Azoxystrobin Targets Prdx1 To Induce Apoptosis In Oral Leukoplakia

The aim of this study is to further elucidate the mechanism of azoxystrobin (AZOX) inducing mitochondrial apoptosis in oral leukoplakia (OLK). The binding conformation of AZOX-Prdx1 was analyzed using PyMol, which also predicted their interaction sites. Cell viability in OLK cell lines (DOK and Leuk-1) was assessed by CCK-8 assays. Apoptosis rates were measured via flow cytometry, while subcellular localization and expression levels of Bax, Bcl-2, and Cyt c were detected using immunofluorescence and Western blotting. Prdx1 directly binds to AZOX, forming four hydrogen bonds with three amino acid residues (Gln94, Thr90, and Thr49) and a pi-pi interaction with Trp87. Mutations at Trp87 and Thr90 in Prdx1 significantly attenuated AZOX-induced mitochondrial apoptosis in OLK cells at 24 h and 48 h (p<0.05) without affecting cell viability, and in mutant-transfected OLK cells, where Bax, Cyt c, and Bcl-2 are predominantly localized in the cytoplasm, the expression of Bax and Cyt c significantly decreased at 24 h (p<0.05), whereas Bcl-2 remained unchanged compared to wild-type controls. These results indicate that these two sites are critical for AZOX-induced mitochondrial apoptosis. Trp87 and Thr90 residues of Prdx1 are identified as critical binding sites for AZOX to trigger mitochondrial apoptosis in OLK. The mutations at the sites impair AZOX binding efficacy, confirming Prdx1 as a druggable target and enabling structure-based optimization of Prdx1 inhibitors for oral precancer therapy.