RESUMO
Engineered multivalent drugs are promising candidates for fighting infection by highly variable viruses, such as HCV. The combination into a single molecule of more than one inhibitory domain, each with its own target specificity and even a different mechanism of action, results in drugs with potentially enhanced therapeutic properties. In the present work, the anti-HCV chimeric inhibitor RNA HH363-10, which has a hammerhead catalytic domain and an aptamer RNA domain, was subjected to an in vitro selection strategy to isolate ten different optimised chimeric inhibitor RNAs. The catalytic domain was preserved while the aptamer RNA domain was evolved to contain two binding sites, one mapping to the highly conserved IIIf domain of the HCV genome's internal ribosome entry site (IRES), and the other either to IRES domain IV (which contains the translation start codon) or the essential linker region between domains I and II. These chimeric molecules efficiently and specifically interfered with HCV IRES-dependent translation in vitro (with IC50 values in the low µM range). They also inhibited both viral translation and replication in cell culture. These findings highlight the feasibility of using in vitro selection strategies for obtaining improved RNA molecules with potential clinical applications.
Assuntos
Antivirais/farmacologia , Aptâmeros de Nucleotídeos/farmacologia , Genoma Viral/efeitos dos fármacos , Hepacivirus/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , RNA Catalítico/farmacologia , Antivirais/química , Aptâmeros de Nucleotídeos/química , Pareamento de Bases , Sequência de Bases , Sítios de Ligação , Linhagem Celular Tumoral , Genes Reporter , Hepacivirus/genética , Hepacivirus/crescimento & desenvolvimento , Hepacivirus/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Sítios Internos de Entrada Ribossomal/efeitos dos fármacos , Luciferases/genética , Luciferases/metabolismo , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Viral/antagonistas & inibidores , RNA Viral/biossíntese , Replicação Viral/efeitos dos fármacosRESUMO
Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV-infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers, and improved survival compared to the untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene-targeting applications in vivo.
Assuntos
Terapia Genética/métodos , RNA Catalítico/administração & dosagem , Ribonuclease P/antagonistas & inibidores , Salmonella/genética , Viroses/terapia , Administração Oral , Animais , Macrófagos/enzimologia , Macrófagos/virologia , Camundongos , RNA Catalítico/farmacologia , Carga Viral/efeitos dos fármacos , Viroses/prevenção & controle , Replicação Viral/efeitos dos fármacosRESUMO
Trinucleotide repeat expansions (TREs) are a recently described class of mutations characterized by a change in the size of the genomic fragment due to amplification of the repeated unit. A number of diseases have been attributed to TRE, including Huntington disease and myotonic dystrophy (DM), but attempts at genetic therapy have yet to prove successful. A potential therapeutic approach would be to repair the expanded repeat using the trans-splicing ability of group I intron ribozymes. We have used DM as a model to test this hypothesis. A group I intron ribozyme (DMPK-RZ1) was designed to modify the TRE at the 3' end of the human myotonic dystrophy protein kinase (DMPK) transcripts. DMPK-RZ1 was shown to ligate a small DMPK mRNA fragment, contained within the ribozyme, to a simple DMPK-target RNA in vitro. It also modified a larger target transcript, leading to replacement of twelve repeats with five repeats, both in vitro and in mammalian cells. Finally, this ribozyme successfully replaced the 3' end of endogenous DMPK mRNA in fibroblasts with a different 3' region. Ribozyme-mediated RNA repair may thus form a novel therapeutic strategy for diseases associated with repeat expansions.
Assuntos
Splicing de RNA/efeitos dos fármacos , RNA Catalítico/farmacologia , Repetições de Trinucleotídeos/genética , Linhagem Celular , Humanos , Miotonina Proteína Quinase , Proteínas Serina-Treonina Quinases/genética , RNA/análise , RNA/genética , RNA Catalítico/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genéticaRESUMO
OBJECTIVE: Hepatitis C virus (HCV) is one of the major pathogens that lead to viral hepatitis. At present, Interferon treatment in combination with ribavirin is the first line clinical therapeutic approach. However, the responses are usually poor and the viral infection reoccurs. Therefore, exploring new antiviral agents and therapies is under urgent needs. METHODS: The sequence and structure of the core coding region of HCV genome were analyzed through the two computer software, DNAMAN and RNA Structure. The cytosine 52 nt downstream of the AUG initiation triplet was identified as the optimal target cleavage site. Based on the flanking sequence of this assumed cleavage site, a guide sequence (GS) was designed and covalently linked to the 3 prime terminus of the M1 RNA, which is catalytic subunit of the RNase P derived from Escherichia coli using PCR. We named this new targeting ribozyme M1GS-HCV/C52 and it antiviral activities were analyzed in cultured cells. RESULTS: In the in vitro cleavage assay, M1GS-HCV/C52 ribozyme could effectively cleave the HCV target RNA into two fragments at the specific cleavage site. Moreover, comparing to the blank control, this engineered M1GS ribozyme could reduce the core protein expression of more than 80% in the HCV-infected host cell and lead to a 1500-fold reduction of HCV RNA copies in the culture supernatant. An another M1GS ribozyme, M1GS-HCV/C52*, which has the same guide sequence but does not contain a 24nt-long bridge sequence, did not exhibit apparent inhibition for the expression of HCV core gene and viral proliferation in our paralleled assay. CONCLUSION: We successfully constructed an M1GS ribozyme showing affective and specific cleavage of target viral RNA. Further results showed that the engineering ribozyme had notably antiviral activity in cultured cells, thus provided a new promising approach for clinical anti-HCV therapeutic strategy.
Assuntos
Antivirais/farmacologia , Proteínas do Capsídeo/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Hepacivirus/genética , Hepatite B/virologia , RNA Catalítico/metabolismo , Ribonuclease P/metabolismo , Ribonuclease P/farmacologia , Antivirais/química , Proteínas do Capsídeo/metabolismo , Linhagem Celular Tumoral , Desenho de Fármacos , Proteínas de Escherichia coli/genética , Hepacivirus/efeitos dos fármacos , Hepacivirus/metabolismo , Hepatite B/tratamento farmacológico , Humanos , RNA Catalítico/química , RNA Catalítico/genética , RNA Catalítico/farmacologia , RNA Viral/genética , RNA Viral/metabolismo , Ribonuclease P/genéticaRESUMO
Enzymes generally are thought to derive their functional activity from conformational motions. The limited chemical variation in RNA suggests that such structural dynamics may play a particularly important role in RNA function. Minimal hammerhead ribozymes are known to cleave efficiently only in â¼ 10-fold higher than physiologic concentrations of Mg(2+) ions. Extended versions containing native loop-loop interactions, however, show greatly enhanced catalytic activity at physiologically relevant Mg(2+) concentrations, for reasons that are still ill-understood. Here, we use Mg(2+) titrations, activity assays, ensemble, and single molecule fluorescence resonance energy transfer (FRET) approaches, combined with molecular dynamics (MD) simulations, to ask what influence the spatially distant tertiary loop-loop interactions of an extended hammerhead ribozyme have on its structural dynamics. By comparing hammerhead variants with wild-type, partially disrupted, and fully disrupted loop-loop interaction sequences we find that the tertiary interactions lead to a dynamic motional sampling that increasingly populates catalytically active conformations. At the global level the wild-type tertiary interactions lead to more frequent, if transient, encounters of the loop-carrying stems, whereas at the local level they lead to an enrichment in favorable in-line attack angles at the cleavage site. These results invoke a linkage between RNA structural dynamics and function and suggest that loop-loop interactions in extended hammerhead ribozymes-and Mg(2+) ions that bind to minimal ribozymes-may generally allow more frequent access to a catalytically relevant conformation(s), rather than simply locking the ribozyme into a single active state.
Assuntos
Movimento (Física) , Conformação de Ácido Nucleico , RNA Catalítico/química , RNA Catalítico/metabolismo , Sequência de Bases , Catálise , Desenho de Fármacos , Interações Medicamentosas/fisiologia , Transferência Ressonante de Energia de Fluorescência , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , RNA Catalítico/síntese química , RNA Catalítico/farmacologia , Projetos de Pesquisa , Manejo de Espécimes/normas , Manejo de Espécimes/estatística & dados numéricos , Relação Estrutura-AtividadeRESUMO
The continuous growth of advanced malignancies almost universally correlates with the reactivation of telomerase. While there is still a great deal of basic and applied research to be done, telomerase remains a very attractive novel target for cancer therapeutics. In this review, we will discuss the challenges and the pros and cons of the most promising antitelomerase approaches currently being investigated.
Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/terapia , Telomerase/antagonistas & inibidores , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Desenho de Fármacos , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Previsões , Humanos , Imunoterapia , Modelos Biológicos , Neoplasias/diagnóstico , Neoplasias/enzimologia , Oligonucleotídeos Antissenso/farmacologia , RNA Catalítico/farmacologia , Inibidores da Transcriptase Reversa/farmacologia , Telomerase/genética , Telomerase/metabolismo , Telômero/química , Telômero/metabolismoRESUMO
BACKGROUND: Hepatitis delta virus (HDV) ribozyme is an attractive molecular tool that can specifically recognize and catalyze the self-cleavage of the viral RNA phosphodiester backbone. However, a major obstacle in the medical application of the HDV ribozyme is the lack of specificity in the delivery of the ribozyme to defined target cells. RESULTS: The objective of this study was to determine whether retroviral vectors can deliver the HDV ribozyme into the target cells and to elucidate whether HDV ribozyme plays a role in hepatitis B virus (HBV) replication. In our study, the transduction of helper-free pseudotyped retrovirus, which showed a broad host range, in human hepatoma cells was performed under 2 conditions, that is, in the presence of polymerized human serum albumin (pHSA) and in the absence of pHSA. The transduction ability in the presence of pHSA was higher than in the absence of pHSA. Moreover, HBsAg and HBeAg levels after transductions with pHSA were significantly lower than those in the absence of pHSA, thus indicating that the recombinant retrovirus had HBV-specific cleavage activity and targeted HepG2215 cells. CONCLUSIONS: These data suggest that this system provides a new approach for targeting hepatocytes and has a great potential in gene therapy for HBV infection.
Assuntos
Antivirais/farmacologia , Produtos Biológicos/farmacologia , Vírus da Hepatite B/efeitos dos fármacos , Vírus Delta da Hepatite/enzimologia , RNA Catalítico/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacocinética , Produtos Biológicos/farmacocinética , Linhagem Celular , Vetores Genéticos , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos E da Hepatite B/biossíntese , Hepatócitos/virologia , Humanos , RNA Catalítico/farmacocinética , Retroviridae/genética , Transdução GenéticaRESUMO
The HER-2 proto-oncogene (also called c-erbB-2/neu) encodes the protein, p185, which is closely related to the growth and metastasis of adenocarcinoma, and is overexpressed in 25-30% of human breast cancers. In this study, we attempt to reverse the malignant phenotype of the breast cancer cell line, MCF-7, using a HER-2-specific hammerhead ribozyme. Two anti-HER-2 hammerhead ribozymes, RZ1 and RZ2, were synthesized, inserted separately into the nonviral eukaryotic expression vector, pcDNA3.1(-), and transfected into MCF-7 cells. Analyses showed that the HER-2 mRNA and p185, as well as oncogene k-ras were down-regulated remarkably in the ribozyme-transfected cells, while the onco-suppressor gene, p53, was up-regulated. Furthermore, the tumorigenicity of the RZ1-stably transfected MCF-7 cells was decreased dramatically in nude mice. These results demonstrate that the use of anti-HER-2 ribozymes may be a beneficial strategy for gene therapy of breast cancer.
Assuntos
Adenocarcinoma/terapia , Neoplasias da Mama/terapia , Regulação Neoplásica da Expressão Gênica/genética , Marcação de Genes/métodos , Terapia Genética/métodos , RNA Catalítico/farmacologia , Receptor ErbB-2/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Primers do DNA/genética , Feminino , Citometria de Fluxo , Vetores Genéticos/genética , Humanos , Camundongos , Camundongos Nus , Proto-Oncogene Mas , RNA Catalítico/genética , Receptor ErbB-2/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
The epidermal growth factor receptor (HER1/EGFR) is known to be disregulated in a large subgroup of glioblastoma multiforme cases. Disregulation of HER1/EGFR is related to malignant transformation and tumor growth in various human cancers, including malignant glioma. One mechanism that may lead to disregulated HER1/EGFR signaling is the intrinsic alteration of the receptor structure due to mutational changes. The most common mutant form of HER1/EGFR, named variant III (EGFRvIII), results from an 801 bp in-frame deletion in the DNA sequence encoding the extracellular ligand-binding domain. Independent of ligand-binding, EGFRvIII is constitutively activated and beyond external control. Since its cellular expression was shown to relate enhanced tumorigenicity, various therapeutic strategies were developed to target EGFRvIII, including monoclonal antibodies, vaccination therapies and small-molecule tyrosine kinase inhibitors. In this review, we focus on ribozyme-mediated inhibition of EGFRvIII messenger RNA expression as a gene therapeutic approach for EGFRvIII-expressing glioblastoma multiforme.
Assuntos
Receptores ErbB/antagonistas & inibidores , Glioblastoma/tratamento farmacológico , RNA Catalítico/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Receptores ErbB/genética , Glioblastoma/genética , Humanos , RNA Catalítico/uso terapêutico , RNA Mensageiro/antagonistas & inibidores , Deleção de SequênciaRESUMO
The 5'-untranslated region (5'UTR) of the HIV-1 RNA is an attractive target for engineered ribozymes due to its high sequence and structural conservation. This region encodes several conserved structural RNA domains essential in key processes of the viral replication and infection cycles. This paper reports the inhibitory effects of catalytic antisense RNAs composed of two inhibitory RNA domains: an engineered ribozyme targeting the 5' UTR and a decoy or antisense domain of the dimerization initiation site (DIS). These chimeric molecules are able to cleave the HIV-1 5'UTR efficiently and prevent viral genome dimerization in vitro. Furthermore, catalytic antisense RNAs inhibited viral production up to 90% measured as p24 antigen levels in ex vivo assays. The use of chimeric RNA molecules targeting different domains represents an attractive antiviral strategy to be explored for the prevention of side effects from current drugs and of the rapid emergence of escape variants of HIV-1.
Assuntos
Fármacos Anti-HIV/síntese química , HIV-1/efeitos dos fármacos , RNA Catalítico/síntese química , Replicação Viral/efeitos dos fármacos , Regiões 5' não Traduzidas , Fármacos Anti-HIV/farmacologia , Dimerização , HIV-1/genética , HIV-1/fisiologia , Humanos , RNA Catalítico/farmacologia , RNA Catalítico/uso terapêutico , RNA Viral/efeitos dos fármacosRESUMO
OBJECTIVE: Severe acute respiratory syndrome (SARS) is a severe pulmonary infectious disease caused by a novel coronavirus. To develop an effective and specific medicine targeting the SARS-coronavirus (CoV), a chimeric DNA-RNA hammerhead ribozyme was designed and synthesized using a sequence homologous with the mouse hepatitis virus (MHV). METHOD: Chimeric DNA-RNA hammerhead ribozyme targeting MHV and SARS-CoV were designed and synthesized.To confirm its activity, in vitro cleavage reactions were performed with the synthesized ribozyme. Effects of the chimeric ribozyme were evaluated on multiplication of MHV. Effects of the chimeric ribozyme on expression of SARS-CoV were evaluated in cultured 3T3 cells. RESULT: The synthetic ribozyme cleaved the synthetic target MHV and SARS-CoV RNA into fragments of predicted length. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited multiplication of MHV in DBT cells by about 60%. The chimeric DNA-RNA hammerhead ribozyme targeting SARS-CoV significantly inhibited the expression of SARS-CoV RNA in 3T3 cells transfected with the recombinant plasmid. The chimeric DNA-RNA ribozyme targeting SARS-CoV significantly inhibited MHV viral activity and expression of recombinant SARS RNA in vitro. CONCLUSION: These findings indicate that the synthetic chimeric DNA-RNA ribozyme could provide a feasible treatment for SARS.
Assuntos
Antivirais/farmacologia , Vírus da Hepatite Murina/efeitos dos fármacos , RNA Catalítico/farmacologia , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/efeitos dos fármacos , Animais , Sequência de Bases , Linhagem Celular , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Hepatite Murina/genética , Vírus da Hepatite Murina/fisiologia , Estabilidade de RNA , RNA Catalítico/genética , RNA Viral/metabolismo , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/genética , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave/fisiologia , Replicação Viral/efeitos dos fármacosRESUMO
BACKGROUND: Ewing family tumours (EFT) are the second most common bone tumours in children and adolescents. In the majority of EFT, EWSR1-FLI1 (Ewing sarcoma breakpoint region 1-Friend leukaemia virus integration 1) fusion proteins can be detected and EWSR1-FLI1 substantially contributes to the malignant phenotype of EFT. Therefore, inactivation of EWSR1-FLI1 is an interesting strategy for EFT therapy. MATERIALS AND METHODS: A ribozyme with specificity for EWSR1-FLI1 was developed and the activity in vitro was investigated. Synthetic RNAs corresponding to EWSR1-FLI1 were used as substrates. In addition, the total RNA from EFT cells was used as substrate and the rapid amplification of cDNA ends method for the detection of the cleavage products was used. RESULTS: The ribozyme cleaved the synthetic RNA in a sequence specific manner with high efficiency in vitro. Furthermore, the expected cleavage products were detected after digestion of the total cellular RNA with this ribozyme. A point mutation in the catalytic centre of the ribozyme abolished enzymatic activity. CONCLUSION: The RNA corresponding to EWSR1-FLI1 is accessible for ribozyme mediated inactivation and ribozymes are able to cleave EWSR1-FLI1 specific RNA in the presence of a high background of normal cellular RNAs.
Assuntos
Neoplasias Ósseas/genética , Proteínas de Ligação a Calmodulina/genética , Proteína Proto-Oncogênica c-fli-1/genética , RNA Catalítico/farmacologia , RNA Mensageiro/genética , RNA Neoplásico/genética , Proteínas de Ligação a RNA/genética , Sarcoma de Ewing/genética , Sequência de Bases , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proteínas de Ligação a Calmodulina/metabolismo , Humanos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteína Proto-Oncogênica c-fli-1/metabolismo , RNA Catalítico/síntese química , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Proteína EWS de Ligação a RNA , Proteínas de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Sarcoma de Ewing/patologia , Homologia de Sequência do Ácido Nucleico , Células Tumorais CultivadasRESUMO
Antisense molecules and ribozymes capture the imagination with their promise of rational drug design and exquisite specificity. However, they are far more difficult to produce than was originally anticipated, and their ability to eliminate the function of a single gene has never been proven. Furthermore, a wide variety of unexpected non-antisense effects have come to light. Although some of these side effects will almost certainly have clinical value, they make it hard to produce drugs that act primarily through true antisense mechanisms and complicate the use of antisense compounds as research reagents. To minimize unwanted non-antisense effects, investigators are searching for antisense compounds and ribozymes whose target sites are particularly vulnerable to attack. This is a challenging quest.
Assuntos
Oligonucleotídeos Antissenso , Animais , Sequência de Bases , Desenho de Fármacos , Marcação de Genes , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , RNA Catalítico/farmacologiaRESUMO
Besides altering its own expression during cell transformation, Annexin A2 is upregulated during the progression of many cancer types and also plays key roles during viral infection and multiplication. Consequently, there has been great interest in Annexin A2 as a potential drug target. The successful design of efficient in vivo delivery systems constitutes an obstacle in full exploitation of antisense and RNA-cleaving technologies for the knock-down of specific targets. Efficiency is dependent on the method of delivery and accessibility of the target. Here, hairpin ribozymes and an antisense RNA against rat annexin A2 mRNA were tested for their efficiencies in a T7-driven coupled transcription/translation system. The most efficient ribozyme and antisense RNA were subsequently inserted into a retroviral vector under the control of a tRNA promoter, in a cassette inserted between retroviral Long Terminal Repeats for stable insertion into host DNA. The Phoenix package system based on defective retroviruses was used for virus-mediated gene transfer into PC12 cells. Cells infected with the ribozyme-containing particles died shortly after infection. However, the same ribozyme showed a very high catalytic effect in vitro in cell lysates, explained by its loose hinge helix 2 region. This principle can be transferred to other ribozymes, such as those designed to cleave the guide RNA in the CRISPR/Cas9 technology, as well as to target specific viral RNAs. Interestingly, efficient down-regulation of the expression of Annexin A2 by the antisense RNA resulted in up-regulation of Annexin A7 as a compensatory effect after several cell passages. Indeed, compensatory effects have previously been observed during gene knock-out, but not during knock-down of protein expression. This highlights the problems in interpreting the phenotypic effects of knocking down the expression of a protein. In addition, these data are highly relevant when considering the effects of the CRISPR/Cas9 approach.
Assuntos
Anexina A2/antagonistas & inibidores , Anexina A2/genética , Técnicas de Silenciamento de Genes/métodos , RNA Antissenso/farmacologia , RNA Catalítico/farmacologia , Animais , Anexina A2/biossíntese , Bovinos , Células PC12 , RatosRESUMO
Survivin is one of the most cancer-specific proteins overexpressed in almost all malignancies, but is nearly undetectable in most normal tissues in adults. Functionally, as a member of the inhibitor of apoptosis family, survivin has been shown to inhibit apoptosis and increase proliferation. The antiapoptotic function of survivin seems to be related to its ability to inhibit caspases directly or indirectly. Furthermore, the role of survivin in cell cycle division control is related to its role in the chromosomal passenger complex. Consistent with its determining role in these processes, survivin plays a crucial role in cancer progression and cancer cell resistance to anticancer drugs and ionizing radiation. On the basis of these findings, recently survivin has been investigated intensively as an ideal tumor biomarker. Thus, multiple molecular approaches such as use of the RNA interfering technique, antisense oligonucleotides, ribozyme, and small molecule inhibitors have been used to downregulate survivin regulation and inhibit its biological function consequently. In this review, all these approaches are explained and other compounds that induced apoptosis in different cell lines through survivin inhibition are also reported.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias/terapia , Survivina/metabolismo , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/efeitos da radiação , Biomarcadores Tumorais/antagonistas & inibidores , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Progressão da Doença , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Neoplasias/genética , Neoplasias/mortalidade , Neoplasias/patologia , Oligonucleotídeos Antissenso/farmacologia , Oligonucleotídeos Antissenso/uso terapêutico , Prognóstico , Interferência de RNA , RNA Catalítico/farmacologia , RNA Catalítico/uso terapêutico , Tolerância a Radiação/efeitos dos fármacos , Tolerância a Radiação/genética , Survivina/antagonistas & inibidores , Survivina/genética , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Expression of calcitonin (CT) and its receptor (CTR) is elevated in advanced prostate cancer (PC). Although the significance of CT-CTR axis in PC cell growth, invasion, and epithelial to mesenchymal transition has been established, its role in tumor metastasis has not been examined. To examine the role of CT-CTR axis in tumor metastasis, we employed stable CT-CTR activated and silenced system of three PC cell lines, LNCaP cells that lack endogenous CT, PC-3 cells that lack endogenous CTR, and PC-3M cells that co-express CT and CTR. Enforced expression of CT in LNCaP cells and CTR in PC-3 cells increased their ability to form orthotopic tumors and distant metastases in multiple organs. By contrast, silencing of CT expression in PC-3M cells not only reduced their tumorigenicity, but also completely abrogated their metastatic potential. To investigate the effect of in vivo silencing of CT expression on tumor growth, we employed recombinant adeno-associated virus (rAAV) to deliver anti-CT ribozymes in preexisting tumors of nude mice and large probasin promoter (LPB)-Tag transgenic mice. rAAV-CT(-) treatment not only abrogated the growth of pre-implanted tumors in nude mice, but also significantly reduced the growth of spontaneous tumors in LPB-Tag mice. Analysis of CT upregulated and silenced PC-3M transcriptomes revealed 105 genes affected by the modulation of CT expression. These CT signature genes generated survival, adhesion, pro-inflammatory, and pro-metastatic pathways. Added together, these data indicate a pivotal role for CT-CTR axis in PC metastasis and may serve as a potential therapeutic target for advanced PC.
Assuntos
Calcitonina/fisiologia , Inflamação/metabolismo , Metástase Neoplásica , Neoplasias da Próstata/metabolismo , Transdução de Sinais/fisiologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Adesão Celular/fisiologia , Dependovirus/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos Transgênicos , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias da Próstata/patologia , RNA Catalítico/farmacologia , Receptores da Calcitonina/metabolismo , Transfecção , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Stathmin is one of the key regulators of the microtubule cytoskeleton and the mitotic spindle in eukaryotic cells. It is expressed at high levels in a wide variety of human cancers and may provide an attractive target for cancer therapy. We had previously shown that stathmin inhibition results in the abrogation of the malignant phenotype. The microtubule-interfering drug, taxol, has both antitumorigenic and antiangiogenic properties. We had also shown that the antitumor activities of taxol and stathmin inhibition are synergistic. We hypothesized that taxol and stathmin inhibition may also have synergistic antiangiogenic activities. A replication-deficient bicistronic adenoviral vector that coexpresses green fluorescent protein and an anti-stathmin ribozyme was used to target stathmin mRNA. Exposure of endothelial cells to anti-stathmin adenovirus alone resulted in a dose-dependent inhibition of proliferation, migration, and differentiation into capillary-like structures. This inhibition was markedly enhanced by exposure of transduced endothelial cells to very low concentrations of taxol, which resulted in a virtually complete loss of proliferation, migration, and differentiation of endothelial cells. In contrast, exposure of nontransduced endothelial cells to taxol alone resulted in a modest inhibition of proliferation, migration, and differentiation. Our detailed analysis showed that the antiangiogenic effects of the combination of stathmin inhibition and taxol exposure are synergistic. Our studies also showed that the mechanism of this synergistic interaction is likely to be mediated through the stabilization of microtubules. Thus, this novel combination may provide an attractive therapeutic strategy that combines a synergistic antitumor activity with a synergistic antiangiogenic activity.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Paclitaxel/farmacologia , RNA Catalítico/farmacologia , Estatmina/antagonistas & inibidores , Adenoviridae/genética , Células Cultivadas , Terapia Combinada , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Humanos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neovascularização Fisiológica , RNA Catalítico/genética , Estatmina/genética , Estatmina/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacos , Veias Umbilicais/metabolismoRESUMO
PURPOSE: WAVE1 belongs to the Wiskott-Aldrich syndrome family of proteins, which have an integral part in cell motility, a crucial step in cancer metastasis. We investigated the expression pattern and the effects of manipulating endogenous WAVE1 expression in prostate cancer cells. MATERIALS AND METHODS: WAVE1 protein expression in normal and cancer specimens, and in prostate cell lines was assessed using immunohistochemistry and reverse transcriptase-polymerase chain reaction, respectively. Hammerhead ribozyme transgenes were synthesized and cloned into the mammalian expression vector pEF6/V5-His TOPO TA, and transfected by electroporation into PC-3(DeltaW1R1/2) and DU-145(DeltaW1R1/2) cell lines. In vitro invasion, adhesion and growth assays were used to assess the impact of WAVE1 knockdown. RESULTS: Immunohistochemistry of prostate tissue specimens showed that the cytoplasm of cancer cells had stronger staining than normal epithelium. Reverse transcriptase-polymerase chain reaction for WAVE1 showed strong expression in the PC-3 (European Collection of Cell Cultures, Salisbury, United Kingdom) and DU-145 (ATCC(R)) cell lines. WAVE1 knockdown was associated with a significant decrease in invasion but not in adhesion. The mean +/- SEM number of invading PC-3(DeltaW1R1) and PC-3(DeltaW1R2) cells was 7.27 +/- 0.38 and 6 +/- 0.29, respectively, compared to 12.27 +/- 0.42 PC-3(WT) cells (p <0.001). Similarly the mean number of invading DU-145(DeltaW1R1) and DU-145(DeltaW1R2) cells was 9.20 +/- 0.70 and 11.60 +/- 0.84 compared to 14.80 +/- 0.24 DU-145(WT) cells (p <0.001). CONCLUSIONS: To our knowledge this is the first report of the expression pattern of WAVE1 in prostate cancer cell lines and tissues, and the functional impact of WAVE1 knockdown. Further investigations to assess WAVE1 as a potential target for anti-metastasis therapy must be explored.
Assuntos
Invasividade Neoplásica/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Análise de Variância , Western Blotting , Linhagem Celular Tumoral , Eletroforese em Gel de Ágar , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Invasividade Neoplásica/patologia , RNA Catalítico/farmacologia , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e Especificidade , Carga Tumoral/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Neuronal migrations along glial fibers provide a primary pathway for the formation of cortical laminae. To examine the mechanisms underlying glial-guided migration, we analyzed the dynamics of cytoskeletal and signaling components in living neurons. Migration involves the coordinated two-stroke movement of a perinuclear tubulin 'cage' and the centrosome, with the centrosome moving forward before nuclear translocation. Overexpression of mPar6alpha disrupts the perinuclear tubulin cage, retargets PKCzeta and gamma-tubulin away from the centrosome, and inhibits centrosomal motion and neuronal migration. Thus, we propose that during neuronal migration the centrosome acts to coordinate cytoskeletal dynamics in response to mPar6alpha-mediated signaling.
Assuntos
Astrócitos/fisiologia , Movimento Celular/fisiologia , Cerebelo/citologia , Neurônios/fisiologia , Proteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Animais Recém-Nascidos , Movimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Polaridade Celular/fisiologia , Extensões da Superfície Celular/efeitos dos fármacos , Extensões da Superfície Celular/fisiologia , Células Cultivadas , Centrossomo/metabolismo , Clonagem Molecular/métodos , Técnicas de Cocultura/métodos , Diagnóstico por Imagem/métodos , Complexo Dinactina , Vetores Genéticos/fisiologia , Proteína Glial Fibrilar Ácida/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica/métodos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/virologia , Técnicas de Cultura de Órgãos , Fotodegradação , Proteína Quinase C/metabolismo , Proteína Quinase C-épsilon , Proteínas/genética , Proteínas/metabolismo , RNA Catalítico/farmacologia , Fatores de Tempo , Transfecção/métodos , Tubulina (Proteína)/metabolismo , Gravação de Videoteipe/métodos , Zidovudina/metabolismoRESUMO
The ability of certain enzymatic RNA molecules, or ribozymes, to site-specifically cleave other RNA molecules opens new vistas in gene therapy. Ribozymes can be designed to target specifically a particular mRNA and inhibit protein expression, permitting 'anti-gene' therapy. Here, we describe the progress towards developing ribozymes for use in gene therapy applications. Significant advances have been made in understanding ribozyme transcription unit design and the first clinical tests of ribozyme safety in humans are soon to be initiated.