Secreted Frizzled‐Related Protein‐2 Inhibits Doxorubicin‐Induced Apoptosis Mediated through the Akt‐mTOR Pathway in Soleus Muscle

Figure 1. Effect of sFRP2 treatment on lipid peroxides, superoxide dismutase, and catalase activity. Figure 1 shows quantitative data from the ELISA kits for lipid peroxides (a) to determine oxidative injury to the muscle, MnSOD (b) to determine the presence of the antioxidant superoxide dismutase, and (c) to determine the presence of the antioxidant, catalase. Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. n = 4-5 for lipid peroxides, n = 5-6 for MnSOD, and n = 6 for catalase activity.

Figure 2. Significant decrease in DHE-positive cells post-sFRP2 treatment. (a) shows DAPI staining to determine the total number of nuclei in (A, D, and G), DHE staining to measure oxidative stress levels in (B, E, and H), and the merged photomicrographs (C, F, and I). (b) shows the quantitative immunohistochemistry data for the DHE staining. Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. Scale for A is 100 μm. n = 4-5.

Figure 3. sFRP2 treatment decreases caspase-3 activity and inhibits apoptosis. (a) shows representative imaging of soleus muscle. The muscle has been stained with antimyosin (A, E and I), TUNEL to confirm apoptosis (B, F, and J), and DAPI to determine total nuclei (C, G, and K), and the merged images of all staining can be seen (with enlargements, denoted by a red arrow) in (D, H, and L). (b) shows a graph of the quantitative data from immunohistochemistry for the percentage of apoptotic nuclei. (c) shows a stain of the soleus muscle using antimyosin, caspase-3, TUNEL, and DAPI, from left to right. (d) shows the quantitative results from an ELISA kit for caspase-3 activity, a key mediator in apoptosis. Units represented in percentage of apoptotic nuclei in (b) and in arbitrary units for caspase-3 activity in (d). ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. Scale for (a) is 100 μm. Scale for (c) is 50 μm. n = 5-6 for apoptotic nucleus percentage, and n = 7-8 for caspase-3 activity.

Figure 4. sFRP2 treatment post-administration of Dox decreases proapoptotic protein BAX and increases antiapoptotic protein BCL2. Figure 4 shows a Western blot analysis of BAX (proapoptotic), BCL2 (antiapoptotic), and β-actin as the control. Qualitative data can be seen in (a), and quantitative data in the form of graphs can be seen below for BAX in (b) and BCL2 in (c). Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. n = 3 for both BAX and BCL2.

Figure 5. sFRP2 treatment decreases pPTEN levels and increases pAKT and p-mTOR/mTOR. Figure 5 shows the quantitative results of the ELISA kits for pPTEN (a) and pAKT (b). (c) shows analysis of p-mTOR over total mTOR activity using Western blot. Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. n = 7 for pPTEN and n = 6-7 for pAKT. n = 4-5 for p-mTOR/mTOR analysis.

Figure 6. Wnt3a and β-catenin decrease with sFRP2 administration. Figure 6 shows graphs for the quantitative data from the ELISA assay kits for wnt3a to determine the involvement of the wnt signaling pathway and β-catenin to determine levels of this signaling molecule (a and b, resp.). Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. n = 6-7 for wnt3a, and n = 6 for β-catenin activity.

Figure 7. Endogenous presence of sFRP2 in soleus muscle. (a) shows a qualitative Western blot of sFPR2 with β-actin as the loading control. To the right of this Western blot picture in (b) is the quantitative results from the sFRP2 densitometry analysis using ImageJ software (NIH), confirming its presence in the soleus muscle. Units represented in arbitrary units. ∗p < 0.05 compared to control, and #p < 0.05 compared to the Dox group. n = 4-5 for sFRP2.

Figure 8. sFRP2 treatment inhibits oxidative stress induced by doxorubicin. Figure 8 is a flow chart that describes the effects of sFRP2 treatment on the two pathways involved in doxorubicin-induced apoptosis: Akt/mTOR and Wnt/β-catenin.