Mechanistic insights into temoporfin-based photodynamic therapy: Ferroptosis as a critical regulator under normoxic and hypoxic conditions in head and neck cancer

Abstract

Temoporfin is a second-generation photosensitizer used in photodynamic therapy (PDT) for the clinical treatment of head and neck cancer. However, its role in inhibiting cancer cell viability under normoxic and hypoxic conditions remains unclear. The oral squamous cell carcinoma (OSCC) cell lines, SAS and OECM-1 were cultured under normoxic or hypoxic conditions to investigate temoporfin-based PDT-induced cell death and the underlying mechanisms. Cell viability was analyzed using the MTT assay. Intracellular reactive oxygen species (ROS) levels, cell apoptosis, intracellular ROS, iron levels, lipid peroxidation, and glutathione (GSH) levels were assessed by flow cytometry. The expression of proteins related to oxidative stress, apoptosis, autophagy, and ferroptosis was verified by western blotting. Results showed that increasing the temoporfin dose, absorption time, and illumination time was positively correlated with the inhibition of oral cancer cells. Hypoxic conditions attenuated the toxicity of temoporfin in cancer cells. OECM-1 cells were more sensitive to temoporfin than SAS cells. Temoporfin-based PDT-induced ROS exhibited similar trends to oxidative stress-inducing enzymes under both normoxic and hypoxic conditions and triggered cell autophagy and ferroptosis. Administration of the ferroptosis inhibitor BRD4770 under normoxic conditions reversed temoporfin-based PDT-induced reductions in glutathione peroxidase 4 (GPx4), increasing in light chain 3-II (LC3-II) and cleaved poly (ADP-ribose) polymerase (cleaved-PARP). This study confirms that hypoxia weakens the anticancer effects of temoporfin-based PDT and that ferroptosis plays a key role in temoporfin-based PDT-mediated cancer cell inhibition.