## 3 Results
### Akt1/Src/Cav1/Catalase Signaling Promotes Radioresistance by Buffering Oxidative Stress in RD Cells
Data are mean \(\pm\) SEM, *\(p\)-value \(<0.05\); **\(p\)-value \(<0.001\); ***\(p\)-value \(<0.0001\); unpaired Student’s \(t\)-test vs. control RD line **(E)** or one-way ANOVA test vs. vehicle-treated cells (F). Statin Treatment Induces Radio- and Chemosensitization through Downregulation of Akt1/Cav1 Signaling and Increased Levels of Oxidative Stress  
Many studies have indicated that oxidative stress is involved in the various toxicities associated with statins [28]. In addition, we recently showed that activation of Akt1 increases susceptibility to statin treatment [9]. Furthermore, the localization and function of Cav1 are strictly dependent on the proper availability of cholesterol [15]. For these reasons, we decided to evaluate the impact of statin treatment on Akt1 signaling. In RD and myrAkt1 cells treated with lovastatin or simvastatin, both total and phosphorylated forms of Akt1 and Cav1 were significantly reduced as early as 4 h, as demonstrated by IB analysis (Figure 4A). Therefore, we decided to evaluate the ROS levels in statin-treated cells. As shown in Figure 4B, lovastatin or simvastatin dose-dependently increased intracellular ROS levels in RD and RH30 lines and in both radioresistant RMS lines as detected after 24 h. This evidence led us to hypothesize that statin treatment may result in attenuation of radioresistance mechanisms by decreasing the Akt1 pathway while simultaneously increasing ROS levels. To this end, the RMS lines were pretreated with lovastatin and simvastatin  
Figure 3: The expression pattern of Akt1, Src, and Cav1 is conserved in the radioresistant myrAkt1 and RDCav1 lines, and its inhibition promotes radiosensitization. (**A,B**) IR treatment was performed 24 h after plating control, myrAkt1 and RDCav1 cells (1.5 \(\times\) 10\({}^{5}\)) in 60 mm dishes. Cells were processed at the indicated time-points for IB (\(n\) = 2). To explain the absence of Src and Cav1 bands in the control cells, it should be noted that detection was performed under low exposure conditions to avoid interference with the strong signals arisen from protein homogenates of radioresistant cells. Tubulin was used as loading control. (**C,D**) Subconfluent myrAkt1 and RDCav1 cells were pretreated for 2 h with the indicated compounds before IR treatment. Cell survival was evaluated by clonogenic assay (\(n\) = 3). Data are mean \(\pm\) SEM, ***\(p\)-value \(<\) 0.0001; one-way ANOVA test vs. vehicle-treated irradiated cells. Representative images were taken after crystal violet incorporation. for 2 h before IR treatment. In the clonogenic assay, we observed that statins significantly reduced the cell viability of post-irradiated RD and RH30 lines as well as of radioresistant myrAkt1 and RDCav1 clones (Figure 5A). Statin-induced radiosensitization was confirmed by assessing \(\gamma\)H2 AX, a marker of DNA double strand breaks (DSBs). In radioresistant myrAkt1 and RDCav1 lines, IR treatment already promoted the appearance of nuclear staining indicative of DSBs after 1 h, which completely disappeared after 24 h, indicative of DNA repair (Figure 5B). However, treatment of cells with statins prior to IR resulted in prolonged \(\gamma\)H2 AX staining which was still detectable 24 h after IR, indicative of unrepaired DNA damage (Figure 5B). Using FACS analysis, we found that statins significantly increased cell apoptosis in all RMS lines (Figure 5C). Notably, statin treatment produced the strongest effect on the radioresistant lines, resulting in a percentage of cell apoptosis between 80 and 90% (Figure 5C). The combination of statins and IR significantly increased cell apoptosis in the RD and RH30 lines, while the additive effect in the radioresistant lines was less evident due to the strong effect already produced by statin treatment alone (Figure 5C).