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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 irradiation-induced DNA damage in U251 cells. DNA damage in U251 cells was examined by alkaline comet assays. The cells were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA, and then the cells were treated with 8 Gy of IR (or unirradiated). 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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fig3: Effect of cathepsin L suppression on irradiation-induced DNA damage in U251 cells. DNA damage in U251 cells was examined by alkaline comet assays. The cells were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA, and then the cells were treated with 8 Gy of IR (or unirradiated). 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: The repair of DNA damage, such as double-strand breaks (DSBs), is an important determinant of cellular radiosensitivity25. We performed single-cell gel electrophoresis assays (alkaline comet assays) to study the DNA damage in U251 cells caused by cathepsin L inhibition. Figure 3 shows that a significantly faster DNA damage repair rate was observed in the Con shRNA cells compared with the Z-FY-CHO-pretreated cells or post-IR cathepsin L shRNA-transfected cells. Over 90% of the repair was completed 30 h following IR in the Con shRNA cells, whereas the cathepsin L-suppressed cells remained unrepaired (65%). These data suggest that cathepsin L inhibition may affect the DNA repair level.


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 irradiation-induced DNA damage in U251 cells. DNA damage in U251 cells was examined by alkaline comet assays. The cells were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA, and then the cells were treated with 8 Gy of IR (or unirradiated). 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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getmorefigures.php?uid=PMC4561966&req=5

fig3: Effect of cathepsin L suppression on irradiation-induced DNA damage in U251 cells. DNA damage in U251 cells was examined by alkaline comet assays. The cells were pretreated with Z-FY-CHO or transfected with Con shRNA or cathepsin L shRNA, and then the cells were treated with 8 Gy of IR (or unirradiated). 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: The repair of DNA damage, such as double-strand breaks (DSBs), is an important determinant of cellular radiosensitivity25. We performed single-cell gel electrophoresis assays (alkaline comet assays) to study the DNA damage in U251 cells caused by cathepsin L inhibition. Figure 3 shows that a significantly faster DNA damage repair rate was observed in the Con shRNA cells compared with the Z-FY-CHO-pretreated cells or post-IR cathepsin L shRNA-transfected cells. Over 90% of the repair was completed 30 h following IR in the Con shRNA cells, whereas the cathepsin L-suppressed cells remained unrepaired (65%). These data suggest that cathepsin L inhibition may affect the DNA repair level.

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