## 4 Discussion
According to our data, the Akt1/Src/Cav1 signaling in RMS that activates in response to high oxidative stress seems to represent a key determinant of radioresistance by increasing ROS detoxification and repair of DNA damage, as previously anticipated in other works [23; 37]. In this regard, the elevated catalase expression observed following activation of the Akt1/Src/Cav1 axis likely reflects an increased need for irradiated RMS cells to detoxify superoxide anions and the dismututation product H2O2 [27]. Interestingly, increased Cav1 expression in lung adenocarcinoma has been reported to attenuate H2O2 production favoring tumor progression [38]. Therefore, understanding the mechanisms through which the Akt1/Src/Cav1 pathway contributes to buffering oxidative stress and DNA repair remains one of the major objectives deserving of further investigation for the development of therapies aimed at overcoming resistance mechanisms in RMS. The mechanisms by which oxidative stress in RMS can preferentially activate intracellular kinases have not been investigated thus far. It has been reported that high ROS levels can lead to activation of Src and Akt by reversible oxidation of conserved cysteine residues [39; 40]. For example, ROS generated by IR therapy or by the activity of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidases (NOX) and mitochondria can reversibly oxidate critical cysteine residues within Src, promoting its activation [41]. Similarly, prolonged phosphorylation of the PI3K/Akt pathway may result from IR-induced oxidative stress that mediates the inactivation of protein tyrosine phosphatases (PTPs) involved in dephosphorylation of RTK [42], such as erythroblastic oncogene B (ErbB)/human epidermal growth factor receptors (HER) [43]. Importantly, Akt can also be activated in response to IR-induced DNA damage by members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related protein (ATR), and DNA-PK, as a part of pro-survival program [44; 45; 46]. Therefore, it is of considerable importance to understand the mechanisms involved in the activation of antioxidant kinases, such as the Akt and Src kinases, in order to develop targeted therapies for RMS. Previous works have shown that statins cause cytotoxicity in RMS cells by increasing autophagy and apoptosis [47; 48; 49]. We have confirmed these data here, further demonstrating that statin treatment generates high oxidative stress in RMS cells, leading to cell apoptosis. In this regard, the excessive or long-term use of statins is known to cause in vitro and in vivo cytotoxicity that seems to be mainly related to oxidative stress [28], as observed in skeletal muscle, where rhabdomy may occur [50]. In RD and myrAkt1 cells, the Akt1 and Cav1 proteins were robustly downregulated after statin treatment. We do not know the mechanism by which statins induce Akt1/Cav1 downregulation; nevertheless, statin-mediated attenuation of the Akt and mTOR signaling pathways has already been correlated with reduction of tumor burden in different cancers [51]. We assume that, after statin treatment, the failure to activate the Akt1/Cav1 pathway determines the inability to buffer oxidative stress, facilitating ROS accumulation and apoptotic events. Interestingly, the radioresistanceM myrAkt1 and RDCav1 lines were more protected from the cytotoxic effects of actinomycin D, a classic antineoplastic agent used in the treatment of RMS that primarily acts as an inhibitor of RNA synthesis but which also induces oxidative stress [29]. statin treatment in combination with IR or actinomycin D was effective to enhance radio- and chemosensitization of RMS lines.