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Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells.

Hollomon MG, Gordon N, Santiago-O'Farrill JM, Kleinerman ES - BMC Cancer (2013)

Bottom Line: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS.The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage.Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. hollomon_mg@tsu.edu.

ABSTRACT

Background: Autophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS.

Methods: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation.

Results: Autophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress.

Conclusions: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

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Autophagy inhibition has an opposing impact on CPT-induced cell death. A, ATG5 protein levels in DLM8 and K7M3 cells following shRNA-mediated knockdown of ATG5. Cells were infected with lentivirus containing empty shRNA vector or lentiviral shRNA targeted against ATG5 mRNA. Following infection, cells were lysed and total protein collected. To confirm ATG5 protein knockdown and autophagy inhibition, total protein was immunoblotted for ATG5 and LC3I/C3II protein levels, respectively. Actin served as a protein loading control. B, Acidic vesicular organelle (AVO) formation. Autophagy-competent and autophagy-inhibited DLM8 cells were treated with CPT for 24 h followed by assessment of AVO formation. Impact of autophagy inhibition on cell death in C, DLM8 and D, K7M3 OS cells. Autophagy-competent and autophagy-inhibited DLM8 and K7M3 cells were treated with CPT as indicated for 48 h. Following drug treatment, cell viability was assessed by trypan blue exclusion. *, p < 0.05, compared with same treatment group. E, Basal levels of autophagy in MC3T3, DLM8 and K7M3 cells. Cells were untreated and allowed to grow to approximately 70% confluency. Cells were then collected, lysed and total protein immunoblotted for LC3I/LC3II. 30ug of protein were loaded for each cell line. Actin served as a protein loading control. F, Phosphorylation of p53 in DLM8 cells. Cells were treated with CPT as indicated for 24 h. Following CPT treatment, cells were lysed and cell lysate probed for phospho p53 and total p53 protein expression. Data represents the results of at least three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from two independent experiments.
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Figure 4: Autophagy inhibition has an opposing impact on CPT-induced cell death. A, ATG5 protein levels in DLM8 and K7M3 cells following shRNA-mediated knockdown of ATG5. Cells were infected with lentivirus containing empty shRNA vector or lentiviral shRNA targeted against ATG5 mRNA. Following infection, cells were lysed and total protein collected. To confirm ATG5 protein knockdown and autophagy inhibition, total protein was immunoblotted for ATG5 and LC3I/C3II protein levels, respectively. Actin served as a protein loading control. B, Acidic vesicular organelle (AVO) formation. Autophagy-competent and autophagy-inhibited DLM8 cells were treated with CPT for 24 h followed by assessment of AVO formation. Impact of autophagy inhibition on cell death in C, DLM8 and D, K7M3 OS cells. Autophagy-competent and autophagy-inhibited DLM8 and K7M3 cells were treated with CPT as indicated for 48 h. Following drug treatment, cell viability was assessed by trypan blue exclusion. *, p < 0.05, compared with same treatment group. E, Basal levels of autophagy in MC3T3, DLM8 and K7M3 cells. Cells were untreated and allowed to grow to approximately 70% confluency. Cells were then collected, lysed and total protein immunoblotted for LC3I/LC3II. 30ug of protein were loaded for each cell line. Actin served as a protein loading control. F, Phosphorylation of p53 in DLM8 cells. Cells were treated with CPT as indicated for 24 h. Following CPT treatment, cells were lysed and cell lysate probed for phospho p53 and total p53 protein expression. Data represents the results of at least three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from two independent experiments.

Mentions: Autophagy was inhibited by knocking down the expression of essential autophagy protein ATG5. Knockdown of ATG5 protein expression and its impact on autophagy inhibition were confirmed by immunoblot of ATG5 and LC3II, respectively (Figure 4A). Knockdown of ATG5 reduced CPT-induced AVO formation, thus validating AVO as a reliable screen for autophagy induction (Figure 4B). Knockdown of ATG5 protein expression in DLM8 cells decreased CPT-induced cell death. In contrast, knockdown of ATG5 protein expression in K7M3 cells increased CPT-induced cell death (Figure 4C and D). Basal levels of autophagy were higher in K7M3 cells compared to DLM8 cells and a nontransformed osteoblast cell line, suggesting increased dependence of K7M3 on autophagy for metabolic homeostasis (Figure 4E). Camptothecin treatment induced similar level of phosphorylation of p53 at Ser15 in both autophagy-competent and autophagy-inhibited DLM8 cells, indicating similar levels of CPT-induced DNA damage (Figure 4F).


Knockdown of autophagy-related protein 5, ATG5, decreases oxidative stress and has an opposing effect on camptothecin-induced cytotoxicity in osteosarcoma cells.

Hollomon MG, Gordon N, Santiago-O'Farrill JM, Kleinerman ES - BMC Cancer (2013)

Autophagy inhibition has an opposing impact on CPT-induced cell death. A, ATG5 protein levels in DLM8 and K7M3 cells following shRNA-mediated knockdown of ATG5. Cells were infected with lentivirus containing empty shRNA vector or lentiviral shRNA targeted against ATG5 mRNA. Following infection, cells were lysed and total protein collected. To confirm ATG5 protein knockdown and autophagy inhibition, total protein was immunoblotted for ATG5 and LC3I/C3II protein levels, respectively. Actin served as a protein loading control. B, Acidic vesicular organelle (AVO) formation. Autophagy-competent and autophagy-inhibited DLM8 cells were treated with CPT for 24 h followed by assessment of AVO formation. Impact of autophagy inhibition on cell death in C, DLM8 and D, K7M3 OS cells. Autophagy-competent and autophagy-inhibited DLM8 and K7M3 cells were treated with CPT as indicated for 48 h. Following drug treatment, cell viability was assessed by trypan blue exclusion. *, p < 0.05, compared with same treatment group. E, Basal levels of autophagy in MC3T3, DLM8 and K7M3 cells. Cells were untreated and allowed to grow to approximately 70% confluency. Cells were then collected, lysed and total protein immunoblotted for LC3I/LC3II. 30ug of protein were loaded for each cell line. Actin served as a protein loading control. F, Phosphorylation of p53 in DLM8 cells. Cells were treated with CPT as indicated for 24 h. Following CPT treatment, cells were lysed and cell lysate probed for phospho p53 and total p53 protein expression. Data represents the results of at least three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from two independent experiments.
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Figure 4: Autophagy inhibition has an opposing impact on CPT-induced cell death. A, ATG5 protein levels in DLM8 and K7M3 cells following shRNA-mediated knockdown of ATG5. Cells were infected with lentivirus containing empty shRNA vector or lentiviral shRNA targeted against ATG5 mRNA. Following infection, cells were lysed and total protein collected. To confirm ATG5 protein knockdown and autophagy inhibition, total protein was immunoblotted for ATG5 and LC3I/C3II protein levels, respectively. Actin served as a protein loading control. B, Acidic vesicular organelle (AVO) formation. Autophagy-competent and autophagy-inhibited DLM8 cells were treated with CPT for 24 h followed by assessment of AVO formation. Impact of autophagy inhibition on cell death in C, DLM8 and D, K7M3 OS cells. Autophagy-competent and autophagy-inhibited DLM8 and K7M3 cells were treated with CPT as indicated for 48 h. Following drug treatment, cell viability was assessed by trypan blue exclusion. *, p < 0.05, compared with same treatment group. E, Basal levels of autophagy in MC3T3, DLM8 and K7M3 cells. Cells were untreated and allowed to grow to approximately 70% confluency. Cells were then collected, lysed and total protein immunoblotted for LC3I/LC3II. 30ug of protein were loaded for each cell line. Actin served as a protein loading control. F, Phosphorylation of p53 in DLM8 cells. Cells were treated with CPT as indicated for 24 h. Following CPT treatment, cells were lysed and cell lysate probed for phospho p53 and total p53 protein expression. Data represents the results of at least three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblots are representative of immunoblots from two independent experiments.
Mentions: Autophagy was inhibited by knocking down the expression of essential autophagy protein ATG5. Knockdown of ATG5 protein expression and its impact on autophagy inhibition were confirmed by immunoblot of ATG5 and LC3II, respectively (Figure 4A). Knockdown of ATG5 reduced CPT-induced AVO formation, thus validating AVO as a reliable screen for autophagy induction (Figure 4B). Knockdown of ATG5 protein expression in DLM8 cells decreased CPT-induced cell death. In contrast, knockdown of ATG5 protein expression in K7M3 cells increased CPT-induced cell death (Figure 4C and D). Basal levels of autophagy were higher in K7M3 cells compared to DLM8 cells and a nontransformed osteoblast cell line, suggesting increased dependence of K7M3 on autophagy for metabolic homeostasis (Figure 4E). Camptothecin treatment induced similar level of phosphorylation of p53 at Ser15 in both autophagy-competent and autophagy-inhibited DLM8 cells, indicating similar levels of CPT-induced DNA damage (Figure 4F).

Bottom Line: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS.The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage.Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Pediatrics, The University of Texas MD Anderson Cancer Center, Houston, TX 77054, USA. hollomon_mg@tsu.edu.

ABSTRACT

Background: Autophagy induction can increase or decrease anticancer drug efficacy. Anticancer drug-induced autophagy induction is poorly characterized in osteosarcoma (OS). In this study, we investigated the impact of autophagy inhibition on camptothecin (CPT)-induced cytotoxicity in OS.

Methods: Autophagy-inhibited DLM8 and K7M3 metastatic murine OS cell lines were generated by infection with lentiviral shRNA directed against the essential autophagy protein ATG5. Knockdown of ATG5 protein expression and inhibition of autophagy was confirmed by immunoblot of ATG5 and LC3II proteins, respectively. Metabolic activity was determined by MTT assay and cell viability was determined by trypan blue exclusion. Acridine orange staining and immunoblotting for LC3II protein expression were used to determine autophagy induction. Oxidative stress was assessed by staining cells with HE and DCFH-DA followed by flow cytometry analysis. Mitochondrial membrane potential was determined by staining cells with TMRE followed by flow cytometry analysis. Immunoblotting was used to detect caspase activation, Parp cleavage and p53 phosphorylation.

Results: Autophagy inhibition caused a greater deficit in metabolic activity and cell growth in K7M3 cells compared to DLM8 cells. K7M3 cells exhibited higher basal autophagy levels than DLM8 cells and non-transformed murine MCT3 osteoblasts. Autophagy inhibition did not affect CPT-induced DNA damage. Autophagy inhibition decreased CPT-induced cell death in DLM8 cells while increasing CPT-induced cell death in K7M3 cells. Autophagy inhibition reduced CPT-induced mitochondrial damage and CPT-induced caspase activation in DLM8 cells. Buthionine sulfoximine (BSO)-induced cell death was greater in autophagy-competent DLM8 cells and was reversed by antioxidant pretreatment. Camptothecin-induced and BSO-induced autophagy induction was also reversed by antioxidant pretreatment. Significantly, autophagy inhibition not only reduced CPT-induced oxidative stress but also reduced basal oxidative stress.

Conclusions: The results of this study indicate that autophagy inhibition can have an opposing effect on CPT-induced cytotoxicity within OS. The cytoprotective mechanism of autophagy inhibition observed in DLM8 cells involves reduced CPT-induced oxidative stress and not reduced DNA damage. Our results also reveal the novel finding that knockdown of ATG5 protein reduces both basal oxidative stress and drug-induced oxidative stress.

Show MeSH
Related in: MedlinePlus