- Calmodulins from Schistosoma mansoni: Biochemical analysis and interaction with IQ-motifs from voltage-gated calcium channels. [Journal Article]
- CCCell Calcium 2018 May 17; 74:1-13
- The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathwa...
The trematode Schistosoma mansoni is a causative agent of schistosomiasis, the second most common parasitic disease of humans after malaria. Calcium homeostasis and calcium-mediated signalling pathways are of particular interest in this species. The drug of choice for treating schistosomiasis, praziquantel, disrupts the regulation of calcium uptake and there is interest in exploiting calcium-mediated processes for future drug discovery. Calmodulin is a calcium sensing protein, present in most eukaryotes. It is a critical regulator of processes as diverse as muscle contraction, cell division and, partly through interaction with voltage-gated calcium channels, intra-cellular calcium concentrations. S. mansoni expresses two highly similar calmodulins - SmCaM1 and SmCaM2. Both proteins interact with calcium, manganese, cadmium (II), iron (II) and lead ions in native gel electrophoresis. These ions also cause conformational changes in the proteins resulting in the exposure of a more hydrophobic surface (as demonstrated by anilinonaphthalene-8-sulfonate fluorescence assays). The proteins are primarily dimeric in the absence of calcium ions, but monomeric in the presence of this ion. Both SmCaM1 and SmCaM2 interact with a peptide corresponding to an IQ-motif derived from the α-subunit of the voltage-gated calcium channel SmCav1B (residues 1923-1945). Both proteins bound with slightly higher affinity in the presence of calcium ions. However, there was no difference between the affinities of the two proteins for the peptide. This interaction could be antagonised by chlorpromazine and trifluoperazine, but not praziquantel or thiamylal. Interestingly no interaction could be detected with the other three IQ-motifs identified in S. mansoni voltage-gated ion calcium channels.
- Treatment of SEC62 over-expressing tumors by Thapsigargin and Trifluoperazine. [Journal Article]
- BCBiomol Concepts 2018 May 19; 9(1):53-63
- Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistan...
Treatment with analogues of the SERCA-inhibitor Thapsigargin is a promising new approach for a wide variety of cancer entities. However, our previous studies on various tumor cells suggested resistance of SEC62 over-expressing tumors to this treatment. Therefore, we proposed the novel concept that e.g. lung-, prostate-, and thyroid-cancer patients should be tested for SEC62 over-expression, and developed a novel therapeutic strategy for a combinatorial treatment of SEC62 over-expressing tumors. The latter was based on the observations that treatment of SEC62 over-expressing tumor cells with SEC62-targeting siRNAs showed less resistance to Thapsigargin as well as a reduction in migratory potential and that the siRNA effects can be mimicked by the Calmodulin antagonist Trifluoperazine. Therefore, the combinatorial treatment of SEC62 over-expressing tumors was proposed to involve Thapsigargin and Trifluoperazine. Here, we addressed the impact of Thapsigargin and Trifluoperazine in separate and combined treatments of heterotopic tumors, induced by inoculation of human hypopharyngeal squamous cell carcinoma (FaDu)-cells into the mouse flank. Seeding of the tumor cells and/or their growth rate were significantly reduced by all three treatments, suggesting Trifluoperazine is a small molecule to be considered for future therapeutic strategies for patients, suffering from Sec62-overproducing tumors.
- Osteocyte-Driven Downregulation of Snail Restrains Effects of Drd2 Inhibitors on Mammary Tumor Cells. [Journal Article]
- CRCancer Res 2018 May 16
- While bone is a frequent target of breast cancer-associated metastasis, little is known about the effects of tumor-bone interactions on the efficacy of tumor-suppressing agents. Here we examined the ...
While bone is a frequent target of breast cancer-associated metastasis, little is known about the effects of tumor-bone interactions on the efficacy of tumor-suppressing agents. Here we examined the effect of two FDA-approved dopamine modulators, Fluphenazine (FP) and Trifluoperazine (TFP), on mammary tumor cells, osteoclasts, osteoblasts, and osteocytes. These agents suppressed proliferation and migration of mammary tumor cells chiefly by antagonizing dopamine receptor D2 and reduced bone resorption by downregulating nuclear factor of activated T-cells, cytoplasmic 1 (Nfatc1). Three-dimensional spheroid formation assays revealed that tumor cells have high affinity to osteocytes and type I collagen, and interactions with osteocytes as well as administration of FP and TFP downregulated Snail and suppressed migratory behaviors. Unlike the inhibitory action of FP and TFP on tumor growth, tumor-osteocyte interactions stimulated tumor proliferation by upregulating NFκB and Akt. In the bone microenvironment, osteocytes downregulated Snail and acted as an attractant as well as a stimulant to mammary tumor cells. These results demonstrate that tumor-osteocyte interactions strengthen dopamine receptor-mediated suppression of tumor migration but weaken its inhibition of tumor proliferation in the osteocyte-rich bone microenvironment.
- Synergistic Enhancement of Enzyme Performance and Resilience via Orthogonal Peptide-Protein Chemistry Enabled Multilayer Construction. [Journal Article]
- BBiomacromolecules 2018 Jun 15
- Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitat...
Protein immobilization is critical to utilize their unique functions in diverse applications. Herein, we report that orthogonal peptide-protein chemistry enabled multilayer construction can facilitate the incorporation of various folded structural domains, including calmodulin in different states, affibody, and dihydrofolate reductase (DHFR). An extended conformation is found to be the most advantageous for steady film growth. The resulting protein thin films exhibit sensitive and selective responsive behaviors to biosignals, such as Ca2+, trifluoperazine, and nicotinamide adenine dinucleotide phosphate (NADPH), and fully maintain the catalytic activity of DHFR. The approach is applicable to different substrates such as hydrophobic gold and hydrophilic silica microparticles. The DHFR enzyme can be immobilized onto silica microparticles with tunable amounts. The multilayer setup exhibits a synergistic enhancement of DHFR activity with increasing numbers of bilayers and also makes the embedded DHFR more resilient to lyophilization. Therefore, this is a convenient and versatile method for protein immobilization with potential benefits of synergistic enhancement in enzyme performance and resilience.
- Calmodulin antagonist enhances DR5-mediated apoptotic signaling in TRA-8 resistant triple negative breast cancer cells. [Journal Article]
- JCJ Cell Biochem 2018; 119(7):6216-6230
- Patients with triple negative breast cancer (TNBC) have no successful "targeted" treatment modality, which represents a priority for novel therapy strategies. Upregulated death receptor 5 (DR5) expre...
Patients with triple negative breast cancer (TNBC) have no successful "targeted" treatment modality, which represents a priority for novel therapy strategies. Upregulated death receptor 5 (DR5) expression levels in breast cancer cells compared to normal cells enable TRA-8, a DR5 specific agonistic antibody, to specifically target malignant cells for apoptosis without inducing normal hepatocyte apoptosis. Drug resistance is a common obstacle in TRAIL-based therapy for TNBC. Calmodulin (CaM) is overexpressed in breast cancer. In this study, we characterized the novel function of CaM antagonist in enhancing TRA-8 induced cytotoxicity in TRA-8 resistant TNBC cells and its underlying molecular mechanisms. Results demonstrated that CaM antagonist(s) enhanced TRA-8 induced cytotoxicity in a concentration and time-dependent manner for TRA-8 resistant TNBC cells. CaM directly bound to DR5 in a Ca2+ dependent manner, and CaM siRNA promoted DR5 recruitment of FADD and caspase-8 for DISC formation and TRA-8 activated caspase cleavage for apoptosis in TRA-8 resistant TNBC cells. CaM antagonist, trifluoperazine, enhanced TRA-8 activated DR5 oligomerization, DR5-mediated DISC formation, and TRA-8 activated caspase cleavage for apoptosis, and decreased anti-apoptotic pERK, pAKT, XIAP, and cIAP-1 expression in TRA-8 resistant TNBC cells. These results suggest that CaM could be a key regulator to mediate DR5-mediated apoptotic signaling, and suggests a potential strategy for using CaM antagonists to overcome drug resistance of TRAIL-based therapy for TRA-8 resistant TNBC.
- Ruthenium(II)-N-alkyl phenothiazine complexes as potential anticancer agents. [Journal Article]
- JBJ Biol Inorg Chem 2018 Apr 11
- In recent years, the search for effective anticancer compounds based on transition metal complexes has been the focus of medical investigations. The synergy between the ruthenium(II) and N-alkylpheno...
In recent years, the search for effective anticancer compounds based on transition metal complexes has been the focus of medical investigations. The synergy between the ruthenium(II) and N-alkylphenothiazine counter-ions (chlorpromazine hydrochloride, thioridazine hydrochloride and trifluoperazine dihydrochloride, respectively) through the formation of three different complexes (1-3) was investigated. We explored whether the selected counter-ions and complexes might affect redox homeostasis and genome integrity of normal human blood cells, and induce an inhibition of Na+/K+-ATPase and AChE at pharmacologically relevant doses. Our results have shown that counter-ions and complexes did not affect the activity of Na+/K+-ATPase, while AChE activity was inhibited in a dose-dependent manner. All investigated compounds disturbed the viability and redox homeostasis of lymphocytes. Complexes 1 and 2 displayed potent cytotoxic and prooxidant action while complex 3 behaved as a weaker genotoxic inducer. Still, the tested complexes appeared to be less genotoxic and more cytostatic than the corresponding counter-ions. The effects of selected complexes were also tested in PC12 and U2OS cancer cells with special attention being given to the ability of phenothiazines to affect dopamine D2 receptors. Using the confocal laser scanning microscopy, we observed that all the complexes reduced cell viability. Although all investigated complexes have been bound to the dopamine receptor D2-eGFP, only complex 3 reduced its surface density and increased its lateral mobility in investigated cell lines. Albeit the role of alternative targets for complex 3 cannot be ruled out, its effects should be further examined as potential treatment strategy against cancer cells that overexpress D2.
- Repositioning of the antipsychotic trifluoperazine: Synthesis, biological evaluation and in silico study of trifluoperazine analogs as anti-glioblastoma agents. [Journal Article]
- EJEur J Med Chem 2018 May 10; 151:186-198
- Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival...
Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival time in mice models of glioblastoma. In attempt to identify an effective trifluoperazine analog, fourteen compounds have been synthesized and biologically in vitro and in vivo assessed. Using MTT assay, compounds 3dc and 3dd elicited 4-5 times more potent inhibitory activity than trifluoperazine with IC50 = 2.3 and 2.2 μM against U87MG glioblastoma cells, as well as, IC50 = 2.2 and 2.1 μM against GBL28 human glioblastoma patient derived primary cells, respectively. Furthermore, they have shown a reasonable selectivity for glioblastoma cells over NSC normal neural cell. In vivo evaluation of analog 3dc confirmed its advantageous effect on reduction of tumor size and increasing the survival time in brain xenograft mouse model of glioblastoma. Molecular modeling simulation provided a reasonable explanation for the observed variation in the capability of the synthesized analogs to increase the intracellular Ca2+ levels. In summary, this study presents compound 3dc as a proposed new tool for the adjuvant chemotherapy of glioblastoma.
- Reprint of: A chemical screen identifies trifluoperazine as an inhibitor of glioblastoma growth. [Journal Article]
- BBBiochem Biophys Res Commun 2018 May 05; 499(2):136-142
- 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 inh...
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.
- Early prediction of olanzapine-induced weight gain for schizophrenia patients. [Journal Article]
- PRPsychiatry Res 2018; 263:207-211
- The aim of this study was to determine whether weight changes at week 2 or other factors predicted weight gain at week 6 for schizophrenia patients receiving olanzapine. This study was the secondary ...
The aim of this study was to determine whether weight changes at week 2 or other factors predicted weight gain at week 6 for schizophrenia patients receiving olanzapine. This study was the secondary analysis of a six-week trial for 94 patients receiving olanzapine (5 mg/d) plus trifluoperazine (5 mg/d), or olanzapine (10 mg/d) alone. Patients were included in analysis only if they had completed the 6-week trial (per protocol analysis). Weight gain was defined as a 7% or greater increase of the patient's baseline weight. The receiver operating characteristic curve was employed to determine the optimal cutoff points of statistically significant predictors. Eleven of the 67 patients completing the 6-week trial were classified as weight gainers. Weight change at week 2 was the statistically significant predictor for ultimate weight gain at week 6. A weight change of 1.0 kg at week 2 appeared to be the optimal cutoff point, with a sensitivity of 0.92, a specificity of 0.75, and an AUC of 0.85. Using weight change at week 2 to predict weight gain at week 6 is favorable in terms of both specificity and sensitivity. Weight change of 1.0 kg or more at 2 weeks is a reliable predictor.
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- Trifluoperazine induces apoptosis through the upregulation of Bax/Bcl‑2 and downregulated phosphorylation of AKT in mesangial cells and improves renal function in lupus nephritis mice. [Journal Article]
- IJInt J Mol Med 2018; 41(6):3278-3286
- The inhibition of mesangial cell (MC) proliferation has become an important therapy in preventing glomerular proliferation diseases. Trifluoperazine (TFP) has been reported to inhibit the proliferati...
The inhibition of mesangial cell (MC) proliferation has become an important therapy in preventing glomerular proliferation diseases. Trifluoperazine (TFP) has been reported to inhibit the proliferation of several types of cancer cell, however, the effects of TFP in renal proliferation diseases remain to be fully elucidated. The present study examined the effects of TFP on the proliferation of MCs and quantified cell apoptosis progression in vivo and in vitro. The effects of various TFP concentrations and treatment durations on cell proliferation and cell apoptosis in vitro were analyzed using flow cytometry in conjunction with a Cell Counting kit‑8 assay. Cell proliferation in vivo was determined using hematoxylin and eosin staining and immunohistochemistry of Ki67. The expression of the two cell apoptosis‑related proteins, B‑cell lymphoma-2 (Bcl‑2) and Bcl‑2‑associated X protein (Bax), were estimated using western blot analysis and immunohistochemistry in vivo and in vitro. TFP‑induced phosphatidylinositol 3‑kinase (PI3K)/protein kinase B (AKT) signaling pathways were also estimated using western blot analysis. These results suggested that TFP inhibited MC proliferation in a dose‑ and time‑dependent manner. It was found that TFP inhibited the abnormal proliferation of MCs, which was stimulated by 20% fetal bovine serum in vitro and in lupus MRL/lpr mice. TFP promoted cell apoptosis, downregulated the expression of Bcl‑2 and upregulated the expression of Bax in a dose‑dependent manner at mRNA and protein levels. In addition, TFP inhibited phosphorylated AKT, potentially leading to the suppressed activation of PI3K/AKT signaling pathways. TFP treatment significantly decreased the levels of blood urea nitrogen and serum creatinine, but had no significant effects on the body weight and liver function of the lupus mice. These results validated and reinforced the potential of TFP in the treatment of mesangial proliferative diseases.