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ECPB 2014, 68(4): 56–63
Clinical physiology and biochemistry

Operation of NO-signaling system in blood lymphocytes of women at norm and while neoplastic ovarian transformation

YAKUBETS O., VOROBETS D., VOROBETS Z., GZEGOTSKI M.
Abstract

The peculiarities of arginase and NO-synthase pathways of L-arginine metabolism in peripheral blood lymphocytes of patients with ovarian cancer were studied. Lymphocytes were extracted from freshly received, heparin treated blood, from patients with ovarian cancer and a control group (clinically healthy female patients) using fikol-triumbrast concentration gradient. To reveal of the latent enzymatic activities, they were treated with 0,2% saponin. It was shown that the development of cancer pathology is associated with an imbalance in the NO synthesis in blood lymphocytes. The blood lymphocytes iNOS activity of healthy individuals was slightly identified and measured as 1, 320,18 nmol NADRH (H+) / min per mg of protein.

The reason for such imbalance is the activation of arginase and inducible isoform of NO-synthase (iNOS) while significant inhibition of its constitutive isoform. In the lymphocytes of patients with ovarian cancer, regardless of a stage, eNOS activity is reduced by 4,1 times. Against the background of inhibition of eNOS in the blood lymphocytes of patients with ovarian cancer there was a sharp increase in iNO-synthase activity (by 167 times) . For assessment the functioning intensity of NOS pathway of L-arginine metabolism the content of stable NO metabolites such as Nitro-(NO2–) and Nitrate-(NO3–) anions in a blood was determined. The concentration of NO metabolites is changed simultaneously. NO2– decreases by 1,6-times and NO3– increases by 1,5-times. Moreover, when ovarian cancer, the ratio NO3–/NO2– increases by 3,4 times. Whereas normally this ratio is equal to 1,4 times.

Our results show that with increasing malignancy (from I to IV ovarian cancer stage), increases the concentration of malondialdehyde in plasma. In parallel with this, from stage I to IV eNOS activity in peripheral blood lymphocytes decreases. At the same time iNOS activity from I to IV stage of disease increases considerably. Thus, the corresponding relationship between the concentration of malondialdehyde and activities of NOS is being traced. With increasing the concentration of MDA the activity of eNOS decreases and iNOS activity increases. Nitric oxide hyperproduction resulting from the activation of iNOS can convert it from a level of adaptation into a pathogenetic level of the disease, which is accompanied by or is the result of lipid peroxidation processes. It is shown that in patients with ovarian cancer iNOS expression correlates with differentiation stage of the tumor, and intracellular NO – with a stage of the disease. It is assumed that inhibition of iNOS may be a potential therapeutic target in the treatment of ovarian cancer.

The analysis of the kinetic properties of NOS of blood lymphocytes of patients with ovarian cancer was carried out. It was shown that the affinity constant of iNOS affinity for L-arginine is 5,4-fold lower than for eNOS of blood lymphocytes of persons in the control group. The inhibition of eNOS occurs via competitive type and is related to the reduction of maximum reaction rate.

During oncopathology development infringement of NOS pathway of L-arginine metabolism, which points at dysmetabolic changes in the NO synthesis system, is observed, namely its hyperproduction. The value of the NOS activity and concentration of NO, along with the other parameters, may show the functional state of the cell and thus be prognostic indices of diagnosis and evaluation of oncotherapy.

Keywords: NO-synthase, arginase, L-arginine, nitric oxide, lymphocytes, ovarian cancer

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