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.

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.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with almost no curative chemotherapeutic treatment. Besides the development of new compounds, repurposing of approved drugs to treat cancer, alone or in combination, has become an attractive strategy, showing many therapeutic and economic advantages. However, it is necessary to improve our knowledge about the mechanism of cell death elicited by approved drugs itself, but also to rationally develop more powerful multidrug treatments. In this work, we focus our attention on determining the mechanism promoting cell death following trifluoperazine (TFP) treatment, which is an antipsychotic drug with strong anticancer activity in PDAC. We demonstrate that TFP induces cell death by apoptosis and necroptosis, which can be partially inhibited by Z-VAD-FMK as well as necrostatin-1, respectively. This cell death promotion is triggered by a poor ATP content, observed in TFP-treated cells as a consequence of a dramatic decrease in OXPHOS metabolism due to mitochondrial stress. Remarkably, mitochondrial homeostasis was seriously affected, and a loss of mitochondrial membrane potential and ROS overproduction was observed. Moreover, this mitochondrial stress was coupled with an ER stress and the activation of the endoplasmic-reticulum-associated protein degradation (ERAD) and the unf olded protein response (UPR) pathways. We took advantage of this information and inhibited this process by using the proteasome inhibitors MG-132 or bortezomib compounds in combination with TFP and found a significant improvement of the anticancer effect of the TFP on primary PDAC-derived cells. In conclusion, this study not only uncovers the molecular mechanisms that are triggered upon TFP-treatment but also its possible combination with bortezomib for the future development of therapies for pancreatic cancer.

Trifluoperazine, a known calmodulin antagonist, belongs to a class of phenothiazine compounds that have multiple sites of action in mycobacteria including lipid synthesis, DNA processes, protein synthesis and respiration. The objective of this study is to evaluate the potential of TFP to be used as a lead molecule for development of novel TB drugs by showing its efficacy on multiple drug resistant (MDR) Mycobacterium tuberculosis (M.tb) and non-replicating dormant M.tb. Wild type and MDR M.tb were treated with TFP under different growth conditions of stress like low pH, starvation, presence of nitric oxide and in THP-1 infection model. Perturbation in growth kinetics of bacilli at different concentrations of TFP was checked to determine the MIC of TFP for active as well as dormant bacilli. Results show that TFP is able to significantly reduce the actively replicating as well as non-replicating bacillary load. It has also shown inhibitory effect on the growth of MDR M.tb. TFP has shown enhanced activity against intracellular bacilli, presumably because phenothiazines are known to get accumulated in macrophages. This concentration was, otherwise, found to be non-toxic to macrophage in vitro. Our results show that TFP has the potential to be an effective killer of both actively growing and non-replicating bacilli including MDR TB. Further evaluation and in vivo studies with Trifluoperazine can finally help us know the feasibility of this compound to be used as either a lead compound for development of new TB drugs or as an adjunct in the current TB chemotherapy.

BACKGROUND It has been reported that trifluoperazine (TFP) inhibits proliferation of cancer cells, however, the effects of TFP in renal proliferation diseases are still unclear. This study examined the effects of TFP on proliferation of human renal mesangial cells and analyzed the underlying mechanisms. MATERIAL AND METHODS Cell proliferation in vivo was determined by HE staining, immunohistochemistry of proliferating cell nuclear antigen (PCNA), and Western blot analysis (Ki-67 and PCNA). Effects of different TFP concentrations and treatment duration on cell proliferation and cell cycle were analyzed using the MTT assay and flow cytometry. Expression of G0/G1 phase cell cycle-related proteins and TFP-induced MAPK and PI3K/AKT signaling pathways was estimated with Western blot analysis. RESULTS Our findings suggest that TFP inhibits cell proliferation in a dose- and time-dependent manner and decreased PCNA and Ki-67 levels in lupus MRL/lpr mice. TFP arrested the cell cycle in the G0/G1 phase, down-regulating cyclin D1, CDK2, and CDK4, and up-regulating p21 expression in a dose-dependent manner. In addition, TFP inhibited p-AKT and p-JNK, possibly by suppressing the activation of PI3K/AKT and JNK/MAPK signaling pathways. TFP treatment remarkably reduced the levels of serum creatinine (Cr) in lupus mice. CONCLUSIONS TFP exhibits inhibitory activity against mesangial cells in vivo and in vitro, which is associated with G1 cell cycle arrest by inactivation of PI3K/AKT and JNK/MAPK signaling pathways. These results suggest the potential of TFP in treatment of mesangial proliferative diseases.

Glioblastoma (GBM) is regarded as the most common malignant brain tumor but treatment options are limited. Thus, there is an unmet clinical need for compounds and corresponding targets that could inhibit GBM growth. We screened a library of 80 dopaminergic ligands with the aim of identifying compounds capable of inhibiting GBM cell line proliferation and survival. Out of 45 active compounds, 8 were further validated. We found that the dopamine receptor D2 antagonist trifluoperazine 2HCl inhibits growth and proliferation of GBM cells in a dose dependent manner. Trifluoperazine's inhibition of GBM cells is cell line dependent and correlates with variations in dopamine receptor expression profile. We conclude that components of the dopamine receptor signaling pathways are potential targets for pharmacological interventions of GBM growth.

Glioma patients constitute the greatest percentage of depressed neoplasm patients. These patients often require antidepressant treatment, but the effect of antidepressant drugs on glioma cells requires further evaluation. In the present study, we evaluated the effect of trifluoperazine (TFP) on the proliferation and apoptosis of glioma cells. Transcriptomic and bioinformatics analysis results suggested that antidepressant drugs, especially TFP, may upregulate the drug-resistant ability of glioma cells. A low concentration of TFP upregulated the viability of glioma cells. Colony formation and EdU assays confirmed that TFP treatment accelerates glioma cell proliferation, but no significant difference was found in the cell cycle distribution of glioma cells after treatment with TFP or control. Flow cytometry and TUNEL staining results suggested that TFP treatment decreased apoptosis in glioma cells. In addition, TFP treatment downregulated the intracellular Ca2+ concentration of glioma cells. In vivo experimental results indicated that TFP treatment promoted proliferation and reduced apoptosis in xenograft tumours in nude mice. Taken together, our results suggest that a low concentration of TFP promotes proliferation and reduces apoptosis in glioma cells both in vitro and in vivo. The potential harmful effects of antidepressant drugs on gliomas require further evaluation before their use in glioma patients.

The aim of this study was to investigate the effects of CaM antagonist, PTZ, and TFP on cell proliferation and migration of colon cancer cells and its impact on POPDC protein expression.

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder. Presently, there is no causal therapy available to slow down or halt disease progression. The presynaptic protein alpha-synuclein aggregates to form intraneuronal Lewy bodies in PD. It is generally believed that intermediates on the way from monomers to the large aggregates would mediate neurotoxicity, but the precise species and mechanism responsible for neuronal death are controversially debated. To study alpha-synuclein-mediated toxicity, we developed a new model in which moderate overexpression of wild-type alpha-synuclein led to gradual death of human postmitotic dopaminergic neurons. In accordance with findings in postmortem PD brains, small oligomeric species occurred and the autophagic flux was impaired in our model. The phenothiazine neuroleptic trifluoperazine, an activator of macroautophagy, selectively reduced one particular alpha-synuclein species and rescued cells. Inversely, blocking of autophagy led to an accumulation of this oligomeric species and increased cell death. These data show that activation of autophagy is a promising approach to protect against alpha-synuclein pathology and likely acts by targeting one specific alpha-synuclein species.

Multiple sclerosis (MS) is one of the most common neurodegenerative diseases. In this disease, the immune system attacks oligodendrocyte cells and the myelin sheath of myelinated neurons in the central nervous system, causing their destruction. These conditions lead to impaired conduction of nerve impulses and are manifested by symptoms such as weakness, fatigue, visual and motor disorders. This study aimed to evaluate the ability of trifluoperazine (TF) to improve cuprizone-induced behavioral and histopathological changes in the prefrontal cortex of C57BL/6 male mice. Demyelination was induced by adding 0.2% cuprizone (CPZ) to the standard animal diet for 6 weeks. Three doses of TF (0.5, 1 and 2 mg/kg/day; i.p.) were given once daily for the last 2 weeks of treatment. Treatment with CPZ induced a weight loss during 6 weeks of treatment compared to the control group, which was reversed by the administration of TF. Behavioral tests (pole test and rotarod performance test) showed a decrease in motor coordination and balance in the group treated with CPZ (P < 0.01). Treatment with TF during the last two weeks was able to improve these motor deficiencies. Histopathological examination also evidenced an increase in demyelination in the CPZ group, which was improved by TF administration. In addition, CPZ intake significantly decreased the cerebral cortex levels of p-Nrf2 (P < 0.001) and increased the levels of p-IKB (P < 0.001) and, these changes were normalized in the TF groups. TF administration also reversed the increased levels of nitrite and the reduced activity of the antioxidant enzyme superoxide dismutase associated with CPZ exposure. TF can to reduce the harmful effects of CPZ by reducing the demyelination and modulating the Nrf2 and NF-kB signaling pathways.

Multiple myeloma (MM) is the second most common hematologic malignancy of immunoglobulin-secreting plasma cells. Recent modern combination therapies have improved survival rates, but many patients develop resistance to novel drugs, leading to relapse. Trifluoperazine (TFP), a typical antipsychotic drug, has been reported to exert antitumor effects by targeting various pathways. Thus far, the role of TFP in MM has not been elucidated. In the current study, we demonstrated that TFP inhibited cell growth and autophagy activity but induced apoptosis of U266 and RPMI 8226 MM cells. Furthermore, cotreatment of these cell lines with TFP and rapamycin, a potent autophagy inducer, reduced cell apoptosis compared with TFP treatment alone. We also found that TFP inhibited nuclear protein 1 (NUPR1) expression. In the presence of TFP, cells stably overexpressing NUPR1 showed a higher viability than cells treated with the nonspecific control. Autophagy suppression and apoptosis induction caused by TFP were also reversed in MM cells upon NUPR1 overexpression. Overall, our results indicate that in the context of MM, TFP targets NUPR1, inhibiting cell growth and inducing apoptosis by autophagy inhibition. Our results could contribute toward efforts for the development of more effective therapies for MM to be tested in future clinical trials.

Resistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo.

Repurposing existing drugs for cancer treatment is an effective strategy. An approved antipsychotic drug, trifluoperazine (TFP), has been reported to have potential anticancer effects against several cancer types. Here, we investigated the effect and molecular mechanism of TFP in colorectal cancer (CRC). In vitro studies showed that TFP induced G0/G1 cell cycle arrest to dramatically inhibit CRC cell proliferation through downregulating cyclin-dependent kinase (CDK) 2, CDK4, cyclin D1, and cyclin E and upregulating p27. TFP also induced apoptosis, decreased mitochondrial membrane potential, and increased reactive oxygen species levels in CRC cells, indicating that TFP induced mitochondria-mediated intrinsic apoptosis. Importantly, TFP significantly suppressed tumor growth in two CRC subcutaneous tumor models without side effects. Interestingly, TFP treatment increased the expression levels of programmed death-1 ligand 1 (PD-L1) in CRC cells and programmed death-1 (PD-1) in tumor-infiltrating CD4+ and CD8+ T cells, implying that the combination of TFP with an immune checkpoint inhibitor, such as an anti-PD-L1 or anti-PD-1 antibody, might have synergistic anticancer effects. Taken together, our study signifies that TFP is a novel treatment strategy for CRC and indicates the potential for using the combination treatment of TFP and immune checkpoint blockade to increase antitumor efficiency.

Sigma-1 receptors are ubiquitous multifunctional ligand-regulated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Sigma-1 receptors bind ligands of different chemical structure and pharmacological action and modulate a wide range of cellular processes in health and disease, including Ca2+ signaling. To elucidate the involvement of sigma-1 receptors in the processes of Ca2+ signaling in macrophages we studied the effect of sigma-1 receptor ligands, phenothiazine neuroleptics chlorpromazine and trifluoperazine, on Ca2+ responses induced by inhibitors of endoplasmic Ca2+-ATPases thapsigargin and cyclopiazonic acid, as well as by disulfide-containing immunomodulators Glutoxim and Molixan in rat peritoneal macrophages. Using Fura-2AM microfluorimetry we showed for the first time that chlorpromazine and trifluoperazine inhibit both phases of Ca2+ responses induced by Glutoxim, Molixan, thapsigargin, and cyclopiazonic acid in rat peritoneal macrophages. The data obtained indicate the participation of sigma-1 receptors in a complex signaling cascade caused by Glutoxim or Molixan and leading to an increase in intracellular Ca2+ concentration in macrophages. The results also indicate the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+entry in macrophages.

Fragile X syndrome (FXS) is a prevailing genetic disorder of intellectual disability and autism. There is no efficacious medication for FXS. Through in silico screening with a public database, computational analysis of transcriptome profile in FXS mouse neurons predicts therapeutic value of an FDA-approved drug trifluoperazine. Systemic administration of low-dose trifluoperazine at 0.05 mg/kg attenuates multiple FXS- and autism-related behavioral symptoms. Moreover, computational analysis of transcriptome alteration caused by trifluoperazine suggests a new mechanism of action against PI3K (Phosphatidylinositol-4,5-bisphosphate 3-kinase) activity. Consistently, trifluoperazine suppresses PI3K activity and its down-stream targets Akt (protein kinase B) and S6K1 (S6 kinase 1) in neurons. Further, trifluoperazine normalizes the aberrantly elevated activity of Akt and S6K1 and enhanced protein synthesis in FXS mouse. Together, our data demonstrate a promising value of transcriptome-based computation in identification of therapeutic strategy and repurposing drugs for neurological disorders, and suggest trifluoperazine as a potential treatment for FXS.

Blood-brain barrier (BBB) disruption following ischemic stroke (IS) can induce significant aftereffects. Elevated calmodulin (CaM) expression following stroke causes calcium overload-a key contributor to BBB collapse. Trifluoperazine (TFP), a CaM inhibitor, reduces CaM overexpression following IS. However, it remains unclear whether TFP participates in BBB repair after IS. We administered TFP to mice subjected to middle cerebral artery occlusion (MCAO) and bEnd.3 cells subjected to oxygen-glucose deprivation (OGD). TFP treatment in MCAO mice reduced cerebral CaM expression and infarct size and decreased BBB permeability. OGD-treated bEnd.3 cells showed significantly increased CaM protein levels and reduced tight junction (TJ) protein levels; these changes were reversed by TFP treatment. Our results found that TFP administration in mice inhibited actin contraction following cerebral ischemia-reperfusion by suppressing the MLCK/p-MLC pathway, thereby attenuating cell retraction, improving TJ protein integrity, and reducing BBB permeability. Consequently, this treatment may promote neurological function recovery after IS.

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.

The hepatotoxicity of acetaminophen (APAP) occurs by initial metabolism to N-acetyl-p-benzoquinone imine which depletes GSH and forms APAP-protein adducts. Subsequently, the reactive nitrogen species peroxynitrite is formed from nitric oxide (NO) and superoxide leading to 3-nitrotyrosine in proteins. Toxicity occurs with inhibited mitochondrial function. We previously reported that in hepatocytes the nNOS (NOS1) inhibitor NANT inhibited APAP toxicity, reactive nitrogen and oxygen species formation, and mitochondrial dysfunction. In this work we examined the effect of trifluoperazine (TFP), a calmodulin antagonist that inhibits calcium induced nNOS activation, on APAP hepatotoxicity and reactive nitrogen formation in murine hepatocytes and in vivo. In freshly isolated hepatocytes TFP inhibited APAP induced toxicity, reactive nitrogen formation (NO, GSNO, and 3-nitrotyrosine in protein), reactive oxygen formation (superoxide), loss of mitochondrial membrane potential, decreased ATP production, decreased oxygen consumption rate, and increased NADH accumulation. TFP did not alter APAP induced GSH depletion in the hepatocytes or the formation of APAP protein adducts which indicated that reactive metabolite formation was not inhibited. Since we previously reported that TFP inhibits the hepatotoxicity of APAP in mice without altering hepatic APAP-protein adduct formation, we examined the APAP treated mouse livers for evidence of reactive nitrogen formation. 3-Nitrotyrosine in hepatic proteins and GSNO were significantly increased in APAP treated mouse livers and decreased in the livers of mice treated with APAP plus TFP. These data are consistent with a hypothesis that APAP hepatotoxicity occurs with altered calcium metabolism, activation of nNOS leading to increased reactive nitrogen formation, and mitochondrial dysfunction.

Cardiotoxicity of doxorubicin frequently complicates treatment outcome. Aberrantly activated calcium/calmodulin pathway can eventually trigger signaling cascades that mediate cardiotoxicity. Therefore, we tested the hypothesis that trifluoperazine, a strong calmodulin antagonist, may alleviate this morbidity.

As a tumor suppressor, Forkhead box O1 (FOXO1) is located in the nucleus where it regulates gene expression and inhibits tumor progression. However, the antitumor effects of FOXO1 are attenuated in several tumors due to its translocation from the nucleus to the cytoplasm. Trifluoperazine (TFP) is able to reverse tumor drug resistance by inhibiting multidrug resistance (MDR), however, the detailed molecular mechanisms by which this occurs remain to be fully elucidated. In the present study, the doxorubicin (DOX)‑resistant SHG44/DOX glioma cell line was established. The results showed that TFP promoted DOX‑induced cytotoxicity, cell cycle arrest and early apoptosis using a Cell Counting Kit‑8 and flow cytometry. In vivo experiments also demonstrated that DOX combined with TFP reduced tumor volumes and proliferation indices, and led to higher protein levels of FOXO1. In addition, TFP inhibited the nuclear exclusion of FOXO1, contributing toward the downregulation of MDR genes and an increase in intracellular DOX concentrations by reverse transcription‑quantitative polymerase chain reaction, western blot analysis, immunofluorescence and spectrophotometer analysis. Therefore, TFP may inhibit DOX resistance by stimulating FOXO1 nuclear translocation and suppressing MDF genes in SHG44/DOX cells, contributing to promising clinical prospects for tumor chemotherapy.