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e21581 Background: Overexpression of the Bcl-2 protein inhibits apoptosis and promotes carcinogenesis. Stress causes signaling leading to cell buffering with Bcl-2 protein above acceptable levels. The purpose of the study was to analyze the influence of comorbidity – chronic neurogenic pain (CNP) – on the Bcl-2 levels in mitochondria of cells of melanoma, the heart, skin and brain in female mice with growing tumors. Methods: Female С57ВL/6 mice were divided into groups: intact group (n = 21); control group with a CNP model – bilateral sciatic nerve ligation (n = 21); group M – B16/F10 melanoma (n = 63); CNP+M group – B16/F10 melanoma was transplanted 3 weeks after the CNP model creation (n = 63). The concentration of Bcl-2 (ng/mg of protein) was determined in mitochondrial samples by ELISA (Thermo Fisher Scientific, Austria). Statictical analysis of results: Statistica 10.0. Results: CNP decreased the Bcl-2 level in heart mitochondria by 1.3 times (p < 0.05), but increased it in skin and brain mitochondria by 5.8 and 1.3 times, respectively. Similar changes were observed in melanoma growth 1 week after its transplantation: Bcl-2 levels decreased in heart mitochondria by 1.3 times, and increased in the skin and brain by 8.9 and 1.3 times, respectively. After 2 weeks of the tumor growth, Bcl-2 in brain mitochondria decreased by 1.7 times, and it started declining in the skin by the 3rd week – by 4 times, compared to intact females. Bcl-2 in tumor mitochondria exceeded the values in the skin by more than 4 times throughout the experiment. Tumor growth in presence of CNP caused a decrease in Bcl-2 in brain mitochondria by 2.4 times after 3 weeks, and in the heart and skin – by 2 and 1.7 times, respectively, after 2 weeks. Bcl-2 in tumor mitochondria in presence of CNP was lower than in the intact skin on average by 1.8 times throughout the experiment. Conclusions: CNP as a comorbidity caused a modulating effect on the mechanisms of survival and apoptosis of cells both in the tumor and in the main organs providing the vital functions of the body - the brain and heart, and also affects the target organ of melanoma - the skin. The results demonstrated the ability of comorbidity to change levels of Bcl-2 in mitochondria depending on the stage of tumor development.

e21580 Background: The thyroid gland is the most important regulator of the body's response to stressful influences, including malignant growth. Iodine is a constituent of thyroid hormones, and it plays an important role in anti-tumor protection. The purpose of the study was to analyze the levels of thyroid hormones in the thyroid gland of male and female mice with B16/F10 melanoma, as well as the effect of an organic iodine-based preparation on the survival of animals with transplanted melanoma. Methods: Levels of total (T3, T4) and free (FT3, FT4) thyroid hormones were measured by RIA in the thyroid of male and female С57ВL/6 mice (n = 80) with transplantable B16/F10 melanoma. The effect of an organic iodine-based preparation on the dynamics of the melanoma growth was studied in the main group of animals (n = 42) receiving per os 1,3-diethylbenzimidazolium triiodide in a single dose of 0.4 mg/kg throughout the experiment; the control group (n = 20) received distilled water. The tumor volume and survival of mice were measured. Results: The lowT3/lowT4 syndrome developed in females with transplantable B16/F10 melanoma starting from week 2 of the experiment, diagnosed by decreasing levels of FТ3 and FТ4 by 3 times and lower and normal levels of T3 and T4. In males, melanoma growth caused the thyroid hypofunction expressed in a decrease in the levels of T3 and T4 by 2 times, and FT3 and FT4 by 1.5 times. The latent period prior to the tumor onset was 10-12 days in females and 5-7 days in males. The survival of females was 5-6 weeks, males – 3-4 weeks. The administration of the organic iodine-based preparation extended the latent period in females up to 14-18 days, in males up to 10-14 days; the survival of females increased by 1.8 times, males by 1.5 times. 9% of females in the main group showed complete tumor resorption without relapses throughout their lives. Conclusions: The growth of transplantable B16/F10 melanoma in male and female С57ВL/6 mice was accompanied by suppression of the thyroid function with the development of uncontrolled hypothyroidism in males and the lowT3/lowT4 syndrome in females, which correlates with the survival of animals.

e15045 Background: Berberine is an alkaloid compound with a structure that is highly similar to that of intercalating agents. It affects numerous cell signaling pathways and is widely studied as potential anticancer drug. It is known that berberine affects cancer cells migration through metalloproteinase-2 inhibition, but this effect was never studied on glioma cells. Anti-migratory drugs are of special interest in brain cancer therapy since glioma's highly invasive nature makes total surgical removal of tumor practically impossible. The aim of the study was to evaluate berberine anti-migratory activity on glioma cells. Methods: Cell migration capacity of T98G and U87MG cell lines, as well as primary glioma cell culture established in our laboratory, was assessed via standard wound healing assay with automated image acquisition and analysis on Lionheart FX (BioTek) cell imager. Prior to assay setting up cell cultures were maintained in DMEM medium with L-glutamine (1 μM) (Gibco) and 10% FBS (Gibco) at 37C0 and 5.0% CO2. Cells were seeded at 250 000 cells per well on 24-well plates and incubated overnight in order to attach to plate bottom. After that a vertical wound was made manually in each well, and berberine was added to experimental wells to final concentration 50 mg/L. Plates with cells were continuously incubated and photographed in cell imager at 37C0 and 5.0% CO2. The extent of cells migration was measured as the percent of wound area decrease after 24 hours of incubation in relation to starting time point. Data are given as: Mean ± 95% confidence interval. Results: In our study we berberine exhibited anti-migratory activity in all cell cultures under study. In rather fast growing primary cell culture wound area decrease was 99.23%±0.62% in control sample and 91.75%±0.28% in experimental sample. The difference was small but significant at p < 0.001 level (df = 30). Popular permanent glioma cell lines T98G and U87MG showed more prominent decrease in studied parameter with higher degree of variance at the same time. In T98G wound area decrease was 71.6%±12.3% in control and 48.8%± 7.6% in experimental samples after 24 hours of cultivation in presence of 50 mg/L berberine. While U87MG demonstrated 60.28%±5.13% and 37.5%± 8.34% wound area decrease accordingly. The obtained difference between control and experimental groups in permanent cell cultures was statistically significant at the 0.05 level (df = 30). Conclusions: Our preliminary research proved berberine to be potent anti-migratory agent in glioma treatment. Further investigations are needed to evaluate its ability to inhibit glioma cell expansion in vivo.

e21557 Background: 10% of cancer patients have comorbidities accompanied by chronic pain. Neurotrophins and fibrinolytic system are involved in carcinogenesis and pain pathogenesis. The purpose of the study was to measure levels of neurotrophins in white matter of the brain of urokinase-deficient (uPA–) mice with B16/F10 melanoma growing in presence of chronic neuropathic pain (CNP). Methods: The study included female mice С57ВL/6 (normal genome uPA+, n = 40) and C57BL/6-Plautm1.1BugThisPlauGFDhu/GFDhu (urokinase gene-knockout uPA–, n = 28) with B16/F10 melanoma (M) implanted subcutaneously 2 weeks after bilateral sciatic nerve ligation (CNP model). Intact mice (I) were controls. Levels of brain-derived neurotrophic factor BDNF, nerve growth factor NGF-β and neurotrophins 3 and 4 (NT3, NT4) were measured by ELISA in white matter of the brain after 3 weeks of tumor growth in presence of CNP. Results: Tumor volume in (uPA–) females by week 3 of carcinogenesis was 0.04 cm3, which was 70 times smaller than in (uPA+) females. Tumor volume in (uPA–) females with CNP was 5.76 cm3, which was 144 times larger than in (uPA–) females without CNP, and in (uPA+) females – 2.5 cm3. The brain of I (uPA–) showed higher levels of NT3 (by 1.3 times, p < 0.05), NT4 (by 2.6 times) and NGF-β (by 1.9 times, p < 0.05) and lower BDNF (by 1.7 times, p < 0.05), compared to I (uPA+). Both strains of mice with M or CNP demonstrated decreased levels of NGF-β, more pronounced in animals with a combination of these factors. (uPA–) females with CNP+M showed a decrease of NT3 and BDNF by 2 times, with NGF-β 2.2 times higher than in (uPA+) mice. Conclusions: The study revealed underlying differences in levels of neurotrophins in the brain of (uPA–) females which could contribute to the creation of conditions for the inhibition of tumor growth. Changes in the levels of NGF-β in mice with melanoma or CNP were nonspecific. Changes in the BDNF, NT3 and NGF-β balance in the brain of (uPA–) mice may be part of the mechanism of greater stimulation of melanoma growth under the influence of CNP.

e22097 Background: Chronic neurogenic pain (CNP) as a comorbid disease is quite frequent, but its effect on malignant diseases is poorly studied. The risk of cutaneous melanoma in women is higher, but the disease course in men is more severe. The purpose of the study was to reveal the effect of CNP on the levels of sex hormones in the blood of patients with melanoma depending on their gender. Methods: Blood levels of estradiol (E2), estrone (E1), progesterone (P4), testosterone (T) and prolactin (PRL) were measured by ELISA in patients with Т3-4NxM0 melanoma (M): 21 women with CNP (pelvic pain - 7, osteochondrosis – 14), mean age 67.2±2.7 years; 17 men with CNP (osteochondrosis), mean age 65.6±3.1 years. The control group included patients with melanoma similar in age, gender and disease stages without CNP. Results: In women with M+CNP, E2 and P4 were decreased by 1.8 times, while E1 was 1.4 times, T – 2 times, PRL – 1.5 times higher, compared to controls. In men with M+CNP, blood levels of estrogens were unchanged, PRL was 2.5 times lower and P4 and T – 1.3 times higher, compared to the corresponding control levels. Conclusions: CNP upregulated blood levels of androgens in both male and female melanoma patients, and caused the inversion of estrogens only in women with the prevalence of E1 over E2. Gender differences in the CNP influence included the elevation of progesterone and a decrease of prolactin in the blood of men, and P4 deficiency with increased prolactin in women with M+CNP. The specific effect of CNP on the hormonal profile in patients with cutaneous melanoma should be considered in choosing antitumor treatment.

e22082 Background: Clusters of tumor cells in blood vessels secrete VEGF family factors necessary for the formation of premetastatic niches. TGF-β activates the epidermal-mesenchymal transition (EMT). The purpose of the study was to analyze levels of angiogenesis and EMT factors in the blood of patients with cutaneous melanoma (M) and chronic neurogenic pain (CNP). Methods: Blood levels of VEGF-A, VEGF-C, VEGFR-1, VEGFR-3, TGF-β1 and TGF-βR2 were measured by ELISA in patients with Т3-4NxM0 melanoma: 21 women with CNP (pelvic pain - 7, osteochondrosis – 14), mean age 67.2±2.7 years; 17 men with CNP (osteochondrosis), mean age 65.6±3.1 years. The control group included patients with melanoma similar in age, gender and disease stages without CNP. Results: VEGF-A in women and men with M+CNP was higher than in controls by 2.7 and 24.9 times respectively; VEGFR-1 was decreased in women by 1.8 times and increased in men by 1.8 times. VEGF-С was unchanged in women and 1.5 times higher in men, and VEGFR-3 was increased by 2.2 times in women and unchanged in men. TGF-β1 was elevated in women and men with M+CNP by 1.4 and 1.8 times, compared to controls, TGF-βR2 – by 1.9 and 2 times respectively. Conclusions: In the blood of women with M+CNP, CNP activates the blood vascular endothelial growth factor VEGF-A and the factor of epidermal-mesenchymal transition TGF-β, while in the blood of men with M+CNP it activates the blood vascular endothelial growth factor VEGF-A, the lymphatic vascular endothelial growth factor VEGF-C and the factor of epidermal-mesenchymal transition TGF-β. VEGF mediates vascular permeability and is associated with the mobilization of endothelial progenitor cells from the bone marrow into premetastatic niches. Activation of TGF-β signaling promotes the induction of the epidermal-mesenchymal transition and supports the properties of cancer stem cells.

e21035 Background: Lack of prognostic markers of melanoma to assess its progression, metastasis (MTS), and treatment effectiveness, determines the feasibility of studying biophysical aspects of the transmission of events from cellular to extracellular environment. Receipt and circulation of pathological proteins of melanoma (M) in the blood carries important information, and its trap is the phase transition of biofluids into a solid state. The purpose of the study was to determine markers of melanoma at the supramolecular level in solid-state samples of the blood serum (BS). Methods: 240 samples of BS obtained prior to the surgery from 60 patients with M were studied. The preparations were prepared by the Shatokhina-Shabalin method by creating a closed "analytic cell": dehydration of a BS drop (10 mcL) under a cover glass at 20–240С, humidity 65–70%, for 72 hours. The folding of M proteins was assessed by a polarized light microscopy (Leica DM LS2 microscope, x400-640) using photo and video control and the Morphotest program. Results: The initial stages of M development were characterized by the early appearance of single combined melanoform crystals — anisotropic microspherolites (Mi) embedded in the center of a large anisomorphone (lAM). As M progressed, Mi aggregation with basic lAMs was enhanced. Anisotropy of Mi increased, revealing clear sectors filled with melanin. In the later stages of M growth, melanoform aggregations dominated with point, filamentous, and chained melanin structures. The manifestation of MTS was accompanied by the destruction of the aggregations and the filling of the analytic cell space with numerous Mi. Conclusions: Correspondence of crystallogenesis of M melanomorphic markers in BS in tumor progression allows using the studied supramolecular factors to predict the development of melanoma.

e21036 Background: The high metastatic potential of melanoma and the need for long-term patient monitoring causes the search for tumor markers of this malignant neoplasm. Our aim was a comparative analysis of levels of tumor-specific proteins CD44 and S100 and protein composition in melanocytic lesions of the skin. Methods: We studied 86 samples of cutaneous melanoma and nevus tissues, their perifocal tissues and resection line tissues obtained during tumor excision from 23 patients with cutaneous melanoma pT1-4N0-1M0 and 14 patients with nevi. Intact skin samples obtained from non-cancer patients during reconstructive plastic surgery were used as the comparison group. All patients gave their written informed consent. Levels of CD44 (BenderMedSystems, USA) and S100 (Fujirebio, Sweden) were determined by ELISA in 10% homogenates of all tissues; total protein levels were determined by standard spectrometry and fractional composition of proteins were studied by turbidimetric method. Statistical processing of results was performed using the Statistika 6.0 program with Student’s t-test for two independent groups. Results: Melanoma was characterized by a sharp increase in S100B levels, 28 and 7 times exceeding the levels in intact tissues and nevi. The level of CD44 in melanoma tissue was increased only by 2 times, in nevus tissue - by 48%. The ratio of albumin and gamma globulins in the tissue of melanoma and nevi was 79% and 29% lower than in healthy skin. A more than twofold increase in the gamma globulin fraction in the melanoma tumor tissue against a decrease in albumin and the absence of changes in other globulins, as well as a moderate but statistically significant increase in the gamma globulin fraction in nevi tissue, indicates that S100B and CD44 proteins belong to the gamma globulin fraction. Conclusions: A highly specific increase of S100 levels and a less specific increase of CD44 levels in supernatant liquid of melanoma tissue homogenates, together with the predominance of the gamma globulin fraction, allow considering such factors as a prognostically unfavorable sign of tumor progression, which can be important when choosing a personalized treatment strategy.

e21625Background: The purpose of the study was the development of a mathematical model of predicting the progression of cutaneous melanoma based on an assessment of its local cytokine microenvironm...

e20110 Background: Methodological aspects of modern technologies of ultrasound scanning (US) are still relevant in oncosurgery of skin cancer as there is need to clarify some parameters of tumor pr...

e20010 Background: For the study of the pathogenesis of melanoma interest is a comparative study of tumor markers in melanoma tissue early stage and melanocytic nevi. Methods: 40 samples of early s...