## 4 Discussion  
Despite being a highly effective anticancer agent, DOX administration is limited by its potent toxicity in various tissues, including skeletal muscle. Recent studies have shown that acute EXE before DOX treatment protects DOX-induced myotoxicity; however, it remains unclear whether EXE should be administered as an adjuvant intervention during multiple DOX treatment cycles to appease unwanted toxicity to non-cancer tissues. Thus, the present study examined the effect of chronic EXE administration as an adjuvant treatment during chronic DOX treatment cycles on phenotypic changes in SOL muscle. The present study provides novel insight into the mechanisms responsible for EXE-induced myocyte protection against chronic DOX treatment cycles. Our data showed that EXE administered during DOX treatment did not prevent DOX-induced loss of body weight, lean mass, and absolute SOL muscle mass, but sustained healthier muscle morphology. Furthermore, our results showed that suppression of proteolytic activation induced by the AKT-FOXO3\(\alpha\)-MuRF-1 axis and potentiation of basal autophagy is linked to the EXE-mediated muscle quality control against DOX-induced myotoxicity. Previous studies have shown that DOX treatment leads to an unfavorable phenotype in skeletal muscle tissues, such as muscle atrophy and myotoxicity [22; 25; 26; 27]. Our data also verified those anomalies as evidenced by loss of body weight, absolute SOL muscle weight, and body composition (reduction of final body weight, lean mass, and fat mass examined by DEXA) in response to DOX treatment (5 mg/kg, biweekly, total dosage = 25 mg/kg). Consistent with our findings, a recent study also showed a significant reduction in body weight and gastrocnemius mass in response to chronic DOX treatment (2.5 mg/kg, twice a week, total dosage = 25 mg/kg) [28]. Interestingly, EXE intervention combined with DOX treatments resulted in a more significant loss of body weight and absolute SOL muscle mass than the only DOX treatment as opposed to our postulation that EXE might be attenuated DOX-induced muscle wasting. We are not sure why this incidence happened in our study; however, considering the possibility that DOX incurs a loss of appetite and that EXE increases caloric expenditure, the combinatory effects of DOX and EXE may elicit the additional loss of body weight and muscle mass. The next question is, does this observation mean that cancer patients receiving DOX chemotherapy should refrain from EXE during the DOX chemotherapy period due to additional muscle loss? According to our results, the answer is \"no\" because the relative SOL muscle mass was indistinguishable between SED-DOX and EXE-DOX groups, and a muscle damage level (e.g., the appearance of degenerated muscle fibers and ragged red fibers) was lower in the EXE-DOX group compared to the SED-DOX group. Consistent with our affirmation, other studies have confirmed that various EXE paradigms provide similar therapeutic potency against DOX-induced myotoxicity. For instance, short-term EXE (\(\leq\)4 weeks) before DOX treatment [7; 14; 29; 30; 31], long-term EXE (\(\geq\)4 weeks) before DOX treatment [4], short-term EXE (\(<\)4 weeks) during/after DOX treatment [28; 32; 33], and long-term EXE (\(\geq\)4 weeks) during/after DOX treatment [5; 20; 34] conferred myocyte protection. Furthermore, a recent clinical study has shown that cancer patients performing EXE during chemotherapy improve a disease-free survival rate compared to sedentary cancer patients [35], indicating that EXE administration during multiple chemotherapy cycles is safe and beneficial. A DOX-mediated TOP2B modulation plays a crucial role in myotoxicity, as the ablation of TOP2B attenuates DOX-induced DNA damage in MEF cells [36]. Besides, cardiac-specific deletion of TOP2B protects the heart against DOX-induced cardiac toxicity [11], demonstrating that DOX-induced TOP2B upregulation is linked to DOX-induced myotoxicity.