Non‑thermal plasma activated media induces ferroptosis in head and neck cancer through HMOX1‑mediated iron accumulation.

OBJECTIVES.: To investigate the anticancer mechanisms of non-thermal plasma activated media (NTPAM) treatment in head and neck cancer (HNC) cells, with a focus on its role in oxidative stress and ferroptosis induction. METHODS.: 4 HNC cell lines were treated with NTPAM, and cell sequencing data were analyzed revealed Ferroptosis as a potential cell death pathway. Intracellular ferrous iron (Fe²⁺) levels, Ferroptosis markers, and gene expression of HMOX1 and NRF2 were measured in vitro on FADU and SNU1041 cell lines. RNA silencing of HMOX1 and NRF2 were performed to assess their roles in NTP - induced ferroptosis. In vivo tumor models were examined for intra-tumor Fe²⁺ accumulation and HMOX1 expression following NTPAM treatment. RESULTS.: NTPAM treatment induced HNC cell death via a non-canonical ferroptosis pathway, primarily through increased Fe²⁺ level. Overexpression of HMOX1, an NRF2 regulated gene, promotes NTPAM induces intracellular Fe²⁺ increase; and silencing HMOX1 rescues HNC from NTPAM induced iron accumulation and ferroptosis. NRF2 knockdown deregulates HMOX1 expression and prevents Fe²⁺ increase but do not improve cell survival or ferroptosis morphology. In vivo, NTPAM-treated tumors showed elevated intratumoral Fe²⁺ and increased HMOX1 expression, confirming their contribution to ferroptosis. CONCLUSION.: Our finding suggests that NTPAM directly induces ferroptosis in HNC cells by HMOX1 - mediated intracellular Fe²⁺ accumulation. This process is facilitated by increased nuclear translocation of NRF2, which upregulates HMOX1 expression and thereby drives iron dysregulation leading to ferroptotic toxicity.