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Herein we present the biocatalysed preparation of a mono-N-carbamate-protected precursor of antitumoral Nutlin-3a through enantioselective alkoxycarbonylation of meso-1,2-disubstituted-1,2-diaminoethane using enzyme lipases and dialkyl carbonates as acylating agents. A series of supported or free lipase enzymes were screened in combination with commercially available diallyl, diethyl and dimethyl carbonates. The reactions were conducted at different temperatures, for different reaction times and with variable co-solvent systems to evaluate the effects on the enzyme catalytic activity. The best results in terms of conversion, enantiomeric excess and yield were obtained when lipase from Candida antarctica B (CAL-B) was used with diallyl carbonate (DAC) when conducting the reaction solventless at 75 °C.
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Lipasa , Lipasa/metabolismo , Lipasa/química , Estereoisomerismo , Proteínas Fúngicas/metabolismo , Biocatálisis , Piperazinas/química , Piperazinas/síntesis química , Enzimas Inmovilizadas/química , Solventes/química , Estructura MolecularRESUMEN
Purpose: Proliferative vitreoretinal diseases (PVDs) represent a heterogeneous group of pathologies characterized by the presence of retinal proliferative membranes, in whose development retinal pigment epithelium (RPE) is deeply involved. As the only effective treatment for PVDs at present is surgery, we aimed to investigate the potential therapeutic activity of Nutlin-3a, a small non-genotoxic inhibitor of the MDM2/p53 interaction, on ARPE-19 cell line and on human RPE primary cells, as in vitro models of RPE and, more importantly, to formulate and evaluate Nutlin-3a loaded liposomes designed for ophthalmic administration. Methods: Liposomes were produced using an innovative approach by a microfluidic device under selection of different conditions. Liposome size distribution was evaluated by photon correlation spectroscopy and centrifugal field flow fractionation, while the liposome structure was studied by transmission electron microscopy and Fourier-transform infrared spectroscopy. The Nutlin-3a entrapment capacity was evaluated by ultrafiltration and HPLC. Nutlin-3a biological effectiveness as a solution or loaded in liposomes was evaluated by viability, proliferation, apoptosis and migration assays and by morphological analysis. Results: The microfluidic formulative study enabled the selection of liposomes composed of phosphatidylcholine (PC) 5.4 or 8.2 mg/mL and 10% ethanol, characterized by roundish vesicular structures with 150-250 nm mean diameters. Particularly, liposomes based on the lower PC concentration were characterized by higher stability. Nutlin-3a was effectively encapsulated in liposomes and was able to induce a significant reduction of viability and migration in RPE cell models. Conclusion: Our results lay the basis for a possible use of liposomes for the ocular delivery of Nutlin-3a.
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Oftalmopatías , Imidazoles , Liposomas , Piperazinas , Humanos , Liposomas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Microfluídica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/farmacología , ApoptosisRESUMEN
The inhibition of the Mdm2-p53 protein-protein interaction is a promising strategy for anticancer therapy. However, the problem of developing secondary chemoresistance in tumors treated with such drugs has not yet been sufficiently studied. In this work, we compared the properties of a drug-resistant cell line obtained during long-term cultivation in the presence of an Mdm2 inhibitor, Nutlin-3a, with a similarly obtained line insensitive to the cytostatic drug paclitaxel. We first confirmed the higher safety levels of Mdm2 inhibitors when compared with cytostatics in terms of the development of secondary chemoresistance. We showed that Nutlin-3a affects both the targeted p53-mediated cellular machinery and the universal ABC-mediated efflux mechanism. While both targeted and general defense mechanisms are activated by the Mdm2 inhibitor, it still increases the susceptibility of tumor cells to other drugs. The results obtained indicate that the risks of developing chemoresistance under the therapy with a targeted agent are fundamentally lower than during cytotoxic therapy.
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Cellular senescence, a state of irreversible growth arrest, is implicated in various age-related pathologies, including skin aging. In this study, we investigated the role of CLCA2, a calcium-activated chloride channel accessory protein, in cellular senescence and its implications for skin aging. Utilizing UVB and Nutlin3a-induced senescence models, we observed the upregulation of CLCA2 at both transcriptomic and proteomic levels, suggesting its involvement in senescence pathways. Further analysis revealed that the depletion of CLCA2 led to accelerated senescence onset, characterized by classic senescence markers and a unique secretome profile. In 3D skin equivalent models, SEs constructed with CLCA2 knockdown fibroblasts exhibited features reminiscent of aged skin, underscoring the importance of CLCA2 in maintaining skin homeostasis. Our findings highlight CLCA2 as a novel regulator of cellular senescence and its potential implications for skin aging mechanisms.
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Inhibitors of the p53-MDM2 interaction such as RG7388 have been developed to exploit latent tumor suppressive properties in p53 in 50% of tumors in which p53 is wild-type. However, these agents for the most part activate cell cycle arrest rather than death, and high doses in patients elicit on-target dose-limiting neutropenia. Recent work from our group indicates that combination of p53-MDM2 inhibitors with the class-I HDAC inhibitor Entinostat (which itself has dose-limiting toxicity issues) has the potential to significantly augment cell death in p53 wild-type colorectal cancer cells. We investigated whether coencapsulation of RG7388 and Entinostat within polymeric nanoparticles (NPs) could overcome efficacy and toxicity limitations of this drug combination. Combinations of RG7388 and Entinostat across a range of different molar ratios resulted in synergistic increases in cell death when delivered in both free drug and nanoencapsulated formats in all colorectal cell lines tested. Importantly, we also explored the in vivo impact of the drug combination on murine blood leukocytes, showing that the leukopenia induced by the free drugs could be significantly mitigated by nanoencapsulation. Taken together, this study demonstrates that formulating these agents within a single nanoparticle delivery platform may provide clinical utility beyond use as nonencapsulated agents.
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Antineoplásicos , Benzamidas , Inhibidores de Histona Desacetilasas , Piridinas , Pirrolidinas , para-Aminobenzoatos , Humanos , Animales , Ratones , Inhibidores de Histona Desacetilasas/farmacología , Inhibidores de Histona Desacetilasas/uso terapéutico , Proteína p53 Supresora de Tumor/metabolismo , Apoptosis , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Combinación de Medicamentos , Proteínas Proto-Oncogénicas c-mdm2RESUMEN
The skin's protective mechanisms, in some cases, are not able to counteract the destructive effects induced by UV radiations, resulting in dermatological diseases, as well as skin aging. Nutlin-3, a potent drug with antiproliferative activity in keratinocytes, can block UV-induced apoptosis by activation of p53. In the present investigation, ethosomes and transethosomes were designed as delivery systems for nutlin-3, with the aim to protect the skin against UV damage. Vesicle size distribution was evaluated by photon correlation spectroscopy and morphology was investigated by cryogenic transmission electron microscopy, while nutlin-3 entrapment capacity was evaluated by ultrafiltration and HPLC. The in vitro diffusion kinetic of nutlin-3 from ethosomes and transethosomes was studied by Franz cell. Moreover, the efficiency of ethosomes and transethosomes in delivering nutlin-3 and its protective role were evaluated in ex vivo skin explants exposed to UV radiations. The results indicate that ethosomes and transethosomes efficaciously entrapped nutlin-3 (0.3% w/w). The ethosome vesicles were spherical and oligolamellar, with a 224 nm mean diameter, while in transethosome the presence of polysorbate 80 resulted in unilamellar vesicles with a 146 nm mean diameter. The fastest nutlin-3 kinetic was detected in the case of transethosomes, with permeability coefficients 7.4-fold higher, with respect to ethosomes and diffusion values 250-fold higher, with respect to the drug in solution. Ex vivo data suggest a better efficacy of transethosomes to promote nutlin-3 delivery within the skin, with respect to ethosomes. Indeed, nutlin-3 loaded transethosomes could prevent UV effect on cutaneous metalloproteinase activation and cell proliferative response.
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BACKGROUND AND AIMS: The specific mechanisms underlying the inhibition of hepatocellular carcinoma (HCC) proliferation and metastasis by mitochondrial apoptosis are not yet fully understood. However, it plays a vital role in suppressing HCC's ability to proliferate and spread. The involvement of MRPL21, a member within the family of mitochondrial ribosomal proteins (MRPs), is well-documented in both cellular apoptosis and energy metabolism. This study aims to explore and unravel the underlying mechanisms through which MRPL21 contributes to mitochondrial apoptosis and resistance against apoptosis in HCC. METHODS: To evaluate the level of MRPL21 expression at the gene and protein expression levels, analysis was performed on human liver samples and blood using techniques for quantification. A knockdown plasmid targeting MRPL21 was constructed to investigate its impact on the growth and apoptosis of hepatocellular carcinoma (HCC). To evaluate the impact of MRPL21 knockdown on hepatocellular carcinoma (HCC) cell proliferation and apoptosis, various assays were performed including CCK-8 assays, flow cytometry analysis, detection of reactive oxygen species (ROS), and assessment of mitochondrial membrane potential (MMP). Furthermore, the role of MRPL21 in TP53 mutation was examined using Nutlin-3. RESULTS: In HCC tissues and blood samples, an upregulation of MRPL21 expression was observed when compared to samples obtained from healthy individuals, and it is correlated with a poor prognosis for HCC. Silencing MRPL21 can effectively suppress Hep3B and HCCLM3 cells proliferation by modulating the mitochondrial membrane potential, it triggers the generation of reactive oxygen species (ROS), thereby leading to G0/G1 cell cycle arrest and initiation of early apoptosis. Furthermore, by inhibiting P53 activity, Nutlin-3 treatment can enhance MRPL21-deficiency-mediated apoptosis in Hep3B and HCCLM3 cells. CONCLUSION: Through its influence on TP53 mutation, MRPL21 promotes HCC proliferation and progression while conferring resistance to apoptosis. These findings suggest that MRPL21 holds promise as a valuable biomarker for the treatment of HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Apoptosis/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Mutación , Especies Reactivas de Oxígeno/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Oncogenic KRAS mutation, the most frequent mutation in non-small cell lung cancer (NSCLC), is an aggressiveness risk factor and leads to the metabolic reprogramming of cancer cells by promoting glucose, glutamine, and fatty acid absorption and glycolysis. Lately, sotorasib was approved by the FDA as a first-in-class KRAS-G12C inhibitor. However, sotorasib still has a derivative barrier, which is not effective for other KRAS mutation types, except for G12C. Additionally, resistance to sotorasib is likely to develop, demanding the need for alternative therapeutic strategies. METHODS: KRAS mutant, and wildtype NSCLC cells were used in vitro cell analyses. Cell viability, proliferation, and death were measured by MTT, cell counting, colony analyses, and annexin V staining for FACS. Cell tracker dyes were used to investigate cell morphology, which was examined by holotomograpy, and confocal microscopes. RNA sequencing was performed to identify key target molecule or pathway, which was confirmed by qRT-PCR, western blotting, and metabolite analyses by UHPLC-MS/MS. Zebrafish and mouse xenograft model were used for in vivo analysis. RESULTS: In this study, we found that nutlin-3a, an MDM2 antagonist, inhibited the KRAS-PI3K/Akt-mTOR pathway and disrupted the fusion of both autophagosomes and macropinosomes with lysosomes. This further elucidated non-apoptotic and catastrophic macropinocytosis associated methuosis-like cell death, which was found to be dependent on GFPT2 of the hexosamine biosynthetic pathway, specifically in KRAS mutant /p53 wild type NSCLC cells. CONCLUSION: These results indicate the potential of nutlin-3a as an alternative agent for treating KRAS mutant/p53 wild type NSCLC cells.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Animales , Ratones , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Espectrometría de Masas en Tándem , Pez Cebra , Apoptosis , Proteínas Proto-Oncogénicas c-mdm2/genética , Muerte Celular , Mutación , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/genética , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismoRESUMEN
In most lymphomas, p53 signaling pathway is inactivated by various mechanisms independent to p53 gene mutations or deletions. In many cases, p53 function is largely regulated by alterations in the protein abundance levels by the action of E3 ubiquitin-protein ligase MDM2, targeting p53 to proteasome-mediated degradation. In the present study, an integrating transcriptomics and proteomics analysis was employed to investigate the effect of p53 activation by a small-molecule MDM2-antagonist, nutlin-3a, on three lymphoma cell models following p53 activation. Our analysis revealed a system-wide nutlin-3a-associated effect in all examined lymphoma types, identifying in total of 4037 differentially affected proteins involved in a plethora of pathways, with significant heterogeneity among lymphomas. Our findings include known p53-targets and novel p53 activation effects, involving transcription, translation, or degradation of protein components of pathways, such as a decrease in key members of PI3K/mTOR pathway, heat-shock response, and glycolysis, and an increase in key members of oxidative phoshosphorylation, autophagy and mitochondrial translation. Combined inhibition of HSP90 or PI3K/mTOR pathway with nutlin-3a-mediated p53-activation enhanced the apoptotic effects suggesting a promising strategy against human lymphomas. Integrated omic profiling after p53 activation offered novel insights on the regulatory role specific proteins and pathways may have in lymphomagenesis.
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Discovery of MDM2 and MDM2-p53 interaction inhibitors changed the direction of anticancer research as it is involved in about 50% of cancer cases globally. Not only the inhibition of MDM2 but also its interaction with p53 proved to be an effective strategy in anticancer drug design and development. Various molecules of natural as well as synthetic origin have been reported to possess excellent MDM2 inhibitory potential. The present review discusses the pathophysiology of the MDM2-p53 interaction loop and MDM2/MDM2-p53 interaction inhibitors from literature covering recent patents. Focus has also been put on characteristic features of the active site of the target and its desired interactions with the currently FDA-approved inhibitor. The designing approach of previously reported MDM2/MDM2-p53 interaction inhibitors, their SAR studies, in silico studies, and the biological efficacy of various inhibitors from natural as well as synthetic origins are also elaborated. An attempt is made to cover recently patented MDM2/MDM2- p53 interaction inhibitors.
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Antineoplásicos , Neoplasias , Humanos , Proteína p53 Supresora de Tumor , Proteínas Proto-Oncogénicas c-mdm2/química , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/uso terapéutico , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Neoplasias/tratamiento farmacológico , Diseño de FármacosRESUMEN
DNA replication of E1-deleted first-generation adenoviruses (AdV) in cultured cancer cells has been reported repeatedly and it was suggested that certain cellular proteins could functionally compensate for E1A, leading to the expression of the early region 2 (E2)-encoded proteins and subsequently virus replication. Referring to this, the observation was named E1A-like activity. In this study, we investigated different cell cycle inhibitors with respect to their ability to increase viral DNA replication of dl70-3, an E1-deleted adenovirus. Our analyses of this issue revealed that in particular inhibition of cyclin-dependent kinases 4/6 (CDK4/6i) increased E1-independent adenovirus E2-expression and viral DNA replication. Detailed analysis of the E2-expression in dl70-3 infected cells by RT-qPCR showed that the increase in E2-expression originated from the E2-early promoter. Mutations of the two E2F-binding sites in the E2-early promoter (pE2early-LucM) caused a significant reduction in E2-early promoter activity in trans-activation assays. Accordingly, mutations of the E2F-binding sites in the E2-early promoter in a virus named dl70-3/E2Fm completely abolished CDK4/6i induced viral DNA replication. Thus, our data show that E2F-binding sites in the E2-early promoter are crucial for E1A independent adenoviral DNA replication of E1-deleted vectors in cancer cells. IMPORTANCE E1-deleted AdV vectors are considered replication deficient and are important tools for the study of virus biology, gene therapy, and large-scale vaccine development. However, deletion of the E1 genes does not completely abolish viral DNA replication in cancer cells. Here, we report, that the two E2F-binding sites in the adenoviral E2-early promoter contribute substantially to the so-called E1A-like activity in tumor cells. With this finding, on the one hand, the safety profile of viral vaccine vectors can be increased and, on the other hand, the oncolytic property for cancer therapy might be improved through targeted manipulation of the host cell.
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Adenoviridae , Ciclo Celular , Replicación del ADN , Replicación Viral , Adenoviridae/genética , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Sitios de Unión , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Células/efectos de los fármacos , Células/virología , Replicación del ADN/efectos de los fármacos , ADN Viral/metabolismo , Regulación Viral de la Expresión Génica/efectos de los fármacos , Mutación , Regiones Promotoras Genéticas/genética , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/fisiología , HumanosRESUMEN
Inactivation of the p53 tumor suppressor, either by mutations or through hyperactivation of repressors such as MDM2 and MDM4, is a hallmark of cancer. Although many inhibitors of the p53-MDM2/4 interaction have been developed, such as Nutlin, their therapeutic value is limited by highly heterogeneous cellular responses. We report here a multi-omics investigation of the cellular response to MDM2/4 inhibitors, leading to identification of FAM193A as a widespread regulator of p53 function. CRISPR screening identified FAM193A as necessary for the response to Nutlin. FAM193A expression correlates with Nutlin sensitivity across hundreds of cell lines. Furthermore, genetic codependency data highlight FAM193A as a component of the p53 pathway across diverse tumor types. Mechanistically, FAM193A interacts with MDM4, and FAM193A depletion stabilizes MDM4 and inhibits the p53 transcriptional program. Last, FAM193A expression is associated with better prognosis in multiple malignancies. Altogether, these results identify FAM193A as a positive regulator of p53.
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Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/farmacología , Apoptosis , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Neoplasias/patología , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Oral squamous cell carcinoma (OSCC) is an epithelial malignant tumor with great challenges of tumor metastasis and drug resistance. Nutlin-3 is a MDM2 inhibitor that can potently activate tumor suppressor gene p53. However, the exact role of Nutlin-3 in OSCC has not been identified yet. SCC-9 cells were treated with 0, 2.5, 5, 10, 20 µM Nutlin3. MDM2 and p53 protein levels were assessed using western blot analysis. Then, CCK8 assay, clone formation assay, TUNEL staining, wound healing and transwell assays were conducted to analyze the influences of Nutlin3 on the proliferation, apoptosis, migration, and invasion in SCC-9 cells. Moreover, SCC-9 cells were co-treated with 0, 0.5, 1, 2.5, 5 µM cisplatin and Nutlin3 to determine the effect of Nutlin3 on cisplatin chemosensitivity in OSCC. As expected, Nutlin-3 inhibited MDM2 but restored p53 in OSCC in a concentration-dependent manner. Meanwhile, Nutlin-3 suppressed the proliferation, clone formation, migration, invasion and epithelial-mesenchymal transition of SCC-9 cells and both boosted the apoptosis. In addition, Nutlin-3 caused a reduced cell viability and an elevated cell apoptosis rate in cisplatin-treated SCC-9 cells, indicating that Nutlin-3 enhanced cisplatin chemosensitivity in OSCC cells. Taken together, Nutlin-3 may suppress tumorigenesis and progression of OSCC and enhance chemosensitivity to cisplatin in OSCC.
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Esophageal squamous cell carcinoma (ESCC) is highly resistant to chemoradiation therapy. We aimed to examine whether Nutlin-3, a molecule that suppresses murine double min 2 (MDM2)-mediated p53 and Retinoblastoma (RB) protein degradation leading to downregulation of DNA methyltransferases (DNMTs), can be a novel therapeutic agent for ESCC. We used wild-type and chemoradiation-resistant ESCC cell lines in this study. The expression of DNMTs, p53 and RB, and methylation level of tumor suppressor genes (TSG) were analyzed upon Nutlin-3 treatment. The antitumor efficacy of Nutlin-3 was investigated in ESCC cell lines and xenograft tumor model. TSG protein expression was checked in the excised tumor tissue. Nutlin-3 induced upregulation of p53 and RB and downregulation of DNMTs proteins in the chemoradiation-resistant and aggressive ESCC cells. The methylation level of TSGs was decreased by Nutlin-3. Nutlin-3 inhibits clonogenic growth of ESCC cells and exerts a synergistic cytotoxic-effect when combined with chemotherapeutic agent cisplatin. Moreover, xenograft tumor growth in SCID mice was suppressed by Nutlin-3. The protein expression level of DNMTs was downregulated, and that of TSGs was upregulated by Nutlin-3 treatment in the excised tumor tissue. In conclusion, Nutlin-3 is a potential therapeutic agent that can potentiate the treatment efficacy of chemoradiation-resistant ESCC.
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Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Animales , Humanos , Ratones , Apoptosis , Línea Celular Tumoral , ADN/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/radioterapia , Metiltransferasas/metabolismo , Metiltransferasas/farmacología , Ratones SCID , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteína de Retinoblastoma/metabolismo , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Targeting MDM2-p53 interaction has emerged as a promising antitumor therapeutic strategy. Several MDM2-p53 inhibitors have advanced into clinical trials, but results are not favorable. The lack of appropriate biomarkers for selecting patients has been assumed as the critical reason for this failure. We previously identified ZER6 isoform p52-ZER6 as an oncogene upregulated in tumor tissues. In this study we investigated whether p52-ZER6 acted as a blocker of MDM2-p53 binding inhibitors, and whether p52-ZER6 could be used as a biomarker of MDM2-p53 binding inhibitors. In p53 wild-type colorectal carcinoma HCT116, hepatocarcinoma HepG2 and breast cancer MCF-7 cells, overexpression of p52-ZER6 enhanced MDM2-p53 binding and promoted p53 ubiquitination/proteasomal degradation. Furthermore, overexpression of p52-ZER6 in the tumor cells dose-dependently reduced their sensitivity to both nutlin and non-nutlin class MDM2-p53 binding inhibitors. We showed that p52-ZER6 restored tumor cell viability, which was suppressed by nutlin-3, through restoring their proliferation potential while suppressing their apoptotic rate, suggesting that MDM2-p53 binding inhibitors might not be effective for patients with high p52-ZER6 levels. We found that nutlin-3 treatment or p52-ZER6 knockdown alone promoted the accumulation of p53 protein in the tumor cells, and their combinatorial treatment significantly increased the accumulation of p53 protein. In HCT116 cell xenograft nude mouse model, administration of shp52-ZER6 combined with an MDM2-p53 binding inhibitor nutlin-3 exerted synergistic antitumor response. In conclusion, this study reveals that p52-ZER6 might be a potential biomarker for determining patients appropriate for MDM2-p53 binding inhibition-based antitumor therapy, and demonstrates the potential of combinatorial therapy using MDM2-p53 binding inhibitors and p52-ZER6 inhibition.
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Antineoplásicos , Proteínas Proto-Oncogénicas c-mdm2 , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Apoptosis , Biomarcadores , Línea Celular Tumoral , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
SARS-CoV viruses have been shown to downregulate cellular events that control antiviral defenses. They adopt several strategies to silence p53, key molecule for cell homeostasis and immune control, indicating that p53 has a central role in controlling their proliferation in the host. Specific actions are the stabilization of its inhibitor, MDM2, and the interference with its transcriptional activity. The aim of our work was to evaluate a new approach against SARS-CoV-2 by using MDM2 inhibitors to raise p53 levels and activate p53-dependent pathways, therefore leading to cell cycle inhibition. Experimental setting was performed in the alveolar basal epithelial cell line A549-hACE2, expressing high level of ACE2 receptor, to allow virus entry, as well as p53 wild-type. Cells were treated with several concentrations of Nutlin-3 or RG-7112, two known MDM2 inhibitors, for the instauration of a cell cycle block steady-state condition before and during SARS-CoV-2 infection, and for the evaluation of p53 activation and impact on virus release and related innate immune events. The results indicated an efficient cell cycle block with inhibition of the virion release and a significant inhibition of IL-6, NF-kB and IFN-λ expression. These data suggest that p53 is an efficient target for new therapies against the virus and that MDM2 inhibitors deserve to be further investigated in this field.
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MDM2 is the principal inhibitor of p53, and MDM2 inhibitors can disrupt the physical interaction between MDM2 and p53. The half-life of p53 is very short in normal cells and tissues, and an uncontrolled increase in p53 levels has potential harmful effects. It has been shown that p53 is frequently mutated in most cancers; however, p53 mutations are rare in retinoblastoma. Therefore, therapeutic strategies aimed at increasing the expression levels of wild-type p53 are attractive. In this minireview, we discuss the potential use of nutlin-3, the prototype small molecule inhibitor that disrupts the MDM2-p53 interaction, for the treatment of retinoblastoma. Although p53 has pleiotropic biological effects, the functions of p53 depend on its sub-cellular localization. In the nucleus, p53 induces the transcription of a vast array of genes, while in mitochondria, p53 regulates mitochondrial metabolism. This review also discusses the relative contribution of p53-mediated gene transcription and mitochondrial perturbation for retinoblastoma treatment.
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OBJECTIVE: Tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL) is a potent anticancer agent, which could specifically target cancerous cells. Nutlin-3, a small-molecular inhibitor of murine double minute 2 (MDM2), shows oncogenic potential in a variety of human cancers. It has also been found to promote the TRAIL-induced apoptosis of cancer cells in esophageal squamous cancer, but its potential role and underlying mechanisms in the TRAIL-treated hepatocellular carcinoma (HCC) remains to be elucidated. METHODS: HSS cell line (Huh7) cells were used as an in vitro model of HCC. TRAIL (100 ng/ml) was used to induce cell apoptosis. Cell viability was measured by CCK-8 assay. Cell apoptosis was detected using TUNEL staining. Mitochondrial activity was evaluated by measuring caspase 3/7 and 9 levels. P53 expression at protein and mRNA level were measured by Western blotting and RT-qPCR, respectively. RESULTS: The combination of Nutlin-3 and TRAIL facilitated the apoptosis and increased the levels of mitochondrial cleaved-caspase3/7 and 9 in HCC cells compared with TRAIL treatment alone, both in a concentration-dependent way. Nutlin-3 also upregulated the expression of p53 and DR5, while knockdown of p53 significantly hindered the pro-apoptotic effect of Nutlin-3. Further studies revealed that Nutlin-3 downregulated the expression of survivin and bcl-2, both of which could be reversed by p53 knockdown. Moreover, survivin suppressant YM155 and bcl-2 inhibitor YM155 further enhanced the apoptosis of HCC cells in the presence of Nutlin-3 and TRAIL. CONCLUSION: These results suggested that Nutlin-3 facilitated TRAIL-induced apoptosis of HCC cells by activating the p53-survivin/bcl-2 pathway, which provided novel insights into the mechanism of Nutlin-3 and confirmed the potential of combination of Nutlin-3 and TRAIL as an adjuvant in HCC therapy.
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Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Antineoplásicos/farmacología , Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Caspasa 3/metabolismo , Línea Celular Tumoral , Humanos , Imidazoles , Ligandos , Neoplasias Hepáticas/patología , Ratones , Piperazinas , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , ARN Mensajero , Survivin/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The MDM2-p53 protein-protein interaction is a promising model for researchers to design, study, and discover new anticancer drugs. The design of therapeutically active compounds that can maintain or restore the binding of MDM2 to p53 has been found to limit the oncogenic activities of both. This led to the current development of a group of xanthone-core and cis-imidazoline analogs compounds, among which γ-Mangostin (GM), α-Mangostin (AM), and Nutlin exhibited their MDM2-p53 interaction inhibitory effects. Therefore, in this study, we seek to determine the mechanisms by which these compounds elicit MDM2-p53 interaction targeting. Unique to the binding of GM, AM, and Nutlin, from our findings, they share the same three active site residues Val76, Tyr50, and Gly41, which represent the top active side residues that contribute to high electrostatic energy. Consequently, the free binding energy contributed enormously to the binding of these compounds, which culminated in the high binding affinities of GM, AM, and Nutlin with high values. Furthermore, GM, AM, and Nutlin commonly interrupted the stable and compact conformation of MDM2 coupled with its active site, where Cα deviations were relatively high. We believe that our findings would assist in the design of more potent active anticancer drugs.
Asunto(s)
Antineoplásicos , Garcinia mangostana , Imidazolinas , Xantonas , Dominio Catalítico , Garcinia mangostana/metabolismo , Imidazoles/química , Imidazoles/metabolismo , Imidazoles/farmacología , Simulación de Dinámica Molecular , Piperazinas/farmacología , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Xantonas/farmacologíaRESUMEN
2,4,3',5'-Tetramethoxystilbene (TMS) is a selective inhibitor of cytochrome P450 1B1 to block the conversion from estradiol to 4-OH-estradiol. Several studies suggested that TMS may act as a potent anti-cancer agent for hormone-related cancer including cervical cancer. Nutlin-3a is a cis-imidazoline analog that interferes with the interaction between mouse double minute 2 homolog (MDM2) and the tumor suppressor p53. The purpose of the study was to compare the cytotoxic effect of TMS and nutlin-3a treatment individually and in combination in HeLa cells. To assess the potential synergistic effects between TMS and nutlin-3a, low concentrations of TMS and nutlin-3a were simultaneously treated in HeLa cells. Based on cell viability, apoptosis assays, and the increase in cleaved caspase-3 and poly (ADP-ribose) polymerase cleavage, it was demonstrated that the combination with TMS and nutlin-3a exerts a synergistic effect on cancer cell death. Isobologram analysis of HeLa cells noted synergism between TMS and nutlin-3a. The combined treatment increased the expression of mitochondrial pro-apoptotic factors such as Bax and Bak, and decreased the expression of the XIAP. In addition, combination treatment significantly enhanced the translocation of AIF to the nucleus in HeLa cells. In conclusion, the results demonstrate that the combination of TMS and nutlin-3a induces synergistic apoptosis in HeLa cells, suggesting the possibility that this combination can be applied as a novel therapeutic strategy for cervical cancer.