## 4 Discussion  
PDT is a technique that is already used for the clinical treatment of cancer. However, the mechanism underlying the cancer-specific accumulation of porphyrin remains unclear. Endocytosis via the low-density lipoprotein (LDL) receptor and the inactivation of ferrochelatase, which is an enzyme that inserts iron ions into porphyrin and synthesizes heme, have been reported to be associated with the accumulation of intracellular porphyrin [17,18]. I previously reported that mitochondrial ROS upregulate the expression of HCP1 and enhance the effect of cancer therapy by PDT [6]. I also reported that HCP1 is a downstream target of hypoxia-inducible factor (HIF)-1\(\alpha\), and that NO stabilizes HIF-1\(\alpha\) by inhibiting prolyl hydroxylase, thereby regulating HCP1 expression [19]. In this study, I showed that the NO induced by LPS treatment and the ROS generated by X-ray irradiation together upregulated HCP1 expression levels and increased cellular porphyrin uptake. It is likely that the effect on HCP1 levels is mediated by peroxynritrite, which is an RNS produced by the reaction with NO and superoxide. NO and ROS together produce peroxynritrite, and the rate constant is quite fast [20]. In support of this, when RAW264 cells stimulated by LPS--which have increased iNOS levels and a resultant increase in NO levels--are irradiated with X-rays, there is an increase in the intracellular production of peroxynritrite (Figure 3). Although high levels of increase were not observed in the present study, the induction of intracellular peroxynritrite production stimulated by LPS or irradiation was recently reported using a novel detection probe [21]. Peroxynritrite is also formed in vivo and plays numerous roles as a signal mediator in the development of many diseases, such as chronic inflammatory diseases, carcinogenesis, and neurodegenerative disorders [11,22]. In the tumor microenvironment, TAMs expressing iNOS promote tumor growth, invasion, immune evasion, and the acquisition of drug resistance [23]. Activated macrophages also produce superoxide via NOX activation and form peroxynritrite in the presence of NO and superoxide. This peroxynritrite could result in the oxidation and nitration of both proteins and lipids, leading to changes in cellular signaling [24]. In PDT, NO- and H\({}_{2}\)O\({}_{2}\)-producing agents have been reported to enhance sensitization to PDT [25,26]. They are thought to directly enhance the effect of PDT, but the effect of the RNS peroxynritrite has not been reported. The non-steroidal anti-inflammatory drug indomethacin has been used as an ROS inducer; it has been reported to enhance the cancer-specific accumulation of porphyrin and the overexpression of manganese superoxide dismutase (MnSOD, a mitochondrial superoxide scavenging enzyme) decreased HCP1 expression and the subsequent PDT effect [27]. These results suggest that the removal of superoxide by MnSOD could prevent the synthesis of peroxynritrite and the accumulation of intracellular porphyrin. In numerous types of cancer cells, elevated expression of iNOS, increased NO production, and the overgeneration of ROS have been reported [28, 29, 30, 31]. In addition, TAMs possibly help to promote the expression of HCP1 in tumor cells via the production of peroxynritrite, as shown in Figures 3 and 5, and may therefore lead to effective phototherapy. In conclusion, a highly reactive nitrogen species, peroxynritrite, is generated in the mouse macrophage cell line RAW264 through the production of NO, which is produced as a result of the upregulated expression of iNOS by LPS treatment, and ROS, which is mainly superoxide derived from mitochondria, produced by irradiation with X-rays. It was observed that the cellular production of peroxynritrite contributed more toward unregulating the expression of HCP1 and enhanced intracellular porphyrin accumulation. This macrophage-mediated mechanism may therefore enhance the effect of PDT to suppress tumor growth.