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.

Cytochrome P450 content in primary tumors and liver metastases of patients with metastatic colorectal cancer.

AIM: To determine the content of low-spin form of cytochrome P450 in primary tumors and liver metastases of the patients with metastatic colorectal cancer (mCRC) and and assess its prognostic significance. MATERIALS AND METHODS: The levels of the oxidized and low-spin forms of cytochrome P450 in the tissues of patients with mCRC were studied by electron paramagnetic resonance. To detect CYP 1A2 and CYP 1B1 isoforms, Western blot analysis was used. The activity of metalloprotreinases was studied by gelatine zymography. RESULTS: In the liver metastases and tissues adjacent to metastasis, the levels of low-spin forms of cytochrome P450 are lower than in the samples of conventionally normal liver tissue. Western blot analysis revealed that low-spin form of cytochrome P450 in primary tumors and liver metastases detected by electron paramagnetic resonance is attributed largely to CYP 1B1 isoform. The content of low-spin form of cytochrome P450 inversely correlated with the the activity of gelatinases (matrix metalloproteinase-2 and -9). The survival of patients with high levels of the low-spin form of cytochrome P450 in tumor tissues was higher than that of patients with low levels of the enzyme (105 months vs 61 months). CONCLUSION: In the primary tumors and liver metastases of patients with mCRC, the content of the low-spin form of cytochrome P450 decreased, which correlated inversely with patient's survival.

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.

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.

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.

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.

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.

Electron paramagnetic resonance study of tumor affected bone marrow.

To study the mechanism of interaction of tumor cells with bone marrow cells continuous wave electron paramagnetic resonance (EPR) experiments at 9 GHz including a spin trapping of superoxide were carried out. The common features of the EPR spectra in healthy and tumor affected tissues of donors and rats as well as their difference are presented and discussed. It is proposed that labile iron pool plays a significant role in mechanisms of tumor invasion. We hope that the observed EPR features could be used to study the mechanisms of invasion and progression of tumor in different organs.

O2*- and NO-associated mechanisms of selective action of redox-active cobalt complexes on tumor tissue.

AIM: To study the influence of redox-active cobalt(III) complex with tetradentate Schiff base and nicotinamide as an axial ligand on the rate of superoxide radical-anions generation and levels of NO in tumor and normal tissues of Lewis lung carcinoma bearing mice as well as activity of matrix metalloproteinases 2 and 9 (MMPs) in tumor. METHODS: The superoxide radical-anions formation and NO level in tissues were assessed by EPR method with the use of 1-hydroxy-2,2,6,6-tetramethyl-4-oxopiperidin and diethyldithiocarbomate spin traps, respectively. MMPs activities were determined by zymography in polyacrylamide gel. RESULTS: It was observed that the rate of superoxide radical-anions generation was selectively increased in tumor tissue (by a factor of 6-7) accompanied with the decrease of NO level (by a factor of 2) due to tested complex administration. Activities of MMPs in tumor were significantly decreased. CONCLUSION: It is supposed that the one of mechanisms of detected earlier antimetastatic effect of complex is based on its ability to induce the formation of high level of superoxide radical-anions selectively in the tumor tissue that results in the damage of its regulatory functions, in particular alteration in the regulation of NO-synthase, decrease of NO generation as well as activities of MMPs.

Effects of radical oxygen species and NO: formation of intracellular hypoxia and activation of matrix metalloproteinases in tumor tissues.

AIM: To establish the association between the radical oxygen species (ROS) and NO levels in the tumor cells mitochondria, between cell hypoxia development and activation of matrix metalloproteinases-2 and -9. MATERIALS AND METHODS: Electron paramagnetic resonance (EPR) at room temperature and at the temperature of liquid nitrogen (77 degrees K), spin traps technology, enzymography in polyacrylamide gel were applied. RESULTS: Redox-centers in the respiration cascade of mitochondria have been revealed, multiple oxidative damage of which in breast and liver cancer tissues of experimental animals as well as in tumor tissue from patients with gastric cancer promote the development of cell hypoxia. Involvement of ROS and NO in activation of latent forms of matrix metalloproteinases in gastric tumor tissues has been shown. CONCLUSION: We hypothesize that superoxide radical-anions participate in development of cell hypoxia in tumors and surrounding normal tissues inducing activation of latent forms of matrix metalloproteinases.

High formation of superoxide anion and nitric oxide, and matrix metalloproteinases activity in vascular wall of rectal carcinoma vessels.

AIM: To study the relationship between the level of generation of reactive oxygen species (ROS) and nitric oxide (NO) and activity of matrix metalloproteinases (MMPs) MMP-2 and MMP-9 in the vessels isolated from rectal tumors and Arteria rectalis superior. METHODS: EPR at the room temperature and 77 degrees K, Spin Traps technology and zymography in polyacrylamide gels were applied. RESULTS: In the vessels isolated from rectal tumors and Arteria rectalis superior high levels of ROS, NO and formation of complexes of NO with FeS-proteins at the sites of electron-transporting chain of mitochondria have been detected. High activities of MMP-2 and MMP-9 in vascular wall were also observed. The direct positive correlation between the rate of NO generation and formation of complexes of NO with FeS-proteins as well as between ROS and NO formation and MMPs activities have been revealed. CONCLUSION: Altered oxidative equilibrium in mitochondria of cells in vascular wall promotes formation of cell hypoxia and its autocatalytic potentiation accompanied with activation of MMPs.

Hypoxia level and matrix metalloproteinases-2 and -9 activity in Lewis lung carcinoma: correlation with metastasis.

AIM: To investigate the relationship between tumor hypoxia in vivo, activity of matrix metalloproteinases (MMPs), and metastatic potential of tumor. MATERIALS AND METHODS: Lewis lung carcinoma (3LL) was used in this study. Total activity of MMP-2 and -9 in tumor was measured biochemically, tumor hypoxia level was assessed by (31)P NMR spectroscopy in tissue perchloric extracts. RESULTS: It was determined that hypoxia level in primary tumor has been concomitantly increasing along with tumor growth and correlated with metastasis level in lung. The positive correlation between hypoxia level and activities of MMP-2 and MMP-9 in primary tumor was registered. Moreover, the activity of MMP-2 and -9 in 3LL (primary tumor) directly correlates with metastasis level in lung. CONCLUSION: This study demonstrated that the growth of primary tumor is distinctly accompanied by an increase of tumor hypoxia level which positively correlates both with the activity of MMP-2 and -9 in primary tumor and metastatic efficiency.

Modifying effect of organocobalt complexes on the tumour response to anticancer treatments.

A new type of agents are proposed for combined cancer therapy. They are organocobalt (III) chelates containing a sigma-bounded organyl group and a mixed tridentate ligand derived from a Schiff base. These complexes generate free radicals due to the action of protons in physiological ranges of pH and temperature, and hence are conceivably capable of selectively attacking a malignant neoplasm that is slightly acidic and can be made even more so by introducing some means intensifying glycolysis. An in vivo examination was performed using transplanted rat tumours (Guerin and Walker 256 carcinomas, Sarcoma 45). The modifying effect of one of these complexes on the tumour response to cis-DDP, radiation and/or local hyperthermia was tested by means of tumour growth delay assay and local tumour control. The potentiating effect of the complex was maximal when it was administered 60-90 minutes prior to other agents (cisDDP, X-irradiation heat). The enhancement ratio was found to be ca. 2.0-4.0 for cisDDP and 2.0 for radiation. In conclusion, in our tumour models, an increase of the antitumour effect was obtained for conventional antitumour agents when they were supplemented with organocobalt complex. It can be hypothesised that DNA in tumour cells may be considered to be the main target for organocobalt complexes.