Limits...
Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1.

Wei L, Surma M, Gough G, Shi S, Lambert-Cheatham N, Chang J, Shi J - PLoS ONE (2015)

Bottom Line: We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug.We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h).These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16-24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

No MeSH data available.


Related in: MedlinePlus

NAC attenuates H2O2-induced caspase activation, but shows limited effect on doxorubicin-induced caspase activation.(A) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with increasing concentrations of H2O2 and/or 2 mM NAC for 4 h. Equal amount of proteins was loaded. (B) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. n = 4–6 for each condition. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 or doxorubicin only condition.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4489912&req=5

pone.0131763.g003: NAC attenuates H2O2-induced caspase activation, but shows limited effect on doxorubicin-induced caspase activation.(A) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with increasing concentrations of H2O2 and/or 2 mM NAC for 4 h. Equal amount of proteins was loaded. (B) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. n = 4–6 for each condition. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 or doxorubicin only condition.

Mentions: NAC, an analog and precursor of glutathione, has been used in the clinical practice for many decades with well characterized molecular mechanisms [36,37]. Because the time needed reaching the plateau of caspases 3, 8, and 9 activations was different under H2O2 and doxorubicin treatments (Fig 2), we assessed the effects of NAC on caspase activations in MEFs after 4 h of H2O2 treatment and 8 or 16 h of doxorubicin treatment. As expected, the treatment with 2 mM of NAC completely suppressed H2O2-induced caspase activations (Fig 3A). In contrast, the treatment with the same concentration of NAC exhibited only a partial reduction of less than 30% on doxorubicin-induced caspase 3 activation (Fig 3B). In addition to the treatment mentioned above, increasing NAC concentrations from 2 to 10 mM, or decreasing doxorubicin concentrations from 3 to 1 μM, the limited effects of NAC on doxorubicin-induced caspase activation were still persistent (<30%) (S3 Fig). These results indicate that doxorubicin-induced caspase activations are largely mediated by ROS-independent mechanisms.


Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1.

Wei L, Surma M, Gough G, Shi S, Lambert-Cheatham N, Chang J, Shi J - PLoS ONE (2015)

NAC attenuates H2O2-induced caspase activation, but shows limited effect on doxorubicin-induced caspase activation.(A) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with increasing concentrations of H2O2 and/or 2 mM NAC for 4 h. Equal amount of proteins was loaded. (B) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. n = 4–6 for each condition. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 or doxorubicin only condition.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489912&req=5

pone.0131763.g003: NAC attenuates H2O2-induced caspase activation, but shows limited effect on doxorubicin-induced caspase activation.(A) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with increasing concentrations of H2O2 and/or 2 mM NAC for 4 h. Equal amount of proteins was loaded. (B) Representative image (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. n = 4–6 for each condition. *P < 0.05 vs. control. #P < 0.05 vs. H2O2 or doxorubicin only condition.
Mentions: NAC, an analog and precursor of glutathione, has been used in the clinical practice for many decades with well characterized molecular mechanisms [36,37]. Because the time needed reaching the plateau of caspases 3, 8, and 9 activations was different under H2O2 and doxorubicin treatments (Fig 2), we assessed the effects of NAC on caspase activations in MEFs after 4 h of H2O2 treatment and 8 or 16 h of doxorubicin treatment. As expected, the treatment with 2 mM of NAC completely suppressed H2O2-induced caspase activations (Fig 3A). In contrast, the treatment with the same concentration of NAC exhibited only a partial reduction of less than 30% on doxorubicin-induced caspase 3 activation (Fig 3B). In addition to the treatment mentioned above, increasing NAC concentrations from 2 to 10 mM, or decreasing doxorubicin concentrations from 3 to 1 μM, the limited effects of NAC on doxorubicin-induced caspase activation were still persistent (<30%) (S3 Fig). These results indicate that doxorubicin-induced caspase activations are largely mediated by ROS-independent mechanisms.

Bottom Line: We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug.We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h).These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

View Article: PubMed Central - PubMed

Affiliation: Riley Heart Research Center, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University, School of Medicine, Indianapolis, Indiana, United States of America.

ABSTRACT
We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16-24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16-24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

No MeSH data available.


Related in: MedlinePlus