Bacterial RNA virus MS2 exposure increases the expression of cancer progression genes in the LNCaP prostate cancer cell line

Bacteriophages effectively counteract diverse bacterial infections, and their ability to treat most types of cancer has been explored using phage engineering or phage-virus hybrid platforms. In the present study, it was demonstrated that the bacteriophage MS2 can affect the expression of genes associated with the proliferation and survival of LNCaP prostate epithelial cells. LNCaP cells were exposed to bacteriophage MS2 at a concentration of 1x10(7) plaque forming units/ml for 24-48 h. After exposure, various cellular parameters, including cell viability, morphology, and changes in gene expression, were examined. MS2 affected cell viability adversely, reducing viability by 25% in the first 4 h of treatment; however, cell viability recovered within 24-48 h. Similarly, the AKT, androgen receptor, integrin alpha 5, integrin beta 1, MAPK1, MAPK3, STAT3, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha genes, which are involved in various normal cellular processes and tumor progression, were significantly upregulated, whereas the expression levels of HSP90, ITGB5, ITGB3, HSP27, ITGAV, and PI3K genes were unchanged. Therefore, based on viability and gene expression changes, bacteriophage MS2 severely impaired LNCaP cells by reducing anchorage-dependent survival and androgen signaling. A caveolin-mediated endocytosis mechanism for MS2-mediated signaling in prostate cancer cells was proposed based on reports involving bacteriophages T4, M13, and MS2, and their interactions with LNCaP and PC3 cell lines

Bacterial RNA virus MS2 exposure increases the expression of cancer progression genes in the LNCaP prostate cancer cell line

Bacteriophages effectively counteract diverse bacterial infections, and their ability to treat most types of cancer has been explored using phage engineering or phage-virus hybrid platforms. In the present study, it was demonstrated that the bacteriophage MS2 can affect the expression of genes associated with the proliferation and survival of LNCaP prostate epithelial cells. LNCaP cells were exposed to bacteriophage MS2 at a concentration of 1x10(7) plaque forming units/ml for 24-48 h. After exposure, various cellular parameters, including cell viability, morphology, and changes in gene expression, were examined. MS2 affected cell viability adversely, reducing viability by 25% in the first 4 h of treatment; however, cell viability recovered within 24-48 h. Similarly, the AKT, androgen receptor, integrin alpha 5, integrin beta 1, MAPK1, MAPK3, STAT3, and peroxisome proliferator-activated receptor-gamma coactivator 1 alpha genes, which are involved in various normal cellular processes and tumor progression, were significantly upregulated, whereas the expression levels of HSP90, ITGB5, ITGB3, HSP27, ITGAV, and PI3K genes were unchanged. Therefore, based on viability and gene expression changes, bacteriophage MS2 severely impaired LNCaP cells by reducing anchorage-dependent survival and androgen signaling. A caveolin-mediated endocytosis mechanism for MS2-mediated signaling in prostate cancer cells was proposed based on reports involving bacteriophages T4, M13, and MS2, and their interactions with LNCaP and PC3 cell lines

Aspectos celulares da cartilagem epifisaria de rãs : elementos envolvidos nos processos de calcificação e crescimento osseo

Orientador: Hernandes Faustino de CarvalhoTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Os anfibios anuros ocupam uma posição inferior na escala evolutiva dos vertebrados e, além disso, apresentam uma postura de repouso e um modo de locomoção bem característico. Estas características certamente influenciam a estrutura, composição e organização da matriz extracelular dos ossos, cartilagens e tendões. Estudos sobre a estrutura da cartilagem epifisária destes animais têm revelado que esta cartilagem difere em vários aspectos do modelo descrito para aves e mamíferos. As duas principais diferenças consistem na localização da cartilagem de crescimento no interior do tubo ósseo metafisário e na existência de uma expansão lateral da cartilagem articular, cobrindo a face externa da extremidade do osso periosteal. Os estudos realizados por diferentes autores sugeriram a existência de um mecanismo de crescimento ósseo menos dependente da cartilagem de crescimento e mais relacionado com a atividade dos osteoblastos no periósteo. Ou seja, o crescimento longitudinal dos ossos longos ocorre por ossificação periosteal e não por ossificação endocondral. Além disso_ foram encontrados_ nos anuros, sítios ectópicos de calcificação na cartilagem articular. Considerando que estas características dos anuros diferem enormemente dos modelos conhecidos para aves e mamíferos, fazia-se relevante um estudo mais detalhado sobre a estrutura da cartilagem epifisária e sobre o crescimento dos ossos longos destes animais. Neste sentido, este projeto teve por objetivo caracterizar os aspectos celulares das diferentes regiões da cartilagem epifisária de Rana catesbeiana envolvidos com o crescimento ósseo e com a calcificação da cartilagem articular. Especial atenção, portanto, foi dispensada às características dos condrócitos da cartilagem de crescimento e dos osteoblastos no periósteo. Para isto, foram empregadas análises citoquímicas, cito químicas enzimáticas, imunocitoquímicas, de incorporação de marcadores de cálcio fluorescentes e de morte celular, além de análises ultra-estruturais. Os resultados obtidos, apresentados na forma de artigos, demonstraram que: 1) em Rana catesbeiana, entre a face interna da projeção lateral da cartilagem articular e a face externa da extremidade do osso periosteal, existe uma estrutura fibrosa complexa e especializada, denominado ligamento osteocondral. Este ligamento é constituído de duas regiões distintas, com células e matriz extracelular diferenciadas, exibindo um arranjo de fibras de colágeno e de células que permite a esta estrutura, ao mesmo tempo, garantir uma firme e flexível ancoragem da cartilagem articular no osso periosteal e promover o crescimento ósseo em comprimento e espessura; 2) a ossificação endocondral é um evento tardio e não desempenha um papel essencial no desenvolvimento e crescimento dos ossos longos nestes animais. Entretanto, quando estes animais crescem e ganham peso, aparentemente, a ossificação endocondral está presente, reforçando as extremidades ósseas. Além disso, a hipertrofia e morte dos condrócitos bem como a atividade de fosfatase alcalina e a calcifícação da matriz extracelular não estão diretamente associadas entre si e nem à formação de trabéculas ósseas; 3) a calcificação da cartilagem articular é um processo fisiológico nestes animais, aparecendo logo após a metamorfose e sendo estimulada por forças de compressão. Além disso, esta calcificação parece estar relacionada à formação de uma estrutura similar ao centro secundário de ossificação, encontrado nos animais com 4 anos pós-metamorfoseAbstract: Anurans are in a lower phylogenetic position when compared to birds and mammals. Furthermore, anurans present very distinct movement and posture behavior, which obviously affect the structure, composition and organization of the extracellular matrix of tendons. Studies about the structure of anuran epiphyseal cartilage have shown that this cartilage differs in several aspects from those found in birds and mammals. The two main differences are: the growth cartilage is localized inside the bone metaphyseal tube and there is a lateral expansion of articular cartilage, covering the externa I surface of the periostal bone. Different authors suggested the existence of abone growth mechanism independent of growth cartilage and more related to the osteoblasts activity on the periosteum. 1t means that the longitudinal growth of long bones occurs by periostea1 ossification rather than by endochondral ossification. Furthermore, in the anuran, ectopic sites of calcification were found in the articular cartilage. Considering that the anuran cartilage differs of the avian and mammalian counterparts, we decided to perform a detailed study on both the epiphyseal cartilage structure and the long bone growth in these animals. In this sense, this work characterized the cellular aspects of different regions of the epiphyseal cartilage of Rana catesbeiana involved with the bone growth and with articular cartilage calcification. Special attention was given to the characteristics of the growth cartilage chondrocytes and to the osteoblasts in the periosteum, using cytochemical analysis, enzymatic cytochemistry, immunocytochemistry, calcium probes, DNA fragmentation test and ultrastructural analysis. The results showed that: 1) there is a complex and specialized fibrous attachment (the osteochondralligament) in Rana catesbeiana, that anchors the inner face of the lateral articular cartilage expansion and the outer face of the periosteal bone end. This ligament has two distinct regions with different arrangements of the collagen fibers and cells, which warrant a strong and flexible anchorage of the articular cartilage to the periosteal bone, besides playing a role of periosteum and contributing to the growth of the long bones; 2) endochondral ossification is a late event and does not play an essential role in the developrnent and growth of long bones in R. catesbeiana. However, as the animals grow older and gain weight, further reinforcement of the bone ends is apparent1y necessary and endochondral ossification takes place. Furthermore, the chondrocyte hypertrophy and death as well as alkaline phosphatase activity and matrix calcification are neither directly associated to each other nor to the formation of endochondral bone; 3) Calcification of the articular cartilage is a non-pathological process that occurs after metamorphosis and is apparently stimulated by mechanical loading. Besides, this calcification precedes the formation of a similar structure to a secondary center of ossification of mammals, found in 4-year-old individualsDoutoradoBiologia CelularDoutor em Biologia Celular e Estrutura

A cartilagem epifisaria e o desenvolvimento dos ossos longos em Rana catesbeiana

Orientador: Hernandes Faustino de CarvalhoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A cartilagem pode ser descrita como um tecido avascular contendo células arredondadas separadas por uma matriz predominantemente basófila. As células da cartilagem, os condrócitos, produzem uma matriz extracelular constituída principalmente de fibrilas de colágeno tipo II e de grandes proteoglicanos agregantes. Em menores quantidades são encontrados outros tipos de colágeno e proteínas não colagênicas. Existem difererltes tipos de cartilagens distintas principalmente, na composição e organização dos componentes da sua matriz extracelular. Durante o desenvolvimento, os ossos longos são precedidos por um modelo cartilaginoso. Deste modelo cartilaginoso inicial, persiste nas extremidades dos ossos um tipo de cartilagem hialina denominada de cartilagem epifisária, que compreende a cartilagem articular e a cartilagem de crescimento. Dos diferentes tipos de cartilagens, a cartilagem de crescimento é talvez a mais dinâmica. Nesta cartilagem ocorrem processos de proliferação, maturação e hipertrofia celular. Estes processos são essenciais para o crescimento longitudinal do osso e formação de osso endocondral. Estas descrições são baseadas principalmente nos aspectos encontrados em aves e mamíferos, que já foram exaustivamente estudados. Pouco se sabe destes processos em anfíbios anuros, sendo que os trabalhos anteriores muitas vezes mostram-se contraditórios no que diz respeito à ossificação endocondral e à calcificação da cartilagem epifisária. Este trabalho teve por objetivos acompanhar o desenvolvimento e o envelhecimento da cartilagem epifisária de Rana catesbeiana, descrevendo os aspectos celulares e da matriz extracelular, com especial referência ao crescimento ósseo e a calcificação da cartilagem. Cartilagens epifisárias da epífise distal do fêmur e proximal da tíbio-fíbula de animais em diferentes fases de desenvolvimento e envelhecimento foram utilizadas e submetidas a testes histoquímicos, citoquímica enzimática e análises ultraestruturais. Os resultados demonstram que o fêmur e a tíbio-fíbula são formados por ossificacão periosteal de um modelo cartilaginoso. Este osso periosteal forma uma estrutura tubular cujas extremidades se encaixam na cartilagem epifisária. Esta cartilagem epifisária possui uma cartilagem articular bem desenvolvida e apresenta projeções laterais que recobrem a face externa do osso periosteal. Entre a cartilagem lateral e o osso periosteal encontra-se o ligamento osteo-condral. Este ligamento osteo-condral contem muita fibras de colágeno do tipo I e é muito vascularizado. Internamente à extremidade do osso periosteal encontra-se a cartilagem de crescimento com as zonas de reserva, proliferação, maturação e hipertrófica. As células da zona de proliferação são achatadas e separam-se no sentido perpendicular ao longo eixo do osso, ao contrário do que acontece nos mamíferos. Os condrócitos hipertróficos mais distais apresentam aspectos de degeneração. Existem trêm sítios distintos de calcificação da cartilagem epifisária. A calcificacão da cartilagem lateral é a mais freqüente e aumenta com o envelhecimento, os outros dois sítios são encontrados na cartilagem de crescimento. Os testes de atividade de fostatase alcalina revelaram intensa atividade desta enzima nos condrócitos próximos às áreas de calcificação e nos osteoblastos dos periósteo. Ossificação endocondral só é encontrada na epífise distal do fêmur de animais velhos, sendo muito reduzida e, aparentemente, associada à presença de canais da cartilagem. É nos animais velhos que aparece também a ossificação endosteal, com a formação de canais de Havers. Células mononucleadas são aparentemente responsáveis pela erosão da matriz da cartilagem hipertrófica não mineralizada e células semelhantes a osteoclastos ou condroclastos erodem a matriz calcificada. Em conjunto, os resultados revelam um tipo especializado de cartilagem epifisária capaz de permitir um crescimento ósseo rápido e de resistir aos impactos impostos às articulações durante os saltosAbstract: Cartilage is described as an avascular tissue with round cells embedded in a rather basophilic matrix. Cartilage cells, the chondrocytes, produce an extracellular matrix containing mainly type II collagen fibrils and proteoglycans. Minor collagens and noncollagens proteins are also present in the cartilage matrix. There are different types of cartilage, differing by the composition and organization of the matrix components. Developmentally, long bones are precceded by a cartilaginous scaffold. Cartilage tissue remains at the bone extremities where it is called epiphyseal cartilage. This cartilage can be divided in articular cartilage and in growth cartilage. Growth cartilage is probably the most dynamic cartilage because it bears processes such as cell proliferation, maturation and hypertrophy. These processes are essential for the growth of long bones and f9r endochondral bone formation. These processes are described following studies developed with birds and mammals, that were already exhaustivelly studied. Little knowledge exists on the growth of long bone in amphibians and previous publications are contradictory with regard to the endochondral ossification and the calcification of the epiphyseal cartilage. The objective of this work was to study the development and aging of the epiphyseal cartilage of Rana catesbeiana, describing the cells and extracellular matrix, with especial attention to their role in bone growth and cartilage calcification. Epiphyseal cartilage from the distal femoral epiphysis and from the proximal tibio-fibula epiphysis of animaIs with different ages were used and subjected to histochemistry, enzyme cytochemistry and ultrastructural analyses. The results showed that the femur and tibio-fibula are constructed by periosteal ossification of a cartilaginous scaffold. This periosteal bone beco me a tubular structure which inserts into the epiphyseal cartilage. Epiphyseal cartilage contains a well developed articular cartilage and presents lateral projections that cover the external face of the periosteal bone. In between the lateral cartilage and the periosteal bone there is a osteochondral ligament. This ligament contains many collagen fibers and blood vessels. The growth cartilage is found within the periosteal bone with its reserve, proliferation, maturation and hypertrophic zones. Cells of the proliferation zone are flat and divide in the longitudinal direction with to respect to the length of the bone, as opposed to what happens in mammals. Hypertrophic chondrocytes present degeneration aspects. There are three distinct calcification sites in the epiphyseal cartilage. Lateral cartilage calcification is more frequent and increase with age, while the other sites are found in the growth cartilage. Alkaline phosphatase activity was found in chondrocytes close to the calcification sites and also in the osteoblasts of the periosteum. Endochondral ossification is only found in the distal femoral epiphysis of old animaIs. However, it was very reduced and apparently associated with cartilage canaIs. Old animaIs also has endosteal bone containing haversian systems. Mononucleated cells are responsible for the erosion of the uncalcified cartilage matrix and osteoclast-like or chondroclast-like cells erode the calcified matrix. Together, the results show that the bullfrog epiphyseal cartilage is very especialized tissue able of allow a fast bone growth and to support impacts on the joint during jumpsMestradoBiologia CelularMestre em Ciências Biológica

Doxazosin Treatment Alters Stromal Cell Behavior And Increases Elastic System Fibers Deposition In Rat Prostate.

Doxazosin (DOX), an α-adrenoceptor antagonist, induces the relaxation of smooth muscle cell tonus and reduces the clinical symptoms of benign prostatic hyperplasia (BPH). However, the effects of DOX in the prostate stromal microenvironment are not fully known. In a previous study, we showed that DOX treatment for 30 days increased deposition of collagen fibers in the three rat prostatic lobes. Herein, we investigated the effects of DOX on stromal cell ultrastructure and elastic fiber deposition. Adult Wistar rats were treated with DOX (25 mg/kg/day); and the ventral, dorsal, and anterior prostates were excised at 30 days of treatment. The prostatic lobes were submitted to histochemical and stereological-morphometric analyze and transmission electron microscopy (TEM). Histochemical staining plus stereological analysis of the elastic fiber system showed that DOX-treated prostatic lobes presented more elaunin and elastic fibers than controls, mainly in the ventral lobe. Ultrastructural analysis showed that fibroblasts and smooth muscle cells from DOX-treated prostates presented active synthetic phenotypes, evidenced by enlarged rough endoplasmic reticulum and Golgi apparatus cisterns, and confirmed the observation of thickened elaunin fibers. Our findings suggest that, under α-adrenergic blockade by DOX, the fibroblasts become more active and smooth muscle cells shift from a predominantly contractile to a more synthetic phenotype. The deposition of collagen and elastic system fibers in the prostatic stroma may counterbalance the absence of smooth muscle tone during α-blockers treatment.731036-4

Doxazosin reduces cell proliferation and increases collagen fibers in rat prostatic lobes

We investigated the effects of doxazosin (Dox), an alpha-adrenoceptor antagonist used clinically for the treatment of benign prostatic hyperplasia (BPH), on the rat prostatic complex by assessing structural parameters, collagen fiber content, cell proliferation, and apoptosis. Adult Wistar rats were treated with Dox (25 mg/kg per day), and the ventral (VP), dorsolateral, and anterior prostate (AP) regions of the prostate complex were excised at 3, 7, and 30 days after treatment. At 24 h before being killed, the rats were injected once with 5-bromodeoxyuridine (BrdU; thymidine analog) to label mitotically active cells. The prostates were weighed and processed for histochemistry, morphometry-stereology, immunohistochemistry for BrdU, Western blotting for proliferating cell nuclear antigen (PCNA), and the TUNEL reaction for apoptosis. Dox-treated prostate lobes at day 3 presented increased weight, an enlarged ductal lumen, low cubical epithelial cells, reduced epithelial folds, and stretched smooth muscle cells. However, at day 30, the prostates exhibited a weight reduction of ∼20% and an increased area of collagen and reticular fibers in the stromal space. Dox also reduced epithelial cell proliferation and increased apoptosis in the three prostatic lobes. Western blotting for PCNA confirmed the reduction of cell proliferation by Dox, with the AP and VP being more affected than the dorsal prostate. Thus, Dox treatment alters epithelial cell behavior and prostatic tissue mechanical demand, inducing tissue remodeling in which collagen fibers assume a major role. © 2007 Springer-Verlag