RESUMO
Treacher Collins syndrome-3 (TCS-3) is a rare congenital craniofacial disorder attributed to variants in the RNA pol I subunit C (POLR1C). The pathogenesis of TCS-3 linked to polr1c involves the activation of apoptosis-dependent p53 pathways within neural crest cells (NCCs). This occurs due to disruptions in ribosome biogenesis, and the restoration of polr1c expression in early embryogenesis effectively rescues the observed craniofacial phenotype in polr1c-deficient zebrafish. Clinical variability in TCS patients suggests interactions between genes and factors like oxidative stress. Elevated production of reactive oxygen species (ROS) in epithelial cells may worsen phenotypic outcomes in TCS individuals. Our study confirmed excessive ROS production in facial regions, inducing apoptosis and altering p53 pathways. Deregulated cell-cycle and epithelial-to-mesenchymal transition (EMT) genes were also detected in the TCS-3 model. Utilizing p53 inhibitor (Pifithrin-α; PFT-α) or antioxidants (Glutathione; GSH and N-Acetyl-L-cysteine; NAC) effectively corrected migrated NCC distribution in the pharyngeal arch (PA), suppressed oxidative stress, prevented cell death, and modulated EMT inducers. Crucially, inhibiting p53 activation or applying antioxidants within a specific time window, notably within 30 h post-fertilization (hpf), successfully reversed phenotypic effects induced by polr1c MO.
Assuntos
Antioxidantes , Benzotiazóis , Modelos Animais de Doenças , Disostose Mandibulofacial , Estresse Oxidativo , Espécies Reativas de Oxigênio , Tolueno/análogos & derivados , Proteína Supressora de Tumor p53 , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Disostose Mandibulofacial/genética , Disostose Mandibulofacial/tratamento farmacológico , Antioxidantes/farmacologia , Benzotiazóis/farmacologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Tolueno/farmacologia , Crista Neural/efeitos dos fármacos , Crista Neural/metabolismo , Apoptose/efeitos dos fármacos , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , RNA Polimerase I/genéticaRESUMO
In cancer therapy, DNA intercalators are mainly known for their capacity to kill cells by inducing DNA damage. Recently, several DNA intercalators have attracted much interest given their ability to inhibit RNA Polymerase I transcription (BMH-21), evict histones (Aclarubicin) or induce chromatin trapping of FACT (Curaxin CBL0137). Interestingly, these DNA intercalators lack the capacity to induce DNA damage while still retaining cytotoxic effects and stabilize p53. Herein, we report that these DNA intercalators impact chromatin biology by interfering with the chromatin stability of RNA polymerases I, II and III. These three compounds have the capacity to induce degradation of RNA polymerase II and they simultaneously enable the trapping of Topoisomerases TOP2A and TOP2B on the chromatin. In addition, BMH-21 also acts as a catalytic inhibitor of Topoisomerase II, resembling Aclarubicin. Moreover, BMH-21 induces chromatin trapping of the histone chaperone FACT and propels accumulation of Z-DNA and histone eviction, similarly to Aclarubicin and CBL0137. These DNA intercalators have a cumulative impact on general transcription machinery by inducing accumulation of topological defects and impacting nuclear chromatin. Therefore, their cytotoxic capabilities may be the result of compounding deleterious effects on chromatin homeostasis.
Assuntos
Cromatina , DNA Topoisomerases Tipo II , Substâncias Intercalantes , RNA Polimerase II , Humanos , Antígenos de Neoplasias/metabolismo , Antígenos de Neoplasias/genética , Carbazóis , Cromatina/metabolismo , Dicetopiperazinas , DNA/metabolismo , DNA/química , Dano ao DNA , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Histonas/metabolismo , Substâncias Intercalantes/farmacologia , Substâncias Intercalantes/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase I/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Inibidores da Topoisomerase II/farmacologia , Transcrição Gênica/efeitos dos fármacos , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Aclarubicina/farmacologiaRESUMO
Increased ribosome biogenesis is a distinguishing feature of cancer cells, and small molecule inhibitors of ribosome biogenesis are currently in clinical trials as single agent therapy. It has been previously shown that inhibiting ribosome biogenesis through the inhibition of nuclear export of ribosomal subunits sensitizes tumor cells to radiotherapy. In this study, the radiosensitizing potential of CX-5461, a small molecule inhibitor of RNA polymerase I, was tested. Radiosensitization was measured by clonogenic survival assay in a panel of four tumor cell lines derived from three different tumor types commonly treated with radiation. 50 nM CX-5461 radiosensitized PANC-1, U251, HeLa, and PSN1 cells with dose enhancement factors in the range of 1.2-1.3. However, 50 nM CX-5461 was not sufficient to inhibit 45S transcription alone or in combination with radiation. The mechanism of cell death with the combination of CX-5461 and radiation occurred through mitotic catastrophe and not apoptosis. CX-5461 inhibited the repair and/or enhanced the initial levels of radiation-induced DNA double strand breaks. Understanding the mechanism of CX-5461-induced radiosensitization should be of value in the potential application of the CX-5461/radiotherapy combination in cancer treatment.
Assuntos
Benzotiazóis , Naftiridinas , RNA Polimerase I , Radiossensibilizantes , Apoptose , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Humanos , Naftiridinas/farmacologia , RNA Polimerase I/antagonistas & inibidores , Radiossensibilizantes/farmacologiaRESUMO
BACKGROUND: Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. METHODS: SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. RESULTS: Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. CONCLUSIONS: SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer.
Assuntos
Benzotiazóis , Leiomiossarcoma , Naftiridinas , Neoplasias Uterinas , Benzotiazóis/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Leiomiossarcoma/tratamento farmacológico , Leiomiossarcoma/genética , Leiomiossarcoma/metabolismo , Naftiridinas/farmacologia , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Transdução de Sinais/efeitos dos fármacos , Neoplasias Uterinas/tratamento farmacológico , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
DNA topoisomerase II (TOP2) is a nuclear protein that resolves DNA topological problems and plays critical roles in multiple nuclear processes. Human cells have two TOP2 proteins, TOP2A and TOP2B, that are localized in both the nucleoplasm and nucleolus. Previously, ATP depletion was shown to augment the nucleolar localization of TOP2B, but the molecular details of subnuclear distributions, particularly of TOP2A, remained to be fully elucidated in relation to the status of cellular ATP. Here, we analyzed the nuclear dynamics of human TOP2A and TOP2B in ATP-depleted cells. Both proteins rapidly translocated from the nucleoplasm to the nucleolus in response to ATP depletion. FRAP analysis demonstrated that they were highly mobile in the nucleoplasm and nucleolus. The nucleolar retention of both proteins was sensitive to the RNA polymerase I inhibitor BMH-21, and the TOP2 proteins in the nucleolus were immediately dispersed into the nucleoplasm by BMH-21. Under ATP-depleted conditions, the TOP2 poison etoposide was less effective, indicating the therapeutic relevance of TOP2 subnuclear distributions. These results give novel insights into the subnuclear dynamics of TOP2 in relation to cellular ATP levels and also provide discussions about its possible mechanisms and biological significance.
Assuntos
Trifosfato de Adenosina/deficiência , Nucléolo Celular/metabolismo , DNA Topoisomerases Tipo II/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , RNA Polimerase I/antagonistas & inibidores , Nucléolo Celular/efeitos dos fármacos , DNA Topoisomerases Tipo II/genética , Inibidores Enzimáticos/farmacologia , Etoposídeo/farmacologia , Células HeLa , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Inibidores da Topoisomerase II/farmacologia , Translocação GenéticaRESUMO
The nucleolus of a cell is a critical cellular compartment that is responsible for ribosome biogenesis and plays a central role in tumor progression. Fisetin, a nutraceutical, is a naturally occurring flavonol from the flavonoid group of polyphenols that has anti-cancer effects. Fisetin negatively impacts several signaling pathways that support tumor progression. However, effect of fisetin on the nucleolus and its functions were unknown. We observed that fisetin is able to physically enter the nucleolus. In the nucleolus, RNA polymerase I (RNA Pol I) mediates the biogenesis of ribosomal RNA. Thus, we investigated the impacts of fisetin on the nucleolus. We observed that breast tumor cells treated with fisetin show a 20-30% decreased nucleolar abundance per cell and a 30-60% downregulation of RNA Pol I transcription activity, as well as a 50-70% reduction in nascent rRNA synthesis, depending on the cell line. Our studies show that fisetin negatively influences MAPK/ERK pathway to impair RNA Pol I activity and rRNA biogenesis. Functionally, we demonstrate that fisetin acts synergistically (CI = 0.4) with RNA Pol I inhibitor, BMH-21 and shows a noteworthy negative impact (60% decrease) on lung colonization of breast cancer cells. Overall, our findings highlight the potential of ribosomal RNA (rRNA) biogenesis as a target for secondary prevention and possible treatment of metastatic disease.
Assuntos
Nucléolo Celular/efeitos dos fármacos , Flavonóis/uso terapêutico , Neoplasias Pulmonares/prevenção & controle , RNA Polimerase I/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Flavonas/farmacologia , Flavonas/uso terapêutico , Flavonóis/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , Neoplasias Pulmonares/secundário , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , RNA Ribossômico/biossínteseRESUMO
Guanine quadruplexes (G4s) are non-canonical nucleic acid structures commonly found in regulatory genomic regions. G4 targeting has emerged as a therapeutic approach in cancer. We have screened naphthalene-diimides (NDIs), a class of G4 ligands, in a cellular model of colorectal cancer (CRC). Here, we identify the leading compound T5 with a potent and selective inhibition of cell growth by high-affinity binding to G4s in ribosomal DNA, impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induces a rapid inhibition of Pol I transcription, nucleolus disruption, proteasome-dependent Pol I catalytic subunit A degradation and autophagy. Moreover, we attribute the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. Finally, we succinctly demonstrate that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Therefore, we report a mode of action for these NDIs involving ribosomal G4 targeting.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Imidas/farmacologia , Naftalenos/farmacologia , RNA Polimerase I/antagonistas & inibidores , Ribossomos/efeitos dos fármacos , Idoso , Antineoplásicos/síntese química , Antineoplásicos/química , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Feminino , Quadruplex G/efeitos dos fármacos , Humanos , Imidas/química , Masculino , Pessoa de Meia-Idade , Naftalenos/química , RNA Polimerase I/metabolismo , Ribossomos/metabolismoRESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer lacking targeted therapy. Here, we evaluated the anti-cancer activity of APR-246, a P53 activator, and CX-5461, a RNA polymerase I inhibitor, in the treatment of TNBC cells. We tested the efficacy of individual and combination therapy of CX-5461 and APR-246 in vitro, using a panel of breast cancer cell lines. Using publicly available breast cancer datasets, we found that components of RNA Pol I are predominately upregulated in basal-like breast cancer, compared to other subtypes, and this upregulation is associated with poor overall and relapse-free survival. Notably, we found that the treatment of breast cancer cells lines with CX-5461 significantly hampered cell proliferation and synergistically enhanced the efficacy of APR-246. The combination treatment significantly induced apoptosis that is associated with cleaved PARP and Caspase 3 along with Annexin V positivity. Likewise, we also found that combination treatment significantly induced DNA damage and replication stress in these cells. Our data provide a novel combination strategy by utilizing APR-246 in combination CX-5461 in killing TNBC cells that can be further developed into more effective therapy in TNBC therapeutic armamentarium.
Assuntos
Benzotiazóis/farmacologia , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Naftiridinas/farmacologia , Quinuclidinas/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Replicação do DNA/genética , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Ribosome biogenesis (RiBi) is one of the most complex and energy demanding processes in human cells, critical for cell growth and proliferation. Strong causal links between inherited and acquired impairment in RiBi with cancer pathogenesis are emerging, pointing to RiBi as an attractive therapeutic target for cancer. Here, we will highlight new knowledge about causes of excessive or impaired RiBi and the impact of these changes on protein synthesis. We will also discuss how new knowledge about secondary consequences of dysregulated RiBi and protein synthesis, including proteotoxic stress, metabolic alterations, adaptive transcriptional and translational programs, and the impaired ribosome biogenesis checkpoint (IRBC) provide a foundation for the development of new anticancer therapies.
Assuntos
Benzotiazóis/farmacologia , Carcinogênese/efeitos dos fármacos , Naftiridinas/farmacologia , Neoplasias/tratamento farmacológico , RNA Polimerase I/antagonistas & inibidores , Ribossomos/metabolismo , Benzotiazóis/uso terapêutico , Carcinogênese/genética , Carcinogênese/patologia , Reparo do DNA/genética , Humanos , Mutação , Naftiridinas/uso terapêutico , Neoplasias/genética , Neoplasias/patologia , Biogênese de Organelas , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/genética , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Polimerase I/metabolismo , Proteínas Ribossômicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Mutações Sintéticas Letais , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
BACKGROUND: Intrinsic and acquired drug resistance represent fundamental barriers to the cure of high-grade serous ovarian carcinoma (HGSC), the most common histological subtype accounting for the majority of ovarian cancer deaths. Defects in homologous recombination (HR) DNA repair are key determinants of sensitivity to chemotherapy and poly-ADP ribose polymerase inhibitors. Restoration of HR is a common mechanism of acquired resistance that results in patient mortality, highlighting the need to identify new therapies targeting HR-proficient disease. We have shown promise for CX-5461, a cancer therapeutic in early phase clinical trials, in treating HR-deficient HGSC. METHODS: Herein, we screen the whole protein-coding genome to identify potential targets whose depletion cooperates with CX-5461 in HR-proficient HGSC. RESULTS: We demonstrate robust proliferation inhibition in cells depleted of DNA topoisomerase 1 (TOP1). Combining the clinically used TOP1 inhibitor topotecan with CX-5461 potentiates a G2/M cell cycle checkpoint arrest in multiple HR-proficient HGSC cell lines. The combination enhances a nucleolar DNA damage response and global replication stress without increasing DNA strand breakage, significantly reducing clonogenic survival and tumour growth in vivo. CONCLUSIONS: Our findings highlight the possibility of exploiting TOP1 inhibition to be combined with CX-5461 as a non-genotoxic approach in targeting HR-proficient HGSC.
Assuntos
Benzotiazóis/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Dano ao DNA/efeitos dos fármacos , Recombinação Homóloga , Naftiridinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , RNA Polimerase I/antagonistas & inibidores , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/patologia , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Pontos de Checagem da Fase G1 do Ciclo Celular , Genes BRCA2 , Humanos , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Gradação de Tumores , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Interferência de RNA , RNA Polimerase I/genéticaRESUMO
Oral squamous cell carcinoma (OSCC) is the major cause of morbidity and mortality in head and neck cancer patients worldwide. This malignant disease is challenging to treat because of the lack of effective curative strategies and the high incidence of recurrence. This study aimed to investigate the efficacy of a single and dual approach targeting ribosome biogenesis and protein translation to treat OSCC associated with the copy number variation (CNV) of ribosomal DNA (rDNA). Here, we found that primary OSCC tumors frequently exhibited a partial loss of 45S rDNA copy number and demonstrated a high susceptibility to CX5461 (a selective inhibitor of RNA polymerase I) and the coadministration of CX5461 and INK128 (a potent inhibitor of mTORC1/2). Combined treatment displayed the promising synergistic effects that induced cell apoptosis and reactive oxygen species (ROS) generation, and inhibited cell growth and proliferation. Moreover, INK128 compromised NHEJ-DNA repair pathway to reinforce the antitumor activity of CX5461. In vivo, the cotreatment synergistically suppressed tumor growth, triggered apoptosis and strikingly extended the survival time of tumor-bearing mice. Additionally, treatment with the individual compounds and coadministration appeared to reduce the incidence of enlarged inguinal lymph nodes. Our study supports that the combination of CX5461 and INK128 is a novel and efficacious therapeutic strategy that can combat this cancer and that 45S rDNA may serve as a useful indicator to predict the efficacy of this cotreatment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Benzotiazóis/farmacologia , Benzoxazóis/farmacologia , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 2 de Rapamicina/antagonistas & inibidores , Neoplasias Bucais/tratamento farmacológico , Naftiridinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , RNA Polimerase I/antagonistas & inibidores , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Ribossômico/genética , DNA Ribossômico/metabolismo , Sinergismo Farmacológico , Humanos , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Bucais/enzimologia , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , RNA Polimerase I/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Targeting RNA polymerase-1 (POL1) machinery is a new strategy for suppression of multiple sclerosis (MS) relapse activity. Oral administration of POL1 inhibitor RAM-589.555, which is characterized by high permeability and bioavailability in naïve mice, ameliorates proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (EAE) by suppressing activated autoreactive lymphocytes. We assessed the accessibility of RAM-589.555 to the central nervous system (CNS) of EAE-mice and further investigated its immunomodulatory effects on CNS-resident astro- and micro-glial cells in-vitro and in-vivo. METHODS: Effects of RAM-589.555 on activated microglia and astrocyte viability, proliferation, and secretion of neurotrophic factors were assessed in-vitro. The pharmacokinetic of RAM-589.555 was evaluated in the blood and central nervous system (CNS) of EAE-affected mice. High-dimensional single-cell mass cytometry was applied to characterize the effect of RAM-589.555 on EAE-affected mice's CNS-resident micro- and astroglial cells and CNS-infiltrating immune cells, which were obtained seven days after RAM-589.555 administration at EAE onset. Simultaneously, the expression level of pre-rRNA, the POL1 end product, was assessed in blood cells, microglia, and astrocytes to monitor RAM-589.555 effects. RESULTS: RAM-589.555 demonstrated blood and CNS permeability in EAE mice. In-vitro, incubation with 400 nM of RAM-589.555 significantly reduced viability and proliferation of lipopolysaccharide (LPS)-activated microglia by 70% and 45% (p < 0.05), respectively, while tumor necrosis factor α (TNFα)-activated astrocytes were not affected. The secretion of neurotrophic factors was preserved. Furthermore, 7 days after administration of RAM-589.555 at EAE onset, the level of pre-rRNA transcript in peripheral blood mononuclear cells (PBMC) was decreased by 38.6% (p = 0.02), while levels of pre-rRNA transcript in microglia and astrocytes remained unchanged. The high-dimensional single-cell mass cytometry analysis showed decreased percentages of CNS-resident microglia and astrocytes, diminished pro-inflammatory cytokines (IL-1ß, IL-6, IL-12, IL-17, TNFα, and IFNγ), and an increase of their anti-inflammatory cytokines (IL-4, IL-10, and TGFß) in RAM-589.555-treated compared to vehicle-treated mice (p < 0.05). CONCLUSIONS: These data correlate RAM-589.555-induced clinical amelioration and its CNS-permeability to decreased CNS-inflammation, and decreased micro- and astrogliosis, while restoring micro- and astroglial anti-inflammatory and neuroprotective capacity.
Assuntos
Anti-Inflamatórios/uso terapêutico , Sistema Nervoso Central/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Neuroglia/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , RNA Polimerase I/antagonistas & inibidores , Animais , Animais Recém-Nascidos , Anti-Inflamatórios/farmacologia , Sistema Nervoso Central/metabolismo , Relação Dose-Resposta a Droga , Encefalomielite Autoimune Experimental/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Feminino , Camundongos , Neuroglia/metabolismo , Neuroproteção/fisiologia , RNA Polimerase I/metabolismo , RecidivaRESUMO
Proteins are manufactured by ribosomes-macromolecular complexes of protein and RNA molecules that are assembled within major nuclear compartments called nucleoli1,2. Existing models suggest that RNA polymerases I and III (Pol I and Pol III) are the only enzymes that directly mediate the expression of the ribosomal RNA (rRNA) components of ribosomes. Here we show, however, that RNA polymerase II (Pol II) inside human nucleoli operates near genes encoding rRNAs to drive their expression. Pol II, assisted by the neurodegeneration-associated enzyme senataxin, generates a shield comprising triplex nucleic acid structures known as R-loops at intergenic spacers flanking nucleolar rRNA genes. The shield prevents Pol I from producing sense intergenic noncoding RNAs (sincRNAs) that can disrupt nucleolar organization and rRNA expression. These disruptive sincRNAs can be unleashed by Pol II inhibition, senataxin loss, Ewing sarcoma or locus-associated R-loop repression through an experimental system involving the proteins RNaseH1, eGFP and dCas9 (which we refer to as 'red laser'). We reveal a nucleolar Pol-II-dependent mechanism that drives ribosome biogenesis, identify disease-associated disruption of nucleoli by noncoding RNAs, and establish locus-targeted R-loop modulation. Our findings revise theories of labour division between the major RNA polymerases, and identify nucleolar Pol II as a major factor in protein synthesis and nuclear organization, with potential implications for health and disease.
Assuntos
Nucléolo Celular/enzimologia , Nucléolo Celular/genética , DNA Ribossômico/genética , RNA Polimerase II/metabolismo , RNA não Traduzido/biossíntese , RNA não Traduzido/genética , Ribossomos/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Linhagem Celular Tumoral , Nucléolo Celular/fisiologia , DNA Helicases/metabolismo , DNA Intergênico/genética , Humanos , Enzimas Multifuncionais/metabolismo , Biossíntese de Proteínas , Estruturas R-Loop , RNA Helicases/metabolismo , RNA Polimerase I/antagonistas & inibidores , RNA Polimerase I/metabolismo , Ribonuclease H/metabolismo , Ribossomos/química , Ribossomos/genética , Sarcoma de Ewing/genética , Sarcoma de Ewing/patologiaRESUMO
Acquired resistance to PARP inhibitors (PARPi) is a major challenge for the clinical management of high grade serous ovarian cancer (HGSOC). Here, we demonstrate CX-5461, the first-in-class inhibitor of RNA polymerase I transcription of ribosomal RNA genes (rDNA), induces replication stress and activates the DNA damage response. CX-5461 co-operates with PARPi in exacerbating replication stress and enhances therapeutic efficacy against homologous recombination (HR) DNA repair-deficient HGSOC-patient-derived xenograft (PDX) in vivo. We demonstrate CX-5461 has a different sensitivity spectrum to PARPi involving MRE11-dependent degradation of replication forks. Importantly, CX-5461 exhibits in vivo single agent efficacy in a HGSOC-PDX with reduced sensitivity to PARPi by overcoming replication fork protection. Further, we identify CX-5461-sensitivity gene expression signatures in primary and relapsed HGSOC. We propose CX-5461 is a promising therapy in combination with PARPi in HR-deficient HGSOC and also as a single agent for the treatment of relapsed disease.
Assuntos
Benzotiazóis/farmacologia , Cistadenocarcinoma Seroso/tratamento farmacológico , Dano ao DNA , Naftiridinas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/metabolismo , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Feminino , Xenoenxertos , Recombinação Homóloga , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Modelos Biológicos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Polimerase I/antagonistas & inibidores , TranscriptomaRESUMO
Health and survival in old age can be improved by changes in gene expression. RNA polymerase (Pol) I is the essential, conserved enzyme whose task is to generate the pre-ribosomal RNA (rRNA). We find that reducing the levels of Pol I activity is sufficient to extend lifespan in the fruit fly. This effect can be recapitulated by partial, adult-restricted inhibition, with both enterocytes and stem cells of the adult midgut emerging as important cell types. In stem cells, Pol I appears to act in the same longevity pathway as Pol III, implicating rRNA synthesis in these cells as the key lifespan determinant. Importantly, reduction in Pol I activity delays broad, age-related impairment and pathology, improving the function of diverse organ systems. Hence, our study shows that Pol I activity in the adult drives systemic, age-related decline in animal health and anticipates mortality.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , RNA Polimerase I/antagonistas & inibidores , Animais , LongevidadeRESUMO
The stabilization of G-quadruplex DNA structures by small molecules with affinity to oncogene promoters has emerged as a promising anticancer strategy, due to a potential role in gene expression regulation. We explored the ability of BMH-21 (1) and its analogue BA-41 (2) to bind the G-quadruplex structure present in the c-KIT promoter by biophysical methods and molecular modeling. We provide evidence that both compounds interact with the c-KIT 21-mer sequence. The stable monomeric intramolecular parallel G-quadruplex obtained by the mutation of positions 12 and 21 allowed the precise determination of the binding mode by NMR and molecular dynamics studies. Both compounds form a complex characterized by one ligand molecule positioned over the tetrad at the 3'-end, stabilized by an extensive network of π-π interactions. The binding constants (Kb) obtained with fluorescence are similar for both complexes (around 106 M-1). Compound BA-41 (2) showed significant antiproliferative activity against a human lymphoma cell line, SU-DHL4, known to express substantial levels of c-KIT. However, the partial inhibition of c-KIT expression by Western blot analysis suggested that the interaction of compound 2 with the c-KIT promoter is not the primary event and that multiple effects provide a contribution as determinants of biological activity.
Assuntos
Quadruplex G , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Proteínas Proto-Oncogênicas c-kit/genética , RNA Polimerase I/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
BACKGROUND: Advanced prostate cancers depend on protein synthesis for continued survival and accelerated rates of metabolism for growth. RNA polymerase I (Pol I) is the enzyme responsible for ribosomal RNA (rRNA) transcription and a rate-limiting step for ribosome biogenesis. We have shown using a specific and sensitive RNA probe for the 45S rRNA precursor that rRNA synthesis is increased in prostate adenocarcinoma compared to nonmalignant epithelium. We have introduced a first-in-class Pol I inhibitor, BMH-21, that targets cancer cells of multiple origins, and holds potential for clinical translation. METHODS: The effect of BMH-21 was tested in prostate cancer cell lines and in prostate cancer xenograft and mouse genetic models. RESULTS: We show that BMH-21 inhibits Pol I transcription in metastatic, castration-resistant, and enzalutamide treatment-resistant prostate cancer cell lines. The genetic abrogation of Pol I effectively blocks the growth of prostate cancer cells. Silencing of p53, a pathway activated downstream of Pol I, does not diminish this effect. We find that BMH-21 significantly inhibited tumor growth and reduced the Ki67 proliferation index in an enzalutamide-resistant xenograft tumor model. A decrease in 45S rRNA synthesis demonstrated on-target activity. Furthermore, the Pol I inhibitor significantly inhibited tumor growth and pathology in an aggressive genetically modified Hoxb13-MYC|Hoxb13-Cre|Ptenfl/fl (BMPC) mouse prostate cancer model. CONCLUSION: Taken together, BMH-21 is a novel promising molecule for the treatment of castration-resistant prostate cancer.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , RNA Polimerase I/antagonistas & inibidores , Animais , Benzamidas , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Camundongos , Camundongos Nus , Terapia de Alvo Molecular , Nitrilas , Células PC-3 , Feniltioidantoína/análogos & derivados , Feniltioidantoína/farmacologia , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/patologia , RNA Polimerase I/genética , RNA Polimerase I/metabolismo , RNA Ribossômico/genética , Distribuição Aleatória , Transcrição Gênica/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
RNA polymerase I (RNA Pol. I) activity is consistently expanded in multiplying cells to continue the expanded interest for ribosome generation and protein synthesis, which are fundamental for cell development and division. Thus, selective inhibitors of RNA Pol. I may offer a general helpful intends to block cancer cell multiplication. Hernandonine, isolated from the root wood of Hernandia nymphaeifolia, causes rearrangement of nucleolar proteins consistent with segregation of the nucleolus, a hallmark of RNA Pol. I transcription stress. Furthermore, the compound destabilizes RPA194, the large catalytic protein of RNA Pol. I, in a proteasome-dependent manner and inhibits nascent rRNA synthesis and expression of the 45S rRNA precursor. Finally, hernandonine induces cellular apoptosis through a p53-dependent or p53-independent process in solid tumor cell lines. These outcomes feature the prevailing effect of RNA Pol. I transcription stress on apoptosis pathway initiation and present a synthetically novel and significant molecule that represses RNA Pol. I, making it a potential objective for malignancy treatment. IMPLICATIONS: Our findings position hernandonine as a potential, particular, and orally administered cancer treatment agent appropriate for use in investigational clinical trials.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Hernandiaceae/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Quinolinas/farmacologia , RNA Polimerase I/antagonistas & inibidores , RNA Ribossômico/genética , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Nucléolo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Proteínas Nucleares/efeitos dos fármacos , RNA Polimerase I/metabolismo , Ribossomos/metabolismoRESUMO
BACKGROUND: Transplant vasculopathy is a major cause of chronic rejection of transplanted organs. In the present study, we examined the effects of CX-5461, a novel selective inhibitor of RNA polymerase I, on development of transplant vasculopathy using a modified model of rat aortic transplantation. METHODS: The thoracic aortas from Fischer rats were transplanted into the abdominal cavity of Lewis rats. CX-5461 was mixed in pluronic gel and administered via perivascular release. RESULTS: Treatment with CX-5461 mitigated the development of neointimal hyperplasia and vascular inflammation. This effect was likely to be attributable in part to inhibition of macrophage-dependent innate immunity reactions. Specifically, CX-5461 exhibited potent inhibitory effects on macrophage migration and lipopolysaccharide-induced activation. Treatment with CX-5461 also prevented macrophage differentiation and maturation from primary bone marrow cells. In macrophages, CX-5461 did not alter the total amount of p53 protein, but significantly increased p53 phosphorylation, which was involved in regulating cytokine-stimulated macrophage proliferation. CONCLUSIONS: In conclusion, our results suggest that pharmacological inhibition of RNA polymerase I may be a novel strategy to treat transplantation-induced arterial remodeling.
Assuntos
Aorta/transplante , Doenças da Aorta/prevenção & controle , Arteriosclerose/prevenção & controle , Benzotiazóis/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Transplante de Coração/efeitos adversos , Naftiridinas/uso terapêutico , Neointima/prevenção & controle , Aloenxertos/citologia , Aloenxertos/efeitos dos fármacos , Aloenxertos/imunologia , Animais , Aorta/citologia , Aorta/efeitos dos fármacos , Aorta/imunologia , Doenças da Aorta/imunologia , Arteriosclerose/imunologia , Benzotiazóis/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Transplante de Coração/métodos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Naftiridinas/farmacologia , Neointima/imunologia , Cultura Primária de Células , Células RAW 264.7 , RNA Polimerase I/antagonistas & inibidores , Ratos , Ratos Endogâmicos F344 , Resultado do Tratamento , Remodelação Vascular/efeitos dos fármacos , Remodelação Vascular/imunologiaRESUMO
Ribosome biogenesis is a complex process orchestrated by a host of ribosome assembly factors. Although it is known that many of the proteins involved in this process have RNA binding activity, the full repertoire of proteins that interact with the precursor ribosomal RNA is currently unknown. To gain a greater understanding of the extent to which RNA-protein interactions have the potential to control ribosome biogenesis, we used RNA affinity isolation coupled with proteomics to measure the changes in RNA-protein interactions that occur when rRNA transcription is blocked. Our analysis identified 211 out of 457 nuclear RNA binding proteins with a >3-fold decrease in RNA-protein interaction after inhibition of RNA polymerase I (RNAPI). We have designated these 211 RNA binding proteins as the RNAPI RNA interactome. As expected, the RNAPI RNA interactome is highly enriched for nucleolar proteins and proteins associated with ribosome biogenesis. Selected proteins from the interactome were shown to be nucleolar in location and to have RNA binding activity that was dependent on RNAPI activity. Furthermore, our data show that two proteins, which are required for rRNA maturation, AATF and NGDN, and which form part of the RNA interactome, both lack canonical RNA binding domains and yet are novel pre-rRNA binding proteins.