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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 expression of Bax and Bcl-2 proteins. (A) Hoechst staining of the cells that were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h. The resulting cells were treated with 8 Gy IR and analyzed at 48 and 72 h post-IR. (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 treated with IR and analyzed at 72 h post-IR by Western blot analysis of Bax and Bcl-2 protein levels. 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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fig6: Effect of cathepsin L inhibition on irradiation (IR)-induced expression of Bax and Bcl-2 proteins. (A) Hoechst staining of the cells that were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h. The resulting cells were treated with 8 Gy IR and analyzed at 48 and 72 h post-IR. (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 treated with IR and analyzed at 72 h post-IR by Western blot analysis of Bax and Bcl-2 protein levels. 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: We assessed the ability of Z-FY-CHO+IR to induce apoptosis to study the mechanism by which cathepsin L inhibition affects the radiosensitivity of glioma cells. Figure 6A shows that U251 cells that were treated with IR underwent morphlogical changes, such as nuclear chromatin condensation, that are consistent with fragmented, apoptotic nuclei. Furthermore, the morphological changes in the Z-FY-CHO+IR-treated cells were more significant than those in the IR-treated cells (Figure 6A). Cotreatment with Z-FY-CHO and IR for 24 h did not result in significant morphological changes compared with the control group (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 expression of Bax and Bcl-2 proteins. (A) Hoechst staining of the cells that were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h. The resulting cells were treated with 8 Gy IR and analyzed at 48 and 72 h post-IR. (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 treated with IR and analyzed at 72 h post-IR by Western blot analysis of Bax and Bcl-2 protein levels. 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

fig6: Effect of cathepsin L inhibition on irradiation (IR)-induced expression of Bax and Bcl-2 proteins. (A) Hoechst staining of the cells that were pretreated with Z-FY-CHO at 10 μmol/L (or untreated) for 12 h. The resulting cells were treated with 8 Gy IR and analyzed at 48 and 72 h post-IR. (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 treated with IR and analyzed at 72 h post-IR by Western blot analysis of Bax and Bcl-2 protein levels. 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: We assessed the ability of Z-FY-CHO+IR to induce apoptosis to study the mechanism by which cathepsin L inhibition affects the radiosensitivity of glioma cells. Figure 6A shows that U251 cells that were treated with IR underwent morphlogical changes, such as nuclear chromatin condensation, that are consistent with fragmented, apoptotic nuclei. Furthermore, the morphological changes in the Z-FY-CHO+IR-treated cells were more significant than those in the IR-treated cells (Figure 6A). Cotreatment with Z-FY-CHO and IR for 24 h did not result in significant morphological changes compared with the control group (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