Methanolic extract of Zataria multiflora and arsenic trioxide combination therapy modulates oncogenic and tumor suppressor MicroRNA expression and RAF1 signaling in NB4 acute promyelocytic leukemia cells.

This study investigates the hypothesis that Zataria multiflora (Shirazi thyme) methanolic extract (ZME) exerts synergistic anti-leukemic effects when combined with low-dose ATO, mediated through modulation of oncogenic and tumor suppressor miRNAs. We treated NB4 acute promyelocytic leukemia cells with ZME (20 µg/mL), ATO (0.25 µM), or their combination for 24 and 48 h. Cell viability was assessed using the MTT assay; apoptosis was evaluated by flow cytometry with Annexin V/propidium iodide staining. Expression of miRNAs (miR-19a-3p, miR-23a-5p, miR-181b-5p, miR-3156-5p, and miR-4498) was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Molecular docking studies evaluated the predicted interactions between thymol/carvacrol (major constituents of ZME) and RAF1 protein, a downstream effector in the mitogen-activated protein kinase (MAPK) signaling pathway. The combination of ZME (20 µg/mL) + ATO (0.25 µM) significantly reduced cell viability and metabolic activity compared to individual treatments, with synergistic effects confirmed by combination index analysis (CI < 1). Apoptosis induction increased to 44% in combined treatment compared to 31.51% with ZME alone and 9.32% with ATO alone. Notably, the combination modulated miRNA expression patterns, particularly reducing miR-19a-3p and miR-23a-5p to near-baseline levels, while miR-181b-5p showed less pronounced changes. Molecular docking analysis predicted favorable interactions between thymol/carvacrol and RAF1 active sites, suggesting potential pathway engagement. These data indicate that ZME acts as a synergistic adjuvant with low-dose ATO, potentially enabling dose reduction while maintaining therapeutic efficacy and reducing toxicity burden in APL treatment. However, functional validation of miRNA targets and RAF1 pathway engagement is required to substantiate mechanistic claims.