RUNX3 is a biomarker for determining sensitivity to pheophorbide a-photodynamic therapy in human oral squamous cell carcinoma

Background & Objective: Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, the screening process for sensitive cancers to PDT should be preceded. For this, we investigated a molecular biomarker to determine the sensitivity to Pa-PDT in immortalized human oral keratinocytes (IHOK) and OSCC cell lines. Methods: For this study, two immortalized oral keratinocytes (IHOK-p, IHOK-s) and 8 oral squamous cell carcinoma (OSCC) cell lines were used. After cells were treated with Pa-PDT, phototoxicity and the level of reactive oxygen species (ROS) were measured. Apoptosis was measured using annexin V/PI staining and western blotting. mRNA and proteins of apoptotic genes were investigated by RT-PCR, real-time PCR, and western blotting. Transfection was performed using RUNX3-small interfering RNA. Results: After Pa-PDT, cell viability was more than 50% reduced and ROS generated in IHOK and OSCC. In addition, apoptosis occurred in all cell lines. Among apoptosis-related genes, Bim expression was altered following Pa-PDT. Therefore, mRNA and protein expression of RUNX3, a gene upstream of Bim were examined by Pa-PDT. We found that RUNX3 was highly responsive to Pa-PDT. Furthermore, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT. In addition, we found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Conclusion: This is the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. These results should be further proven by animal studies to apply to clinical trials. Nevertheless, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT in oral precancerous and cancerous lesions.