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Uveal melanoma (UM) is the most common primary intraocular tumor in adults, with no standardized treatment for advanced disease. Based on preliminary bioinformatical analyses DTYMK and PARP1 were selected as potential therapeutic targets. High levels of both proteins were detected in uveal melanoma cells and correlated with increased tumor growth and poor prognosis. In vitro tests on MP41 (BAP1 positive) and MP46 (BAP1 negative) cancer cell lines using inhibitors pamiparib (PARP1) and Ymu1 (DTYMK) demonstrated significant cytotoxic effects. Combined treatment had synergistic effects in MP41 and additive in MP46 cell lines, reducing cell proliferation and inhibiting the mTOR signaling pathway. Furthermore, the applied inhibitors in combination decreased cell motility and migration speed, especially for BAP1-negative cell lines. Our hypothesis of the double hit into tumoral DNA metabolism as a possible therapeutic option in uveal melanoma was confirmed since combined targeting of DTYMK and PARP1 affected all tested cytophysiological parameters with the highest efficiency. Our in vitro findings provide insights into novel therapeutic avenues for managing uveal melanoma, warranting further exploration in preclinical and clinical settings.
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Proliferação de Células , Melanoma , Poli(ADP-Ribose) Polimerase-1 , Neoplasias Uveais , Humanos , Neoplasias Uveais/tratamento farmacológico , Neoplasias Uveais/patologia , Neoplasias Uveais/metabolismo , Melanoma/tratamento farmacológico , Melanoma/patologia , Melanoma/metabolismo , Linhagem Celular Tumoral , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.
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Esclerose Lateral Amiotrófica , Caenorhabditis elegans , Histonas , Mutação , Poli(ADP-Ribose) Polimerase-1 , Proteína FUS de Ligação a RNA , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Humanos , Histonas/metabolismo , Proteína FUS de Ligação a RNA/metabolismo , Proteína FUS de Ligação a RNA/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Animais , Mutação/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Poli ADP Ribosilação , Células-Tronco Pluripotentes Induzidas/metabolismo , Ligação ProteicaRESUMO
Structural modification based on natural privileged scaffolds has proven to be an attractive approach to generate potential antitumor candidates with high potency and specific targeting. As a continuation of our efforts to identify potent PARP-1 inhibitors, natural 3-arylcoumarin scaffold was served as the starting point for the construction of novel structural unit for PARP-1 inhibition. Herein, a series of novel 8-carbamyl-3-arylcoumarin derivatives were designed and synthesized. The antiproliferative activities of target compounds against four BRCA-mutated cancer cells (SUM149PT, HCC1937, MDA-MB-436 and Capan-1) were evaluated. Among them, compound 9b exhibited excellent antiproliferative effects against SUM149PT, HCC1937 and Capan-1 cells with IC50 values of 0.62, 1.91 and 4.26 µM, respectively. Moreover, 9b could significantly inhibit the intracellular PARP-1/2 activity in SUM149PT cells with IC50 values of 2.53 nM and 6.45 nM, respectively. Further mechanism studies revealed that 9b could aggravate DNA double-strand breaks, increase ROS production, decrease mitochondrial membrane potential, arrest cell cycle at G2/M phase and ultimately induce apoptosis in SUM149PT cells. In addition, molecular docking study demonstrated that the binding mode of 9b with PARP-1 was similar to that of niraparib, forming multiple hydrogen bond interactions with the active site of PARP-1. Taken together, these findings suggest that 8-carbamyl-3-arylcoumarin scaffold could serve as an effective structural unit for PARP-1 inhibition and offer a valuable paradigm for the structural modification of natural products.
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BACKGROUND: Poly (ADP-ribose) polymerase 1 and 2 (PARP1/2) inhibitors (PARPi) are targeted therapies approved for homologous recombination repair (HRR)-deficient breast, ovarian, pancreatic, and prostate cancers. Since inhibition of PARP1 is sufficient to cause synthetic lethality in tumors with homologous recombination deficiency (HRD), PARP1 selective inhibitors such as saruparib (AZD5305) are being developed. It is expected that selective PARP1 inhibition leads to a safer profile that facilitates its combination with other DNA damage repair inhibitors. Here, we aimed to characterize the antitumor activity of AZD5305 in patient-derived preclinical models compared to the first-generation PARP1/2 inhibitor olaparib and to identify mechanisms of resistance. METHODS: Thirteen previously characterized patient-derived tumor xenograft (PDX) models from breast, ovarian, and pancreatic cancer patients harboring germline pathogenic alterations in BRCA1, BRCA2, or PALB2 were used to evaluate the efficacy of AZD5305 alone or in combination with carboplatin or an ataxia telangiectasia and Rad3 related (ATR) inhibitor (ceralasertib) and compared it to the first-generation PARPi olaparib. We performed DNA and RNA sequencing as well as protein-based assays to identify mechanisms of acquired resistance to either PARPi. RESULTS: AZD5305 showed superior antitumor activity than the first-generation PARPi in terms of preclinical complete response rate (75% vs. 37%). The median preclinical progression-free survival was significantly longer in the AZD5305-treated group compared to the olaparib-treated group (> 386 days vs. 90 days). Mechanistically, AZD5305 induced more replication stress and genomic instability than the PARP1/2 inhibitor olaparib in PARPi-sensitive tumors. All tumors at progression with either PARPi (39/39) showed increase of HRR functionality by RAD51 foci formation. The most prevalent resistance mechanisms identified were the acquisition of reversion mutations in BRCA1/BRCA2 and the accumulation of hypomorphic BRCA1. AZD5305 did not sensitize PDXs with acquired resistance to olaparib but elicited profound and durable responses when combined with carboplatin or ceralasertib in 3/6 and 5/5 models, respectively. CONCLUSIONS: Collectively, these results show that the novel PARP1 selective inhibitor AZD5305 yields a potent antitumor response in PDX models with HRD and delays PARPi resistance alone or in combination with carboplatin or ceralasertib, which supports its use in the clinic as a new therapeutic option.
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Proteína BRCA1 , Proteína BRCA2 , Inibidores de Poli(ADP-Ribose) Polimerases , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Camundongos , Proteína BRCA1/genética , Proteína BRCA2/genética , Feminino , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Indóis/uso terapêutico , Indóis/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Carboplatina/farmacologia , Carboplatina/uso terapêutico , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
Poly (ADP-ribose) polymerase 1 (PARP1) is a multifunctional nuclear enzyme that catalyzes poly-ADP ribosylation in eukaryotic cells. In addition to maintaining genomic integrity, this nuclear enzyme is also involved in transcriptional regulation. PARP1 can trigger and maintain changes in the chromatin structure and directly recruit transcription factors. PARP1 also prevents DNA methylation. However, most previous reviews on PARP1 have focused on its involvement in maintaining genome integrity, with less focus on its transcriptional regulatory function. This article comprehensively reviews the transcriptional regulatory function of PARP1 and its application in disease treatment, providing new ideas for targeting PARP1 for the treatment of diseases other than cancer.
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Poli(ADP-Ribose) Polimerase-1 , Transcrição Gênica , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Animais , Neoplasias/genética , Neoplasias/metabolismo , Regulação da Expressão Gênica , Metilação de DNA , Cromatina/metabolismoRESUMO
Introduction: Glioblastoma, which affects a large number of patients every year and has an average overall lifespan of around 14.6 months following diagnosis stands out as the most lethal primary invasive brain tumor. Currently, surgery, radiation, and chemotherapy with temozolomide (TMZ) are the three major clinical treatment approaches. However, the ability to treat patients effectively is usually limited by TMZ resistance. Naringin, a bioflavonoid with anti-cancer, antioxidant, metal-chelating, and lipid-lowering effects, has emerged as a promising therapeutic option. Methods: To explore the targets and pathways of naringin and TMZ in glioblastoma network pharmacology, cell line-based ELISA, flow cytometry, immunocytochemistry, western blotting, and LC-HRMS based metabolomics study were used. Results: The findings through the network pharmacology suggested that the key targets of naringin in the chemosensitization of glioblastoma would be Poly [ADP-ribose] polymerase 1 (PARP-1), O-6-Methylguanine-DNA Methyltransferase (MGMT), and caspases. The functional enrichment analysis revealed that these targets were significantly enriched in important pathways such as p53 signaling, apoptosis, and DNA sensing. Further, the results of the in-vitro study in U87-MG and T98-G glioblastoma cells demonstrated that TMZ and naringin together significantly reduced the percentage of viability and inhibited the DNA repair enzymes PARP-1 and MGMT, and PI3K/AKT which led to chemosensitization and, in turn, induced apoptosis, which was indicated by increased p53, caspase-3 expression and decreased Bcl2 expression. Additionally, a metabolomics study in T98-G glioblastoma cells using liquid chromatography high-resolution mass spectrometry (LC-HRMS) revealed downregulation of C8-Carnitine (-2.79), L-Hexanoylcarnitine (-4.46), DL-Carnitine (-2.46), Acetyl-L-carnitine (-3.12), Adenine (-1.3), Choline (-2.07), Propionylcarnitine (-1.69), Creatine (-1.33), Adenosine (-0.84), Spermine (-1.42), and upregulation of Palmitic Acid (+1.03) and Sphingosine (+0.89) in the naringin and TMZ treatment groups. Discussion: In conclusion, it can be said that naringin in combination with TMZ chemosensitized TMZ antiglioma response and induced apoptosis in tumor cells.
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Cyclic GMP-AMP synthase (cGAS), a cytosolic DNA sensor, also exhibits nuclear genomic localization and is involved in DNA damage signaling. In this study, we investigated the impact of cGAS crotonylation on the regulation of the DNA damage response, particularly homologous recombination repair, following exposure to ionizing radiation (IR). Lysine 254 of cGAS is constitutively crotonylated by the CREB-binding protein; however, IR-induced DNA damage triggers sirtuin 3 (SIRT3)-mediated decrotonylation. Lysine 254 decrotonylation decreased the DNA-binding affinity of cGAS and inhibited its interaction with PARP1, promoting homologous recombination repair. Moreover, SIRT3 suppression led to homologous recombination repair inhibition and markedly sensitized cancer cells to IR and DNA-damaging chemicals, highlighting SIRT3 as a potential target for cancer therapy. Overall, this study revealed the crucial role of cGAS crotonylation in the DNA damage response. Furthermore, we propose that modulating cGAS and SIRT3 activities could be potential strategies for cancer therapy.
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Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.
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Neoplasias da Mama , Núcleo Celular , Poli(ADP-Ribose) Polimerase-1 , Proteínas Proto-Oncogênicas c-akt , Humanos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Feminino , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Núcleo Celular/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Linhagem Celular Tumoral , PTEN Fosfo-Hidrolase/metabolismo , PTEN Fosfo-Hidrolase/genética , Aminoacil-tRNA Sintetases/metabolismo , Aminoacil-tRNA Sintetases/genética , Transporte Ativo do Núcleo Celular , Sinais de Localização Nuclear/metabolismoRESUMO
Performing accurate Fluorescence Correlation Spectroscopy (FCS) measurements in cells can be challenging due to cellular motion or other intracellular processes. In this respect, it has recently been shown that analysis of FCS data in short temporal segments (segmented FCS) can be very useful to increase the accuracy of FCS measurements inside cells. Here, we demonstrate that segmented FCS can be performed on a commercial laser scanning microscope (LSM), even in the absence of the dedicated FCS module. We show how data can be acquired on a Leica SP8 confocal microscope and then exported and processed with a custom software in MATLAB. The software performs segmentation of the data to extract an average ACF and measure the diffusion coefficient in specific subcellular regions. First of all, we measure the diffusion of fluorophores of different size in solution, to show that good-quality ACFs can be obtained in a commercial LSM. Next, we validate the method by measuring the diffusion coefficient of GFP in the nucleus of HeLa cells, exploiting variations of the intensity to distinguish between nucleoplasm and nucleolus. As expected, the measured diffusion coefficient of GFP is slower in the nucleolus relative to nucleoplasm. Finally, we apply the method to HeLa cells expressing a PARP1 chromobody to measure the diffusion coefficient of PARP1 in different subcellular regions. We find that PARP1 diffusion is slower in the nucleolus compared to the nucleoplasm.
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Microscopia Confocal , Espectrometria de Fluorescência , Humanos , Células HeLa , Microscopia Confocal/métodos , Espectrometria de Fluorescência/métodos , Proteínas de Fluorescência Verde/metabolismo , Difusão , Núcleo Celular/metabolismo , Software , Poli(ADP-Ribose) Polimerase-1/metabolismoRESUMO
Heme oxygenase-1 (HO-1, HMOX1) degrades heme protecting cells from heme-induced oxidative damage. Beyond its well-established cellular functions, heme has emerged as a stabilizer of G-quadruplexes. These secondary DNA structures interfere with DNA replication. We recently revealed that nuclear HO-1 colocalizes with DNA G-quadruplexes and promotes their removal. Here, we investigate whether HO-1 safeguards cells against replication stress. Experiments were conducted in control and HMOX1-deficient HEK293T cell lines. Immunostaining unveiled that DNA G-quadruplexes accumulated in the absence of HO-1, the effect that was further enhanced in response to δ-aminolevulinic acid (ALA), a substrate in heme synthesis. This was associated with replication stress, as evidenced by an elevated proportion of stalled forks analyzed by fiber assay. We observed the same effects in hematopoietic stem cells isolated from Hmox1 knockout mice and in a lymphoblastoid cell line from an HMOX1-deficient patient. Interestingly, in the absence of HO-1, the speed of fork progression was higher, and the response to DNA conformational hindrance less stringent, indicating dysfunction of the PARP1-p53-p21 axis. PARP1 activity was not decreased in the absence of HO-1. Instead, we observed that HO-1 deficiency impairs the nuclear import and accumulation of p53, an effect dependent on the removal of excess heme. We also demonstrated that administering ALA is a more specific method for increasing intracellular free heme compared to treatment with hemin, which in turn induces strong lipid peroxidation. Our results indicate that protection against replication stress is a universal feature of HO-1, presumably contributing to its widely recognized cytoprotective activity.
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Replicação do DNA , Heme Oxigenase-1 , Humanos , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Animais , Camundongos , Células HEK293 , Estresse Oxidativo , Heme/metabolismo , Camundongos Knockout , Quadruplex G , Ácido Aminolevulínico/farmacologia , Ácido Aminolevulínico/metabolismo , Dano ao DNARESUMO
Persistent DNA-protein crosslinks formed by human topoisomerase IIIα (TOP3A-DPCs) interfere with DNA metabolism and lead to genome damage and cell death. Recently, we demonstrated that such abortive TOP3A-DPCs are ubiquitylated and proteolyzed by Spartan (SPRTN). Here, we identify transient poly(ADP-ribosylation) (PARylation) in addition to ubiquitylation as a signaling mechanism for TOP3A-DPC repair and provide evidence that poly(ADP-ribose) polymerase 1 (PARP1) drives the repair of TOP3A-DPCs by recruiting flap endonuclease 1 (FEN1) to the TOP3A-DPCs. We find that blocking PARylation attenuates the interaction of FEN1 and TOP3A and that TOP3A-DPCs accumulate in cells with compromised PARP1 activity and in FEN1-deficient cells. We also show that PARP1 suppresses TOP3A-DPC ubiquitylation and that inhibiting the ubiquitin-activating enzyme E1 (UBE1) increases TOP3A-DPCs, consistent with ubiquitylation serving as a signaling mechanism for TOP3A-DPC repair mediated by SPRTN and TDP2. We propose that two concerted pathways repair TOP3A-DPCs: PARylation-driven FEN1 excision and ubiquitylation-driven SPRTN-TDP2 excision.
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Reparo do DNA , DNA Topoisomerases Tipo I , Endonucleases Flap , Poli(ADP-Ribose) Polimerase-1 , Ubiquitinação , Humanos , Endonucleases Flap/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Poli ADP Ribosilação , DNA/metabolismoRESUMO
DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.
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Neural stem cells play a crucial role in maintaining brain homeostasis. Neural stem cells senescence can lead to the decline of nerve repair and regeneration, causing brain aging and neurodegenerative diseases. However, the mechanism underlying neural stem cells senescence remains poorly understood. In this study, we report a novel HO-1/PARP1 non-canonical pathway highlighting how oxidative stress triggers the DNA damage response, ultimately leading to premature cellular senescence in neural stem cells. HO-1 acts as a sensor for oxidative stress, while PARP1 functions as a sensor for DNA damage. The simultaneous expression and molecular interaction of these two sensors can initiate a crosstalk of oxidative stress and DNA damage response processes, leading to the vicious cycle. The persistent activation of this pathway contributes to the senescence of neural stem cells, which in turn plays a crucial role in the progression of neurodegenerative diseases. Consequently, targeting this novel signaling pathway holds promise for the development of innovative therapeutic strategies and targets aimed at mitigating neural stem cells senescence-related disorders.
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In recent years, poly(ADP-ribose)polymerase-1 (PARP-1) and histone deacetylase (HDAC) have emerged as significant targets in tumor therapy, garnering widespread attention. In this study, we designed and synthesized two novel phthalazinone PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors, named DLC-1-46 containing dithiocarboxylate fragments and DLC-47-63 containing hydroxamic acid fragments, and evaluated their inhibitory activities on enzymes and cells. Among the PARP-1 inhibitors, most compounds exhibited high inhibitory activity against the PARP-1 enzyme, with DLC-1-6 being particularly notable, showing IC50 values <0.2â¯nM. Notably, DLC-1 demonstrated significant anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells reaching 0.08, 26.39, and 1.01⯵M, respectively. Further investigation revealed that DLC-1 arrested MDA-MB-231 cells in the G1 phase and induced apoptosis in a dose-dependent manner. Among the designed dual PARP-1/HDAC-1 inhibitors, several compounds exhibited potent dual-target inhibitory activity, with DLC-49 displaying IC50 values of 0.53â¯nM and 17â¯nM for PARP-1 and HDAC-1, respectively. DLC-50 demonstrated the most potent anti-proliferative activity, with IC50 values for inhibiting the proliferation of MDA-MB-436, MDA-MB-231, and MCF-7 cells at 0.30, 2.70, and 2.41⯵M, respectively. Cell cycle arrest and apoptosis assays indicated that DLC-50 arrested the cell cycle in the G2 phase and induced apoptosis in HCT-116 cells. Our findings present a novel avenue for further exploration of PARP-1 inhibitors and dual PARP-1/HDAC-1 inhibitors.
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Antineoplásicos , Proliferação de Células , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Histona Desacetilase 1 , Inibidores de Histona Desacetilases , Ftalazinas , Poli(ADP-Ribose) Polimerase-1 , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Histona Desacetilase 1/antagonistas & inibidores , Histona Desacetilase 1/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Relação Estrutura-Atividade , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Ftalazinas/farmacologia , Ftalazinas/síntese química , Ftalazinas/química , Estrutura Molecular , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Apoptose/efeitos dos fármacos , Simulação de Acoplamento MolecularRESUMO
PARP1 is a highly conserved nuclear protein in multicellular organisms that by modulating chromatin opening facilitates gene expression during development. All reported Parp1 null knockout mice strains are viable with no developmental anomalies. It was believed that functional redundancy with other PARP family members, mainly PARP2, explain such a controversy. However, while PARP2 has similar catalytic domain to PARP1, it lacks other domains, making the absence of developmental problems in Parp1 mice knockouts unlikely. Contrary to prior assumptions, in our analysis of the best-investigated Parp1 knockout mice strain we identified persistent mRNA expression, albeit at reduced levels. Transcript analysis revealed an alternatively spliced Parp1 variant lacking exon two. Subsequent protein analysis confirmed the existence of a truncated PARP1 protein in knockout mice. The decreased level of pADPr was detected in Parp1 knockout ES cells with Western Blotting analysis, but immunofluorescence staining did not detect any difference in distribution or level of pADPr in nuclei of knockout ES cells. pADPr level in double Parp1 Parg mutant ES cells greatly exceeded its amount in normal and even in hypomorph Parg mutant ES cells, suggesting the presence of functionally active PARP1. Therefore, our findings challenge the conventional understanding of PARP1 depletion effects.
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The emergence of PARP inhibitors as a therapeutic strategy for tumors with high genomic instability, particularly those harboring BRCA mutations, has advanced cancer treatment. However, recent advances have illuminated a multifaceted role of PARP1 beyond its canonical function in DNA damage repair. This review explores the expanding roles of PARP1, highlighting its crucial interplay with the immune system during tumorigenesis. We discuss PARP1's immunomodulatory effects in macrophages and T cells, with a particular focus on cytokine expression. Understanding these immunomodulatory roles of PARP1 not only holds promise for enhancing the efficacy of PARP inhibitors in cancer therapy but also paves the way for novel treatment regimens targeting immune-mediated inflammatory diseases.
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Most clinical PARP inhibitors (PARPis) trap PARP1 in a chromatin-bound state, leading to PARPi-mediated cytotoxicity. PARPi resistance impedes the treatment of ovarian cancer in clinical practice. However, the mechanism by which cancer cells overcome PARP1 trapping to develop PARPi resistance remains unclear. Here, it is shown that high levels of KAT6A promote PARPi resistance in ovarian cancer, regardless of its catalytic activity. Mechanistically, the liquid-liquid phase separation (LLPS) of KAT6A, facilitated by APEX1, inhibits the cytotoxic effects of PARP1 trapping during PARPi treatment. The stable KAT6A-PARP1-APEX1 complex reduces the amount of PARP1 trapped at the DNA break sites. In addition, inhibition of KAT6A LLPS, rather than its catalytic activity, impairs DNA damage repair and restores PARPi sensitivity in ovarian cancer both in vivo and in vitro. In conclusion, the findings demonstrate the role of KAT6A LLPS in fostering PARPi resistance and suggest that repressing KAT6A LLPS can be a potential therapeutic strategy for PARPi-resistant ovarian cancer.
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BACKGROUND: Heart failure (HF) is characterized by oxidative stress and mitochondrial dysfunction. This study investigates the therapeutic potential of Necrostatin-1 (Nec-1) delivered through exosomes derived from induced pluripotent stem cells (iPSCs) to address these pathologies in HF. METHODS: An HF rat model was established, and comprehensive assessments were performed using echocardiography, hemodynamics, and ventricular mass index measurements. iPSCs were used to isolate exosomes, loaded with Nec-1, and characterized for efficient delivery into cardiomyocytes. The interaction between Nec-1-loaded exosomes (Nec-1-Exos), poly (ADP-ribose) polymerase 1 (PARP1), and apoptosis-inducing factor mitochondria-associated 1 (AIFM1) was explored. Gain-of-function experiments assessed changes in cardiomyocyte parameters, and histological analyses were conducted on myocardial tissues. RESULTS: Cardiomyocytes successfully internalized Nec-1-loaded exosomes, leading to downregulation of PARP1, inhibition of AIFM1 nuclear translocation, increased ATP and superoxide dismutase levels, reduced reactive oxygen species and malonaldehyde levels, and restored mitochondrial membrane potential. Histological examinations confirmed the modulation of the PARP1/AIFM1 axis by Nec-1, mitigating HF. CONCLUSIONS: iPSC-derived exosomes carrying Nec-1 attenuate oxidative stress and mitochondrial dysfunction in HF by targeting the PARP1/AIFM1 axis. This study proposes a promising therapeutic strategy for HF management and highlights the potential of exosome-mediated drug delivery.
Assuntos
Exossomos , Insuficiência Cardíaca , Imidazóis , Indóis , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Estresse Oxidativo , Poli(ADP-Ribose) Polimerase-1 , Exossomos/metabolismo , Animais , Estresse Oxidativo/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Insuficiência Cardíaca/metabolismo , Indóis/farmacologia , Masculino , Imidazóis/farmacologia , Cardiotônicos/farmacologia , Ratos Sprague-Dawley , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Fator de Indução de Apoptose/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , RatosRESUMO
BACKGROUND: Endometriosis is a gynecological disease characterized by the presence of endometrial tissue in abnormal locations, leading to severe symptoms, inflammation, pain, organ dysfunction, and infertility. Surgical removal of endometriosis lesions is crucial for improving pain and fertility outcomes, with the goal of complete lesion removal. This study aimed to analyze the location and expression patterns of poly (ADP-ribose) polymerase 1 (PARP-1), epithelial cell adhesion molecule (EpCAM), and folate receptor alpha (FRα) in endometriosis lesions and evaluate their potential for targeted imaging. METHODS: Gene expression analysis was performed using the Turku endometriosis database (EndometDB). By immunohistochemistry, we investigated the presence and distribution of PARP-1, EpCAM, and FRα in endometriosis foci and adjacent tissue. We also applied an ad hoc platform for the analysis of images to perform a quantitative immunolocalization analysis. Double immunofluorescence analysis was carried out for PARP-1 and EpCAM, as well as for PARP-1 and FRα, to explore the expression of these combined markers within endometriosis foci and their potential simultaneous utilization in surgical treatment. RESULTS: Gene expression analysis revealed that PARP-1, EpCAM, and FOLR1 (FRα gene) are more highly expressed in endometriotic lesions than in the peritoneum, which served as the control tissue. The results of the immunohistochemical study revealed a significant increase in the expression levels of all three biomarkers inside the endometriosis foci compared to the adjacent tissues. Additionally, the double immunofluorescence analysis consistently demonstrated the presence of PARP-1 in the nucleus and the expression of EpCAM and FRα in the cell membrane and cytoplasm. CONCLUSION: Overall, these three markers demonstrate significant potential for effective imaging of endometriosis. In particular, the results emphasize the importance of PARP-1 expression as a possible indicator for distinguishing endometriotic lesions from adjacent tissue. PARP-1, as a potential biomarker for endometriosis, offers promising avenues for further investigation in terms of both pathophysiology and diagnostic-therapeutic approaches.
Assuntos
Endometriose , Molécula de Adesão da Célula Epitelial , Receptor 1 de Folato , Poli(ADP-Ribose) Polimerase-1 , Endometriose/metabolismo , Endometriose/cirurgia , Endometriose/genética , Endometriose/diagnóstico , Endometriose/patologia , Feminino , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Receptor 1 de Folato/genética , Receptor 1 de Folato/metabolismo , Molécula de Adesão da Célula Epitelial/genética , Molécula de Adesão da Célula Epitelial/metabolismo , Adulto , Biomarcadores/metabolismo , Imuno-Histoquímica , Endométrio/metabolismo , Endométrio/patologia , Endométrio/cirurgiaRESUMO
Innate immune cells show enhanced responsiveness to secondary challenges after an initial non-related stimulation (Trained Innate Immunity, TII). Acute NOD2 activation by Muramyl-Dipeptide (MDP) promotes TII inducing the secretion of pro-inflammatory mediators, while a sustained MDP-stimulation down-regulates the inflammatory response, restoring tolerance. Here we characterized in-vitro the response of murine macrophages to lipopolysaccharide (LPS) challenge under NOD2-chronic stimulation. RAW264.7 cells were trained with MDP (1 µg/ml, 48 h) and challenged with LPS (5 µg/ml, 24 h). Trained cells formed multinucleated giant cells with increased phagocytosis rates compared to untrained/challenged cells. They showed a reduced mitochondrial activity and a switch to aerobic glycolysis. TNF-α, ROS and NO were upregulated in both trained and untrained cultures (MDP+, MDP- cells, p > 0.05); while IL-10, IL-6 IL-12 and MHCII were upregulated only in trained cells after LPS challenge (MDP + LPS+, p < 0.05). A slight upregulation in the expression of B7.2 was also observed in this group, although differences were not statistically significant. MDP-training induced resistance to LPS challenge (p < 0.01). The relative expression of PARP-1 was downregulated after the LPS challenge, which may contribute to the regulatory milieu and to the innate memory mechanisms exhibited by MDP-trained cells. Our results demonstrate that a sustained MDP-training polarizes murine macrophages towards a M2b profile, inhibiting parthanatos. These results may impact on the development of strategies to immunomodulate processes in which inflammation should be controlled.