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Combination of taxol and Bcl-2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumour growth.

George J, Banik NL, Ray SK - J. Cell. Mol. Med. (2009)

Bottom Line: We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth.In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination.In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, USA.

ABSTRACT
Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti-apoptotic molecule Bcl-2 is up-regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl-2 siRNA or both agents together for 72 h. Knockdown of Bcl-2 potentiated efficacy of taxol for cell death. Fluorescence-activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl-2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl-2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl-2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.

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FACS analysis for detection of apoptotic cells and DNA fragmentation. Treatment (72 hrs) of cells: transfection with a plasmid vector expressing scrambled siRNA (treated control), 100 nM taxol, transfection with a plasmid vector expressing Bcl-2 siRNA and taxol + Bcl-2 siRNA. (A) FACS dot plots of U251MG cells. Before FACS analysis, the cells were treated with 50 μg/ml propidium iodide for 30 min. at 4°C in dark. Population in the M1 area represents the apoptotic/dead cells. (B) FACS histograms of U251MG cells. The prominent increase in population of cells in the sub-G1 phase (M1) indicated increase in apoptosis after treatment with taxol or Bcl-2 siRNA or both. (C) Quantitative presentation of DNA fragmentation data from FACS analysis to indicate percent changes in live and apoptotic cells. Data are representative of four independent experiments (*P < 0.001 when compared with scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
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fig02: FACS analysis for detection of apoptotic cells and DNA fragmentation. Treatment (72 hrs) of cells: transfection with a plasmid vector expressing scrambled siRNA (treated control), 100 nM taxol, transfection with a plasmid vector expressing Bcl-2 siRNA and taxol + Bcl-2 siRNA. (A) FACS dot plots of U251MG cells. Before FACS analysis, the cells were treated with 50 μg/ml propidium iodide for 30 min. at 4°C in dark. Population in the M1 area represents the apoptotic/dead cells. (B) FACS histograms of U251MG cells. The prominent increase in population of cells in the sub-G1 phase (M1) indicated increase in apoptosis after treatment with taxol or Bcl-2 siRNA or both. (C) Quantitative presentation of DNA fragmentation data from FACS analysis to indicate percent changes in live and apoptotic cells. Data are representative of four independent experiments (*P < 0.001 when compared with scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).

Mentions: FACS analysis is a useful tool for detection of apoptotic cells and DNA fragmentation. So, we performed FACS analysis for determining apoptosis and DNA fragmentation in U251MG cells after treatment with taxol or Bcl-2 siRNA or both agents together (Fig. 2). An increase in cell population in the M1 area indicated apoptotic cells, as shown in the FACS dot plot graphs (Fig. 2A). A significant increase (P < 0.001) in cell population in the M1 area occurred due to treatment with either taxol or Bcl-2 siRNA, compared with the scrambled siRNA transfected cells. The cell population in the M1 area was doubled after the treatment with combination of taxol and Bcl-2 siRNA. Further, we monitored DNA fragmentation in the cells after the treatments, as shown in the FACS histograms (Fig. 2B). The M1 area represented the population of apoptotic or dead cells with DNA fragmentation and the M2 area denoted the population of healthy cells. The apoptotic DNA fragmentation was remarkably increased after the treatment with combination of taxol and Bcl-2 siRNA (Fig. 2B). Concomitantly, the population of healthy cells was decreased. We presented the quantitative FACS data for percentage of live and apoptotic cells based on DNA fragmentation (Fig. 2C). Quantitation of data in the M1 area for DNA fragmentation demonstrated 28%, 30% and 65.5% apoptotic cells after treatment with taxol, Bcl-2 siRNA and combination of both, respectively (Fig. 2C). Results indicated that the treatment with combination of taxol and Bcl-2 siRNA caused more apoptosis in U251MG cells than either treatment alone. The untreated controls were not shown.


Combination of taxol and Bcl-2 siRNA induces apoptosis in human glioblastoma cells and inhibits invasion, angiogenesis and tumour growth.

George J, Banik NL, Ray SK - J. Cell. Mol. Med. (2009)

FACS analysis for detection of apoptotic cells and DNA fragmentation. Treatment (72 hrs) of cells: transfection with a plasmid vector expressing scrambled siRNA (treated control), 100 nM taxol, transfection with a plasmid vector expressing Bcl-2 siRNA and taxol + Bcl-2 siRNA. (A) FACS dot plots of U251MG cells. Before FACS analysis, the cells were treated with 50 μg/ml propidium iodide for 30 min. at 4°C in dark. Population in the M1 area represents the apoptotic/dead cells. (B) FACS histograms of U251MG cells. The prominent increase in population of cells in the sub-G1 phase (M1) indicated increase in apoptosis after treatment with taxol or Bcl-2 siRNA or both. (C) Quantitative presentation of DNA fragmentation data from FACS analysis to indicate percent changes in live and apoptotic cells. Data are representative of four independent experiments (*P < 0.001 when compared with scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496127&req=5

fig02: FACS analysis for detection of apoptotic cells and DNA fragmentation. Treatment (72 hrs) of cells: transfection with a plasmid vector expressing scrambled siRNA (treated control), 100 nM taxol, transfection with a plasmid vector expressing Bcl-2 siRNA and taxol + Bcl-2 siRNA. (A) FACS dot plots of U251MG cells. Before FACS analysis, the cells were treated with 50 μg/ml propidium iodide for 30 min. at 4°C in dark. Population in the M1 area represents the apoptotic/dead cells. (B) FACS histograms of U251MG cells. The prominent increase in population of cells in the sub-G1 phase (M1) indicated increase in apoptosis after treatment with taxol or Bcl-2 siRNA or both. (C) Quantitative presentation of DNA fragmentation data from FACS analysis to indicate percent changes in live and apoptotic cells. Data are representative of four independent experiments (*P < 0.001 when compared with scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
Mentions: FACS analysis is a useful tool for detection of apoptotic cells and DNA fragmentation. So, we performed FACS analysis for determining apoptosis and DNA fragmentation in U251MG cells after treatment with taxol or Bcl-2 siRNA or both agents together (Fig. 2). An increase in cell population in the M1 area indicated apoptotic cells, as shown in the FACS dot plot graphs (Fig. 2A). A significant increase (P < 0.001) in cell population in the M1 area occurred due to treatment with either taxol or Bcl-2 siRNA, compared with the scrambled siRNA transfected cells. The cell population in the M1 area was doubled after the treatment with combination of taxol and Bcl-2 siRNA. Further, we monitored DNA fragmentation in the cells after the treatments, as shown in the FACS histograms (Fig. 2B). The M1 area represented the population of apoptotic or dead cells with DNA fragmentation and the M2 area denoted the population of healthy cells. The apoptotic DNA fragmentation was remarkably increased after the treatment with combination of taxol and Bcl-2 siRNA (Fig. 2B). Concomitantly, the population of healthy cells was decreased. We presented the quantitative FACS data for percentage of live and apoptotic cells based on DNA fragmentation (Fig. 2C). Quantitation of data in the M1 area for DNA fragmentation demonstrated 28%, 30% and 65.5% apoptotic cells after treatment with taxol, Bcl-2 siRNA and combination of both, respectively (Fig. 2C). Results indicated that the treatment with combination of taxol and Bcl-2 siRNA caused more apoptosis in U251MG cells than either treatment alone. The untreated controls were not shown.

Bottom Line: We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth.In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination.In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, Columbia, SC 29209, USA.

ABSTRACT
Taxol is a powerful chemotherapeutic agent that binds to microtubules to prevent tumour cell division. However, a traditional high dose of taxol may also induce apoptosis in normal cells. The anti-apoptotic molecule Bcl-2 is up-regulated in tumour cells to prevent apoptosis. We designed this study to determine whether use of a low dose of taxol and anti-apoptotic Bcl-2 gene silencing would effectively induce apoptosis in human glioblastoma U251MG cells and also inhibit invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. We treated the cells with either 100 nM taxol or transfected with a plasmid vector expressing Bcl-2 siRNA or both agents together for 72 h. Knockdown of Bcl-2 potentiated efficacy of taxol for cell death. Fluorescence-activated cell sorting analysis, double immunofluorescent staining and TUNEL assay demonstrated apoptosis in about 70% of the cells after treatment with the combination of taxol and Bcl-2 siRNA. In vitro Matrigel invasion assay demonstrated dramatic decrease in glioblastoma cell invasion and in vivo angiogenesis assay showed complete inhibition of neovascularization in athymic nude mice after treatment with the combination. Further, treatment with the combination of taxol and Bcl-2 siRNA caused suppression of intracranial tumour growth and subcutaneous solid tumour development. In conclusion, our results indicate that the combination of taxol and Bcl-2 siRNA effectively induces apoptosis and inhibits glioblastoma cell invasion, angiogenesis and intracranial as well as subcutaneous tumour growth. Therefore, the combination of a low dose of taxol and Bcl-2 siRNA is a promising therapeutic strategy for controlling the aggressive growth of human glioblastoma.

Show MeSH
Related in: MedlinePlus