ИСТИНА

Photodynamic therapy (PDT) of cancer, which mechanism is associated with the development of free-radical reactions, has been intensively used in recent years. However, the search for new means to increase the anti-tumor efficacy of PDT and reduce the genotoxic effect on normal cells of the body remains relevant [1]. In spite of the significant achievements of multidisciplinary research in the field of PDT, one of the drawbacks of the method is still the dependence of the efficacy of the procedure on the blood supply and the degree of oxygenation of the tumor. Considering that the key anti-tumor effects of ozone are increased oxygenation of the tumor tissue, inhibition of the proliferation of tumor cells, and increased immune responses [2], we believe that the combined application of ozone and PDT can enhance the destruction of the tumor tissue. The purpose of the study was to evaluate the combined effect of PDT and ozonized physiological solution (OPS) on the growth of kidney carcinoma in laboratory animals, the intensity of free radical oxidation of lipids, proteins and DNA, and the state of the antioxidant defense system of the tumor-bearing organism. Materials and Methods The experiments were performed with 40 white inbred male rats aged 2.5 months. A model of neoplasia was created by subcutaneous inoculation of the strain of the RA kidney carcinoma acquired in the Scientific Research Institute of Experimental Diagnosis and Therapy of Tumors of the N.N. Blokhin Russian Oncology Center of the Russian Academy of Medical Sciences (Moscow). The animals with tumor volumes of 0.07-0.22 cm3 were included into the experiments. The animals were divided into four groups: (1) intact healthy rats (n=10); (2) tumor-bearing rats (n=10); (3) tumor-bearing rats treated with PDT (n=10); (4) tumor-bearing rats treated with PDT and OPS (n=10). With PDT, a 0.3% solution of "Photosens" preparation (SSC "NIOPIK", Russia) was introduced intratumorally and the tumor region was exposed to a LED laser (λ = 660 ± 10 nm, intensity of 100 mW/cm2) with the help of a physiotherapeutic LED device APS (Polironik LLC, Russia) at 10 h after the injection of the photosensitizer. A total of two PDT sessions were performed on the 15th and 19th days after the tumor inoculation. An additional intraperitoneal injections of 0.5 ml of OPS were introduced into the tumor-bearing animals treated with PDT and OPS with ozone concentration in the ozone-oxygen mixture of 400 μg/l (10 days course) [3]. The anti-tumor effect was assessed on the 20th day of neoplasia growth by the absolute tumor growth rate, reflecting the change in the volume of the tumor focus in relation to the volume before applying therapy (K≥0 - continued tumor growth, -1≤К<0 - tumor regression) [4]. The state of free radical homeostasis was assessed using the method of induced chemiluminescence [5], the comet assay [6], the modified method of determining oxidative modification of proteins (OMP) by the level of carbonyl derivatives, the concentration of malonic acid was determined by thiobarbituric acid dialdehyde (MDA), superoxide dismutase (SOD) activity was determined by the reaction of reduction of nitro-blue tetrazolium, catalase activity was determined according to the rate of destruction of hydrogen peroxide in a neutral medium [7]. Plasma, erythrocytes, homogenates of the liver, spleen and tumor tissue were examined. The homogenization of the organs was carried out in liquid nitrogen. Statistical analysis was performed using the Mann-Whitney U-test. Results On the 20th day after transplantation, the tumor in the group of tumor-bearing rats without treatments was characterized by non-aggressive growth (K = 1.37). The statistically significant increase in the activity of SOD in erythrocytes and a tendency to an increase in the concentration of MDA in the blood plasma were also observed in tumor-bearing rats compared with healthy rats. An increase in SOD and catalase activity was recorded in liver tissue homogenates. On the 20th day of the growth of kidney carcinoma, the level of spontaneous DNA damage increased 2.5 times, and the rate of DNA repair in whole blood leukocytes decreased 3 times as compared with intact animals. After PDT procedures, an inhibition of tumor growth, a decrease in SOD activity in erythrocytes, a decrease in concentration of MDA in the blood plasma and a decrease in SOD and catalase activity in the liver were observed in most cases. However, in one animal, an increase in tumor growth was observed against the background of pronounced oxidative stress of the whole organism. The combined effect of PDT and intraperitoneal injections of OPS led to the tumor regression in all experimental rats (K = -1). The biochemical parameters of free radical processes and the antioxidant defense system of the body were restored to the level of intact animals: the activity of SOD in erythrocytes decreased statistically significantly, the hemoglobin content in erythrocytes increased. An increase in the intensity of chemiluminescence and the level of OMP in liver tissues was also observed. It is important that the ozone-photodynamic effect significantly reduced the spontaneous level of DNA damage in whole blood leukocytes of tumor-bearing animals, and the number of dead cells with deep DNA degradation (class C3/C4) was 13 times less than after PDT alone. Conclusions The experimental scheme of ozone-photodynamic therapy developed causes tumor regression and has a reduced risk of stimulating the tumor growth compared to mono-PDT. The protection effect of the combined action of PDT and OPS is manifested as a decrease in the level of DNA damage in normal cells. The work was supported by the Russian Foundation for Basic Research (project 19-02-00667).