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Silencing of R-Spondin1 increases radiosensitivity of glioma cells.

Gu X, Wang X, Xiao H, Ma G, Cui L, Li Y, Zhou H, Liang W, Zhao B, Li K - Oncotarget (2015)

Bottom Line: Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma.We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival.In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurology, Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma. We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival. Rspo1 expression was also associated with reduced survival rates in glioma patients after treatment with radiotherapy and temozolomide (RT-TMZ). Importantly, Rspo1 was dramatically upregulated after radiation treatment in patients with glioma. Rspo1 silencing by shRNA potentiated glioma cell death upon radiation treatment. In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition. Thus, combining radiotherapy with silencing of Rspo1 is a potential therapeutic approach.

No MeSH data available.


Related in: MedlinePlus

Silencing of Rspo1 sensitizes U87 cells to radiation(A) U87 cells transfected with either nonsense control shRNA (Ctrl shRNA) or Rspo1 shRNA1 and Rspo1 shRNA2. The levels of Rspo1 were detected by immunostaining with the anti-Rspo1 antibody, and actin was used as a loading control. (B, C) Silencing of Rspo1 sensitizes tumor cells to radiation. U87 cells were transfected with either a nonsense control shRNA (Ctrl shRNA) or one of two specific shRNAs targeting Rspo1. At 24 h after transfection, the cells were treated with increasing doses of radiation. The cell death rate (B), measured using the ToxiLight kit as described in the Materials and Methods section, and survival (C) were evaluated 48 h after treatment. The results were normalized against those obtained with control (untreated) cells. Whereas control shRNA-transfected cells showed a general resistance to radiation treatment, Silencing of Rspo1 strongly induced cell death and decreased cell survival in the presence of radiation. (D) Survival fraction curves of U87/control shRNA cells and U87/Rspo1 shRNA cells tested by clonogenic survival assay. (E, F) Silencing of Rspo1 triggered apoptosis in combination with radiation treatment. U87 cells were transfected as in B and C and treated with the indicated dose of radiation for 24 h. Active caspase 3 levels (E), normalized to those in untreated cells, and DNA fragmentation (F) were evaluated as described in the Materials and Methods section. Whereas radiation failed to induce apoptosis in control shRNA-transfected cells, Rspo1-silenced cells showed a strong increase in the apoptotic rate. The results represent at least three separate experiments. Error bars: ± S.D. *p < 0.05. **p < 0.01.
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Figure 4: Silencing of Rspo1 sensitizes U87 cells to radiation(A) U87 cells transfected with either nonsense control shRNA (Ctrl shRNA) or Rspo1 shRNA1 and Rspo1 shRNA2. The levels of Rspo1 were detected by immunostaining with the anti-Rspo1 antibody, and actin was used as a loading control. (B, C) Silencing of Rspo1 sensitizes tumor cells to radiation. U87 cells were transfected with either a nonsense control shRNA (Ctrl shRNA) or one of two specific shRNAs targeting Rspo1. At 24 h after transfection, the cells were treated with increasing doses of radiation. The cell death rate (B), measured using the ToxiLight kit as described in the Materials and Methods section, and survival (C) were evaluated 48 h after treatment. The results were normalized against those obtained with control (untreated) cells. Whereas control shRNA-transfected cells showed a general resistance to radiation treatment, Silencing of Rspo1 strongly induced cell death and decreased cell survival in the presence of radiation. (D) Survival fraction curves of U87/control shRNA cells and U87/Rspo1 shRNA cells tested by clonogenic survival assay. (E, F) Silencing of Rspo1 triggered apoptosis in combination with radiation treatment. U87 cells were transfected as in B and C and treated with the indicated dose of radiation for 24 h. Active caspase 3 levels (E), normalized to those in untreated cells, and DNA fragmentation (F) were evaluated as described in the Materials and Methods section. Whereas radiation failed to induce apoptosis in control shRNA-transfected cells, Rspo1-silenced cells showed a strong increase in the apoptotic rate. The results represent at least three separate experiments. Error bars: ± S.D. *p < 0.05. **p < 0.01.

Mentions: The upregulation of Rspo1 upon radiation treatment suggests that the dependence on Rspo1 for survival is amplified in radiotherapy-treated cancer cells. Thus, we analyzed the effect of silencing Rspo1 by shRNA on radiation-induced cell death. U87 cells were transfected with plasmids expressing Rspo1 shRNAs and treated with increasing doses of radiation. Both Rspo1 shRNA1 and Rspo1 shRNA2 silenced Rspo1 protein expression by over 80% compared with nonsense control shRNA (Fig. 4A). Silencing of Rspo1 was associated with a marked potentiation of radiation-induced cell death, as shown by the loss of cell permeability (Fig. 4B) and decrease in cell survival (Fig. 4C). Furthermore, a clonogenic survival assay was performed to measure radiation sensitivity. We found that the survival fraction was significantly decreased when Rspo1 was knocked down by Rspo1 shRNA in U87 cells (Fig. 4D). Activation of the cell apoptosis marker caspase 3 (Fig. 4E) and DNA fragmentation (Fig. 4F) were increased accordingly.


Silencing of R-Spondin1 increases radiosensitivity of glioma cells.

Gu X, Wang X, Xiao H, Ma G, Cui L, Li Y, Zhou H, Liang W, Zhao B, Li K - Oncotarget (2015)

Silencing of Rspo1 sensitizes U87 cells to radiation(A) U87 cells transfected with either nonsense control shRNA (Ctrl shRNA) or Rspo1 shRNA1 and Rspo1 shRNA2. The levels of Rspo1 were detected by immunostaining with the anti-Rspo1 antibody, and actin was used as a loading control. (B, C) Silencing of Rspo1 sensitizes tumor cells to radiation. U87 cells were transfected with either a nonsense control shRNA (Ctrl shRNA) or one of two specific shRNAs targeting Rspo1. At 24 h after transfection, the cells were treated with increasing doses of radiation. The cell death rate (B), measured using the ToxiLight kit as described in the Materials and Methods section, and survival (C) were evaluated 48 h after treatment. The results were normalized against those obtained with control (untreated) cells. Whereas control shRNA-transfected cells showed a general resistance to radiation treatment, Silencing of Rspo1 strongly induced cell death and decreased cell survival in the presence of radiation. (D) Survival fraction curves of U87/control shRNA cells and U87/Rspo1 shRNA cells tested by clonogenic survival assay. (E, F) Silencing of Rspo1 triggered apoptosis in combination with radiation treatment. U87 cells were transfected as in B and C and treated with the indicated dose of radiation for 24 h. Active caspase 3 levels (E), normalized to those in untreated cells, and DNA fragmentation (F) were evaluated as described in the Materials and Methods section. Whereas radiation failed to induce apoptosis in control shRNA-transfected cells, Rspo1-silenced cells showed a strong increase in the apoptotic rate. The results represent at least three separate experiments. Error bars: ± S.D. *p < 0.05. **p < 0.01.
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Related In: Results  -  Collection

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Figure 4: Silencing of Rspo1 sensitizes U87 cells to radiation(A) U87 cells transfected with either nonsense control shRNA (Ctrl shRNA) or Rspo1 shRNA1 and Rspo1 shRNA2. The levels of Rspo1 were detected by immunostaining with the anti-Rspo1 antibody, and actin was used as a loading control. (B, C) Silencing of Rspo1 sensitizes tumor cells to radiation. U87 cells were transfected with either a nonsense control shRNA (Ctrl shRNA) or one of two specific shRNAs targeting Rspo1. At 24 h after transfection, the cells were treated with increasing doses of radiation. The cell death rate (B), measured using the ToxiLight kit as described in the Materials and Methods section, and survival (C) were evaluated 48 h after treatment. The results were normalized against those obtained with control (untreated) cells. Whereas control shRNA-transfected cells showed a general resistance to radiation treatment, Silencing of Rspo1 strongly induced cell death and decreased cell survival in the presence of radiation. (D) Survival fraction curves of U87/control shRNA cells and U87/Rspo1 shRNA cells tested by clonogenic survival assay. (E, F) Silencing of Rspo1 triggered apoptosis in combination with radiation treatment. U87 cells were transfected as in B and C and treated with the indicated dose of radiation for 24 h. Active caspase 3 levels (E), normalized to those in untreated cells, and DNA fragmentation (F) were evaluated as described in the Materials and Methods section. Whereas radiation failed to induce apoptosis in control shRNA-transfected cells, Rspo1-silenced cells showed a strong increase in the apoptotic rate. The results represent at least three separate experiments. Error bars: ± S.D. *p < 0.05. **p < 0.01.
Mentions: The upregulation of Rspo1 upon radiation treatment suggests that the dependence on Rspo1 for survival is amplified in radiotherapy-treated cancer cells. Thus, we analyzed the effect of silencing Rspo1 by shRNA on radiation-induced cell death. U87 cells were transfected with plasmids expressing Rspo1 shRNAs and treated with increasing doses of radiation. Both Rspo1 shRNA1 and Rspo1 shRNA2 silenced Rspo1 protein expression by over 80% compared with nonsense control shRNA (Fig. 4A). Silencing of Rspo1 was associated with a marked potentiation of radiation-induced cell death, as shown by the loss of cell permeability (Fig. 4B) and decrease in cell survival (Fig. 4C). Furthermore, a clonogenic survival assay was performed to measure radiation sensitivity. We found that the survival fraction was significantly decreased when Rspo1 was knocked down by Rspo1 shRNA in U87 cells (Fig. 4D). Activation of the cell apoptosis marker caspase 3 (Fig. 4E) and DNA fragmentation (Fig. 4F) were increased accordingly.

Bottom Line: Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma.We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival.In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Institute of Neurology, Guangdong Medical College, Zhanjiang 524001, China.

ABSTRACT
Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma. We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival. Rspo1 expression was also associated with reduced survival rates in glioma patients after treatment with radiotherapy and temozolomide (RT-TMZ). Importantly, Rspo1 was dramatically upregulated after radiation treatment in patients with glioma. Rspo1 silencing by shRNA potentiated glioma cell death upon radiation treatment. In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition. Thus, combining radiotherapy with silencing of Rspo1 is a potential therapeutic approach.

No MeSH data available.


Related in: MedlinePlus