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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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Camptothecin increases autophagic activity. Following 48 h CPT treatment, cells were incubated with the lysosomotropic agent acridine orange and fluorescence analyzed by flow cytometry. A, Representative flow cytometry analysis of acidic vesicular organelle (AVO) formation in wildtype DLM8 cells. B, Graph representation of CPT-induced AVO formation in wildtype DLM8 and K7M3 cells. *, p < 0.05, compared with same treatment group. C, LC3I/LC3II protein expression. Following 48 h CPT treatment, cells were lysed and cell lysate immunoblotted for LC3I/LC3II protein expression. Increased LC3II expression is indicative of autophagy induction. The expression of treatment group LC3II/actin ratio was determined by densitometry and compared to control group LC3II/actin ratio which was normalized to the arbitrary value of one. Treatment group LC3II expression was normalized to control actin levels as needed. 30ug of protein were loaded for DLM8 LC3I/LC3II determination while only 7.5ug of protein were loaded for K7M3 LC3I/LC3II determination. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblot is representative of immunoblots from three independent experiments.
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Figure 3: Camptothecin increases autophagic activity. Following 48 h CPT treatment, cells were incubated with the lysosomotropic agent acridine orange and fluorescence analyzed by flow cytometry. A, Representative flow cytometry analysis of acidic vesicular organelle (AVO) formation in wildtype DLM8 cells. B, Graph representation of CPT-induced AVO formation in wildtype DLM8 and K7M3 cells. *, p < 0.05, compared with same treatment group. C, LC3I/LC3II protein expression. Following 48 h CPT treatment, cells were lysed and cell lysate immunoblotted for LC3I/LC3II protein expression. Increased LC3II expression is indicative of autophagy induction. The expression of treatment group LC3II/actin ratio was determined by densitometry and compared to control group LC3II/actin ratio which was normalized to the arbitrary value of one. Treatment group LC3II expression was normalized to control actin levels as needed. 30ug of protein were loaded for DLM8 LC3I/LC3II determination while only 7.5ug of protein were loaded for K7M3 LC3I/LC3II determination. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblot is representative of immunoblots from three independent experiments.

Mentions: To determine CPT-induced apoptosis we assessed markers of apoptosis. Cleaved caspase-3 and cleaved PARP (Figure 2A) with accompanying cell death indicated CPT-induced apoptotic cell death. Pre-treatment with pan-caspase inhibitor blocked caspase-3 activation in both cell lines (Figure 2B) and reversed CPT-induced cell death in DLM8 cells but not in K7M3 cells (Figure 2C). Acidic vesicular organelle accumulation was determined to screen for increased autophagic activity following CPT treatment. Camptothecin treatment significantly increased AVO production in DLM8 and K7M3 cells (Figure 3A and B). Autophagy induction was confirmed by LC3II immunoblot. During autophagy induction, LC3I is converted to LC3II. LC3II protein expression increased in both cell lines following CPT treatment, confirming increased autophagic activity (Figure 3C). It is important to note that to measure LC3II protein levels, 30 ug of total protein from DLM8 were loaded to a SDS-PAGE gel, while only 7.5 ug of total protein from K7M3 were loaded. Thirty micrograms of total protein from K7M3 resulted in saturation of the membrane which prevented detection of differences in protein expression between treatment groups. Camptothecin-induced autophagy induction was also confirmed by assessment of a second autophagy marker p62 (Additional file 1: Figure S1). Reduced p62 protein expression is indicative of autophagy induction. Wildtype cells were treated with Bafilomycin A1 to determine the functional status of autophagy. Bafilomycin A1 inhibits autophagosome and lysosome fusion causing an increase in LC3II accumulation. Bafilomycin A1 caused an increase in LC3II accumulation compared to non-treated cells in both cell lines (Additional file 2: Figure S2), indicating that autophagy flux was functional in both cell lines.


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)

Camptothecin increases autophagic activity. Following 48 h CPT treatment, cells were incubated with the lysosomotropic agent acridine orange and fluorescence analyzed by flow cytometry. A, Representative flow cytometry analysis of acidic vesicular organelle (AVO) formation in wildtype DLM8 cells. B, Graph representation of CPT-induced AVO formation in wildtype DLM8 and K7M3 cells. *, p < 0.05, compared with same treatment group. C, LC3I/LC3II protein expression. Following 48 h CPT treatment, cells were lysed and cell lysate immunoblotted for LC3I/LC3II protein expression. Increased LC3II expression is indicative of autophagy induction. The expression of treatment group LC3II/actin ratio was determined by densitometry and compared to control group LC3II/actin ratio which was normalized to the arbitrary value of one. Treatment group LC3II expression was normalized to control actin levels as needed. 30ug of protein were loaded for DLM8 LC3I/LC3II determination while only 7.5ug of protein were loaded for K7M3 LC3I/LC3II determination. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblot is representative of immunoblots from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Camptothecin increases autophagic activity. Following 48 h CPT treatment, cells were incubated with the lysosomotropic agent acridine orange and fluorescence analyzed by flow cytometry. A, Representative flow cytometry analysis of acidic vesicular organelle (AVO) formation in wildtype DLM8 cells. B, Graph representation of CPT-induced AVO formation in wildtype DLM8 and K7M3 cells. *, p < 0.05, compared with same treatment group. C, LC3I/LC3II protein expression. Following 48 h CPT treatment, cells were lysed and cell lysate immunoblotted for LC3I/LC3II protein expression. Increased LC3II expression is indicative of autophagy induction. The expression of treatment group LC3II/actin ratio was determined by densitometry and compared to control group LC3II/actin ratio which was normalized to the arbitrary value of one. Treatment group LC3II expression was normalized to control actin levels as needed. 30ug of protein were loaded for DLM8 LC3I/LC3II determination while only 7.5ug of protein were loaded for K7M3 LC3I/LC3II determination. Actin served as a protein loading control. Data represents the results of three independent experiments, ± SE. p < 0.05 was considered significant. Immunoblot is representative of immunoblots from three independent experiments.
Mentions: To determine CPT-induced apoptosis we assessed markers of apoptosis. Cleaved caspase-3 and cleaved PARP (Figure 2A) with accompanying cell death indicated CPT-induced apoptotic cell death. Pre-treatment with pan-caspase inhibitor blocked caspase-3 activation in both cell lines (Figure 2B) and reversed CPT-induced cell death in DLM8 cells but not in K7M3 cells (Figure 2C). Acidic vesicular organelle accumulation was determined to screen for increased autophagic activity following CPT treatment. Camptothecin treatment significantly increased AVO production in DLM8 and K7M3 cells (Figure 3A and B). Autophagy induction was confirmed by LC3II immunoblot. During autophagy induction, LC3I is converted to LC3II. LC3II protein expression increased in both cell lines following CPT treatment, confirming increased autophagic activity (Figure 3C). It is important to note that to measure LC3II protein levels, 30 ug of total protein from DLM8 were loaded to a SDS-PAGE gel, while only 7.5 ug of total protein from K7M3 were loaded. Thirty micrograms of total protein from K7M3 resulted in saturation of the membrane which prevented detection of differences in protein expression between treatment groups. Camptothecin-induced autophagy induction was also confirmed by assessment of a second autophagy marker p62 (Additional file 1: Figure S1). Reduced p62 protein expression is indicative of autophagy induction. Wildtype cells were treated with Bafilomycin A1 to determine the functional status of autophagy. Bafilomycin A1 inhibits autophagosome and lysosome fusion causing an increase in LC3II accumulation. Bafilomycin A1 caused an increase in LC3II accumulation compared to non-treated cells in both cell lines (Additional file 2: Figure S2), indicating that autophagy flux was functional in both cell lines.

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