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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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Expression of Bcl-2 at mRNA and protein levels in U251MG cells. Treatment (72 hrs) of cells: control, 100 nM taxol, transfection with a plasmid vector carrying the scrambled siRNA cDNA, transfection with a plasmid vector carrying the Bcl-2 siRNA cDNA and taxol + Bcl-2 siRNA. In determination of both RNA and protein levels, GAPDH was used as an internal control. (A) Semi-quantitative RT-PCR for examination of Bcl-2 mRNA. (B) Western blotting for examination of Bcl-2 protein. (C) MTT assay for determination of percent changes in cell viability. For MTT assay, the cells were treated for 48 hrs. Data are representative of six independent experiments in duplicate (*P < 0.001 when compared with the control mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA mean values).
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fig01: Expression of Bcl-2 at mRNA and protein levels in U251MG cells. Treatment (72 hrs) of cells: control, 100 nM taxol, transfection with a plasmid vector carrying the scrambled siRNA cDNA, transfection with a plasmid vector carrying the Bcl-2 siRNA cDNA and taxol + Bcl-2 siRNA. In determination of both RNA and protein levels, GAPDH was used as an internal control. (A) Semi-quantitative RT-PCR for examination of Bcl-2 mRNA. (B) Western blotting for examination of Bcl-2 protein. (C) MTT assay for determination of percent changes in cell viability. For MTT assay, the cells were treated for 48 hrs. Data are representative of six independent experiments in duplicate (*P < 0.001 when compared with the control mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA mean values).

Mentions: Remarkable knockdown of Bcl-2 at mRNA and protein levels occurred in both U138MG and U251MG cells after transfection with the plasmid vector expressing Bcl-2 siRNA. The data were presented only for U251MG cells (Fig. 1). We found no appreciable alterations in Bcl-2 mRNA (Fig. 1A) and Bcl-2 protein (Fig. 1B) levels after transfection with a scrambled siRNA vector or treatment with taxol alone. Treatment with Bcl-2 siRNA alone or combination of taxol and Bcl-2 siRNA resulted in approximately 80% knockdown of Bcl-2 mRNA (Fig. 1A). Bcl-2 siRNA alone resulted in almost 60% down-regulation of cognate protein and the combination of taxol and Bcl-2 siRNA resulted in about 70% down-regulation of Bcl-2 (Fig. 1B). Expression of GAPDH was used as an internal control to demonstrate equal loading of both RNA and protein samples.


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)

Expression of Bcl-2 at mRNA and protein levels in U251MG cells. Treatment (72 hrs) of cells: control, 100 nM taxol, transfection with a plasmid vector carrying the scrambled siRNA cDNA, transfection with a plasmid vector carrying the Bcl-2 siRNA cDNA and taxol + Bcl-2 siRNA. In determination of both RNA and protein levels, GAPDH was used as an internal control. (A) Semi-quantitative RT-PCR for examination of Bcl-2 mRNA. (B) Western blotting for examination of Bcl-2 protein. (C) MTT assay for determination of percent changes in cell viability. For MTT assay, the cells were treated for 48 hrs. Data are representative of six independent experiments in duplicate (*P < 0.001 when compared with the control mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA mean values).
© Copyright Policy
Related In: Results  -  Collection

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

fig01: Expression of Bcl-2 at mRNA and protein levels in U251MG cells. Treatment (72 hrs) of cells: control, 100 nM taxol, transfection with a plasmid vector carrying the scrambled siRNA cDNA, transfection with a plasmid vector carrying the Bcl-2 siRNA cDNA and taxol + Bcl-2 siRNA. In determination of both RNA and protein levels, GAPDH was used as an internal control. (A) Semi-quantitative RT-PCR for examination of Bcl-2 mRNA. (B) Western blotting for examination of Bcl-2 protein. (C) MTT assay for determination of percent changes in cell viability. For MTT assay, the cells were treated for 48 hrs. Data are representative of six independent experiments in duplicate (*P < 0.001 when compared with the control mean values and #P < 0.001 when compared with taxol or Bcl-2 siRNA mean values).
Mentions: Remarkable knockdown of Bcl-2 at mRNA and protein levels occurred in both U138MG and U251MG cells after transfection with the plasmid vector expressing Bcl-2 siRNA. The data were presented only for U251MG cells (Fig. 1). We found no appreciable alterations in Bcl-2 mRNA (Fig. 1A) and Bcl-2 protein (Fig. 1B) levels after transfection with a scrambled siRNA vector or treatment with taxol alone. Treatment with Bcl-2 siRNA alone or combination of taxol and Bcl-2 siRNA resulted in approximately 80% knockdown of Bcl-2 mRNA (Fig. 1A). Bcl-2 siRNA alone resulted in almost 60% down-regulation of cognate protein and the combination of taxol and Bcl-2 siRNA resulted in about 70% down-regulation of Bcl-2 (Fig. 1B). Expression of GAPDH was used as an internal control to demonstrate equal loading of both RNA and protein samples.

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