Active oxygen intermediates in the degradation of hematoporphyrin derivative in tumor cells subjected to photodynamic therapy.

Hematoporphyrin derivative (HPD), a sensitizer used in photodynamic therapy (PDT) of malignancies, is progressively destroyed during the treatment. Prior studies suggested that upon PDT the photobleaching of HPD in tumor tissues is largely mediated by self-sensitized singlet oxygen. However, little is known about the role of other reactive oxygen species (ROS). The main aim of this work was to clarify the significance of H2O2, superoxide (O2.(-)) and hydroxyl (OH.) radicals in bleaching of HPD in tumor cells subjected to PDT. Experiments were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in PBS and then irradiated with red light at 630 nm in the same buffer. Studies showed that photosensitization of EAC cells by HPD led to the formation of significant amounts of H2O2, O2.(-) and OH., and that these ROS could be involved in the photobleaching of HPD during PDT. In fact, we found that addition of catalase (CAT, a scavenger H2O2), Cu/Zn-superoxide dismutase (Cu/Zn-SOD) and Tiron (scavengers of O2.(-)), Na-benzoate, mannitol and deferoxamine (scavengers of OH.) caused a substantial decrease in the rate of HPD photobleaching in EAC cells. In these cells, the inhibitory effects of Na-benzoate, mannitol and deferoxamine on the photodegradation of HPD correlated well with suppression of the OH. generation, a highly active oxidizer. In EAC cells, the glutathione redox cycle and CAT (scavengers of H2O2) as well as Cu/Zn-SOD was found to suppress the photoinduced degradation of HPD. It was also established that HPD can directly scavenge H2O2 and oxygen free radicals; in a phosphate buffer its second-order rate constants were measured as 5.51+/-0.32 x 10(3)M(-1)s(-1) (for the reaction with O2.(-)), 5.08+/-0.31 x 10(4)M(-1)s(-1) (for H2O2), and 3.44+/-0.08 x 10(10)M(-1)s(-1) (for OH.). Thus, our data suggest that OH. could be one of the main oxidants mediating the photobleaching behavior of HPD in malignancies. Studies showed that photoexcited moieties of HPD can oxidize cell proteins with the formation of protein peroxides (PPO), which currently are regarded as a new form of ROS. Model experiments suggest that PPO could also participate in bleaching of HPD in tumors treated with PDT. It was found that HPD may destroy in tumor cells after cessation of photoirradiation and that this event is largely mediated by the presence of H2O2, a precursor of OH(.).

Hydrogen peroxide, superoxide, and hydroxyl radicals are involved in the phototoxic action of hematoporphyrin derivative against tumor cells.

This study was aimed to estimate the participation of reactive oxygen species (ROS), other than singlet oxygen (1O2), in the antitumor effect of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD) as well as to determine the ability of photoexcited HPD to the formation of protein peroxides that currently are regarded as a new form of ROS. Studies were performed on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in phosphate-buffered saline and then irradiated with red light at 630 run in the same buffer. Experiments indicated that H2O2 and oxygen radicals could mediate the tumoricidal action of HPD-PDT; we found that photosensitization of EAC cells with HPD leads to the formation of significant amounts of H2O2, superoxide (O2-.), and hydroxyl (OH.) radicals, which along with 1O2 were involved in photoinactivation of the cells in vitro. Our data showed that in EAC cells subjected to HPD-PDT, the generation H2O2, O2-., and OH. could be largely mediated by: (i) an increase in the activity of xanthine oxidase (XOD), due most probably to the conversion of xanthine dehydrogenase (XDH) to XOD via a Ca2+-dependent proteolytic process as well as oxidation of SH groups in XDH; and (ii) photooxidation of some cellular constituents (proteins). Another interesting finding of our studies is that in tumor cells subjected to HPD-PDT the Fenton-like reactions could play an important role in the generation of OH., and that cell-bound Cu/Zn-superoxide dismutase as well as catalase can protect tumor cells against the phototoxic action of HPD. In addition, we clearly demonstrated the ability of photoexcited HPD to the generation of protein peroxides in tumor cells. Studies suggest that 1O2 is the main agent responsible for the generation of protein peroxides in EAC cells treated with HPD-PDT, although other ROS (H2O2, O2-., and OH.) were also implicated in this process. However, further work is needed to clarify the significance of these peroxides in the antitumor effect of PDT with HPD.

Influence of temperature on the efficiency of photodestruction of Ehrlich ascites carcinoma cells sensitized by hematoporphyrin derivative.

AIM: To elucidate the mechanism of the potentiating influence of heating associated with photoirradiation on the antitumor efficiency of photodynamic therapy (PDT) with hematoporphyrin derivative (HPD). METHODS: The study was carried out on Ehrlich ascites carcinoma (EAC) cells, which were loaded with HPD in a serum-free medium and then irradiated with red light (lambda max=630 nm) at various temperatures. Cytotoxicity was estimated by the trypan blue exclusion assay. RESULTS: Our data support the view that in PDT the hyperthermia (around 44 degrees C) produced by irradiation can enhance synergistically the HPD-photoinduced tumor eradication; it was found that raising the irradiation temperature from 30 to 44 degrees C caused a substantial (approximately 1.5 fold) increase in the rate of HPD-photosensitized inactivation of EAC cells, while hyperthermia (44 degrees C) itself showed little toxic effects towards the cells. CONCLUSION: Studies indicated that the potentiating effect of heating on the antitumor efficiency of HPD-PDT could be largely explained by the stimulation of reactive oxygen species formation such as H2O2, superoxide and hydroxyl radicals. It was also found that photosensitization of EAC cells by HPD caused a strong fall in the activity of catalase (CAT) and glutathione (GSH) peroxidase, and that heating sensitized the H2O2-detoxifying enzymes to HPD-photoinduced inactivation. Under HPD-PDT, these events could result in loss of protection against accumulating H2O2; we revealed that cell-bound CAT and the GSH redox cycle play an important role in the protection of EAC cells against HPD-PDT. Moreover, our findings suggest that during PDT with HPD, an increase in the temperature of tumors could enhance the efficiency of this therapy via the stimulation of a chlorin-type photoproduct formation.