Influence of heating on the activity of xanthine oxidase in tumor cells subjected to the phototoxic action of hematoporphyrin derivative.

The aim of this study was to clarify the mechanism of the stimulatory effect of heat stress on generation of superoxide radical (O2-*) in tumors subjected to photodynamic therapy (PDT) with hematoporphyrin derivative (HPD). For this purpose, the effect of heating on the activity of xanthine oxidase (XOD) in tumor cells upon their photosensitization with HPD was examined; this enzyme is participated in purine catabolism and has the ability to generate O2-*, a precursor of H2O2 and very cytotoxic hydroxyl radical. 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 3 different temperatures (30, 37 and 44 degrees C). In the cells, the activity of XOD was assayed fluorometrically, using pterine as the substrate, whereas the production of O2-* by the nitro blue tetrazolium method. It was found that increasing of the temperature from 30 to 44 degrees C strongly (by approximately 2.5-fold) enhanced the generation of O2-* in EAC cells that correlated well with an increase in the rate of their photosensitized killing. Experiments showed that the intensification of O2-* formation could be mediated by the stimulatory effects of heating on the activity of XOD; namely, the 12 min treatment of EAC cells by HPD-PDT at a control (30 degrees C) temperature caused an about 2-fold growth in the activity of XOD, whereas the same light exposure at 44 degrees C led already to a 2.7-fold increase in the activity of this enzyme. However, incubation of EAC cells in the dark even at a hyperthermic (44 degrees C) temperature had no effect on their XOD activity. Thus, our findings strongly suggest that upon PDT with HPD the mild hyperthermia (approximately 44 degrees C) produced by photoirradiation might enhance the PDT-induced oxidative stress and, as a result, its tumoricidal effect via a rise in the activity of XOD. Besides, the obtained results suggest that severe hyperthermia (> 45 degrees C) could induce, contrary to mild hyperthermia, a reduction in the efficiency of HPD-PDT; we found that in EAC cells the raising temperature of an environment from 30 to 44 degrees C induced more than 2-fold increase in the activity of XOD, whereas further heating from 44 to 60 degrees C led to inactivation of this enzyme.