ABSTRACT
The methylcytosine dioxygenases TET proteins (TET1, TET2, and TET3) play important regulatory roles in neural function. In this study, we investigated the role of TET proteins in neuronal differentiation using Neuro2a cells as a model. We observed that knockdown of TET1, TET2 or TET3 promoted neuronal differentiation of Neuro2a cells, and their overexpression inhibited VPA (valproic acid)-induced neuronal differentiation, suggesting all three TET proteins negatively regulate neuronal differentiation of Neuro2a cells. Interestingly, the inducing activity of TET protein is independent of its enzymatic activity. Our previous studies have demonstrated that srGAP3 can negatively regulate neuronal differentiation of Neuro2a cells. Furthermore, we revealed that TET1 could positively regulate srGAP3 expression independent of its catalytic activity, and srGAP3 is required for TET-mediated neuronal differentiation of Neuro2a cells. The results presented here may facilitate better understanding of the role of TET proteins in neuronal differentiation, and provide a possible therapy target for neuroblastoma.
Subject(s)
Animals , Mice , Catalytic Domain , Cell Differentiation , Physiology , Cell Line, Tumor , DNA-Binding Proteins , Genetics , Metabolism , Enzyme Inhibitors , Pharmacology , GTPase-Activating Proteins , Genetics , Metabolism , Immunohistochemistry , Microscopy, Fluorescence , Neuroblastoma , Metabolism , Pathology , Protein Isoforms , Genetics , Metabolism , Proto-Oncogene Proteins , Genetics , Metabolism , RNA Interference , RNA, Messenger , Metabolism , RNA, Small Interfering , Metabolism , Valproic Acid , PharmacologyABSTRACT
Gene therapy with adenoviral vectors is a promising new approach for the treatment of refractory advanced breast cancer. Strategies to restrict adenoviral-mediated therapeutic gene expression are important to avoid harming normal cells. Fatty acid synthase (FAS) is overexpressed in several human cancers. FAS is highly expressed in infiltrating breast cancer tissue, and always associated with malignant phenotypes and poor prognosis. In this study, expression of the FAS was evaluated in three breast cancer cell lines. A 680 bp-FAS promoter was cloned and its transcriptional activity was analyzed in breast cancer cell lines. We made a recombinant adenovirus construct carrying herpes simplex virus thymidine kinase (HSV-TK) driven by human FAS promoter (Ad-FAS-TK) and analyzed its target cytotoxicity in vitro and in vivo against human breast cancer cells combined with prodrug ganciclovir (GCV). The results show that the expression of FAS varies in the three breast cancer cell lines examined (respectively, SK-Br3>MCF-7>MDA-MB-231), but FAS promoter can initiate relative high transcriptional activities in all three kinds of cancer cells while little in normal fibroblast cells. Furthermore, FAS promoter can drive the therapeutic gene in a wider range of human breast cancers than cerbB2 promoter and exhibit a stronger activity than midkine (MK) promoter. Combination of Ad-FAS-TK and GCV treatment exhibited strong-targeted cytotoxic effect on breast cancer cells but showed little activity in normal fibroblast cells. The tumorigenic capability of breast cancer cells treated with Ad-FAS-TK/GCV was completely inhibited in vitro and in vivo assays. In conclusion, adenoviral-mediated suicide gene therapy controlled by tumor associated-FAS promoter can induce specific cytotoxic effect on human breast cancer cells in vitro and in vivo. So it is a promising target for the development of gene therapy against breast cancers.
Subject(s)
Adenoviruses, Human , Breast Neoplasms/genetics , Fatty Acid Synthases/therapeutic use , Genetic Therapy , Recombinant Fusion Proteins/therapeutic use , Tumor Suppressor Proteins/therapeutic use , Antineoplastic Agents/therapeutic use , Breast Neoplasms/drug therapy , Breast Neoplasms/therapy , DNA-Binding Proteins/administration & dosage , DNA-Binding Proteins/therapeutic use , Fatty Acid Synthases/drug effects , Female , Ganciclovir/therapeutic use , Gene Expression , Genes, Transgenic, Suicide , Genetic Vectors , Humans , In Vitro Techniques , Nuclear Proteins/administration & dosage , Nuclear Proteins/therapeutic use , Phenotype , Prognosis , Recombinant Fusion Proteins/administration & dosage , Treatment Failure , Treatment Outcome , Tumor Suppressor Proteins/administration & dosageABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of sodium ferulate (SF), an intravenous drug made from traditional Chinese herbs, on activation of postsynaptic density-95 (PSD-95) and neuroprotection after transient cerebral artery occlusion in rats.</p><p><b>METHODS</b>Forty-six male Sprague-Dawley rats were randomized into 2 groups (n=23 in each group): the control group and the SF group. After anesthesia, the middle cerebral artery occlusion (MCAO) was conducted with the intraluminal filament technique. The neurological deficit was assessed with the method devised by Bederson et al. The 2,3,4-triphenyltetrazolium chloride staining was used to assess the infarct volume. We adopted a modified six-point scale to conduct neurobehavioral evaluation. Immediately the activation of postsynaptic density-95 (PSD-95) was studied with Western blot analysis system in the cortex and striatum of rat brain.</p><p><b>RESULTS</b>The neurologic deficit score of the SF group decreased substantially compared with that of the control group (P<0.05). The infarct volume of the control group (168.1 mm3 +/- 42.2 mm3) was significantly larger than that of the SF group (61.5 mm3 +/- 28.7 mm3) at 24 hours after reperfusion (P<0.01). And the rats showed some neurological deficit. The activity of PSD-95 in the SF group at most timepoints was less than that in the control group. No upregulation of PSD-95 protein could be detected in the contralateral cortex.</p><p><b>CONCLUSIONS</b>Sodium ferulate can induce a neuroprotective effect against the transient focal cerebral ischemic injury and weaken the activation of PSD-95 in ischemic area after MCAO.</p>
