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Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage.

Zhang QQ, Wang WJ, Li J, Yang N, Chen G, Wang Z, Liang ZQ - Acta Pharmacol. Sin. (2015)

Bottom Line: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells.Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells.Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215000, China.

ABSTRACT

Aim: Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro.

Methods: Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy.

Results: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

Conclusion: Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.

No MeSH data available.


Related in: MedlinePlus

Effect of cathepsin L suppression on the response of glioma cells to irradiation. (A) Survival curves from the clonogenic assays of U251 cells. Cells were treated with Z-FY-CHO at 0, 1.25, 2.5, 5, and 10 μmol/L for 12 h, the cells were then treated with 4 Gy irradiation (or unirradiated) (left). Cells were treated with Z-FY-CHO at 10 μmol/L, stably transfected cell clones were designated Con shRNA and cathepsin L shRNA, the cells were then irradiated with 2, 4, 6, or 8 Gy (right). (B) RT-PCR and Western blot analyses were performed to determine the expression levels of cathepsin L in U251 cells that were transfected with cathepsin L shRNA or Con shRNA. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. fP<0.01 compared with the IR group.
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fig2: Effect of cathepsin L suppression on the response of glioma cells to irradiation. (A) Survival curves from the clonogenic assays of U251 cells. Cells were treated with Z-FY-CHO at 0, 1.25, 2.5, 5, and 10 μmol/L for 12 h, the cells were then treated with 4 Gy irradiation (or unirradiated) (left). Cells were treated with Z-FY-CHO at 10 μmol/L, stably transfected cell clones were designated Con shRNA and cathepsin L shRNA, the cells were then irradiated with 2, 4, 6, or 8 Gy (right). (B) RT-PCR and Western blot analyses were performed to determine the expression levels of cathepsin L in U251 cells that were transfected with cathepsin L shRNA or Con shRNA. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. fP<0.01 compared with the IR group.

Mentions: As mentioned previously, cathepsin L expression is exclusively elevated in U251 cells and in the conditioned medium after IR, and cathepsin L expression levels may correlate with the degree of radioresistance. The crucial role of cathepsin L in the malignancy of brain tumors has led to the development of novel cathepsin L inhibition strategies. A specific cathepsin L inhibitor, Z-FY-CHO, was used to corroborate our findings. The effects of IR and Z-FY-CHO treatments on cell survival were determined by conducting clonogenic assays to identify the function of cathepsin L in cell viability. As shown in Figure 2A, the inhibition rate was lower than 10% when the cells were treated with Z-FY-CHO (2.5–10 μmol/L) alone, while the clonogenicity of U251 cells that were treated with both Z-FY-CHO and IR was significantly reduced. The analysis was performed by comparing the SF values for cells left untreated (SF=1), cells treated with the cathepsin L inhibitor Z-FY-CHO (SF=0.85), cells treated with radiation (SF=0.83) and cells treated with Z-FY-CHO+4 Gy IR (SF=0.22). The cells treated with both Z-FY-CHO and IR generated a reduced number of colonies compared with the Z-FY-CHO-treated cells (P<0.01). As shown in Figure 2A, our results indicated that the clonogenicity of U251 cells treated with IR and Z-FY-CHO was significantly reduced in an ionization dose-dependent manner compared with IR treatment alone (treatment with Z-FY-CHO+IR resulted in a SER of 1.31, P<0.01). To demonstrate the function of cathepsin L in U251 glioma cells after IR, endogenous cathepsin L was knocked down in cells using short hairpin RNA (shRNA) interference, and then RT-PCR and Western blot analyses were conducted to confirm that cathepsin L expression was reduced in the cathepsin L knockdown (cathepsin L shRNA) cells (Figure 2B). Next, the cells were exposed to increasing IR doses (0, 2, 4, 6, and 8 Gy), and a dose-dependent reduction in clonogenic survival was observed. The cathepsin L shRNA cells also exhibited higher radiosensitivity than the Con shRNA U251 cells (Figure 2A). The results were analyzed by comparing the SF values of cells left untreated (SF=1), cells treated with radiation (SF=0.140) and cells treated with cathepsin L shRNA+8 Gy IR (SF=0.018), and the difference in these values was statistically significant (P<0.01). The SER of cathepsin L shRNA U251 cells was 1.54 (Figure 2A). These results indicated that cathepsin L inhibition could sensitize U251 glioma cells to IR.


Cathepsin L suppression increases the radiosensitivity of human glioma U251 cells via G2/M cell cycle arrest and DNA damage.

Zhang QQ, Wang WJ, Li J, Yang N, Chen G, Wang Z, Liang ZQ - Acta Pharmacol. Sin. (2015)

Effect of cathepsin L suppression on the response of glioma cells to irradiation. (A) Survival curves from the clonogenic assays of U251 cells. Cells were treated with Z-FY-CHO at 0, 1.25, 2.5, 5, and 10 μmol/L for 12 h, the cells were then treated with 4 Gy irradiation (or unirradiated) (left). Cells were treated with Z-FY-CHO at 10 μmol/L, stably transfected cell clones were designated Con shRNA and cathepsin L shRNA, the cells were then irradiated with 2, 4, 6, or 8 Gy (right). (B) RT-PCR and Western blot analyses were performed to determine the expression levels of cathepsin L in U251 cells that were transfected with cathepsin L shRNA or Con shRNA. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. fP<0.01 compared with the IR group.
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Related In: Results  -  Collection

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fig2: Effect of cathepsin L suppression on the response of glioma cells to irradiation. (A) Survival curves from the clonogenic assays of U251 cells. Cells were treated with Z-FY-CHO at 0, 1.25, 2.5, 5, and 10 μmol/L for 12 h, the cells were then treated with 4 Gy irradiation (or unirradiated) (left). Cells were treated with Z-FY-CHO at 10 μmol/L, stably transfected cell clones were designated Con shRNA and cathepsin L shRNA, the cells were then irradiated with 2, 4, 6, or 8 Gy (right). (B) RT-PCR and Western blot analyses were performed to determine the expression levels of cathepsin L in U251 cells that were transfected with cathepsin L shRNA or Con shRNA. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. fP<0.01 compared with the IR group.
Mentions: As mentioned previously, cathepsin L expression is exclusively elevated in U251 cells and in the conditioned medium after IR, and cathepsin L expression levels may correlate with the degree of radioresistance. The crucial role of cathepsin L in the malignancy of brain tumors has led to the development of novel cathepsin L inhibition strategies. A specific cathepsin L inhibitor, Z-FY-CHO, was used to corroborate our findings. The effects of IR and Z-FY-CHO treatments on cell survival were determined by conducting clonogenic assays to identify the function of cathepsin L in cell viability. As shown in Figure 2A, the inhibition rate was lower than 10% when the cells were treated with Z-FY-CHO (2.5–10 μmol/L) alone, while the clonogenicity of U251 cells that were treated with both Z-FY-CHO and IR was significantly reduced. The analysis was performed by comparing the SF values for cells left untreated (SF=1), cells treated with the cathepsin L inhibitor Z-FY-CHO (SF=0.85), cells treated with radiation (SF=0.83) and cells treated with Z-FY-CHO+4 Gy IR (SF=0.22). The cells treated with both Z-FY-CHO and IR generated a reduced number of colonies compared with the Z-FY-CHO-treated cells (P<0.01). As shown in Figure 2A, our results indicated that the clonogenicity of U251 cells treated with IR and Z-FY-CHO was significantly reduced in an ionization dose-dependent manner compared with IR treatment alone (treatment with Z-FY-CHO+IR resulted in a SER of 1.31, P<0.01). To demonstrate the function of cathepsin L in U251 glioma cells after IR, endogenous cathepsin L was knocked down in cells using short hairpin RNA (shRNA) interference, and then RT-PCR and Western blot analyses were conducted to confirm that cathepsin L expression was reduced in the cathepsin L knockdown (cathepsin L shRNA) cells (Figure 2B). Next, the cells were exposed to increasing IR doses (0, 2, 4, 6, and 8 Gy), and a dose-dependent reduction in clonogenic survival was observed. The cathepsin L shRNA cells also exhibited higher radiosensitivity than the Con shRNA U251 cells (Figure 2A). The results were analyzed by comparing the SF values of cells left untreated (SF=1), cells treated with radiation (SF=0.140) and cells treated with cathepsin L shRNA+8 Gy IR (SF=0.018), and the difference in these values was statistically significant (P<0.01). The SER of cathepsin L shRNA U251 cells was 1.54 (Figure 2A). These results indicated that cathepsin L inhibition could sensitize U251 glioma cells to IR.

Bottom Line: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells.Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells.Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215000, China.

ABSTRACT

Aim: Cathepsin L is a lysosomal cysteine protease that plays important roles in cancer tumorigenesis, proliferation and chemotherapy resistance. The aim of this study was to determine how cathepsin L regulated the radiosensitivity of human glioma cells in vitro.

Methods: Human glioma U251 cells (harboring the mutant type p53 gene) and U87 cells (harboring the wide type p53 gene) were irradiated with X-rays. The expression of cathepsin L was analyzed using Western blot and immunofluorescence assays. Cell survival and DNA damage were evaluated using clonogenic and comet assays, respectively. Flow cytometry was used to detect the cell cycle distribution. Apoptotic cells were observed using Hoechst 33258 staining and fluorescence microscopy.

Results: Irradiation significantly increased the cytoplasmic and nuclear levels of cathepsin L in U251 cells but not in U87 cells. Treatment with the specific cathepsin L inhibitor Z-FY-CHO (10 μmol/L) or transfection with cathepsin L shRNA significantly increased the radiosensitivity of U251 cells. Both suppression and knockdown of cathepsin L in U251 cells increased irradiation-induced DNA damage and G2/M phase cell cycle arrest. Both suppression and knockdown of cathepsin L in U251 cells also increased irradiation-induced apoptosis, as shown by the increased levels of Bax and decreased levels of Bcl-2.

Conclusion: Cathepsin L is involved in modulation of radiosensitivity in human glioma U251 cells (harboring the mutant type p53 gene) in vitro.

No MeSH data available.


Related in: MedlinePlus