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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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In vitro Matrigel invasion assay using U138MG and U251MG cells. 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. Invasion assays were carried out in 12-well transwell inserts of polycarbonate filters. After 48 hrs incubation at 37°C in a CO2 incubator, the membranes were collected and stained with HEMA. The number of cells that migrated to the undersurface of the membrane were examined under a microscope, counted and photographed. (A) The changes in capability of invasion of U138MG cells after the treatments. (B) Quantitative evaluation of invading U138MG cells. (C) The changes in capability of invasion of U251MG cells after the treatments. (D) Quantitative evaluation of invading U251MG cells. The quantitative data are presented as mean ± S.D. of cells from 10 randomly selected microscopic fields from three independent wells (*P < 0.001 when compared with the scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
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fig04: In vitro Matrigel invasion assay using U138MG and U251MG cells. 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. Invasion assays were carried out in 12-well transwell inserts of polycarbonate filters. After 48 hrs incubation at 37°C in a CO2 incubator, the membranes were collected and stained with HEMA. The number of cells that migrated to the undersurface of the membrane were examined under a microscope, counted and photographed. (A) The changes in capability of invasion of U138MG cells after the treatments. (B) Quantitative evaluation of invading U138MG cells. (C) The changes in capability of invasion of U251MG cells after the treatments. (D) Quantitative evaluation of invading U251MG cells. The quantitative data are presented as mean ± S.D. of cells from 10 randomly selected microscopic fields from three independent wells (*P < 0.001 when compared with the scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).

Mentions: We examined the effects of taxol, Bcl-2 siRNA and combination of both agents on the invasive properties of both U138MG and U251MG cells using the Matrigel invasion assay (Fig. 4). The staining of invading cells through the polycarbonate membranes demonstrated that the invasion of cells was very low after treatment with taxol or Bcl-2 siRNA, compared with invasion of untreated control cells (not shown) or scrambled siRNA vector transfected cells (Fig. 4A and C). Taxol was more effective than Bcl-2 siRNA to prevent cell invasion through the Matrigel. The treatment with combination of taxol and Bcl-2 siRNA resulted in a remarkable decrease in the number of invading cells. Quantitation of the invading cells using Image-Pro Plus software showed that the U138MG cells after treatment with taxol, Bcl-2 siRNA and both agents together resulted in only 31%, 57% and 12% invasion, respectively, compared with cells after transfection with scrambled siRNA (Fig. 4B). In U251MG cells, we observed only 35%, 49% and 10% invasion after treatment with taxol, Bcl-2 siRNA and both agents together, respectively (Fig. 4D). The scrambled siRNA treatments were considered as treated controls for both cell lines.


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)

In vitro Matrigel invasion assay using U138MG and U251MG cells. 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. Invasion assays were carried out in 12-well transwell inserts of polycarbonate filters. After 48 hrs incubation at 37°C in a CO2 incubator, the membranes were collected and stained with HEMA. The number of cells that migrated to the undersurface of the membrane were examined under a microscope, counted and photographed. (A) The changes in capability of invasion of U138MG cells after the treatments. (B) Quantitative evaluation of invading U138MG cells. (C) The changes in capability of invasion of U251MG cells after the treatments. (D) Quantitative evaluation of invading U251MG cells. The quantitative data are presented as mean ± S.D. of cells from 10 randomly selected microscopic fields from three independent wells (*P < 0.001 when compared with the scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4496127&req=5

fig04: In vitro Matrigel invasion assay using U138MG and U251MG cells. 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. Invasion assays were carried out in 12-well transwell inserts of polycarbonate filters. After 48 hrs incubation at 37°C in a CO2 incubator, the membranes were collected and stained with HEMA. The number of cells that migrated to the undersurface of the membrane were examined under a microscope, counted and photographed. (A) The changes in capability of invasion of U138MG cells after the treatments. (B) Quantitative evaluation of invading U138MG cells. (C) The changes in capability of invasion of U251MG cells after the treatments. (D) Quantitative evaluation of invading U251MG cells. The quantitative data are presented as mean ± S.D. of cells from 10 randomly selected microscopic fields from three independent wells (*P < 0.001 when compared with the scrambled siRNA treatment mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA treatment mean values).
Mentions: We examined the effects of taxol, Bcl-2 siRNA and combination of both agents on the invasive properties of both U138MG and U251MG cells using the Matrigel invasion assay (Fig. 4). The staining of invading cells through the polycarbonate membranes demonstrated that the invasion of cells was very low after treatment with taxol or Bcl-2 siRNA, compared with invasion of untreated control cells (not shown) or scrambled siRNA vector transfected cells (Fig. 4A and C). Taxol was more effective than Bcl-2 siRNA to prevent cell invasion through the Matrigel. The treatment with combination of taxol and Bcl-2 siRNA resulted in a remarkable decrease in the number of invading cells. Quantitation of the invading cells using Image-Pro Plus software showed that the U138MG cells after treatment with taxol, Bcl-2 siRNA and both agents together resulted in only 31%, 57% and 12% invasion, respectively, compared with cells after transfection with scrambled siRNA (Fig. 4B). In U251MG cells, we observed only 35%, 49% and 10% invasion after treatment with taxol, Bcl-2 siRNA and both agents together, respectively (Fig. 4D). The scrambled siRNA treatments were considered as treated controls for both cell lines.

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