ABSTRACT
Lewy body (LB), which mainly consists of abnormal α-synuclein (αS) aggregates, is a histological hallmark of Parkinson's disease (PD). αS aggregation and LB inclusions are induced by spreading αS fibrils to neurons; therefore, the formation and transmission of αS fibrils to neurons may play an essential role in initiating LB formation in neurons. αS expressed in neurons is released into the extracellular space and taken up by macrophages and microglia; therefore, we hypothesized that macrophages/microglia play a role in the formation and spread of αS fibrils. In this study, we aimed to investigate the involvement of macrophages/microglia in the formation and spread of αS fibrils using transgenic animals that express human αS in macrophages/microglia. Transgenic zebrafish expressing A53T mutated αS (αS_A53T) in macrophages/microglia revealed αS accumulation in neurons. Transcriptome analysis by RNA-seq of human αS and αS_A53T expressing zebrafish revealed that kinase genes and E3 ubiquitin protein ligase genes were significantly high, and neuronal activity and transport-related Gene Ontology terms were also isolated. Meanwhile, αS_A53T monomers were taken up by A-THP-1 cells; processed to larger molecules, which could be αS fibrils; and released from macrophage cells. Furthermore, the ubiquitin-proteasome system modulated αS fibrils in A-THP-1 cells. αS fibrils suggest being formed from monomers in macrophages and spread to neurons to induce αS aggregates. Therefore, macrophages may play an essential role in the formation of αS aggregates and the pathogenesis of PD.
Subject(s)
Macrophages , Neurons , alpha-Synuclein , Animals , Animals, Genetically Modified , Humans , Inclusion Bodies/metabolism , Macrophages/metabolism , Neurons/metabolism , Parkinson Disease/pathology , THP-1 Cells , Zebrafish/genetics , Zebrafish/metabolism , alpha-Synuclein/genetics , alpha-Synuclein/metabolismABSTRACT
A majority of mesothelioma specimens were defective of p14 and p16 expression due to deletion of the INK4A/ARF region, and the p53 pathway was consequently inactivated by elevated MDM2 functions which facilitated p53 degradaton. We investigated a role of p53 elevation by MDM2 inhibitors, nutlin-3a and RG7112, in cytotoxicity of replication-competent adenoviruses (Ad) lacking the p53-binding E1B55kDa gene (Ad-delE1B). We found that a growth inhibition by p53-activating Ad-delE1B was irrelevant to p53 expression in the infected cells, but combination of Ad-delE1B and the MDM2 inhibitor produced synergistic inhibitory effects on mesothelioma with the wild-type but not mutated p53 genotype. The combination augmented p53 phosphorylation, activated apoptotic but not autophagic pathway, and enhanced DNA damage signals through ATM-Chk2 phosphorylation. The MDM2 inhibitors facilitated production of the Ad progenies through augmented expression of nuclear factor I (NFI), one of the transcriptional factors involved in Ad replications. Knocking down of p53 with siRNA did not increase the progeny production or the NFI expression. We also demonstrated anti-tumor effects by the combination of Ad-delE1B and the MDM2 inhibitors in an orthotopic animal model. These data collectively indicated that upregulation of wild-type p53 expression contributed to cytotoxicity by E1B55kDa-defective replicative Ad through NFI induction and suggested that replication-competent Ad together with augmented p53 levels was a therapeutic strategy for p53 wild-type mesothelioma.
Subject(s)
Adenoviridae/genetics , Adenovirus E1 Proteins/genetics , Antineoplastic Agents/pharmacology , Imidazoles/pharmacology , Imidazolines/pharmacology , Mesothelioma/therapy , Neurofibromin 1/metabolism , Oncolytic Virotherapy , Oncolytic Viruses/genetics , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Tumor Suppressor Protein p53/metabolism , Adenoviridae/growth & development , Animals , Apoptosis/drug effects , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Cell Line, Tumor , Chemotherapy, Adjuvant , Gene Expression Regulation, Neoplastic , Mesothelioma/genetics , Mesothelioma/metabolism , Mesothelioma/virology , Mice, Inbred BALB C , Mice, Nude , Neurofibromin 1/genetics , Oncolytic Viruses/growth & development , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , Tumor Burden/drug effects , Tumor Suppressor Protein p53/genetics , Virus Replication , Xenograft Model Antitumor AssaysABSTRACT
A majority of mesothelioma had the wild-type p53 genotype but was defective of p53 functions primarily due to a genetic defect in INK4A/ARF region. We examined a growth suppressive activity of CP-31398 which was developed to restore the p53 functions irrespective of the genotype in mesothelioma with wild-type or mutated p53. CP-31398 up-regulated p53 levels in cells with wild-type p53 genotype but induced cell growth suppression in a p53-independent manner. In contrasts, nutlin-3a, an MDM2 inhibitor, increased p53 and p21 levels in mesothelioma with the wild-type p53 genotype and produced growth suppressive effects. We investigated a combinatory effect of CP-31398 and nutlin-2a and found the combination produced synergistic growth inhibition in mesothelioma with the wild-type p53 but not with mutated p53. Western blot analysis showed that the combination increased p53 and the phosphorylation levels greater than treatments with the single agent, augmented cleavages of PARP and caspase-3, and decreased phosphorylated FAK levels. Combination of CP-31398 and defactinib, a FAK inhibitor, also achieved synergistic inhibitory effects and increased p53 with FAK dephosphorylation levels greater than the single treatment. These data indicated that a p53-activating CP-31398 achieved growth inhibitory effects in combination with a MDM2 or a FAK inhibitor and suggested a possible reciprocal pathway between p53 elevation and FAK inactivation.
Subject(s)
Antineoplastic Agents/pharmacology , Focal Adhesion Kinase 1/genetics , Imidazoles/pharmacology , Piperazines/pharmacology , Proto-Oncogene Proteins c-mdm2/genetics , Pyrimidines/pharmacology , Tumor Suppressor Protein p53/genetics , Antineoplastic Combined Chemotherapy Protocols , Ataxia Telangiectasia Mutated Proteins/genetics , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Cycle/drug effects , Cell Cycle/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Checkpoint Kinase 1/genetics , Checkpoint Kinase 1/metabolism , Checkpoint Kinase 2/genetics , Checkpoint Kinase 2/metabolism , Drug Synergism , Epithelium/drug effects , Epithelium/metabolism , Epithelium/pathology , Focal Adhesion Kinase 1/antagonists & inhibitors , Focal Adhesion Kinase 1/metabolism , Gene Expression Regulation, Neoplastic , Genotype , Humans , Phosphorylation/drug effects , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-mdm2/antagonists & inhibitors , Proto-Oncogene Proteins c-mdm2/metabolism , Signal Transduction , Tumor Suppressor Protein p53/agonists , Tumor Suppressor Protein p53/metabolism , Ubiquitination/drug effectsABSTRACT
Pemetrexed (PEM) inhibits DNA and RNA synthesis and is currently one of the first-line agents for mesothelioma. PEM suppresses the activities of several enzymes involved in purine and pyrimidine synthesis, and elevated activity of these enzymes in tumors is often linked with resistance to PEM. The agent also stimulates AMP-activated protein kinase (AMPK) and consequently influences the mammalian target of rapamycin complex 1 (mTORC1) pathways. Nevertheless, it remains unclear whether PEM resistance is linked to the AMPK or mTORC1 pathways. Here, we established two independent PEM-resistant mesothelioma cell lines in which expression of the PEM-target enzymes was not elevated, and found that levels of phosphorylated AMPK and p70S6K and, to a lesser extent, levels of phosphorylated AKT and p53, were increased in these cells as compared with the respective parent cells. PEM stimulation also augmented phosphorylation of AMPK, p70S6K, AKT and p53 in most cases. An AMPK activator increased phosphorylation and PEM resistance in parental cells, and the inhibitor decreased the resistance of PEM-resistant cells. In contrast, inhibitors for p70S6K and AKT did not influence PEM resistance; furthermore, increased levels of endogenous p53 did not affect PEM sensitivity. These data collectively indicate that constitutive activation of AMPK is associated with PEM resistance, and that this is unconnected with elevated DNA and RNA synthesis.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Cell Survival/drug effects , Mesothelioma/metabolism , Pemetrexed/pharmacology , Blotting, Western , Cell Line, Tumor , Humans , Mechanistic Target of Rapamycin Complex 1/metabolism , Ribosomal Protein S6 Kinases, 70-kDa/metabolism , Signal Transduction/drug effects , Tumor Suppressor Protein p53/metabolismABSTRACT
Restoration of p53 functions is one of the therapeutic strategies for esophageal carcinoma which is often defective of the p53 pathway. We examined effects of CP-31398 which potentially increased expression of wild-type p53 or converted mutated p53 to the wild-type. We used 9 kinds of human squamous esophageal carcinoma cells with different p53 genotypes and examined expression of p53 and the related molecules in CP-31398-treated cells. Cisplatin, a DNA damaging agent, induced cleavages of PARP and caspase-3 without increase of p53 levels, indicating that the p53 down-stream pathway was disrupted in these cells. CP-31398 induced growth retardation but the cytotoxic effects were irrelevant to p53 genotype. CP-31398 influenced expression of p53 and the downstream molecules in a cell-dependent manner, but constantly increased p21 expression at the transcriptional level with decreased YY1 expression. Knockdown experiments with siRNA demonstrated that the CP-31398-mediated p21 up-regulation was unrelated with p53 expression but was associated with YY1 expression. We also showed that CP-31398-induced cell cycle changes including increase of G2/M populations was attributable to the up-regulated p21. These data collectively indicated that CP-31398 augmented endogenous p21 levels and induced cell cycle changes through regulation of YY1, and that YY1 was a novel target of CP-31398 in p53 dysfunctional cells.
ABSTRACT
BACKGROUND: Pemetrexed (PEM) is an anti-cancer agent targeting DNA and RNA synthesis, and clinically in use for mesothelioma and non-small cell lung carcinoma. A mechanism of resistance to PEM is associated with elevated activities of several enzymes involved in nucleic acid metabolism. METHODS: We established two kinds of PEM-resistant mesothelioma cells which did not show any increase of the relevant enzyme activities. We screened genes enhanced in the PEM-resistant cells with a microarray analysis and confirmed the expression levels with Western blot analysis. A possible involvement of the candidates in the PEM-resistance was examined with a WST assay after knocking down the expression with si-RNA. We also analyzed a mechanism of the up-regulated expression with agents influencing AMP-activated protein kinase (AMPK) and p53. RESULTS: We found that expression of cardiac ankyrin repeat protein (CARP) was elevated in the PEM-resistant cells with a microarray and Western blot analysis. Down-regulation of CARP expression with si-RNA did not however influence the PEM resistance. Parent and PEM-resistant cells treated with PEM increased expression of CARP, AMPK, p53 and histone H2AX. The CARP up-regulation was however irrelevant to the p53 genotypes and not induced by an AMPK activator. Augmented p53 levels with nutlin-3a, an inhibitor for p53 degradation, and DNA damages were not always associated with the enhanced CARP expression. CONCLUSIONS: These data collectively suggest that up-regulated CARP expression is a potential marker for development of PEM-resistance in mesothelioma and that the PEM-mediated enhanced expression is not directly linked with immediate cellular responses to PEM.
ABSTRACT
BACKGROUND: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity. METHODS: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses. RESULTS: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level. CONCLUSIONS: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.