Phototoxic Effect of Na-pheophorbide a Toward Osteosarcoma Cells in Vitro Using a Laser Diode

Objective: The purpose of this study was to investigate the effectiveness of photodynamic therapy (PDT) with Na-pheophorbide A in anticancer treatment, using osteosarcoma cells in vitro.

Background Data: It has been reported that PDT with chlorophyll derivatives inhibits the proliferation of various cancer cells. However, there have been no reports that have evaluated the effectiveness of PDT in suppressing osteosarcoma cells.

Materials And Methods: Uptake of Na-pheophorbide A into Hu09 cells (osteosarcoma cells) was assayed using fluorescence microscopy following incubation of the cells with 28 μmol/L of Na-pheophorbide A. The viability of Hu09 cells after PDT treatment was assessed using trypan blue dye staining and MTS assays. PDT-induced apoptosis was determined by evaluation of the activity of selected members of the caspase family and by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of cells.

Results: Na-pheophorbide A uptake by cells was rapid, being observed after 60 min of treatment, and Na-pheophorbide A persisted in cells for >24 h. PDT treatment decreased cell viability compared with the control group, indicating high cytocidal activity of PDT. This cytocidal effect was dependent upon drug concentration, light dose, and the number of irradiation times. An increase in the number of cells positive for TUNEL staining and increases in the activity of caspases-3, -8 and -9 were observed in the first 2 h after PDT treatment.

Conclusions: A cytotoxic effect of PDT with Na-pheophorbide A on an osteosarcoma cell line in vitro was shown. Caspase activity assays suggested that PDT with Na-pheophorbide A induced an apoptotic change in HuO9 cells, mainly via activation of mitochondrial caspase -9 and -3 pathways.