulating the RAF/ERK signaling pathway and might activate cAMP-induced autophagy to promote apoptosis and it may be a potential drug for breast cancer treatment.
Subject(s)
Antipsychotic Agents , Breast Neoplasms , Humans , Female , MAP Kinase Signaling System , Breast Neoplasms/drug therapy , Antipsychotic Agents/pharmacology , Antipsychotic Agents/therapeutic use , Pimozide/pharmacology , Cell Proliferation , Apoptosis , Autophagy , Cell Line, TumorABSTRACT
Of the two putative amino acid binding periplasmic receptors of ABC transporter family in Candidatus Liberibacter asiaticus (CLas), cystine binding receptor (CLasTcyA) has been shown to mainly express in phloem of citrus plant and is a target for inhibitor development. The crystal structure of CLasTcyA in complex with substrates has been reported earlier. The present work reports the identification and evaluation of potential candidates for their inhibitory potential against CLasTcyA. Among many compounds, selected through virtual screening, and MD simulation, pimozide, clidinium, sulfasalazine and folic acid showed significantly higher affinities and stability in complex with CLasTcyA. The SPR studies with CLasTcyA revealed significantly higher binding affinities for pimozide and clidinium (Kd, 2.73 nM and 70 nM, respectively) as compared to cystine (Kd, 1.26 µM). The higher binding affinities could be attributed to significantly increased number of interactions in the binding pocket as evident from the crystal structures of CLasTcyA in complex with pimozide and clidinium as compared to cystine. The CLasTcyA possess relatively large binding pocket where bulkier inhibitors fit quite well. In planta studies, carried out to assess the effect of inhibitors on HLB infected Mosambi plants, showed significant reduction in CLas titre in plants treated with inhibitors as compared to control plants. The results showed that pimozide exhibited higher efficiency as compared to clidinium in reducing CLas titre in treated plants. Our results showed that the inhibitor development against critical proteins like CLasTcyA can be an important strategy in management of HLB.
Subject(s)
Rhizobiaceae , Cystine/pharmacology , Pimozide/pharmacology , Plant DiseasesABSTRACT
In spite of the higher nosocomial and community-acquired infections caused by Staphylococcus aureus, emerging drug resistance is a leading cause of increased mortality and morbidity associated with the overuse of antimicrobials. It is an emergent need to find out new molecules to combat such infections. In the present study, we analyzed the antibacterial effect of pimozide (PMZ) against gram-positive and gram-negative bacterial strains, including methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus. The growth of MSSA and MRSA was completely inhibited at concentrations of 12.5 and 100 µg/mL, respectively, which is referred to as 1× minimum inhibitory concentration (MIC). The cell viability was completely eliminated within 90 min of PMZ treatment (2× MIC) through reactive oxygen species (ROS)-mediated killing without affecting cell membrane permeability. It suppressed α-hemolysin production and biofilm formation of different S. aureus strains by almost 50% at 1× MIC concentration, and was found to detach matured biofilm. PMZ treatment effectively eliminates S. aureus infection in Caenorhabditis elegans and improves its survival by 90% and is found safe to use with no hemolytic effect on human and chicken blood tissues. Taken together, it is concluded that PMZ may turn out to be an effective antibacterial for treating bacterial infections including MSSA and MRSA.
Subject(s)
Anti-Infective Agents , Methicillin-Resistant Staphylococcus aureus , Staphylococcal Infections , Humans , Staphylococcus aureus , Pimozide/pharmacology , Reactive Oxygen Species , Anti-Bacterial Agents/pharmacology , Staphylococcal Infections/drug therapy , Methicillin/pharmacology , Anti-Infective Agents/pharmacology , Microbial Sensitivity TestsABSTRACT
Pimozide is an antipsychotic drug used to treat chronic psychosis, such as Tourette's syndrome. Despite its widespread clinical use, pimozide can cause unexpected adverse effects, including arrhythmias. However, the adverse effects of pimozide on vascular K+ channels have not yet been determined. Therefore, we investigated the effects of pimozide on voltage-gated K+ (Kv) channels in rabbit coronary arterial smooth muscle cells. Pimozide concentration-dependently inhibited the Kv currents with an IC50 value of 1.78 ± 0.17 µM and a Hill coefficient of 0.90 ± 0.05. The inhibitory effect on the Kv current by pimozide was highly voltage-dependent in the voltage range of Kv channel activation, and additive inhibition of the Kv current by pimozide was observed in the full activation voltage range. The decay rate of inactivation was significantly accelerated by pimozide. Pimozide shifted the inactivation curve to a more negative potential. The recovery time constant from inactivation increased in the presence of pimozide. Furthermore, pimozide-induced inhibition of the Kv current was augmented by applying train pulses. Although pretreatment with the Kv2.1 subtype inhibitor guangxitoxin and the Kv7 subtype inhibitor linopirdine did not alter the degree of pimozide-induced inhibition of the Kv currents, pretreatment with the Kv1.5 channel inhibitor DPO-1 reduced the inhibitory effects of pimozide on Kv currents. Pimozide induced membrane depolarization. We conclude that pimozide inhibits Kv currents in voltage-, time-, and use (state)-dependent manners. Furthermore, the major Kv channel target of pimozide is the Kv1.5 channel.
Subject(s)
Antipsychotic Agents , Potassium Channels, Voltage-Gated , Animals , Rabbits , Antipsychotic Agents/toxicity , Pimozide/pharmacology , Potassium Channel Blockers/pharmacology , Muscle, Smooth, Vascular , Potassium Channels, Voltage-Gated/pharmacology , Myocytes, Smooth MuscleABSTRACT
Despite significant progress in understanding the pathogenesis of type 2 diabetes (T2D), the condition remains difficult to manage. Hence, new therapeutic options targeting unique mechanisms of action are required. We have previously observed that elevated skeletal muscle succinyl CoA:3-ketoacid CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone oxidation, contributes to the hyperglycemia characterizing obesity and T2D. Moreover, we identified that the typical antipsychotic agent pimozide is a SCOT inhibitor that can alleviate obesity-induced hyperglycemia. We now extend those observations here, using computer-assisted in silico modeling and in vivo pharmacology studies that highlight SCOT as a noncanonical target shared among the diphenylbutylpiperidine (DPBP) drug class, which includes penfluridol and fluspirilene. All three DPBPs tested (pimozide, penfluridol, and fluspirilene) improved glycemia in obese mice. While the canonical target of the DPBPs is the dopamine 2 receptor, studies in obese mice demonstrated that acute or chronic treatment with a structurally unrelated antipsychotic dopamine 2 receptor antagonist, lurasidone, was devoid of glucose-lowering actions. We further observed that the DPBPs improved glycemia in a SCOT-dependent manner in skeletal muscle, suggesting that this older class of antipsychotic agents may have utility in being repurposed for the treatment of T2D.
Subject(s)
Antipsychotic Agents , Diabetes Mellitus, Type 2 , Hyperglycemia , Animals , Mice , Antipsychotic Agents/pharmacology , Antipsychotic Agents/therapeutic use , Coenzyme A-Transferases , Diabetes Mellitus, Type 2/drug therapy , Dopamine , Fluspirilene/pharmacology , Hyperglycemia/drug therapy , Mice, Obese , Penfluridol/pharmacology , Pimozide/pharmacology , Receptors, Dopamine/metabolismABSTRACT
Signal Transducer and Activator of Transcription 5 (STAT5) is a transcription factor that plays a key role in neoplasia, triggered by the fusion oncogene BCR-ABL1; it is not only an essential protein for the pathogenesis of chronic myeloid leukemia (CML), but also its overexpression is associated with drug resistance developed toward various generations of Tyrosine Kinase Inhibitors (TKIs); these are still accepted as gold standard therapeutics for the treatment of CML. In this study, it was investigated whether suppression of STAT5 via a "STAT5 inhibitor" Pimozide resulted in any regain of chemosensitivity to third-generation TKI Ponatinib. Accordingly, the experimental work was designed on both parental CML cell line K562WT and its 1 nM Ponatinib-resistant counterpart, indicated as K562-Pon1. Based on the experimental results, Pimozide was more effective in resistant cells compared to wild-type cells for inducing apoptosis and block cell arrest. Combination therapy of Pimozide and Ponatinib demonstrated that STAT5 was a significant protein for regaining chemosensitivity to Ponatinib when its expression was suppressed both at mRNA and protein level. In conclusion, we consider that STAT5 inhibitor Pimozide can be a good alternative or combination therapy with TKIs for patients suffering from chemotherapeutic drug resistance. Communicated by Ramaswamy H. Sarma.
Subject(s)
Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Pyridazines , Humans , K562 Cells , Fusion Proteins, bcr-abl , Pimozide/pharmacology , Pimozide/therapeutic use , STAT5 Transcription Factor/genetics , STAT5 Transcription Factor/metabolism , STAT5 Transcription Factor/pharmacology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Drug Resistance, Neoplasm/genetics , Pyridazines/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Proteins/metabolism , Apoptosis , Aryldialkylphosphatase/metabolism , Aryldialkylphosphatase/pharmacology , Aryldialkylphosphatase/therapeutic useABSTRACT
BACKGROUND: The taxane cabazitaxel (CBZ) is a promising treatment for docetaxel-resistant castration-resistant prostate cancer (CRPC). However, the survival benefit with CBZ for patients with CRPC is limited. This study used screening tests for candidate drugs targeting CBZ-resistant-related gene expression and identified pimozide as a potential candidate for overcoming CBZ resistance in CRPC. METHODS: We established CBZ-resistant cell lines, DU145CR and PC3CR by incubating DU145 cells and PC3 cells with gradually increasing concentrations of CBZ. We performed in silico drug screening for candidate drugs that could reprogram the gene expression signature of a CBZ-resistant prostate cancer cells using a Connectivity Map. The in vivo effect of the drug combination was tested in xenograft mice models. RESULTS: We identified pimozide as a promising candidate drug for CBZ-resistant CRPC. Pimozide had a significant antitumor effect on DU145CR cells. Moreover, combination treatment with pimozide and CBZ had a synergic effect for DU145CR cells in vitro and in vivo. Microarray analysis identified AURKB and KIF20A as potential targets of pimozide in CBZ-resistant CRPC. DU145CR had significantly higher AURKB and KIF20A expression compared with a non-CBZ-resistant cell line. Inhibition of AURKB and KIF20A had an antitumor effect in DU145CR xenograft tumors. Higher expression of AURKB and KIF20A was a poor prognostic factor of TGCA prostate cancer cohort. CBZ-resistant prostate cancer tissues in our institution had higher AURKB and KIF20A expression. CONCLUSIONS: Pimozide appears to be a promising drug to overcome CBZ resistance in CRPC by targeting AURKB and KIF20A.
Subject(s)
Antineoplastic Agents , Prostatic Neoplasms, Castration-Resistant , Male , Humans , Animals , Mice , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/genetics , Prostatic Neoplasms, Castration-Resistant/metabolism , Drug Resistance, Neoplasm/genetics , Pimozide/pharmacology , Pimozide/therapeutic use , Early Detection of Cancer , Taxoids/pharmacology , Taxoids/therapeutic use , Cell Line, TumorABSTRACT
Ubiquitin-specific protease 1 (USP1) is a deubiquitinase involved in DNA damage repair by modulating the ubiquitination of major regulators, such as PCNA and FANCD2. Because USP1 is highly expressed in many cancers, dysregulation of USP1 contributes to cancer therapy. However, the role of USP1 and the mechanisms underlying chemotherapy remain unclear. In this study, we found high USP1 expression in tumor tissues and that it correlated with poor prognosis in RCC. Mechanistically, USP1 enhanced survivin stabilization by removing ubiquitin. Pharmacological inhibitors (ML23 and pimozide) and siRNA targeting USP1 induced downregulation of survivin expression. In addition, ML323 upregulated DR5 expression by decreasing miR-216a-5p expression at the post-transcriptional level, and miR-216a-5p mimics suppressed the upregulation of DR5 by ML323. Inhibition of USP1 sensitized cancer cells. Overexpression of survivin or knockdown of DR5 markedly prevented the co-treatment with ML323 and TRAIL-induced apoptosis. These results of in vitro were proved in a mouse xenograft model, in which combined treatment significantly reduced tumor size and induced survivin downregulation and DR5 upregulation. Furthermore, USP1 and survivin protein expression showed a positive correlation, whereas miR-216a-5p and DR5 were inversely correlated in RCC tumor tissues. Taken together, our results suggest two target substrates of USP1 and demonstrate the involvement of survivin and DR5 in USP1-targeted chemotherapy.
Subject(s)
Antineoplastic Agents , Carcinoma, Renal Cell , Kidney Neoplasms , MicroRNAs , Ubiquitin-Specific Proteases , Animals , Antineoplastic Agents/pharmacology , Apoptosis/genetics , Carcinoma, Renal Cell/metabolism , Cell Line, Tumor , Deubiquitinating Enzymes/genetics , Down-Regulation/genetics , Humans , Mice , MicroRNAs/metabolism , Pimozide/pharmacology , Proliferating Cell Nuclear Antigen/metabolism , RNA, Small Interfering/pharmacology , Receptors, TNF-Related Apoptosis-Inducing Ligand , Survivin/genetics , Survivin/metabolism , Ubiquitin-Specific Proteases/antagonists & inhibitors , Ubiquitin-Specific Proteases/genetics , Ubiquitins/metabolism , Up-Regulation/geneticsABSTRACT
T-type Ca2+ channels (T-channels), particularly Cav3.2 and Cav3.1 isoforms, are promising targets for treating various diseases including intractable pain. Given the potent inhibitory activity of pimozide, an antipsychotic, against T-channels, we conducted structure-activity relationship studies of pimozide derivatives, and identified several compounds including 3a, 3s, and 4 that had potency comparable to that of pimozide in inhibiting T-channels, but little binding affinity to dopamine D2 receptors. The introduction of a phenylbutyl group on the benzoimidazole nuclei of pimozide was considered a key structural modification to reduce the binding affinity to D2 receptors. Those pimozide derivatives potently suppressed T-channel-dependent somatic and visceral pain in mice, without causing any motor dysfunctions attributable to D2 receptor blockade, including catalepsy. The present study thus provides an avenue to develop novel selective T-channel inhibitors available for pain management via the structural modification of existing medicines.
Subject(s)
Calcium Channels, T-Type , Visceral Pain , Mice , Animals , Pimozide/pharmacology , Pimozide/therapeutic use , Calcium Channels, T-Type/metabolism , Visceral Pain/drug therapy , Dopamine , Calcium Channel Blockers/chemistry , Receptors, Dopamine/metabolismABSTRACT
The Warburg effect is a promising target for the diagnosis and treatment of cancer, referring to the ability of cancer cells to generate energy through high levels of glycolysis even in the presence of oxygen, allowing them to grow and proliferate rapidly. The antipsychotic Pimozide has strong anti-breast cancer effects both in vivo and in vitro, whether Pimozide has an inhibitory effect on aerobic glycolysis has not been elucidated. In this study, Pimozide inhibited the Warburg effect of breast cancer cells by hindering glucose uptake, ATP level and lactate production; reducing the extracellular acidification rate (ECAR); suppressing the expression of PKM2, a rate-limiting enzyme in glycolysis. Intriguingly, Pimozide was significantly involved in reprogramming glucose metabolism in breast cancer cells through a p53-dependent manner. Mechanistic studies demonstrated Pimozide increased the expression of p53 through inhibition of the PI3K/Akt/MDM2 signaling pathway, which in turn downregulated the expression of PKM2. In sum, our results suggest that Pimozide mediates the p53 signaling pathway through PI3K/AKT/MDM2 to inhibit the Warburg effect and breast cancer growth, and it may be a potential aerobic glycolysis inhibitor for the treatment of breast cancer.
Subject(s)
Breast Neoplasms , Tumor Suppressor Protein p53 , Breast Neoplasms/drug therapy , Cell Line, Tumor , Cell Proliferation , Female , Glycolysis , Humans , Phosphatidylinositol 3-Kinases/metabolism , Pimozide/pharmacology , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Tumor Suppressor Protein p53/metabolismABSTRACT
The present study focuses on the interconnected functional network of altered metabolism and EMT (epithelial to mesenchymal transition) signaling in breast cancer. We have interlinked the metabolic and EMT signaling circuits and selected Insulin receptor (IR), Integrin beta 1 (ITGB1), and CD36 as target proteins based on network analysis. Extensive computational approaches discerned the potential drug molecules from the library of 1293 FDA-approved drugs to block all three target proteins. Using molecular docking, molecular dynamics simulation, and MMPBSA binding free energy studies, Capmatinib, Ponatinib, Naldemedine, and Pimozide were identified as potential repurposed drugs to block the function of all three target proteins. Among in silico selected candidate drugs, Pimozide, a known anti-psychotic drug, was further validated using in-vitro studies for its anti-cell proliferative potential on breast cancer cell lines (namely, MCF7, MDAMB231 and MDAMB468). The inhibitory concentration (IC50 ) values of MCF7, MDAMB231 and MDAMB468 was found to be 16.26â µM, 20.82â µM and 13.10â µM, respectively. The effect of Pimozide on EMT-induced MDAMB231 and MDAMB468 cells was evident from their IC50 values of 7.85â µM and 6.83â µM, respectively. The potent anti-cancer property of Pimozide has opened up avenues for drug repurposing towards 'multi-targeted therapy' in EMT dynamics.
Subject(s)
Breast Neoplasms , Drug Repositioning , Breast Neoplasms/drug therapy , Cell Line, Tumor , Epithelial-Mesenchymal Transition , Female , Humans , Molecular Docking Simulation , Pimozide/pharmacologyABSTRACT
The persistent increase of multidrug-resistant (MDR) Mycobacterium tuberculosis (Mtb) infections negatively impacts Tuberculosis treatment outcomes. Host-directed therapies (HDT) pose an complementing strategy, particularly since Mtb is highly successful in evading host-defense by manipulating host-signaling pathways. Here, we screened a library containing autophagy-modulating compounds for their ability to inhibit intracellular Mtb-bacteria. Several active compounds were identified, including two drugs of the diphenylbutylpiperidine-class, Fluspirilene and Pimozide, commonly used as antipsychotics. Both molecules inhibited intracellular Mtb in pro- as well as anti-inflammatory primary human macrophages in a host-directed manner and synergized with conventional anti-bacterials. Importantly, these inhibitory effects extended to MDR-Mtb strains and the unrelated intracellular pathogen, Salmonella enterica serovar Typhimurium (Stm). Mechanistically Fluspirilene and Pimozide were shown to regulate autophagy and alter the lysosomal response, partly correlating with increased bacterial localization to autophago(lyso)somes. Pimozide's and Fluspirilene's efficacy was inhibited by antioxidants, suggesting involvement of the oxidative-stress response in Mtb growth control. Furthermore, Fluspirilene and especially Pimozide counteracted Mtb-induced STAT5 phosphorylation, thereby reducing Mtb phagosome-localized CISH that promotes phagosomal acidification. In conclusion, two approved antipsychotic drugs, Pimozide and Fluspirilene, constitute highly promising and rapidly translatable candidates for HDT against Mtb and Stm and act by modulating the autophagic/lysosomal response by multiple mechanisms.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antipsychotic Agents/pharmacology , Antitubercular Agents/pharmacology , Drug Repositioning , Mycobacterium tuberculosis/drug effects , Salmonella enterica/drug effects , Autophagy/drug effects , Cell Line , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , Lysosomes/metabolism , Microbial Sensitivity Tests , Models, Biological , Phagosomes/metabolism , Pimozide/pharmacology , Salmonella Infections/drug therapy , Salmonella Infections/microbiology , Small Molecule Libraries , Tuberculosis/drug therapy , Tuberculosis/microbiologyABSTRACT
Colorectal cancer (CRC) is a recurring cancer that is often resistant to conventional therapies and therefore requires the development of molecular-based therapeutic approaches. Dopamine receptor D2 (DRD2) is associated with the growth of many types of tumors, but its oncogenic role in CRC is unclear. Here, we observed that elevated DRD2 expression was associated with a poor survival rate among patients with CRC. Depletion of DRD2 suppressed CRC cell growth and motility by downregulating ß-catenin/ZEB signaling in vitro and in vivo, whereas overexpression of DRD2 promoted CRC cell progression. Inhibition of DRD2 by the antagonist pimozide inhibited tumor growth and lymph node metastasis in vivo and enhanced the cytotoxic effects of conventional agents in vitro. Taken together, our findings indicate that targeting the DRD2/ß-catenin/ZEB1 signaling axis is a potentially promising therapeutic strategy for patients with CRC.
Subject(s)
Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Disease Progression , Receptors, Dopamine D2/metabolism , Zinc Finger E-box-Binding Homeobox 1/metabolism , beta Catenin/metabolism , Aged , Animals , Cell Movement/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Dopamine Antagonists/pharmacology , Female , HCT116 Cells , HT29 Cells , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Middle Aged , Pimozide/pharmacology , RNA Interference , Receptors, Dopamine D2/genetics , Signal Transduction , Survival Rate , Transfection , Tumor Burden/drug effects , Tumor Burden/genetics , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
Increasing evidence suggests that induction of lethal macroautophagy/autophagy carries potential significance for the treatment of glioblastoma (GBM). In continuation of previous work, we demonstrate that pimozide and loperamide trigger an ATG5- and ATG7 (autophagy related 5 and 7)-dependent type of cell death that is significantly reduced with cathepsin inhibitors and the lipid reactive oxygen species (ROS) scavenger α-tocopherol in MZ-54 GBM cells. Global proteomic analysis after treatment with both drugs also revealed an increase of proteins related to lipid and cholesterol metabolic processes. These changes were accompanied by a massive accumulation of cholesterol and other lipids in the lysosomal compartment, indicative of impaired lipid transport/degradation. In line with these observations, pimozide and loperamide treatment were associated with a pronounced increase of bioactive sphingolipids including ceramides, glucosylceramides and sphingoid bases measured by targeted lipidomic analysis. Furthermore, pimozide and loperamide inhibited the activity of SMPD1/ASM (sphingomyelin phosphodiesterase 1) and promoted induction of lysosomal membrane permeabilization (LMP), as well as release of CTSB (cathepsin B) into the cytosol in MZ-54 wild-type (WT) cells. Whereas LMP and cell death were significantly attenuated in ATG5 and ATG7 knockout (KO) cells, both events were enhanced by depletion of the lysophagy receptor VCP (valosin containing protein), supporting a pro-survival function of lysophagy under these conditions. Collectively, our data suggest that pimozide and loperamide-driven autophagy and lipotoxicity synergize to induce LMP and cell death. The results also support the notion that simultaneous overactivation of autophagy and induction of LMP represents a promising approach for the treatment of GBM.Abbreviations: ACD: autophagic cell death; AKT1: AKT serine/threonine kinase 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG14: autophagy related 14; CERS1: ceramide synthase 1; CTSB: cathepsin B; CYBB/NOX2: cytochrome b-245 beta chain; ER: endoplasmatic reticulum; FBS: fetal bovine serum; GBM: glioblastoma; GO: gene ontology; HTR7/5-HT7: 5-hydroxytryptamine receptor 7; KD: knockdown; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LAP: LC3-associated phagocytosis; LMP: lysosomal membrane permeabilization; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MTOR: mechanistic target of rapamycin kinase; RB1CC1: RB1 inducible coiled-coil 1; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SMPD1/ASM: sphingomyelin phosphodiesterase 1; VCP/p97: valosin containing protein; WT: wild-type.
Subject(s)
Autophagy/drug effects , Autophagy/physiology , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Glioblastoma/drug therapy , Glioblastoma/pathology , Loperamide/pharmacology , Pimozide/pharmacology , Autophagy-Related Protein 5/antagonists & inhibitors , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 7/antagonists & inhibitors , Autophagy-Related Protein 7/genetics , Autophagy-Related Protein 7/metabolism , Brain Neoplasms/metabolism , Cathepsins/metabolism , Cell Death/drug effects , Cell Death/physiology , Cell Line, Tumor , Ceramides/metabolism , Gene Knockout Techniques , Glioblastoma/metabolism , Humans , Lipid Metabolism/drug effects , Lysosomes/drug effects , Lysosomes/metabolism , Permeability/drug effects , Proteome/metabolism , Sphingomyelin Phosphodiesterase/antagonists & inhibitors , Sphingomyelin Phosphodiesterase/metabolismABSTRACT
As hyperprolactinemia is observed in patients with bromocriptineresistant prolactinoma, prolactin (PRL) has been implicated in the development of bromocriptine resistance. Since PRL primarily mediates cell survival and drug resistance via the Janus kinase2 (JAK2)/signal transducer and activator of transcription 5A (STAT5) signaling pathway, the STAT5 inhibitor, pimozide, may inhibit cell proliferation and reverse bromocriptine resistance in prolactinoma cells. In the present study, compared with bromocriptine or pimozide alone, the combination of pimozide and bromocriptine exerted enhanced reduction in cell growth and proliferation, and increased apoptosis and cell cycle arrest in bromocriptineresistant prolactinoma cells. A reduction in phosphoSTAT5, cyclin D1 and Bcell lymphoma extralarge (BclxL) expression levels were observed in cells treated with the combination of drugs. In addition, pimozide suppressed spheroid formation of human pituitary adenoma stemlike cells, and reduced the protein expression of the cancer stem cell markers, CD133 and nestin. Pimozide did not exert any additional antitumor activity in STAT5knockdown primary culture cells of human bromocriptineresistant prolactinomas. Furthermore, Pimozide combined with bromocriptine treatment significantly reduced human prolactinoma xenograft growth. Western blot and immunohistochemical analyses also demonstrated significant inhibition of cell proliferation and stem cell marker proteins in vivo. Collectively, these data indicated that pimozide treatment reduced prolactinoma growth by targeting both proliferating cells and stem cells, at least in part, by inhibiting the STAT5/BclxL and STAT5/cyclin D1 signaling pathways.
Subject(s)
Bromocriptine/pharmacology , Cyclin D1/metabolism , Pimozide/pharmacology , Pituitary Neoplasms , Prolactinoma , STAT5 Transcription Factor/metabolism , Signal Transduction/drug effects , bcl-X Protein/metabolism , Animals , Cell Line, Tumor , Humans , Mice , Mice, Nude , Pituitary Neoplasms/drug therapy , Pituitary Neoplasms/metabolism , Pituitary Neoplasms/pathology , Prolactinoma/drug therapy , Prolactinoma/metabolism , Prolactinoma/pathology , Rats , Xenograft Model Antitumor AssaysABSTRACT
Brain tumors are considered as one of the most aggressive and incurable forms of cancer. The majority of the patients with brain tumors have a median survival rate of 12%. Brain tumors are lethal despite the availability of advanced treatment options such as surgical removal, chemotherapy, and radiotherapy. In this study, we have evaluated the anti-cancer effects of pimozide, which is a neuroleptic drug used for the treatment of schizophrenia and chronic psychosis. Pimozide significantly reduced the proliferation of U-87MG, Daoy, GBM 28, and U-251MG brain cancer cell lines by inducing apoptosis with IC50 (Inhibitory concentration 50) ranging from 12 to 16 µM after 48 h of treatment. Our Western blotting analysis indicated that pimozide suppressed the phosphorylation of STAT3 at Tyr705 and Src at Tyr416, and it inhibited the expression of anti-apoptotic markers c-Myc, Mcl-1, and Bcl-2. Significant autophagy induction was observed with pimozide treatment. LC3B, Beclin-1, and ATG5 up-regulation along with autolysosome formation confirmed the induction of autophagy with pimozide treatment. Inhibiting autophagy using 3-methyladenine or LC3B siRNA significantly blocked the apoptosis-inducing effects of pimozide, suggesting that pimozide mediated its apoptotic effects by inducing autophagy. Oral administration of 25 mg/kg pimozide suppressed the intracranially implanted U-87MG tumor growth by 45% in athymic nude mice. The chronic administration of pimozide showed no general signs of toxicity, and the behavioral activity of the mice remained unchanged. Taken together, these results indicate that pimozide inhibits the growth of brain cancer by autophagy-mediated apoptosis.
Subject(s)
Antineoplastic Agents/pharmacology , Autophagy/drug effects , Brain Neoplasms/drug therapy , Gene Expression Regulation, Neoplastic/drug effects , Pimozide/pharmacology , STAT3 Transcription Factor/genetics , Adenine/analogs & derivatives , Adenine/pharmacology , Animals , Antipsychotic Agents/pharmacology , Autophagy/genetics , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Drug Repositioning , Female , Humans , Mice , Mice, Nude , Microtubule-Associated Proteins/antagonists & inhibitors , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , STAT3 Transcription Factor/metabolism , Signal Transduction , Xenograft Model Antitumor AssaysABSTRACT
Considerable evidence indicates that adenosine and dopamine systems interact in the regulation of basal ganglia function. Nonselective adenosine antagonists such as the methylxanthine caffeine as well as selective adenosine A2A antagonists have been shown to produce antiparkinsonian and antidepressant effects in animal models. The present studies were conducted to assess if another methylxantine, theophylline, can reverse motor and motivational impairments induced by dopamine antagonism in rats. RESULTS: Theophylline (3.75-30.0 mg/kg, IP) reversed tremulous jaw movements (TJMs), catalepsy, and locomotor suppression induced by the dopamine D2 antagonist pimozide. It also reversed TJMs induced by the muscarinic receptor agonist pilocarpine, which is a well-known tremorogenic agent. Parallel studies assessed the ability of theophylline (5.0-20.0 mg/kg, IP) to reverse the changes in effort-related choice behavior induced by the dopamine D1 antagonist ecopipam (0.2 mg/kg, IP) and the D2 antagonist haloperidol (0.1 mg/kg, IP). Rats were tested on two different operant choice tasks which assess the tendency to work for a preferred reinforcer by lever pressing (for palatable pellets or a high 5% sucrose solution) vs. approaching and consuming a less preferred reinforcer (freely available lab chow or a less concentrated 0.3% sucrose solution). Theophylline restored food and sucrose-reinforced lever pressing in animals treated with the D2 antagonist. However, it was unable to reverse the effects of the D1 antagonist. Overall, the effects of theophylline resembled those previously reported for adenosine A2A antagonists, and suggest that theophylline could be clinically useful for the treatment of motor and motivational symptoms in humans.
Subject(s)
Choice Behavior/drug effects , Dopamine Antagonists/pharmacology , Motor Activity/drug effects , Purinergic P1 Receptor Antagonists/pharmacology , Theophylline/pharmacology , Tremor/metabolism , Animals , Behavior, Animal/drug effects , Benzazepines/pharmacology , Catalepsy/drug therapy , Catalepsy/metabolism , Conditioning, Operant/drug effects , Haloperidol/pharmacology , Humans , Male , Motivation/drug effects , Muscarinic Agonists/pharmacology , Pilocarpine/pharmacology , Pimozide/pharmacology , Rats , Rats, Sprague-Dawley , Sucrose/pharmacology , Tremor/drug therapyABSTRACT
Drug repurposing is an attractive and pragmatic way offering reduced risks and development time in the complicated process of drug discovery. In the past, drug repurposing has been largely accidental and serendipitous. The most successful examples so far have not involved a systematic approach. Nowadays, remarkable advances in drugs, diseases and bioinformatic knowledge are offering great opportunities for designing novel drug repurposing approach through comprehensive understanding of drug information. In this study, we introduced a novel drug repurposing approach based on transcriptomic data and chemical structures using deep learning. One strong candidate for repurposing has been identified. Pimozide is an anti-dyskinesia agent that is used for the suppression of motor and phonic tics in patients with Tourette's Disorder. However, our pipeline proposed it as a strong candidate for treating non-small cell lung cancer. The cytotoxicity of pimozide against A549 cell lines has been validated.
Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Computational Biology/methods , Drug Repositioning/methods , A549 Cells , Deep Learning , Drug Discovery , Gene Expression Profiling/methods , Humans , Pimozide/metabolism , Pimozide/pharmacology , Transcriptome/geneticsABSTRACT
BACKGROUND: Mast cells (MCs) mediate a key role in allergic diseases. Detailed studies of how the neuroleptic drug pimozide affects MC activity are lacking. The aim of this study was to investigate pimozide inhibition of immunoglobulin E (IgE)-mediated MC activation and MC-mediated allergic responses. METHOD: MCs were stimulated with anti-dinitrophenyl (DNP) IgE antibodies and DNP-horse serum albumin (HSA) antigen (Ag), and anti-allergic pimozide effects were detected by measuring ß-hexosaminidase levels. Morphological changes were observed histologically. In vivo pimozide effects were assessed in passive cutaneous anaphylaxis (PCA) and ovalbumin (OVA)-sensitized active systemic anaphylaxis mouse (ASA) model experiments. Levels of phosphorylated (p-) SYK (spleen tyrosine kinase) and MAPKs (mitogen-activated protein kinases) were detected in western blots. RESULTS: We found that pimozide inhibited MC degranulation, reduced MC release of ß-hexosaminidase dose-dependently in activated RBL-2H3 (IC50: 13.52 µM) and bone marrow derived MC (BMMC) (IC50: 42.42 µM), and reduced MC morphological changes. The IgE/Ag-induced migration effect was suppressed by pimozide treatment dose-dependently. Pimozide down-regulated IgE/Ag-induced phosphorylation of SYK and MAPKs in activated MCs. Moreover, pimozide attenuated allergic reactions in PCA and ASA model mice, and decreased MC populations among splenic cells. CONCLUSIONS: The antipsychotic drug pimozide can suppress IgE-mediated MC activation in vitro and in vivo and should be considered for repurposing to suppress MC-mediated diseases.