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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 expression of r-H2AX, Ku70, and Rad51 in U251 cells. 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), and harvested for Western blot analysis of γ-H2AX, Ku70, and Rad51 protein levels at 6 h post-IR (A) and for Western blot analysis of γ-H2AX at the indicated time after IR(B). 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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fig4: Effect of cathepsin L suppression on irradiation-induced expression of r-H2AX, Ku70, and Rad51 in U251 cells. 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), and harvested for Western blot analysis of γ-H2AX, Ku70, and Rad51 protein levels at 6 h post-IR (A) and for Western blot analysis of γ-H2AX at the indicated time after IR(B). 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: Phosphorylated H2AX (histone H2AX protein) is designated γ-H2AX; γ-H2AX, as an early response to DNA damage, is considered a marker of DSBs. Normally, the level of γ-H2AX is low in mammals, unless a DSB is introduced, eg, by ionizing radiation. As a key regulator of IR-induced nuclear foci formation, γ-H2AX functions as a barrier for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Thus, high levels of γ-H2AX suppress DNA damage repair26,27. Considering the important function of this protein in DNA repair, particularly after DNA damage is induced, γ-H2AX was quantified by Western blot. The level of γ-H2AX was increased in the IR alone group and particularly in the combined treatment group (Figure 4A and 4B) compared with the untreated group. Ku70, which is one of the key regulatory subunits of the DNA-dependent protein kinase, has an important function in the repair of DNA DSBs28. Our results showed that the Ku70 protein level of the combined treatment group was significantly decreased compared with the IR alone group. These results suggest that the increased level of γ-H2AX and decreased level of Ku70 may be involved in the increased radiosensitivity induced by cathepsin L suppression.


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 expression of r-H2AX, Ku70, and Rad51 in U251 cells. 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), and harvested for Western blot analysis of γ-H2AX, Ku70, and Rad51 protein levels at 6 h post-IR (A) and for Western blot analysis of γ-H2AX at the indicated time after IR(B). 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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fig4: Effect of cathepsin L suppression on irradiation-induced expression of r-H2AX, Ku70, and Rad51 in U251 cells. 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), and harvested for Western blot analysis of γ-H2AX, Ku70, and Rad51 protein levels at 6 h post-IR (A) and for Western blot analysis of γ-H2AX at the indicated time after IR(B). 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: Phosphorylated H2AX (histone H2AX protein) is designated γ-H2AX; γ-H2AX, as an early response to DNA damage, is considered a marker of DSBs. Normally, the level of γ-H2AX is low in mammals, unless a DSB is introduced, eg, by ionizing radiation. As a key regulator of IR-induced nuclear foci formation, γ-H2AX functions as a barrier for the accumulation and retention of the central components of the signaling cascade initiated by DNA damage. Thus, high levels of γ-H2AX suppress DNA damage repair26,27. Considering the important function of this protein in DNA repair, particularly after DNA damage is induced, γ-H2AX was quantified by Western blot. The level of γ-H2AX was increased in the IR alone group and particularly in the combined treatment group (Figure 4A and 4B) compared with the untreated group. Ku70, which is one of the key regulatory subunits of the DNA-dependent protein kinase, has an important function in the repair of DNA DSBs28. Our results showed that the Ku70 protein level of the combined treatment group was significantly decreased compared with the IR alone group. These results suggest that the increased level of γ-H2AX and decreased level of Ku70 may be involved in the increased radiosensitivity induced by cathepsin L suppression.

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