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2.
Yakugaku Zasshi ; 140(10): 1259-1268, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32999205

RESUMO

RNA interference (RNAi) is the standard method of suppressing gene expression because of its target specificity, potency, and ability to silence the expression of virtually any gene. Using 21-mer small interfering RNA (siRNA) is the general approach for inducing RNAi, as siRNA can be easily prepared using a DNA/RNA synthesizer. Synthetic siRNA can be chemically modified to increase the potency of RNAi activity and abrogate innate immune stimulation. However, designing chemically modified siRNA requires substantial experimentation. A practical method for understanding the interaction of siRNA and RNAi-related proteins and how modifications affect RNA-protein interactions is therefore needed. Plasmid DNA (pDNA) expressing short hairpin RNA (shRNA) can also be used to induce RNAi. pDNA produces numerous shRNAs that induce RNAi with potent and longterm RNAi activity, even if only one pDNA molecule is delivered to the nucleus. However, this approach has some drawbacks with regard to its therapeutic application, such as a low pDNA transfection efficiency due to its huge molecular size and innate immune responses induced by extra genes, such as CpG motifs. To overcome these issues with RNAi inducers (siRNA and pDNA), our group developed some chemical approaches using chemically modified oligonucleotides. This article focuses on our two original approaches. The first involves the groove modification of siRNA duplexes to understand siRNA-protein interactions using 7-bromo-7-deazaadenosine and 3-bromo-3-deazaadenosine as chemical probes, while the second involves the generation of RNAi medicine using chemically modified DNA, known as an intelligent shRNA expression device (iRed).


Assuntos
Desenvolvimento de Medicamentos/métodos , Interferência de RNA , RNA Interferente Pequeno/síntese química , DNA , Imunidade Inata , Oligonucleotídeos/química , Domínios e Motivos de Interação entre Proteínas , RNA Interferente Pequeno/química , Terapêutica com RNAi , Tubercidina/química
3.
Anticancer Res ; 40(9): 4895-4905, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878777

RESUMO

BACKGROUND/AIM: Nicotinamide phosphoribosyl-transferase (NAMPT) is a rate-limiting enzyme in the pathway synthesizing nicotinamide adenine dinucleotide (NAD (+)) from nicotinamide (NAM). Glioma tissues exhibit up-regulated NAMPT expression associated with a poor prognosis of patients. To determine if NAMPT can be a molecular therapeutic target, we investigated the effects of short hairpin RNA (shRNA)-mediated NAMPT down-regulation. MATERIALS AND METHODS: We designed shRNA to NAMPT and transfected to T98G cells. The characteristics of these cells were analyzed. RESULTS: The NAMPT shRNA-transfected cells exhibited delayed cell growth. However, there was no difference in the increase of sensitivity to temozolomide (TMZ) or X-ray irradiation between the NAMPT and scramble shRNA-transfected cells. The expression of NAMPT in the NAMPT shRNA-transfected cells increased with cell passage. Additionally, the shRNA-mediated transfection was associated with enhanced expression of quinolinic acid phosphoribo-syltransferase (QPRT). CONCLUSION: shRNA-mediated NAMPT down-regulation may not decrease the NADt to a sufficient level to increase TMZ/radiation sensitivity.


Assuntos
Citocinas/metabolismo , Regulação para Baixo , Glioma/enzimologia , Nicotinamida Fosforribosiltransferase/metabolismo , Antineoplásicos Alquilantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/genética , Glioma/metabolismo , Glioma/patologia , Humanos , NAD/metabolismo , Nicotinamida Fosforribosiltransferase/genética , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , RNA Interferente Pequeno/genética , Temozolomida/farmacologia
4.
Pharm Res ; 37(10): 196, 2020 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-32944844

RESUMO

PURPOSE: Hypoxia-inducible factor (HIF) is one of the critical components of the tumor microenvironment that is involved in tumor development. HIF-1α functionally and physically interacts with CDK1, 2, and 5 and stimulates the cell cycle progression and Cyclin-Dependent Kinase (CDK) expression. Therefore, hypoxic tumor microenvironment and CDK overexpression lead to increased cell cycle progression and tumor expansion. Therefore, we decided to suppress cancer cell expansion by blocking HIF-1α and CDK molecules. METHODS: In the present study, we used the carboxylated graphene oxide (CGO) conjugated with trimethyl chitosan (TMC) and hyaluronate (HA) nanoparticles (NPs) loaded with HIF-1α-siRNA and Dinaciclib, the CDK inhibitor, for silencing HIF-1α and blockade of CDKs in CD44-expressing cancer cells and evaluated the impact of combination therapy on proliferation, metastasis, apoptosis, and tumor growth. RESULTS: The results indicated that the manufactured NPs had conceivable physicochemical properties, high cellular uptake, and low toxicity. Moreover, combination therapy of cancer cells using CGO-TMC-HA NPs loaded with HIF-1α siRNA and Dinaciclib (SCH 727965) significantly suppressed the CDKs/HIF-1α and consequently, decreased the proliferation, migration, angiogenesis, and colony formation in tumor cells. CONCLUSIONS: These results indicate the ability of CGO-TMC-HA NPs for dual drug/gene delivery in cancer treatment. Furthermore, the simultaneous inhibition of CDKs/HIF-1α can be considered as a novel anti-cancer treatment strategy; however, further research is needed to confirm this treatment in vivo. Graphical Abstract The suppression of HIF-1α and CDKs inhibits cancer growth. HIF-1α is overexpressed by the cells present in the tumor microenvironment. The hypoxic environment elevates mitochondrial ROS production and increases p38 MAP kinase, JAK/STAT, ERK, JNK, and Akt/PI3K signaling, resulting in cyclin accumulation and aberrant cell cycle progression. Furthermore, the overexpression of HIF-1α/CDK results in increased expression of genes such as BCL2, Bcl-xl, Ki-67, TGFß, VEGF, FGF, MMP2, MMP9, and, HIF-1α and consequently raise the survival, proliferation, angiogenesis, metastasis, and invasion of tumor cells. In conclusion, HIF-1α-siRNA/Dinaciclib-loaded CGO-TMC-HA NPs can inhibit the tumor expansion by blockage of CDKs and HIF-1α (JAK: Janus kinase, STAT: Signal transducer and activator of transcription, MAPK: mitogen-activated protein kinase, ERK: extracellular signal-regulated kinase, JNK: c-Jun N-terminal kinase, PI3K: phosphatidylinositol 3-kinase).


Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/administração & dosagem , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Experimentais/terapia , Compostos de Piridínio/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/química , Compostos Bicíclicos Heterocíclicos com Pontes/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Quitosana/química , Grafite/química , Ácido Hialurônico/química , Camundongos , Nanopartículas/química , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Compostos de Piridínio/química , Compostos de Piridínio/farmacocinética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinética
5.
Nat Commun ; 11(1): 4709, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32948765

RESUMO

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.


Assuntos
Reparo do DNA , Instabilidade Genômica , Glioblastoma/genética , Células-Tronco Neoplásicas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Apoptose , Proteína BRCA1 , Neoplasias Encefálicas/genética , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Inativação Gênica , Glioblastoma/patologia , Células HEK293 , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Células-Tronco Neoplásicas/patologia , RNA Interferente Pequeno/genética , Tolerância a Radiação , Radiação Ionizante , Transcriptoma
6.
Anticancer Res ; 40(10): 5545-5556, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32988878

RESUMO

BACKGROUND/AIM: The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -ß, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38ß in human pancreatic cancer. MATERIALS AND METHODS: Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38ß, in vitro growth and migration as well as in vivo tumorigenicity were assessed. RESULTS: All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38ß inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38ß knockdown. While in vivo inhibition of p38ß decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. CONCLUSION: p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.


Assuntos
Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Neoplasias Pancreáticas/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Carcinogênese/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Neoplasias Pancreáticas/patologia , Fosforilação , Isoformas de Proteínas/genética , RNA Interferente Pequeno/genética
7.
Nat Commun ; 11(1): 4825, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973178

RESUMO

Short regulatory RNA molecules underpin gene expression and govern cellular state and physiology. To establish an alternative layer of control over these processes, we generated chimeric regulatory RNAs that interact reversibly and light-dependently with the light-oxygen-voltage photoreceptor PAL. By harnessing this interaction, the function of micro RNAs (miRs) and short hairpin (sh) RNAs in mammalian cells can be regulated in a spatiotemporally precise manner. The underlying strategy is generic and can be adapted to near-arbitrary target sequences. Owing to full genetic encodability, it establishes optoribogenetic control of cell state and physiology. The method stands to facilitate the non-invasive, reversible and spatiotemporally resolved study of regulatory RNAs and protein function in cellular and organismal environments.


Assuntos
Expressão Gênica , Células Fotorreceptoras/metabolismo , RNA/metabolismo , Animais , Células HEK293 , Humanos , MicroRNAs/metabolismo , RNA/genética , RNA Interferente Pequeno
8.
Pharmacol Rev ; 72(4): 862-898, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32929000

RESUMO

RNA-based therapies, including RNA molecules as drugs and RNA-targeted small molecules, offer unique opportunities to expand the range of therapeutic targets. Various forms of RNAs may be used to selectively act on proteins, transcripts, and genes that cannot be targeted by conventional small molecules or proteins. Although development of RNA drugs faces unparalleled challenges, many strategies have been developed to improve RNA metabolic stability and intracellular delivery. A number of RNA drugs have been approved for medical use, including aptamers (e.g., pegaptanib) that mechanistically act on protein target and small interfering RNAs (e.g., patisiran and givosiran) and antisense oligonucleotides (e.g., inotersen and golodirsen) that directly interfere with RNA targets. Furthermore, guide RNAs are essential components of novel gene editing modalities, and mRNA therapeutics are under development for protein replacement therapy or vaccination, including those against unprecedented severe acute respiratory syndrome coronavirus pandemic. Moreover, functional RNAs or RNA motifs are highly structured to form binding pockets or clefts that are accessible by small molecules. Many natural, semisynthetic, or synthetic antibiotics (e.g., aminoglycosides, tetracyclines, macrolides, oxazolidinones, and phenicols) can directly bind to ribosomal RNAs to achieve the inhibition of bacterial infections. Therefore, there is growing interest in developing RNA-targeted small-molecule drugs amenable to oral administration, and some (e.g., risdiplam and branaplam) have entered clinical trials. Here, we review the pharmacology of novel RNA drugs and RNA-targeted small-molecule medications, with a focus on recent progresses and strategies. Challenges in the development of novel druggable RNA entities and identification of viable RNA targets and selective small-molecule binders are discussed. SIGNIFICANCE STATEMENT: With the understanding of RNA functions and critical roles in diseases, as well as the development of RNA-related technologies, there is growing interest in developing novel RNA-based therapeutics. This comprehensive review presents pharmacology of both RNA drugs and RNA-targeted small-molecule medications, focusing on novel mechanisms of action, the most recent progress, and existing challenges.


Assuntos
RNA/efeitos dos fármacos , RNA/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Aptâmeros de Nucleotídeos/uso terapêutico , Betacoronavirus , Técnicas de Química Analítica/métodos , Técnicas de Química Analítica/normas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Infecções por Coronavirus/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Desenvolvimento de Medicamentos/organização & administração , Descoberta de Drogas , Humanos , MicroRNAs/farmacologia , MicroRNAs/uso terapêutico , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Pandemias , Pneumonia Viral/tratamento farmacológico , RNA/efeitos adversos , RNA Antissenso/farmacologia , RNA Antissenso/uso terapêutico , RNA Guia/farmacologia , RNA Guia/uso terapêutico , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/farmacologia , RNA Ribossômico/efeitos dos fármacos , RNA Ribossômico/farmacologia , RNA Interferente Pequeno/farmacologia , RNA Interferente Pequeno/uso terapêutico , RNA Viral/efeitos dos fármacos , Ribonucleases/metabolismo , Riboswitch/efeitos dos fármacos
9.
mBio ; 11(5)2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934084

RESUMO

Bats are primary reservoirs for multiple lethal human viruses, such as Ebola, Nipah, Hendra, rabies, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), and, most recently, SARS-CoV-2. The innate immune systems of these immensely abundant, anciently diverged mammals remain insufficiently characterized. While bat genomes contain many endogenous retroviral elements indicative of past exogenous infections, little is known about restrictions to extant retroviruses. Here, we describe a major postentry restriction in cells of the yinpterochiropteran bat Pteropus alecto Primate lentiviruses (HIV-1, SIVmac) were potently blocked at early life cycle steps, with up to 1,000-fold decreases in infectivity. The block was specific, because nonprimate lentiviruses such as equine infectious anemia virus and feline immunodeficiency virus were unimpaired, as were foamy retroviruses. Interspecies heterokaryons demonstrated a dominant block consistent with restriction of incoming viruses. Several features suggested potential TRIM5 (tripartite motif 5) or myxovirus resistance protein 2 (MX2) protein restriction, including postentry action, cyclosporine sensitivity, and reversal by capsid cyclophilin A (CypA) binding loop mutations. Viral nuclear import was significantly reduced, and this deficit was substantially rescued by cyclosporine treatment. However, saturation with HIV-1 virus-like particles did not relieve the restriction at all. P. alecto TRIM5 was inactive against HIV-1 although it blocked the gammaretrovirus N-tropic murine leukemia virus. Despite major divergence in a critical N-terminal motif required for human MX2 activity, P. alecto MX2 had anti-HIV activity. However, this did not quantitatively account for the restriction and was independent of and synergistic with an additional CypA-dependent restriction. These results reveal a novel, specific restriction to primate lentiviruses in the Pteropodidae and advance understanding of bat innate immunity.IMPORTANCE The COVID-19 pandemic suggests that bat innate immune systems are insufficiently characterized relative to the medical importance of these animals. Retroviruses, e.g., HIV-1, can be severe pathogens when they cross species barriers, and bat restrictions corresponding to retroviruses are comparatively unstudied. Here, we compared the abilities of retroviruses from three genera (Lentivirus, Gammaretrovirus, and Spumavirus) to infect cells of the large fruit-eating bat P. alecto and other mammals. We identified a major, specific postentry restriction to primate lentiviruses. HIV-1 and SIVmac are potently blocked at early life cycle steps, but nonprimate lentiviruses and foamy retroviruses are entirely unrestricted. Despite acting postentry and in a CypA-dependent manner with features reminiscent of antiretroviral factors from other mammals, this restriction was not saturable with virus-like particles and was independent of P. alecto TRIM5, TRIM21, TRIM22, TRIM34, and MX2. These results identify a novel restriction and highlight cyclophilin-capsid interactions as ancient species-specific determinants of retroviral infection.


Assuntos
Quirópteros/imunologia , Gammaretrovirus/imunologia , Imunidade Inata/imunologia , Lentivirus de Primatas/imunologia , Spumavirus/imunologia , Células 3T3 , Animais , Aotidae , Gatos , Linhagem Celular , Quirópteros/virologia , Ciclofilina A/metabolismo , Furões , Gammaretrovirus/crescimento & desenvolvimento , Células HEK293 , Humanos , Lentivirus de Primatas/crescimento & desenvolvimento , Camundongos , Interferência de RNA , RNA Interferente Pequeno/genética , Spumavirus/crescimento & desenvolvimento , Proteínas com Motivo Tripartido/metabolismo
10.
Nan Fang Yi Ke Da Xue Xue Bao ; 40(5): 727-732, 2020 May 30.
Artigo em Chinês | MEDLINE | ID: mdl-32897220

RESUMO

OBJECTIVE: To investigate the changes in the exosomes secreted by mouse dendritic cell line DC2.4 after infection with Toxoplasma gondii and to analyze the possible regulatory mechanisms underlying such changes. METHODS: The exosomes were extracted by ultracentrifugation from DC2.4 cells at 28 h after infection with Toxoplasma gondii. The morphology of the exosomes was examined with transmission electron microscopy, and the exosome size and density were determined using a nanoparticle tracker. High-throughput sequencing was carried out to identify the differentially expressed small RNAs in the exosomes derived from the infected cells. RESULTS: T. gondii infection resulted in a significantly increased density of exosomes secreted by DC2.4 cells. Small RNA sequencing revealed that Toxoplasma infection caused an increase in the number of miRNAs and piRNAs in the exosomes. The significantly up-regulated piRNAs after the infection included piR-mmu-159, piR-mmu-1526, piR-mmu-9082, piR-mmu-17405, and piR-mmu-25576. CONCLUSIONS: Toxoplasma infection causes accumulation and enrichment of exosomes secreted by DC2.4 cells with increased miRNAs and piRNAs in the exosomes.


Assuntos
Exossomos , Toxoplasma , Animais , Linhagem Celular , Células Dendríticas , Camundongos , MicroRNAs , RNA Interferente Pequeno
11.
Pestic Biochem Physiol ; 170: 104700, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32980067

RESUMO

Argonautes (Ago) are important core proteins in RNA interference (RNAi) pathways of eukaryotic cells. Generally, it is thought that Ago1, Ago2 and Ago3 are involved in the miRNA (microRNA), siRNA (small interfering RNA) and piRNA (Piwi-interacting RNA)-mediated RNAi pathways, respectively. As a main component of the RNA-induced silencing complex (RISC), Ago2 plays an indispensable role in using siRNA to recognize and cut target messenger RNAs resulting in suppression of transcript levels, but the contributions of Ago1 and Ago3 to the siRNA-mediated RNAi pathway remain to be explored in many insect species. In this study, we investigated the contributions of four Ago genes (named LmAgo1, LmAgo2a and LmAgo2b and LmAgo3) to RNAi efficiency in Locusta migratoria by using both in vivo and in vitro experiments. Our results showed that suppression of each of the Ago genes significantly impaired RNAi efficiency when targeting Lmß-tubulin transcripts, resulting in recovery of 48, 43.3, 61.4 or 26% of Lmß-tubulin transcripts following RNAi-mediated suppression of LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3, respectively. Furthermore, overexpression of LmAgo1, LmAgo2a, LmAgo2b, or LmAgo3 in a PAc5.1-V5/HisB vector and co-transfection with psicheck2 fluorescence vector in S2 cells reduced luciferase fluorescence by 38.3, 58.9, 53.3 or 55.6%, respectively. Taken together, our results showed that LmAgo1, LmAgo2a, LmAgo2b, and LmAgo3 each make significant contributions to RNAi efficiency in L. migratoria and suggest that the involvement of all four enzymes could be one of the major factors supporting robust RNAi responses observed in this species.


Assuntos
Locusta migratoria/genética , MicroRNAs/genética , Animais , Proteínas Argonauta/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , RNA Interferente Pequeno/genética
12.
Ecotoxicol Environ Saf ; 203: 110928, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32888618

RESUMO

Hexavalent chromium [Cr(VI)] is seriously harmful to ecosystems and living organisms due to its strong toxicity. Role of dynamin-related protein 1 (Drp1) and Drp1-associated mitochondrial fragmentation in mitophagy and cytotoxicity after Cr(VI) exposure has not been clarified so far. We confirmed that Cr(VI) caused mitochondrial fission by up-regulating Drp1 expression and enhancing Drp1 mitochondrial translocation. By applying the intracellular Ca2+ antagonist BAPTA-AM and mitochondrial Ca2+ antagonist Ru360, we demonstrated that Cr(VI)-induced excessive mitochondrial fission was in a Ca2+-Drp1 dependent manner. The administration of Drp1 siRNA significantly suppressed the overactivation of mitophagy in Cr(VI)-induced hepatotoxicity. The specific Drp1 inhibitor mitochondrial division inhibitor-1 (Mdivi-1) blocked the overactive mitophagy and subsequently ameliorated hepatotoxicity caused by Cr(VI) in vivo. We reached the conclusion that Drp1-dependent mitochondrial fission contributes to Cr(VI)-induced mitophagy and hepatotoxicity, which may provide experimental basis for the study of chromium-associated toxicity, especially for the prevention of health damage in chromium-exposed population.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/etiologia , Cromo/toxicidade , Dinaminas/metabolismo , Poluentes Ambientais/toxicidade , Hepatócitos/efeitos dos fármacos , Dinâmica Mitocondrial/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Animais , Linhagem Celular , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Induzida por Substâncias e Drogas/patologia , Ecossistema , Hepatócitos/metabolismo , Hepatócitos/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Hepáticas/efeitos dos fármacos , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/patologia , RNA Interferente Pequeno/metabolismo
13.
Anticancer Res ; 40(9): 5035-5041, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32878791

RESUMO

BACKGROUND/AIM: Based on the cytotoxic agent (-)-zampanolide, N,N'-(arylmethylene)bisamides were designed and synthesized as candidate anti-cancer agents. Among them, N,N'-[(3,4-dimethoxyphenyl)methylene]biscinnamide (DPMBC) was identified as the most potent cytotoxic analog against cancer cells. In this study, we investigated the mechanisms underlying DPMBC-induced cell death in HL-60 human promyelocytic leukemia and PC-3 human prostate cancer cells. MATERIALS AND METHODS: Cell growth was assessed by the WST-8 assay. Induction of apoptosis was assessed by nuclear morphology, DNA ladder formation, and flow cytometry using Annexin V staining. Activation of factors in the apoptotic signaling pathway was assessed by western blot analyses. Knockdown of death receptor 5 (DR5) was performed using siRNA. RESULTS: DPMBC up-regulated expression levels of DR5 protein and induced apoptosis through the extrinsic apoptotic pathway mediated by DR5 and caspases. CONCLUSION: DPMBC is an extrinsic apoptosis inducer, which has potential as a therapeutic agent for cancer therapy.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Macrolídeos/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Antineoplásicos/química , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Relação Dose-Resposta a Droga , Humanos , Macrolídeos/química , Estrutura Molecular , RNA Interferente Pequeno/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1278-1282, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798412

RESUMO

OBJECTIVE: To investigate the effects of down-regulating of c-Met expression to the proliferation, invasiveness and apoptosis of human multiple myeloma RPMI 8226 cells. METHODS: According to transfection the RPMI8226 cells were dividide into RPMI 8226 (untreated RPMI 8226), RPMI 8226 /shRNA-Met and RPMI8226/shRNA-control group, respectively. Protein expression level of c-Met was detected by Western blot so as to evaluate transfection condition; the proliferation of the cells was detected by MTT; apoptosis and cycle of the cells were detected by flow cytometry; effect of c-Met/shRNA on RPMI 8226 cell adhesion was detected by RPMI 8226 cell adherence to ECM (Fn and Matrigel) and ECV304 cells. Invasiveness of RPMI 8226 cell was detected by Transwell assay. RESULTS: The c-Met short hairpin RNA (shRNA) was successfully transfected into RPMI 8226 cells, and could inhibit the expression of c-Met significantly. The down-regulation of c-Met could inhibit the proliferation of RPMI 8226 cells significantly. The percentage of cells in the G0/G1 phase and apoptotic rate (sub-G1) in the RPMI 8226/shRNA-Met group were higher than those in the control group, the adhesion rate and the number of migrated RPMI 8226/shRNA-Met cells were decreased significantly as compared with control group. There were no significant differences in each indexes between RPMI 8226/shRNA-control and control group. CONCLUSION: Knockdown of c-Met can affect the proliferation, adherence, invasiveness and apoptosis of human multiple myeloma RPMI 8226 cells.


Assuntos
Mieloma Múltiplo , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Humanos , RNA Interferente Pequeno
15.
Nat Commun ; 11(1): 4124, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807787

RESUMO

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Células HCT116 , Humanos , Immunoblotting , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
16.
Nat Commun ; 11(1): 3806, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732922

RESUMO

Most triple-negative breast cancer (TNBC) patients fail to respond to T cell-mediated immunotherapies. Unfortunately, the molecular determinants are still poorly understood. Breast cancer is the disease genetically linked to a deficiency in autophagy. Here, we show that autophagy defects in TNBC cells inhibit T cell-mediated tumour killing in vitro and in vivo. Mechanistically, we identify Tenascin-C as a candidate for autophagy deficiency-mediated immunosuppression, in which Tenascin-C is Lys63-ubiquitinated by Skp2, particularly at Lys942 and Lys1882, thus promoting its recognition by p62 and leading to its selective autophagic degradation. High Tenascin-C expression is associated with poor prognosis and inversely correlated with LC3B expression and CD8+ T cells in TNBC patients. More importantly, inhibition of Tenascin-C in autophagy-impaired TNBC cells sensitizes T cell-mediated tumour killing and improves antitumour effects of single anti-PD1/PDL1 therapy. Our results provide a potential strategy for targeting TNBC with the combination of Tenascin-C blockade and immune checkpoint inhibitors.


Assuntos
Autofagia/imunologia , Linfócitos T CD8-Positivos/imunologia , Tenascina/metabolismo , Neoplasias de Mama Triplo Negativas/imunologia , Evasão Tumoral/imunologia , Animais , Antineoplásicos Imunológicos/farmacologia , Autofagia/genética , Antígeno B7-H1/antagonistas & inibidores , Linfócitos T CD8-Positivos/transplante , Linhagem Celular Tumoral , Feminino , Células HEK293 , Humanos , Imunoterapia Adotiva , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Prognóstico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/terapia , Evasão Tumoral/genética
17.
PLoS Genet ; 16(8): e1008915, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32776928

RESUMO

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactivate into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association. In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been described in vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines.


Assuntos
Genoma Humano , Herpesvirus Humano 6/genética , Integração Viral , Grupo com Ancestrais do Continente Asiático/genética , Cromossomos Humanos Par 22/genética , Evolução Molecular , Mutação em Linhagem Germinativa , Humanos , Polimorfismo de Nucleotídeo Único , RNA Interferente Pequeno/genética
18.
Nat Commun ; 11(1): 3940, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769985

RESUMO

R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, here, we map RNA:DNA hybrids, replication stress markers and DNA double-strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids are found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR is only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also accumulate at TTS, leading to persistent checkpoint activation, spreading of γ-H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.


Assuntos
Replicação do DNA , DNA Topoisomerases Tipo I/metabolismo , Estruturas R-Loop/genética , Regiões Terminadoras Genéticas/genética , Transcrição Genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Fosforilação , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo
19.
PLoS One ; 15(8): e0237015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32760098

RESUMO

Graves' orbitopathy (GO) is characterised in early stages by orbital fibroblast inflammation, which can be aggravated by oxidative stress and often leads to fibrosis. Protein tyrosine protein 1B (PTP1B) is a regulator of inflammation and a therapeutic target in diabetes. We investigated the role of PTP1B in the GO mechanism using orbital fibroblasts from GO and healthy non-GO subjects. After 24 hours of transfection with PTPN1 siRNA, the fibroblasts were exposed to interleukin (IL)-1ß, cigarette smoke extract (CSE), H2O2, and transforming growth factor (TGF)-ß stimulations. Inflammatory cytokines and fibrosis-related proteins were analysed using western blotting and/or enzyme-linked immunosorbent assay (ELISA). Reactive oxygen species (ROS) release was detected using an oxidant-sensitive fluorescent probe. IL-1ß, tumor necrosis factor (TNF)-α, bovine thyroid stimulating hormone (bTSH), high-affinity human stimulatory monoclonal antibody of TSH receptor (M22), and insulin-like growth factor-1 (IGF-1) significantly increased PTP1B protein production in GO and non-GO fibroblasts. PTPN1 silencing significantly blocked IL-1ß-induced inflammatory cytokine production, CSE- and H2O2-induced ROS synthesis, and TGF-ß-induced expression of collagen Iα, α-smooth muscle actin (SMA), and fibronectin in GO fibroblasts. Silencing PTPN1 also decreased phosphorylation levels of Akt, p38, and c-Jun N-terminal kinase (JNK) and endoplasmic reticulum (ER)-stress response proteins in GO cells. PTP1B may be a potential therapeutic target of anti-inflammatory, anti-oxidant and anti-fibrotic treatment of GO.


Assuntos
Oftalmopatia de Graves/enzimologia , Oftalmopatia de Graves/terapia , Proteína Tirosina Fosfatase não Receptora Tipo 1/antagonistas & inibidores , Adulto , Animais , Apoptose , Bovinos , Sobrevivência Celular , Citocinas/biossíntese , Estresse do Retículo Endoplasmático , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Inativação Gênica , Oftalmopatia de Graves/patologia , Humanos , Técnicas In Vitro , Mediadores da Inflamação/metabolismo , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Córtex Pré-Frontal/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/genética , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , RNA Interferente Pequeno/genética , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
20.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 28(4): 1144-1151, 2020 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-32798389

RESUMO

OBJECTIVE: To explore the effect of regulating A20 expression on NF-κB and biological characteristics of Jurkat cells with glucocorticoid (GC) resistance. METHODS: CCRF CEM and Jurkat cells were treated with dexamethasone (DEX) at concentrations of 100、10、1、0.1、0.01 and 0.001 µmol/L, and cultured for 24、48 and 72 h. The proliferation inhibition rate of Jurkat cell was detected by CCK-8. A20 plasmid was constructed, A20-siRNA was designed and synthesized, and transfected into Jurkat cells by liposome. CCK-8 was used to detect the proliferation rates of Jurkat cells in different concentrations of DEX group, DEX combined with A20 plasmid group and A20-siRNA group. The mRNA expression level of NF-κB was detected by RT-qPCR, the protein expression level of NF-κB was detected by Western blot, and the apoptosis of Jurkat cells was examined by flow cytometry. RESULTS: The inhibitory effects of DEX at different concentrations on the growth of CCRF CEM cells were time-dependent (r=0.984, P<0.05) and concentration-dependent (r=0.966, P<0.05). At the point of 24 hour, the IC50 approached 1 µmol/L in CCRF CEM cells. Great large differences began to appear between 1 and 10 µmol/L, the proliferation rate of Jurkat cells treated with 1 µmol/L DEX did not show a significant change. Therefore, 1 µmol/L was selected as control group. The cell proliferation rate of A20 plasmid transfection combined with different concentrations of DEX group was lower than that of DEX group and A20-siRNA combined with DEX group. After transfection of A20 plasmid, the expression level of NF-κB was significantly lower than that of control group (P<0.05), and the apoptotic rate was significantly higher than that of control group (P<0.05). After transfection of Jurkat cells with A20-siRNA, the expression level of NF-κB was significantly higher than that of control group (P<0.05). The apoptotic rate of cells in A20-siRNA group was not significantly changed (P>0.05). CONCLUSION: Jurkat cells are resistant to DEX. A20 overexpression combined with DEX can increase sensitivity of Jurkat cells with GC resistance and decrease the proliferation rate of Jurkat cells, down-regulate the expression level of NF-κB and promote the apoptosis of Jurkat cells.


Assuntos
Apoptose , NF-kappa B , Proliferação de Células , Humanos , Células Jurkat , RNA Interferente Pequeno , Transfecção , Proteína 3 Induzida por Fator de Necrose Tumoral alfa
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