Mentha-derived green synthesized ZnO nanoparticles potentiate methotrexate induced molecular and migratory inhibition via gene expression modulation in T24 bladder cancer cells.

Bladder cancer is characterized by high recurrence rates and limited responsiveness to conventional chemotherapy, including Methotrexate (MTX). Nanotechnology-based strategies may enhance therapeutic efficacy while reducing drug resistance. In this study, zinc oxide nanoparticles (ZnO-NPs) were green-synthesized using Mentha sp. extract and evaluated alone and in combination with MTX against T24 bladder cancer cells. The synthesized ZnO-NPs exhibited stable physicochemical properties and pronounced cytotoxic activity compared with MTX monotherapy. Combination treatment produced an additive cytotoxic effect but demonstrated enhanced mechanistic activity, including increased apoptosis, cell-cycle arrest, and DNA damage. Molecular analysis revealed significant upregulation of pro-apoptotic genes (TP53, BAX, CASP3) and suppression of the anti-apoptotic gene BCL2. In addition, the combination treatment significantly modulated TGF-β1 expression, interculture reactive oxygen species and altered cell migratory behavior. Collectively, these findings indicate that green-synthesized ZnO-NPs can potentiate MTX-induced molecular responses, supporting their potential as a nanomedicine-based adjuvant strategy for bladder cancer treatment.