thiourea and tenovin-1

Introduction of hydrophilic groups can improve the solubility of leading drugs but inevitably affect their interaction with proteins. This study selected sirtuin inhibitors Tenovin-1 (T1) and Tenovin-6 (T6) as drug models to determine differences in binding mode to human serum albumin (HSA). T1 and T6 quenched the endogenous fluorescence of HSA via static quenching mechanism. Introduction of hydrophilic groups greatly reduced the binding constant, i.e., from 1.302 × 10

Topics: Acetanilides; Benzamides; Binding Sites; Circular Dichroism; Humans; Hydrophobic and Hydrophilic Interactions; Molecular Docking Simulation; Pharmaceutical Preparations; Protein Binding; Serum Albumin, Human; Sirtuins; Spectrometry, Fluorescence; Thermodynamics; Thiourea

Dengue fever is a common arbovirus disease, which has been spread to the entire tropical world. At present, effective drugs for the treatment of dengue fever have not yet appeared, and the dengue vaccines studied in various countries have also experienced severe adverse reactions. Thus it is urgent to find new chemicals against dengue virus. Now we found Sirtuins (SIRTs) were increased during dengue virus infection and tenovin-1, a SIRT1/2 inhibitor, showed an impressive antiviral ability in vitro. In BHK-21 cells, tenovin-1 inhibited the replication of DENV2 with an EC50 at 3.41 ± 1.10 μM, also inhibited other three types of dengue viruses with EC50 at 0.97 ± 1.11 μM, 1.81 ± 1.08 μM, 3.81 ± 1.34 μM respectively. Moreover, the cytopathic effect-induced DENV2 was largely improved by tenovin-1 treatment and the release of progeny viruses was inhibited by tenovin-1 treatment. At the same time, the viral protein level and mRNA level were decreased with tenovin-1 treatment after dengue virus infection. From the drug-addition assay, the tenovin-1 played its antiviral after viral infection, which indicated tenovin-1 was not a microbicide. Apart from its antiviral effect, tenovin-1 inhibited the inflammatory response caused by DENV2, reducing the release of inflammatory factors during viral infection. The antiviral effect of tenovin-1 was abrogated with SIRT agonist or SIRT2 knockdown treatment, which indicated the effect of tenovin-1 was on-target. In conclusion, tenovin-1 was proved to be a promising compound against flavivirus infection through SIRT2, which should be pay more attention for further study.

Topics: Acetanilides; Dengue Virus; Sirtuin 2; Thiourea; Virus Replication

Studies indicate that the pattern of shear stress determines the direction of endothelial progenitor cells (EPCs) differentiation. However, the mechanism remains largely unknown. Herein, we try to identify the role of oscillatory shear stress (OSS) in the transdifferentiation of EPCs into mesenchymal cells and the mechanism involved.. OSS was applied to EPCs using the flow chamber system in vitro. Matrigel, Boyden chamber, and healing assay were used to observe the changes in EPCs function. Further, 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe and/or western blot were performed to detect the expression of reactive oxygen species (ROS), p53 and PKCζ in EPCs. EPCs transduced with Lentivirus carrying Tp53 were implanted into the arterial vessel in the balloon injured rat model, and neointimal thickening was verified by HE staining.. OSS enhanced the expression of mesenchymal cell markers alpha-smooth muscle actin (α-SMA) and smooth muscle 22 alpha (SM22α) on EPCs. In the meantime, OSS time-dependently decreased p53 expression in EPCs, which was partially abolished by treatment with ROS scavenger N-acetylcysteine (NAC) or protein kinase C zeta (PKCζ) inhibitor Go6983. Moreover, the p53 agonist tenovin-1 attenuated the changes of OSS-mediated the mesenchymal cell markers and EPCs function. Besides, we also found that transplanting EPCs transfected with LV-Tp53 significantly inhibited neointimal thickening and promoted reendothelialization in vivo.. This study demonstrates OSS-induced EPC transdifferentiation into mesenchymal cells and ROS/PKCζ/p53 pathway play an essential role in it. It may serve as a promising therapeutic target for cardiovascular disease in the future.

Topics: Acetanilides; Animals; Cell Differentiation; Cell Transdifferentiation; Cells, Cultured; Endothelial Progenitor Cells; Male; Mesenchymal Stem Cells; Protein Kinase C; Rats; Reactive Oxygen Species; Stress, Mechanical; Thiourea; Time Factors; Tumor Suppressor Protein p53

The tenovins are a frequently studied class of compounds capable of inhibiting sirtuin activity, which is thought to result in increased acetylation and protection of the tumor suppressor p53 from degradation. However, as we and other laboratories have shown previously, certain tenovins are also capable of inhibiting autophagic flux, demonstrating the ability of these compounds to engage with more than one target. In this study, we present two additional mechanisms by which tenovins are able to activate p53 and kill tumor cells in culture. These mechanisms are the inhibition of a key enzyme of the

Topics: Acetanilides; Autophagy; Cell Proliferation; Dihydroorotate Dehydrogenase; Enzyme Inhibitors; Humans; Neoplasms; Oxidoreductases Acting on CH-CH Group Donors; Polypharmacology; Sirtuin 1; Thiourea; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Arthropod-borne viruses are diverse pathogens and are often associated with human disease. These viruses span multiple genera, including flaviviruses, alphaviruses, and bunyaviruses. In a high-throughput drug screen, we found that tenovin-1 was antiviral against the flaviviruses Zika virus and dengue virus. Tenovin-1 is a sirtuin inhibitor, and here we found that inhibition of sirtuins, but not inhibition of the related histone deacetylases, is potently antiviral against diverse arboviruses. Sirtuin inhibitors block infection of arboviruses in multiple human cell types. We found that sirtuin inhibitors arrest infection downstream of entry but that they do so at an early step, preventing the accumulation of viral RNA and protein. However, sirtuin inhibitors had no impact on the replication of flaviviral replicons, suggesting a defect in the establishment of replication. Consistent with this, we found that sirtuin inhibitors impacted double-stranded RNA (dsRNA) accumulation during flaviviral infection. Since these viruses infect vector insects, we also tested whether sirtuin inhibitors impacted infection of adult flies and found that these inhibitors blocked infection; therefore, they target highly conserved facets of replication. Taken together, these results suggest that sirtuin inhibitors represent a new class of potent host-targeting antivirals.

Topics: Acetanilides; Animals; Antiviral Agents; Arboviruses; Dengue Virus; Diptera; Drug Discovery; Female; HEK293 Cells; High-Throughput Screening Assays; Host Microbial Interactions; Humans; Sirtuins; Thiourea; Virus Replication; Zika Virus

The sirtuin 1/2 inhibitor tenovin-1 activates p53 and may have potential in the management of cancer. Here, we investigated the responsiveness of Ewing's sarcoma cells to tenovin-1. We examined its effects in two Ewing's sarcoma cell lines with different p53 status, i.e. in p53 wild-type and p53 null cells. Effects were assessed by flow cytometric analyses of cell death, mitochondrial membrane depolarization and reactive oxygen species (ROS) generation, by caspase 3/7 activity measurement, by mRNA expression profiling and by immunoblotting. Tenovin-1 elicited caspase-mediated cell death in p53 wild-type cells, but caspase-independent cell death in p53 null cells. Remarkably, it induced a nonlinear concentration response in the latter: low concentrations of tenovin-1 were much more effective than were higher concentrations. Tenovin-1's effects in p53 null cells involved gene expression changes of Bcl-2 family members, mitochondrial membrane depolarization, nuclear translocation of apoptosis-inducing factor, ROS formation and DNA damage; all these effects followed a bell-shaped pattern. In conclusion, our results provide new insights into tenovin-1's mode of action by demonstrating that it can induce different pathways of cell death.

Topics: Acetanilides; Antineoplastic Agents; Apoptosis; Apoptosis Inducing Factor; Caspases; Cell Line, Tumor; DNA Damage; Gene Expression Regulation, Neoplastic; Humans; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Sarcoma, Ewing; Sirtuin 1; Sirtuin 2; Thiourea; Tumor Suppressor Protein p53

The chromatin contains the genetic and the epigenetic information of a eukaryotic organism. Posttranslational modifications of histones, such as acetylation and methylation, regulate their structure and control gene expression. Histone acetyltransferases (HATs) acetylate lysine residues in histones while histone deacetylases (HDACs) remove this modification. HDAC inhibitors (HDACi) can alter gene expression patterns and induce cytotoxicity in cancer cells. Here we provide an overview of methods to determine the cytotoxic effects of HDACi treatment. Our chapter describes colorimetric methods, like trypan blue exclusion test, crystal violet staining, lactate dehydrogenase assay, MTT and Alamar Blue assays, as well as fluorogenic methods like TUNEL staining and the caspase-3/7 activity assay. Moreover, we summarize flow cytometric analysis of propidium iodide uptake, annexin V staining, cell cycle status, ROS levels, and mitochondrial membrane potential as well as detection of apoptosis by Western blot.

Topics: Acetanilides; Antineoplastic Agents; Apoptosis; Blotting, Western; Caspase 3; Caspase 7; Cell Cycle; Chromatin; Colorimetry; Coloring Agents; Enzyme Activation; Flow Cytometry; Fluoresceins; Fluorescent Dyes; Gene Expression Regulation, Neoplastic; HCT116 Cells; Histone Acetyltransferases; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Protein Processing, Post-Translational; Reactive Oxygen Species; Thiourea

Since altered energy metabolism is a hallmark of cancer, many drugs targeting metabolic pathways are in active clinical trials. The tumor suppressor p53 is often inactivated in cancer, either through downregulation of protein or loss-of-function mutations. As such, stabilization of p53 is considered as one promising approach to treat those cancers carrying wild type (WT) p53. Herein, SIRT1 inhibitor Tenovin-1 and polo-like kinase 1 (Plk1) inhibitor BI2536 were used to stabilize p53. We found that both Tennovin-1 and BI2536 increased the anti-neoplastic activity of metformin, an inhibitor of oxidative phosphorylation, in a p53 dependent manner. Since p53 has also been shown to regulate metabolic pathways, we further analyzed glycolysis and oxidative phosphorylation upon drug treatments. We showed that both Tennovin-1 and BI2536 rescued metformin-induced glycolysis and that both Tennovin-1 and BI2536 potentiated metformin-associated inhibition of oxidative phosphorylation. Of significance, castration-resistant prostate cancer (CRPC) C4-2 cells show a much more robust response to the combination treatment than the parental androgen-dependent prostate cancer LNCaP cells, indicating that targeting energy metabolism with metformin plus p53 stabilizers might be a valid approach to treat CRPC carrying WT p53.

Topics: Acetanilides; Antineoplastic Agents; Apoptosis; Cell Cycle Proteins; Cell Line, Tumor; Drug Synergism; Drug Therapy, Combination; Gene Expression Regulation, Neoplastic; Glycolysis; Humans; Male; Metformin; Mitochondria; Mitosis; Oxidative Phosphorylation; Polo-Like Kinase 1; Prostate; Prostatic Neoplasms, Castration-Resistant; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Pteridines; Signal Transduction; Sirtuin 1; Thiourea; Tumor Suppressor Protein p53

Sirtuin 1 (SIRT1) and sirtuin 2 (SIRT2) are NAD+-dependent protein deacetylases involved in the regulation of key cancer-associated genes. In this study we evaluated the relevance of these deacetylases in lung cancer biology.. Protein levels of SIRT1 and SIRT2 were determined in non-small cell lung cancer (NSCLC) cell lines and primary tumors from 105 patients. Changes in proliferation were assessed after SIRT1 and SIRT2 downregulation in lung cancer cell lines using siRNA-mediated technology or tenovin-1, a SIRT1 and SIRT2 inhibitor.. High SIRT1 and SIRT2 protein levels were found in NSCLC cell lines compared with non-tumor lung epithelial cells. The expression of SIRT1 and SIRT2 proteins was also significantly higher in lung primary tumors than in normal tissue (P<0.001 for both sirtuins). Stronger nuclear SIRT1 staining was observed in adenocarcinomas than in squamous cell carcinomas (P=0.033). Interestingly, in NSCLC patients, high SIRT1 and SIRT2 expression levels were associated with shorter recurrence-free survival (P=0.04 and P=0.007, respectively). Moreover, the combination of high SIRT1 and SIRT2 expression was an independent prognostic factor for shorter recurrence-free survival (P=0.002) and overall survival (P=0.022). In vitro studies showed that SIRT1 and/or SIRT2 downregulation significantly decreased proliferation of NSCLC.. Our results support the hypothesis that SIRT1 and SIRT2 have a protumorigenic role in lung cancer, promoting cell proliferation. Moreover, the expression of these proteins is associated with poor prognosis in NSCLC patients and may help to identify those NSCLC patients with high risk of recurrence that could benefit from adjuvant therapy after resection.

Topics: Acetanilides; Aged; Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression; Humans; Lung Neoplasms; Male; Middle Aged; Neoplasm Grading; Neoplasm Staging; Prognosis; RNA Interference; RNA, Small Interfering; Sirtuin 1; Sirtuin 2; Thiourea; Tumor Suppressor Protein p53

Estrogen is synthesized from cholesterol and high cholesterol levels are suggested to be associated with increased risk of estrogen receptor(ER)-positive breast cancer. The cholesterol metabolite 27-hydroxycholesterol (27-OHC) was recently identified as a selective estrogen receptor modulator (SERM) and may therefore impact breast cancer progression. However, the mechanisms by which 27-OHC may contribute to breast cancer are not all known. We determined the extent to which 27-OHC regulates cell proliferation in MCF7 ER-positive breast cancer cell line involving the tumor suppressor protein p53. We found that treatment of MCF7 cells with 27-OHC resulted reduced p53 transcriptional activity. Conversely, treatment of the ER-negative MDA-MB 231 cells with 27-OHC induced no significant change in p53 activity. Exposure of MCF7 cells to 27-OHC was also associated with increased protein levels of the E3 ubiquitin protein ligase MDM2 and decreased levels of p53. Moreover, 27-OHC also enhanced physical interaction between p53 and MDM2. Furthermore, 27-OHC-induced proliferation was attenuated using either the p53 activator Tenovin-1 or the MDM2 inhibitor Nutlin-3 and Mdm2 siRNA. Taken together, our results indicate that 27-OHC may contribute to ER-positive breast cancer progression by disrupting constitutive p53 signaling in an MDM2-dependent manner.

Topics: Acetanilides; Breast Neoplasms; Cell Proliferation; Dose-Response Relationship, Drug; Female; Gene Expression Regulation, Neoplastic; Humans; Hydroxycholesterols; Imidazoles; MCF-7 Cells; Piperazines; Protein Binding; Proto-Oncogene Proteins c-mdm2; Receptors, Estrogen; RNA Interference; Selective Estrogen Receptor Modulators; Signal Transduction; Thiourea; Transcription, Genetic; Transfection; Tumor Suppressor Protein p53

Silent information regulator type-1 (SIRT1) is the best-studied member of the Sirtuin (Sir2) family of nicotinamide dinucleotide (NAD)-dependent class III histone deacetylases (HDACs), but has not yet been explored in cutaneous T-cell lymphoma (CTCL). We analyzed five CTCL cell lines and lesional tissues using flow cytometry, immunostaining, immunoblotting, cell death, viability, and apoptosis assays, small-molecule inhibitors, and shRNA knockdown. We found strong SIRT1 expression among CTCL lines relative to normal lymphocytes. CTCL cells in lesional tissues also expressed SIRT1 strongly. SIRT1 knockdown resulted in reduced cellular metabolism and proliferation, increased apoptosis, and PARP cleavage products. Tenovin-1, which reversibly inhibits class III HDACs (SIRT1 and SIRT2), reduced SIRT enzymatic activity and SIRT1 expression and led to increased apoptosis. These alterations were accompanied by increased forkhead box O3 (FoxO3) in several cell lines and increased nuclear p53, as well as acetylated p53 in wtp53 MyLa CTCL line. A combination of class I/II and class III HDACIs (vorinostat and tenovin-1) produced significantly greater growth inhibition, cell death via apoptosis, as well as superior p53 promoter upregulation in wtp53 MyLa cells as compared with either agent alone. This occurred in a partially p53-dependent manner, as these effects were blunted by p53 knockdown. Our results indicate that SIRT1 is strongly expressed in CTCL. Its inhibition results in reduced growth and increased apoptosis of CTCL cells. Furthermore, our findings suggest that some CTCL patients, such as those with wtp53, might benefit more from treatment with a combination of different classes of HDACIs than with a single agent.

Topics: Acetanilides; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Forkhead Box Protein O3; Forkhead Transcription Factors; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Lymphoma, T-Cell, Cutaneous; Sirtuin 1; Thiourea; Tumor Suppressor Protein p53; Up-Regulation; Vorinostat

Melanoma is one of the most lethal forms of skin cancer and its incidence is continuing to rise in the United States. Therefore, novel mechanism and target-based strategies are needed for the management of this disease. SIRT1, a NAD(+)-dependent class III histone deacetylase, has been implicated in a variety of physiological processes and pathological conditions. We recently demonstrated that SIRT1 is upregulated in melanoma and its inhibition by a small-molecule, tenovin-1, inhibits cell proliferation and clonogenic survival of melanoma cells, possibly via activating p53. Here, we employed a gel free quantitative proteomics approach to identify the downstream effectors and targets of SIRT1 in melanoma. The human malignant melanoma, G361 cells were treated with tenovin-1 followed by protein extraction, in liquid trypsin digestion, and peptide analyses using nanoLC-MS/MS. A total of 1091 proteins were identified, of which 20 proteins showed significant differential expression with 95% confidence interval. These proteins were subjected to gene ontology and Ingenuity Pathway Analysis (IPA) to obtain the information regarding their biological and molecular functions. Real-Time qRT-PCR validation showed that five of these (PSAP, MYO1B, MOCOS, HIS1H4A and BUB3) were differentially expressed at mRNA levels. Based on their important role in cell cycle regulation, we selected to focus on BUB family proteins (BUB3, as well as BUB1 and BUBR1) for subsequent validation. The qRT-PCR and immunoblot analyses showed that tenovin-1 inhibition of SIRT1 resulted in a downregulation of BUB3, BUB1 and BUBR1 in multiple melanoma cell lines. Since tenovin-1 is an inhibitor of both SIRT1 and SIRT2, we employed lentivirus mediated silencing of SIRT1 and SIRT2 in G361 cells to determine if the observed effects on BUB family proteins are due to SIRT1- or SIRT2- inhibition. We found that only SIRT1 inhibition resulted in a decrease in BUB3, BUB1 and BUBR1. Our study identified the mitotic checkpoint regulator BUB family proteins as novel downstream targets of SIRT1. However, further validation is needed in appropriate models to confirm our findings and expand on our observations.

Topics: Acetanilides; Cell Cycle Proteins; Cell Line, Tumor; Down-Regulation; Enzyme Activation; Gene Ontology; Gene Silencing; Histone Deacetylase Inhibitors; Histones; Humans; Melanoma; Myosin Type I; Poly-ADP-Ribose Binding Proteins; Protein Serine-Threonine Kinases; Proteomics; RNA, Messenger; Saposins; Sirtuin 1; Sulfurtransferases; Thiourea

Melanoma causes more deaths than any other skin cancer, and its incidence in the US continues to rise. Current medical therapies are insufficient to control this deadly neoplasm, necessitating the development of new target-based approaches. The objective of this study was to determine the role and functional significance of the class III histone deacetylase SIRT1 in melanoma. We have found that SIRT1 is overexpressed in clinical human melanoma tissues and human melanoma cell lines (Sk-Mel-2, WM35, G361, A375, and Hs294T) compared to normal skin and normal melanocytes, respectively. In addition, treatment of melanoma cell lines A375, Hs294T, and G361 with Tenovin-1, a small molecule SIRT1 inhibitor, resulted in a significant decrease in cell growth and cell viability. Further, Tenovin-1 treatment also resulted in a marked decrease in the clonogenic survival of melanoma cells. Further experiments showed that the anti-proliferative response of Tenovin-1 was accompanied by an increase in the protein as well as activity of the tumor suppressor p53. This increase in p53 activity was substantiated by an increase in the protein level of its downstream target p21. Overall, these data suggest that small molecule inhibition of SIRT1 causes anti-proliferative effects in melanoma cells. SIRT1 appears to be acting through the activity of the tumor suppressor p53, which is not mutated in the majority of melanomas. However, future detailed studies are needed to further explore the role and mechanism of SIRT1 in melanoma development and progression and its usefulness in melanoma treatment.

Topics: Acetanilides; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Histone Deacetylase Inhibitors; Humans; Melanocytes; Melanoma; Regulatory Elements, Transcriptional; RNA, Messenger; RNA, Neoplasm; Sirtuin 1; Skin Neoplasms; Thiourea; Tumor Stem Cell Assay; Tumor Suppressor Protein p53; Up-Regulation

In the present work we have studied the effects of p53 on the proliferation and differentiation of neural progenitor cells (NPC) in mouse hippocampal organotypic culture. To study the role of p53 the selective p53 inhibitor pifithrin-alpha (PFT) and activator tenovin 1 (TEN) were used in the experiments. Obtained data demonstrated that the injections of PFT did not affect on the amount of phospho-H3 positive cells in the subgranular zone of hippocampus. This data revealed that p53 inhibition does not change the proliferation level of the NPC. In opposite, at the TEN treatments we observed increased of the proliferation activity. Analysis of Pim-1 and Phb 1, which regulate cell cycle progression, demonstrated that p53 activation led to increased level of Pim-1 as well as the proliferation. Thus, our data correlate with published ones and proposed that Pim-1 positively regulates NPC cell cycle progression. In opposite to Pim-1, Phb 1 has anti-proliferative action. Our obtained data demonstrated that TEN diminished Phb 1 expression. Primarily PFT injections led to the increasing Phbl level, but then dramatically decreased it that accompanied with unchanged proliferation level. In other words, increased proliferation level after TEN treatments, which we observed, can be partly depend from the inhibition of anti-proliferative activity of Phb. In our study we demonstrated that both TEN and in a greater degree PFT stimulates neuronal differentiation by activation of CRMP-2 expression, but do not affect on gliogenesis. Thus, obtained data revealed that p53 is an important factor of neuronal differentiation and, probably, p53 action is mediated by cell cycle regulator protein such as Pim-1 and Phbl.

Topics: Acetanilides; Animals; Antineoplastic Agents; Benzothiazoles; Cell Proliferation; Cells, Cultured; Hippocampus; Intercellular Signaling Peptides and Proteins; Mice; Nerve Tissue Proteins; Neural Stem Cells; Neurogenesis; Prohibitins; Proto-Oncogene Proteins c-pim-1; Repressor Proteins; Thiourea; Toluene; Tumor Suppressor Protein p53