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).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.

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 attenuates doxorubicin- and H2O2-induced necrotic cell death.(A). Representative images of Sytox Green (Green) and Hoechst 33342 staining (blue) of WT MEFs treated with 200 μM of H2O2 or 3 μM doxorubicin for 0 or 4 h. Bar, 200 μm. (B-D) Necrotic cells measured by Sytox Green staining in attached WT and/or ROCK1-/- MEFs at indicated time points and dosages of H2O2 and doxorubicin. The ratio of Sytox Green positive cells was expressed as percentage of attached cells. At least 10,000 cells were analyzed for each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. (E) Representative scatter plots of necrosis and apoptosis quantified by FACS analysis after staining with annexin V and 7-AAD in attached WT and ROCK1-/- cells collected after treatment for 4 h with 200 μM of H2O2 or 16 h with 3 μM doxorubicin. Viable cells are annexin V−/7-AAD−; annexin V+/7-AAD− cells are in early apoptosis; annexin V+/7-AAD+ cells are in late apoptosis; necrotic cells are annexin V−/7-AAD+. Necrotic cells induced by H2O2 are predominantly annexin V negative, whereas the majority of necrotic cells induced doxorubicin are annexin V positive (late apoptosis).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131763.g008: ROCK1 deletion attenuates doxorubicin- and H2O2-induced necrotic cell death.(A). Representative images of Sytox Green (Green) and Hoechst 33342 staining (blue) of WT MEFs treated with 200 μM of H2O2 or 3 μM doxorubicin for 0 or 4 h. Bar, 200 μm. (B-D) Necrotic cells measured by Sytox Green staining in attached WT and/or ROCK1-/- MEFs at indicated time points and dosages of H2O2 and doxorubicin. The ratio of Sytox Green positive cells was expressed as percentage of attached cells. At least 10,000 cells were analyzed for each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. (E) Representative scatter plots of necrosis and apoptosis quantified by FACS analysis after staining with annexin V and 7-AAD in attached WT and ROCK1-/- cells collected after treatment for 4 h with 200 μM of H2O2 or 16 h with 3 μM doxorubicin. Viable cells are annexin V−/7-AAD−; annexin V+/7-AAD− cells are in early apoptosis; annexin V+/7-AAD+ cells are in late apoptosis; necrotic cells are annexin V−/7-AAD+. Necrotic cells induced by H2O2 are predominantly annexin V negative, whereas the majority of necrotic cells induced doxorubicin are annexin V positive (late apoptosis).

Mentions: In addition to apoptosis, it has been reported that necrotic cell death also contributes to doxorubicin or H2O2 cytotoxicity [38,39]. We assessed early necrotic cell death induced by these two stresses within 4 h of treatment by measuring cellular uptake of Sytox Green dye, which is cell membrane impermeable in live cells but permeable in necrotic cells due to compromised cell membrane (Fig 8A). H2O2 treatment induced necrotic cell death in WT MEFs in a dose and time-dependent manner. The maximal levels of necrotic cell ratio were reached within 4 h (Fig 8B), and the time course of necrotic cell death is similar to that of caspase activation induced by H2O2 (Fig 2). The rate of necrosis increased when treated with H2O2 at 50 μM or higher and reached 8–10% at 200 μM H2O2. This dose-dependent increase of necrosis during the early time window (4 h) suggests that H2O2 treatment induces primary necrosis. To support this notion, double staining of annexin V and 7-AAD was performed in H2O2-treated cells followed by flow cytometry analysis, and the results showed that the majority of necrotic cells (7-AAD positive) were annexin V negative, therefore an event independent of apoptosis (Fig 8E). On the other hand, doxorubicin at 3 μM produced a much smaller scale of necrotic cell death, less than 3%, after 4 h, and reached 8–10% at 16 h (Fig 8D). Double staining of annexin V and 7-AAD followed by flow cytometry analysis showed that the majority of necrotic cells (7-AAD positive) were also annexin V positive (late apoptosis), suggesting that necrotic cell death largely occurs secondary to apoptosis after doxorubicin treatment. These results indicate that H2O2 is a stronger inducer for primary necrotic cell death than doxorubicin in MEFs. These results support that H2O2 and doxorubicin induce necrotic cell death through different mechanisms in MEFs.


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 attenuates doxorubicin- and H2O2-induced necrotic cell death.(A). Representative images of Sytox Green (Green) and Hoechst 33342 staining (blue) of WT MEFs treated with 200 μM of H2O2 or 3 μM doxorubicin for 0 or 4 h. Bar, 200 μm. (B-D) Necrotic cells measured by Sytox Green staining in attached WT and/or ROCK1-/- MEFs at indicated time points and dosages of H2O2 and doxorubicin. The ratio of Sytox Green positive cells was expressed as percentage of attached cells. At least 10,000 cells were analyzed for each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. (E) Representative scatter plots of necrosis and apoptosis quantified by FACS analysis after staining with annexin V and 7-AAD in attached WT and ROCK1-/- cells collected after treatment for 4 h with 200 μM of H2O2 or 16 h with 3 μM doxorubicin. Viable cells are annexin V−/7-AAD−; annexin V+/7-AAD− cells are in early apoptosis; annexin V+/7-AAD+ cells are in late apoptosis; necrotic cells are annexin V−/7-AAD+. Necrotic cells induced by H2O2 are predominantly annexin V negative, whereas the majority of necrotic cells induced doxorubicin are annexin V positive (late apoptosis).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131763.g008: ROCK1 deletion attenuates doxorubicin- and H2O2-induced necrotic cell death.(A). Representative images of Sytox Green (Green) and Hoechst 33342 staining (blue) of WT MEFs treated with 200 μM of H2O2 or 3 μM doxorubicin for 0 or 4 h. Bar, 200 μm. (B-D) Necrotic cells measured by Sytox Green staining in attached WT and/or ROCK1-/- MEFs at indicated time points and dosages of H2O2 and doxorubicin. The ratio of Sytox Green positive cells was expressed as percentage of attached cells. At least 10,000 cells were analyzed for each condition. *P < 0.05 vs. control of the same genotype. #P < 0.05 vs. WT under the same treatment condition. (E) Representative scatter plots of necrosis and apoptosis quantified by FACS analysis after staining with annexin V and 7-AAD in attached WT and ROCK1-/- cells collected after treatment for 4 h with 200 μM of H2O2 or 16 h with 3 μM doxorubicin. Viable cells are annexin V−/7-AAD−; annexin V+/7-AAD− cells are in early apoptosis; annexin V+/7-AAD+ cells are in late apoptosis; necrotic cells are annexin V−/7-AAD+. Necrotic cells induced by H2O2 are predominantly annexin V negative, whereas the majority of necrotic cells induced doxorubicin are annexin V positive (late apoptosis).
Mentions: In addition to apoptosis, it has been reported that necrotic cell death also contributes to doxorubicin or H2O2 cytotoxicity [38,39]. We assessed early necrotic cell death induced by these two stresses within 4 h of treatment by measuring cellular uptake of Sytox Green dye, which is cell membrane impermeable in live cells but permeable in necrotic cells due to compromised cell membrane (Fig 8A). H2O2 treatment induced necrotic cell death in WT MEFs in a dose and time-dependent manner. The maximal levels of necrotic cell ratio were reached within 4 h (Fig 8B), and the time course of necrotic cell death is similar to that of caspase activation induced by H2O2 (Fig 2). The rate of necrosis increased when treated with H2O2 at 50 μM or higher and reached 8–10% at 200 μM H2O2. This dose-dependent increase of necrosis during the early time window (4 h) suggests that H2O2 treatment induces primary necrosis. To support this notion, double staining of annexin V and 7-AAD was performed in H2O2-treated cells followed by flow cytometry analysis, and the results showed that the majority of necrotic cells (7-AAD positive) were annexin V negative, therefore an event independent of apoptosis (Fig 8E). On the other hand, doxorubicin at 3 μM produced a much smaller scale of necrotic cell death, less than 3%, after 4 h, and reached 8–10% at 16 h (Fig 8D). Double staining of annexin V and 7-AAD followed by flow cytometry analysis showed that the majority of necrotic cells (7-AAD positive) were also annexin V positive (late apoptosis), suggesting that necrotic cell death largely occurs secondary to apoptosis after doxorubicin treatment. These results indicate that H2O2 is a stronger inducer for primary necrotic cell death than doxorubicin in MEFs. These results support that H2O2 and doxorubicin induce necrotic cell death through different mechanisms in MEFs.

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).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.

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