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.

A Farewell to Flat Biology. Three-dimensional Cell Culture Models in Cancer Drug Target Identification and Validation

Cells of epithelial origin, e.g. from breast and prostate cancers, effectively differentiate into complex multicellular structures when cultured in three-dimensions (3D) instead of conventional two-dimensional (2D) adherent surfaces. The spectrum of different organotypic morphologies is highly dependent on the culture environment that can be either non-adherent or scaffold-based. When embedded in physiological extracellular matrices (ECMs), such as laminin-rich basement membrane extracts, normal epithelial cells differentiate into acinar spheroids reminiscent of glandular ductal structures. Transformed cancer cells, in contrast, typically fail to undergo acinar morphogenic patterns, forming poorly differentiated or invasive multicellular structures. The 3D cancer spheroids are widely accepted to better recapitulate various tumorigenic processes and drug responses. So far, however, 3D models have been employed predominantly in the Academia, whereas the pharmaceutical industry has yet to adopt a more widely and routine use. This is mainly due to poor characterisation of cell models, lack of standardised workflows and high throughput cell culture platforms, and the availability of proper readout and quantification tools. In this thesis, a complete workflow has been established entailing well-characterised 3D cell culture models for prostate cancer, a standardised 3D cell culture routine based on high-throughput-ready platform, automated image acquisition with concomitant morphometric image analysis, and data visualisation, in order to enable large-scale high-content screens. Our integrated suite of software and statistical analysis tools were optimised and validated using a comprehensive panel of prostate cancer cell lines and 3D models. The tools quantify multiple key cancer-relevant morphological features, ranging from cancer cell invasion through multicellular differentiation to growth, and detect dynamic changes both in morphology and function, such as cell death and apoptosis, in response to experimental perturbations including RNA interference and small molecule inhibitors. Our panel of cell lines included many non-transformed and most currently available classic prostate cancer cell lines, which were characterised for their morphogenetic properties in 3D laminin-rich ECM. The phenotypes and gene expression profiles were evaluated concerning their relevance for pre-clinical drug discovery, disease modelling and basic research. In addition, a spontaneous model for invasive transformation was discovered, displaying a highdegree of epithelial plasticity. This plasticity is mediated by an abundant bioactive serum lipid, lysophosphatidic acid (LPA), and its receptor LPAR1. The invasive transformation was caused by abrupt cytoskeletal rearrangement through impaired G protein alpha 12/13 and RhoA/ROCK, and mediated by upregulated adenylyl cyclase/cyclic AMP (cAMP)/protein kinase A, and Rac/ PAK pathways. The spontaneous invasion model tangibly exemplifies the biological relevance of organotypic cell culture models. Overall, this thesis work underlines the power of novel morphometric screening tools in drug discovery.

Libyan breast cancer: Health services and biology. Diagnosis delay and prognostic value of DNA pploidy, S-phase fraction, and Ki-67 and Bcl-2 immunohistochemistry

The aim of this study was to investigate the diagnosis delay and its impact on the stage of disease. The study also evaluated a nuclear DNA content, immunohistochemical expression of Ki-67 and bcl-2, and the correlation of these biological features with the clinicopathological features and patient outcome. 200 Libyan women, diagnosed during 2008–2009 were interviewed about the period from the first symptoms to the final histological diagnosis of breast cancer. Also retrospective preclinical and clinical data were collected from medical records on a form (questionnaire) in association with the interview. Tumor material of the patients was collected and nuclear DNA content analysed using DNA image cytometry. The expression of Ki-67 and bcl-2 were assessed using immunohistochemistry (IHC). The studies described in this thesis show that the median of diagnosis time for women with breast cancer was 7.5 months and 56% of patients were diagnosed within a period longer than 6 months. Inappropriate reassurance that the lump was benign was an important reason for prolongation of the diagnosis time. Diagnosis delay was also associated with initial breast symptom(s) that did not include a lump, old age, illiteracy, and history of benign fibrocystic disease. The patients who showed diagnosis delay had bigger tumour size (p<0.0001), positive lymph nodes (p<0.0001), and high incidence of late clinical stages (p<0.0001). Biologically, 82.7% of tumors were aneuploid and 17.3% were diploid. The median SPF of tumors was 11% while the median positivity of Ki-67 was 27.5%. High Ki-67 expression was found in 76% of patients, and high SPF values in 56% of patients. Positive bcl-2 expression was found in 62.4% of tumors. 72.2% of the bcl-2 positive samples were ER-positive. Patients who had tumor with DNA aneuploidy, high proliferative activity and negative bcl-2 expression were associated with a high grade of malignancy and short survival. The SPF value is useful cell proliferation marker in assessing prognosis, and the decision cut point of 11% for SPF in the Libyan material was clearly significant (p<0.0001). Bcl-2 is a powerful prognosticator and an independent predictor of breast cancer outcome in the Libyan material (p<0.0001). Libyan breast cancer was investigated in these studies from two different aspects: health services and biology. The results show that diagnosis delay is a very serious problem in Libya and is associated with complex interactions between many factors leading to advanced stages, and potentially to high mortality. Cytometric DNA variables, proliferative markers (Ki-67 and SPF), and oncoprotein bcl-2 negativity reflect the aggressive behavior of Libyan breast cancer and could be used with traditional factors to predict the outcome of individual patients, and to select appropriate therapy.

Floral biology and breeding system of three Ipomoea weeds

The floral biology of three weeds, Ipomoea cairica, I. grandifolia and I. nil (Convolvulaceae), was studied in Botucatu and Jaboticabal, São Paulo, in southeastern Brazil. The three species are melittophilous, with a varied set of floral visitors, but with some overlapping. Cluster analysis using Jacquard similarity index indicated a greater similarity among different plant species in the same locality than among the populations at different places, in relation to floral visitor sets. The promiscuous and opportunistic features of the flowers were shown, with such type of adaptation to pollination being advantageous to weeds since pollinator availability is unpredictable at ruderal environments.

Phenology, fruit set and dispersal of Cordia multispicata Cham., an important weed shrub of abandoned pastures in eastern Amazonia

ABSTRACT - (Phenology, fruit set and dispersal of Cordia multispicata Cham., an important weed shrub of abandoned pastures in eastern Amazonia). The reproductive ecology of the distylous tropical shrub Cordia multispicata was studied in an abandoned pasture in Paragominas County , Pará state, Brazil. It is a common species in the Amazon basin where it occurs as a weed in open and disturbed habitats. C. multispicata has many flowers per inflorescence (85 ± 12) but 84% abort before fertilization. Flowering occurs throughout the year. Fruits are small, with a red fleshy pericarp (skin-pulp) attractive to birds. Fruit set is lower during the dry season (less than 30%) and higher during the rainy season when there are many visits of insects to the flowers. Fruiting has a peak between the end of the dry season and the middle of the rainy season. Nineteen bird species were observed foraging for the fruits of C. multispicata, and 79% of those species can be considered as potential dispersal agents. The efficient seed dispersal and aggregated spatial distribution associated with some characteristics of the dispersors greatly contributed to the success of this species in abandoned pastures of eastern Amazonia.

Biologia floral de cinco espécies de Passiflora L. (Passifloraceae) em mata semidecídua

(Biologia floral de cinco espécies de Passiflora L. (Passifloraceae) em mata semidecídua). O estudo da biologia floral de cinco espécies de Passiflora foi feito em uma mata de planalto em Campinas, São Paulo. Passiflora alata, P. amethystina e P. miersii apresentam flores de cor púrpura a violeta e corona variegada. As flores são diurnas, perfumadas, autoincompatíveis e polinizadas por abelhas de grande porte. Passiflora amethystina e P. miersii diferem de P. alata por apresentarem filamentos livres no opérculo, que em P. alata é horizontal e denticulado. Estas diferenças no opérculo promovem comportamentos característicos das abelhas durante as visitas. Passiflora suberosa possui flores verde-amareladas e opérculo plicado. As flores são diurnas, inodoras, autocompatíveis e polinizadas por vespas. Em P. capsularis as flores são brancas e o opérculo é plicado. As flores são noturnas, perfumadas, autocompatíveis e possivelmente polinizadas por mariposas. O opérculo plicado das duas últimas espécies permite que os visitantes tenham fácil acesso ao néctar.

Monostromatic green algae (Ulvales, Chlorophyta) of São Paulo and Paraná states (Brazil): distribution, growth, and reproduction

(Monostromatic green algae (Ulvales, Chlorophyta) of São Paulo and Paraná states (Brazil): distribution, growth, and reproduction). Culture studies were used for taxa identification and to understand aspects of the biology and physiology of monostromatic green blades growing in various sites along the coast of São Paulo state (23º30'-25ºl2'S, 45º10'-48ºW) and one site in Paraná state (25º35'S, 48º21'W), southeast and south Brazil, respectively. Possible variations of the growth rate, age of reproduction and life history were tested under different conditions of temperature, salinity and day length. Two species were found: Ulvaria oxysperma (Kützing) Bliding and Monostroma sp. The first one has been previously reported for many temperate and tropical estuaries around the world. Green monostromatic blades with the same life-history and ontogeny as Monostroma sp. have been reported so far only for the tropical coast of Brazil. Species are distinct in their ontogeny of the thallus (constant under different conditions) and limiting temperatures of survival. U. oxysperma grows and reproduces from 10 to 25ºC and dies when maintained at 30ºC; Monostroma sp. does not reproduce at 15ºC and survives at 30ºC. The different salinities and day lengths that were tested had no significant effect on either species.

Biologia floral e sistema de reprodução de Jacquemontia multiflora (Choisy) Hallier f. (Convolvulaceae)

Aspectos da fenologia, biologia da polinização e reprodução de Jacquemontia multiflora foram estudados na Fazenda Catalunha, Santa Maria da Boa Vista-PE. J. multiflora é uma liana anual, que apresenta floração do tipo cornucópia, com pico desta fenofase no bimestre março/abril, que corresponde ao final da estação chuvosa. As flores estão reunidas em cimeiras que apresentam eixo principal desenvolvido, expondo as flores acima da folhagem. As flores são raso-campanuladas, azuis, inodoras e secretam pequenas quantidades de néctar. A antese é diurna, ocorrendo por volta da 5:30h., e a duração das flores é de aproximadamente nove horas, podendo ser consideradas como efêmeras. Abelhas Apidae e Halictidae são os visitantes mais frequentes. Apis mellifera e Trigona spinipes são consideradas como principais polinizadores desta espécie. Quanto ao sistema de reprodução J. multiflora é autógama facultativa, produzindo frutos e sementes por autopolinização manual (30%) e também por polinização cruzada (60%).

Fly pollination and pollinator sharing in two synchronopatric species: Cordia multispicata (Boraginaceae) and Borreria alata (Rubiaceae)

The floral biology of Cordia multispicata (Boraginaceae) and Borreria alata (Rubiaceae) was studied in natural populations in a fragment of the Atlantic forest in Pernambuco, northeastern Brazil. Both species flower during almost the whole year. Cordia multispicata is a shrubby species with white, distylous and tubular flowers. Borreria alata is a herbaceous species. Its flowers are whitish, tubular and have a polymorphism in relation to the size of their style. Floral anthesis in both species begins at 6:00 a.m. Sugar concentration in the nectar was about 16% in C. multispicata and 30% in B. alata. Nine species of flies, mainly of the genus Palpada (Syrphidae), were observed visiting flowers of the two species. Seven of them were observed visiting and pollinating flowers of both C. multispicata and B. alata. Two species visited only flowers of C. multispicata, whereas no fly was exclusive to B. alata flowers. Both species have similar flower morphology, flowering time, habitats in the forest and establish populations very close to each other. These facts can favour the pollinators’ sharing and increase pollinator attraction.

Late-acting self-incompatibility in Capparis retusa (Capparaceae), a species of Chaco woodland in NE Argentina

The reproductive biology of Capparis retusa was studied by means of controlled pollination experiments and fluorescence microscopy observations of post-pollination events. Self-pollinated flowers mostly failed to form fruits despite the fact that self-pollen tubes grew to the ovary and penetrated ovules within 24 h. Since embryo development to globular stage was observed in some self-fertilized ovules it was concluded that control of self-fertilization in this species occurs by some kind of post-zygotic mechanism.

Biologia floral de Unonopsis guatterioides (A. D.C.) R.E. Fr., uma Annonaceae polinizada por Euglossini

A biologia floral e a polinização por abelhas Euglossini de uma Annonaceae é descrita pela primeira vez e as adaptações a esse modo de polinização são comparadas às de outras espécies da família, que é predominantemente polinizada por coleópteros. A espécie Unonopsis guatterioides foi estudada em dois fragmentos de mata de terra firme no município de Manaus (AM). As flores são protogínicas e permanecem abertas expondo os estigmas e estames durante a antese que ocorre entre dois dias. São visitadas por machos de Eulaema bombiformis (Euglossini) que coletam o odor o qual se assemelha ao de "capim cidreira" (Andropogum nardus L.). Visitas de abelhas Meliponidae e coleópteros Chrysomelidae também foram registradas. Unonopsis guatterioides apresenta dicogamia sincronizada.

Biologia reprodutiva de Calibrachoa elegans (Miers) Stehmann & Semir (Solanaceae)

Calibrachoa elegans é uma espécie anual, endêmica da região da canga do quadrilátero ferrífero de Minas Gerais, Brasil. O estudo da sua biologia reprodutiva envolveu experimentos de cruzamentos, teste do sistema de auto-incompatibilidade e observação do processo de polinização. Os experimentos realizados através de polinizações manuais indicam que a população estudada é preferencialmente alógama. A interrupção do crescimento do tubo polínico no estilete das flores autopolinizadas, verificada através da microscopia de fluorescência, confirmou a ocorrência de reação de auto-incompatibilidade. A polinização é realizada apenas pelas fêmeas de Hexantheda missionica (Colletinae, Apoideae), uma abelha com alta fidelidade de visita às flores de C. elegans. Os machos desta espécie utilizam as flores como fonte de néctar, como local para pouso entre os vôos de patrulhamento a procura de fêmeas para acasalamento e como abrigo noturno. Para se acomodarem na flor, geralmente cortam e removem os estames e o estilete. As populações de Hexantheda missionica encontradas em Minas Gerais são o primeiro registro da espécie para a região sudeste do Brasil e, provavelmente, representam remanescentes que chegaram a esta região no passado e que agora se encontram disjuntas e isoladas das populações da região sul do Brasil.

Pollination ecology of Tabebuia aurea (Manso) Benth. & Hook. and T. ochracea (Cham.) Standl. (Bignoniaceae) in Central Brazil cerrado vegetation

The pollination ecology and breeding systems of Tabebuia aurea (Manso) Benth. & Hook., and T. ochracea (Cham.) Standl. were investigated in an area of cerrado vegetation in the Federal District of Brazil. These species occur sympatrically, flower massively and synchronously for a month, during the dry season (July to September). Both have diurnal anthesis, with similar floral structures, a yellow tubular corolla and produce nectar. Fourteen species of bees visited both Tabebuia species, but, only three Centris species and Bombus morio, were considered potential pollinators, because of their high frequency on the flowers and their efficiency in carrying pollen. Tests on the breeding systems of T. aurea and T. ochracea demonstrated that boths species are self-incompatible, with late-acting self-incompatibility. The proportion of fruit set from cross pollination (T. aurea 17.2% and T. ochracea 12.3%) in both species was low considering the great number of flowers displayed. This suggests a lack of maternal resources for fruit-set. The great amount of seeds per fruit (about 92 in T. aurea and 285 in T. ochracea) may represent an investment of maternal resources allocated on higher quality of fertilized ovules.

Thermal-biological aspects on the seed germination of Cucumis anguria L.: influence of the seed coat

The seed coat influences the early stages of germination of many seeds and sometimes maintains seed dormancy. Early reports have shown that the testa influences the germination response of Cucumis anguria seeds to light although the response to temperature as influenced by the tegument is not well understood. The main purpose of this study was to observe the influence of the testa on the germination of Cucumis anguria by using parameters as germinability and isothermal germination rate. The assays were carried out in a thermal-gradient block with water imbibed seeds kept in darkness. Estimates of the activation enthalpies (deltaH) show |deltaH| < 50 kJ.mol-1 between 26.1 °C and 35.2 °C (intact seeds) and between 25.4 °C and 35.2 °C (scarified seeds), whereas at temperatures greater than 35.2 °C the germination may be limited by processes with |deltaH| > 125 kJ.mol-1. It is suggested that the testa limits embryo expansion rather than interfering with diffusion processes.

Ruellia brevifolia (Pohl) Ezcurra (Acanthaceae): fenologia da floração, biologia da polinização e reprodução

A biologia reprodutiva de Ruellia brevifolia foi estudada em populações naturais do sudeste brasileiro. R. brevifolia é um subarbusto que forma agrupamentos densos em áreas abertas mas sombreadas e floresce o ano todo, produzindo flores casmógamas (CH) e cleistógamas (CL) nas estações úmida e seca, respectivamente. A cleistogamia é do tipo climática, originando sementes no período de maior estresse ambiental. Flores CH são inodoras e possuem corola tubulosa e vermelha, sendo hercogâmicas ou não; apresentam protoginia, antese diurna e duram dois dias. Néctar é o principal recurso floral, sendo produzido em baixa quantidade (5,2 mil ± 2,36) e com concentração de açúcares em torno de 30%. Flores CL assemelham-se a botões jovens de flores CH e apresentam redução de tamanho principalmente no androceu e na corola. A razão pólen/óvulo das flores CL é ca. de 60% menor que a das flores CH devido a menor quantidade de grãos de pólen. R. brevifolia é autocompatível embora alogamia seja favorecida pela protoginia e pelas flores hercogâmicas. Autopolinização espontânea parece estar relacionada às flores CH não hercogâmicas. Beija-flores, principalmente Amazilia lactea, e borboletas, em especial Heliconius etilla narcaea e Phoebis argante, são os polinizadores de R. brevifolia. Os beija-flores visitam R. brevifolia apenas durante os picos de floração, quando há elevado número de flores. A baixa quantidade de néctar por flor, bem como as visitas de A. lactea em "linha-de-captura", provavelmente, promovem a polinização cruzada. Embora as borboletas visitem especialmente dentro e entre agrupamentos próximos, também visitam as flores nos períodos de baixa densidade floral, quando se deslocam a maiores distâncias, favorecendo a polinização cruzada.