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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

Changes in cellular cathepsin L following irradiation (IR). (A) Western blot analysis of cathepsin L protein levels in the nuclear fraction, cytosol fraction and total cell lysate at 48 h post-IR. (B) Cells were treated with 8 Gy IR and analyzed at 24 h post-IR by immunofluorescence using an anti-cathepsin L antibody. (C) The cathepsin L level in the conditioned medium at 24 h after irradiation was measured in triplicate, and the experiment was repeated at least twice to confirm the results. bP<0.05, cP<0.01 compared with the control group. The amount of secreted cathepsin L is presented in pg/mL. Con, control; IR, irradiation-treated samples.
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fig1: Changes in cellular cathepsin L following irradiation (IR). (A) Western blot analysis of cathepsin L protein levels in the nuclear fraction, cytosol fraction and total cell lysate at 48 h post-IR. (B) Cells were treated with 8 Gy IR and analyzed at 24 h post-IR by immunofluorescence using an anti-cathepsin L antibody. (C) The cathepsin L level in the conditioned medium at 24 h after irradiation was measured in triplicate, and the experiment was repeated at least twice to confirm the results. bP<0.05, cP<0.01 compared with the control group. The amount of secreted cathepsin L is presented in pg/mL. Con, control; IR, irradiation-treated samples.

Mentions: To study the association between radiotherapy and cathepsin L expression, we measured the activity of this protease in human glioblastoma cell lines harboring the wild type (U87 cell line) and the mutant p53 gene (U251 cell line) after IR. Compared with U251 cells, a higher cathepsin L protein level was observed in U87 cells (Figure 1A). This result is consistent with the results of another group, who observed that the U87 cell line expressing wild type p53 exhibited significantly higher enzymatic activity and mRNA levels of cathepsin L compared with the mutant p53 glioblastoma cell line U37322. This group demonstrated that the p53 gene upregulates cathepsin L gene transcription directly by binding to the cathepsin L gene promoter and indirectly by increasing the expression of another transcription factor, C/EBPα. Interestingly, we found that cathepsin L was present in the nuclei of certain U251 cells; after IR, cathepsin L expression increased in both nuclear and cytosolic fractions (Figure 1B). Moreover, the cathepsin L that accumulates in the nucleus of U251 cells is much more catalytically active23, suggesting that gliomas with mutant p53 expression have extremely different cathepsin L activity after IR. Mutant p53 genes may drive a mutator phenotype, resulting in radioresistance through differential p53 transactivation24. We hypothesized that upregulated cathepsin L expression may be related to radioresistance in a mutant p53 glioblastoma cell line. Upon malignant transformation, cathepsin L often translocates to the cell surface and is secreted into the surrounding medium to modify the extracellular matrix. Therefore, we examined whether IR can influence cathepsin L secretion in glioma cells and thereby interfere with radioresistance. U251 and U87 cells were treated with IR, and the levels of cathepsin L secreted in the conditioned media were measured using an ELISA kit. The levels of secreted cathepsin L in the conditioned media of U251 cells treated with radiotherapy were higher compared to the levels in the untreated group. However, cathepsin L secretion in the conditioned medium of U87 cells treated with IR showed no significant correlation with the levels in the untreated group (Figure 1C). According to these results, we chose U251 cells for the following experiments.


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)

Changes in cellular cathepsin L following irradiation (IR). (A) Western blot analysis of cathepsin L protein levels in the nuclear fraction, cytosol fraction and total cell lysate at 48 h post-IR. (B) Cells were treated with 8 Gy IR and analyzed at 24 h post-IR by immunofluorescence using an anti-cathepsin L antibody. (C) The cathepsin L level in the conditioned medium at 24 h after irradiation was measured in triplicate, and the experiment was repeated at least twice to confirm the results. bP<0.05, cP<0.01 compared with the control group. The amount of secreted cathepsin L is presented in pg/mL. Con, control; IR, irradiation-treated samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4561966&req=5

fig1: Changes in cellular cathepsin L following irradiation (IR). (A) Western blot analysis of cathepsin L protein levels in the nuclear fraction, cytosol fraction and total cell lysate at 48 h post-IR. (B) Cells were treated with 8 Gy IR and analyzed at 24 h post-IR by immunofluorescence using an anti-cathepsin L antibody. (C) The cathepsin L level in the conditioned medium at 24 h after irradiation was measured in triplicate, and the experiment was repeated at least twice to confirm the results. bP<0.05, cP<0.01 compared with the control group. The amount of secreted cathepsin L is presented in pg/mL. Con, control; IR, irradiation-treated samples.
Mentions: To study the association between radiotherapy and cathepsin L expression, we measured the activity of this protease in human glioblastoma cell lines harboring the wild type (U87 cell line) and the mutant p53 gene (U251 cell line) after IR. Compared with U251 cells, a higher cathepsin L protein level was observed in U87 cells (Figure 1A). This result is consistent with the results of another group, who observed that the U87 cell line expressing wild type p53 exhibited significantly higher enzymatic activity and mRNA levels of cathepsin L compared with the mutant p53 glioblastoma cell line U37322. This group demonstrated that the p53 gene upregulates cathepsin L gene transcription directly by binding to the cathepsin L gene promoter and indirectly by increasing the expression of another transcription factor, C/EBPα. Interestingly, we found that cathepsin L was present in the nuclei of certain U251 cells; after IR, cathepsin L expression increased in both nuclear and cytosolic fractions (Figure 1B). Moreover, the cathepsin L that accumulates in the nucleus of U251 cells is much more catalytically active23, suggesting that gliomas with mutant p53 expression have extremely different cathepsin L activity after IR. Mutant p53 genes may drive a mutator phenotype, resulting in radioresistance through differential p53 transactivation24. We hypothesized that upregulated cathepsin L expression may be related to radioresistance in a mutant p53 glioblastoma cell line. Upon malignant transformation, cathepsin L often translocates to the cell surface and is secreted into the surrounding medium to modify the extracellular matrix. Therefore, we examined whether IR can influence cathepsin L secretion in glioma cells and thereby interfere with radioresistance. U251 and U87 cells were treated with IR, and the levels of cathepsin L secreted in the conditioned media were measured using an ELISA kit. The levels of secreted cathepsin L in the conditioned media of U251 cells treated with radiotherapy were higher compared to the levels in the untreated group. However, cathepsin L secretion in the conditioned medium of U87 cells treated with IR showed no significant correlation with the levels in the untreated group (Figure 1C). According to these results, we chose U251 cells for the following experiments.

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