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.

Enhancement of the efficiency of photodynamic therapy of tumours by t-butyl-4-hydroxyanisole.

The effects of photodynamic therapy (PDT) alone and in combination with 3(2)-t-butyl-4-hydroxyanisole (BHA) on Ehrlich ascites carcinoma (EAC) cells have been investigated. BHA, a widely used food antioxidant, administered to the cells prior to light exposure is found to cause concentration-dependent alterations of the haematoporphyrin derivative (HpD)-based PDT. BHA (0.15 mM) causes a small (about 10%) inhibition in the rate of HpD-photosensitized injury of EAC cells. In contrast, upon increasing the concentration of BHA from 0.15 to 0.5 mM, a 1.3-fold enhancement in HpD-PDT efficiency is achieved. The cytotoxic effect on the cells treated with HpD-PDT and a higher concentration of BHA (0.5 mM) is additive. When BHA (0.5 mM) is given immediately after HpD-PDT, the combination is found to be three to four times more effective than when BHA is added to EAC cells before phototherapy. In this treatment regimen BHA acts synergistically with HpD-PDT. Such a difference in the action of BHA on the efficiency of HpD-PDT might be explained by the ability of BHA to inhibit the HpD-photosensitized destruction of some biomolecules. An enhancing action of BHA on the intensity of HpD-photosensitized death of tumour cells is also observed in vivo. Even a single dose of BHA (0.6 mM kg-1, 15 min after irradiation) causes (in an additive manner) an approximately two-fold increase in the efficiency of HpD-PDT of mice bearing Ehrlich ascites tumour (intraperitoneal transplantation). The results obtained indicate that the potentiating effect of BHA on the HpD-PDT could be caused by the impairment of the mitochondrial respiration, since there is a good correspondence between the concentration of BHA that increases the efficiency of PDT and the concentration that inhibits the oxygen consumption and dehydrogenase activity of EAC cells. The influence of BHA on the efficiency of PDT does not depend on the nature of the photosensitizer used; the effects with chlorin-e6 trimethyl ester are similar to that seen for HpD.

Study of the photochemical and phototoxic properties of lonidamine [1-(2,4-dichlorobenzyl)-1H-indazol-3-carboxylic acid].

Lonidamine (LND) is an antispermatogenic and antitumour agent acting via inhibition of the energy metabolism. According to our results LND in vitro acted as a photosensitizer enhancing synergistically the lethal action of UV radiation (lambda max = 330 nm, the range between 260-390 nm) towards Ehrlich carcinoma cells (EAC). The primary targets of phototoxic action of LND probably were cell membranes and mitochondria. UV irradiation of EAC in the presence of LND increased the permeability of the plasma membranes, stimulated the photoperoxidation of lipids, enhanced the inhibition of dehydrogenase activity and oxygen consumption of the cells. Deficiency of oxygen substantially decreased phototoxicity of LND. LND may induce photosensitized destruction of biomolecules by acting through type 1 and 2 reactions. It could be supposed that negative side effects of LND (e.g., photophobia and photosensitivity that have been reported for some cancer patients treated with LND) could be associated with its photosensitizing properties.