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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

ROCK1 deletion has no inhibition on H2O2-induced caspase activation.(A). Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT and ROCK1-/- MEFs treated with increasing concentrations of H2O2 for 4 h. Equal amount of proteins was loaded. (B) Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached ROCK1-/- MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. Similar experiments performed with the WT MEFs were presented in Fig 3B. n = 4–6 in each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. ¶P < 0.05 vs. the same genotype under doxorubicin only condition.
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pone.0131763.g007: ROCK1 deletion has no inhibition on H2O2-induced caspase activation.(A). Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT and ROCK1-/- MEFs treated with increasing concentrations of H2O2 for 4 h. Equal amount of proteins was loaded. (B) Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached ROCK1-/- MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. Similar experiments performed with the WT MEFs were presented in Fig 3B. n = 4–6 in each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. ¶P < 0.05 vs. the same genotype under doxorubicin only condition.

Mentions: To further validate the notion that different mechanisms mediate the cytotoxic effects induced by doxorubicin and H2O2, we examined the effects of ROCK1 deletion on caspase activations induced by these two stresses. As previously described [22], ROCK1 deletion efficiently reduced the activations of caspases 3, 8, and 9 by more than 70% in the treatment of 3 μM doxorubicin for 16 h: the maximal levels of caspases 3 and 8 activations were in the range of 4–6 folds in ROCK1-/- MEFs (Fig 7B) versus 15–25 folds in WT MEFs (Fig 2). Efficient inhibition by ROCK1 deletion on doxorubicin-induced caspase activations could be observed in a dose-dependent study (0.5 to 10 μM) and a time-course study (8 to 24 h) (S6 Fig). Moreover, NAC treatment was more effective in reducing caspase activations in ROCK1-/- MEFs compared to WT MEFs: NAC at 2 mM reduced doxorubicin-induced caspase 3 activation by more than 70% in ROCK1-/- MEFs (Fig 7B), but less than 30% in WT MEFs (Fig 3B) supporting the additive protection of NAC and ROCK1 deletion. On the other hand, ROCK1 deletion had no apparent effect on H2O2-induced caspase activation, our data showed similar levels of caspase activations between ROCK1 deletion and WT MEFs after 4 h of H2O2 treatment with different concentrations from 50 to 200 μM (Fig 7A). These results indicate that H2O2-induced caspase activation is mediated by ROCK1-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)

ROCK1 deletion has no inhibition on H2O2-induced caspase activation.(A). Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT and ROCK1-/- MEFs treated with increasing concentrations of H2O2 for 4 h. Equal amount of proteins was loaded. (B) Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached ROCK1-/- MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. Similar experiments performed with the WT MEFs were presented in Fig 3B. n = 4–6 in each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. ¶P < 0.05 vs. the same genotype under doxorubicin only condition.
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pone.0131763.g007: ROCK1 deletion has no inhibition on H2O2-induced caspase activation.(A). Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached WT and ROCK1-/- MEFs treated with increasing concentrations of H2O2 for 4 h. Equal amount of proteins was loaded. (B) Representative image of Western blot of cleaved caspases 3, 8, and 9 (top) and quantitative analysis (bottom) of Western blot of cleaved caspase 3 in cell lysates from attached ROCK1-/- MEFs treated with 3 μM doxorubicin and/or 2 mM NAC for 8 or 16 h. Similar experiments performed with the WT MEFs were presented in Fig 3B. n = 4–6 in each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. ¶P < 0.05 vs. the same genotype under doxorubicin only condition.
Mentions: To further validate the notion that different mechanisms mediate the cytotoxic effects induced by doxorubicin and H2O2, we examined the effects of ROCK1 deletion on caspase activations induced by these two stresses. As previously described [22], ROCK1 deletion efficiently reduced the activations of caspases 3, 8, and 9 by more than 70% in the treatment of 3 μM doxorubicin for 16 h: the maximal levels of caspases 3 and 8 activations were in the range of 4–6 folds in ROCK1-/- MEFs (Fig 7B) versus 15–25 folds in WT MEFs (Fig 2). Efficient inhibition by ROCK1 deletion on doxorubicin-induced caspase activations could be observed in a dose-dependent study (0.5 to 10 μM) and a time-course study (8 to 24 h) (S6 Fig). Moreover, NAC treatment was more effective in reducing caspase activations in ROCK1-/- MEFs compared to WT MEFs: NAC at 2 mM reduced doxorubicin-induced caspase 3 activation by more than 70% in ROCK1-/- MEFs (Fig 7B), but less than 30% in WT MEFs (Fig 3B) supporting the additive protection of NAC and ROCK1 deletion. On the other hand, ROCK1 deletion had no apparent effect on H2O2-induced caspase activation, our data showed similar levels of caspase activations between ROCK1 deletion and WT MEFs after 4 h of H2O2 treatment with different concentrations from 50 to 200 μM (Fig 7A). These results indicate that H2O2-induced caspase activation is mediated by ROCK1-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