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Efficacy and safety of dendrimer nanoparticles with coexpression of tumor necrosis factor-α and herpes simplex virus thymidine kinase in gene radiotherapy of the human uveal melanoma OCM-1 cell line.

Wang Y, Mo L, Wei W, Shi X - Int J Nanomedicine (2013)

Bottom Line: The cellular morphology of OCM-1 cells altering was observed by TEM, and a decrease in cell proliferation was revealed in cell-growth curves.Flow cytometry of annexin V/propidium iodide double-dyeing apoptosis and caspase-3 fluorescence staining showed that this treatment method could turn transfected OCM-1 cells into apoptosis and necrosis by the effects of the gene expression.This study indicated that the dendrimer nanoparticles with coexpression of TNF-α and HSV1-TK gene therapy are effective and safe and can provide us with a novel strategy to treat human uveal melanoma in the future.

View Article: PubMed Central - PubMed

Affiliation: Beijing Tongren Eye Center, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT
Human uveal melanoma is the most common primary intraocular tumor, and brachytherapy is one of the most common and effective treatment strategies. In order to find a safer and more effective way to increase the radio sensitivity of the tumor, we tried to use the dendrimer nanoparticle performing coexpression gene radiotherapy. In this study, we constructed recombinant DNA plasmids (early growth response-1 tumor necrosis factor-α [pEgr1-TNFα], pEgr1 thymidine kinase [TK], and pEgr1-TNFα-TK) according to the Egr1 promoter sequence. The sequences of human TNFα and herpes simplex virus (HSV) TK that were published by GenBank. Agarose gel electrophoresis and DNA sequencing had proven that we constructed the double-gene recombined plasmids pEgr1-TNF-TK correctly, as well as the plasmids pEgr1-TNFα and pEgr1-TK. The dendrimer nanoparticles combined with plasmid DNA as dendriplexes were verified with agarose gel electrophoresis and observed by transmission electron microscopy (TEM) and scanning electron microscopy to define size and shape. Zeta potential was measured using a Zetasizer analyzer. Optimal size and neutral zeta-potential characteristics of dendriplexes were achieved for the transfection studies. DNase I examination proved that the dendriplexes could protect plasmid DNA for at least 6 hours. The recombinant plasmids were transfected with dendrimer nanoparticles into the human choroidal melanoma OCM-1 cell line, followed by exposure to iodine-125 ((125)I) after transfection. After transfection with dendrimer nanoparticles and the irradiation of (125)I, the gene expressions of TNFα and HSV1-TK were significantly increased at the protein level by enzyme-linked immunosorbent assay and Western blot analysis in OCM-1 cells. The cellular morphology of OCM-1 cells altering was observed by TEM, and a decrease in cell proliferation was revealed in cell-growth curves. Flow cytometry of annexin V/propidium iodide double-dyeing apoptosis and caspase-3 fluorescence staining showed that this treatment method could turn transfected OCM-1 cells into apoptosis and necrosis by the effects of the gene expression. This study indicated that the dendrimer nanoparticles with coexpression of TNF-α and HSV1-TK gene therapy are effective and safe and can provide us with a novel strategy to treat human uveal melanoma in the future.

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(A–D) Agarose gel electrophoresis of dendriplexes. (A) Dendripexes of TNF-TK, TNF, and TK were verified with gel electrophoresis. (B–D) DNase sensitivity examination results in 30 minutes, 4 hours, and 6 hours, respectively. The “d”s and “p”s refer to dendriplexes and plasmid DNA, respectively. We can see there are still bands around the original place.Abbreviations: TNF-TK, tumor necrosis factor-thymidine kinase; TNF, tumor necrosis factor; TK, thymidine kinase.
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f2-ijn-8-3805: (A–D) Agarose gel electrophoresis of dendriplexes. (A) Dendripexes of TNF-TK, TNF, and TK were verified with gel electrophoresis. (B–D) DNase sensitivity examination results in 30 minutes, 4 hours, and 6 hours, respectively. The “d”s and “p”s refer to dendriplexes and plasmid DNA, respectively. We can see there are still bands around the original place.Abbreviations: TNF-TK, tumor necrosis factor-thymidine kinase; TNF, tumor necrosis factor; TK, thymidine kinase.

Mentions: The dendriplexes of TNF-TK, TNF, and TK were verified with agarose gel electrophoresis, as shown in Figure 2A. Each group showed similar bands with the plasmid DNA, but the bands were larger due to agglomeration. DNase sensitivity examination showed that dendriplexes and plasmid DNA both had apparent bands in the original place, and a little trailing and extra bands showed as DNA degradation in 30 minutes. In 6 hours, dendriplexes could still be observed with bands in the original place, but not the plasmid DNA (Figure 2B, C and D). The electron microscopy sizing data of dendrimer nanoparticles and dendriplexes demonstrated particle sizes of about 20 nm and 100–200 nm, and the dendriplex agglomeration was about 500 nm (Figure 3). The zeta potentials of TNF-TK, TNF, and TK were 6.49 ± 0.83 mV, 6.71 ± 0.77 mV, and 7.91 ± 1.60 mV, respectively. There was no significantly statistical difference between them.


Efficacy and safety of dendrimer nanoparticles with coexpression of tumor necrosis factor-α and herpes simplex virus thymidine kinase in gene radiotherapy of the human uveal melanoma OCM-1 cell line.

Wang Y, Mo L, Wei W, Shi X - Int J Nanomedicine (2013)

(A–D) Agarose gel electrophoresis of dendriplexes. (A) Dendripexes of TNF-TK, TNF, and TK were verified with gel electrophoresis. (B–D) DNase sensitivity examination results in 30 minutes, 4 hours, and 6 hours, respectively. The “d”s and “p”s refer to dendriplexes and plasmid DNA, respectively. We can see there are still bands around the original place.Abbreviations: TNF-TK, tumor necrosis factor-thymidine kinase; TNF, tumor necrosis factor; TK, thymidine kinase.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3795008&req=5

f2-ijn-8-3805: (A–D) Agarose gel electrophoresis of dendriplexes. (A) Dendripexes of TNF-TK, TNF, and TK were verified with gel electrophoresis. (B–D) DNase sensitivity examination results in 30 minutes, 4 hours, and 6 hours, respectively. The “d”s and “p”s refer to dendriplexes and plasmid DNA, respectively. We can see there are still bands around the original place.Abbreviations: TNF-TK, tumor necrosis factor-thymidine kinase; TNF, tumor necrosis factor; TK, thymidine kinase.
Mentions: The dendriplexes of TNF-TK, TNF, and TK were verified with agarose gel electrophoresis, as shown in Figure 2A. Each group showed similar bands with the plasmid DNA, but the bands were larger due to agglomeration. DNase sensitivity examination showed that dendriplexes and plasmid DNA both had apparent bands in the original place, and a little trailing and extra bands showed as DNA degradation in 30 minutes. In 6 hours, dendriplexes could still be observed with bands in the original place, but not the plasmid DNA (Figure 2B, C and D). The electron microscopy sizing data of dendrimer nanoparticles and dendriplexes demonstrated particle sizes of about 20 nm and 100–200 nm, and the dendriplex agglomeration was about 500 nm (Figure 3). The zeta potentials of TNF-TK, TNF, and TK were 6.49 ± 0.83 mV, 6.71 ± 0.77 mV, and 7.91 ± 1.60 mV, respectively. There was no significantly statistical difference between them.

Bottom Line: The cellular morphology of OCM-1 cells altering was observed by TEM, and a decrease in cell proliferation was revealed in cell-growth curves.Flow cytometry of annexin V/propidium iodide double-dyeing apoptosis and caspase-3 fluorescence staining showed that this treatment method could turn transfected OCM-1 cells into apoptosis and necrosis by the effects of the gene expression.This study indicated that the dendrimer nanoparticles with coexpression of TNF-α and HSV1-TK gene therapy are effective and safe and can provide us with a novel strategy to treat human uveal melanoma in the future.

View Article: PubMed Central - PubMed

Affiliation: Beijing Tongren Eye Center, Capital Medical University, Beijing, People's Republic of China.

ABSTRACT
Human uveal melanoma is the most common primary intraocular tumor, and brachytherapy is one of the most common and effective treatment strategies. In order to find a safer and more effective way to increase the radio sensitivity of the tumor, we tried to use the dendrimer nanoparticle performing coexpression gene radiotherapy. In this study, we constructed recombinant DNA plasmids (early growth response-1 tumor necrosis factor-α [pEgr1-TNFα], pEgr1 thymidine kinase [TK], and pEgr1-TNFα-TK) according to the Egr1 promoter sequence. The sequences of human TNFα and herpes simplex virus (HSV) TK that were published by GenBank. Agarose gel electrophoresis and DNA sequencing had proven that we constructed the double-gene recombined plasmids pEgr1-TNF-TK correctly, as well as the plasmids pEgr1-TNFα and pEgr1-TK. The dendrimer nanoparticles combined with plasmid DNA as dendriplexes were verified with agarose gel electrophoresis and observed by transmission electron microscopy (TEM) and scanning electron microscopy to define size and shape. Zeta potential was measured using a Zetasizer analyzer. Optimal size and neutral zeta-potential characteristics of dendriplexes were achieved for the transfection studies. DNase I examination proved that the dendriplexes could protect plasmid DNA for at least 6 hours. The recombinant plasmids were transfected with dendrimer nanoparticles into the human choroidal melanoma OCM-1 cell line, followed by exposure to iodine-125 ((125)I) after transfection. After transfection with dendrimer nanoparticles and the irradiation of (125)I, the gene expressions of TNFα and HSV1-TK were significantly increased at the protein level by enzyme-linked immunosorbent assay and Western blot analysis in OCM-1 cells. The cellular morphology of OCM-1 cells altering was observed by TEM, and a decrease in cell proliferation was revealed in cell-growth curves. Flow cytometry of annexin V/propidium iodide double-dyeing apoptosis and caspase-3 fluorescence staining showed that this treatment method could turn transfected OCM-1 cells into apoptosis and necrosis by the effects of the gene expression. This study indicated that the dendrimer nanoparticles with coexpression of TNF-α and HSV1-TK gene therapy are effective and safe and can provide us with a novel strategy to treat human uveal melanoma in the future.

Show MeSH
Related in: MedlinePlus