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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 vivo angiogenesis (dorsal skinfold) assay. 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. The U251MG cells (2 × 105) from each treatment were suspended in 200 μl of serum-free medium and injected into a diffusion chamber. Then, diffusion chambers were surgically implanted under the dorsal skin of nude mice and left for 10 days. (A) In vivo angiogenesis in terms of development of neovasculature. Strong development of tumour-induced neovasculature (TN) with curved thin structures (as indicated by TN arrows) arising from pre-existing vasculature (PV) with relatively straight structures (as indicated by PV arrows) was observed in nude mice with U251MG cells untreated (not shown) and transfected with scrambled siRNA vector. The formation of such neovasculature was considerably reduced in nude mice with U251MG cells treated with taxol or Bcl-2 siRNA and completely inhibited in nude mice with U251MG cells treated with combination of taxol and Bcl-2 siRNA. (B) Quantitative presentation of neovasculature to indicate the extent of in vivo angiogenesis. The measurement of the TN was performed with the help of an ocular micrometer. Values are presented as mean ± S.D. of TN in six animals from each treatment group (*P <0.001 when compared with the scrambled siRNA treatment mean values).
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fig05: In vivo angiogenesis (dorsal skinfold) assay. 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. The U251MG cells (2 × 105) from each treatment were suspended in 200 μl of serum-free medium and injected into a diffusion chamber. Then, diffusion chambers were surgically implanted under the dorsal skin of nude mice and left for 10 days. (A) In vivo angiogenesis in terms of development of neovasculature. Strong development of tumour-induced neovasculature (TN) with curved thin structures (as indicated by TN arrows) arising from pre-existing vasculature (PV) with relatively straight structures (as indicated by PV arrows) was observed in nude mice with U251MG cells untreated (not shown) and transfected with scrambled siRNA vector. The formation of such neovasculature was considerably reduced in nude mice with U251MG cells treated with taxol or Bcl-2 siRNA and completely inhibited in nude mice with U251MG cells treated with combination of taxol and Bcl-2 siRNA. (B) Quantitative presentation of neovasculature to indicate the extent of in vivo angiogenesis. The measurement of the TN was performed with the help of an ocular micrometer. Values are presented as mean ± S.D. of TN in six animals from each treatment group (*P <0.001 when compared with the scrambled siRNA treatment mean values).

Mentions: We examined the effect of taxol, Bcl-2 siRNA and both agents together on in vivo angiogenesis in terms of neovascularization in the dorsal skin of nude mice (Fig. 5). The implantation of diffusion chambers containing U251MG untreated cells (not shown) and scrambled siRNA vector transfected cells (treated control) maintained the formation of well-developed blood microvessels as indicated by the thin and curved structures arising from the pre-existing vessels in a zigzag manner (Fig. 5A). The formation of similar structures was partially inhibited in cells treated with taxol or Bcl-2 siRNA but completely inhibited in cells treated with both agents together (Fig. 5A). Quantitative measurements of the tumour-induced neovasculature revealed inhibition of 75% and 70% neuvasculature in cells treated with taxol alone and Bcl-2 siRNA alone, respectively, while inhibition of 100% neovasculature in cells treated with both agents together (Fig. 5B). These results established that combination of taxol and Bcl-2 siRNA could completely inhibit in vivo angiogenesis.


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 vivo angiogenesis (dorsal skinfold) assay. 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. The U251MG cells (2 × 105) from each treatment were suspended in 200 μl of serum-free medium and injected into a diffusion chamber. Then, diffusion chambers were surgically implanted under the dorsal skin of nude mice and left for 10 days. (A) In vivo angiogenesis in terms of development of neovasculature. Strong development of tumour-induced neovasculature (TN) with curved thin structures (as indicated by TN arrows) arising from pre-existing vasculature (PV) with relatively straight structures (as indicated by PV arrows) was observed in nude mice with U251MG cells untreated (not shown) and transfected with scrambled siRNA vector. The formation of such neovasculature was considerably reduced in nude mice with U251MG cells treated with taxol or Bcl-2 siRNA and completely inhibited in nude mice with U251MG cells treated with combination of taxol and Bcl-2 siRNA. (B) Quantitative presentation of neovasculature to indicate the extent of in vivo angiogenesis. The measurement of the TN was performed with the help of an ocular micrometer. Values are presented as mean ± S.D. of TN in six animals from each treatment group (*P <0.001 when compared with the scrambled siRNA treatment mean values).
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fig05: In vivo angiogenesis (dorsal skinfold) assay. 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. The U251MG cells (2 × 105) from each treatment were suspended in 200 μl of serum-free medium and injected into a diffusion chamber. Then, diffusion chambers were surgically implanted under the dorsal skin of nude mice and left for 10 days. (A) In vivo angiogenesis in terms of development of neovasculature. Strong development of tumour-induced neovasculature (TN) with curved thin structures (as indicated by TN arrows) arising from pre-existing vasculature (PV) with relatively straight structures (as indicated by PV arrows) was observed in nude mice with U251MG cells untreated (not shown) and transfected with scrambled siRNA vector. The formation of such neovasculature was considerably reduced in nude mice with U251MG cells treated with taxol or Bcl-2 siRNA and completely inhibited in nude mice with U251MG cells treated with combination of taxol and Bcl-2 siRNA. (B) Quantitative presentation of neovasculature to indicate the extent of in vivo angiogenesis. The measurement of the TN was performed with the help of an ocular micrometer. Values are presented as mean ± S.D. of TN in six animals from each treatment group (*P <0.001 when compared with the scrambled siRNA treatment mean values).
Mentions: We examined the effect of taxol, Bcl-2 siRNA and both agents together on in vivo angiogenesis in terms of neovascularization in the dorsal skin of nude mice (Fig. 5). The implantation of diffusion chambers containing U251MG untreated cells (not shown) and scrambled siRNA vector transfected cells (treated control) maintained the formation of well-developed blood microvessels as indicated by the thin and curved structures arising from the pre-existing vessels in a zigzag manner (Fig. 5A). The formation of similar structures was partially inhibited in cells treated with taxol or Bcl-2 siRNA but completely inhibited in cells treated with both agents together (Fig. 5A). Quantitative measurements of the tumour-induced neovasculature revealed inhibition of 75% and 70% neuvasculature in cells treated with taxol alone and Bcl-2 siRNA alone, respectively, while inhibition of 100% neovasculature in cells treated with both agents together (Fig. 5B). These results established that combination of taxol and Bcl-2 siRNA could completely inhibit in vivo angiogenesis.

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