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
<p><b>OBJECTIVE</b>To evaluate the effects of polyamidoamine dendrimer (PAMAM) liposome as gene carriers on the cellular uptake and its cytotoxicity in colonic cancer cell.</p><p><b>METHODS</b>The liposome modified PAMAM was synthesized with liposome and polyamidoamine dendrimer. Plasmid PEGFP-N1 was mixed with the liposome-modified PAMAM or unmodified PAMAM to form nanoparticle complexes. The shape and size of the nanoparticle complexes were observed by transmission electron microscope and the zeta potential was measured by analytical tool. The encapsulating efficiency was determined by ultraviolet spectrophotometer in centrifuging method. After the cell lines SW620 (colonic cancer cell), MCF-7 (breast cancer cell), ECV304 (vascular endothelial cell) were transfected by the two kinds of PAMAM nanoparticle complexes, the flow cytometry was used to determine the uptake of enhanced green fluorescent protein (EGFP) gene. The cytotoxicity of PAMAM liposome nanoparticles and PAMAM nanoparticles was evaluated by MTT assay.</p><p><b>RESULTS</b>The diameter of liposome modified PAMAM complex was (192 ± 16) nm, and that of PAMAM complex was (189 ± 19) nm (P > 0.05); and the zeta potential of liposome modified PAMAM complex was higher than that of PAMAM complex [(42 ± 7) mV vs. (32 ± 7) mV, P < 0.05]. There was no significant difference in envelopment rate between the two groups [(82 ± 7)% vs. (84 ± 6)%, P > 0.05]. After the colonic cancer cell line SW620 was transfected with the two kinds of PAMAM nanoparticle complexes, the cellular uptake of the cells with the liposome-modified PAMAM complex was significantly higher than that of the cell with PAMAM complex (P < 0.05). The cellular survival rate of the cell lines with liposome-modified PAMAM complex was significantly higher than that of cell lines with PAMAM complex (P < 0.05).</p><p><b>CONCLUSION</b>The liposome modified PAMAM can improve gene transfection efficiency and suppress its cytotoxicity.</p>
Subject(s)
Humans , Cell Line, Tumor , Cell Survival , Colonic Neoplasms , Metabolism , Pathology , Dendrimers , Pharmacokinetics , Toxicity , Genetic Vectors , Pharmacokinetics , Toxicity , Liposomes , Pharmacokinetics , Toxicity , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To evaluate the inhibitory effects of survivin antisense oligonucleotide (survivin-ASODN) mediated by polyamidoamine dendrimer (PAMAM) against the growth of subcutaneously transplanted colorectal cancer in nude mice.</p><p><b>METHODS</b>Nude mouse models bearing colorectal cancer was established by subcutaneous injection of SW620 cells. Survivin- OSADN (300 microg/L) was mixed with 4.06 microg/L PAMAM or liposome to prepare two transfection complexes, and their morphologies were observed by transmission electron microscope. The particle size of the prepared complexes was determined by laser particle size analyzer, and the zeta potential was measured. The encapsulation efficiency and the DNA release rate in vitro were determined by ultraviolet spectrophotometer. The transfection complexes were then directly injected into the xenografts of the tumor-bearing nude mice. The tumor volume changes were observed, and the expression of survivin in the transplanted tumor was measured by Western blotting.</p><p><b>RESULTS</b>The PAMAM-survivin-ASODN complex had a significantly smaller diameter and greater zeta potential than liposome-survivin-ASODN (P<0.01 and 0.05, respectively). The encapsulation efficiency was comparable between the two complexes. In in vitro condition, PAMAM-survivin-ASODN allowed sustained survivin-ASODN release for as long as 14 days, as compared with the 5 days for the liposome complex. After injection into the tumor xenografts, PAMAM-survivin- ASODN resulted in significantly lower expression of survivin protein in the transplanted tumors (P<0.05), and also in significantly greater reduction of the tumor volume than the liposome complex (P<0.05).</p><p><b>CONCLUSION</b>PAMAM can effectively deliver survivin-ASODN into transplanted colorectal tumor cells to reduce the expression of survivin and inhibit the tumor growth.</p>
Subject(s)
Animals , Humans , Mice , Cell Proliferation , Colorectal Neoplasms , Pathology , Dendrimers , Inhibitor of Apoptosis Proteins , Mice, Inbred BALB C , Mice, Nude , Microtubule-Associated Proteins , Genetics , Pharmacology , Neoplasm Transplantation , Oligonucleotides, Antisense , Pharmacology , Polyamines , Pharmacology , Repressor Proteins , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To evaluate the specific killing effects of combination of recombinant adenovirus mediated double suicide gene driven by KDR promoter and survivin antisense oligonucleotide(ASODN) on colorectal cancer cells and vascular endothelial cells.</p><p><b>METHODS</b>The 293 packaging cells were transfected with the plasmids of pAdEasy-CDglyTK and the recombinant adenovirus were generated. The KDR expressive cells of SW620, ECV304 were infected with adenovirus, meanwhile survivinASODN was transferred into the same cells. The infection rate of adenovirus and transfection efficiency of survivinASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on SW620, ECV304 were examined through MTT method.</p><p><b>RESULTS</b>The cells which were infected with the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection efficiency. The high expression of CDglyTK gene was found in SW620, ECV304 cells infected with recombinant adenovirus and survivin ASODN decreased the survivin protein level. The survival rate of gene therapy group was significantly lower than that of negative group. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate of SW620 and ECV304 cells as compared with the AdKDR-CDglyTK or survivin ASODN used alone (P<0.05). The survival rate was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that AdKDR-CDglyTK used alone (P>0.05). The combined therapy of AdKDR-CDglyTK and survivin ASODN showed synergistic killing efficacy and more significant bystander effects.</p><p><b>CONCLUSION</b>The combined gene therapy of AdKDR-CDglyTK system and survivin ASODN has stronger specific killing effects on colorectal cancer cells and vein endothelial cells.</p>
Subject(s)
Humans , Adenoviridae , Genetics , Cell Line, Tumor , Colorectal Neoplasms , Genetics , Metabolism , Endothelial Cells , Metabolism , Genes, Transgenic, Suicide , Genetics , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins , Genetics , Oligonucleotides, Antisense , Genetics , Receptors, Vascular Endothelial Growth Factor , Genetics , Transcription Initiation SiteABSTRACT
<p><b>OBJECTIVE</b>To compare the durability of quantum dots with that of green fluorescein for labeling survivin antisense oligonucleotide (ASODN) and investigate the difference in growth and apoptosis of cells transfected with the labeled survivin ASODN.</p><p><b>METHODS</b>Survivin ASODN labeled with quantum dots or green fluorescein was transfected into MCF-7 cells via Lipolifectmain(TM2000). The proliferation of MCF-7 cells was assessed with MTT assay, survivin mRNA expression determined by RT-PCR and its protein expression measured by Western blot analysis. The apoptosis rate of the transfected cells was estimated by flow cytometry, and the fluorescence distribution in the cells observed under fluorescent inverted microscope.</p><p><b>RESULTS</b>The mRNA and protein expressions of survivin were significantly decreased in the MCF-7 cells after cell transfection with survivin ASODN labeled with quantum dots or green fluorescein, and no significant difference was noted between the two labeling methods (P>0.05). Nor did survivin ASODN transfection with different labeling methods produced significant difference in cell proliferation and apoptotic rate (P>0.05). For green fiuorescein labeling, the fluorescence disappeared 4 days after transfection, whereas the fluorescence sustained for 1 week for quantum dots labeling.</p><p><b>CONCLUSION</b>Survivin ASODNs labeled with quantum dots and green fiuorescein do not significantly differ in survivin expression or the transfected cell proliferation and apoptosis rate, but quantum dot labeling can be more stable with longer maintcnance of the labeling.</p>
Subject(s)
Humans , Apoptosis , Blotting, Western , Cell Line, Tumor , Cell Proliferation , Flow Cytometry , Fluorescein , Chemistry , Gene Expression , Inhibitor of Apoptosis Proteins , Microscopy, Fluorescence , Microtubule-Associated Proteins , Genetics , Metabolism , Oligonucleotides, Antisense , Chemistry , Genetics , Quantum Dots , Reverse Transcriptase Polymerase Chain Reaction , Staining and Labeling , Methods , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To evaluate the selectively killing effects of combination of adenovirus mediated double suicide gene driven by kinase-domain insert containing receptor (KDR) promoter and survivin antisense oligonucleotide on breast tumor cells and vein endothelial cells.</p><p><b>METHODS</b>Human embryonal kidney cells 293 were transfected with the plasmids of pAdEasy-KDR-CDglyTK and the adenovirus was generated. The KDR expressive cells of MCF-7, ECV304 were infected by adenovirus and survivin ASODN was transferred into the same cells meanwhile. The infection rates of adenovirus and transfection efficiency of survivin ASODN were observed and the expression of CDglyTK was detected by RT-PCR. The expression of survivin was measured by Western blot. The killing effects and bystander effects on cells were assessed by MTT assay.</p><p><b>RESULTS</b>The cells infected by the adenovirus mediated double suicide gene could be transfected with the survivin ASODN and the infection rate was not affected as well as the transfection rate. The high expression of CDglyTK gene was found in the two kinds of cells and survivin ASODN decreased the survivin protein level. When survivin ASODN was transferred into MCF-7, ECV304 cells, the survival rates were 56.4% +/- 3.8% and 55.9% +/- 3.6% respectively. The combination of survivin ASODN and AdKDR-CDglyTK gene therapy showed significantly lower survival rate comparing with using each treatment alone (P < 0.05) and the survival rate decreased gradually with the increasing of the concentration of GCV and 5-FC. But the survival rate for combined gene therapy group was slightly lower in GCV 100 microg/ml, 5-FC 2000 microg/ml than that of AdKDR-CDglyTK group (P > 0.05). The combination of survivin ASODN and AdKDR-CDglyTK therapy showed synergistic killing efficacy and more significant bystander effects.</p><p><b>CONCLUSION</b>The combined therapy with AdKDR-CDglyTK system and survivin ASODN shows obvious killing effects on breast tumor cells and vein endothelial cells.</p>
Subject(s)
Female , Humans , Adenoviridae , Genetics , Breast Neoplasms , Genetics , Metabolism , Pathology , Cell Line , Cell Line, Tumor , Cell Survival , Endothelial Cells , Metabolism , Pathology , Genes, Transgenic, Suicide , Genetics , Genetic Therapy , Methods , Genetic Vectors , Inhibitor of Apoptosis Proteins , Microtubule-Associated Proteins , Genetics , Neoplasm Proteins , Genetics , Oligonucleotides, Antisense , Genetics , Plasmids , Promoter Regions, Genetic , Receptors, Vascular Endothelial Growth Factor , Genetics , TransfectionABSTRACT
Objective:To use polyamidoamine(PAMAM)dendrimer as gene delivery system for survivin gene anti- sense oligodeoxynucleotide(asODN)transfection for inhibition of HepG2 cancer cell growth.Methods:The first to the fifth generation of PAMAM and asODN were used to prepare a complex:PAMAM-asODN.The morphology of PAMAM- asODN was observed using agrose electrophoresis and atomic force microscope(AFM).PAMAM-asODN was then used to transfect HepG2 cells and cells transfected with asODN served as control.The transfection efficacy of PAMAM-asODN into HepG2 cells was observed under confocol microscope,the surviving mRNA expression was analyzed by RT-PCR,and the inhibition of HepG2 cell growth was determined by MTT assay.Results:Agrose electrophoresis showed strong complexing action between PAMAM and asODN and they formed a complex with a diameter of 25 nm.Confocol microscope showed the transfection efficacy of PAMAM-asODN was higher than that of asODN.RT-PCR showed a decreased expression of sur- vivin mRNA in PAMAM-asODN transfected cells.MTF results demonstrated that the growth of HepG2 cell was obviously inhibited after transfection of PAMAM-asODN and the inhibition rate increased with culture time,concentration of com- plex,the generation of PAMAM.PAMAM-asODN at 6.0?mol/L G4.0 resulted in a 55% inhibition of HepG2 cells 96 h after culture.Conclusion:PAMAM dendrimers can efficiently mediate the entry of survivin asODN into HepG2 cells,re- sulting in inhibition of HepG2 cells.PAMAM might be a promising gene carrier for potential molecular therapy of cancer.