The present study aimed to investigate the effect and elucidate the potential mechanisms of 9-hydroxypheophorbide α-based photodynamic therapy (9-HPbD-PDT) on apoptosis and necrosis induction and migration suppression of laryngeal cancer AMC-HN-3 (HN-3) cells. staining and an upregulated expression of poly ADP-ribose polymerase was detected through western blotting. A disruption of the mitochondrial membrane potential was detected 2 h following PDT. Significant suppression of cell migration and downregulation of epidermal growth factor receptor (EGFR) expression were recorded following PDT. These results indicate that this distribution of photosensitizer leads to differences in the generation of ROS which subsequently determines the type of cell death. Overall mitochondrial activation under oxidative stress is important in the 9-HPbD-PDT-induced apoptosis of HN-3 cells. Migration suppression of HN-3 cells following PDT may be associated with LCZ696 the inhibited expression of EGFR due to oxidative stress. and (1); however the factors involved in the overall process and the contribution to either mechanism are not completely elucidated. PDT has previously exhibited novel effects in the treatment of oncologic diseases; however the limitations of laser penetration into normal tissues and long-lasting cutaneous photosensitivity following irradiation are unavoidable and may affect the applicability of PDT to malignant tumor therapy (2). As a novel chlorophyll derived photosensitizer 9 α (9-HPbD) has a relatively longer absorption wavelength (664 nm) and a shorter half-life in the body compared with other LCZ696 photosentisizers (3). In addition 9 exhibited an apoptosis-inducing effect and growth suppression in MCF-7 human breast malignancy cells (3). A previous study concerning combination treatment with 9-HPbD-PDT and carboplatin resulted in an enhanced photocytotoxicity and apoptosis induction in laryngeal AMC-HN-3 (HN-3) cancer cells (4). Oxidative stress-directed cell death and migration suppression of HN-3 cells using 9-HPbD-PDT is additionally investigated in the present study. The present study aimed to investigate the effect and elucidate the potential mechanisms of 9-HPbD-PDT on apoptosis and necrosis induction as well as migration suppression of HN-3 cells. Materials and methods Reagents and antibodies All media and supplements for cell culture were supplied by Hyclone? (GE Healthcare Life Sciences UTP14C Logan UT USA). Dimethyl sulfoxide (DMSO) 3 5 5 bromide (MTT) RIPA buffer protease [glutathione (GSH)] and phosphatase (ascorbic acid) inhibitors Hoechst 33342 dye and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis MO USA). 2′ 7 diacetate (H2DCFDA; D399) and rhodamine 123 were purchased from Molecular Probes (Thermo Fisher Scientific Inc. Waltham MA USA). Rabbit anti-GAPDH polyclonal antibody (cat. no. ab9485; dilution 1:2 0 was supplied by Abcam (Cambridge UK); mouse anti-poly ADP-ribose polymerase (PARP) polyclonal antibody (cat. no. AM30; dilution 1:200) was obtained from Merck Millipore (Darmstadt Germany); and goat anti-epidermal growth factor receptor (EGFR) polyclonal antibody (cat. no. sc-03-G; dilution 1:200) was purchased from Santa Cruz Biotechnology Inc. (Dallas TX USA). Cell culture The HN-3 cell line (5) was developed from a 63-year-male patient with previously untreated laryngeal squamous cell carcinoma and was kindly provided by Asan Medical Center (Seoul Korea). The cell line was cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum penicillin (50 models/ml) and streptomycin (50 μg/ml) at 37°C in a 5% CO2 and LCZ696 95% air atmosphere in a humidified incubator. PDT protocol 9-HPbD (3) was kindly donated by Kumho Life and Environmental Science Laboratory (Kwangju Korea). 9-HPbD LCZ696 was stored in ethanol (1.5 mg/ml) and aluminum foil at ?20°C. HN-3 cells at 90% confluence were incubated with 9-HPbD in the dark for 6 h at 37°C. Subsequent to changing the culture medium 9 cells were subsequently exposed to a 664 nm diode laser (Hi-Tech Optoelectronics Co. Ltd. Beijing China) at 2.0 J/cm2 for 15 min. In order to assess the antioxidant effect HN-3 cells were incubated simultaneously with either 5 mM GSH or 2.5 mM ascorbic acid and 0.59 μg/ml 9-HPbD. Subsequently the incubated cells were irradiated by laser according to the aforementioned conditions. Cell viability assay A MTT assay was used to measure cell viability. The treated cells at 90% confluence were incubated with 50 μl MTT answer (2 mg/ml) for 2 h. The MTT answer was exchanged with 100 μl DMSO and the absorbance at 540 LCZ696 nm was measured following 20 sec of shaking. Cell viability was calculated according LCZ696 to the following equation: Cell viability (%).