Angiogenic potential of circulating blood neutrophils in endometrial cancer

Neutrophils play an important role in carcinogenesis, mediating inflammation, immunosuppression and metastasis. A dual role of neutrophils in regulation of angiogenesis has been shown. Endometrial cancer is the most common malignant neoplasm of the female reproductive system. To assess the cellular angiogenic potential, we determined the levels of inflammatory and angiogenic cytokines (VEGF-A, IL-17A, IL-1β, IL-6) in cell lysate of peripheral blood neutrophils in the patients with endometrial cancer, and with uterine myoma (comparison group). Expression of the nuclear factor-kB was determined. In nuclear fraction of neutrophils. The neutrophil-to-lymphocyte ratio (NLR) was assessed in the studied groups. Statistical processing of the obtained data was carried out using Statistica 10 software. We have not found any significant changes in NLR in endometrial cancer, compared with controls and the uterine myoma groups. Expression of NF-kB and VEGF was increased as compared to the control for all the studied stages of endometrial cancer and in uterine myoma. There was a change in NF-kB level in neutrophils, depending on the tumor differentiation grade. A regression relationship was found between the content of VEGF and NF-kB in neutrophils. We have found increased IL-1β and IL-6 levels in neutrophils of the uterine myoma patients, and at different stages of endometrial cancer compared with control. The IL-1β level was higher in neutrophils of the patients with intermediate and high tumor grade, compared to low-grade cases. IL-17A expression in the neutrophil lysate was significantly reduced in uterine myoma and at different stages of endometrial cancer, as compared with controls. There was a moderate inverse correlation between the contents of VEGF and IL-6 in neutrophils (r = -0.426, p = 0.001); a remarkable inverse relationship between VEGF and IL-17A (r = -0.615, p < 0.001). The combination of IL-6 and IL-17A levels in the neutrophils lysate (according to the results of multivariate analysis) may be used for the differential diagnosis of endometrial cancer and uterine myoma. Thus, the differences in the expression of inflammatory and angiogenic cytokines included in NF-kB-dependent signaling, may point to acquisition of pro-tumor functions by neutrophils during the endometrial cancer progression

Influence of proinflammatory cytokines on membrane rigidity and morphofunctional state of circulating neutrophils in ovarian tumors

Priming and activation mediated by cytokines cause transient reactions of actin polymerization in neutrophils (Nph), expansion and softening of cells, changes in receptor status, phagocytic ability, and generation of extracellular traps (NET), thus ultimately determining pro- or antitumor phenotype of Nph. To assess the effects of pro-inflammatory cytokines on membrane rigidity and morphofunctional state of neutrophils in benign tumors and ovarian cancer, the levels of circulating cytokines (IL-2, IL-18, MCP-1, TIMP-1), expression of adhesion markers (CD11b), degranulation (CD63), as well as FcãRIIIb receptors promoting phagocytosis (CD16). Ability of neutrophils to phagocytosis and the formation of NET was determined. Statistical evaluation of the data obtained was carried out using Statistica 13.0, Jamovi 1.6.5.0 software. An increase in membrane rigidity was found in benign and malignant ovarian tumors. In benign ovarian tumors, phagocytic activity and expression of CD11b were enhanced. In ovarian cancer, the number of CD11b+Nph and CD63+Nph were increased. Meaanwhile, at initial cancer stage, the ability to form NET predominates, and phagocytic activity increases with advancing ovarian cancer. Serum MCP-1 levels are elevated in benign tumors and at all stages of ovarian cancer. IL-2 is elevated at early stage and in advanced ovarian cancer. The level of IL-18 and TIMP-1 in the serum of patients with benign ovarian tumor did not differ significantly from the norm. Using multiple regression approach, the dependence of neutrophil membrane rigidity on the levels of circulating IL-2, TIMP-1, MCP-1, IL-18 was revealed in benign ovarian tumors, and a direct correlation was found between the neutrophil membrane rigidity and CD11b expression. Only IL-2 was associated with neutrophil membrane rigidity in ovarian cancer. At the same time, the rigidity of the neutrophil membrane directly correlated with CD16, CD63, expression like as with phagocytic index and inversely correlates with the number of traps. A combination of IL-2, MCP-1 and membrane rigidity of circulating neutrophils (based on multivariate analysis) could be used for differential diagnosis of ovarian cancer. Thus, in a benign ovarian tumor, circulating proinflammatory cytokines are associated with increased rigidity of neutrophil membrane and increase in their adhesion capacity. In ovarian cancer, only IL-2 is associated with altered rigidity of circulating neutrophils. Increase of the latter index is accompanied by elevated phagocytic activity and decreased ability to form NET

Phenotype of circulating neutrophils at different stages of cervical neoplasia

At the present time, there is no common point of view to the role of circulating neutrophils (NP) in emergence and development of neoplasia. It is suggested that due to high functional plasticity, the neutrophils may exhibit both pro- and antitumor activity. In order to study the NP phenotype at different stages of cervical neoplasia (CN), we have evaluated their absolute and relative amounts, myeloperoxidase activity, spontaneous and induced NST-test markers, and the level of intracellular cationic proteins. Spontaneous production of elastase and active forms of matrix metalloproteinases, the levels of IL-2, IL-8, IL-18, IFNy, G-CSF were determined in the NP cell lysates and in blood serum. The formation of extracellular traps (NET) was evaluated using 1-day cultures of Saccharomyces cerevisiae as an inducer. We examined 31 patients with cervical intraepithelial neoplasia (CIN) and 21 primary patients with cervical cancer (CC, Ia stage according to FIGO scale), as well as 25 practically healthy women. We revealed increased spontaneous and inducible oxygen-dependent cytolytic and phagocytic activity and spontaneous production of NET if compared to normal values, along with decreased absolute NP numbers in patients with CIN, thus suggesting the antitumor activity of NP. The levels of “pro-tumor” cytokines (MMP-9, IL-2 and G-CSF) become increased over normal levels as early as at the CIN stage, both for the neutrophils and blood plasma. High levels of regulatory IFNy and neutrophil-priming IL-8 in blood plasma do not presume any use of exogenous NP-activating factors at the stage of cervical dysplasia. At the initial stage of cervical cancer, the absolute NP amounts are significantly increased compared to normal counts. However, despite increased spontaneous oxygen-dependent cytolytic activity, the NPs have a significantly reduced activity of phagocytosis and sharply increased spontaneous production of NET, thus, generally, being characteristic to the “pro-tumorous” NP phenotype. IL-2 levels are elevated, and MMP-9 values are still increased in NP and blood plasma of patients with CC (stage Ia). Hence, the obtained results suggest some changes of NP phenotype to a pro-tumorous pattern during transition from intraepithelial dysplasia to cervical cancer. These results allowed us to design an algorithm for examining women with suspected cervical cancer, including IL-2 measurement in blood serum, and MMP-9 amounts in the NP lysates

IL-4 and its polymorphism (<i>IL4</i>-589C/T) in cervical neoplasia

The transition of cervical neoplasia (CIN) to cervical cancer occurs with the active participation of IL-4, for which both pro- and antitumor effects have been shown with tumors of various localizations. The expression of cytokines is regulated at the transcriptional level in the promoter region of the gene. It has been shown that the genotype IL4 (589C/T) (rs2243250) is associated with the development of gastric and breast cancer. The contribution of IL-4 genotypic variations to the development of CIN has not yet been studied. The aim of the study was to assess the risk of developing cervical neoplasia by the presence polymorphism of IL4 (589C/T) and the level of IL-4. The object of the study was circulating neutrophils, serum and genomic DNA of 36 patients with CIN and 20 women without dysplasia (comparison group). Using ELISA, the level of IL-4 was determined in neutrophil lysate and serum. Phagocytic activity and adhesive ability (CD11b) of neutrophils were assessed. Allele-specific real-time PCR using Taq-Man probes was used to analyze of the IL4 589C/T (rs2243250). Statistical processing was carried out using Statistica 13 and Jamovi 1.6.5.0. As a result of the study, it was found that the level of IL-4 in serum and circulating neutrophils in patients with CIN is significantly higher than in the comparison group. The -589C* allele of the IL4 gene and the TT genotype are more common in the group with CIN (55.5%) than in the control (25%). At the same time, a direct relationship was established between the presence of polymorphism and increased adhesive ability and with indicators of the phagocytic number of circulating neutrophils. Analysis of the incidence of IL4 C589T by the «case-control» method showed that the chances of CIN formation in carriers of the -589C allele and the TT genotype were 3.75 (95% CI: 1.013 - 13.880, Chi-square = 4.161, p = 0.042). The -589C* allele and TT IL4 genotype, neutrophil and serum IL-4 levels are associated with HPV infection. Using a binary logistic regression model, we demonstrated the possibility of using IL-4 levels in circulating neutrophils and IL-4 gene polymorphism (589C/T) for the differential diagnosis of patients with CIN (χ2 = 15.6, p = 0.001). Significant significance for their combination was assessed by ROC-curve analysis (IL-4 in neutrophils; IL4 (-589С*), 75% probability. Thus, the IL4 (589C/T) is associated with the adhesive and phagocytic activity of circulating neutrophils. In HPV-infected patients, IL4 gene polymorphism (589C/T) can serve as a marker for early detection and prognosis of CIN

Angiogenic potential of circulating peripheral blood neutrophils in kidney cancer

The role of neutrophils in kidney cancer is currently being studied. Their role in carcinogenesis is ambiguous. As one of the most abundant blood leukocytes, neutrophils play an important role in cancer progression through multiple mechanisms, including promotion of angiogenesis, immunosuppression, and cancer metastasis. Neutrophils synthesize and release pro-angiogenic factors that are able to directly or indirectly stimulate the growth and migration of endothelial cells, which in turn causes the formation of new blood vessels from pre-existing ones. The production of various factors by neutrophils, including proangiogenic ones, is mediated by the expression of the genes of these molecules. Functional heterogeneity is characterized by differences in neutrophil gene expression patterns. The aim of this study was to evaluate the angiogenic potential of circulating neutrophils in kidney cancer. The object of the study were blood neutrophils of patients with verified clear cell kidney cancer at stage I (T1N0M0G1, n = 28, median age 60), stage II (T2N0M0G2, n = 15, median age 61) and stage III (T3N0M0G2, n = 15, median age 63) before surgery. The control group consisted of apparently healthy donors (n = 15, median age 54). Serum levels of IL-8 and VEGF-A were assessed by enzyme immunoassay. Expression of the CXCL8 and VEGF-A genes in circulating neutrophils was determined by reverse transcription quantitative PCR. As a result of our study, an increase in the level of IL-8 and VEGF-A in the blood serum of patients with kidney cancer in all studied groups compared with the control group was revealed. We observed a direct correlation between serum levels of IL-8 and VEGF-A in patients with kidney cancer (r = 0.429; p = 0.016), which confirms the relationship of these angiogenic factors. A significant increase in CXCL8 gene expression by circulating neutrophils was found in patients on II (2.91, Q0.25-Q0.75: (1.296-4.99), p = 0.02) and III (1.93, Q0.25-Q0.75: (0.755-11.36, p = 0.014) stages of kidney cancer compared with the control group (1.50, Q0.25-Q0.75: (0.80-4.05)). However, VEGF-A gene expression by circulating neutrophils did not differ from those in the control group. Blood neutrophils in kidney cancer exercise their angiogenic potential through the production of IL-8

Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts

ВЗАИМОСВЯЗЬ УРОВНЕЙ ВОСПАЛИТЕЛЬНЫХ ЦИТОКИНОВ КРОВИ И ЧИСЛА ЦИРКУЛИРУЮщИХ ОПУХОЛЕВЫХ КЛЕТОК С ОТВЕТОМ НА СТАНДАРТНУЮ ХИМИОТЕРАПИЮ У БОЛЬНЫХ РАКОМ ЯИЧНИКОВ

Introduction. Serum chemokines are inflammatory mediators, which role is shown in the occurrence and progression of a number of malignant tumors. Produced by white blood cells, stem cells, tumor and endothelial cells, chemokines control their movement and positioning. Chronic inflammation underlies the progression of ovarian cancer (OC ). This increases the likelihood of chemokines stimulating or blocking tumor progression.The aim of the study was to evaluate the relationship between the blood levels of inflammatory cytokines in blood and the number of circulating tumor cells (CTC s) with the response to standard chemotherapy (CT ) in patients with cancer.Material and Methods. In patients with primary OC before and after 2–4 courses of chemotherapy and in patients with benign ovarian tumors (as a control), serum levels of CCL 2, CCL 3, CCL 4, CXCL 8 and CX3CL 1 were evaluated by multiplex xMAP analysis. The amount of CTC s (population CD 45-/ Epcam+/CK+) was determined using a flow cytometer. Patients with ovarian cancer were divided into 3 groups according to the platinum sensitivity criterion of GC JG 4th, and progression-free interval (PFI) was determined. Results. It was found that the levels of CCL 2, CCL 3, CCL 4, CXCL 8, and CX3CL 1 in case of OC did not significantly differ from that in the control, strongly negatively correlated with age (except for the CCL 2 level). CT significantly increased the level of CCL 2 in the group of refractory OC ; of CCL 3 – in the group of sensitive OC , of CCL 4 – in the groups of resistant and sensitive OC , and C XCL 8 level increased in the groups with resistant and sensitive OC and decreased in the group of refractory OC . The number of CTC s in patients with OC was significantly higher than in the control. After CT , a decrease in the amount of CTC s strongly and significantly correlated with a decrease in the level of CX3CL 1 in the groups of refractory andsensitive OC . The maximum PFI occurred with an increase in serum levels of CCL 3, CXCL 8, a decrease in CCL 4 and a constant level of CX3CL 1.Conclusion. Thus, no significant differences in the levels of CCL 2, CCL 3, CCL 4, and IL -8 between patients with OC and control groups were found. The levels of chemokines studied and the amount of CTC s differed in the groups divided by the tumor sensitivity to CT . We observed significant correlations between the amount of CTC s and the level of CX3CL 1 in the group of platinumsensitive OC .Введение. Сывороточные хемокины – медиаторы воспаления, роль которых доказана в возникновении и прогрессировании злокачественных опухолей ряда локализаций.  Продуцируясь лейкоцитами, стволовыми, опухолевыми и эндотелиальными клетками, хемокины контролируют их движение и позиционирование. Хроническое воспаление лежит в основе прогрессирования рака яичников (РЯ), и это повышает вероятность стимулирования или блокирования хемокинами прогрессирования опухоли. Целью исследования было изучение взаимосвязи уровней воспалительных цитокинов крови и числа циркулирующих опухолевых клеток (ЦОК) с ответом на стандартную химиотерапию у больных РЯ. Материал и методы. У пациенток с первичным РЯ до и после 2–4 курсов химиотерапии (ХТ) и пациенток с доброкачественными опухолями яичников (контроль) в сыворотке крови уровни ccl2, ccl3, ccl4, cXcl8 и cX3cl1 оценивали методом мультиплексного анализа xmap. Количество ЦОК (популяция cd45-/ epcam+/cK+) определяли на проточном цитометре. Пациентки с РЯ были разделены на 3 группы по критерию  платиночувствительности согласно gcJg 4th, было определено время до прогрессирования (pFi). Результаты. Установлено, что уровень ccl2, ccl3, ccl4, cXcl8 и cX3cl1 при РЯ значимо не отличался от такового в контроле, отрицательно коррелировал с возрастом (за  исключением уровня ccl2). Химиотерапия значимо повышала уровень ccl2 в группе  рефрактерного РЯ; ccl3 – в группе чувствительного РЯ, ccl4 – в группах резистентного и чувствительного РЯ, cXcl8 – повышала в группах с резистентным и чувствительным РЯ и снижала в группе рефрактерного РЯ. Количество ЦОК у больных с РЯ значимо выше, чем в контроле. После ХТ снижение количества ЦОК достоверно коррелировало со снижением уровня cX3cl1 в группах рефрактерного и чувствительного РЯ. Максимальная продолжительность периода без прогрессирования отмечена при повышении уровней ccl3, cXcl8, снижении уровня ccl4 и неизменном уровне cX3cl1. Выводы. Уровни ccl2, ccl3, ccl4 и il-8 значимо не различаются у больных РЯ и группы контроля. Уровни изученных хемокинов и количество ЦОК изменялись в группах с  различной чувствительностью опухоли к ХТ. При платиночувствительном РЯ наблюдаются значимые корреляции количества ЦОК и уровня cX3cl1 в сыворотке крови до лечения

Comparative characterization of oxidative stress in cervical cancer at different clinical stages of disease

Parameters of oxidative stress on the level of malondialdehyde (MDA), activity of glutathione-reductase, glutathione-transferase, superoxide- dismutase (SOD) and catalase in tumor tissue, plasma and red blood cells of patients with cervical cancer (CC) w as evaluated a t various clinical stages of the disease. It is revealed that an increase in tumor tissue MD A and glutathione enzymes while reducing SOD and catalase, which may indicate the development of oxidative stress in the dynamics of tumor progression. The data obtained suggest the increase of expres- sion of oxidative stress in red blood cells and tumor tissue in CC in the dynamics of tumor progression

ПРОЦЕССЫ ПЕРЕКИСНОГО ОКИСЛЕНИЯ ЛИПИДОВ И ЗАЩИТНАЯ РОЛЬ АНТИОКСИДАНТНОЙ СИСТЕМЫ В ПЕЧЕНИ И ЭРИТРОЦИТАХ В УСЛОВИЯХ ОСТРОЙ КРОВОПОТЕРИ LIPID PEROXIDATION PROCESSES AND PROTECTIVE ROLE OF THE ANTIOXIDANT SYSTEM IN LIVER AND IN ERYTHROCYTES IN THE CASE OF ACUT

Механизм взаимодействия печени и системы эритрона остается не до конца изученным. Рассматрива-ется данная проблема в 2-х аспектах: во-первых -оценка роли печени в функциях эритроцитарной системы, во-вторых -раскрытие патогенеза нарушений со стороны красной крови при поражениях печени. В условиях острой кровопотери как печень, так и эритроциты несут большую функциональную нагрузку, в связи с чем представляет интерес изучение их антиоксидантной системы, выполняющей защитную функцию. Изуче-но влияние острой кровопотери на показатели системы «перекисное окисление липидов -антиоксиданты» в печени и эритроцитах. Работа выполнена на белых беспородных крысах. Исследовали содержание мало-нового диальдегида и активность каталазы в печени и эритроцитах, а также активность глутатионредуктазы в эритроцитах. Определяли общее содержание белка и процентное содержание фракций белков в сыворотке крови. Показано, что возникшая гипоксия активирует процессы перекисного окисления липидов в гепатоци-тах и эритроцитах, о чем свидетельствует увеличение в них уровня малонового диальдегида. Одновременно повышается активность каталазы в печени и эритроцитах и антиоксидантный потенциал сыворотки крови, что может свидетельствовать об активации антиоксидантной защитной системы. Ключевые слова: кровопотеря, гипоксия, печень, эритроциты, синтез белка, перекисное окисление липидов (ПОЛ), антиоксидантная система LIPID PEROXIDATION PROCESSES AND PROTECTIVE ROLE OF THE ANTIOXIDANT SYSTEM IN LIVER AND IN ERYTHROCYTES IN THE CASE OF ACUTE BLOOD LOSS Kseyko D.A., Gening T.P. Ulyanovsk State University, Ulyanovsk, e-mail: [email protected] The mechanism of liver and erythron system interaction is not fully understood. This problem is considered in two aspects: fi rstly, the role of liver function in erythrocyte system; secondly, the disclosure of the pathogenesis of disorders of the red blood cells in liver lesions. In acute blood loss both the liver and erythrocytes have an important functional load. Therefore the study of their antioxidant system that performs a protective function is of great interest. The effect of acute blood loss on the performance of the «lipid peroxidation-antioxidant» system in the liver and erythrocytes was studied. Тhe work was done on white rats. The content of malondialdehyde and catalase activity in the liver and red blood cells and the activity of glutathione reductase in erythrocytes were studied. The total protein content and the percentage of protein fractions in blood serum were determined. It is shown that emerged hypoxia activates the processes of lipid peroxidation in hepatocytes and erythrocytes what is evidenced by an increase in their level of malondialdehyde. Simultaneously the activity of catalase in the liver and erythrocytes and antioxidant potential of blood serum are increasing. It can indicate activation of the antioxidant protective system. Keywords: blood loss, hypoxia, liver, erythrocytes, protein synthesis, lipids peroxidation, antioxidant system Важное место, занимаемое печенью в об-мене веществ, делает ее причастной к функ-циям почти всех органов и систем. Особую роль печень играет в работе системы эритро-на Кроме того, известно, что состояние мембран эритроцитов отражает состояние и других клеточных мембран организма. Получены данные о корреляциях электри-ческих и вязкоупругих свойств эритроцитов с функциональным состоянием печени при ее диффузных заболеваниях и изменениями липидного состава их мембран [6]. В условиях острой кровопотери как печень, так и эритроциты несут большую функциональную нагрузку в связи с чем представляет интерес изучение их антиок-сидантной системы, выполняющей защит-ную функцию. Цель исследования -изучение системы «перекисное окисление липидов (ПОЛ) -антиоксиданты» в печени и эритроцитах в условиях острой кровопотери. Материалы и методы исследования Работа выполнена на белых беспородных кры-сах массой 240-280 г. Гипоксию моделировали кро-вопусканием через катетер [15]. Объем кровопотери составил 2 % от массы животного. Животные были разделены на следующие группы: 1 группа -интакт-ные животные, 2-я группа -крысы через 6 ч после кровопотери и 3-я группа -крысы через 24 ч после кровопотери. Исследовали содержание малонового диальде-гида (МДА) [1] и активность каталаз

The effect of raman-laser irradiation on erythrocytes of rats

The effect of RAMAN-laser irradiation on erythrocytes of rats was investigated. It was established that laser irradiation with doses used increases lipid peroxidation in membranes of red blood cells and stimulates the activity of cellular antioxidant enzymes