Arsenic Sulfide EnhancesRadiosensitization in Rhabdomyosarcoma via Activating NFATc3-RAG1 Mediated DNA Double Strand Break (DSB)

Abstract

Abstract Background: Due to the high level of recurrence and metastasis, rhabdomyosarcoma (RMS) represents one of the most lethal soft-tissue sarcomas in children. It is thus imperative to explore a novel radiosensitizer to enhance the curative of radiotherapy in RMS patients. The trace element arsenic has been reported to function as a radiosensitizer in sarcomas. To determine whether arsenic sulfide (As4S4) potentiates radiation sensitization in RMS, we carried out this study to investigate the mechanism of As4S4 in radiotherapy. Methods: RMS cell line (A-673) was treated with As4S4 and radiotherapy. Cell viability and drug-induced apoptosis were detected by cell counting kit-8 (CCK-8) and flow cytometry, respectively. Western blot and real-time qPCR were carried out to detect the nuclear factor of activated T-cells 3 (NFATc3) and recombination activating 1 (RAG1). DNA damage-associated proteins were also determined. For in vivo experiments, the therapeutic efficacy of As4S4-induced radiosensitization was evaluated via xenograft tumors in mice. To identify NFATc3 and RAG1, which were mostly involved in the mechanism of radiosensitization, we established a clinical cohort of 59 RMS patients. Immunohistochemistry (IHC) staining was applied to detect the expression of NFATc3 and RAG1 in RMS tissues in order to analyze the relationship with prognosis. We further developed a prediction model using stepwise logistic regression. Results: As4S4 combined with radiotherapy exhibited predominant inhibition in RMS cells through CCK-8 and flow cytometry. We revealed that As4S4 as well as the knockdown of NFATc3 resulted in DSB in RMS cells by the increased expression of RAG1. Our in vivo experiment confirmed that co-treatment exerted efficient inhibition of RMS growth. In a clinical cohort of 59 RMS patients, survival analysis showed that NFATc3 and RAG1 were related to overall survival (OS). Cox regression analysis further indicated that NFATc3, RAG1, and Risk level could be regarded as independent prognostic factors for RMS patients. Conclusions: In summary, As4S4 enhances radiosensitization in RMS via activating NFATc3-RAG1 mediated DNA DSB. NFATc3 and RAG1 are potential therapeutic targets in treating RMS. Our findings led us to conclude that As4S4 could be considered a radio-sensitizing agent for treating RMS.