Dihydroartemisinin downregulates the expression of COX-2 in esophageal squamous cell carcinoma through ROS/MAPK/Sp1 axis

Background Esophageal squamous cell carcinoma (ESCC) is associated with a poor clinical prognosis due to its highly invasive and metastatic characteristics, as well as chemotherapy resistance, highlighting an urgent need to explore novel therapeutic targets and drugs. Dihydroartemisinin (DHA), a derivative of artemisinin, has been demonstrated to possess broad anti-tumor activity, while its specific molecular mechanisms in ESCC remain incompletely elucidated. Methods Transcriptomic sequencing and The Cancer Genome Atlas (TCGA) database analysis were employed to identify differentially expressed genes following DHA treatment. Cyclooxygenase-2 (COX-2) was found to be significantly downregulated after DHA treatment and highly expressed in ESCC tissues. The regulatory effect of DHA on COX-2 expression was validated using RT-qPCR and Western blot. Gain- and loss-of-function experiments were conducted to assess the impact of COX-2 on ESCC cell proliferation and migration. Dual-luciferase reporter assays and rescue experiments were performed to verify the transcriptional regulation of COX-2 by Sp1 and its role in the malignant phenotype of ESCC. Gene Ontology (GO) enrichment analysis revealed that genes downregulated by DHA were significantly enriched in the MAPK signaling pathway. Intracellular reactive oxygen species (ROS) levels and the expression of key MAPK pathway proteins were assessed via flow cytometry, ROS rescue experiments, and Western blot, respectively. A xenograft nude mouse model was established to evaluate the effects of Sp1 overexpression and DHA treatment on tumor growth in vivo. Results DHA downregulated COX-2 expression in ESCC cells in a time- and dose-dependent manner and significantly inhibited cell proliferation and migration. Sp1, as a transcription factor, directly bound to the COX-2 promoter and positively regulated its expression, thereby influencing the malignant behavior of ESCC. Transcriptomic analysis indicated that the MAPK pathway was the most significantly enriched among the pathways affected by DHA. DHA induced ROS accumulation, which subsequently inhibited MAPK (ERK/MSK) phosphorylation and downregulated the protein expression of Sp1 and COX-2. These effects were reversible by the antioxidant N-acetylcysteine (NAC) or Sp1 overexpression. In vivo experiments further confirmed that DHA significantly suppressed tumor growth by inhibiting the Sp1/COX-2 axis. Conclusion This study demonstrates that DHA inhibits the proliferation and metastasis of ESCC by inducing ROS accumulation, suppressing MAPK (ERK/MSK) signaling activation, downregulating the expression of Sp1 and its target gene COX-2, and ultimately acting through the ROS–MAPK (ERK/MSK)–Sp1–COX-2 signaling axis. These findings provide a new strategy for the targeted therapy of ESCC and underscore the potential value of Sp1 and COX-2 as therapeutic targets.