Тканевая экспрессия аутофагиального маркера LC3B как потенциальный биомаркер рецидива рака предстательной железы после лечения высокоинтенсивным сфокусированным ультразвуком (пилотное исследование)

Background. The role of autophagy markers in prostate tumor recurrence has not been sufficiently investigated. We hypothesized that autophagy activation may be one mechanism by which prostate cancer cells survive exposure to high-intensity focused ultrasound (HIFU).Aim. To compare tissue expression of autophagic LC3B marker in prostate biopsies before and after treatment of localized prostate cancer by HIFU ablation.Materials and methods. 45 patients with localized morphologically confirmed prostate cancer were examined: group 1 – 25 patients of 65.6 ± 8.4 years without signs of recurrence or progression of the disease; group 2 – 20 patients of 67.5 ± 7.9 years with tumor recurrence proven during morphological examination. Immunohistochemical examination was performed by streptavidin-biotin method. In all cases, Anti-LC3B antibody ab48394 was used. The reaction results were quantified using the Histochemical score (Hs) system.Results. Prior to treatment, all patients of group 1 showed moderate cytoplasmic expression (Hs = 111 [111; 115]) of antibodies against LC3B in prostate adenocarcinoma cells, 5 % of patients – weak cytoplasmic expression in muscle connective stromal cells (Hs = 47 [43; 50]), 10 % of patients – weak positive LC3B reaction in the vessel wall (Hs = 28 [20; 35]). After treatment, the expression of LC3B in adenocarcinoma cells became negative, in the cytoplasm of muscle connective stromal cells weak (Hs = 75 [67.5; 80.0]), in the endothelium of the vascular wall even weaker (Hs = 55 [45.5; 60.0]) (p <0.001). Prior to treatment in group 2, LC3B expression in tumor tissue was moderate in 89 % of patients (Hs = 151.5 [137.5; 160.0]), weak in muscle connective stromal cells in 12 % of patients (Hs = 44 [35; 51.5]), and weak in the vascular wall in 5 % of patients (Hs = 30 [25; 35]). After treatment, LC3B expression in adenocarcinoma cells became pronounced (Hs = 260 [250; 285]), in muscle connective stromal cells – moderate (Hs = 118 [100; 130]), in the vascular wall – weak (Hs = 45 [30; 55]) (p <0.001). There was a significant correlation between tumor recurrence and LC3B overexpression (r = 0.51; p <0.001).Conclusion. The development of prostate cancer recurrence is associated with increased expression of autophagic LC3B protein. Increased LC3B expression, which is interpreted as evidence of autophagy activation and correlates with the risk of disease progression, is used by the tumor as an oncogenic advantage.Введение. Роль маркеров аутофагии при рецидиве опухоли предстательной железы недостаточно исследована. Мы предположили, что активация аутофагии может являться одним из механизмов, с помощью которого клетки рака предстательной железы выживают после воздействия высокоинтенсивным сфокусированным ультразвуком (HIFU).Цель исследования – сравнить тканевую экспрессию аутофагиального маркера LC3B в биоптатах предстательной железы до и после лечения локализованного рака предстательной железы методом HIFU-аблации.Материалы и методы. Обследованы 45 пациентов с локализованным раком предстательной железы, подтвержденным морфологически: 1-я группа – 25 пациентов (средний возраст 65,6 ± 8,4 года) без признаков рецидива или прогрессирования заболевания; 2-я группа – 20 пациентов (средний возраст 67,5 ± 7,9 года) с доказанным при морфологическом исследовании рецидивом опухоли. Иммуногистохимическое исследование проводили стрептавидин-биотиновым методом. Во всех случаях использовали антитело Anti-LC3B antibody ab48394. Результаты реакции подсчитывали по системе Histochemical score (Hs).Результаты. До лечения у всех пациентов 1-й группы зафиксирована умеренная цитоплазматическая экспрессия (Hs = 111 [111; 115]) антител к LC3B в клетках аденокарциномы предстательной железы, у 5 % – слабая цитоплазматическая экспрессия в клетках мышечно-соединительнотканной стромы (Hs = 47 [43; 50]), у 10 % – слабая положительная реакция LC3B в стенке сосудов (Hs = 28 [20; 35]). После лечения экспрессия LC3B в клетках аденокарциномы стала отрицательной, в цитоплазме клеток мышечно-соединительнотканной стромы – слабой (Hs = 75 [67,5; 80,0]), в эндотелии сосудистой стенки – еще слабее (Hs = 55 [45,5; 60,0]) (р <0,001). До лечения во 2-й группе у 89 % пациентов выявлена умеренная экспрессия LC3B в опухолевой ткани(Hs = 151,5 [137,5; 160,0]), у 12 % – слабая экспрессия в клетках мышечно-соединительнотканной стромы (Hs = 44 [35; 51,5]), у 5 % – слабая экспрессия в стенке сосудов (Hs = 30 [25; 35]). После лечения экспрессия LC3B в клетках аденокарциномы стала выраженной (Hs = 260 [250; 285]), в клетках мышечно-соединительнотканной стромы – умеренной (Hs = 118 [100; 130]), в сосудистой стенке – слабой (Hs = 45 [30; 55]) (р <0,001). Зафиксирована значимая ассоциация между рецидивом опухоли и гиперэкспрессией LC3B (r = 0,51; p <0,001).Заключение. Развитие рецидива рака предстательной железы связано с увеличением экспрессии аутофагиального белка LC3B. Повышенная экспрессия LC3B, которая интерпретируется как свидетельство активации аутофагии и коррелирует с риском прогрессирования заболевания, используется опухолью как онкогенное преимущество

Effect of increased nitrogen depositions and drought stress on the development of young Norway spruce Picea abies (L.) Karst. stands

The effects of drought stress, stress by increased nitrogen depositions and the combined effect of the two stress factors on the growth of Norway spruce Picea abies (L.) Karst. were studied in two stands. The drought stress was induced by reducing atmospheric precipitations by 60% and the increased nitrogen depositions were simulated by repeated applications of ammonium sulphate at a rate corresponding to 100 kg N ha–1 year–1. All stress factors under study affected the height increment of the above-ground part, the length and colour of needles, and the biomass, vertical distribution, functionality and mycorrhizal infection of fine roots. The root system responded to the simulated stresses right from the the very first year of their action, exhibiting a greater damage than the above-ground part of the plant. Drought acted as a stress factor stronger than the nitrogen depositions themselves. The strongest impact was recorded in the simultaneous influence of the stress factors

Root system development in two provenances of Picea abies at two different sites

The paper examines the development of the root system (both skeletal and fine roots) in 19-year-old Norway spruce (Picea abies (L.) Karst.) of two provenances (from altitudes 320 m and 1100 m), growing on two plots (540 m a.s.l., lowland, modal Cambisol; 820 m a.s.l., slope, ranker podzol), by comparing 34 parameters. The results show that the root system emergence is not affected by provenance but rather by site, namely by soil type and terrain slope. At an altitude of 540 m, both provenances produced an anchoring root system of circular floor projection with a rooting depth of 80 cm, while at an altitude of 820 m they had an elliptical superficial root system with a rooting depth of 45 cm. At the higher-situated plot, the provenance from an altitude of 1100 m showed a higher biomass, vitality, and specific length of fine roots

Tree growing space and acorn production of Quercus robur

Pedunculate oak (Quercus robur L.) is considered the main commercial tree species in conditions of floodplain forests of central Europe. In the Czech Republic clear-cutting silvicultural system represents a prevailing regeneration method of oak stands in floodplain forest. The natural regeneration is limited only in stands and years with sufficient acorn production. We analysed an acorn crop periodicity and an influence of canopy opening to the acorn production in South Moravian floodplain forest, Czech Republic between 2008 and 2011, The acorn crop from trees with different growth spaces in four stand conditions (closed canopy (A), moderate open canopy (B), open canopy (C) and single growing trees (D) with unlimited growth space was analysed. The amount of acorns was determined using seed traps placed under the crowns of sample trees. The average amount of acorns in 2009 and 2011 ranged from 13 (A) to 102 (C)/m2 and from 2 (A) to 124 (D)/m2, respectively. At the same time, a considerable difference was recorded in the fructification of sample trees in the various stand situations. Significant correlations were confirmed between the amount of acorns and crown size (r2 2009=0.27, r2 2011=0.07) of parent trees and their growth space – release area (r2 2009=0.42, r2 2011=0.25) and social area (r2 2009=0.31, r2 2011=0.26). The production of acorns could be affected by establishing conditions for the pedunculate oak with a minimum crown projection area in adult trees of at least 70 m2 and/or with a minimum release area of 300 m2, it means spacing between parent trees about 10 m or 100 trees per 1 ha

Quality of beechnuts from different crop years

Fresh and stored beechnuts collected in 1992, 1995, 1998, 1999 and 2000 were tested for viability and in 1998, 1999 and 2000 also for germination. Germination parameters such as peakvalue, mean weekly germination (modified mean daily germination), germination capacity, and germination value (Czabator 1962) were calculated from the germination test data and these indicators were also used for comparing beechnut quality. The highest viability for pre-stored beechnuts occurred in 1998 (80%) and this increased by 4% when these seedlots were stored for 1.5 years. Viability of fresh beechnuts collected in 1992, 1995 and 1999 was 64, 73 and 77%, respectively. Viability of 1995-collected beechnuts decreased after 3 years storage. Germination of fresh seeds was only done for the 1998, 1999 and 2000 collections where the best germination occurred for the 1998 collection. Germination of beechnuts collected in 1992 and 1995 was significantly reduced after 3 and 6 years of storage, respectively, while beechnuts collected in 1998 and stored for 1.5 years germinated about 15% better than fresh seeds of the 1999 crop. The peakvalue, mean weekly germination and the time required for viable seeds to reach 80% germination showed that the 1998 crop had the highest, overall quality. Eighty percent of the viable seeds collected in 1998 germinated in 9-10 weeks while fresh beechnuts from 1999 needed nearly 13 weeks to germinate, as did beechnuts collected in 1992 and stored for 7 years. Besides the germination capacity the germination value seems to be the very good indicator for determining the quality of stored beechnuts