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1.
Mol Pharmacol ; 99(3): 226-241, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33446509

RESUMO

An essential function of DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is to resolve DNA topologic entanglements during chromosome disjunction by introducing transient DNA double-stranded breaks. TOP2α/170 is an important target for DNA damage-stabilizing anticancer drugs, whose clinical efficacy is compromised by drug resistance often associated with decreased TOP2α/170 expression. We recently demonstrated that an etoposide-resistant K562 clonal subline, K/VP.5, with reduced levels of TOP2α/170, expresses high levels of a novel C-terminal truncated TOP2α isoform (90 kDa, TOP2α/90). TOP2α/90, the translation product of a TOP2α mRNA that retains a processed intron 19 (I19), heterodimerizes with TOP2α/170 and is a resistance determinant through a dominant-negative effect on drug activity. We hypothesized that genome editing to enhance I19 removal would provide a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. To enhance I19 removal in K/VP.5 cells, CRISPR/Cas9 was used to make changes (GAG//GTAA AC →GAG//GTAA GT ) in the TOP2α gene's suboptimal exon 19/intron 19 5' splice site (E19/I19 5' SS). Gene-edited clones were identified by quantitative polymerase chain reaction and verified by sequencing. Characterization of a clone with all TOP2α alleles edited revealed improved I19 removal, decreased TOP2α/90 mRNA/protein, and increased TOP2α/170 mRNA/protein. Sensitivity to etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition was restored to levels comparable to those in parental K562 cells. Together, the results indicate that our gene-editing strategy for optimizing the TOP2α E19/I19 5' SS in K/VP.5 cells circumvents resistance to etoposide and other TOP2α-targeted drugs. SIGNIFICANCE STATEMENT: Results presented here indicate that CRISPR/Cas9 gene editing of a suboptimal exon 19/intron 19 5' splice site in the DNA topoisomerase IIα (TOP2α) gene results in circumvention of acquired drug resistance to etoposide and other TOP2α-targeted drugs in a clonal K562 cell line by enhancing removal of intron 19 and thereby decreasing formation of a truncated TOP2α 90 kDa isoform and increasing expression of full-length TOP2α 170 kDa in these resistant cells. Results demonstrate the importance of RNA processing in acquired drug resistance to TOP2α-targeted drugs.


Assuntos
DNA Topoisomerases Tipo II/genética , Regulação para Baixo , Etoposídeo/farmacologia , Edição de Genes/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Sistemas CRISPR-Cas , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Humanos , Íntrons , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/enzimologia , Sítios de Splice de RNA
2.
Int J Nanomedicine ; 15: 7601-7613, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116490

RESUMO

Introduction: Etoposide refers to a derivative of podophyllotoxin, which plays an important role in the treatment of cancer due to its prominent anti-tumor effect. As a BCS IV drug, etoposide exhibits insufficient aqueous solubility and permeability, thereby limiting its oral absorption. To enhance the oral bioavailability of etoposide, this study developed an amorphous nanopowder. Methods: Based on preliminary screening and experimental design, the stabilizer and preparation process of etoposide nanosuspension were explored. Subsequently, using a Box-Behnken design, the effects of independent factors (ultrasonication time, ratio of two phases and stabilizer concentration) on response variables (particle size and polydispersity index) were studied, and then the formulation was optimized. Finally, nanosuspension was further freeze dried with 1% of mannitol resulting in the formation of etoposide amorphous nanopowder. Results: The optimized etoposide nanopowder showed as spherical particles with an average particle size and polydispersity index of 211.7 ± 10.4 nm and 0.125 ± 0.028. X-ray powder diffraction and differential scanning calorimetry confirmed the ETO in the nanopowder was amorphous. Compared with coarse powder and physical mixture, etoposide nanopowder achieved significantly enhanced saturated solubility and dissolution in various pH environments. The Cmax and AUC0-t of etoposide nanopowder after oral administration in rats were respectively 2.21 and 2.13 times higher than the crude etoposide suspension. Additionally, the Tmax value of nanopowder was 0.25 h, compared with 0.5 h of reference group. Discussion: In the present study, the optimized amorphous nanopowder could significantly facilitate the dissolution and oral absorption of etoposide and might act as an effective delivery method to enhance its oral bioavailability.


Assuntos
Composição de Medicamentos , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Nanopartículas/química , Administração Oral , Análise de Variância , Animais , Disponibilidade Biológica , Varredura Diferencial de Calorimetria , Cristalização , Etoposídeo/química , Etoposídeo/farmacocinética , Liofilização , Masculino , Modelos Estatísticos , Tamanho da Partícula , Permeabilidade , Pós , Ratos Sprague-Dawley , Solubilidade , Solventes , Suspensões , Difração de Raios X
3.
Mutat Res ; 856-857: 503232, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32928372

RESUMO

miR-34a has been identified as a tumor suppressor microRNA (miRNA) involved in the P53 network. Its expression levels correlate to carcinogenesis, which are generally lower in tumor tissue and higher in response to DNA damage. In this study, the response of miR-34a from exposure to genotoxic agents in human lymphoblastoid TK6 cells was evaluated to assess whether the expression of this miRNA could be used as an early indicator for genotoxic damage in mammalian cells. TK6 cells were treated with seven genotoxic agents with different mode-of-actions (cisplatin, N-ethyl-N-nitrosourea, etoposide, mitomycin C, methyl methanesulphonate, taxol, and X-ray radiation) and a non-genetic toxin (usnic acid) at different concentrations for four hours (except for X-rays) and the expression levels of miR-34a were measured 24 h after the beginning of the treatments. The expression levels of miR-34a were significantly increased by these genetic toxins in a dose-dependent manner, while no significant change in miRNA expression was found in the usnic acid-treated cells. These results suggest that miR-34a can respond to genotoxic insults sensitively; thus, miR-34a expression has the potential to be used to evaluate genotoxicity of agents.


Assuntos
Carcinogênese/genética , Dano ao DNA/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , MicroRNAs/genética , Apoptose/efeitos dos fármacos , Biomarcadores/metabolismo , Carcinogênese/efeitos dos fármacos , Cisplatino/farmacologia , Cisplatino/toxicidade , Etilnitrosoureia/farmacologia , Etoposídeo/farmacologia , Etoposídeo/toxicidade , Genes Supressores de Tumor , Humanos , Mutagênicos/farmacologia , Mutagênicos/toxicidade
4.
Int J Nanomedicine ; 15: 4105-4123, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606667

RESUMO

Background: Magnetic nanoparticles (MNPs) hold promise for enhancing delivery of therapeutic agents, either through direct binding or by functioning as miniature propellers. Fluid-filled conduits and reservoirs within the body offer avenues for MNP-enhanced drug delivery. MNP clusters can be rotated and moved across surfaces at clinically relevant distances in response to a rotating magnet. Limited data are available regarding issues affecting MNP delivery by this mechanism, such as adhesion to a cellular wall. Research reported here was initiated to better understand the fundamental principles important for successful implementation of rotational magnetic drug targeting (rMDT). Methods: Translational movements of four different iron oxide MNPs were tested, in response to rotation (3 Hz) of a neodymium-boron-iron permanent magnet. MNP clusters moved along biomimetic channels of a custom-made acrylic tray, by surface walking. The effects of different distances and cellular coatings on MNP velocity were analyzed using videography. Dyes (as drug surrogates) and the drug etoposide were transported by rotating MNPs along channels over a 10 cm distance. Results: MNP translational velocities could be predicted from magnetic separation times. Changes in distance or orientation from the magnet produced alterations in MNP velocities. Mean velocities of the fastest MNPs over HeLa, U251, U87, and E297 cells were 0.24 ± 0.02, 0.26 ± 0.02, 0.28 ± 0.01, and 0.18 ± 0.03 cm/sec, respectively. U138 cells showed marked MNP adherence and an 87.1% velocity reduction at 5.5 cm along the channel. Dye delivery helped visualize the effects of MNPs as microdevices for drug delivery. Dye delivery by MNP clusters was 21.7 times faster than by diffusion. MNPs successfully accelerated etoposide delivery, with retention of chemotherapeutic effect. Conclusion: The in vitro system described here facilitates side-by-side comparisons of drug delivery by rotating MNP clusters, on a human scale. Such microdevices have the potential for augmenting drug delivery in a variety of clinical settings, as proposed.


Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Nanopartículas de Magnetita/química , Microtecnologia/instrumentação , Rotação , Transporte Biológico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Difusão , Etoposídeo/farmacologia , Humanos , Microesferas , Tamanho da Partícula , Tomografia Computadorizada por Raios X
5.
Oncogene ; 39(27): 5068-5081, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32528131

RESUMO

Topoisomerase II poisons are one of the most common class of chemotherapeutics used in cancer. We and others had shown that a subset of glioblastomas, the most malignant of all primary brain tumors in adults, is responsive to TOP2 poisons. To identify genes that confer susceptibility to this drug in gliomas, we performed a genome-scale CRISPR knockout screen with etoposide. Genes involved in protein synthesis and DNA damage were implicated in etoposide susceptibility. To define potential biomarkers for TOP2 poisons, CRISPR hits were overlapped with genes whose expression correlates with susceptibility to this drug across glioma cell lines, revealing ribosomal protein subunit RPS11, 16, and 18 as putative biomarkers for response to TOP2 poisons. Loss of RPS11 led to resistance to etoposide and doxorubicin and impaired the induction of proapoptotic gene APAF1 following treatment. The expression of these ribosomal subunits was also associated with susceptibility to TOP2 poisons across cell lines from gliomas and multiple other cancers.


Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Etoposídeo/farmacologia , Glioblastoma/tratamento farmacológico , Proteínas Ribossômicas/metabolismo , Inibidores da Topoisomerase II/farmacologia , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Neoplasias Encefálicas/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , DNA Topoisomerases Tipo II/metabolismo , Doxorrubicina/farmacologia , Técnicas de Inativação de Genes , Glioblastoma/genética , Humanos
6.
Nucleic Acids Res ; 48(12): 6654-6671, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32501506

RESUMO

DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.


Assuntos
Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Topoisomerases Tipo II/ultraestrutura , Conformação de Ácido Nucleico/efeitos dos fármacos , Sítios de Ligação/genética , Fator de Ligação a CCCTC/genética , DNA/genética , DNA/ultraestrutura , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Etoposídeo/farmacologia , Genoma Humano/genética , Instabilidade Genômica/genética , Humanos , Sítio de Iniciação de Transcrição/efeitos dos fármacos
7.
Am J Hematol ; 95(8): 937-943, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32311140

RESUMO

Relapsed or refractory acute myeloid leukemia (R/R AML) has a poor prognosis and is best treated with salvage chemotherapy as a bridge to allogeneic stem cell transplant (alloSCT). However, the optimal salvage therapy remains unknown. Here we compared two salvage regimens; mitoxantrone, etoposide, and cytarabine (MEC) and mitoxantrone and high-dose Ara-C (Ara-C couplets). We analyzed 155 patients treated at three academic institutions between 1998 and 2017; 87 patients received MEC and 68 received Ara-C couplets. The primary endpoint was overall response (OR). Secondary endpoints included progression-free survival (PFS), overall survival (OS), duration of hospitalization, hematologic and nonhematologic toxicities, and success in proceeding to alloSCT. Baseline characteristics of the cohorts were well matched, though patients receiving Ara-C couplets had more co-morbidities (48.5% vs 33%; P = .07). OR was achieved in 43.7% of MEC and 54.4% of Ara-C couplets patients (P = .10). Ara-C couplets patients also trended towards a longer OS and PFS, more frequently proceeded to alloSCT (31% vs 54.4%; P = .003), and experienced less febrile neutropenia (94% vs 72%; P < .001) and grade 3/4 gastrointestinal toxicities (17.2% vs 2.94%; P = .005). No significant differences in other toxicities or median duration of hospitalization were noted. This is the first multi-institutional study directly comparing these regimens in a racially diverse population of R/R AML patients. Although these regimens have equivalent efficacy in terms of achieving OR, Ara-C couplets use is associated with significant reductions in toxicities, suggesting it should be used more frequently in these patients.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citarabina/envenenamento , Citarabina/uso terapêutico , Etoposídeo/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Mitoxantrona/uso terapêutico , Adulto , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Citarabina/farmacologia , Etoposídeo/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mitoxantrona/farmacologia , Recidiva , Adulto Jovem
8.
Nat Commun ; 11(1): 1754, 2020 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-32273498

RESUMO

Alternative autophagy is an autophagy-related protein 5 (Atg5)-independent type of macroautophagy. Unc51-like kinase 1 (Ulk1) is an essential initiator not only for Atg5-dependent canonical autophagy but also for alternative autophagy. However, the mechanism as to how Ulk1 differentially regulates both types of autophagy has remained unclear. In this study, we identify a phosphorylation site of Ulk1 at Ser746, which is phosphorylated during genotoxic stress-induced alternative autophagy. Phospho-Ulk1746 localizes exclusively on the Golgi and is required for alternative autophagy, but not canonical autophagy. We also identify receptor-interacting protein kinase 3 (RIPK3) as the kinase responsible for genotoxic stress-induced Ulk1746 phosphorylation, because RIPK3 interacts with and phosphorylates Ulk1 at Ser746, and loss of RIPK3 abolishes Ulk1746 phosphorylation. These findings indicate that RIPK3-dependent Ulk1746 phosphorylation on the Golgi plays a pivotal role in genotoxic stress-induced alternative autophagy.


Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/fisiologia , Dano ao DNA , Complexo de Golgi/metabolismo , Serina/metabolismo , Sequência de Aminoácidos , Animais , Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Sítios de Ligação/genética , Células Cultivadas , Embrião de Mamíferos/citologia , Etoposídeo/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos Knockout , Microscopia Confocal , Fosforilação , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Homologia de Sequência de Aminoácidos , Serina/genética
9.
Anticancer Res ; 40(3): 1619-1624, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32132065

RESUMO

BACKGROUND: Prognosis of metastatic malignant peripheral nerve sheath tumor (MPNST) is poor and the role of chemotherapy is controversial. There has been no report of metastatic MPNST with a good prognosis without surgery for metastases. CASE REPORT: A 40-year-old man with neurofibromatosis type 1 (NF1)-related MPNST on his shoulder with multiple lung metastases visited our hospital. After two cycles of chemotherapy with ifosfamide, carboplatin and etoposide (ICE), the primary lesion and lung metastases had shrunk. The primary lesion was resected with negative margins. Subsequently, 'gradual subtraction' ICE was administered, wherein the dose was reduced and the treatment interval was increased. After 14 courses of ICE over a period of 2 years, the lung metastases disappeared; there has been no recurrence for over 12 years. CONCLUSION: ICE can be an excellent, inexpensive treatment for NF1-related MPNST. 'Gradual subtraction' chemotherapy allowed us to maintain long-term efficacy, induce tumor dormancy, and reduce side-effects.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Etoposídeo/uso terapêutico , Ifosfamida/uso terapêutico , Neoplasias da Bainha Neural/tratamento farmacológico , Neurofibromatose 1/patologia , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carboplatina/farmacologia , Etoposídeo/farmacologia , Humanos , Ifosfamida/farmacologia , Masculino , Metástase Neoplásica , Neoplasias da Bainha Neural/patologia , Prognóstico
10.
Eur J Pharm Biopharm ; 149: 218-228, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32112893

RESUMO

Multidrug resistance (MDR) and the spread of cancer cells (metastasis) are major causes leading to failure of cancer treatment. MDR can develop in two main ways, with differences in their mechanisms for drug resistance, first drug-selected MDR developing after chemotherapeutic treatment, and metastasis-associated MDR acquired by cellular adaptation to microenvironmental changes during metastasis. This study aims to use a nanoparticle-mediated photodynamic therapy (NPs/PDT) approach to overcome both types of MDR. A photosensitizer, 5,10,15,20-Tetrakis(4-hydroxy-phenyl)-21H,23H-porphine (pTHPP) was loaded into poly(D,L-lactide-co-glycolide) (PLGA)-lipid hybrid nanoparticles. The photocytotoxic effect of the nanoparticles was evaluated using two different MDR models established from one cell line, A549 human lung adenocarcinoma, including (1) A549RT-eto, a MDR cell line derived from A549 cells by drug-selection, and (2) detachment-induced MDR acquired by A549 cells when cultured as floating cells under non-adherent conditions, which mimic metastasizing cancer cells in the blood/lymphatic circulation. In the drug-selected MDR model, A549RT-eto cells displayed 17.4- and 1.8-fold resistance to Etoposide and Paclitaxel, respectively, compared to the A549 parental cells. In contrast to treatment with anticancer drugs, NPs/PDT with pTHPP-loaded nanoparticles resulted in equal photocytotoxic effect in A549RT-eto and parental cells. Intracellular pTHPP accumulation and light-induced superoxide anion generation were observed at similar levels in the two cell lines. The NPs/PDT killed A549RT-eto and parental cells through apoptosis as revealed by flow cytometry. In the metastasis-associated MDR model, A549 floating cells exhibited resistance to Etoposide (11.6-fold) and Paclitaxel (57.8-fold) compared to A549 attached cells, but the floating cells failed to show resistance against the photocytotoxic effect of the NPs/PDT. The MDR overcoming activity of NPs/PDT is mainly due to delivery ability of the PLGA-lipid hybrid nanoparticles. In conclusion, this work suggests that PLGA-lipid hybrid nanoparticles have potential in delivering photosensitizer or chemotherapeutic drug for treating both drug-selected and metastasis-associated MDR lung cancer cells.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Fotoquimioterapia/métodos , Células A549 , Adenocarcinoma de Pulmão/patologia , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/administração & dosagem , Etoposídeo/farmacologia , Humanos , Lipídeos/química , Neoplasias Pulmonares/patologia , Nanopartículas , Paclitaxel/administração & dosagem , Paclitaxel/farmacologia , Fármacos Fotossensibilizantes/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Porfirinas/administração & dosagem
11.
Cancer Sci ; 111(5): 1851-1855, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32216001

RESUMO

Gene rearrangements of MLL/KMT2A or RUNX1 are the major cause of therapy-related leukemia. Moreover, MLL rearrangements are the major cause of infant leukemia, and RUNX1 rearrangements are frequently detected in cord blood. These genes are sensitive to topoisomerase II inhibitors, and various genes have been identified as potential fusion partners. However, fetal exposure to these inhibitors is rare. Therefore, we postulated that even a proliferation signal itself might induce gene rearrangements in hematopoietic stem cells. To test this hypothesis, we detected gene rearrangements in etoposide-treated or non-treated CD34+ cells cultured with cytokines using inverse PCR. In the etoposide-treated cells, variable-sized rearrangement bands were detected in the RUNX1 and MLL genes at 3 hours of culture, which decreased after 7 days. However, more rearrangement bands were detected in the non-treated cells at 7 days of culture. Such gene rearrangements were also detected in peripheral blood stem cells mobilized by cytokines for transplantation. However, none of these rearranged genes encoded the leukemogenic oncogene, and the cells with rearrangements did not expand. These findings suggest that MLL and RUNX1 rearrangements, which occur with very low frequency in normal hematopoietic progenitor cells, may be induced under cytokine stimulation. Most of the cells with gene rearrangements are likely eliminated, except for leukemia-associated gene rearrangements, resulting in the low prevalence of leukemia development.


Assuntos
Subunidade alfa 2 de Fator de Ligação ao Core/genética , Citocinas/farmacologia , Rearranjo Gênico/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Idoso , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Etoposídeo/farmacologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfoma Difuso de Grandes Células B/patologia , Pessoa de Meia-Idade , Células-Tronco de Sangue Periférico/efeitos dos fármacos , Células-Tronco de Sangue Periférico/metabolismo , Inibidores da Topoisomerase II/farmacologia
12.
J Biosci ; 452020.
Artigo em Inglês | MEDLINE | ID: mdl-32098913

RESUMO

As a tumor suppressor, p53 preserves genomic integrity in eukaryotes. However, limited evidence is available for the p53 shuttling between the cytoplasm and nucleus. Previous studies have shown that ß-actin polymerization negatively regulates p53 nuclear import through its interaction with p53. In this study, we found that DNA damage induces both ß-actin and p53 accumulation in the nucleus. ß-actin knockdown impaired the nuclear transport of p53. Additionally, ß-actin could interact with p53 which was enhanced in response to genotoxic stress. Furthermore, N terminal deletion mutants of p53 shows reduced levels of association with ß-actin. We further identified Ser15, Thr18 and Ser20 of p53 are critical to the ß-actin: p53 interaction, which upon mutation into alanine abrogates the binding. Taken together, this study reveals that ß-actin regulates the nuclear import of p53 through protein-protein interaction.


Assuntos
Actinas/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antineoplásicos Fitogênicos/farmacologia , Etoposídeo/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Actinas/genética , Transporte Ativo do Núcleo Celular/fisiologia , Linhagem Celular Tumoral , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína Supressora de Tumor p53/genética
13.
Cell Rep ; 30(4): 1235-1245.e4, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31995761

RESUMO

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair.


Assuntos
Reagentes para Ligações Cruzadas/farmacologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Camptotecina/farmacologia , Reparo do DNA/genética , Etoposídeo/farmacologia , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Técnicas de Inativação de Genes , Técnicas Genéticas , Células HeLa , Humanos , Inibidores da Topoisomerase I/farmacologia
14.
Int J Biol Macromol ; 146: 497-507, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923489

RESUMO

Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a predominant DNA sensor inducing the activation of the innate immune responses that produce proinflammatory cytokines and type I interferons, which has been well-investigated in mammals. However, chicken cGAS (chcGAS), which participates in avian innate immunity, has not been well-investigated. Here, we cloned the complete open reading frame sequence of chcGAS. Multiple sequence alignment and phylogenetic analysis revealed that chcGAS was homologous to mammalian cGAS. The chcGAS mRNA was highly expressed in the bone marrow and ileum. The subcellular localization of chcGAS was mainly in the cytoplasm, and partial co-localization was observed in the endoplasmic reticulum. Through overexpression and RNA interference, we demonstrated that chcGAS responded to exogenous dsDNA, HS-DNA, and poly(dA:dT), and to self dsDNA from the DNA damage response, thereby triggering the activation of STING/TBK1/IRF7-mediated innate immunity in both chicken embryonic fibroblasts and chicken liver cancer cells. Furthermore, downregulation of chcGAS enhanced the infection of fowl adenovirus serotype 4 in LMH cells. Our results demonstrated that chcGAS was an important cytosolic DNA sensor activating innate immune responses and may shed light on a strategy for preventing infectious diseases in the poultry industry.


Assuntos
Adenoviridae/imunologia , Galinhas/imunologia , Galinhas/virologia , Citosol/metabolismo , DNA/metabolismo , Imunidade Inata , Nucleotídeos Cíclicos/metabolismo , Sorogrupo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dano ao DNA , Etoposídeo/farmacologia , Perfilação da Expressão Gênica , Fator Regulador 7 de Interferon/metabolismo , Interferon beta/metabolismo , Interleucina-1beta/metabolismo , Nucleotídeos Cíclicos/química , Filogenia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/metabolismo
15.
Mol Pharmacol ; 97(3): 159-170, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31836624

RESUMO

DNA topoisomerase IIα protein (TOP2α) 170 kDa (TOP2α/170) is an important target for anticancer agents whose efficacy is often attenuated by chemoresistance. Our laboratory has characterized acquired resistance to etoposide in human leukemia K562 cells. The clonal resistant subline K/VP.5 contains reduced TOP2α/170 mRNA and protein levels compared with parental K562 cells. The aim of this study was to determine whether microRNA (miRNA)-mediated mechanisms play a role in drug resistance via decreased expression of TOP2α/170. miRNA-sequencing revealed that human miR-9-3p and miR-9-5p were among the top six of those overexpressed in K/VP.5 compared with K562 cells; validation by quantitative polymerase chain reaction demonstrated overexpression of both miRNAs. miRNA recognition elements (MREs) for both miRNAs are present in the 3'-untranslated region (UTR) of TOP2α/170. Transfecting K562 cells with a reporter plasmid harboring the TOP2α/170 3'-UTR together with either miR-9-3p or miR-9-5p mimics resulted in a statistically significant decrease in luciferase expression. Mutating the miR-9-3p or miR-9-5p MREs prevented this decrease, demonstrating direct interaction between these miRNAs and TOP2α/170 mRNA. Transfection of K562 cells with miR-9-3p or miR-9-5p mimics led to decreased TOP2α/170 protein levels without a change in TOP2α/170 mRNA and resulted in attenuated etoposide-induced DNA damage (gain-of-miRNA-inhibitory function). Conversely, transfection of miR-9-3p or miR-9-5p inhibitors in K/VP.5 cells (overexpressed miR-9 and low TOP2α/170) led to increased TOP2α/170 protein expression without a change in TOP2α/170 mRNA levels and resulted in enhancement of etoposide-induced DNA damage (loss-of-miRNA-inhibitory function). Taken together, these results strongly suggest that these miRNAs play a role in and are potential targets for circumvention of acquired resistance to etoposide. SIGNIFICANCE STATEMENT: Results presented here indicate that miR-9-3p and miR-9-5p decrease DNA topoisomerase IIα protein 170 kDa expression levels in acquired resistance to etoposide. These findings contribute new information about and potential strategies for circumvention of drug resistance by modulation of microRNA levels. Furthermore, increased expression of miR-9-3p and miR-9-5p in chemoresistant cancer cells may support their validation as biomarkers of responsiveness to DNA topoisomerase II-targeted therapy.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , DNA Topoisomerases Tipo II/biossíntese , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Etoposídeo/farmacologia , MicroRNAs/biossíntese , DNA Topoisomerases Tipo II/genética , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/fisiologia , Humanos , Células K562 , MicroRNAs/genética , Transcrição Genética/efeitos dos fármacos , Transcrição Genética/fisiologia
16.
Pediatr Blood Cancer ; 67(4): e28133, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31876116

RESUMO

BACKGROUND: The aim of this study was to improve the predictive power of patient-derived xenografts (PDXs, also known as mouse avatars) to more accurately reflect outcomes of clofarabine-based treatment in pediatric acute lymphoblastic leukemia (ALL) patients. PROCEDURE: Pharmacokinetic (PK) studies were conducted using clofarabine at 3.5 to 15 mg/kg in mice. PDXs were established from relapsed/refractory ALL patients who exhibited good or poor responses to clofarabine. PDX engraftment and response to clofarabine (either as a single agent or in combinations) were assessed based on stringent objective response measures modeled after the clinical setting. RESULTS: In naïve immune-deficient NSG mice, we determined that a clofarabine dose of 3.5 mg/kg resulted in systemic exposures equivalent to those achieved in pediatric ALL patients treated with clofarabine-based regimens. This dose was markedly lower than the doses of clofarabine used in previously reported preclinical studies (typically 30-60 mg/kg) and, when scheduled consistent with the clinical regimen (daily × 5), resulted in 34-fold lower clofarabine exposures. Using a well-tolerated clofarabine/etoposide/cyclophosphamide combination regimen, we then found that the responses of PDXs better reflected the clinical responses of the patients from whom the PDXs were derived. CONCLUSIONS: This study has identified an in vivo clofarabine treatment regimen that reflects the clinical responses of relapsed/refractory pediatric ALL patients. This regimen could be used prospectively to identify patients who might benefit from clofarabine-based treatment. Our findings are an important step toward individualizing prospective patient selection for the use of clofarabine in relapsed/refractory pediatric ALL patients and highlight the need for detailed PK evaluation in murine PDX models.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Medicina de Precisão/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antimetabólitos Antineoplásicos/farmacologia , Clofarabina/farmacologia , Ciclofosfamida/farmacologia , Etoposídeo/farmacologia , Humanos , Camundongos
17.
DNA Cell Biol ; 39(1): 69-77, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31702387

RESUMO

Chemoresistance is one of the major obstacles for cancer therapy. Abnormal expression of long noncoding RNAs (lncRNAs) was broadly implicated in chemoresistance of multiple cancers. This study was aimed to investigate the function of urothelial cancer associated 1 (UCA1) in multidrug resistance of retinoblastoma and its potential molecular mechanism. In this study, we observed that UCA1 was significantly upregulated in chemoresistant retinoblastoma tissues and multidrug resistant retinoblastoma cell lines and predicted an unfavorable overall survival. Functionally, knockdown of UCA1 remarkably inhibited proliferation and sensitized retinoblastoma cells to multiple chemotherapy drugs, including vincristine (VCR), carboplatin (CBP), cisplatin (DDP), VP-16 (etoposide), and 5-fluorouracil (5-Fu). Mechanistic studies demonstrated that UCA1 functioned as a miRNA sponge to increase stathmin 1 (STMN1) expression through sponging miR-513a-5p. In addition, silence of miR-513a-5p or STMN1 overexpression could partly reverse UCA1 knockdown-induced inhibitory effects on proliferation and multidrug resistance of retinoblastoma cells. Overall, this study is the first to demonstrate that UCA1 plays a critical role in retinoblastoma chemoresistance, and UCA1 may serve as a potential diagnostic biomarker and therapeutic target of retinoblastoma.


Assuntos
Proliferação de Células/genética , Resistência a Múltiplos Medicamentos/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Retinoblastoma/genética , Antineoplásicos/farmacologia , Carboplatina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Resistência a Múltiplos Medicamentos/efeitos dos fármacos , Etoposídeo/farmacologia , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Retinoblastoma/tratamento farmacológico , Retinoblastoma/patologia , Vincristina/farmacologia
18.
Oncol Rep ; 43(1): 337-345, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746397

RESUMO

Ewing sarcomas (ES) are highly malignant mesenchymal tumors, which most often occur in children and adolescents. The current treatment of choice comprises wide resection in combination with multimodal chemotherapy including etoposide (Eto). Due to the serious side effects associated with common chemotherapeutics and prevalent multidrug resistance in recurrent and metastatic ES, there is a growing demand for alternative strategies and add­on drugs. Previous research has demonstrated efficient cell death induction by Eto in combination with arsenic trioxide (ATO) in ES cell lines. The aim of the present study was to investigate the effect of different temporal sequences of ATO and Eto administration on apoptosis induction and to explore the effect of both drugs on inhibitory glycogen synthase kinase­3ß (GSK3­ß) phosphorylation as well as multidrug transporter gene expression. The intensity of caspase activation was mainly determined by the Eto doses in A673 and TC­71 cells, whereas in RD­ES cells ATO application actively suppressed Eto­induced apoptosis. This coincided with an increase in inhibitory GSK­3ß phosphorylation in ATO­treated RD­ES cells. Inherent mRNA expression of multidrug resistance­associated protein 1 (MRP1) was low in the ES cell lines compared to that observed in the mesenchymal stem cells (MSC), whereas multidrug resistance protein 1 (MDR1) gene expression was considerably increased in the ES cell lines. ATO treatment reduced MRP1 mRNA expression in the A673 and TC­71 cells, while expression was induced in the MSC and RD­ES cells. In contrast, MDR1 mRNA expression was specifically induced by ATO in the A673 and TC­71 cells, reinforcing the expression differences between MSC and the ES cell lines. Although a reliable cell death induction by the combination of ATO and Eto has been previously shown in ES cell lines, the present study showed marked heterogeneity of the ES cell response to ATO and Eto treatment, illustrating the difficulty of prediction of individual treatment outcome in ES.


Assuntos
Trióxido de Arsênio/farmacologia , Neoplasias Ósseas/metabolismo , Etoposídeo/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Sarcoma de Ewing/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Fosforilação/efeitos dos fármacos , Sarcoma de Ewing/tratamento farmacológico , Sarcoma de Ewing/genética , Proteína GLI1 em Dedos de Zinco/genética
19.
Hum Exp Toxicol ; 39(4): 464-476, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31823663

RESUMO

Glioblastoma multiforme (GBM) is one of the most aggressive astrocytic tumors; it is resistant to most chemotherapeutic agents currently available and is associated with a poor patient survival. Thus, the development of new anticancer compounds is urgently required. Herein, we studied the molecular mechanisms of cell death induced by the experimental drugs resveratrol and MG132 or the antineoplastic drugs cisplatin and etoposide on a human GBM cell line (D54) and on primary cultured mouse astrocytes (PCMAs). Caspases, Bcl-2, inhibitors of apoptosis proteins (IAP) family members, and p53 were identified as potential molecular targets for these drugs. All drugs had a cytotoxic effect on D54 cells and PCMAs, with a similar inhibitory concentration (IC50) after 24 h. However, MG132 and cisplatin were more effective to induce apoptosis and autophagy than resveratrol and etoposide. Cell death by apoptosis involved the activation of caspases-3/7, -8, and -9, increased lysosomal permeability, LC3 lipidation, poly-(ADP-ribose) polymerase (PARP)-1 fragmentation, and a differential expression of genes related with apoptosis and autophagy like Mcl-1, Survivin, Noxa, LC3, and Beclin. In addition, apoptosis activation was partially dependent on p53 activation. Since experimental and antineoplastic drugs yielded similar results, further work is required to justify their use in clinical protocols.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Glioblastoma/patologia , Leupeptinas/farmacologia , Resveratrol/farmacologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Caspases/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Etoposídeo/farmacologia , Humanos , Camundongos , Proteína Supressora de Tumor p53/metabolismo
20.
Nucleosides Nucleotides Nucleic Acids ; 39(1-3): 170-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31608820

RESUMO

Topoisomerase II (TOP2) resolves topologically entwined duplex DNA. It generates a transient DNA double-strand break intermediate, known as TOP2 cleavage complex (TOP2cc) that contains a covalent link between TOP2 and the 5'-terminus of the incised DNA duplex. Etoposide, a frontline anticancer drug, freezes the intermediate and forms irreversible TOP2ccs. Tyrosyl-DNA phosphodiesterase 2 (TDP2) is thought to repair irreversible TOP2ccs by hydrolyzing the phosphodiester bond between TOP2 and DNA after the proteasomal degradation of trapped TOP2ccs. However, the functional cooperation between TOP2 and proteasome in the repair of trapped TOP2ccs in vivo remains unknown. In this study, we analyze the repair of etoposide-induced TOP2ccs in wild-type and TDP2-deficient (TDP2-/-) TK6 cells in the absence and presence of MG132, a potent proteasome inhibitor. The results suggested that TOP2ccs were repaired by proteasome-dependent and proteasome-independent pathways. Both proteasome-dependent and proteasome-independent pathways were further subdivided into TDP2-dependent and TDP2-independent pathways, indicating that four pathways operate in the repair of TOP2ccs. In cell survival assays, MG132 increased the etoposide sensitivity of TDP2-/- cells, supporting the TDP2-independent and proteasome-dependent pathway among these multiple repair pathways. We also demonstrated that TDP2 released TOP2 from DNA that contained etoposide-induced TOP2cc without proteolytic degradation in vitro. Taken together, the present findings uncover novel proteasome-independent mechanisms for the repair of TOP2ccs.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Linhagem Celular , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/farmacologia , Técnicas de Inativação de Genes , Humanos , Hidrólise , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Ligação Proteica , Proteólise
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