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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 inhibition on irradiation (IR)-induced G2/M phase arrest. (A) Cell cycle phase distribution of cells that were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA. Then, the cells were treated with 8 Gy IR or unirradiated. (B) The cells were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h or transfected with cathepsin L shRNA or Con shRNA. The cells were analyzed at 48 h post-IR by Western blot to determine cyclin B1 and A protein levels. At least 3 independent experiments were performed. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. eP<0.05, fP<0.01 compared with the IR group.
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fig5: Effect of cathepsin L inhibition on irradiation (IR)-induced G2/M phase arrest. (A) Cell cycle phase distribution of cells that were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA. Then, the cells were treated with 8 Gy IR or unirradiated. (B) The cells were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h or transfected with cathepsin L shRNA or Con shRNA. The cells were analyzed at 48 h post-IR by Western blot to determine cyclin B1 and A protein levels. At least 3 independent experiments were performed. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. eP<0.05, fP<0.01 compared with the IR group.

Mentions: Our previous study demonstrated that nuclear translocated cathepsin L can process the CDP/Cux transcription factor and revealed an important role for cathepsin L in controlling cell cycle progression7. We analyzed the cell cycle phase distribution by performing flow cytometry to assess whether cathepsin L inhibition-induced radiosensitivity in glioma cells is also associated with altered cell cycle distribution. Unlike the unirradiated controls, the cell percentages at G2/M phase were higher in the cathepsin L-suppressed U251 cells. Moreover, the G2/M cell cycle arrest in the cells treated with both cathepsin L inhibition and IR was more evident, particularly at the 48 h time point (Figure 5A), compared with that in the irradiated cells. These results may explain the cathepsin L suppression-induced radiosensitivity in glioma cells. However, the combined treatments for 24 and 72 h did not result in significant increases in the cell percentages at G2/M phase compared with IR alone (data not shown).


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 inhibition on irradiation (IR)-induced G2/M phase arrest. (A) Cell cycle phase distribution of cells that were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA. Then, the cells were treated with 8 Gy IR or unirradiated. (B) The cells were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h or transfected with cathepsin L shRNA or Con shRNA. The cells were analyzed at 48 h post-IR by Western blot to determine cyclin B1 and A protein levels. At least 3 independent experiments were performed. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. eP<0.05, fP<0.01 compared with the IR group.
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Related In: Results  -  Collection

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fig5: Effect of cathepsin L inhibition on irradiation (IR)-induced G2/M phase arrest. (A) Cell cycle phase distribution of cells that were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA. Then, the cells were treated with 8 Gy IR or unirradiated. (B) The cells were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h or transfected with cathepsin L shRNA or Con shRNA. The cells were analyzed at 48 h post-IR by Western blot to determine cyclin B1 and A protein levels. At least 3 independent experiments were performed. Mean±SD. n=3. bP<0.05, cP<0.01 compared with the control group. eP<0.05, fP<0.01 compared with the IR group.
Mentions: Our previous study demonstrated that nuclear translocated cathepsin L can process the CDP/Cux transcription factor and revealed an important role for cathepsin L in controlling cell cycle progression7. We analyzed the cell cycle phase distribution by performing flow cytometry to assess whether cathepsin L inhibition-induced radiosensitivity in glioma cells is also associated with altered cell cycle distribution. Unlike the unirradiated controls, the cell percentages at G2/M phase were higher in the cathepsin L-suppressed U251 cells. Moreover, the G2/M cell cycle arrest in the cells treated with both cathepsin L inhibition and IR was more evident, particularly at the 48 h time point (Figure 5A), compared with that in the irradiated cells. These results may explain the cathepsin L suppression-induced radiosensitivity in glioma cells. However, the combined treatments for 24 and 72 h did not result in significant increases in the cell percentages at G2/M phase compared with IR alone (data not shown).

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