Down-regulation of murine beta-defensin-2 in Lewis lung carcinoma cells results in accelerated growth of tumor cells in vitro and in vivo

To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. Materials and Methods: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma (3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic ability of obtained cell lines cultivated in vitro were analyzed using 3H-incorporation technique and colony formation in semi-soft medium, respectively. Expression of mBD-2 mRNA was studied by semiquantative RT-PCR analysis. Also, transfected cells were transplanted to C57B mice, and the patterns of tumor growth in vivo were analyzed by routine techniques. Results: We have found out that in the 3LL cells transfected with pcDNA3-mBD-2 and pcDNA3-Igk-mBD-2, the expression of mBD-2 mRNA is significantly down regulated compared to wild-type cells and 3LL cells transfected with blank vector. The cells with suppressed mBD-2 expression differed from parental cells and cells transfected with blank vector by higher proliferation rate (p < 0.001) and higher clonogenic ability. The 3LL-mBD-2 and 3LL-Igk-mBD-2 cells that are transplanted to C57B mice gave rise to more aggressive tumors that possessed significantly higher growth rate (p < 0.01) than those that arise from wild-type 3LL cells. Conclusion: The obtained results are evidencing on a possible tumor-suppressing role of mBD-2 expression.Цель: настоящая работа посвящена анализу противоопухолевых свойств бета-дефенсина-2 мыши (mBD-2) in vitro and in vivo. Материалы и методы: на основе pcDNA3.1+ вектора были созданы 2 плазмидных конструкта, кодирующих зрелую форму mBD-2, содержащие или несодержащие сигнальную последовательность Igk (pcDNA3mBD-2 и pcDNA3Igk-mBD-2 соответственно). Путем трансфекции клеток 3LL полученными векторами, а также контрольным вектором pcDNA3.1+ были получены клеточные линии (3LL-mBD-2, 3LL-Igk-mBD-2 и 3LL-pcDNA3), для которых были проведены исследования их пролиферативной активности, определенной по уровню включения 3 Н-тимидина в ДНК, и способности к колониеобразованию в среде, содержащей метилцеллюлозу. Экспрессию гена mBD-2 исследовали с помощью полуколичественного ОТ-ПЦР-анализа. Трансфецированные клетки были имплантированы мышам линии C57BL, после чего была проанализирована динамика роста опухоли. Результаты: установлено, что в трансфектных клеточных линиях 3LL-mBD-2 и 3LL-Igk-mBD-2 уровень экспрессии mBD-2 снижен по сравнению с контрольными. Эти клетки характеризовались достоверным повышением уровня пролиферации (р < 0,001) и способности к колониеобразованию. Клетки сублиний 3LLmBD-2 и 3LL-Igk-mBD-2, трансплантированные мышам линии C57BL, вызывали развитие более агрессивных опухолей, обладающих значительно более высокой скоростью роста (p < 0,01), чем таковые, вызванные перевивкой клеток 3LL. Выводы: полученные результаты свидетельствуют о возможной роли mBD-2 как опухолевого супрессора

Expression of human beta-defensins-1, 2 and 4 mRNA in human lung tumor tissue: a pilot study

To analyze the patterns of human beta-defensin-1, 2, 4 (hBDs) expression in human lung tumors. Materials and Methods: Tissue samples of surgically resected human lung tumors (squamous cell carcinoma (SCC), n = 10; adenocarcinoma (AC), n = 10) paired with conditionally normal tissue samples were analized for expression of hBD-1, 2, 4 mRNA by semiquantitive RT-PCR. Results: In a number of studied lung cancer tissue samples, overexpression of defensin mRNA was registered: hBD-1 mRNA (50% of SCC and 60% AC), hBD-2 mRNA (60% of SCC and 50% of AC) or hBD-4 (40% of SCC and 20% AC). No correlation was detected between the levels of hBD-1, hBD-2 and hBD-4 mRNA and histological type, differentiation grade of the tumor, and the stage of the disease, as well as the content of hBD-2 peptide in blood serum of lung cancer patients. Conclusion: Human beta-defensins-1 and -2 are often up-regulated in human lung tumors.Цель: проанализировать особенности экспрессии мРНК бета-дефенсинов-1, 2, 4 (hBDs) в ткани опухоли легкого человека. Материалы и методы: с помощью метода полуколичественного RT-PCR-анализа изучали уровень экспрессии мРНК hBD-1, 2, 4 в образцах ткани хирургически удаленных опухолей легкого человека (плоскоклеточный рак — ПКР, n = 10; аденокарцинома — AК, n = 10) по сравнению с образцами условно-нормальной ткани легкого тех же пациентов. Результаты: в ряде исследованных образцов опухолей легкого выявлена повышенная экспрессия мРНК hBD-1 (50% ПКР и 60% AК), hBD-2 (60% ПКР и 50% АК) или hBD-4 (40% ПКР и 20% AК). Зависимости между уровнем экспрессии бета-дефенсинов и гистологическим типом опухоли, стадией заболевания и содержанием пептида hBD-2 в сыворотке крови больных не установлено. Выводы: в ткани опухоли легкого человека часто активирована экспрессия hBD-1 и hBD-2

Human beta-defensin-2 controlS cell cycle in malignant epithelial cells: in vitro study

Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. Materials and Methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting. Results: According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 µM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)2D3 has shown similar cell growth suppression effect of native endogenously produced hBD-2. Conclusion: The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB

Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma

Aim: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL). Materials and Methods: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and used for intradermal vaccination. Experimental cancer vaccines introduced i. d. at therapeutic and prophylactic regimens to 3LLbearing C57Bl mice, were applied alone or in combination with VD3 (administered per os) and/or low-dose cyclophosphamide (CP, administered intraperitoneal). Efficacy of treatments was analyzed by primary tumor growth dynamics indexes and by metastasis rate in vaccinated animals. Results: As it has been shown, administration of the protein-based vaccine composed from mBD-2 and 3LL cell lysate in combination with VD3 and CP, but not in VD3 free setting, led to significant suppression of primary tumor growth (p < 0.005) and had significant antimetastatic effect. Introduction of VD3 with or without CP in the scheme of treatment with mBD-2-Muc1-DNA vaccine at therapeutic regimen has led to significant suppression of primary tumor (p < 0.05) and metastasis volumes (p < 0.005), while in the groups of animals treated with DNA-vaccine + VD3 with or without CP at prophylactic regimen, significant antimetastatic effect (p < 0.05) and elevation of average life-span (p < 0.05) have been registered. Conclusion: The results of this pilot study have shown promising clinical effects of VD3 administration in combination with cancer vaccinotherapy in vivo

Fusion expression of recombinant human beta-defensin-3 and analysis of its biological activity

Human beta-defensins (hBDs) are small cationic antimicrobial peptides with multiple biologic activities. The aim of the study was cloning, expression in E.coli, purification and in vitro analysis of biological activity of recombinant human beta-defensin-3 (rec-hBD-3). hBD-3 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E.coli BL21(DE3) cells. Rec-hBD-3 was expressed in bacterial cells as GST-hBD-3 fusion protein, and purified by 3-step procedure via affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography on Sep-Pack C18. Analysis of biological activity of rec-hBD-3 has shown that the peptide is active against Pseudomonas aeruginosa in micromolar concentrations in radial diffusion test. Rec-hBD-3 did not affect proliferation and viability of cultured human cancer cells of A431, A549, and TPC-1 lines, but was capable to potentiate cytotoxic effects of rec-hBD-2 and docetaxel in vitro

Lexical and syntactic features of academic Russian texts: a discriminant analysis

This article presents three mathematical models to differentiate academic texts from three subject discourses written in Russian (i.e., Philological, Mathematical, and Natural Sciences) which further enable design and automated profiling of corresponding typologie

Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells

Aim: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. Materials and Methods: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques. Results: T-rex HEK-HBD2-m cell subline was shown to express both mRNA and hBD-2m protein upon the presence of 1 µg/ml tetracycline in culture medium as it was demonstrated by RT-PCR and immunocytochemical approach. Upon prolonged expression of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. Conclusion: Expression of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival.Цель: создать линию клеток с регулируемой экспрессией бета-дефенсина-2 человека (hBD-2) и проанализировать влияние экспрессии этого пептида на особенности фенотипа и рост клеток. Материалы и методы: клеточная сублиния T-rex HEK hBD2-m, экспрессирующая биологически активную зрелую форму hBD-2 при индукции клеток тетрациклином, получена путем клонирования на основе эмбриональных клеток почки человека линии HEK293T. Морфологические особенности, характеристики роста и показатели колониеобразования в полужидкой среде исследовали стандартными методами. Результаты: с помощью методов РТ-ПЦР и иммуноцитохимии показано, что сублиния клеток T-rex HEK-HBD2- экспрессирует hBD2 в присутствии 1 µг/мл тетрациклина в среде инкубации. Продолжительная экспрессия hBD-2 приводила к тому, что клетки утрачивали способность образовывать монослой при культивировании in vitro и образовывать колонии в полужидкой среде, но характеризовались более высокой скоростью роста и способностью к более продолжительному выживанию в бессывороточной среде, чем исходная линия клеток HEK293T. Выводы: экспрессия hBD-2 в сублинии клеток T-rex HEK-HBD2- cell обусловливает специфические биологические эфеекты, способствующие выживанию клеток

Involvement of human beta-defensin-2 in intracellular signaling: in vitro study

Aim: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. Materials and Methods: A431cells were cultured in the presence of 1 µg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. Results: 30 min incubation of A431 cells with 1 µg/ml of hBD-2 didn’t influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment – in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment – in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. Conclusion: Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells.Цель: проанализировать участие бета-дефенсина-2 человека (hBD-2) в механизмах передачи внутриклеточных сигналов в модели in vitro. Материалы и методы: клетки линии A431 культивировали в присутствии 1 µг/мл рекомбинантного hBD-2 и/или 10 нг/мл ЭФР. Экспрессию мРНК альфа- и бета-изоформ p70S6 киназы оценивали методом полуколичественного ОТ-ПЦР анализа. Экспрессию и активность p70S6K, фосфорилирование PDK1, ERK, JNK, p38 киназ и рецептора ЭФР (ЭФРР) исследовали методом Вестерн-блот анализа. Результаты: 30 мин инкубация клеток A431 с 1 µг/мл hBD-2 не оказывала влияния на уровень аутофосфорилирования ЭФРР, но препятствовала образованию димеров рецептора в присутствии ЭФР. В то же время 30 мин обработка клеток hBD-2 приводила к активации p70S6K, 12 ч — к значительному повышению уровня мРНК альфа- и бета-изоформ p70S6 киназы, а 24 ч — к повышению синтеза p70S6K на уровне белка. PDK1-киназа, фосфорилирующая p70S6K, также подвергалась фосфорилированию в присутствии экзогенного hBD-2 in vitro. Выводы: данные свидетельствуют об участии каскада PDK1-p70S6K в опосредовании действия hBD-2 в клетках A431

Immunohistochemical analysis of beta-defensin-2 expression in human lung tumors

The aim of this paper is to present research was directed on analysis of the expression patterns of human beta-defensin-2 (hBD-2) in human lung tumors

Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells

Background and Aim: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. Methods: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. Results: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100–1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21WAF1 expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. Conclusion: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21WAF1 expression and activation of pRB. Key Words: human beta-defensin-2, melanoma, proliferation, viability, cell cycle, B-Raf, anchorage-independent growth