CORONAVIRUS SARS-COV-2 MODIFIES ANTITUMOR REDOX STATUS OF BLOOD AND INTERCELLULAR MATRIX IN METASTATIC COLORECTAL CANCER PATIENTS (A PILOT STUDY).

BACKGROUND: The current studies demonstrate that SARS-CoV-2 infection results in increasing complications incidence and the total risk of death in cancer patients. SARS-CoV-2 infection triggers oxidative stress representing one of the major factors of the inflammation contributing to the complicated course of the diseases including cancer. AIM: To assess the effect of hypoxia caused by SARS-CoV-2 infection on the redox status of blood in patients with metastatic colorectal cancer (mCRC). MATERIALS AND METHODS: 10 patients with SARS-CoV-2, 11 mCRC patients with metachronous liver disease, and 14 mCRC patients with preceding SARS-CoV-2 infection were included in the study. The data on blood biochemistry (C-reactive protein, ferritin, transferrin, and free iron) were analyzed. The levels of superoxide radicals (ROS) in blood cells were determined by electron paramagnetic resonance (EPR) using the spin trap technique. The metalloproteinase activity was measured by polyacrylamide gel zymography with the addition of gelatin as a substrate. RESULTS: In mCRC patients with prior SARS-CoV-2 infection, a 1.26-fold increase in ROS-generating activity of blood neutrophils was observed compared to mCRC patients with no history of SARS-CoV-2 infection. The blood content of C-reactive protein, transferrin, and free iron in mCRC patients with prior SARS-CoV-2 infection increased by 2, 6, and 1.4 times, respectively. The total activity of gelatinases in platelets and neutrophils in the blood of mCRC patients with prior SARS-CoV-2 infection was 1.4 and 1.2 times higher compared to mCRC patients with no history of SARS-CoV-2 infection. CONCLUSION: mCRC patients with prior COVID-19 have a higher risk of exacerbation of inflammatory reactions. SARS-CoV-2 infection results in redox dіsbalance, which may contribute to the unfavorable course of the disease.

THE CHANGES OF NO LEVEL AND RNase ACTIVITY IN TUMOR TISSUE ACCOMPANYING THE PROGRESSION OF PROSTATE CANCER.

AIM: To assess the inducible NO-synthase activity and the total RNase activity in tissue samples and blood neutrophils of the patients with prostate intraepithelial neoplasia (PIN) and prostate cancer (PCa) of different stages. MATERIALS AND METHODS: NO level was measured in tumor tissue and neutrophils of patients with PIN and PCa of different stages by electron paramagnetic resonance using the spin traps technology. RNase activity in tumor tissue of patients with PIN and PCa was measured by the method of zymography. RESULTS: We have found that NO levels in prostate tumor tissue were significantly higher than in the PIN and increased along with the disease progression. Analysis of NO level in neutrophils of the PCa patients demonstrated that the values were not dispersed and did not depend on the stage of disease. NO level in neutrophils of the PCa patients increased manifold as compared with that in healthy donors. At the same time, the RNase activity in the prostate tumor tissue gradually decreased with PCa progression. CONCLUSION: Activities of inducible NO-synthase and RNases change significantly with progression of PCa.

Redox dependent features of tumors, adipose tissue, neutrophiles and platelets in patients with metastatic colorectal cancer.

AIM: To study the levels of redox-forming molecules generated in tumor, adipose tissue (AT), neutrophils and platelets of patients with colorectal cancer (CRC) with normal body weight or obesity. MATERIALS AND METHODS: 83 samples of tumor tissue and 83 samples of the AT taken at the distance of 5 cm from the tumor as well as 83 blood samples have been analyzed in the patients with CRC with metachronous lesions in liver. The comparison group consisted of 18 patients who were surgically treated for ulcer. The rates of free radical generation were measured by electron paramagnetic resonance. RESULTS: Free radical generation rate in tumor tissue and AT increased and nitrogen oxide (NO) levels decreased in patients with body mass index ≥ 25 kg/m2 as compared to the patients with normal body weight (p < 0.05). The superoxide generation activity in neutrophils of CRC patients exceeded about 7-fold the values obtained in neutrophils from patients with ulcer while the NO levels produced by neutrophils of CRC patients decreased significantly (p < 0.01). The same trends held true for the platelets. CONCLUSIONS: In obese patients with CRC, superoxide-generating activity increases in cancer cells, AT, neutrophils and platelets. The obesity should be considered as the additional oncological risk representing the potent predictor for the events realized in obese patients such as oxidative stress, inflammation, insulin resistance and endothelial dysfunction.

Drag reducing polymers attenuate adverse effects of ischemia-reperfusion upon resection of liver metastases modeled by MC38 mouse colon adenocarcinomama.

BACKGROUND: The resection of metastases within healthy parenchyma improves significantly the long-term outcome in metastatic colorectal cancer. Until now, the resection technique involves Pringle maneuver, which allows reducing blood loss during transsection of liver parenchyma. However, the classical Pringle maneuver has restrictions due to ischemia/reperfusion (I/R) effect, in particular increasing risk of tumor recurrence after liver surgery. AIM: To study the pathological impact of surgical intervention and I/R effect on healthy liver tissue in the experimental setting by evaluating the markers of redox-homeostasis and oxidatively induced mutage-nesis, and also to assess the current possibilities of their correction by application of drag-reducing polymers (DRPs). MATERIALS AND METHODS: MC38 mouse colon adenocarcinoma cells were transplanted intrahepatically to C57Bl/6 mice. The influence of warm ischemia on metastatic potential of MC38 cells, the speed of superoxide radicals (SR) generation and 8-hydroxydeoxyguanosine content were studied. RESULTS: In case of modeled liver metastases, the surgery initiates an increase in the rate of SR generation into the remaining liver tissue and, consequently, provokes its ischemic injury. The application of DRPs protects liver tissue under I/R conditions. CONCLUSIONS: The warm I/R can promotes metastatic lesions in the healthy part of the organ with underlying increase in the rate of SR generation and oxidatively induced damage of guanine in DNA. The hemorheological effects of DRPs ensure increase of microcirculatory perfusion and oxygenation of liver tissues with the reduction of the rate of SR generation and decrease of 8-hydroxydeoxyguanosine as a marker of oxidatively induced mutations in DNA of hepatocytes. The intraperitoneal administration of nanomolar doses of DRPs prevents the activation of the growth of dormant metastatic MC38 cells in the liver. Further experimental and clinical study of these substances will allow reducing the risks of activation of uncontrolled tumor growth in the liver due to the pathological effect of post-operative I/R.

Redox state of adipose tissue for patients with gastric cancer and its connection with the body mass index and distance from the tumor.

Excess body weight has been causally linked to an increased risk of different cancer types, including gastric cancer but the mechanisms underlying this relationship are not well understood. Superoxide generation rate, activity of complex I in electron transport chain of mitochondria, activity of matrix metalloproteinase (MMP-2 and 9) of adipose tissues (AT) of patients with gastric cancer in AT located adjacent to tumor (ATAT) and at a distance of 3 cm (ATD) are measured to follow the connection of the redox state with some of the microenvironment indicators (HIF-1α, CD68, Plin5), body mass index (BMI) and cancer metastasis. Superoxide generation rate in ATAT positively correlates with BMI (r = 0.59, p < 0.05) being 4 times higher than in control (p < 0.05). MMP-2, 9 activities in ATAT positively correlate with BMI (r = 0.67, p < 0.05) being 3.3-4.0 higher than in control (p < 0.05). In ATD a statistically significant increase of MMP-2 activity is found. In ATAT for the group of patients with distant metastasis (M1) the superoxide generation rate, MMP-2, 9 activities are about 2 times higher (p < 0.05) than in the subgroup without distant metastases (M0). M1 is also characterized by the increased values of HIF-1α+ (factor 1.25), CD68+ (factor 1.4) and Plin5+ (factor 2.1) compared to M0 category in tumor tissues (p < 0.05). The results can be used for better understanding the mechanism(s) of symbiosis of tumor and adipose tissues as well as serve as a basis for new therapeutic approaches.

Molecular mechanisms of oxidation damage and liver cell dysfunction in patients with metastatic colorectal cancer.

BACKGROUND: Interaction between tumor cells and tumor microenvironment is critical for homeostasis of normal cells and tumor growth. Tumor cell - stroma interaction represents the potent factor able to initiate cancer and affect tumor progression and disease outcome. The tumors vary by their origin and microenvironment (proportion of stromal cells, their composition and activation state). The surgical stress and tumor microenvironment may potentiate acute hepatic failure in the patients with metastatic colorectal cancer (mCRC). Pathological effect of ischemia-reperfusion (I/R) consists in the increased generation of superoxide radicals (SR) and nitrogen oxide (NO) affecting the postresectional regeneration of liver tissue. Redox state of hepatic tissue in I/R setting upon resection of metastases may trigger the aggressiveness of residual cancer cells and regeneration or degradation of hepatic tissue. The aim of the study was to analyze redox state of hepatic tissue following surgery with Pringle maneuver (PM) in the patients with mCRC. MATERIALS AND METHODS: mCRC samples from 145 patients treated at National Cancer Institute, Ministry of Health of Ukraine, were analyzed. The patients obtained chemotherapy according to the approved international and national standards as well as clinical protocols. Two groups of patients were delineated according to the duration of the interruption of blood inflow due to PM, namely ≤ 45 min and > 45 min. The activity of FeS proteins in the electron transport chain (ETC) in mitochondria and lactoferrin (LF) level in the tissues were assessed by EPR (77К). The rates of SR and NO generation were determined with spin traps. The activity of matrix metalloproteinase (MMP)-2 and -9 was measured by gelatin zymography using SDS-polyacrylamide gel electrophoresis. RESULTS: In tissue of liver resected in the setting of > 45 min ischemia, ETC function in mitochondria was impaired (decreased activity of FeS protein of N-2 ETC complex I due to interaction with NO). This results in the hypoxia state and glycolysis with uncontrolled SR generation. In addition, the efficiency of detoxification system in hepatocytes is reduced substantially with increase in semiquinone and LF levels as well as MMP-2 and -9 activity as compared with liver without metastatic lesions that was not affected by I/R. CONCLUSIONS: The ischemic injury of liver in the setting of metastasis resection results from cell response to interruption of blood flow followed by reperfusion. The key factor in the genesis of reperfusion damage is uncontrolled increase of the levels of SR and their metabolites - reactive oxygen species as well as the increased MMP activity. Also, liver tissue affected by I/R contains high levels of xanthine oxidase metabolizing hypoxanthine and monoamine oxidase deaminizing biogenic amines. Both processes are the sources of SR.

Tumor-associated redox state in metastatic colorectal cancer.

The high incidence of recurrence and metastasizing in colorectal cancer (CRC) poses the challenge for the improvement in long-term treatment outcome. AIM: To determine the major indicators of redox-formative molecules in the tissue of metastatic CRC (mCRC), stages Т2-4N0-2M0G2-3, namely the rate of superoxide radical (SR) generation, nitric oxide (NO) content, the activity of matrix metalloproteinases (MMP), lactoferrin (LF) content, and "free" iron and their association with some clinical and pathological characteristics of the patients. MATERIALS AND METHODS: mCRC samples from 51 patients were analyzed (stage II, 31 patients; stage III, 20 patients). The LF and "free" iron were assessed by electron paramagnetic resonance (EPR) at the temperature of 77 °K. The rate of SR and NO generation was determined with spin traps (ТЕМРО-Н, diethyl dithiocarbamate). The activity of MMP-2 and -9 was measured by gelatin zymography using SDS-polyacrylamide gel electrophoresis. Ki-67 expression was analyzed by immunofluorescence technique. RESULTS: In tumors with metastases into the regional lymph nodes (N1-2 category), SR generation rate was 2.2-fold higher than in the tumors categorized as N0. In G3 mCRC, SR generation rate was 1.7-fold higher than in G2-tumors (p < 0.05). The rate of SR generation correlated inversely with differentiation grade of the tumor (r=-0.61; p < 0.05). MMP-2 and -9 activities in mCRC tissue correlated with SR generation rate and NO level (r = 0.44 ÷ 0.53, p < 0.05). The direct correlation between LF content and the stage of the disease (r = 0.42) and "free" iron content (r = 0.61) was demonstrated while the correlation between LF content and tumor differentiation grade was inverse (r = -0.57; p < 0.05). CONCLUSIONS: The altered tumor-associated redox state in mCRC tissue contributes to the increased cell proliferation and formation of aggressive phenotype of the tumor. The assays for the content of redox-formative components in mCRC may be used as additional prognostic markers of the course of the disease in CRC patients.

DNA oxidation in patients with metastastic colorectal cancer: clinical significance of 8-hydroxy-deoxyguanosine as an independent prognostic factor.

Prognosis of metastatic colorectal cancer (mCRC) patients nowadays is an important subject in the field of oncology. R0-resection of colon with primary tumor and liver metastasis remains the only treatment which significantly improves survival rate. However, recent experimental data show that surgical trauma can indirectly stimulate tumor growth due to mitochondrial dysfunction and unregulated superoxide radical (O2-) generation. AIM: To study the clinical significance of 8-oxo-2'-deoxyguanosine (8-oxodGu) marker, to assess the oncological effects of warm ischemia of liver parenchyma on disease prognosis in patients with mCRC. MATERIAL AND METHODS: 69 urine 24-hour volume tests of patients with mCRC and 17 healthy individuals were studied. Urine 8-oxodGu level was measured using spectrophotometric method with pre-solid phase DNA extraction. The energy system and hepatocyte detoxification system state, levels of O2- in tumor tissue were determined using the method of electron paramagnetic resonance (EPR) and SpinTraps technology at room temperature. Experiments were carried out on a computerized EPR spectrometer RE-1307. EPR spectra were recorded at temperature of liquid nitrogen (196C) in paramagnetically pure quartz dewar on a computerized spectrometer PE-1307 with resonator H011. Error of the method of spectrum integration and spread of spectrum reproduction of one sample was not more than 3%. RESULTS: The average level of marker in healthy individuals was 0.244 day, whereas before the resection and on day 3 after the R0-resection of liver in mCRC patients was 3.42 day and 2.12 day (p < 0.05), respectively. On day 3 after the liver resection due to its metastatic lesions with a total duration of warm ischemia period < 30 min and > 30 min have had marker at level 2.108 day and 2.9883 day (p < 0.0001), respectively. The volume of metastatic tissue significantly and directly correlated with the level of urine 8-oxodGu (R2=0.54, 95% CI 0.0370.0991, p < 0.0001), also duration of surgical intervention (300 min) and duration of worm liver ischemia ( 30 min) during the surgery significantly increased urine level of 8-oxodGu (R2=0.54, 95% CI 0.001 0.004, p < 0.001). CONCLUSIONS: Warm liver ischemia (> 30 min), long-term surgical intervention ( 300 min) and metastatic tissue volume ( 12 cm3) in liver parenchyma in mCRC patients significantly increase urine 8-oxodGu levels. R0-resection of liver metastases in mCRC patients decreases urine 8-oxodGu levels already on day 3 after the surgery. 8-oxodGu is a new factor of oncological prognosis in patients with mCRC.

The influence of lactoferrin on elemental homeostasis and activity of metal-containing enzymes in rats with Walker-256 carcinosarcoma.

AIM: To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes - ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma (BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF). MATERIALS AND METHODS: The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256 carcinosarcoma treated with LF at the doses of 1 and 10 mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO - by unified biochemical method. RESULTS: The introduction of LF at the doses of 1 and 10 mg/kg resulted in a decrease in the ratio of Cu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10 mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10 mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content. CONCLUSIONS: The administration of LF, especially at a dose of 10 mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

Colorectal Cancer and Mitochondrial Dysfunctions of the Adjunct Adipose Tissues: A Case Study.

Excess body weight has been causally linked to an increased risk of different cancer types, including colorectal cancer (CRC) but the mechanisms underlying this association are practically unknown. We investigate redox state-superoxide (SO) generation rate, activity of complex I in electron transport chain (ETC) of mitochondria and of dinitrosyl iron complexes by electron paramagnetic resonance; activity of matrix metalloproteinase (gelatinase) MMP-2 and MMP-9 by gel zymography of adipose tissues (AT) from 46 patients (64.0 ± 1.6 y.o.) with CRC (II-III stages, pT2-3N0-2M0) in the AT adjacent to tumor (ATAT) and at a distance of 3 cm from the tumor (ATD) to follow the connection of the AT redox state with some of the tumor microenvironment indicators. We have incubated the AT species with the tumor necrosis factor α (TNF-α) to follow its influence on the measured values. As a control, normal AT (NAT) obtained during the liposuction is used. Tumor-induced changes in mitochondrial ETC of ATAT, particularly for Complex I, lead to the enhanced SO generation and consequent oxidative modifications of DNA in ATAT (up to 6.1 times higher than that in NAT and 3.7 times higher than that in ATD, p < 0.05). Gelatinase activity in ATAT is significantly higher than in ATD. A considerable effect of TNF-α on ATAT and ATD (but not on NAT, i.e., only on the tissues where the reprogramming of metabolism has already occurred under the influence of tumor) manifested in increase of cellular hypoxia, gelatinase activity, and SO generation rate is observed. The results can be used for better understanding the mechanism(s) of metabolic symbiosis of tumor and AT as well as serving as a basis for new therapeutic approaches.

Influence of ferromagnetic nanocomposite (Ferroplat) on human breast cancer cells of different malignancy degrees: pro/antioxidant balance and energy metabolism.

AIM: To study the effect of Ferroplat (FrP) on the indexes of pro/antioxidant balance and energy metabolism in breast cancer cells of different malignancy degree and different sensitivity to drug therapy. MATERIALS AND METHODS: The study was carried out on breast cancer cells of low (T47D, MCF-7) and high malignancy degree (MCF-7/DDP (cisplatin-resistant), MDA-MB-231, MDA-MB-468) using cell culture techniques, immunocytochemical, biochemical, biophysical methods, flow cytometry and polarography. RESULTS: We established that the addition of FrP to the culture medium reduces the activity of glucose-6-phosphate dehydrogenase (G6PDH), superoxide dismutase (SOD) and the level of non-protein thiols by 32-41% (p < 0.05). At the same time, there was an increase of the total level of ROS and the rate of NO generation by inducible NO synthase by 1.7-2.5 times (p < 0.05). This testifies that FrP disturbs the antioxidant balance in cells, resulting in their death. Also, the use of FrP led to a decrease in the rate of oxygen absorption in MCF-7 and T47D cells by 26% and 25%, respectively (p < 0.05). In cells of high malignancy degree this index decreased by 38-40% under the influence of FrP. Incubation of MCF-7 and T47D cells with the indicated agent also reduced the content of phospholipid cardiolipin by 15-16% (p < 0.05), and in MDA-MB-231, MCF-7/DDP, MDA-MB-468 cells - by 29%, 30% and 32%, respectively. In addition, the effect of FrP caused a decrease in the levels of Mg2+ and lactate in MCF-7 and T47D cells by 21-29% and 14-24%, respectively, whereas in MDA-MB-231, MDA-MB-468, MCF-7/DDP cells - by 34-38% and 32-35%, respectively. In this case, the agent raised the level of glucose in the cells of low malignancy degree by 20-23% (p < 0.05), and in the cells of high malignancy degree and with the phenotype of drug resistance - by 31-36%. However, the nanocomposite did not affect the activity of lactate dehydrogenase in all studied breast cancer cells. CONCLUSION: The study has shown that FrP has an effect on the pro/antioxidant balance and energy metabolism of cancer cells. In addition, the denoted effect of FrP was more pronounced in the breast cancer cells with a high malignancy degree and the phenotype of drug resistance.

Rectal Cancer: Redox State of Venous Blood and Tissues of Blood Vessels from Electron Paramagnetic Resonance and Its Correlation with the Five-Year Survival.

A role of pro- and antioxidants for reducing rectal cancer (RC) incidence in operative, preoperative, and postoperative treatments is still disputable and controversial. The redox state of venous blood and tissues of blood vessels of 60 patients with RC (T2-4N0-2M0G2) and 20 donors is studied by means of the conventional and spin-trapping electron paramagnetic resonance (EPR). The intensity of the signals from ceruloplasmin (CP), transferrin (TF), and labile iron pool (LIP) at temperature T = 77 K as well as superoxide generation rate and nitric oxide (NO) levels at T = 300 K is measured. The reduced CP and TF activity and decreased NO levels increased LIP levels and superoxide-generating rates are detected in blood species. Correlation analysis for the five-year survival rate as a function of the extracted values is done. The results show that the intensities of the corresponding EPR signals from the "native" and "trapped" paramagnetic centers can be potentially used for the understanding of the molecular mechanisms underlying the RC progression and treatment.

Influence of exogenous lactoferrin on the oxidant/antioxidant balance and molecular profile of hormone receptor-positive and -negative human breast cancer cells in vitro.

AIM: To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. MATERIALS AND METHODS: The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. RESULTS: Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of "free" iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231 and MDA-MB-468 cells. These changes resulted in increased expression of proapoptotic Bax protein (p < 0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4-1.7-fold, p < 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14-24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G2/M phase, especially in receptor-negative cell lines. CONCLUSION: The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, "free" iron and NO generation rate, resalting in the blocking of cell cycle at G2/M-phase and apoptosis of malignant cells.

Superoxide- and NO-dependent mechanisms of antitumor and antimetastatic effect of L-arginine hydrochloride and coenzyme Q10.

AIM: To study the redox-dependent mechanism of antiradical, antitumor and antimetastatic action of L-arginine hydrochloride (L-Arg) and coenzyme Q10 (CoQ10) in vivo. MATERIALS AND METHODS: The study was performed on С(57)Вl mice with transplanted Lewis lung carcinoma treated by intraperitoneal injections of L-Arg at low or high doses (60 and 360 mg/kg body weight), CoQ10 (0.2 and 1.2 mg/kg body weight) or their combinations. Electron paramagnetic resonance was applied for analysis of mitochondrial electron transport chain, СoQ10 levels, free iron (FI), the level of NO, and the rate of superoxide radical generation. The activity of matrix metalloproteinase (MMP)-2 and -9 in tumor tissue was determined by zymography method in polyacrylamide gel. RESULTS: Administration of L-Arg at high doses caused an inhibition of tumor growth by 48 ± 8.0%, increase of superoxide radical generation rate and NO levels to a value of 1.23 ± 0.14 and 2.26 ± 0.31 nm/g tissue · min, and decreased activity of MMP-2 and -9 (3.55 ± 0.8 and 4.8 ± 1.0 r.u., respectively). Treatment with L-Arg at low doses stimulated tumor growth and increased the levels of MMP-2 and -9 activities (8.44 ± 2.7 and 9.8 ± 3.1 r.u., respectively). Administration of СoQ10 at high doses significantly decreased superoxide radical generation rate to the values of 0.44 ± 0.09 nm/g tissue · min, levels of free iron and NO, and caused tumor growth inhibition by 54 ± 11.3%. The combined use of L-Arg and СoQ10 at high doses caused tumor growth inhibition by 51 ± 7.4% compared to Lewis lung carcinoma-bearing untreated animals (p<0.05). CONCLUSIONS: Administration of L-Arg and СoQ10 caused the dose-dependent effect on the rate of generation of superoxide radicals, level of ubisemyquinone, complexes NOFeS-proteins, levels of FI and NO. L-Arg at low doses positively modulated MMP-9 activity that promoted tumor progression. Upon combined use of L-Arg and СoQ10, superoxide radicals and NO form the redox state that causes decrease of MMP-2, -9 activities with consequent inhibition of tumor invasion and metastasis.

Disordered redox metabolism of brain cells in rats exposed to low doses of ionizing radiation or UHF electromagnetic radiation.

AIM: To investigate the changes of redox-state of mammalian brain cells as the critical factor of initiation and formation of radiation damage of biological structures in setting of continuous exposure to low doses of ionizing radiation or fractionated ultra high frequency electromagnetic radiation (UHF EMR) at non-thermal levels. MATERIALS AND METHODS: The influence of low-intensity ionizing radiation was studied on outbred female rats kept for 1.5 years in the Chernobyl accident zone. The effects of total EMR in the UHF band of non-thermal spectrum were investigated on Wistar rats. The rate of formation of superoxide radicals and the rate of NO synthesis in mitochondria were determined by the EPR. RESULTS: After exposure to ionizing or UHF radiation, the levels of ubisemiquinone in brain tissue of rats decreased by 3 and 1.8 times, respectively. The content of NO-FeS-protein complexes in both groups increased significantly (р < 0.05). In the conditions of ionizing or EMR the rates of superoxide radical generation in electron-transport chain of brain cell mitochondria increased by 1.5- and 2-fold, respectively (р < 0.05). In brain tissue of rats kept in the Chernobyl zone, significant increase of NO content was registered; similar effect was observed in rats treated with UHFR (р < 0.05). CONCLUSIONS: The detected changes in the electron transport chain of mitochondria of brain cells upon low-intensity irradiation or UHF EMR cause the metabolic reprogramming of cell mitochondria that increases the rate of superoxide radical generation and nitric oxide, which may initiate the development of neurodegenerative diseases and cancer. This article is part of a Special Issue entitled "The Chornobyl Nuclear Accident: Thirty Years After".

Modifying effects of 5-azacytidine on metal-containing proteins profile in Guerin carcinoma with different sensitivity to cytostatics.

AIM: To assess the influence of the treatment with 5-azacytidine (5-aza) on the profile of metal-containing proteins and factors of their regulation in Guerin carcinoma cells in vivo. MATERIALS AND METHODS: The study was conducted on Wistar rats transplanted with wild-type Guerin carcinoma (Guerin/WT) and its strains resistant to cisplatin (Guerin/CP) or doxorubicin (Guerin/Dox). Animals were distributed in 6 groups treated with 5-aza and control animals without treatment. 5-Aza was injected by i.v. route (1 injection in 4 days at a dose of 2 mg/kg starting from the 4th day after tumor transplantation, 4 injections in total). Ferritin levels in blood serum and tumor tissue were measured by ELISA, transferrin and free iron complexes - by low-temperature EPR, miRNA-200b, -133a and -320a levels and promoter methylation - by real-time quantitative reverse transcription polymerase chain reaction. RESULTS: The study has shown that 5-aza treatment caused demethylation of promoter regions of fth1 and tfr1 genes in all studied Guerin carcinoma strains. 5-Aza treatment resulted in a significant decrease of ferritin levels in tumor tissue (by 32.1% in Guerin/WT strain, by 29.8% in Guerin/Dox and by 69.1% in Guerin/CP). These events were accompanied by 3.5-fold and 2-fold increase of free iron complexes levels in tumor tissue of doxorubicin and cisplatin resistant strains, respectively. Also, 5-aza treatment resulted in significantly elevated levels of miR-200b, -133a, 320a expression in tumor tissue. After 5-aza treatment, ferritin levels in blood serum of animals with Guerin/Dox were increased by 23.9%, while in Guerin/Wt and Guerin/CP they were decreased by 17 and 16%, respectively. CONCLUSION: Alterations of epigenetic regulation upon in vivo treatment with 5-aza change the levels of metal-containing proteins due to DNA demethylation and altered miRNA expression profiles in Guerin carcinoma cells.

Remodulating effect of doxorubicin on the state of iron-containing proteins, and redox characteristics of tumor with allowance for its sensitivity to cytostatic agents.

The study was aimed at determining the changes of metal-containing proteins in blood serum and tumor tissue of animals with parental and doxorubicin-resistant strains of Walker-256 carcinosarcoma before and after the cytostatic administration. It has been shown that upon doxorubicin action the levels of total iron and transferrin in the tissues from the both groups of animals decreased while that of ferritine simultaneously increased with more pronounced pattern in the group of animals with resistant tumor strain. It has been shown that upon the action of doxorubicin in tumor tissue of animals with different sensitivity to the cytostatic there could be observed oppositely directed changes in the redox state of these cells that in turn determined the content of " free iron" complexes, RO S generation and concentration of active forms of matrix metaloproteinase- 2 and matrix metaloproteinase-9, namely, the increase of these indexes in animals with parental strain and their decrease in animals with the resistant one. So, our study has demonstrated the remodulating effect of doxorubicin on the state of metal-containing proteins and redox characteristics of tumor dependent on its sensitivity to cytostatic, at the levels of the tumor and an organism. These data may serve as a criterion for the development of programs for the correction of malfunction of iron metabolism aimed at elevating tumor sensitivity to cytostatic agents.

METALLOPROTEINS DURING DEVELOPMENT OF WALKER-256 CARCINOSARCOMA RESISTANT PHENOTYPE.

The study was focused on the detection of changes in serum and tumor metal-containing proteins in animals during development ofdoxorubicin-resistant phenotype in malignant cells after 12 courses of chemotherapy. We found that on every stage of resistance development there was a significant increase in content of ferritin and transferrin proteins (which take part in iron traffick and storage) in Walker-256 carc'inosarcoma tissue. We observed decreased serumferritin levels at the beginning stage of the resistance development and significant elevation of this protein levels in the cases withfully developed resistance phenotype. Transferrin content showed changes opposite to that offerritin. During the development of resistance phenotype the tumor tissue also exhibited increased 'free iron' concentration that putatively correlate with elevation of ROS generation and levels of MMP-2 and MMP-9 active forms. The tumor non-protein thiol content increases gradually as well. The serum of animals with early stages of resistance phenotype development showed high ceruloplasmin activity and its significant reduction after loss of tumor sensitivity to doxorubicin. Therefore, the development of resistance phenotype in Walker-256 carcinosarcoma is accompanied by both the deregulation of metal-containing proteins in serum and tumor tissue and by the changes in activity of antioxidant defense system. Thus, the results of this study allow us to determine the spectrum of metal-containing proteins that are involved in the development of resistant tumor phenotype and that may be targeted for methods for doxorubicin sensitivity correction therapy.

Pharmacological effect of aminoferrocene in mice with L1210 leukemia.

AIM: To study the cytostatic and some biological effects of aminoferrocene using mice with L1210 lymphoid leukemia. MATERIALS AND METHODS: Experiments were performed on BDF1 male mice (DBA/2, female × C57Bl/6, male) with transplantable L1210 lymphoid leukemia. Determination of antitumor activity of Benzyl-Fc Boron (Bn), it was injected intraperitoneally 6 times daily, starting on day 2 after L1210 leukemia cell transplantation. Doses of Bn such as 26; 260 and 2600 µg/kg were used. The determination of intracellular content of cardiolipin, thiols, reactive oxygen species (ROS) and also analysis of Annexin V positivity and mitochondrial transmembrane potential (JC-1 staining) were performed with use of flow cytometry. The levels of "free iron" complexes, transferrin active forms and the rate of NO generation were measured by EPR-specroscopy. RESULTS: Six daily injections of Bn at a dose of 26 µg/kg resulted in an increased survival of mice with L1210 leukemia by 28% (p < 0.05). Bn led to an increase of apoptotic cells number and ROS amount in leukemia cells. Besides, Bn caused a decrease of cardiolipin and nonprotein thiol compounds content. The membrane electrochemical potential of cell mitochondria was decreased also after Bn administration. Studies using EPR-spectroscopy revealed a significant increase in a level of "free iron", content of transferrin active species and generation rate of NO by inducible NO-synthase in L1210 cells after aminoferrocene administration. CONCLUSION: Our data indicate that Benzyl-Fc Boron can be promising candidate for realizing a new strategy of anticancer therapy with the use of ROS-inducing agents.

Redox-regulation of gelatinases during growth of cisplatin-sensitive and resistant Guerin carcinoma.

UNLABELLED: Study was aimed to analyze the dynamics of changes and study interrelations between content of ferritin, transferrin, active gelatinases (MMP-2 and -9) in blood serum and tumor tissue, free iron, rate of superoxide radicals generation in tumor, activity of NADPH-oxidase and iNOS in neutrophils rats with sensitive and resistant strains of Guerin carcinoma (GC). MATERIALS AND METHODS: In order to obtain resistant tumor, 12 courses of cisplatin chemotherapy have been carried out on rats bearing GC. Levels of transferrin and free iron were determined by analysis of EPR spectra from computerized radiospectrometer EPR -RE-1307 at temperature of liquid nitrogen. Rate of superoxide radicals and nitric oxide generation in tumor and neutrophils of blood was determined by EPR using spin traps at room temperature. Content of ferritin in tumor homogenate and blood serum of rats with GC was determined by ELISA method using corresponding kits. Concentration of active forms of MMP-2 and -9 in obtained samples was determined using method of zymography. RESULTS: Unregulated generation of superoxide radicals and NO by mitochondria of tumor cells and NADPH-oxidase and iNOS neutrophils via oxidation of iron-containing proteins causes the accumulation of "free iron" complexes in blood and tumor tissue of rats able to evoke oxide-induced damages of macromolecules. It has been shown that for resistant strain of carcinoma, as compared with sensitive one, significantly higher concentrations of active forms of MMP-2 and -9 in blood serum of rats are typical. Dynamics of gelatinases activity changes in tumor tissue corresponds in general with dynamics of changes in serum. In tumor tissue of rats the indices of gelatinases activity positively correlate with rate of superoxide radicals generation, content of "free iron" complexes, ferritin and activity of transferrin. Cytostatic agent increased levels of reactive oxygen species (ROS) and self-amplify rate of superoxide radicals generation. In turn, activation of MMPs via superoxide-depending regulation allows tumor cells to facilitate migration, invasion and finally - formation of metastatic centers. Mentioned above tumor "oxide phenotype" determines high level of its aggressiveness and forms corresponding level of drug resistance. CONCLUSIONS: Thus, high levels of superoxide radicals oxidize transport proteins and form free iron pool. Iron ions, via Haber - Weiss mechanism, initiate generation of the hydroxyl radicals, which also enhance oxidation processes.