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1.
Cancer Lett ; 548: 215882, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-35988818

RESUMO

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis.


Assuntos
Neoplasias , Biomarcadores , Carboxipeptidase H/genética , Carboxipeptidase H/metabolismo , Linhagem Celular Tumoral , Humanos , Masculino , Neoplasias/genética , RNA Mensageiro/genética , RNA Interferente Pequeno
2.
Int J Mol Sci ; 23(6)2022 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-35328535

RESUMO

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.


Assuntos
Carcinoma Hepatocelular , Exossomos , Neoplasias Hepáticas , MicroRNAs , Carboxipeptidase H/genética , Carcinogênese/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Ciclina D1/metabolismo , Exossomos/metabolismo , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , MicroRNAs/genética , Fenótipo , Projetos Piloto , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
3.
Elife ; 102021 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-34854810

RESUMO

The immune mechanisms underlying hypersensitivity to pain after nerve injury are different in male and female mice.


Assuntos
Microglia , Traumatismos dos Nervos Periféricos , Animais , Feminino , Masculino , Camundongos , Dor , Medula Espinal
4.
Artigo em Inglês | MEDLINE | ID: mdl-34368812

RESUMO

Exosomes are a subtype of extracellular vesicles released from different cell types including those in the nervous system, and are enriched in a variety of bioactive molecules such as RNAs, proteins and lipids. Numerous studies have indicated that exosomes play a critical role in many physiological and pathological activities by facilitating intercellular communication and modulating cells' responses to external environments. Particularly in the central nervous system, exosomes have been implicated to play a role in many neurological disorders such as abnormal neuronal development, neurodegenerative diseases, epilepsy, mental disorders, stroke, brain injury and brain cancer. Since exosomes recapitulate the characteristics of the parental cells and have the capacity to cross the blood-brain barrier, their cargo can serve as potential biomarkers for early diagnosis and clinical assessment of disease treatment. In this review, we describe the latest findings and current knowledge of the roles exosomes play in various neurological disorders and brain cancer, as well as their application as promising biomarkers. The potential use of exosomes to deliver therapeutic molecules to treat diseases of the central nervous system is also discussed.

5.
Nat Commun ; 12(1): 2471, 2021 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-33931636

RESUMO

In vertebrates, motor control relies on cholinergic neurons in the spinal cord that have been extensively studied over the past hundred years, yet the full heterogeneity of these neurons and their different functional roles in the adult remain to be defined. Here, we develop a targeted single nuclear RNA sequencing approach and use it to identify an array of cholinergic interneurons, visceral and skeletal motor neurons. Our data expose markers for distinguishing these classes of cholinergic neurons and their rich diversity. Specifically, visceral motor neurons, which provide autonomic control, can be divided into more than a dozen transcriptomic classes with anatomically restricted localization along the spinal cord. The complexity of the skeletal motor neurons is also reflected in our analysis with alpha, gamma, and a third subtype, possibly corresponding to the elusive beta motor neurons, clearly distinguished. In combination, our data provide a comprehensive transcriptomic description of this important population of neurons that control many aspects of physiology and movement and encompass the cellular substrates for debilitating degenerative disorders.


Assuntos
Neurônios Colinérgicos/citologia , Interneurônios/citologia , Neurônios Motores/citologia , Análise de Célula Única/métodos , Núcleo Solitário/metabolismo , Medula Espinal/metabolismo , Transcriptoma/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Neurônios Colinérgicos/metabolismo , Neurônios Colinérgicos/fisiologia , Feminino , Hibridização In Situ , Interneurônios/metabolismo , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Neurônios Motores/fisiologia , RNA-Seq , Medula Espinal/citologia , Medula Espinal/fisiologia
6.
Int J Mol Sci ; 20(22)2019 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-31731578

RESUMO

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.


Assuntos
Carboxipeptidase H/metabolismo , Proliferação de Células/fisiologia , Neoplasias Pancreáticas/metabolismo , Carboxipeptidase H/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Expressão Gênica/genética , Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Imuno-Histoquímica , Imunoprecipitação , Neoplasias Pancreáticas/genética , Plasmídeos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Eur J Immunol ; 46(1): 154-66, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26443873

RESUMO

Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy.


Assuntos
Dependovirus/imunologia , Terapia Genética/efeitos adversos , Vetores Genéticos/efeitos adversos , Fígado/imunologia , NF-kappa B/antagonistas & inibidores , Poli(ADP-Ribose) Polimerases/imunologia , Animais , Western Blotting , Modelos Animais de Doenças , Regulação para Baixo , Sinergismo Farmacológico , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática , Terapia Genética/métodos , Vetores Genéticos/imunologia , Células HeLa , Hemofilia B/terapia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , Poli(ADP-Ribose) Polimerase-1 , Reação em Cadeia da Polimerase em Tempo Real , Transdução Genética , Transfecção
8.
Curr Pharm Biotechnol ; 14(12): 1072-82, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24678652

RESUMO

Gene therapy has become a clinical reality as demonstrated by remarkable benefits seen in Phase I/II clinical trials for hemophilia B, lipoprotein lipase deficiency and Leber's congenital amarousis. The choice of, and the improved understanding in vector characteristics have contributed significantly to this success. The adeno-associated virus (AAV) vectors used in these trials have been long known to be relatively safe and efficacious. However, certain factors, most notably host immunity to the vector, prevent their widespread use. In patients who have pre-existing antibodies to AAV, these vectors will be rapidly cleared. Administration of a relatively high initial dose of vector to achieve and sustain a higher margin of therapeutic benefit is limited by concerns of vector dose-dependent T cell response. Frequent vector administration necessitated by the non-integrating nature of the virus is difficult due to the variable, yet significant host immunological memory. Thus generation of AAV vectors that are immunologically inert is pivotal for the long-term success with this promising vector system. Several strategies, that aim targeted disruption of antigenic sites or those that chemically modify the vectors have been proposed for host immune evasion. While these approaches have been successful in the pre-clinical model systems, this continues to be a field of intense experimentation and constant improvisation due to limited information available on vector immunology or data from human studies. This review forms a comprehensive report on current strategies available to generate immunologically inert AAV vectors and their potential in mediating longterm gene transfer.


Assuntos
Dependovirus/genética , Dependovirus/imunologia , Técnicas de Transferência de Genes , Terapia Genética , Vetores Genéticos/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Capsídeo/química , Capsídeo/imunologia , Evolução Molecular Direcionada , Humanos , Memória Imunológica , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Linfócitos T/imunologia
9.
Rev Med Virol ; 23(6): 399-413, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24023004

RESUMO

AAV-based gene transfer protocols have shown remarkable success when directed to immune-privileged sites such as for retinal disorders like Lebers congenital amaurosis. In contrast, AAV-mediated gene transfer into liver or muscle tissue for diseases such as hemophilia B, α1 anti-trypsin deficiency and muscular dystrophy has demonstrated a decline in gene transfer efficacy over time. It is now known that in humans, AAV triggers specific pathways that recruit immune sensors. These factors initiate an immediate reaction against either the viral capsid or the vector encoded protein as part of innate immune response or to produce a more specific adaptive response that generates immunological memory. The vector-transduced cells are then rapidly destroyed due to this immune activation. However, unlike other viral vectors, AAV is not immunogenic in murine models. Its immunogenicity becomes apparent only in large animal models and human subjects. Moreover, humans are natural hosts to AAV and exhibit a high seroprevalence against AAV vectors. This limits the widespread application of AAV vectors into patients with pre-existing neutralising antibodies or memory T cells. To address these issues, various strategies are being tested. Alternate serotype vectors (AAV1-10), efficient expression cassettes, specific tissue targeting, immune-suppression and engineered capsid variants are some approaches proposed to minimise this immune stimulation. In this review, we have summarised the nature of the immune response documented against AAV in various pre-clinical and clinical settings and have further discussed the strategies to evade them.


Assuntos
Dependovirus/imunologia , Portadores de Fármacos , Terapia Genética/métodos , Vetores Genéticos/imunologia , Imunidade Adaptativa , Infecções por Adenoviridae/imunologia , Animais , Anticorpos Antivirais/sangue , Dependovirus/genética , Humanos , Imunidade Inata , Estudos Soroepidemiológicos
10.
Hum Gene Ther Methods ; 24(2): 80-93, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23379478

RESUMO

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T→alanine [A] or 9 K→arginine [R] single mutant or 2 double K→R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T→A and 7 K→R vectors were significantly higher (~63-90%) than the AAV2-WT vectors (~30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated ~8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.


Assuntos
Substituição de Aminoácidos , Proteínas do Capsídeo/genética , Dependovirus/genética , Vetores Genéticos/genética , Transdução Genética , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Sequência Conservada , Dependovirus/classificação , Dependovirus/imunologia , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Vetores Genéticos/administração & dosagem , Vetores Genéticos/química , Vetores Genéticos/imunologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Lisina , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Alinhamento de Sequência , Serina , Treonina , Ubiquitinação , Carga Viral
11.
PLoS One ; 8(1): e53845, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23320106

RESUMO

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold) and PERK (up to 8 fold) genes 12-48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.


Assuntos
Dependovirus/genética , Resposta a Proteínas não Dobradas/genética , Fator 6 Ativador da Transcrição/genética , Animais , Dependovirus/classificação , Dependovirus/imunologia , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Vetores Genéticos/imunologia , Células HeLa , Humanos , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Transdução Genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/genética
12.
Cytotechnology ; 62(5): 389-402, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20835846

RESUMO

Adipose tissue is an easily accessible and abundant source of stem cells. Adipose stem cells (ASCs) are currently being researched as treatment options for repair and regeneration of damaged tissues. The standard culture conditions used for expansion of ASCs contain fetal bovine serum (FBS) which is undefined, could transmit known and unknown adventitious agents, and may cause adverse immune reactions. We have described a novel culture condition which excludes the use of FBS and characterised the resulting culture. Human ASCs were cultured in the novel culture medium, which included complement protein C3. These cultures, called C-ASCs, were compared with ASCs cultured in medium supplemented with FBS. Analysis of ASCs for surface marker profile, proliferation characteristics and differentiation potential indicated that the C-ASCs were similar to ASCs cultured in medium containing FBS. Using a specific inhibitor, we show that C3 is required for the survival of C-ASCs. This novel composition lends itself to being developed into a defined condition for the routine culture of ASCs for basic and clinical applications.

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