Fig 1.
Doxorubicin is more potent than H2O2 at inducing caspase activation.
(A)Representative image showing a Western blot of cleaved caspases 3, 8, and 9 in cell lysates from attached WT MEFs treated with H2O2 or doxorubicin at indicated concentrations for 16 h. Equal amount of proteins were loaded. (B-D) Quantitative analysis of immunoreactive bands of cleaved caspases 3, 8, and 9 expressed as fold change relative to WT control. n = 4–6 for each condition. * P < 0.05 vs. control.
Fig 2.
Different time courses of caspase activation by H2O2 versus doxorubicin.
(A) Representative image of Western blot of cleaved caspases 3, 8, and 9 in cell lysates from attached WT MEFs treated with 200 μM of H2O2 or 3 μM of doxorubicin at different time points as indicated. Equal amount of proteins were loaded. (B) Quantitative analysis of immunoreactive bands of cleaved caspase 3 expressed as fold change relative to WT control. n = 4–6 for each condition. * P < 0.05 vs. control.
Fig 3.
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.
Fig 4.
NAC attenuates H2O2-induced disruption of actin cytoskeleton and cell detachment, but shows limited effect on doxorubicin-induced actin cytoskeleton alteration and cell detachment.
(A) Representative images of rhodamine-phalloidin staining for F-actin (red) and DAPI for nucleus (blue) of WT MEFs treated with 200 μM of H2O2 and/or 2 mM NAC for 4 h or with 3 μM of doxorubicin and/or 2 mM NAC for 8 h. Cells showing enriched cortical actin cytoskeleton are indicated with white arrows. Bar, 25 μm. (B) Representative images of bright field photography of WT MEFs treated with 200 μM of H2O2 and/or 2 mM NAC for 16 h showing H2O2-induced cell detachment. Bar, 400 μm. (C) Both floating and attached cells were collected after 16 h treatment with 200 μM of H2O2 and/or 2 mM NAC. Both floating and attached cells were also collected after 16 h treatment with 3 μM doxorubicin and/or increasing concentration of NAC as indicated. Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under each treatment condition. * P < 0.05 vs. control. # P < 0.05 vs. H2O2 or doxorubicin only condition.
Fig 5.
ROCK1 deletion has limited effects on H2O2-induced disruption of actin cytoskeleton, cell detachment and cell death.
(A) Representative images of stained F-actin (red) and nucleus (blue) in WT and ROCK1-/- MEFs treated with 200 μM of H2O2 for 4 h or with 3 μM of doxorubicin for 8 h. Cells showing enriched cortical actin cytoskeleton are indicated with white arrows. Bar, 25 μm. (B) Both floating and attached WT and ROCK1-/- MEFs were collected after 16 h treatment with increasing concentrations of H2O2 as indicated. Both floating and attached cells were also collected after 16 h treatment with 1 or 3 μM doxorubicin. Floating cell ratio was expressed as percentage of total cells (floating plus attached cells) under each treatment condition. (C) MTT assay was performed on WT and ROCK1-/- MEFs treated with increasing concentrations of H2O2 or with 1–3 μM doxorubicin for 16 h. Cell viability was expressed as percentage of control cells without treatment. * P < 0.05 vs. control of the same genotype. # P < 0.05 vs. WT under the same treatment condition.
Fig 6.
H2O2 and doxorubicin treatments show different alteration on MLC phosphorylation.
Representative images of F-actin staining (red), p-MLC (green), and nucleus (blue) in WT and ROCK1-/- MEFs treated with 200 μM of H2O2 for 4 h or with 3 μM of doxorubicin for 8 h. Cells showing enriched cortical p-MLC staining are indicated with white arrows. Bar, 25 μm. Doxorubicin induces an increase in p-MLC translocation to cortical actin cytoskeleton in WT MEFs but to a much smaller extent in ROCK1-/- MEFs. WT and ROCK1-/- MEFs treated with H2O2 both show diffused cytoplasmic p-MLC staining.
Fig 7.
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.
Fig 8.
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).
Fig 9.
NAC and ROCK1 deletion reduce ROS levels induced by doxorubicin and H2O2 treatments.
(A). Representative images of CM-H2DCFDA staining of WT and ROCK1-/- MEFs treated with 200 μM of H2O2 or 3 μM doxorubicin, and/or 2 mM NAC for 0 or 4h, and then exposed to 13 μM CM-H2DCFDA. Coverslip was mounted with AntiFade Mountant containing DAPI and imaged immediately. Bar, 50 μm. (B). Quantitative analysis of CM-H2DCFDA staining of WT and ROCK1-/- MEFs treated as above followed by the measurement with microplate reader and image analytic system. At least 10,000 cells were analyzed in each condition. Fluorescence levels in WT cells at baseline were arbitrarily set at 1. * 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 or H2O2 only condition.
Fig 10.
The diagram summarizes the effects of antioxidant (NAC) and ROCK1 deficiency in opposing apoptosis, necrosis, ROS production and actin cytoskeleton alterations induced by H2O2 and doxorubicin. Antioxidant treatment shows a stronger protection than ROCK1 deletion against H2O2-induced cytotoxic effects while ROCK1 deletion shows a stronger protection than antioxidant treatment against doxorubicin-induced cytotoxicity. These results support the notion that doxorubicin induces apoptosis, necrosis, and actin cytoskeleton alterations predominantly through a ROS-independent and ROCK1-dependent mechanisms. Additional results supporting this concept include different temporal patterns and magnitudes of caspase activations and necrotic cell death, different levels of ROS production and different alteration of actin cytoskeleton induced by H2O2 compared to doxorubicin.