RESUMO
Swelling of the brain or spinal cord (CNS edema) affects millions of people every year. All potential pharmacological interventions have failed in clinical trials, meaning that symptom management is the only treatment option. The water channel protein aquaporin-4 (AQP4) is expressed in astrocytes and mediates water flux across the blood-brain and blood-spinal cord barriers. Here we show that AQP4 cell-surface abundance increases in response to hypoxia-induced cell swelling in a calmodulin-dependent manner. Calmodulin directly binds the AQP4 carboxyl terminus, causing a specific conformational change and driving AQP4 cell-surface localization. Inhibition of calmodulin in a rat spinal cord injury model with the licensed drug trifluoperazine inhibited AQP4 localization to the blood-spinal cord barrier, ablated CNS edema, and led to accelerated functional recovery compared with untreated animals. We propose that targeting the mechanism of calmodulin-mediated cell-surface localization of AQP4 is a viable strategy for development of CNS edema therapies.
Assuntos
Aquaporina 4/metabolismo , Edema/metabolismo , Edema/terapia , Animais , Aquaporina 4/fisiologia , Astrócitos/metabolismo , Encéfalo/metabolismo , Edema Encefálico/metabolismo , Calmodulina/metabolismo , Sistema Nervoso Central/metabolismo , Edema/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Medula Espinal/metabolismo , Traumatismos da Medula Espinal/metabolismo , Trifluoperazina/farmacologiaRESUMO
BACKGROUND: Although not approved for the treatment of anxiety disorders (except trifluoperazine) there is ongoing off-label, unapproved use of first-generation antipsychotics (FGAs) and second-generation antipsychotics (SGAs) for anxiety disorders. There have been systematic reviews and meta-analyses on the use of antipsychotics in anxiety disorders, most of which focused on SGAs. OBJECTIVE: The specific aims of this umbrella review are to: (1) Evaluate the evidence of efficacy of FGAs and SGAs in anxiety disorders as an adjunctive treatment to traditional antidepressant treatments and other nonantipsychotic medications; (2) Compare monotherapy with antipsychotics to first-line treatments for anxiety disorders in terms of effectiveness, risks, and side effects. The review protocol is registered on PROSPERO (CRD42021237436). METHODS: An initial search was undertaken to identify systematic reviews and meta-analyses from inception until 2020, with an updated search completed August 2021 and January 2023. The searches were conducted in PubMed, MEDLINE (Ovid), EMBASE (Ovid), APA PsycInfo (Ovid), CINAHL Complete (EBSCOhost), and the Cochrane Library through hand searches of references of included articles. Review quality was measured using the AMSTAR-2 (A MeaSurement Tool to Assess Systematic Reviews) scale. RESULTS: The original and updated searches yielded 1796 and 3744 articles respectively, of which 45 were eligible. After final review, 25 systematic reviews and meta-analyses were included in the analysis. Most of the systematic reviews and meta-analyses were deemed low-quality through AMSTAR-2 with only one review being deemed high-quality. In evaluating the monotherapies with antipsychotics compared with first-line treatments for anxiety disorder there was insufficient evidence due to flawed study designs (such as problems with randomization) and small sample sizes within studies. There was limited evidence suggesting efficacy of antipsychotic agents in anxiety disorders other than quetiapine in generalized anxiety disorder (GAD). CONCLUSIONS: This umbrella review indicates a lack of high-quality studies of antipsychotics in anxiety disorders outside of the use of quetiapine in GAD. Although potentially effective for anxiety disorders, FGAs and SGAs may have risks and side effects that outweigh their efficacy, although there were limited data. Further long-term and larger-scale studies of antipsychotics in anxiety disorders are needed.
Assuntos
Antipsicóticos , Transtornos de Ansiedade , Humanos , Antipsicóticos/efeitos adversos , Transtornos de Ansiedade/tratamento farmacológico , PubMed , Fumarato de Quetiapina , Trifluoperazina , Revisões Sistemáticas como Assunto , Metanálise como AssuntoRESUMO
Many therapeutics for cardiomyopathy treat the symptoms of the disease rather than the underlying mechanism. The mechanism of cardiomyopathy onset is believed to include two means: calcium sensitivity changes and myosin activity alteration. Trifluoperazine is a compound that binds troponin, and other components of the calcium pathway, which impacts calcium regulation of contraction. Here, the ability of TFP to shift calcium sensitivity was examined in vitro with purified proteins and the impact of TFP on heart function was assessed in vivo using embryonic zebrafish. The binding of TFP to troponin was modeled in silico and a model of zebrafish troponin was generated. TFP increased regulated cardiac actomyosin activity in vitro and elevated embryonic zebrafish heart rates at effective drug concentrations. Troponin structural changes predicted in silico suggest altered protein interactions within thin filaments that would affect the regulation of heart function.
Assuntos
Cálcio , Cardiomiopatias , Animais , Cálcio/metabolismo , Trifluoperazina/farmacologia , Peixe-Zebra/metabolismo , Tropomiosina/química , Troponina/metabolismo , Cardiomiopatias/metabolismo , Sarcômeros/metabolismo , Actinas/metabolismoRESUMO
Calmodulin (CaM) is a calcium sensor which binds and regulates a wide range of target-proteins. This implicitly enables the concentration of calcium to influence many downstream physiological responses, including muscle contraction, learning and depression. The antipsychotic drug trifluoperazine (TFP) is a known CaM inhibitor. By binding to various sites, TFP prevents CaM from associating to target-proteins. However, the molecular and state-dependent mechanisms behind CaM inhibition by drugs such as TFP are largely unknown. Here, we build a Markov state model (MSM) from adaptively sampled molecular dynamics simulations and reveal the structural and dynamical features behind the inhibitory mechanism of TFP-binding to the C-terminal domain of CaM. We specifically identify three major TFP binding-modes from the MSM macrostates, and distinguish their effect on CaM conformation by using a systematic analysis protocol based on biophysical descriptors and tools from machine learning. The results show that depending on the binding orientation, TFP effectively stabilizes features of the calcium-unbound CaM, either affecting the CaM hydrophobic binding pocket, the calcium binding sites or the secondary structure content in the bound domain. The conclusions drawn from this work may in the future serve to formulate a complete model of pharmacological modulation of CaM, which furthers our understanding of how these drugs affect signaling pathways as well as associated diseases.
Assuntos
Antipsicóticos , Calmodulina , Calmodulina/metabolismo , Trifluoperazina/farmacologia , Trifluoperazina/química , Trifluoperazina/metabolismo , Antipsicóticos/química , Cálcio/metabolismo , Ligação Proteica , Sítios de LigaçãoRESUMO
Currently, no specific and standard treatment for traumatic brain injury (TBI) has been developed. Therefore, studies on new therapeutic drugs for TBI treatment are urgently needed. Trifluoperazine (TFP) is a therapeutic agent for the treatment of psychiatric disorders that reduces edema of the central nervous system. However, the specific working mechanism of TFP is not fully understood in TBI. In this study, the immunofluorescence co-localization analysis revealed that the area and intensity covered by Aquaporin4 (AQP4) on the surface of brain cells (astrocyte endfeet) increased significantly after TBI. In contrast, TFP treatment reversed these phenomena. This finding showed that TFP inhibited AQP4 accumulation on the surface of brain cells (astrocyte endfeet). The tunel fluorescence intensity and fluorescence area were lower in the TBI+TFP group compared to the TBI group. Additionally, the brain edema, brain defect area, and modified neurological severity score (mNSS) were lower in the TBI+TFP. The RNA-seq was performed on the cortical tissues of rats in the Sham, TBI, and TBI+TFP groups. A total of 3774 genes differently expressed between the TBI and the Sham group were identified. Of these, 2940 genes were up-regulated and 834 genes were down-regulated. A total of 1845 differently expressed genes between the TBI+TFP and TBI group were also identified, in which 621 genes were up-regulated and 1224 genes were down-regulated. Analysis of the common differential genes in the three groups showed that TFP could reverse the expression of apoptosis and inflammation genes. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the differentially expressed genes (DEGs) were highly enriched in the signaling pathways regulating inflammation. In conclusion, TFP alleviates brain edema after TBI by preventing the accumulation of AQP4 on the surface of brain cells. Generally, TFP alleviates apoptosis and inflammatory response induced by TBI, and promotes the recovery of nerve function in rats after TBI. Thus, TFP is a potential therapeutic agent for TBI treatment.
Assuntos
Edema Encefálico , Lesões Encefálicas Traumáticas , Animais , Ratos , Apoptose/genética , Aquaporina 4/antagonistas & inibidores , Aquaporina 4/genética , Aquaporina 4/metabolismo , Encéfalo , Edema Encefálico/etiologia , Edema Encefálico/genética , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/genética , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Trifluoperazina/farmacologia , Trifluoperazina/uso terapêutico , Trifluoperazina/metabolismoRESUMO
Glioblastoma (GBM) is the deadliest adult brain cancer, and all patients ultimately succumb to the disease. Radiation therapy (RT) provides survival benefit of 6 mo over surgery alone, but these results have not improved in decades. We report that radiation induces a glioma-initiating cell phenotype, and we have identified trifluoperazine (TFP) as a compound that interferes with this phenotype conversion. TFP causes loss of radiation-induced Nanog mRNA expression, and activation of GSK3 with consecutive posttranslational reduction in p-Akt, Sox2, and ß-catenin protein levels. TFP did not alter the intrinsic radiation sensitivity of glioma-initiating cells (GICs). Continuous treatment with TFP and a single dose of radiation reduced the number of GICs in vivo and prolonged survival in syngeneic and patient-derived orthotopic xenograft (PDOX) mouse models of GBM. Our findings suggest that the combination of a dopamine receptor antagonist with radiation enhances the efficacy of RT in GBM by preventing radiation-induced phenotype conversion of radiosensitive non-GICs into treatment-resistant, induced GICs (iGICs).
Assuntos
Antagonistas de Dopamina/farmacologia , Glioblastoma/metabolismo , Fenótipo , Receptores Dopaminérgicos/efeitos dos fármacos , Trifluoperazina/farmacologia , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/radioterapia , Modelos Animais de Doenças , Antagonistas de Dopamina/uso terapêutico , Regulação Neoplásica da Expressão Gênica , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/radioterapia , Glioma/tratamento farmacológico , Glioma/metabolismo , Glioma/patologia , Glioma/radioterapia , Quinase 3 da Glicogênio Sintase/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Mensageiro/metabolismo , Tolerância a Radiação , Fatores de Transcrição SOXB1 , Trifluoperazina/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , beta CateninaRESUMO
Rv1211 is a conserved hypothetical protein in Mycobacterium tuberculosis and is required for the growth and pathogenesis of the bacteria. The protein has been suggested as a calmodulin-like calcium-binding protein with an EF-hand motif and as a target of trifluoperazine, a calmodulin antagonist in eukaryotes that inhibits mycobacterial growth. Here, we expressed the recombinant protein of Rv1211 and performed structural and biochemical studies of Rv1211 and its interaction with Ca2+ or trifluoperazine. Surprisingly, Rv1211 exhibited an elution property typical of a natively unfolded protein. Subsequent circular dichroism experiments with temperature elevation and trifluoroethanol treatment showed that Rv1211 has unfolded structure. Additional NMR experiment confirmed the unfolded state of the protein and further showed that it does not bind to Ca2+. Still, Rv1211 did bind to trifluoperazine, as evidenced by the two-dimensional NMR spectra of 15N-labeled Rv1211. However, there were no peak shifts upon binding, showing that Rv1211 retained its unfolded state even after the trifluoperazine binding. The residues involved in the binding were clustered in the C-terminal region, as identified by the sequence assignment. Isothermal titration calorimetry showed that the Kd of trifluoperazine-Rv1211 binding is 41 µM and that the stoichiometry is 1 : 2 (Rv1211: trifluoperazine). Our results argue against the suggestion of Rv1211 as a Ca2+-binding calmodulin-like protein, and show that Rv1211 is a natively unfolded protein that binds to trifluoperazine. In addition, our results suggest the evidence of the "Fuzziness" in the Rv1211-trifluoperazine interaction that differs from the conventional binding-induced folding of natively unfolded proteins.
Assuntos
Proteínas Intrinsicamente Desordenadas , Mycobacterium tuberculosis , Cálcio/metabolismo , Calmodulina/metabolismo , Motivos EF Hand , Proteínas Intrinsicamente Desordenadas/metabolismo , Mycobacterium tuberculosis/metabolismo , Trifluoperazina/química , Trifluoperazina/farmacologiaRESUMO
Renal interstitial fibrosis (RIF) is a pathological process that fibrotic components are excessively deposited in the renal interstitial space due to kidney injury, resulting in impaired renal function and chronic kidney disease. The molecular mechanisms controlling renal fibrosis are not fully understood. In this present study, we identified Nuclear protein 1 (Nupr1), a transcription factor also called p8, as a novel regulator promoting renal fibrosis. Unilateral ureteral obstruction (UUO) time-dependently induced Nupr1 mRNA and protein expression in mouse kidneys while causing renal damage and fibrosis. Nupr1 deficiency (Nupr1-/- ) attenuated the renal tubule dilatation, tubular epithelial cell atrophy, and interstitial collagen accumulation caused by UUO. Consistently, Nupr1-/- significantly decreased the expression of type I collagen, myofibroblast markers smooth muscle α-actin (α-SMA), fibroblast-specific protein 1 (FSP-1), and vimentin in mouse kidney that were upregulated by UUO. These results suggest that Nupr1 protein was essential for fibroblast activation and/or epithelial-mesenchymal transition (EMT) during renal fibrogenesis. Indeed, Nupr1 was indispensable for TGF-ß-induced myofibroblast activation of kidney interstitial NRK-49F fibroblasts, multipotent mesenchymal C3H10T1/2 cells, and the EMT of kidney epithelial NRK-52E cells. It appears that Nupr1 mediated TGF-ß-induced α-SMA expression and collagen synthesis by initiating Smad3 signaling pathway. Importantly, trifluoperazine (TFP), a Nupr1 inhibitor, alleviated UUO-induced renal fibrosis. Taken together, our results demonstrate that Nupr1 promotes renal fibrosis by activating myofibroblast transformation from both fibroblasts and tubular epithelial cells.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Transição Epitelial-Mesenquimal , Fibroblastos/fisiologia , Rim/patologia , Proteínas de Neoplasias/fisiologia , Animais , Células Cultivadas , Colágeno/metabolismo , Matriz Extracelular/metabolismo , Fibrose , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/fisiologia , Ratos , Transdução de Sinais/fisiologia , Proteína Smad3/fisiologia , Fatores de Transcrição da Família Snail/fisiologia , Trifluoperazina/farmacologiaRESUMO
Dengue virus (DENV), a member of the genus Flavivirus, family Flaviviridae, is the most widespread viral pathogen transmitted to humans by mosquitoes. Despite the increased incidence of DENV infection, there are no antiviral drugs available for treatment or prevention. Phenothiazines are heterocyclic compounds with various pharmacological properties that are very adaptable for drug repurposing. In the present report, we analyzed the antiviral activity against DENV and the related Zika virus (ZIKV) of trifluoperazine (TFP), a phenothiazine derivative in clinical use as an antipsychotic and antiemetic agent. TFP exhibited dose-dependent inhibitory activity against the four DENV serotypes and ZIKV in monkey Vero cells at non-cytotoxic concentrations with 50% effective concentration values in the range 1.6-6.4 µM. A similar level of antiviral efficacy was exhibited by TFP against flavivirus infection in the human cell lines A549 and HepG2. Mechanistic studies, performed using time-dependent infectivity assays, real-time RT-PCR, Western blot, and immunofluorescence techniques, indicated that uncoating of the virus during penetration into the cell was the main target for TFP in infected cells, but the compound also exerted a minor effect on a late stage of the virus multiplication cycle. This study demonstrates that TFP, a pharmacologically active phenothiazine, is a selective inhibitor of DENV multiplication in cell culture. Our findings open perspectives for the repositioning of phenothiazines like TFP with a wide spectrum of antiviral efficacy as potential agents for the control of pathogenic flaviviruses.
Assuntos
Antieméticos , Antipsicóticos , Vírus da Dengue , Dengue , Infecção por Zika virus , Zika virus , Animais , Antieméticos/farmacologia , Antieméticos/uso terapêutico , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Chlorocebus aethiops , Dengue/tratamento farmacológico , Humanos , Fenotiazinas/farmacologia , Fenotiazinas/uso terapêutico , Trifluoperazina/farmacologia , Trifluoperazina/uso terapêutico , Células Vero , Replicação ViralRESUMO
AIMS: Calmodulin (CaM), acts as a kind of multifunctional Ca2+ sensing protein, which is ubiquitous in fungi, is highly conserved across eukaryotes and is involved in the regulation of a range of physiological processes, including morphogenesis, reproduction and secondary metabolites biosynthesis. Our aim was to understand the characteristics and functions of AaCaM in Alternaria alternata, the causal agent of pear black spot. METHODS AND RESULTS: A 450 bp cDNA sequence of AaCaM gene of A. alternata was cloned by the PCR homology method. Sequence analysis showed that this protein encoded by AaCaM was a stable hydrophilic protein and had a high similarity to Neurospora crassa (CAA50271.1) and other fungi. RT-qPCR analysis determined that AaCaM was differentially upregulated during infection structural differentiation of A. alternata both on hydrophobic and pear wax extract-coated surface, with a 3.37-fold upregulation during the hydrophobic induced appressorium formation period (6 h) and a 1.46-fold upregulation during the infection hyphae formation period (8 h) following pear wax induction. Pharmaceutical analysis showed that the CaM-specific inhibitor, trifluoperazine (TFP), inhibited spore germination and appressorium formation, and affected toxins and melanin biosynthesis in A. alternata. CONCLUSIONS: AaCaM plays an important role in regulating infection structure differentiation and secondary metabolism of A. alternata. SIGNIFICANCE AND IMPACT OF STUDY: Our study provides a theoretical basis for further in-depth investigation of the specific role of AaCaM in the calcium signalling pathway underlying hydrophobic and pear wax-induced infection structure differentiation and pathogenicity of A. alternata.
Assuntos
Pyrus , Alternaria/metabolismo , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , DNA Complementar/metabolismo , Melaninas/metabolismo , Preparações Farmacêuticas , Doenças das Plantas/microbiologia , Pyrus/genética , Pyrus/metabolismo , Pyrus/microbiologia , Trifluoperazina/metabolismoRESUMO
A sensitive, selective and rapid bioanalytical method using liquid chromatography-tandem mass spectrometry has been developed for the quantification of trifluoperazine in human plasma. Trifluoperazine-D8 was used as the internal standard and the extraction from human plasma was performed by liquid-liquid extraction technique using tertiary butyl methyl ether as the solvent. Chromatographic separation was carried out on Zodiac C18 column (50 × 4.6 mm, 3 µm) employing a mixture of acetonitrile, methanol and 5 mm ammonium bicarbonate buffer in water (85:10:5, v/v/v) at a flow rate of 0.55 ml/min. The linearity was established within the concentration range of 5-1,250 pg/ml with r2 > 0.99. The results of all of the validation parameters as per the US Food and Drug Administration guidelines were within the acceptance limits. The pharmacokinetics of trifluoperazine after oral administration of a syrup of 1 mg dose under fasting conditions was determined by successful application of the present method.
Assuntos
Espectrometria de Massas em Tandem , Trifluoperazina , Humanos , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Cinética , Reprodutibilidade dos Testes , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Multiple myeloma (MM) is a common hematological malignancy. Bortezomib (BTZ) is a traditional medicine for MM treatment, but there are limitations for current treatment methods. Trifluoperazine (TFP) is a clinical drug for acute and chronic psychosis therapy. Lately, researchers have found that TFP can suppress tumor growth in many cancers. We attempted to study the effects of BTZ and TFP on MM in vivo and in vitro. We concentrated on the individual and combined impact of BTZ and TFP on the proliferation and apoptosis of MM cells via Cell Counting kit-8 assay, EdU assay, western blot, and flow cytometry. We found that combination therapy has a strong synergistic impact on MM cells. Combination therapy could induce cell arrest during G2/M phase and induce apoptosis in MM cells. Meanwhile, BTZ combined with TFP could play a better role in the anti-MM effect in vivo through MM.1s xenograft tumor models. Furthermore, we explored the mechanism of TFP-induced apoptosis in MM, and we noticed that TFP might induce MM apoptosis by inhibiting p-P38 MAPK/NUPR1. In summary, our findings suggest that TFP could synergistically enhance the BTZ-induced anti-cancer effect in multiple myeloma, which might be a promising therapeutic strategy for MM treatment.
Assuntos
Antineoplásicos , Mieloma Múltiplo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Mieloma Múltiplo/tratamento farmacológico , Proteínas de Neoplasias/metabolismo , Trifluoperazina/farmacologia , Trifluoperazina/uso terapêutico , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Vaccination programs have been launched worldwide to halt the spread of COVID-19. However, the identification of existing, safe compounds with combined treatment and prophylactic properties would be beneficial to individuals who are waiting to be vaccinated, particularly in less economically developed countries, where vaccine availability may be initially limited. METHODS: We used a data-driven approach, combining results from the screening of a large transcriptomic database (L1000) and molecular docking analyses, with in vitro tests using a lung organoid model of SARS-CoV-2 entry, to identify drugs with putative multimodal properties against COVID-19. RESULTS: Out of thousands of FDA-approved drugs considered, we observed that atorvastatin was the most promising candidate, as its effects negatively correlated with the transcriptional changes associated with infection. Atorvastatin was further predicted to bind to SARS-CoV-2's main protease and RNA-dependent RNA polymerase, and was shown to inhibit viral entry in our lung organoid model. CONCLUSIONS: Small clinical studies reported that general statin use, and specifically, atorvastatin use, are associated with protective effects against COVID-19. Our study corroborrates these findings and supports the investigation of atorvastatin in larger clinical studies. Ultimately, our framework demonstrates one promising way to fast-track the identification of compounds for COVID-19, which could similarly be applied when tackling future pandemics.
Assuntos
Antivirais/farmacologia , Atorvastatina/farmacologia , Tratamento Farmacológico da COVID-19 , Pulmão/efeitos dos fármacos , Organoides/efeitos dos fármacos , SARS-CoV-2/efeitos dos fármacos , Antivirais/química , Atorvastatina/química , COVID-19/prevenção & controle , Linhagem Celular , Proteases 3C de Coronavírus/química , RNA-Polimerase RNA-Dependente de Coronavírus/química , Doxiciclina/farmacologia , Aprovação de Drogas , Reposicionamento de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pulmão/virologia , Modelos Biológicos , Simulação de Acoplamento Molecular , Organoides/virologia , Cloridrato de Raloxifeno/química , Cloridrato de Raloxifeno/farmacologia , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/genética , Trifluoperazina/química , Trifluoperazina/farmacologia , Estados Unidos , United States Food and Drug Administration , Vesiculovirus/genética , Internalização do Vírus/efeitos dos fármacosRESUMO
P-glycoprotein, member of the B-subfamily of the ATP-binding cassette (ABC) superfamily (e.g., ABCB1), has been demonstrated to confer resistance to clinically relevant anticancer drugs. Paradoxically, ABCB1-expressing multidrug resistant (MDR) cells are hypersensitivity or collateral sensitivity to non-toxic drugs. In this report, we demonstrate the capacity of trifluoperazine (TFP), a calmodulin inhibitor, to confer a collateral sensitivity onto ABCB1-overexpressing MDR cells. We show TFP-induced collateral sensitivity to be linked to ABCB1 expression and ATPase activity, as such phenotype is abolished in ABCB1-knockout MDR cells (CHORC5ΔABCB1 clones A1-A3) or with inhibitors of ABCB1 ATPase. TFP-induced collateral sensitivity is mediated by apoptotic cell death, due to enhanced oxidative stress. The findings in this study show for first time the use TFP as a collateral sensitivity drug, at clinically relevant concentrations. Moreover, given the use of trifluoperazine in the treatment for symptoms of schizophrenia and the role of ABCB1 transporter in tissue blood barriers and other physiologic functions, the finding in this study may have implications beyond cancer chemotherapy.
Assuntos
Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Fenotiazinas/farmacologia , Trifluoperazina/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Linhagem Celular , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Macrolídeos/farmacologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.
Assuntos
Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Cloroquina/farmacologia , Lisossomos/efeitos dos fármacos , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Células HeLa , Humanos , Lisossomos/metabolismo , Macrolídeos/farmacologia , Masculino , Mefloquina/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Promazina/farmacologia , Quinacrina/farmacologia , Trifluoperazina/farmacologiaRESUMO
Targeted therapies and immunotherapy have brought substantial benefits to patients with melanoma. However, brain metastases remain the biggest threat to the survival and quality of life of melanoma patients. One of the major challenges to an effective therapy is the inability of drugs to penetrate the blood-brain barrier (BBB). Anti-schizophrenic drugs can cross the BBB, and many of them have demonstrated anti-cancer effects. Repurposing existing drugs for new clinical indications is an alluring strategy for anticancer drug discovery. Herein, we applied this strategy and screened a small collection of existing anti-schizophrenic drugs to use as anti-melanoma agents. Among them, trifluoperazine dihydrochloride (TFP) exhibited promising potencies for suppressing the growth and metastasis of melanoma, both in vitro and in vivo. TFP obviously suppressed the viability of melanoma cells within the micromolar range and inhibited the growth of melanoma in the subcutaneous mice models. Notably, intraperitoneal (i.p.) administration of TFP (40 mg/kg/day) obviously inhibited the growth of intra-carotid-injection established melanoma brain metastasis and extended the survival of brain metastasis-bearing mice. Moreover, TFP significantly suppressed lung metastasis and bone metastasis of melanoma in preclinical metastasis models. Mechanistically, TFP caused G0/G1 cell cycle arrest and mitochondrial-dependent intrinsic apoptosis of melanoma cells. In addition, TFP treatment increased the expression of microtubule associated protein 1 light chain 3 beta-II (LC3B-II) and p62 in vitro, suggesting an inhibition of autophagic flux. TFP decreased LysoTracker Red uptake after treatment, indicating impaired acidification of lysosomes. Moreover, the colocalization of LC3 with lysosomal-associated membrane protein 1 (LAMP1), a lysosome marker, was also suppressed after TFP treatment, suggesting that TFP might block the fusion of autophagosomes with lysosomes, which led to autophagosome accumulation. Taken together, our data highlight the potential of repurposing TFP as a new adjuvant drug for treating melanoma patients with brain, lung, and bone metastases.
Assuntos
Antineoplásicos/uso terapêutico , Antipsicóticos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Trifluoperazina/uso terapêutico , Animais , Autofagia/efeitos dos fármacos , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/secundário , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Feminino , Humanos , Neoplasias Pulmonares/secundário , Melanoma/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Neoplasias Cutâneas/patologiaRESUMO
Trifluoperazine (TFP), an antipsychotic drug approved by the Food and Drug Administration, has been show to exhibit anti-cancer effects. Pulmonary arterial hypertension (PAH) is a devastating disease characterized by a progressive obliteration of small pulmonary arteries (PAs) due to exaggerated proliferation and resistance to apoptosis of PA smooth muscle cells (PASMCs). However, the therapeutic potential of TFP for correcting the cancer-like phenotype of PAH-PASMCs and improving PAH in animal models remains unknown. PASMCs isolated from PAH patients were exposed to different concentrations of TFP before assessments of cell proliferation and apoptosis. The in vivo therapeutic potential of TFP was tested in two preclinical models with established PAH, namely the monocrotaline and sugen/hypoxia-induced rat models. Assessments of hemodynamics by right heart catheterization and histopathology were conducted. TFP showed strong anti-survival and anti-proliferative effects on cultured PAH-PASMCs. Exposure to TFP was associated with downregulation of AKT activity and nuclear translocation of forkhead box protein O3 (FOXO3). In both preclinical models, TFP significantly lowered the right ventricular systolic pressure and total pulmonary resistance and improved cardiac function. Consistently, TFP reduced the medial wall thickness of distal PAs. Overall, our data indicate that TFP could have beneficial effects in PAH and support the view that seeking new uses for old drugs may represent a fruitful approach.
Assuntos
Fármacos Cardiovasculares/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Hipóxia/prevenção & controle , Miócitos de Músculo Liso/efeitos dos fármacos , Trifluoperazina/farmacologia , Animais , Antipsicóticos/farmacologia , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Reposicionamento de Medicamentos , Feminino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Hemodinâmica/efeitos dos fármacos , Humanos , Hipertensão Pulmonar/induzido quimicamente , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipóxia/induzido quimicamente , Hipóxia/genética , Hipóxia/fisiopatologia , Indóis/administração & dosagem , Monocrotalina/administração & dosagem , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/metabolismo , Pirróis/administração & dosagem , Ratos , Ratos Sprague-Dawley , Survivina/genética , Survivina/metabolismoRESUMO
Isocitrate dehydrogenase (IDH) mutations are found in low-grade gliomas, and the product of the IDH mutant (MT), 2-hydroxyglutarate (2-HG), is the first known oncometabolite. However, the roles of the IDH wild type (WT) in high-grade glioblastoma, which rarely has the IDH mutation, are still unknown. To investigate possible pathways related to IDH WT in gliomas, we carried out bioinformatics analysis, and found that IDH1 has several putative calmodulin (CaM) binding sites. Pull-down and quantitative dissociation constant (Kd) measurements using recombinant proteins showed that IDH1 WT indeed binds to CaM with a higher affinity than IDH1 R132H MT. This biochemical interaction was demonstrated also in the cellular environment by immunoprecipitation with glioblastoma cell extracts. A synthetic peptide for the suggested binding region interfered with the interaction between CaM and IDH1, confirming the specificity of the binding. Direct binding between the synthetic peptide and CaM was observed in an NMR binding experiment, which additionally revealed that the peptide initially binds to the C-lobe of CaM. The physiological meaning of the CaM-IDH1 WT binding was shown with trifluoperazine (TFP), a CaM antagonist, which disrupted the binding and inhibited survival and migration of glioblastoma cells with IDH1 WT. As CaM signaling is activated in glioblastoma, our results suggest that IDH1 WT may be involved in the CaM-signaling pathway in the tumorigenesis of high-grade gliomas.
Assuntos
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Calmodulina/metabolismo , Movimento Celular , Glioblastoma/metabolismo , Glioblastoma/patologia , Isocitrato Desidrogenase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Isocitrato Desidrogenase/química , Modelos Moleculares , Ligação Proteica/efeitos dos fármacos , Trifluoperazina/farmacologiaRESUMO
The development of cancer in patients with schizophrenia is affected by genetic and environmental factors and antipsychotic medication. Several studies found that schizophrenia was associated with decreased risk of some cancers, and the neuroleptic medication might help to reduce the risk of colorectal cancer (CRC). Phenothiazine drugs including trifluoperazine (TFP) are widely used antipsychotic drugs and showed some antitumor effects, we here investigated the potential application of TFP in the treatment of colon cancer. A series doses of TFP were treated to the colon cancer cell line HCT116 and the inhibitory concentration (IC50 ) of TFP for HCT116 was determined by cell counting kit-8. The results indicated that the treatment of TFP impaired the cell vitality of HCT116 in a dose- and time-dependent manner. Meanwhile, the Edu assay demonstrated that the proliferation was also inhibited by TFP, which was accompanied with the induction of apoptosis and autophagy. The expression of CCNE1, CDK4, and antiapoptosis factor BCL-2 was downregulated but the proapoptosis factor BAX was upregulated. The autophagy inhibitor chloroquine could significantly reverse the TFP-induced apoptosis. Moreover, the ability of migration and invasion of HCT116 was found to be suppressed by TFP, which was associated with the inhibition of epithelial-mesenchymal transition (EMT). The function of TFP in vivo was further confirmed. The results showed that the administration of TFP remarkably abrogated the tumor growth with decreased tumor volume and proliferation index Ki-67 level in tumor tissues. The EMT phenotype was also confirmed to be inhibited by TFP in vivo, suggesting the promising antitumor effects of TFP in CRC.
Assuntos
Antineoplásicos/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Trifluoperazina/administração & dosagem , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Fatores de Tempo , Trifluoperazina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma (UC). Most patients inevitably encounter drug resistance and resultant disease relapse. Reduced apoptosis plays a critical role in chemoresistance. Trifluoperazine (TFP), an antipsychotic agent, has demonstrated antitumor effects on various cancers. This study investigated the efficacy of TFP in inhibiting cisplatin-resistant bladder UC and explored the underlying mechanism. Our results revealed that cisplatin-resistant UC cells (T24/R) upregulated the antiapoptotic factor, B-cell lymphoma-extra large (Bcl-xL). Knockdown of Bcl-xL by siRNA resensitized cisplatin-resistant cells to the cisplatin cytotoxic effect. TFP (10-45 µM) alone elicited dose-dependent cytotoxicity, apoptosis, and G0/G1 arrest on T24/R cells. Co-treatment of TFP potentiated cisplatin-induced cytotoxicity in T24/R cells. The phenomenon that TFP alleviated cisplatin resistance to T24/R was accompanied with concurrent suppression of Bcl-xL. In vivo models confirmed that TFP alone effectively suppressed the T24/R xenograft in nude mice. TFP co-treatment enhanced the antitumor effect of cisplatin on the T24/R xenograft. Our results demonstrated that TFP effectively inhibited cisplatin-resistant UCs and circumvented cisplatin resistance with concurrent Bcl-xL downregulation. These findings provide a promising insight to develop a therapeutic strategy for chemoresistant UCs.