Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

Functional Study of Oncogenic Transcription Factor ERG and its Signaling in Prostate Cancer

TMPRSS2–ERG is the most frequent type of genomic rearrangement present in prostate tumors, in which the 5- prime region of the TMPRSS2 gene is fused to the ERG oncogene. TMPRSS2, containing androgen response elements (AREs), is regulated by androgens in the prostate. The truncated TMPRSS2-ERG fusion transcript is overexpressed in half of the prostate cancer patients. The formation of TMPRSS2-ERG transcript is an early event in prostate carcinogenesis and previous in vivo and in vitro studies have shown ectopic ERG expression to be associated with increased cell invasion. However, the molecular function of ERG and its role in cell signaling is poorly understood. In this study, genomic rearrangement of ERG with TMPRSS2 was studied by using comparative genomic hybridization (CGH) in prostate cancer samples. The biological processes associated with the ERG oncogene expression in prostate epithelial cells were studied, and the results were compared with findings observed in clinical prostate tumor samples. The gene expression data indicated that increased WNT signaling and loss of cell adhesion were a characteristic of TMPRSS2- ERG fusion positive prostate tumor samples. Up- regulation of WNT pathway genes were present in ERG positive prostate tumors, with frizzled receptor 4 (FZD4) presenting with the highest association with ERG overexpression, as verified by quantitative reverse transcription-PCR, immunostaining, and immunoblotting in TMPRSS2-ERG positive VCaP prostate cancer cells. Furthermore, ERG and FZD4 silencing increased cell adhesion by inducing active β1-integrin and E-cadherin expression in VCaP cells. Furthermore, we found a novel inhibitor, 4-(chloromethyl) benzoyl chloride which inhibited the WNT signaling and induced similar phenotypic effects as observed after ERG or FZD4 down regulation in VCaP cells. In conclusion, this work deepens our understanding on the complex oncogenic mechanisms of ERG in prostate cancer that may help in developing drugs against TMPRSS2-ERG positive tumors.

Efeito da inundação lateral sobre a distribuição da vegetação ripária em um trecho do rio Cuiabá, MT

Estudos hidrológicos e fitossociológicos foram realizados num perfil topográfico de 550 m instalado perpendicularmente ao rio Cuiabá, no Município de Rosário Oeste, MT, para analisar a influência da inundação sobre a distribuição da vegetação ripária. Utilizando os dados fluviométricos da estação de Rosário Oeste de 1966 a 2003, um modelo hidrológico de remanso em regime subcrítico foi ajustado, permitindo estabelecer a série hidrológica na área de estudo. A partir dessa série, os intervalos de recorrência nessa área foram obtidos. Os resultados sugeriram que a margem do canal principal, o canal secundário e o dique marginal, a planície de inundação e o terraço alagam a cada ~1 1,7 ano, ~1,77 2,8 anos, ~3 anos e de ~3,25 a ~39 anos, respectivamente. A espécie Combretum leprosum Mart. (Combretaceae) apresentou maior VI na margem do canal principal, Callisthene fasciculata (Spr.) Mart. (Vochysiaceae) na margem do canal principal e no terraço e Licania parvifolia Huber (Chrysobalanaceae), Cariniana estrellensis (Raddi) Kuntze. (Lecythidaceae) e Vochysia divergens Pohl. (Vochysiaceae) na planície de inundação. Os resultados indicaram que a frequência e, principalmente, o tempo de alagamento são os principais determinantes ecológicos da distribuição das espécies vegetais ao longo do perfil topográfico.

High-Throughput Screening for Novel Prostate Cancer Drug Targets –Getting Personal

Prostate cancers form a heterogeneous group of diseases and there is a need for novel biomarkers, and for more efficient and targeted methods of treatment. In this thesis, the potential of microarray data, RNA interference (RNAi) and compound screens were utilized in order to identify novel biomarkers, drug targets and drugs for future personalized prostate cancer therapeutics. First, a bioinformatic mRNA expression analysis covering 9873 human tissue and cell samples, including 349 prostate cancer and 147 normal prostate samples, was used to distinguish in silico prevalidated putative prostate cancer biomarkers and drug targets. Second, RNAi based high-throughput (HT) functional profiling of 295 prostate and prostate cancer tissue specific genes was performed in cultured prostate cancer cells. Third, a HT compound screen approach using a library of 4910 drugs and drug-like molecules was exploited to identify potential drugs inhibiting prostate cancer cell growth. Nine candidate drug targets, with biomarker potential, and one cancer selective compound were validated in vitro and in vivo. In addition to androgen receptor (AR) signaling, endoplasmic reticulum (ER) function, arachidonic acid (AA) pathway, redox homeostasis and mitosis were identified as vital processes in prostate cancer cells. ERG oncogene positive cancer cells exhibited sensitivity to induction of oxidative and ER stress, whereas advanced and castrate-resistant prostate cancer (CRPC) could be potentially targeted through AR signaling and mitosis. In conclusion, this thesis illustrates the power of systems biological data analysis in the discovery of potential vulnerabilities present in prostate cancer cells, as well as novel options for personalized cancer management.

Chemical Biology Screen for Prostate Cancer Therapeutics

Prostate cancer initially responds to hormone-based therapeutics such as anti-androgen treatment or chemotherapeutics but eventually becomes resistant. Novel treatment options are therefore urgently needed. This thesis study applied a high-throughput screen of 4910 known drugs and drug-like small molecules to identify compounds that selectively inhibit growth of prostate cancer cells. In addition, the mechanisms underlying the cellular sensitivity to potent cancer selective compounds were addressed. Surprisingly, many of the compounds currently used in the clinics or studied in clinical trials were not cancer-selective. Only four drugs, aldehyde dehydrogenase inhibitor disulfiram (Antabus), antibiotic ionophore monensin, histone deacetylase inhibitor tricostatin A and fungicide thiram inhibited prostate cancer cell growth at nanomolar concentrations without major effects on non-malignant prostate epithelial cells. Disulfiram, monensin and a structurally similar compound to monensin, salinomycin, induced oxidative stress and inhibited aldehyde dehydrogenase activity. Moreover, monensin and salinomycin reduced androgen receptor signalling and steroidogenesis, enforced cell differentiation and reduced the overall levels of cancer stem cells. Taken together, novel and potentially prostate cancer-selective therapeutic agents were identified in this study, including the description of a multitude of intoxicating mechanisms such as those relating to oxidative stress. The results provide novel insights into prostate cancer biology and exemplify useful means of considering novel approaches to cancer treatment.

Tratamento cirúrgico da ginecomastia com pedículos lateral e medial

Ginecomastia é o aumento da mama masculina que pode acometer até 65% dos indivíduos deste sexo na fase infanto-puberal, compreendida entre 13 e 16 anos. Tem como principais causas hepatite ou cirrose hepática, carcinoma ou doenças inflamatórias pulmonares crônicas, carcinomas ou disfunções testiculares, tumores glandulares (pituitária, supra-renal), alterações dos níveis séricos de testosterona, síndromes genéticas (síndrome de Klinefelter, p.ex.), uso de drogas como heroína, maconha ou anabolizantes e hanseníase. Podemos classificar a ginecomastia quanto ao volume, quanto aos tecidos que a compõem (gordurosa ou pseudoginecomastia, glandular e mista), ou quanto ao tratamento necessário para sua correção cirúrgica (pequena, moderada e grave). O tratamento das formas mais graves de ginecomastia é muito diferente daquele aplicado às formas mais suaves, pois nas formas graves, além da ressecção dos tecidos gorduroso e glandular, existe a necessidade de ressecção da pele em excesso e o reposicionamento do complexo aréolo-mamilar. O objetivo deste trabalho é descrever uma técnica cirúrgica específica para estes pacientes portadores de formas graves de ginecomastia, através de dois pedículos dermogordurosos, um lateral e um medial, com aproximadamente 2cm de espessura, mantendo assim a nutrição do complexo aréolo-mamilar. Esses pedículos são delimitados entre as bissetrizes dos quadrantes súpero-lateral e ínfero-lateral, e súpero-medial e ínfero-medial, tendo o mamilo como vértice. Na área de pele excessiva periareolar, obtida através do pinçamento interdigital, é realizada a desepidermização dos pedículos lateral e medial e ressecção de toda pele e tecido celular subcutâneo até a fáscia peitoral nas regiões superior e inferior aos pedículos; a síntese é realizada em dois planos, sendo periareolar a cicatriz resultante. Foram operados com esta técnica vinte pacientes com forma grave de ginecomastia, com média etária de 23,3 anos; sendo seis pacientes da raça negra. O bom posicionamento do complexo aréolo-mamilar e uma cicatriz periareolar resultante, bem como a retirada de conteúdo suficiente, foram as principais vantagens observadas. Como complicações, tivemos assimetria das placas aréolo-mamilares em dois casos, nos quais havia acentuada diferença entre os dois lados na avaliação pré-operatória; cicatrização hipertrófica em um paciente da raça negra, cuja cicatriz foi atenuada com injeções intracicatriciais de triancinolona; necrose parcial de aréola em um caso, cuja ferida cicatrizou por segunda intenção, dispensando qualquer tratamento local posterior; deiscência de sutura periareolar em um caso, no qual foi feita a ressutura, com bom resultado, e quatro pacientes apresentaram coleção sero-hemática subcutânea, que foram drenadas e não apresentaram recidiva.

Tratamento cirúrgico do megacólon chagásico: retocolectomia abdominal com anastomose colorretal mecânica término-lateral

Trinta e cinco doentes portadores de megacólon chagásico foram operados pela técnica da retocolectomia abdominal com anastomose colorretal mecânica término-lateral durante o período de 1993 a 1997. Vinte (57,14%) doentes eram do sexo feminino e 15 (42,85%) do masculino. A idade variou de 27 a 76 anos, com média de 51 anos. A operação constou de ressecção do segmento dilatado, sepultamento do coto retal na altura da reflexão peritoneal com grampeador, dissecção do espaço retrorretal até o plano dos músculos elevadores e anastomose colorretal mecânica término-lateral posterior. Em quatro (11,42%) doentes a anastomose foi anterior. Em três (8,57%) doentes, o teste de escape da anastomose foi positivo, o que obrigou a complementação manual da sutura em dois (5,71 %) e sutura e ostomia derivativa em um (2,85%). Ocorreram sete (20,00%) complicações pós-operatórias precoces, sendo quatro consideradas relevantes (11,42%) e quatro (11,42%) complicações tardias. Houve um (2,85%) óbito por complicação clínica. Os doentes submetidos a colostomia foram reoperados para fechamento da mesma sem intercorrências. A totalidade dos doentes apresenta hábito intestinal normal. Não houve referências a alterações gênito-urinárias, nem a incontinência fecal. A anastomose foi tocada ou visibilizada em todos os pacientes examinados, durante o seguimento ambulatorial. Não houve casos de fecaloma no coto retal. Embora os resultados iniciais sejam bastante satisfatórios, é necessário maior tempo de observação para se avaliar a possibilidade de recidiva.

Free Prostate-specific Antigen Forms and Kallikrein-related Peptidase 2: Tools for Prostate Cancer Diagnostics

Prostate-specific antigen (PSA) is a marker that is commonly used in estimating prostate cancer risk. Prostate cancer is usually a slowly progressing disease, which might not cause any symptoms whatsoever. Nevertheless, some cases of cancer are aggressive and need to be treated before they become life-threatening. However, the blood PSA concentration may rise also in benign prostate diseases and using a single total PSA (tPSA) measurement to guide the decision on further examinations leads to many unnecessary biopsies, over-detection, and overtreatment of indolent cancers which would not require treatment. Therefore, there is a need for markers that would better separate cancer from benign disorders, and would also predict cancer aggressiveness. The aim of this study was to evaluate whether intact and nicked forms of free PSA (fPSA-I and fPSA-N) or human kallikrein-related peptidase 2 (hK2) could serve as new tools in estimating prostate cancer risk. First, the immunoassays for fPSA-I and free and total hK2 were optimized so that they would be less prone to assay interference caused by interfering factors present in some blood samples. The optimized assays were shown to work well and were used to study the marker concentrations in the clinical sample panels. The marker levels were measured from preoperative blood samples of prostate cancer patients scheduled for radical prostatectomy. The association of the markers with the cancer stage and grade was studied. It was found that among all tested markers and their combinations especially the ratio of fPSA-N to tPSA and ratio of free PSA (fPSA) to tPSA were associated with both cancer stage and grade. They might be useful in predicting the cancer aggressiveness, but further follow-up studies are necessary to fully evaluate the significance of the markers in this clinical setting. The markers tPSA, fPSA, fPSA-I and hK2 were combined in a statistical model which was previously shown to be able to reduce unnecessary biopsies when applied to large screening cohorts of men with elevated tPSA. The discriminative accuracy of this model was compared to models based on established clinical predictors in reference to biopsy outcome. The kallikrein model and the calculated fPSA-N concentrations (fPSA minus fPSA-I) correlated with the prostate volume and the model, when compared to the clinical models, predicted prostate cancer in biopsy equally well. Hence, the measurement of kallikreins in a blood sample could be used to replace the volume measurement which is time-consuming, needs instrumentation and skilled personnel and is an uncomfortable procedure. Overall, the model could simplify the estimation of prostate cancer risk. Finally, as the fPSA-N seems to be an interesting new marker, a direct immunoassay for measuring fPSA-N concentrations was developed. The analytical performance was acceptable, but the rather complicated assay protocol needs to be improved until it can be used for measuring large sample panels.

Anastomose esôfago-jejunal látero-lateral com grampeador linear cortante complementada por sutura manual

One of the most difficult procedures in digestive-tract surgery is esophago-jejunal anastomosis following total gastrectomy. Cost/benefit analysis of this procedure justifies the use of mechanical staplers, in spite of their high cost. A technical variant of the side-to-side esophago-jejunal anastomosis is presented, which incorporates the use of a cutting linear stapler. Technical maneuvers are easy to perform, the cost of the cutting linear stapler is smaller than the circular ones, the amplitude of the anastomosis is wider and the likelihood of fistulae is smaller when compared to other techniques. The side-to-side esophago-jejunal anastomosis with the cutting linear stapler is always complemented by a manual suture.

Duodenojejunostomia látero-lateral: uma alternativa cirúrgica para o tratamento do "dumping" pós gastrectomia

Most patients with partiaI gastrectomy have no postoperative problems. Some of them, however, develop severe nutritional disturbance, particularly those with Bilroth II reconstruction, which can be treated clinically in the majority of the cases. Those with important mal absorptive problems require surgery. In this article we present a technical alternative for those patients who need reintervention over a BII operation. Our technique has the advantage of not touching the duodenum previously sutured.

Modificação técnica da coledocoduodenostomia látero-lateral - análise dos resultados

OBJETIVO: Avaliar se a oclusão do colédoco distal se mantém no decorrer do tempo, nos pacientes submetidos a coledocoduodenostomia látero lateral com oclusão do colédoco distal. MÉTODO: Foram analisados 14 doentes submetidos à coledocoduodenostomia látero-lateral modificada por Fava, para prevenir a "Síndrome do Colédoco Distal", tratados de coledocolitíase não complicada. Os doentes avaliados encontravam-se em pós-operatório que variou de três meses até dez anos após a derivação bílio-digestiva. Os doentes foram analisados do ponto de vista clínico através da classificação de Visick, submetidos à dosagem de transanimases (AST e ALT), enzimas canaliculares (gama-GT e fosfatase alcalina), bilirrubinas e ao exame de colangioressonância. Os doentes que apresentaram alguma alteração nos exames citados, foram submetidos à CPRE para avaliação definitiva da via biliar, identificação e tratamento de eventuais complicações da coledocoduodenostomia RESULTADOS: Em 11 doentes (78,6%) foi identificada abertura da oclusão do colédoco distal. Quatro doentes com menos de um ano de pós-operatório; quatro, entre um e cinco anos; e três doentes com mais de cinco anos de coledocoduodenostomia. Três doentes (21,4%) apresentaram "Sump Syndrome" no período de três meses, nove meses e oito anos de pós-operatório respectivamente, sendo tratados com sucesso através de papilotomia endoscópica. CONCLUSÕES: Nos doentes tratados de coledocolitíase não complicada a oclusão do colédoco distal na coledocoduodenostomia modificada por Fava et al, não se mantém patente no decorrer do tempo, não evitando o aparecimento da "Síndrome do Colédoco Distal".

Hérnia encarcerada em orifício de trocarte lateral diagnosticada por tomografia computadorizada

Incisional hernia is an uncommon complication in laparoscopic surgery. The majority of the hernias are located in the umbilical site. Nevertheless, they can occur in the lateral trocar site, although they are rarely diagnosed. We report a case of a 55 year-old patient who underwent a videolaparoscopic hysterectomy and developed small bowel obstruction on the third postoperative day. This initially gave rise to the diagnosis of paralytic ileum. The definitive diagnosis of incarcerated hernia in the lateral trocar site was established after an abdominal computed tomography was performed.

Variação anatômica da artéria torácica interna lateral: relato de caso

The objective of this report was to describe a variation in the origin of the lateral internal thoracic artery (LITA), a variable large-caliber artery in the thoracic wall. This report presents a case in which a trunk coming from the subclavian artery (SCA) bifurcates and gives origin to the LITA and internal thoracic artery (ITA). This case demonstrates an unusual bilateral origin for the LITA, which emerges together with the ITA rather than directly from the SCA, as could be expected. Although such presentation is uncommon, the possibility that it could be damaged during surgical interventions such as thoracotomy and pleural drainage justifies our report .

Fatores prognósticos e impacto da comorbidade na laringectomia fronto-lateral

OBJETIVO: Avaliar sobrevida, impacto da comorbidade, complicações e fatores de falha da laringectomia como tratamento de tumores malignos glóticos. MÉTODOS: Foram analisadas 38 pacientes com tumor glótico sob estadiamento clínico T1b/T2N0M0 submetidos à laringectomia fronto-lateral com reconstrução, de janeiro de 1995 a dezembro de 2006. Foram avaliados os resultados oncológicos, comorbidades (através da escala Adult Comorbidity Evaluation - 27 ACE-27) e complicações, sendo correlacionados com dados demográficos e características do tumor. RESULTADOS: Oito pacientes apresentaram recidiva local e foram resgatados cirurgicamente. Complicações não foram verificadas em 33 pacientes. Não houve diferença significativa das sobrevidas global em cinco anos e livre de doença ao considerarem-se as diferentes categorias de comorbidades. Somente o envolvimento patológico das margens mostrou diferenças significativas na sobrevida global (p=0,0033) e sobrevida livre de doença (p<0,0001). CONCLUSÃO: A sobrevida global em cinco anos foi de 67,6% e a sobrevida livre de doença de 73,7%; a comorbidade não representou fator prognóstico independente; o índice de complicações pós-operatórias foi de 13,2% e somente o envolvimento patológico das margens mostrou diferenças significativas na sobrevida global e livre de doença.

Ressecção laparoscópica do segmento lateral esquerdo do fígado em doador para transplante hepático inter-vivos

Laparoscopic resection of the left lateral segment of the liver in donors of living liver transplantation. The authors present a case of laparoscopic resection of the left lateral segment of the liver in a donor of living liver transplantation. The procedure was done in six hours and the left lateral segment of the liver was removed through a 15 cm right subcostal incision. The patient was discharged on the 5th post-operative day. A 40 mL intrabdominal collection of bile was percutaneously drained guided by ultra-sonography. The drain was removed after five days. Afterwards, the patient had good recovery with no other complication.