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1.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445574

RESUMO

Osteosarcoma is a frequent and extremely aggressive type of pediatric cancer. New therapeutic approaches are needed to improve the overall survival of osteosarcoma patients. Our previous results suggest that NMNAT1, a key enzyme in nuclear NAD+ synthesis, facilitates the survival of cisplatin-treated osteosarcoma cells. A high-throughput cytotoxicity screening was performed to identify novel pathways or compounds linked to the cancer-promoting role of NMNAT1. Nine compounds caused higher toxicity in the NMNAT1 KO U2OS cells compared to their wild type counterparts, and actinomycin D (ActD) was the most potent. ActD-treatment of NMNAT1 KO cells increased caspase activity and secondary necrosis. The reduced NAD+ content in NMNAT1 KO cells was further decreased by ActD, which partially inhibited NAD+-dependent enzymes, including the DNA nick sensor enzyme PARP1 and the NAD+-dependent deacetylase SIRT1. Impaired PARP1 activity increased DNA damage in ActD-treated NMNAT1 knockout cells, while SIRT1 impairment increased acetylation of the p53 protein, causing the upregulation of pro-apoptotic proteins (NOXA, BAX). Proliferation was decreased through both PARP- and SIRT-dependent pathways. On the one hand, PARP inhibitors sensitized wild type but not NMNAT1 KO cells to ActD-induced anti-clonogenic effects; on the other hand, over-acetylated p53 induced the expression of the anti-proliferative p21 protein leading to cell cycle arrest. Based on our results, NMNAT1 acts as a survival factor in ActD-treated osteosarcoma cells. By inhibiting both PARP1- and SIRT1-dependent cellular pathways, NMNAT1 inhibition can be a promising new tool in osteosarcoma chemotherapy.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Ósseas/prevenção & controle , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , Osteossarcoma/prevenção & controle , Antibióticos Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Humanos , Nicotinamida-Nucleotídeo Adenililtransferase/antagonistas & inibidores , Nicotinamida-Nucleotídeo Adenililtransferase/genética , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Células Tumorais Cultivadas
2.
Int J Mol Sci ; 22(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361059

RESUMO

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility.


Assuntos
Núcleo Celular/metabolismo , Dactinomicina/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Guanosina Trifosfato/metabolismo , Proteínas Nucleares/metabolismo , Inibidores da Síntese de Ácido Nucleico/farmacologia , Transcrição Genética , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/genética , Proteínas de Ligação ao GTP/antagonistas & inibidores , Proteínas de Ligação ao GTP/genética , Células HeLa , Humanos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética
3.
RNA ; 27(10): 1241-1256, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34244458

RESUMO

Stress granules (SGs) are membraneless organelles composed of mRNAs and RNA binding proteins which undergo assembly in response to stress-induced inactivation of translation initiation. In general, SG recruitment is limited to a subpopulation of a given mRNA species and RNA-seq analyses of purified SGs revealed that signal sequence-encoding (i.e., endoplasmic reticulum [ER]-targeted) transcripts are significantly underrepresented, consistent with prior reports that ER localization can protect mRNAs from SG recruitment. Using translational profiling, cell fractionation, and single molecule mRNA imaging, we examined SG biogenesis following activation of the unfolded protein response (UPR) by 1,4-dithiothreitol (DTT) and report that gene-specific subsets of cytosolic and ER-targeted mRNAs can be recruited into SGs. Furthermore, we demonstrate that SGs form in close proximity to or directly associated with the ER membrane. ER-associated SG assembly was also observed during arsenite stress, suggesting broad roles for the ER in SG biogenesis. Recruitment of a given mRNA into SGs required stress-induced translational repression, though translational inhibition was not solely predictive of an mRNA's propensity for SG recruitment. SG formation was prevented by the transcriptional inhibitors actinomycin D or triptolide, suggesting a functional link between gene transcriptional state and SG biogenesis. Collectively these data demonstrate that ER-targeted and cytosolic mRNAs can be recruited into ER-associated SGs and this recruitment is sensitive to transcriptional inhibition. We propose that newly transcribed mRNAs exported under conditions of suppressed translation initiation are primary SG substrates, with the ER serving as the central subcellular site of SG formation.


Assuntos
Grânulos Citoplasmáticos/genética , Retículo Endoplasmático/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Resposta a Proteínas não Dobradas , Biomarcadores/metabolismo , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Citosol/efeitos dos fármacos , Citosol/metabolismo , Dactinomicina/farmacologia , Diterpenos/farmacologia , Ditiotreitol/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Compostos de Epóxi/farmacologia , Expressão Gênica , Células HeLa , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Biogênese de Organelas , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Fenantrenos/farmacologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Imagem Individual de Molécula , Estresse Fisiológico/efeitos dos fármacos , Transcrição Genética/efeitos dos fármacos , Microglobulina beta-2/genética , Microglobulina beta-2/metabolismo
4.
Nucleic Acids Res ; 49(11): e65, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-33693821

RESUMO

RNA-protein interactions play key roles in epigenetic, transcriptional and posttranscriptional regulation. To reveal the regulatory mechanisms of these interactions, global investigation of RNA-binding proteins (RBPs) and monitor their changes under various physiological conditions are needed. Herein, we developed a psoralen probe (PP)-based method for RNA tagging and ribonucleic-protein complex (RNP) enrichment. Isolation of both coding and noncoding RNAs and mapping of 2986 RBPs including 782 unknown candidate RBPs from HeLa cells was achieved by PP enrichment, RNA-sequencing and mass spectrometry analysis. The dynamics study of RNPs by PP enrichment after the inhibition of RNA synthesis provides the first large-scale distribution profile of RBPs bound to RNAs with different decay rates. Furthermore, the remarkably greater decreases in the abundance of the RBPs obtained by PP-enrichment than by global proteome profiling suggest that PP enrichment after transcription inhibition offers a valuable way for large-scale evaluation of the candidate RBPs.


Assuntos
Proteômica/métodos , Ribonucleoproteínas/metabolismo , Dactinomicina/farmacologia , Furocumarinas/química , Células HeLa , Humanos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Proteoma , Ribonucleoproteínas/química , Ribonucleoproteínas/isolamento & purificação , Análise de Sequência de RNA , Transcrição Genética/efeitos dos fármacos
5.
Int J Oncol ; 58(2): 266-274, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33491749

RESUMO

Rhabdomyosarcoma (RMS), the most common pediatric soft tissue sarcoma, has an unfavorable outcome in advanced tumor stages with less than 30% failure­free survival. Curcumin (CUR) is a promising drug in complementary oncology with few side effects but proven efficacy in various adult oncological entities. The present study analyzed the effects of CUR on pediatric (RMS) cell lines in vitro. RMS cell lines (RD and RH30), and skeletal muscle cells (SKMC) were treated with different doses of CUR (1.5­30 µM) alone, with phototherapy (PDT, 488 nm) or in combination with vincristine (VCR) or dactinomycin (DAC). MTT assays were used for analysis of RMS tumor cell viability. Clonal cell growth was assessed via colony forming assays and migration of the cells was analyzed with scratch tests. Annexin V staining was used to determine apoptosis in flow cytometry. Possible RMS resistance towards CUR after long­term treatment was analyzed with MTT assays. CUR decreased cell viability in all assessed RMS cell lines in a concentration­dependent manner with IC50=14­20 µM. CUR enhanced the effects of the cytotoxic drugs VCR or DAC, and led to reduced migration and increased cell apoptosis. In combination with PDT, CUR decreased the cell viability in minute quantities with up to a 10­fold lower IC50 than without PDT. CUR effectively inhibited the malignant properties of pediatric RMS cells and should be focused on as a useful additional agent in standard chemotherapy of RMS in children.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Curcumina/farmacologia , Fototerapia/métodos , Rabdomiossarcoma/terapia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Criança , Terapia Combinada/métodos , Curcumina/uso terapêutico , Dactinomicina/farmacologia , Dactinomicina/uso terapêutico , Sinergismo Farmacológico , Humanos , Concentração Inibidora 50 , Rabdomiossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Vincristina/farmacologia , Vincristina/uso terapêutico
6.
Biochem Cell Biol ; 99(4): 508-518, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33507833

RESUMO

G protein coupled receptor kinase 5 (GRK5) is localized within the nucleus and moderates functions such as DNA transcription, in addition to its localization at the plasma membrane. In this report, we show that GRK5 modifies the nucleolar stress response activated by the DNA polymerase inhibitor, actinomycin D (ActD). We show an increased sensitivity to the apoptotic effects of ActD on cervical HeLa cells and the breast cancer cell line MDA MB 231 with reduced protein expression of GRK5. We also tested two types of breast cancer cells (MDA MB 231 and MCF7 cells) and found that the rate of response to ActD varied between them because they have innate differences in the protein expression of GRK5. We also found that GRK5 phosphorylates nucleophosmin (NPM1) at T199 before and during the early stages of ActD treatment. Phosphorylation at T199 increases the ability of NPM1 to interact with p14ARF in vitro, which may affect the protein expression levels of p14ARF. We found that the expression levels of p14ARF were lower in the cells transfected with the control shRNA, but higher in cells transfected with GRK5 shRNA. Collectively, this suggests that GRK5 modifies the nucleolar stress response associated with ActD.


Assuntos
Antibióticos Antineoplásicos/farmacologia , Nucléolo Celular/patologia , Dactinomicina/farmacologia , Quinase 5 de Receptor Acoplado a Proteína G/metabolismo , Neoplasias/patologia , Proteínas Nucleares/metabolismo , Apoptose , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Quinase 5 de Receptor Acoplado a Proteína G/genética , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Células Tumorais Cultivadas
7.
Nat Prod Res ; 35(11): 1869-1873, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31305144

RESUMO

In the course of searching for cytotoxic metabolites from insects associated actinomyces, two new natural p-terphenyl glycosides, strepantibin D (1) and strepantibin E (2), along with terferol (3), actinomycin D (4), actinomycin V (5) and actinomycin V0ß (6), were identified from the fermentation medium of a Streptomyces sp. which was obtained from the larva body of mud dauber wasp. Strepantibin D (1), previously reported as a synthetic derivative of terfestatin A, is firstly isolated as a natural p-terphenyl in this research. Strepantibin D (1) and terferol (3) showed medium cytotoxic activity against breast cancer cells MCF-7, MDA-MB-231 and BT-474. Actinomycins (4-6), especially actinomycin V (5), displayed remarkable cytotoxicity against breast cancer cells, with IC50 values ranging from 0.83 nM to 369.90 nM.


Assuntos
Dactinomicina/farmacologia , Streptomyces/química , Compostos de Terfenil/farmacologia , Vespas/microbiologia , Animais , Antineoplásicos/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dactinomicina/química , Humanos , Larva/microbiologia , Compostos de Terfenil/química
8.
Molecules ; 26(1)2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379243

RESUMO

Ferulic Acid (FA) is a highly abundant phenolic phytochemical which is present in plant tissues. FA has biological effects on physiological and pathological processes due to its anti-apoptotic and anti-oxidative properties, however, the detailed mechanism(s) of function is poorly understood. We have identified FA as a molecule that inhibits apoptosis induced by hydrogen peroxide (H2O2) or actinomycin D (ActD) in rat pheochromocytoma, PC12 cell. We also found that FA reduces H2O2-induced reactive oxygen species (ROS) production in PC12 cell, thereby acting as an anti-oxidant. Then, we analyzed FA-mediated signaling responses in rat pheochromocytoma, PC12 cells using antibody arrays for phosphokinase and apoptosis related proteins. This FA signaling pathway in PC12 cells includes inactivation of pro-apoptotic proteins, SMAC/Diablo and Bad. In addition, FA attenuates the cell injury by H2O2 through the inhibition of phosphorylation of the extracellular signal-regulated kinase (ERK). Importantly, we find that FA restores expression levels of brain-derived neurotrophic factor (BDNF), a key neuroprotective effector, in H2O2-treated PC12 cells. As a possible mechanism, FA increases BDNF by regulating microRNA-10b expression following H2O2 stimulation. Taken together, FA has broad biological effects as a neuroprotective modulator to regulate the expression of phosphokinases, apoptosis-related proteins and microRNAs against oxidative stress in PC12 cells.


Assuntos
Apoptose/efeitos dos fármacos , Ácidos Cumáricos/farmacologia , Peróxido de Hidrogênio/farmacologia , Substâncias Protetoras/farmacologia , Neoplasias das Glândulas Suprarrenais/tratamento farmacológico , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Antioxidantes/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Linhagem Celular Tumoral , Dactinomicina/farmacologia , MicroRNAs/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Células PC12 , Feocromocitoma/tratamento farmacológico , Feocromocitoma/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Int J Mol Sci ; 21(24)2020 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-33322202

RESUMO

Alzheimer's disease (AD) is a neurodegenerative disease characterized by neurological dysfunction, including memory impairment, attributed to the accumulation of amyloid ß (Aß) in the brain. Although several studies reported possible mechanisms involved in Aß pathology, much remains unknown. Previous findings suggested that a protein regulated in development and DNA damage response 1 (REDD1), a stress-coping regulator, is an Aß-responsive gene involved in Aß cytotoxicity. However, we still do not know how Aß increases the level of REDD1 and whether REDD1 mediates Aß-induced synaptic dysfunction. To elucidate this, we examined the effect of Aß on REDD1-expression using acute hippocampal slices from mice, and the effect of REDD1 short hairpin RNA (shRNA) on Aß-induced synaptic dysfunction. Lastly, we observed the effect of REDD1 shRNA on memory deficit in an AD-like mouse model. Through the experiments, we found that Aß-incubated acute hippocampal slices showed increased REDD1 levels. Moreover, Aß injection into the lateral ventricle increased REDD1 levels in the hippocampus. Anisomycin, but not actinomycin D, blocked Aß-induced increase in REDD1 levels in the acute hippocampal slices, suggesting that Aß may increase REDD1 translation rather than transcription. Aß activated Fyn/ERK/S6 cascade, and inhibitors for Fyn/ERK/S6 or mGluR5 blocked Aß-induced REDD1 upregulation. REDD1 inducer, a transcriptional activator, and Aß blocked synaptic plasticity in the acute hippocampal slices. REDD1 inducer inhibited mTOR/Akt signaling. REDD1 shRNA blocked Aß-induced synaptic deficits. REDD1 shRNA also blocked Aß-induced memory deficits in passive-avoidance and object-recognition tests. Collectively, these results demonstrate that REDD1 participates in Aß pathology and could be a target for AD therapy.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/farmacologia , Hipocampo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Transtornos da Memória/metabolismo , Sinapses/metabolismo , Fatores de Transcrição/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anisomicina/farmacologia , Dactinomicina/farmacologia , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Transtornos da Memória/genética , Transtornos da Memória/patologia , Testes de Memória e Aprendizagem , Camundongos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , RNA Interferente Pequeno , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/genética , Sinapses/patologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismo , Fatores de Transcrição/genética , Regulação para Cima
10.
Cells ; 9(11)2020 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153169

RESUMO

Ribosomal RNA (rRNA), the most abundant non-coding RNA species, is a major component of the ribosome. Impaired ribosome biogenesis causes the dysfunction of protein synthesis and diseases called "ribosomopathies," including genetic disorders with cancer risk. However, the potential role of rRNA gene (rDNA) alterations in cancer is unknown. We investigated germline and somatic single-nucleotide variants (SNVs) in the rDNA promoter region (positions -248 to +100, relative to the transcription start site) in 82 lung adenocarcinomas (LUAC). Twenty-nine tumors (35.4%) carried germline SNVs, and eight tumors (9.8%) harbored somatic SNVs. Interestingly, the presence of germline SNVs between positions +1 and +100 (n = 12; 14.6%) was associated with significantly shorter recurrence-free survival (RFS) and overall survival (OS) by univariate analysis (p < 0.05, respectively), and was an independent prognostic factor for RFS and OS by multivariate analysis. LUAC cell line PC9, carrying rDNA promoter SNV at position +49, showed significantly higher ribosome biogenesis than H1650 cells without SNV. Upon nucleolar stress induced by actinomycin D, PC9 retained significantly higher ribosome biogenesis than H1650. These results highlight the possible functional role of SNVs at specific sites of the rDNA promoter region in ribosome biogenesis, the progression of LUAC, and their potential prognostic value.


Assuntos
Adenocarcinoma de Pulmão/genética , Grupo com Ancestrais do Continente Asiático/genética , Mutação em Linhagem Germinativa/genética , Neoplasias Pulmonares/genética , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , RNA Ribossômico/genética , Idoso , Sequência de Bases , Linhagem Celular Tumoral , Dactinomicina/farmacologia , Bases de Dados Genéticas , Feminino , Loci Gênicos , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Recidiva Local de Neoplasia/patologia , Modelos de Riscos Proporcionais , Reprodutibilidade dos Testes , Análise de Sobrevida
11.
Arch Insect Biochem Physiol ; 105(3): e21741, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-33002240

RESUMO

Apoptosis is a process of programmed cell death that is regulated by genes independently. The Bm30kc6 gene is a kind of small molecular lipoprotein about 30 kDa, expressed highly in the late stage of the silkworm hemolymph. Our study showed that overexpression of Bm30kc6 could decrease caspase-3 activation. Meanwhile, activation of caspase-3 increased when Bm30kc6 expression was disturbed by small interfering RNA (siRNA). Cell apoptosis was decreased when Bm30kc6 was overexpressed under UV treatment. The apoptosis rate induced by actinomycin D is similar to the trend by UV. It was inferred that Bm30kc6 has an inhibitory effect on the apoptosis of silkworm cells. The apoptosis-related genes, such as BmFadd, BmDredd, and BmDaxx were increased after overexpression of Bm30kc6 or decreased after interference of siRNA. It was speculated that there was an interactive relationship between Bm30kc6, BmDaxx, BmFadd, and BmDredd in the apoptosis signaling pathways. We investigated the transcription expression of the Bm30kc6 gene in different growth stages and tissues of the silkworm. The results showed that Bm30kc6 reached its peak in the hemolymph during the 6th to 7th days of the 5th instar, or in spinning post 24 h of the silk gland. In the silkworm BmN cells treated with caspase-3/7 inhibitor, the caspase-3 enzyme activity, and the expression levels of Bm30kc6, BmFadd, BmDredd, and BmDaxx were significantly reduced. The expression levels of Bm30kc6 increased sharply when silkworms were treated by molting hormone at Day 3 or 5 of the 5th instar. The results indicated that the expression of the Bm30kc6 gene was affected by the molting hormone and was likely to be its downstream target. In conclusion, the results suggest that the Bm30kc6 gene is involved in the regulation of the apoptotic signaling pathway and plays a role in the apoptotic process.


Assuntos
Apoptose/genética , Bombyx/crescimento & desenvolvimento , Bombyx/genética , Animais , Bombyx/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Inibidores de Caspase/farmacologia , Dactinomicina/farmacologia , Ecdisona/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Hemolinfa/metabolismo , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , RNA Interferente Pequeno , Transdução de Sinais , Raios Ultravioleta
12.
Cancer Res ; 80(23): 5393-5407, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33046443

RESUMO

Medulloblastoma is among the most common malignant brain tumors in children. Recent studies have identified at least four subgroups of the disease that differ in terms of molecular characteristics and patient outcomes. Despite this heterogeneity, most patients with medulloblastoma receive similar therapies, including surgery, radiation, and intensive chemotherapy. Although these treatments prolong survival, many patients still die from the disease and survivors suffer severe long-term side effects from therapy. We hypothesize that each patient with medulloblastoma is sensitive to different therapies and that tailoring therapy based on the molecular and cellular characteristics of patients' tumors will improve outcomes. To test this, we assembled a panel of orthotopic patient-derived xenografts (PDX) and subjected them to DNA sequencing, gene expression profiling, and high-throughput drug screening. Analysis of DNA sequencing revealed that most medulloblastomas do not have actionable mutations that point to effective therapies. In contrast, gene expression and drug response data provided valuable information about potential therapies for every tumor. For example, drug screening demonstrated that actinomycin D, which is used for treatment of sarcoma but rarely for medulloblastoma, was active against PDXs representing Group 3 medulloblastoma, the most aggressive form of the disease. Functional analysis of tumor cells was successfully used in a clinical setting to identify more treatment options than sequencing alone. These studies suggest that it should be possible to move away from a one-size-fits-all approach and begin to treat each patient with therapies that are effective against their specific tumor. SIGNIFICANCE: These findings show that high-throughput drug screening identifies therapies for medulloblastoma that cannot be predicted by genomic or transcriptomic analysis.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Cerebelares/tratamento farmacológico , Meduloblastoma/tratamento farmacológico , Medicina de Precisão/métodos , Animais , Linhagem Celular Tumoral , Neoplasias Cerebelares/genética , Criança , Dactinomicina/farmacologia , Regulação Neoplásica da Expressão Gênica , Ensaios de Triagem em Larga Escala , Humanos , Masculino , Meduloblastoma/genética , Camundongos Endogâmicos NOD , Mutação , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Anal Chem ; 92(18): 12498-12508, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32790289

RESUMO

Single-cell DNA analysis technology has provided unprecedented insights into many physiological and pathological processes. In contrast, technologies that allow protein analysis in single cells have lagged behind. Herein, a method called single-cell Plasmonic ImmunoSandwich Assay (scPISA) that is capable of measuring signaling proteins and protein complexes in single living cells is described. scPISA is straightforward, comprising specific in-cell extraction and ultrasensitive plasmonic detection. It is applied to evaluate the efficacy and kinetics of cytotoxic drugs. It reveals that different drugs exhibit distinct proapoptotic properties at the single-cell level. A set of new parameters is thus proposed for comprehensive and quantitative evaluation of the efficacy of anticancer drugs. It discloses that metformin can dramatically enhance the overall anticancer efficacy when combined with actinomycin D, although it itself is significantly less effective. Furthermore, scPISA reveals that survivin interacts with cytochrome C and caspase-3 in a dynamic fashion in single cells during continuous drug treatment. As compared with conventional assays, scPISA exhibits several significant advantages, such as ultrahigh sensitivity, single-cell resolution, fast speed, and so on. Therefore, this approach may provide a powerful tool for wide, important applications from basic research to clinical applications, particularly precision medicine.


Assuntos
Antineoplásicos/farmacologia , Caspase 3/análise , Citocromos c/análise , Dactinomicina/farmacologia , Imunoensaio , Metformina/farmacologia , Análise de Célula Única , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocromos c/metabolismo , Dactinomicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cinética , Metformina/química , Tamanho da Partícula , Propriedades de Superfície
14.
Sci Rep ; 10(1): 13870, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807803

RESUMO

Although many advances have been achieved to treat aggressive tumours, cancer remains a leading cause of death and a public health problem worldwide. Among the main approaches for the discovery of new bioactive agents, the prospect of microbial secondary metabolites represents an effective source for the development of drug leads. In this study, we investigated the actinobacterial diversity associated with an endemic Antarctic species, Deschampsia antarctica, by integrated culture-dependent and culture-independent methods and acknowledged this niche as a reservoir of bioactive strains for the production of antitumour compounds. The 16S rRNA-based analysis showed the predominance of the Actinomycetales order, a well-known group of bioactive metabolite producers belonging to the Actinobacteria phylum. Cultivation techniques were applied, and 72 psychrotolerant Actinobacteria strains belonging to the genera Actinoplanes, Arthrobacter, Kribbella, Mycobacterium, Nocardia, Pilimelia, Pseudarthrobacter, Rhodococcus, Streptacidiphilus, Streptomyces and Tsukamurella were identified. The secondary metabolites were screened, and 17 isolates were identified as promising antitumour compound producers. However, the bio-guided assay showed a pronounced antiproliferative activity for the crude extracts of Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653. The TGI and LC50 values revealed the potential of these natural products to control the proliferation of breast (MCF-7), glioblastoma (U251), lung/non-small (NCI-H460) and kidney (786-0) human cancer cell lines. Cinerubin B and actinomycin V were the predominant compounds identified in Streptomyces sp. CMAA 1527 and Streptomyces sp. CMAA 1653, respectively. Our results suggest that the rhizosphere of D. antarctica represents a prominent reservoir of bioactive actinobacteria strains and reveals it as an important environment for potential antitumour agents.


Assuntos
Actinobacteria , Técnicas de Cultura/métodos , Descoberta de Drogas , Neoplasias/patologia , Actinobacteria/metabolismo , Actinomycetales/metabolismo , Regiões Antárticas , Antraciclinas/isolamento & purificação , Antraciclinas/metabolismo , Antraciclinas/farmacologia , Antineoplásicos/isolamento & purificação , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Fatores Biológicos/biossíntese , Fatores Biológicos/isolamento & purificação , Fatores Biológicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dactinomicina/biossíntese , Dactinomicina/isolamento & purificação , Dactinomicina/farmacologia , Humanos , Streptomyces/metabolismo
15.
Biosci Biotechnol Biochem ; 84(10): 1975-1985, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32594848

RESUMO

Pyoverdines, a group of peptide siderophores produced by Pseudomonas species, function not only in iron acquisition, but also in their virulence in hosts. Thus, chemical inhibition of pyoverdine production may be an effective strategy to control Pseudomonas virulence. In the plant pathogen Pseudomonas cichorii SPC9018 (SPC9018), pyoverdine production is required for virulence on eggplant. We screened microbial culture extracts in a pyoverdine-production inhibition assay of SPC9018 and found Streptomyces sp. RM-32 as a candidate-producer. We isolated two active compounds from RM-32 cultures, and elucidated their structures to be actinomycins X2 and D. Actinomycins X2 and D inhibited pyoverdine production by SPC9018 with IC50 values of 17.6 and 29.6 µM, respectively. Furthermore, pyoverdine production in other Pseudomonas bacteria, such as the mushroom pathogen P. tolaasii, was inhibited by the actinomycins. Therefore, these actinomycins may be useful as chemical tools to examine pyoverdine functions and as seed compounds for anti-Pseudomonas virulence agents.


Assuntos
Dactinomicina/farmacologia , Oligopeptídeos/biossíntese , Pseudomonas/efeitos dos fármacos , Pseudomonas/metabolismo , Sideróforos/biossíntese , Pseudomonas/patogenicidade , Virulência
16.
Int J Mol Sci ; 21(11)2020 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-32471032

RESUMO

Tumor-infiltrating CD8+ T cells (TIL) are of the utmost importance in anti-tumor immunity. CD103 defines tumor-resident memory T cells (TRM cells) associated with improved survival and response to immune checkpoint blockade (ICB) across human tumors. Co-expression of CD39 and CD103 marks tumor-specific TRM with enhanced cytolytic potential, suggesting that CD39+CD103+ TRM could be a suitable biomarker for immunotherapy. However, little is known about the transcriptional activity of TRM cells in situ. We analyzed CD39+CD103+ TRM cells sorted from human high-grade endometrial cancers (n = 3) using mRNA sequencing. Cells remained untreated or were incubated with PMA/ionomycin (activation), actinomycin D (a platinum-like chemotherapeutic that inhibits transcription), or a combination of the two. Resting CD39+CD103+ TRM cells were transcriptionally active and expressed a characteristic TRM signature. Activated CD39+CD103+ TRM cells differentially expressed PLEK, TWNK, and FOS, and cytokine genes IFNG, TNF, IL2, CSF2 (GM-CSF), and IL21. Findings were confirmed using qPCR and cytokine production was validated by flow cytometry of cytotoxic TIL. We studied transcript stability and found that PMA-responsive genes and mitochondrial genes were particularly stable. In conclusion, CD39+CD103+ TRM cells are transcriptionally active TRM cells with a polyfunctional, reactivation-responsive repertoire. Secondly, we hypothesize that differential regulation of transcript stability potentiates rapid responses upon TRM reactivation in tumors.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias do Endométrio/imunologia , Neoplasias do Endométrio/patologia , Antígenos CD/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Dactinomicina/farmacologia , Neoplasias do Endométrio/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genótipo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Interleucinas/metabolismo , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Gradação de Tumores , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Genética/efeitos dos fármacos
17.
Int J Mol Sci ; 21(9)2020 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-32366058

RESUMO

In this study we explored the efficacy of combining low dose photodynamic therapy using a porphyrin photosensitiser and dactinomycin, a commonly used chemotherapeutic agent. The studies were carried out on compressed collagen 3D constructs of two human ovarian cancer cell lines (SKOV3 and HEY) versus their monolayer counterparts. An amphiphilc photosensitiser was employed, disulfonated tetraphenylporphine, which is not a substrate for ABC efflux transporters that can mediate drug resistance. The combination treatment was shown to be effective in both monolayer and 3D constructs of both cell lines, causing a significant and synergistic reduction in cell viability. Compared to dactinomycin alone or PDT alone, higher cell kill was found using 2D monolayer culture vs. 3D culture for the same doses. In 3D culture, the combination therapy resulted in 10 and 22 times higher cell kill in SKOV3 and HEY cells at the highest light dose compared to dactinomycin monotherapy, and 2.2 and 5.5 times higher cell kill than PDT alone. The combination of low dose PDT and dactinomycin appears to be a promising way to repurpose dactinomycin and widen its therapeutic applications.


Assuntos
Antineoplásicos/farmacologia , Dactinomicina/farmacologia , Neoplasias Ovarianas/metabolismo , Fotoquimioterapia/métodos , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos
18.
Eur J Haematol ; 105(3): 302-307, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32452083

RESUMO

OBJECTIVES: Complete responses have been observed in NPM1-mutated AML patients with dactinomycin, a nucleolar stress-inducing drug. Here, we report a single-center experience of compassionate use of dactinomycin in untreated or relapsed/ refractory NPM1-mutated AML. METHODS: From September 2015 to February 2019, 26 adult patients with NPM1-mutated AML received dactinomycin in different situations: front-line treatment in 4 unfit patients (16%); morphologic (n = 16, 62%), molecular relapses (n = 4, 16%), refractory disease (n = 1, 13%), or postremission therapy in second complete response (n = 1, 13%). RESULTS: Median age was 62.5 years. Median number of dactinomycin cycle was 1 (1-8), and 7 patients (27%) received more than 3 cycles. Three out of 17 patients (18%) in morphologic relapse or refractory to chemotherapy achieved complete remission after the first cycle of dactinomycin. None of the 4 patients unfit for intensive chemotherapy responded to dactinomycin as front-line therapy. Grade 3-4 adverse events were thrombocytopenia (n = 11, 42%), neutropenia (n = 11, 42%), GI toxicity (n = 6, 23%), mucositis (n = 5, 19%), lung infection (n = 5, 19%), and skin rash (n = 2, 7.6%). CONCLUSIONS: Dactinomycin is an inexpensive and easily available drug that may induce significant responses in few AML patients with NPM1 mutations with an acceptable safety profile.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Dactinomicina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Mutação , Proteínas Nucleares/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibióticos Antineoplásicos/farmacologia , Biomarcadores Tumorais , Dactinomicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Leucemia Mieloide Aguda/diagnóstico , Leucemia Mieloide Aguda/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Recidiva , Indução de Remissão , Retratamento , Estudos Retrospectivos , Resultado do Tratamento , Adulto Jovem
19.
Neuro Oncol ; 22(9): 1289-1301, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32227096

RESUMO

BACKGROUND: Glioblastoma (GBM) has been extensively researched over the last few decades, yet despite aggressive multimodal treatment, recurrence is inevitable and second-line treatment options are limited. Here, we demonstrate how high-throughput screening (HTS) in multicellular spheroids can generate physiologically relevant patient chemosensitivity data using patient-derived cells in a rapid and cost-effective manner. Our HTS system identified actinomycin D (ACTD) to be highly cytotoxic over a panel of 12 patient-derived glioma stemlike cell (GSC) lines. ACTD is an antineoplastic antibiotic used in the treatment of childhood cancers. Here, we validate ACTD as a potential repurposed therapeutic for GBM in 3-dimensional GSC cultures and patient-derived xenograft models of recurrent glioblastoma. METHODS: Twelve patient-derived GSC lines were screened at 10 µM, as multicellular spheroids, in a 384-well serum-free assay with 133 FDA-approved compounds. GSCs were then treated in vitro with ACTD at established half-maximal inhibitory concentrations (IC50). Downregulation of sex determining region Y-box 2 (Sox2), a stem cell transcription factor, was investigated via western blot and through immunohistological assessment of murine brain tissue. RESULTS: Treatment with ACTD was shown to significantly reduce tumor growth in 2 recurrent GBM patient-derived models and significantly increased survival. ACTD is also shown to specifically downregulate the expression of Sox2 both in vitro and in vivo. CONCLUSION: These findings indicate that, as predicted by our HTS, ACTD could deplete the cancer stem cell population within the tumor mass, ultimately leading to a delay in tumor progression. KEY POINTS: 1. High-throughput chemosensitivity data demonstrated the broad efficacy of actinomycin D, which was validated in 3 preclinical models of glioblastoma.2. Actinomycin D downregulated Sox2 in vitro and in vivo, indicating that this agent could target the stem cell population of GBM tumors.


Assuntos
Neoplasias Encefálicas , Glioblastoma , Glioma , Animais , Neoplasias Encefálicas/tratamento farmacológico , Linhagem Celular Tumoral , Criança , Dactinomicina/farmacologia , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Células-Tronco Neoplásicas , Fatores de Transcrição SOXB1/genética
20.
Mol Cell Endocrinol ; 510: 110801, 2020 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-32278021

RESUMO

Androgens in asthmatic men may be linked to asthma severity, acting via nongenomic and genomic effects. This ailment affects boys more than girls during infancy, and this proportion reverses in puberty. Plasmatic androgen concentration in young men increases at this age and might be related to lower asthma symptoms. Nongenomic actions occur in a brief period and are independent of the androgen receptor (AR), while genomic effects depend on AR, take hours-days and are modified by transcription or protein synthesis inhibitors. Guinea pig tracheas chronic incubation with testosterone (TES, 40 nM, 48 h) potentiates salbutamol-induced relaxation, an effect that was reversed by flutamide, not observed when tissues were pre-incubated with TES-bovine serum albumin (TES-BSA) nor when tissues were preincubated with TES for 15-60 min. In tracheal myocytes, TES chronic incubation increases salbutamol-induced K+ currents (IK+), an effect that was also reversed by flutamide, actinomycin D and cycloheximide and not seen with TES-BSA. The increment in IK+ was blocked by 4-aminopyridine and iberiotoxin, indicating that delayed rectifier K+ and high-conductance Ca2+ activated K+ channels were involved in the TES potentiation effect. Immunofluorescence studies showed that chronic TES augmented the ß2 adrenergic receptor (ß2-AR) expression in ASM and this finding was corroborated by q-PCR and Western blot assays. ß2-AR affinity for salbutamol after TES incubation was increased. In conclusion, chronic exposure to physiological TES concentration of the guinea pig ASM promotes ß2-AR upregulation favoring ß2 adrenergic responses and probably limiting the severity of the asthmatic exacerbations in teenage boys and men.


Assuntos
Albuterol/farmacologia , Genoma , Pulmão/fisiologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/fisiologia , Receptores Adrenérgicos beta 2/genética , Testosterona/farmacologia , Transcrição Genética/efeitos dos fármacos , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Cobaias , Pulmão/efeitos dos fármacos , Masculino , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso/efeitos dos fármacos , Canais de Potássio/metabolismo , Propanolaminas/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/fisiologia , Regulação para Cima/efeitos dos fármacos
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