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Abstract Introduction: Ewing's sarcoma (ES) is a highly malignant tumor that commonly occurs in children and adolescents. The therapeutic strategy is based on a multi-drug chemotherapy regimen, in combination with radiotherapy and surgery. Unfortunately, ES cells exhibit a high sensitivity to rapid changes in intracellular redox environment resulting in an elevated incidence of drug resistance. In recent years, β3-adrenergic receptor (β3-AR) has gained growing attention due to its implication in tumor onset, progression and metastasis, alongside its physiological functions. Indeed, it has been found to be overexpressed in various tumors, especially pediatric malignancies, including ES. β3-adrenergic receptor acts as the main regulator of the cellular response to redox environment, ultimately by influencing the expression of UCP2, well-known transporter that decreases mitochondrial activity and ROS content. The β3-AR antagonist, SR59230A (SR), demonstrated to reverted these effects. Therefore, we exploited the effects of SR59230A in ES cells that increases the intracellular ROS content and enhancing their sensitivity to treatment with doxorubicin (DOX) drug, which among its functions also influences UCP2 protein. As a result, the combination of both compounds may represent a putative novel therapeutic strategy able to overcome drug resistance in ES cancer. Aim: In the attempt of overcoming chemotherapy resistance in Ewing’s sarcoma, we studied the effects of DOX compound in combination with SR, the main β3-AR antagonist, which enhances the cell’s sensitivity to treatments. Methods: We performed proliferation assays to asses IC50 of both compounds in A673 cells. After 3h and 24h of treatment with DOX and SR, alone and in combination, the ROS levels and mitochondrial membrane potential were evaluated using flow cytometry analysis. The metabolic profile of A673 in the same conditions was measured using Seahorse XF Analyzer. The data was confirmed by Western Blotting investigation. Statistical analysis was performed using one-way and two-way ANOVA with Tukey’s post-hoc test (*p0.05). Results and conclusions: Our data shows an increase in ROS content in A673 cells after 3h and 24h of treatment with DOX and SR in combination compared to treatments with the single drug, in accordance with the modulation of UCP2 exerted by the drugs. This result is confirmed by western blot analysis, which shows an increased level of expression of SOD2 after 24h of treatment with a combination of the compounds. The metabolic real time analysis of A673 after 3h and 24h with the same treatments shows a decreased in ATP mitochondrial production, supporting the flow cytometry investigation that demonstrates a decrease in mitochondrial membrane potential after 3h of treatment with both compounds and with SR alone compared to control. In conclusion the β3-AR/UCP2 axis decreased mitochondrial activity by reducing ATP synthesis and mitochondrial ROS content and this effect is reverted by β3-AR antagonist, allowing to overcome drug resistance in combination treatments with DOX. Citation Format: Megan Lotti, Rachele Amato, Maria Ascone, Cristina Banella, Francesco Carrozzo, Amada Pasha, Annalisa Tondo, Maura Calvani. Overcoming chemotherapic resistance caused by β3-AR in Ewing’s sarcoma with the receptor’s antagonist abstract. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr A061.
Lotti et al. (Thu,) studied this question.