RESUMEN
The human cytomegalovirus (HCMV), one of eight human herpesviruses, establishes lifelong latent infections in most people worldwide. Primary or reactivated HCMV infections cause severe disease in immunosuppressed patients and congenital defects in children. There is no vaccine for HCMV, and the currently approved antivirals come with major limitations. Most approved HCMV antivirals target late molecular processes in the viral replication cycle including DNA replication and packaging. "Bright and early" events in HCMV infection have not been exploited for systemic prevention or treatment of disease. Initiation of HCMV replication depends on transcription from the viral major immediate-early (IE) gene. Alternative transcripts produced from this gene give rise to the IE1 and IE2 families of viral proteins, which localize to the host cell nucleus. The IE1 and IE2 proteins are believed to control all subsequent early and late events in HCMV replication, including reactivation from latency, in part by antagonizing intrinsic and innate immune responses. Here we provide an update on the regulation of major IE gene expression and the functions of IE1 and IE2 proteins. We will relate this insight to experimental approaches that target IE gene expression or protein function via molecular gene silencing and editing or small chemical inhibitors.
Asunto(s)
Citomegalovirus/genética , Regulación Viral de la Expresión Génica , Genes Inmediatos-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Antivirales/uso terapéutico , Sistemas CRISPR-Cas , Citomegalovirus/efectos de los fármacos , Infecciones por Citomegalovirus/terapia , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Interferencia de ARN , ARN Catalítico/efectos de los fármacos , ARN Catalítico/genética , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Herpes simplex virus 1 (HSV-1) has infected more than 80% of the population. Reactivation of the virus causes diseases ranging in severity from benign cold sores to fatal encephalitis. Current treatments involve viral DNA replication inhibitors, but the emergence of drug-resistant mutants is observed frequently, highlighting the need for novel antiviral therapies. Infected cell protein 0 (ICP0) of HSV-1 is encoded by an immediate early gene and plays a fundamental role during infection, because it enables viral gene expression and blocks antiviral responses. One mechanism by which ICP0 functions is through an E3 ubiquitin ligase activity that induces the degradation of targeted proteins. A ΔICP0 virus or mutants with deficiencies in E3 ligase activity cannot counteract beta interferon (IFN-ß)-induced restriction of viral infection, are highly immunogenic, are avirulent, and fail to spread. Thus, small molecules interfering with essential and conserved ICP0 functions are expected to compromise HSV-1 infection. We have developed a high-throughput screening assay, based on the autoubiquitination properties of ICP0, to identify small-molecule inhibitors of ICP0 E3 ubiquitin ligase activity. Through a pilot screening procedure, we identified nine compounds that displayed dose-dependent inhibitory effects on ICP0 but not on Mdm2, a control E3 ubiquitin ligase. Following validation, one compound displayed ICP0-dependent inhibition of HSV-1 infection. This compound appeared to bind ICP0 in a cellular thermal shift assay, it blocked ICP0 self-elimination, and it blocked wild-type but not ICP0-null virus gene expression. This scaffold displays specificity and could be used to develop optimized ICP0 E3 ligase inhibitors.IMPORTANCE Since acyclovir and its derivatives were launched for herpesviruses control almost four decades ago, the search for novel antivirals has waned. However, as human life expectancy has increased, so has the number of immunocompromised individuals who receive prolonged treatment for HSV recurrences. This has led to an increase in unresponsive patients due to acquired viral drug resistance. Thus, novel treatments need to be explored. Here we explored the HSV-1 ICP0 E3 ligase as a potential antiviral target because (i) ICP0 is expressed before virus replication, (ii) it is essential for infection in vivo, (iii) it is required for efficient reactivation of the virus from latency, (iv) inhibition of its E3 ligase activity would sustain host immune responses, and (v) it is shared by other herpesviruses. We report a compound that inhibits HSV-1 infection in an ICP0-dependent manner by inhibiting ICP0 E3 ligase activity.
Asunto(s)
Herpesvirus Humano 1/efectos de los fármacos , Herpesvirus Humano 1/metabolismo , Ensayos Analíticos de Alto Rendimiento , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Línea Celular , Replicación del ADN , Regulación Viral de la Expresión Génica , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Proteínas Inmediatas-Precoces/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: Synovial sarcoma (SS) is an aggressive soft-tissue sarcoma with a poor prognosis owing to its resistance to radiation and chemotherapy. Thus, novel therapeutic strategies for SS are urgently required. Anlotinib, a new oral tyrosine kinase inhibitor, is designed to primarily inhibit multi-targets in vasculogenesis and angiogenesis. This study was designed to characterize its antitumor efficacy and possible mechanism in patients with advanced refractory synovial sarcoma. METHODS: Anlotinib's antitumor effect was evaluated in vivo and vitro. Downstream targets of anlotinib in treating synovial sarcoma were analyzed through microarray assay. Cell proliferation and apoptosis analyses were performed to evaluate the impact of candidate downstream gene depletion in synovial sarcoma cells. Microarray assay were carried out to investigate potential signal network related with candidate downstream gene. RESULTS: Anlotinib significantly suppresses synovial sarcoma proliferation in PDTX model and cell lines. Additionally, GINS1 (also named as PSF1, Partner of SLD Five 1), rather than other conventional gene target, was demonstrated to be a vital target of anlotinib's antitumor effect in synovial sarcoma through microarray assay. Expression of GINS1 was remarkably higher in synovial sarcoma tumor samples and related with poor outcome. Knockdown of GINS1 expression could remarkably inhibit proliferation and promote apoptosis in vitro. Meanwhile, through microarray assay, CITED2, EGR1, SGK1 and SPP1 were identified and further validated by qPCR/WB as downstream targets of GINS1. CONCLUSION: Anlotinib might suppress proliferation of SS through a novel downstream GINS1-regulated network which plays a vital function in SS proliferation and also demonstrated that targeting the GINS1-regulated signal pathway could be a potential strategy for management of SS.
Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Proteínas de Unión al ADN/efectos de los fármacos , Indoles/uso terapéutico , Proteínas de Neoplasias/efectos de los fármacos , Inhibidores de Proteínas Quinasas/uso terapéutico , Quinolinas/uso terapéutico , Sarcoma Sinovial/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Neoplasias Óseas/genética , Proliferación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Progresión de la Enfermedad , Proteína 1 de la Respuesta de Crecimiento Precoz/efectos de los fármacos , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteopontina/efectos de los fármacos , Osteopontina/genética , Osteopontina/metabolismo , Análisis por Matrices de Proteínas , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/análisis , Proteínas Represoras/efectos de los fármacos , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Sarcoma Sinovial/genética , Transactivadores/efectos de los fármacos , Transactivadores/genética , Transactivadores/metabolismoRESUMEN
Depression is a common psychiatric disorder that affects almost 10% of children and adolescents worldwide. Numerous synthetic chemical antidepressants used to treat depression have adverse side effects. Therefore, new therapeutic approaches for depression treatment are urgently needed. Leonurus cardiaca has recently been shown to be effective for the treatment of nervous system diseases such as depression, but its mechanism is not clear. In this study, we aimed to reveal the mechanism underlying leonurine's antidepressant activity. Leonurine was used to treat corticosterone-induced PC12 cells to examine its effect on neurite outgrowth and neurotrophic factors after treatment with the inhibitor of glucocorticoid receptor (GR) and serum-inducible and glucocorticoid-inducible kinase 1 (SGK1). Methyl thiazolyl tetrazolium assays were used to evaluate the viability of cells. High content analysis was used to detect cell area, total neurite length, maximum neurite length, and expression of GR, SGK1, brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and B-cell lymphoma-2 (BCL-2). The results showed that leonurine increased cell viability in a concentration-dependent manner, with the maximal prosurvival effect at 60 µM. Leonurine increased cell area, total neurite length, and maximum neurite length of corticosterone-induced PC12 cells, increased the expression of GR, BDNF, NT-3, and BCL-2, and decreased the expression of SGK1. After treatment with GR inhibitor RU486, the expressions of GR, BDNF, NT-3, and BCL-2 were significantly decreased and SGK1 was increased. In contrast, treatment with GSK650394 had the opposite effect of RU486. Our data indicate that leonurine promotes neurite outgrowth and neurotrophic activity in cultured PC12 cells, and its potential mechanism may involve the GR/SGK1 signaling pathway.
Asunto(s)
Antidepresivos/farmacología , Ácido Gálico/análogos & derivados , Proyección Neuronal/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Ácido Gálico/farmacología , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Células PC12 , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/metabolismoRESUMEN
Nitrones (e.g. α-phenyl-N-tert-butyl nitrone; PBN) are cerebroprotective in experimental stroke. Free radical trapping is their proposed mechanism. As PBN has low radical trapping potency, we tested Sgk1 induction as another possible mechanism. PBN was injected (100 mg/kg, i.p.) into adult male rats and mice. Sgk1 was quantified in cerebral tissue by microarray, quantitative RT-PCR and western analyses. Sgk1+/+ and Sgk1-/- mice were randomized to receive PBN or saline immediately following transient (60 min) occlusion of the middle cerebral artery. Neurological deficit was measured at 24 h and 48 h and infarct volume at 48 h post-occlusion. Following systemic PBN administration, rapid induction of Sgk1 was detected by microarray (at 4 h) and confirmed by RT-PCR and phosphorylation of the Sgk1-specific substrate NDRG1 (at 6 h). PBN-treated Sgk1+/+ mice had lower neurological deficit ( p < 0.01) and infarct volume ( p < 0.01) than saline-treated Sgk1+/+ mice. PBN-treated Sgk1-/- mice did not differ from saline-treated Sgk1-/- mice. Saline-treated Sgk1-/- and Sgk1+/+ mice did not differ. Brain Sgk3:Sgk1 mRNA ratio was 1.0:10.6 in Sgk1+/+ mice. Sgk3 was not augmented in Sgk1-/- mice. We conclude that acute systemic treatment with PBN induces Sgk1 in brain tissue. Sgk1 may play a part in PBN-dependent actions in acute brain ischemia.
Asunto(s)
Óxidos N-Cíclicos/uso terapéutico , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Animales , Encéfalo/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Óxidos N-Cíclicos/farmacología , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/farmacología , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Masculino , Ratones , Ratones Noqueados , Óxidos de Nitrógeno/farmacología , Óxidos de Nitrógeno/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/farmacología , Ratas , Accidente Cerebrovascular/tratamiento farmacológico , Activación Transcripcional/efectos de los fármacosRESUMEN
BACKGROUND: Recent studies reported increased (Pro)renin receptor (PRR) expression during low-salt intake. We hypothesized that PRR plays a role in regulation of renal epithelial sodium channel (ENaC) through serum and glucocorticoid-inducible kinase isoform 1 (SGK-1)-neural precursor cell expressed, developmentally downregulated 4-2 (Nedd4-2) signaling pathway. METHOD: Male Sprague-Dawley rats on normal-sodium diet and mouse renal inner medullary collecting duct cells treated with NaCl at 130 âmmol/l (normal salt), or 63 âmmol/l (low salt) were studied. PRR and α-ENaC expressions were evaluated 1 week after right uninephrectomy and left renal interstitial administration of 5% dextrose, scramble shRNA, or PRR shRNA (nâ=â6 each treatment). RESULTS: In-vivo PRR shRNA significantly reduced expressions of PRR throughout the kidney and α-ENaC subunits in the renal medulla. In inner medullary collecting duct cells, low salt or angiotensin II (Ang II) augmented the mRNA and protein expressions of PRR (Pâ<â0.05), SGK-1 (Pâ<â0.05), and α-ENaC (Pâ<â0.05). Low salt or Ang II increased the phosphorylation of Nedd4-2. In cells treated with low salt or Ang II, PRR siRNA significantly downregulated the mRNA and protein expressions of PRR (Pâ<â0.05), SGK-1 (Pâ<â0.05), and α-ENaC expression (Pâ<â0.05). CONCLUSION: We conclude that PRR contributes to the regulation of α-ENaC via SGK-1-Nedd4-2 signaling pathway.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio/efectos de los fármacos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Riñón/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Receptores de Superficie Celular/efectos de los fármacos , Cloruro de Sodio/farmacología , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Angiotensina II/farmacología , Animales , Células Cultivadas , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Riñón/metabolismo , Túbulos Renales Colectores/citología , Masculino , Ratones , Ubiquitina-Proteína Ligasas Nedd4 , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Renina/metabolismo , Sistema Renina-Angiotensina/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Ubiquitina-Proteína Ligasas/metabolismo , Vasoconstrictores/farmacología , Receptor de ProreninaRESUMEN
A high-salt diet (HSD) in humans is linked to a number of complications, including hypertension and cardiovascular events. Whether a HSD affects the immune response in transplantation is unknown. Using a murine transplantation model, we investigated the effect of NaCl on the alloimmune response in vitro and in vivo. Incremental NaCl concentrations in vitro augmented T cell proliferation in the settings of both polyclonal and allospecific stimulation. Feeding a HSD to C57BL/6 wild-type recipients of bm12 allografts led to accelerated cardiac allograft rejection, despite similar mean BP and serum sodium levels in HSD and normal salt diet (NSD) groups. The accelerated rejection was associated with a reduction in the proportion of CD4(+)Foxp3(+) regulatory T cells (Tregs) and a significant decrease in Treg proliferation, leading to an increased ratio of antigen-experienced CD4(+) T cells to Tregs in mice recipients of a HSD compared with mice recipients of a NSD. Because serum- and glucocorticoid-regulated kinase-1 (SGK1) has been proposed as a potential target of salt in immune cells, we fed a HSD to CD4(Cre)SGK1(fl/fl) B6-transplanted recipients and observed abrogation of the deleterious effect of a HSD in the absence of SGK1 on CD4(+) cells. In summary, we show that NaCl negatively affects the regulatory balance of T cells in transplantation and precipitates rejection in an SGK1-dependent manner.
Asunto(s)
Rechazo de Injerto/inducido químicamente , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/fisiología , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/fisiología , Cloruro de Sodio Dietético/efectos adversos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/fisiología , Animales , Ratones , Ratones Endogámicos C57BL , Factores de TiempoRESUMEN
Human cytomegalovirus (HCMV) is a major cause of morbidity and mortality in newborn infants, immunocompromised individuals with HIV/AIDS and organ transplant recipients. In order to identify a novel antiviral candidate for HCMV-related diseases, crude ethanol extracts from plants were screened for their potential inhibitory activity on HCMV replication in vitro. Ethanol (70%) extract of Elaeocarpus sylvestris leaves (ESE) markedly inhibited the replication of the HCMV Towne strain without exhibiting any significant adverse effects on the viability of human foreskin fibroblasts (HFF). In addition, ESE significantly downregulated HCMV immediate early (IE) gene expression. Taken together, this is the first study, to the best of our knowledge, demonstrating that ESE has a potent antiviral activity against HCMV by downregulating HCMV IE gene expression and replication.
Asunto(s)
Citomegalovirus/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Extractos Vegetales/farmacología , Replicación Viral/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citomegalovirus/patogenicidad , Elaeocarpaceae/química , Fibroblastos/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Extractos Vegetales/químicaRESUMEN
Hypertension is a leading cause of morbidity and mortality worldwide, and disordered sodium balance has long been implicated in its pathogenesis. Aldosterone is perhaps the key regulator of sodium balance and thus blood pressure. The sodium chloride cotransporter (NCC) in the distal convoluted tubule of the kidney is a major site of sodium reabsorption and plays a key role in blood pressure regulation. Chronic exposure to aldosterone increases NCC protein expression and function. However, more acute effects of aldosterone on NCC are unknown. In our salt-abundant modern society where chronic salt deprivation is rare, understanding the acute effects of aldosterone is critical. Here, we examined the acute effects (12-36 h) of aldosterone on NCC in the rodent kidney and in a mouse distal convoluted tubule cell line. Studies demonstrated that aldosterone acutely stimulated NCC activity and phosphorylation without affecting total NCC abundance or surface expression. This effect was dependent upon the presence of the mineralocorticoid receptor and serum- and glucocorticoid-regulated kinase 1 (SGK1). Furthermore, STE20/SPS-1-related proline/alanine-rich kinase (SPAK) phosphorylation also increased, and gene silencing of SPAK eliminated the effect of aldosterone on NCC activity. Aldosterone administration via a minipump in adrenalectomized rodents confirmed an increase in NCC phosphorylation without a change in NCC total protein. These data indicate that acute aldosterone-induced SPAK-dependent phosphorylation of NCC increases individual transporter activity.
Asunto(s)
Aldosterona/farmacología , Proteínas Serina-Treonina Quinasas/fisiología , Simportadores del Cloruro de Sodio/fisiología , Adrenalectomía , Animales , Células Cultivadas , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Masculino , Ratones , Fosforilación/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Mineralocorticoides/efectos de los fármacos , Simportadores del Cloruro de Sodio/efectos de los fármacos , Miembro 3 de la Familia de Transportadores de Soluto 12/efectos de los fármacosRESUMEN
BACKGROUND: Genital herpes, caused by herpes simplex virus type-2 (HSV-2), is a recurrent, lifelong disease affecting tens of millions of people in the USA alone. HSV-2 can be treated therapeutically with acyclovir (ACV) and its derivatives; however, no treatment can prevent HSV reactivation. Novel topical anti-HSV microbicides are much needed to reduce HSV-2 transmission and to treat primary or reactivated infections, especially for ACV-resistant strains. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are single-stranded DNA analogues that enter cells readily and can reduce target gene expression through steric blockage of complementary messenger RNA (mRNA). METHODS: We investigated the antiviral activities of PPMOs targeted to the translation start-site regions of the mRNA for two HSV-2 immediate early genes, immediate early protein (ICP)0 and ICP27, and two early genes, unique long gene (UL)30 and UL39. RESULTS: In cell cultures, PPMOs targeting ICP0 or ICP27 mRNA were found to be highly effective against two strains of HSV-2, one of which was ACV-resistant. In vivo, daily topical applications of up to 1 mM ICP27 PPMO caused no gross or microscopic damage to the genital tract of uninfected BALB/c mice or cotton rats. Cotton rats receiving topical application of ICP27 PPMO 24 h after HSV-2 inoculation showed a reduction in genital lesions and a 37.5% reduction in mortality at 14 days post-infection. Mice receiving topical application of 100 µM of an ICP27 and ICP0 PPMO combination before HSV-2 inoculation had no detectable viral replication in the genital tract at 3-5 days post-infection. CONCLUSIONS: These results demonstrate that topically applied PPMOs hold promise as candidate antiviral microbicides against HSV-2 genital infection.
Asunto(s)
Herpes Genital/tratamiento farmacológico , Herpesvirus Humano 2/efectos de los fármacos , Morfolinas/farmacología , Replicación Viral/efectos de los fármacos , Aciclovir/farmacología , Aciclovir/uso terapéutico , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , Chlorocebus aethiops , Modelos Animales de Enfermedad , Farmacorresistencia Viral , Femenino , Herpes Genital/virología , Herpesvirus Humano 2/genética , Herpesvirus Humano 2/fisiología , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Ratones , Ratones Endogámicos BALB C , Morfolinas/síntesis química , Morfolinas/uso terapéutico , Morfolinos , Péptidos/metabolismo , Prevención Secundaria , Sigmodontinae , Células Vero , Proteínas Virales/efectos de los fármacos , Proteínas Virales/genética , Activación Viral/efectos de los fármacosRESUMEN
In order to develop a gene therapy to human cytomegalovirus (HCMV), RNA interference (RNAi) was employed to inhibit the expression of HCMV UL122 gene in vitro. Recombinant vector pUL122-EGFP, which expressed UL122-EGFP fusion protein, and recombinant vectors psi122-1, psi122-2 and psi122-3, which expressed small interfering RNAs (siRNAs) targeted to UL122 were contransfected into AD293 cells. The fluorescence signal of pUL122-EGFP was greatly suppressed by psi122-1 and psi122-2, with an inhibitory rate of 82.0% +/- 1.0% and 79.5% +/- 2.5%, respectively. The mRNA of pUL122-EGFP of the cells transfected with psi122-1 and psi122-2 was decreased 97.3% +/- 0.6% and 98.0% +/- 0.1%, respectively. Vector psi122-3 showed a slightly low suppression rate. Therefore, it may be concluded that plasmids encoding siRNAs targeted to UL122 is able to in vitro reduce markedly the expression of UL122-EGFP. And it is very likely that the psi122-1 and psi122-2 are potentially efficacious siRNAs in the gene therapy of HCMV infection in vivo, in which further investigations are required. This study is expected to greatly facilitate the use of the RNAi technology for the anti-HCMV studies.
Asunto(s)
Citomegalovirus/efectos de los fármacos , Terapia Genética , Proteínas Inmediatas-Precoces/efectos de los fármacos , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Transactivadores/efectos de los fármacos , Línea Celular , Citomegalovirus/genética , Infecciones por Citomegalovirus/prevención & control , Regulación Viral de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Proteínas Fluorescentes Verdes/farmacología , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Recombinantes de Fusión/farmacología , Transactivadores/genética , TransfecciónRESUMEN
Alternative therapies are needed for HSV-1 infections in patients refractory to treatment with Acyclovir (ACV) and its derivatives. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) are single-stranded DNA analogues that enter cells readily and reduce target gene expression through steric blockage of complementary RNA. When applied before or soon after infection PPMO targeting the translation-start-site regions of HSV-1 ICP0 or ICP27 mRNA reduced HSV-1 plaque formation by 70-98% in vitro. The ICP0 PPMO also reduced ACV-resistant HSV-1 (strain 615.9) plaque formation by 70-90%, while an equivalent dose of ACV produced only 40-50% inhibition when the treatment was applied between 1 and 3hpi. Seven daily topical treatments of 100microg ICP0 PPMO caused no gross or microscopic damage to the corneas of uninfected mice. Topical application of 10microg ICP0 PPMO to the eyes of HSV-1 infected mice reduced the incidence of eye disease by 37.5-50% compared to controls. This study demonstrates that topically applied PPMO holds promise as an antiviral drug candidate against HSV-1 ocular infection.
Asunto(s)
Antivirales/uso terapéutico , Herpesvirus Humano 1/efectos de los fármacos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Queratitis Herpética/tratamiento farmacológico , Morfolinas/uso terapéutico , Ubiquitina-Proteína Ligasas/efectos de los fármacos , Aciclovir/farmacología , Animales , Antivirales/administración & dosificación , Antivirales/efectos adversos , Secuencia de Bases , Chlorocebus aethiops , Farmacorresistencia Viral , Herpesvirus Humano 1/fisiología , Humanos , Queratitis Herpética/virología , Ratones , Datos de Secuencia Molecular , Morfolinas/efectos adversos , Morfolinas/síntesis química , Morfolinas/química , Morfolinos , Células Vero , Proteínas Virales/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Many of the herbal extracts used in the Chinese clinical medical routine inhibit the growth of tumor cells. In the present work, extracts of 12 selected herbs were prepared with methanol, chloroform, ethyl acetate and water, and the effects of these on the multidrug resistance (MDR) and P-glycoprotein of mouse lymphoma cells transfected with the human mdr1 gene and on a human lung alveolar epithelial cell line were investigated. The extracts were tested for antiproliferative effects, and the reversal of MDR in mouse lymphoma cells. The possible chemopreventive effect of the chloroform extracts was studied on the expression of cytomegalovirus (CMV) immediate-early (IE) antigen in human lung cancer cells (A549). The antimicrobial effects of the extracts were tested on some representative micro-organisms. Certain of the chloroform extracts of the plant materials were the most effective compounds on the reversal of MDR. Two of the chloroform extracts enhanced the antiproliferative effect of doxorubicin on MDR mouse lymphoma cells. The selected extracts did not show any antibacterial effect with the agar diffusion method. Certain chloroform extracts decreased the intermediate IE antigen expression of CMV in A459 cells.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos Fitogénicos/farmacología , Medicamentos Herbarios Chinos/farmacología , Extractos Vegetales/farmacología , Animales , Antibacterianos/farmacología , Antígenos Virales/efectos de los fármacos , Antígenos Virales/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Quimioprevención , Citomegalovirus/genética , Citomegalovirus/metabolismo , Doxorrubicina/uso terapéutico , Genes MDR/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Medicina Tradicional China , Ratones , Pruebas de Sensibilidad Microbiana , FitoterapiaRESUMEN
PURPOSE: The transdifferentiation of Tenon fibroblasts to myofibroblasts is a pivotal step in filtering bleb scarring. It is mediated by the cytokine TGF-beta, Rho-dependent contractility, and cell-matrix interactions in an interdependent fashion. HMG-CoA-reductase inhibitors (statins) have been shown to inhibit Rho-GTPase signaling; therefore, the authors studied the influence of lovastatin on TGF-beta-mediated myofibroblast transdifferentiation to assess the potential use of statins in wound healing modulation. METHODS: Human Tenon fibroblasts were grown in culture, pretreated with lovastatin, lovastatin and mevalonate, or specific inhibitors of farnesyl transferase or geranylgeranyl transferase and were stimulated with TGF-beta1. alpha-Smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) transcription were assessed by real-time PCR. alpha-SMA protein expression and localization were studied by Western blot and confocal immunofluorescence microscopy. Cell contractility was determined in collagen gel contraction assays. Phosphorylation of the signaling proteins Smad-2/3 and p38 were detected by Western blot, and Smad-2/3 localization was determined by confocal immunofluorescence microscopy. RESULTS: Lovastatin inhibited TGF-beta-induced CTGF transcription, alpha-SMA expression and incorporation into actin stress fibers, and subsequent collagen gel contraction. These effects were reversed by mevalonate. The inhibition of geranylgeranyl transferase but not farnesyl transferase blocked TGF-beta-induced alpha-SMA expression. Lovastatin decreased TGF-beta-induced p38 activation, whereas Smad-2/3 phosphorylation and nuclear translocation were preserved. CONCLUSIONS: Lovastatin inhibits TGF-beta-induced myofibroblast transdifferentiation in human Tenon fibroblasts, most likely by interfering with Rho-signaling. Statins may, therefore, serve to inhibit scarring after filtering glaucoma surgery.
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Diferenciación Celular/fisiología , Fibroblastos/fisiología , Lovastatina/farmacología , Miofibrillas/fisiología , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/fisiología , Cicatrización de Heridas/fisiología , Actinas/efectos de los fármacos , Actinas/metabolismo , Diferenciación Celular/efectos de los fármacos , Transdiferenciación Celular , Factor de Crecimiento del Tejido Conjuntivo , Fibroblastos/efectos de los fármacos , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Miofibrillas/efectos de los fármacos , ARN/genética , ARN/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The complex mechanisms by which transforming growth factor beta (TGFbeta) regulate re-epithelialisation following injury of stratified epithelia are not fully understood. TGFbeta signals via binding to distinct receptors activating downstream effectors, including Smads which initiate transcription of target genes. However, studies have shown that TGFbeta can also signal independently of Smads through MAPK pathways, demonstrating the diversity of TGFbeta signalling. Connective tissue growth factor (CTGF) is strongly induced by and acts downstream of TGFbeta causing pathophysiology in tissues by inducing matrix deposition, conversion of fibroblasts into contractile myofibroblasts (e.g. dermis and corneal stroma) and stimulation of epithelial-to-mesenchymal transition (e.g. kidney and lung) all of which are known to cause fibrosis. However, a role for CTGF in epithelial cell function which does not involve direct contribution to fibrosis has not been demonstrated. We show for the first time that synthesis of CTGF in cultures of human corneal epithelial cells is induced by TGFbeta through the Ras/MEK/ERK MAPK signalling pathway and that this is required for re-epithelialisation to occur through cell migration. These data reveal a novel function for CTGF in the regulation of epithelial tissue repair beyond its established role in fibrosis, and further highlight the complexity of TGFbeta regulation of epithelial cell function.
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Córnea/metabolismo , Células Epiteliales/metabolismo , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Factor de Crecimiento Transformador beta/metabolismo , Cicatrización de Heridas/fisiología , Línea Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Córnea/citología , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , MAP Quinasa Quinasa 1/efectos de los fármacos , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/efectos de los fármacos , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Regeneración/efectos de los fármacos , Regeneración/fisiología , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología , Cicatrización de Heridas/efectos de los fármacos , Proteínas ras/efectos de los fármacos , Proteínas ras/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Gene expression of connective tissue growth factor (CTGF) is induced in activated hepatic stellate cells (HSC), the major effectors in hepatic fibrosis, and production of extracellular matrix (ECM) is consequently increased. We previously reported that curcumin, the yellow pigment in curry, suppressed ctgf expression, leading to decreased production of ECM by HSC. The purpose of this study is to evaluate signal transduction pathways involved in the curcumin suppression of ctgf expression in HSC. EXPERIMENTAL APPROACHES: Transient transfection assays were performed to evaluate effects of activation of signalling pathways on the ctgf promoter activity. Real-time PCR and Western blotting analyses were conducted to determine expression of genes. RESULTS: Suppression of ctgf expression by curcumin was dose-dependently reversed by lipopolysaccharide (LPS), an NF-kappaB activator. LPS increased the abundance of CTGF and type I collagen in HSC in vitro. Activation of NF-kappaB by dominant active IkappaB kinase (IKK), or inhibition of NF-kappaB by dominant negative IkappaBalpha, caused the stimulation, or suppression of the ctgf promoter activity, respectively. Curcumin suppressed gene expression of Toll-like receptor-4, leading to the inhibition of NF-kappaB. On the other hand, interruption of ERK signalling by inhibitors or dominant negative ERK, like curcumin, reduced NF-kappaB activity and in ctgf expression. In contrast, the stimulation of ERK signalling by constitutively active ERK prevented the inhibitory effects of curcumin. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that the interruption of NF-kappaB and ERK signalling by curcumin results in the suppression of ctgf expression in activated HSC in vitro.
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Curcumina/farmacología , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Animales , Western Blotting , Factor de Crecimiento del Tejido Conjuntivo , Quinasas MAP Reguladas por Señal Extracelular/antagonistas & inhibidores , Proteínas Inmediatas-Precoces/metabolismo , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hígado/citología , Hígado/metabolismo , Masculino , FN-kappa B/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Receptor Toll-Like 4/efectos de los fármacos , Receptor Toll-Like 4/metabolismo , TransfecciónRESUMEN
BACKGROUND: Chronic renal hypoxia is suspected to play a pathogenic role in the genesis of diabetic nephropathy (DN). Cobalt enhances the activity of the hypoxia-inducible factor (HIF), a key factor in the defence against hypoxia. Its long-term effect on DN is evaluated. METHODS: Cobalt chloride was given to hypertensive, type 2 diabetic rats with nephropathy (SHR/NDmcr-cp). Treatment was initiated at the age of 13 weeks and continued for 26 weeks. RESULTS: Cobalt did not correct hypertension and metabolic abnormalities (obesity, hyperglycaemia and hyperlipidaemia) but reduced proteinuria as well as histological kidney injury. Cobalt upregulated renal HIF-1alpha and HIF-2alpha expression and increased the expression of HIF-regulated genes, including erythropoietin, vascular endothelial growth factor and heme oxygenase-1. The renal expression of transforming growth factor (TGF)-beta and connective tissue growth factor (CTGF) was significantly reduced by cobalt. The renal expression of NADPH oxidase, a marker of oxidative stress, and the renal content of pentosidine, a marker of advanced glycation, were also significantly reduced by cobalt. CONCLUSIONS: Cobalt achieved renal protection independently of metabolic status and blood pressure. Its effect was attributed to the upregulation of HIF and HIF-regulated genes and to a mitigated advanced glycation and oxidative stress.
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Antimutagênicos/uso terapéutico , Cobalto/uso terapéutico , Diabetes Mellitus Tipo 2/complicaciones , Nefropatías Diabéticas/prevención & control , Hipertensión/complicaciones , Hipoxia/tratamiento farmacológico , Riñón/irrigación sanguínea , Animales , Arginina/análogos & derivados , Arginina/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/biosíntesis , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Presión Sanguínea/fisiología , Western Blotting , Cromatografía Líquida de Alta Presión , Factor de Crecimiento del Tejido Conjuntivo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Nefropatías Diabéticas/etiología , Nefropatías Diabéticas/metabolismo , Progresión de la Enfermedad , Expresión Génica/efectos de los fármacos , Productos Finales de Glicación Avanzada/metabolismo , Hipertensión/metabolismo , Hipertensión/fisiopatología , Hipoxia/complicaciones , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Riñón/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , NADPH Oxidasas/biosíntesis , NADPH Oxidasas/genética , Obesidad , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Ratas , Ratas Endogámicas SHR , Factor de Crecimiento Transformador beta/biosíntesis , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Shear stress changes play an important role in atheroma formation. This study focussed on atherogenic protein expression under non-uniform shear stress and the pharmacological modulation of shear-related endothelial dysfunction. Bifurcating flow-through cell culture slides were used to expose HUVECs to steady laminar or non-uniform shear stress for 18 h at 10 dyn/cm(2). Protein expression was determined by immunofluorescence, and quantified using MetaVue software. Laminar shear stress resulted in cell alignment, reduced F-actin fibers, and significant induction of endothelial nitric oxide synthase expression. Under non-uniform shear stress at bifurcations, minor upregulation of adhesion molecules was observed. Connective tissue growth factor (CTGF) was significantly downregulated by laminar shear stress and induced in cells exposed to non-uniform shear stress. CTGF upregulation by non-uniform shear stress was RhoA-dependent, because it was almost completely inhibited in cells transfected with dominant negative RhoA-N19, and when cells were treated with 1 micromol/L simvastatin during flow. Pre-incubation of HUVECs with inhibitors of Rho-associated kinase before exposure to flow significantly suppressed the CTGF induction in regions of non-uniform shear stress. In conclusion, non-uniform shear stress-dependent CTGF expression requires active RhoA and can be prevented pharmacologically. Interference with shear stress-induced protein expression may inhibit endothelial dysfunction in atheroprone vessel regions.
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Células Endoteliales/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Proteínas Inmediatas-Precoces/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Simvastatina/farmacología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/fisiopatología , Células Cultivadas , Factor de Crecimiento del Tejido Conjuntivo , Células Endoteliales/fisiología , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Reología , Transducción de Señal/efectos de los fármacos , Venas Umbilicales/citología , Quinasas Asociadas a rho/efectos de los fármacos , Proteína de Unión al GTP rhoA/efectos de los fármacos , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
BACKGROUND: Formaldehyde (FA) is classified as a human carcinogen and has been linked to increased leukemia rates in some epidemiologic studies. Inhalation of FA induces sensory irritation at relatively low concentrations. However, little is known concerning the cellular alterations observed after FA exposure in humans. OBJECTIVES: Our aim was to profile global gene expression in Hs 680.Tr human tracheal fibroblasts exposed to FA and to develop biomarkers for the evaluation of FA exposure in humans. METHODS AND RESULTS: We used gene expression analysis, and identified 54 genes designated as FA responsive. On the basis of these data, we conducted an exploratory analysis of the expression of these genes in human subjects exposed to high or low levels of FA. We monitored FA exposure by measuring the urinary concentration of thiazolidine-4-carboxylate (TZCA), a stable and quantitative cysteinyl adduct of FA. Nine genes were selected for real-time PCR analysis; of these, BHLHB2, CCNL1, SE20-4, C8FW, PLK2, and SGK showed elevated expression in subjects with high concentrations of TZCA. CONCLUSION: The identification of gene marker candidates in vitro using microarray analysis and their validation using human samples obtained from exposed subjects is a good tool for discovering genes of potential mechanistic interest and biomarkers of exposure. Thus, these genes are differentially expressed in response to FA and are potential effect biomarkers of FA exposure.
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Fibroblastos/efectos de los fármacos , Formaldehído/efectos adversos , Perfilación de la Expresión Génica , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores , Línea Celular , Complemento C8/efectos de los fármacos , Complemento C8/genética , Ciclinas/efectos de los fármacos , Ciclinas/genética , Proteínas de Unión al ADN , Exposición a Riesgos Ambientales , Formaldehído/metabolismo , Proteínas de Homeodominio/efectos de los fármacos , Proteínas de Homeodominio/genética , Humanos , Proteínas Inmediatas-Precoces/efectos de los fármacos , Proteínas Inmediatas-Precoces/genética , Proteínas Nucleares/efectos de los fármacos , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , Tiazolidinas/orinaRESUMEN
BACKGROUND/AIMS: Connective tissue growth factor (CTGF/CCN2) has been implicated in the pathogenesis of hepatic fibrosis and suggested as a downstream mediator of the fibrogenic master cytokine TGF-beta. METHODS: We investigated the effect of TGF-beta1 on CTGF/CCN2 expression in cultured rat hepatic stellate cells and hepatocytes by means of Western and Northern blotting, immunocytochemistry, reporter gene analysis, and metabolic labelling. RESULTS: We found that the expression of CTGF/CCN2 in hepatic stellate cells is (i) only marginally (if at all) stimulated by TGF-beta and by a constitutively active type I TGF-beta receptor, (ii) independent from Smad2/3 phosphorylation, (iii) not reduced by TGF-beta1 antagonists or ALK5-receptor inhibitors and (iv) not upregulated during transdifferentiation to myofibroblasts in culture. However, expression and secretion of CTGF/CCN2 in cultured hepatocytes increased spontaneously during culture and was strongly stimulated by TGF-beta1. In bile-duct ligated and CCl(4)-treated rat livers, a strong CTGF/CCN2 expression in hepatocytes was noticed. Endothelin-1 stimulated CTGF/CCN2 expression in stellate cells but not in hepatocytes. Pathway specific signalling inhibitors point to the involvement of non-Smad signalling cascades but their contribution to CTGF/CCN2 regulation is different in both cell types. CONCLUSIONS: The results do not reveal a relevant interrelation between TGF-beta function and CTGF/CCN2 expression in hepatic stellate cells, which is in contrast to hepatocytes.