Objectives: Until recently, a conventional chemotherapy regimen for Acute lymphoblastic leukemia (ALL) is considered an efficient therapeutic method in children. However, suboptimal long-term survival rates in adults, disease relapse, and drug-induced toxicities require novel therapeutic agents for ALL treatments. Today, natural products with pharmacological benefits play a significant role in treating different cancers. Among the most valued natural products, honey bees' royal jelly (RJ) is one of the most appreciated which has revealed anti-tumor activity against different human cancers. This study aimed to evaluate anti-leukemic properties and the molecular mechanisms of RJ cytotoxicity on ALL-derived Nalm-6 cells.

Materials and methods: The metabolic activity was measured by MTT assay. Apoptosis, cell distribution in the cell cycle, and intracellular reactive oxygen species (ROS) level were investigated using flow cytometry analysis. Moreover, quantitative real-time PCR (qRT-PCR) was performed to scrutinize the expression of various regulatory genes.

Results: RJ significantly decreased the viability of Nalm-6 cells but had no cytotoxic effect on normal cells. In addition, RJ induced ROS-mediated apoptosis by up-regulating pro-apoptotic genes while decreasing anti-apoptotic gene expression. The results outlined that ROS-dependent up-regulation of FOXO4 and Sirt1 inhibits the cells' transition to the S phase of the cell cycle through p21 up-regulation. The qRT-PCR analysis of autophagy-related gene expression also demonstrated that RJ induced BECN1 mediated autophagy in Naml-6 cells.

Conclusion: Taken together, this study showed that RJ can be utilized as a potent natural substance to induce ALL cells' programmed cell death. However, further studies are required to examine this compound's pharmaceutical application.