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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–C) Apoptosis analysis of OCM-1 cell lines after treatment for 48 and 72 hours by flow cytometry. (A) Apoptosis analysis using annexin V-FITC/PI double-staining after 48 hours. The negative control group compared with the control group showed no statistical significance (P > 0.05). (B) Apoptosis analysis using Annexin V-FITC/PI double-staining after 72 hours. The apoptosis ratios in pEgr-TNFα-TK treatment, irradiation, and combined groups were 7.86% ± 0.15%, 5.9% ± 0.17%, and 13.77% ± 0.76%, respectively; n = 3 replicates per condition. (C) Caspase-3 fluorescent stain after 48 hours. pEgr-TNFα-TK treatment, irradiation, and combined treatments significantly induced caspase-3 arrest in OCM-1 cells. The negative control group compared with the control group showed no statistical significance (P = 0.531).Notes: *P < 0.005; #P < 0.01 compared with the negative control group and the control group by one-way analysis of variance; n = 3 replicates per condition.Abbreviations: OCM-1, human choroidal melanoma; FITC, fluorescein isothiocyanate; PI, propidium iodide; pEgr-TNFα-TK, plasmid early growth response-1 tumor necrosis factor α thymidine kinase.
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f8-ijn-8-3805: (A–C) Apoptosis analysis of OCM-1 cell lines after treatment for 48 and 72 hours by flow cytometry. (A) Apoptosis analysis using annexin V-FITC/PI double-staining after 48 hours. The negative control group compared with the control group showed no statistical significance (P > 0.05). (B) Apoptosis analysis using Annexin V-FITC/PI double-staining after 72 hours. The apoptosis ratios in pEgr-TNFα-TK treatment, irradiation, and combined groups were 7.86% ± 0.15%, 5.9% ± 0.17%, and 13.77% ± 0.76%, respectively; n = 3 replicates per condition. (C) Caspase-3 fluorescent stain after 48 hours. pEgr-TNFα-TK treatment, irradiation, and combined treatments significantly induced caspase-3 arrest in OCM-1 cells. The negative control group compared with the control group showed no statistical significance (P = 0.531).Notes: *P < 0.005; #P < 0.01 compared with the negative control group and the control group by one-way analysis of variance; n = 3 replicates per condition.Abbreviations: OCM-1, human choroidal melanoma; FITC, fluorescein isothiocyanate; PI, propidium iodide; pEgr-TNFα-TK, plasmid early growth response-1 tumor necrosis factor α thymidine kinase.

Mentions: By using flow cytometry, it was possible to measure quantitatively and analyze the effect of coexpression plasmid on promoting apoptosis. In the transfected groups, which were either exposed or not exposed to 2 Gy 125I, results for signs of early cellular apoptosis between the two groups (annexin V-FITC+/PI cells, early apoptotic cells) showed pEgr-TNFα-TK treatment, irradiation, and combined treatments could evoke OCM-1 cell apoptosis (Figure 8A) when compared with the control group and the negative control group. However, 2 Gy 125I treatment did not induce OCM-1 cell apoptosis in 48 hours (P = 0.057); a statistically significant difference was revealed in 72 hours (P = 0.00). The apoptosis rate of the transfected groups with irradiation was higher than the unexposed transfection groups in 48 hours, while there was statistical significance in the TNF-radiation and TK-radiation groups in comparison with the unexposed transfection group (P < 0.05). In 72 hours (Figure 8B), the apoptosis rate of the transfected groups with irradiation was significantly higher than the unexposed transfection groups, and the difference in early apoptotic rate for cells in the TNF-radiation group was more significant when compared to others groups (P < 0.01).


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–C) Apoptosis analysis of OCM-1 cell lines after treatment for 48 and 72 hours by flow cytometry. (A) Apoptosis analysis using annexin V-FITC/PI double-staining after 48 hours. The negative control group compared with the control group showed no statistical significance (P > 0.05). (B) Apoptosis analysis using Annexin V-FITC/PI double-staining after 72 hours. The apoptosis ratios in pEgr-TNFα-TK treatment, irradiation, and combined groups were 7.86% ± 0.15%, 5.9% ± 0.17%, and 13.77% ± 0.76%, respectively; n = 3 replicates per condition. (C) Caspase-3 fluorescent stain after 48 hours. pEgr-TNFα-TK treatment, irradiation, and combined treatments significantly induced caspase-3 arrest in OCM-1 cells. The negative control group compared with the control group showed no statistical significance (P = 0.531).Notes: *P < 0.005; #P < 0.01 compared with the negative control group and the control group by one-way analysis of variance; n = 3 replicates per condition.Abbreviations: OCM-1, human choroidal melanoma; FITC, fluorescein isothiocyanate; PI, propidium iodide; pEgr-TNFα-TK, plasmid early growth response-1 tumor necrosis factor α thymidine kinase.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3795008&req=5

f8-ijn-8-3805: (A–C) Apoptosis analysis of OCM-1 cell lines after treatment for 48 and 72 hours by flow cytometry. (A) Apoptosis analysis using annexin V-FITC/PI double-staining after 48 hours. The negative control group compared with the control group showed no statistical significance (P > 0.05). (B) Apoptosis analysis using Annexin V-FITC/PI double-staining after 72 hours. The apoptosis ratios in pEgr-TNFα-TK treatment, irradiation, and combined groups were 7.86% ± 0.15%, 5.9% ± 0.17%, and 13.77% ± 0.76%, respectively; n = 3 replicates per condition. (C) Caspase-3 fluorescent stain after 48 hours. pEgr-TNFα-TK treatment, irradiation, and combined treatments significantly induced caspase-3 arrest in OCM-1 cells. The negative control group compared with the control group showed no statistical significance (P = 0.531).Notes: *P < 0.005; #P < 0.01 compared with the negative control group and the control group by one-way analysis of variance; n = 3 replicates per condition.Abbreviations: OCM-1, human choroidal melanoma; FITC, fluorescein isothiocyanate; PI, propidium iodide; pEgr-TNFα-TK, plasmid early growth response-1 tumor necrosis factor α thymidine kinase.
Mentions: By using flow cytometry, it was possible to measure quantitatively and analyze the effect of coexpression plasmid on promoting apoptosis. In the transfected groups, which were either exposed or not exposed to 2 Gy 125I, results for signs of early cellular apoptosis between the two groups (annexin V-FITC+/PI cells, early apoptotic cells) showed pEgr-TNFα-TK treatment, irradiation, and combined treatments could evoke OCM-1 cell apoptosis (Figure 8A) when compared with the control group and the negative control group. However, 2 Gy 125I treatment did not induce OCM-1 cell apoptosis in 48 hours (P = 0.057); a statistically significant difference was revealed in 72 hours (P = 0.00). The apoptosis rate of the transfected groups with irradiation was higher than the unexposed transfection groups in 48 hours, while there was statistical significance in the TNF-radiation and TK-radiation groups in comparison with the unexposed transfection group (P < 0.05). In 72 hours (Figure 8B), the apoptosis rate of the transfected groups with irradiation was significantly higher than the unexposed transfection groups, and the difference in early apoptotic rate for cells in the TNF-radiation group was more significant when compared to others groups (P < 0.01).

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