We have shown herein that β1 integrin stably depleted PC3 sub-clonal cells confer a trend towards increased survival of mice compared to β1 integrin expressing counterparts when tested in an intracardial bone metastasis model. Therefore, we sought to investigate novel factors that mediate β1 integrin-dependent cellular migration and three dimensional growth of prostate cancer PC3 cells in vitro. We show herein that depletion of β1 integrin using siRNA directed techniques results in increased SPARC protein expression. We further show that suppression of SPARC by β1 integrin appears to occur through a JNK dependent mechanism. Moreover, siRNA mediated depletion of β1 integrin results in impaired sphere formation in 3D BME assays. This was mediated in part by the increased production of SPARC. β1 integrin-depleted cells also diminished the enhanced migration of cells on the predominant bone matrix, collagen I. Concomitant SPARC depletion in β1 integrin-depleted cells did not rescue this enhanced migration. These findings suggests that the role of β1 integrin in mediating 3D growth of PC3 cells occurs at least in part through the suppression of SPARC protein expression.

Orientadoesr: Wagner Eduardo Matheus, Ubirajara Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T13:05:18Z (GMT). No. of bitstreams: 1 Silva_ElcioDias_D.pdf: 1974038 bytes, checksum: e1e7b084d346a50a85fec60666fbf526 (MD5) Previous issue date: 2012 / Resumo: Objetivo: avaliar a eficácia relativa do leuprolide 3,75 mg, leuprolide 7,5 mg e goserelina 3,6 mg quanto à redução da testosterona sérica, em níveis de castração. Método: foram avaliados 60 pacientes com carcinoma avançado de próstata, com indicação de bloqueio hormonal. Os pacientes foram divididos em 3 grupos de 20: Grupo 1) pacientes que receberam leuprolide na dose 3,75 mg; Grupo 2) receberam leuprolide 7,5 mg, Grupo 3) receberam goserelina 3,6 mg. Todos os grupos foram tratados por aplicação mensal das respectivas medicações. Os pacientes foram avaliados com dosagens de testosterona sérica em 2 momentos: pré-tratamento e após 3 meses de tratamento. Resultados: a idade dos pacientes foi semelhante nos três grupos, com mediana de 72, 70 e 70 anos, respectivamente aos grupos 1, 2 e 3. Dos pacientes que receberam leuprolide 3,75 mg, leuprolide 7,5 mg e goserelina 3,6 mg, 26,3%, 25% e 35%, respectivamente, não obtiveram nível de castração, considerando nível de corte de testosterona ? 50 ng/dl; e 68,4%, 30% e 45%, respectivamente, não obtiveram nível de castração, considerando nível de corte de testosterona ? 20 ng/dl. Conclusão: não houve diferença significativa na obtenção de níveis de castração com leuprolide 3,75 mg, leuprolide 7,5 mg e goserelina 3,6 mg, quando comparadas as três substâncias em conjunto. Quando comparados os grupos dois a dois, houve diferença estatisticamente significativa na análise do leuprolide 3,75 mg com o leuprolide 7,5 mg, havendo maior obtenção de nível de castração com 7,5 mg, quando se considera este nível como igual ou menor que 20 ng/dl. A importância dessa eventual diferença, no entanto, deve ser vista com cautela, já que a comparação dos três grupos simultaneamente, muito embora próximo, não atingiu o nível de significância estabelecido / Abstract: Purpose: to evaluate the relative efficiency of leuprolide 3.75 mg, leuprolide 7.5 mg, and goserelin 3.6 mg in relation to the reduction of serum testosterone, regarding the levels of castration. Methods: we evaluated prospectively 60 randomized patients with advanced prostate carcinoma, with indication for hormone blockade. The patients were divided into 3 groups of 20: Group 1) received leuprolide 3.75 mg; Group 2) received leuprolide 7.5 mg; group 3) received goserelin 3.6 mg. All groups were treated with monthly application of the respective drugs. Results: the patients' ages were similar within the three groups, with a median of 72, 70, and 70 in groups 1, 2, and 3, respectively. Of the patients that received leuprolide 3.75 mg, leuprolide 7.5 mg, and goserelin 3.6 mg, 26.3%, 25%, and 35%, respectively, did not reach castration levels, considering a testosterone cut of ?50 ng/dl. 68.4%, 30%, and 45%, respectively, did not reach castration levels, considering a testosterone cut of ?20 ng/dl. Conclusions: There were no statiscally significant differences in the levels of castration when comparing with leuprolide 3.75 mg, leuprolide 7.5 mg, and goserelin 3.6 mg, when comparing the three substances altogether. When compared in groups of two, there was a statiscally significant difference between leuprolide 3.75 mg and leuprolide 7.5 mg, in which the latter presented better results in reaching castration levels, when considered equal or less than 20ng/dl. The importance of this difference, however, must be measured with caution, since the comparison of the three groups simultaneously did not reach the established significance level, even though it came close / Doutorado / Fisiopatologia / Doutor em Ciências

Magister Scientiae (Medical Bioscience) - MSc(MBS) / This study investigated the effect of the C. capensis extract (0.001, 0.01, 0.1, 1, 10, 100 1000 μg/ml) on LNCaP prostate cancer, TM3 Leydig and TM4 Sertoli cells for 24 and 96 hours. The following parameters were investigated: morphology, cell viability (XTT), testosterone modulation, DNA fragmentation (TUNEL), lactate dehydrogenase activity (LDH), testosterone production, anti-cancer drug combination. In a separate set of experiments, parameters affecting the initiation, progression and metastasis of cancer were investigated. These included the ability of the aqueous C. capensis rhizome extract to inhibit of reactive oxygen species (ROS) and reactive nitrogen species (RNS) production, and to inhibit collagenase and elastase activity.

Philosophiae Doctor - PhD / There are over 100 different types of cancer, and each of these cancers are classified by the type of cell that it initially affects. For the purpose of this research we will be focussing on prostate cancer (PC). Prostate cancer is the second most common form of cancer in men around the world and annually approximately 4500 men in South Africa are diagnosed making PC a global epidemic. Prostate cancer is a type of cancer which starts in the prostate it is normally a walnut-sized gland found right below the bladder. PC follows a natural course, starting as a tiny group of cancer cells that can grow into a tumour. In some men if PC is not treated it may spread to surrounding tissue by a process called direct invasion/ spread and could lead to death. Current diagnostic tests for prostate cancer have low specificity and poor sensitivity. Although many PC's are slow growing there is currently no test to distinguish between these and cancers that will become aggressive and life threatening. Therefore the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity of current available diagnostic test is required. Biomarkers have recently been identified as a viable option for early detection of disease for example biological indicators ie. DNA, RNA, proteins and microRNAs (miRNAs). Since first described in the 1990s, circulating miRNAs have provided an active and rapidly evolving area of research that has the potential to transform cancer diagnostics and prognostics. In particular, miRNAs could provide potentially new biomarkers for PC as diagnostic molecules. Circulating miRNAs are highly stable and are both detectable and quantifiable in a range of accessible bio-fluids, having the potential to be useful as diagnostic, prognostic and predictive biomarkers. In this study we aimed to identify miRNAs as potential biomarkers to detect and distinguish between various types of PC in its earliest stage. The major objectives of the study were to identify miRNAs and their gene targets that play a critical role in disease onset and progression to further understand their mechanism of action in PC using several in silico methods, and to validate the potential diagnostic miRNAs using qRT-PCR in several cell lines. The identification of specific miRNAs and their targets was done using an "in-house" designed pipeline. Bioinformatic analyses was done using a number of databases including STRING, DAVID, DIANA and mFold database, and these combined with programming and statistical analyses was used for the identification of potential miRNAs specific to PC. Our study identified 40 miRNAs associated with PC using our "in-house" parameters in comparison to the 20-30 miRNAs known to be involved in PC found in public databases e.g. miRBase. A comparison between our parameters and those used in public databases showed a higher degree of specificity for the identification PC-associated miRNAs. These selected miRNAs were analysed using different bioinformatics tools, and were confirmed to be novel miRNAs associated with PC. The identified miRNAs were experimentally validated using qRT-PCR to generate expression profiles for PC as well as various other cancers. Prostate lines utilised in this study included PNT2C2 (normal) which was compared to BPH1 (Benign) and LNCaP (Metastatic). In the study the expression profiles of eight potential miRNA biomarkers for the detection of PC was determined using qRT-PCR, and to distinguish PC from other cancers. QRT-PCR data showed that miRNA-3 and -5 were up-regulated in the BPH1 and LNCaP when compared to PNT2C2. In addition miRNA-8 was also shown to be up-regulated in LNCaP. Based on these results it was shown that a miRNA profile could be established to distinguish between BPH1 and the LNCaP prostate cell lines. The results suggest that one miRNA as a diagnostic marker may be sufficient to differentiate between different cancer cell lines. Furthermore by creating a unique profile for each cancer cell line by using a combination of miRNAs could be a suitable approach as well. Finally, it was shown that through the use of a single or combination of all eight miRNAs a unique profile for all the cancer cell lines tested in this study can be created. This is an important finding which could have potential diagnostic or prognostic implications in clinical practice.

>Magister Scientiae - MSc / Prostate cancer (PCa) is the second most common form of cancer in men around the world. In many parts of Africa, data on prostate cancer is sparse. This is attributed to poor access to testing and diagnostics. The International Agency for Research on Cancer (GLOBOCAN) estimated that 28,000 deaths occurred as a result of PCa in Africa in 2008, 4500 of which were in South Africa. This figure (28,000) is predicated a rise to 57,000 over the next two decades. Currently, the most commonly used diagnostic tests for PCa are the DRE and PSA tests. The former is highly invasive and both have low specificity and poor sensitivity. Therefore, the need for a less invasive early detection method with the ability to overcome the lack of specificity and sensitivity is required. Biomarkers have recently been identified as a viable option for early detection of disease. Examples of biological indicators for disease are miRNAs. miRNAs are small non-coding RNA molecules which play a key role in controlling gene expression and certain biological processes. Studies have shown that aberrantly expressed miRNAs are a hallmark of several diseases like cancer. miRNA expression has been shown to be associated with tumour development, progression and response to therapy, suggesting their possible use as diagnostic, prognostic and predictive biomarkers. The study aimed to investigate the potential of miRNAs implicated in prostate cancer as putative biomarkers for the disease and evaluating these miRNAs in a panel of prostate as well as several other cancer cell lines using qRT-PCR. An in silico approach was used to identify 13 putative miRNAs implicated in prostate cancer of which 8 were further analysed in a parallel study and 5 in this study. Two publicly available target prediction software were used for target gene prediction of the 5 identified miRNAs. The target genes were subjected to functional analysis using web-based software, DAVID. Functions which were clustered with an enrichment score of 1.3 and greater were considered significant. Targets with gene ontologies linked to “transcription regulation”, “regulation of “apopotosis”, “extracellular region” and “metal ion binding” were considered for further analyses. Protein gene interaction analysis was performed to determine the pathways the target genes are involved in using STRING. Expression profile analysis of the genes in various tissues was also carried out using in silico methods through the TiGER and GeneHub-GEPIS databases. Analysis using DAVID resulted in 9 gene targets for the 5 miRNAs. It was found that miR3 seemed the most promising miRNA for biomarker validation based on the in silico analyses. Its target gene MNT was found to be abundantly expressed in prostate tissue from the TiGER results. The GeneHub-GEPIS results also indicated that the gene’s expression is up-regulated during prostate cancer. The expression levels of the miRNAs analysed using qRT-PCR indicated that miR3 is significantly over-expressed in prostate cancer cells when compared to the other cancer cell lines used in this study, corroborating the results observed from the in silico analyses. Another miRNA with interesting results was miR5. It was predicted to target two genes, YWHAZ and TNFSF13B. In TiGER, both were found to be expressed in prostate tissue. The genes were also found to be up regulated during prostate cancer in GeneHub-GEPIS. The expression level of miR5 in LNCaP was 15.32; it was significantly up-regulated in the cell line using qRT-PCR. However, miR5 was also present in HEPG2-7.06, MCF7-0.79, HT29-1.61 and H157-3.59. Thus, it was concluded it can be used as a biomarker in combination with other miRNAs. The miRNA miR2 was found to target the actin filament protein encoding gene AFAP1. The gene was predicted to be upregulated with a DEU of 33.25 in GeneHuB-GEPIS. The qRT-PCR analysis showed that the expression ratio in LNcaP was 8.79. However, miR2 expression was up-regulated in MCF7-0.85 and HT29-1.09 as well. The expression level of miR1 in BHP1 was found to be 4.85. It can be considered as an indicator for benign prostate hyperplasia. Future work would include investigating the expression of miR3 in a larger panel of cancer cells as well as in patient samples. In addition, analysis of the UTR sequences of the miRNAs targets experimentally to prove that the target genes identified using in silico methods, are indeed regulated by these miRNAs. Furthermore, performing gene “knock-out” studies on the genes that code for the miRNAs to study their roles in prostate cancer development.

Enhanced resistance to radiation could be caused by both chronic hypoxia and acute hypoxic which has been reported in prostate cancer in various studies. Therefore currently used dose prescriptions (70Gy in 35 fractions) for external beam radiation therapy (EBRT) of prostate cancer has been suggested insufficient to provide optimum clinical outcome. In this study, we propose a Biologically Guided Radiation Therapy approach to boost dose in hypoxic prostate tumor regions while sparing the urethra. A previously proposed hypoxia model was modified for prostate cancer and incorporated into treatment plan optimization. The concept of equivalent uniform dose (EUD) was used in the optimization and evaluation of results. CT data from 25 prostate cancer patients who recently received EBRT at the British Columbia Cancer Agency (BCCA) and hypothetical hypoxic regions manually drawn on these CT scans were selected for this study. The results show that our methods could boost dose in target volume to substantially higher levels. EUD of planning target volume increased to more than 80Gy, despite accounting for effects of hypoxia. This increase was achieved with only minor changes in dose in normal tissues, typically less than 5Gy. Notably, urethra sparing was excellent with a EUD around 64Gy. Robustness of the proposed approach is verified against various hypoxic settings. EUD comparison between RT plans in biological guided and conventional approaches using the same RT technique (Volumetric Modulated Arc Therapy) also suggests that biologically guided radiation therapy (BGRT) approach is more suitable for dose painting purposes with the advantage of delivering sufficient dose to hypoxia region in different scenarios and sparing normal tissue better. Furthermore, we also investigated the impact of inter-fraction patient set-up error and intra-fraction organ motion on the high dose gradients achieved with this proposed dose painting method and explored the feasibility of adapting geometrical uncertainties (represented as systematic error and random error) into treatment planning. Image error obtained from EPID images are used to derive systematic uncertainty and random uncertainty. During the geometrical uncertainty adapted optimization, dose matrix in PTV is shifted based on systematic error and convolved with a Gaussian kernel which is pre-calculated using random error. CT sets and organ contours from five patients who enrolled in the previous dose painting i study are selected. For each of them, seven plans are generated using cumulated uncertainty data which was collected after every five fractions. We also present the outcome in terms of equivalent uniform dose (EUD). For four of the patients, EUD history of all seven plans suggests using the proposed optimization method with uncertainty data from the first five fractions, it is possible to achieve the same target coverage of static treatment plans (difference in EUD less than 1Gy). Meanwhile, the elimination of PTV margin also leads to a significant dose reduction (more than 15Gy) in rectum. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Le cancer de la prostate est la troisième cause de mortalité par cancer chez l’homme après les cancers colorectaux et broncho-pulmonaires avec, en 2010, près de 150 000 décès. On voit donc tout l’intérêt d’élaborer de nouvelles stratégies thérapeutiques en essayant de prendre en compte à la fois : i) la métabolisation qui peut modifier in vivo l’efficacité de la molécule initiale et ii) les différences individuelles intervenant à ce niveau du fait de la nature de l’environnement du patient.Nous avons porté notre étude, en collaboration avec l’Ecole HEI de Lille, sur l’intérêt de la Phenstatine (molécule en développement pré-clinique) et de ses métabolites puisque qu’au cours de ces dernières années des résultats thérapeutiques prometteurs ont été obtenus, dans les cancers prostatiques hormono-résistants avec les taxanes, dont le rôle est de stabiliser les microtubules en inhibant leur dépolymérisation.L’objectif de ce travail de thèse a donc été dans un premier temps d’étudier l’effet pharmacologique de la Phenstatine et de ses métabolites issus de sa biotransformation par des microsomes hépatiques humains sur la polymérisation de la tubuline ainsi que sur la prolifération de la lignée cancéreuse prostatique humaine PC3. Nous avons ensuite essayé de déterminer les CYPs responsables de la formation des métabolites identifiés et quantifiés. Enfin, nous avons abordé l’influence de certaines conditions environnementales, plus particulièrement la présence de cadmium, sur le métabolisme de ces composés originaux et donc sur leur activité vis-à-vis de cellules cancéreuses prostatiques. Pour tenter de comprendre la réponse ou la non réponse à ces différents traitements, nous avons suivi les profils d’expression de gènes impliqués dans la prise en charge cellulaire des xénobiotiques en utilisant une technologie fonctionnant à haut débit et basée sur le principe de la PCR quantitative en temps réel (TaqmanTM Low Density Arrays). Un modèle d’étude a pu ainsi être élaboré afin de montrer l’implication de l’environnement sur l’efficacité d’une molécule à visée anticancéreuse. Ce modèle devrait par la suite pouvoir être élargi à d’autres agents participant à la métabolisation des xénobiotiques et déboucher sur la mise en évidence des mécanismes génétiques ou épigénétiques responsables de ces réponses particulières aux traitements anticancéreux. / Prostate cancer is ranked third among fatal cancers, after bronchial and colorectal cancers, with 150,000 deaths recorded in 2010. The design of new prostate cancer drugs should take into account : i) the metabolism which can alter the efficiency of an active compound in vivo, and ; ii) the individual differences in metabolism resulting from patients’ environments. Our study, in partnership with the HEI School of Lille, is concerned with Phenstatin (a compound in pre-clinical development) and its metabolites : indeed, promising therapeutic results have recently been obtained with taxanes in hormone-resistant prostate cancers which stabilize microtubules by inhibiting their depolymerization.The aim of this work was firstly to study the pharmacological effect of Phenstatin and its metabolites, obtained after biotransformation by human liver microsomes, on tubulin polymerization and the proliferation of human prostate cancer cells (PC3 cell line). Secondly, we attempted to determinate the CYPs responsible for the formation of the Phenstatin metabolites we have identified and quantified. Thirdly, we addressed the influence of environmental conditions, more specifically the presence of cadmium, on the metabolism of these novel compounds and their activity towards prostate cancer cells. In order to understand the response or non-response of different treatments, we determined expression profiles of genes involved in the metabolism of xenobiotics by using high throughput technology based on real time quantitative PCR (TaqmanTM Low Density Arrays). A study model was designed in order to show the effect of environmental factors on anticancer compounds’ efficiency. This model should subsequently be transposable to other agents involved in xenobiotic metabolism and reveal genetic or epigenetic mechanisms responsible for individuals’ responses to anticancer treatments.

<div>Prostate cancer (PCa) is the second leading cause of cancer related deaths in American men. In this study, I identify two combinational therapeutics to treat PCa – the combination of enzalutamide and simvastatin, and the combination of GSK126 and metformin, both of which strongly suppress PCa cell growth in vitro and in vivo via inhibiting androgen receptor (AR), an important oncogenic driver for the PCa progression. Simvastatin leads to more AR degradation when combined with enzalutamide. For the combination of GSK126 and metformin, the interaction between enhance of zeste homolog2 (EZH2) and AR is interrupted by GSK126, re-sensitizing EZH2 to metformin. Meanwhile, GSK126 inhibits EZH2’s activity. </div><div><br></div><div>Polo-like kinase 1 (PLK1), a cell cycle regulator, is usually overexpressed in non-small-cell lung cancer (NSCLC). Here, we report that PLK1 overexpression promotes the development of Kras^G12D and Trp53^fl/fl (KP)-driven lung adenocarcinoma (LADC). KP mice harboring transgenic PLK1 (KPPI) display heavier tumor burden, poorer tumor differentiation, and lower survival than KP mice. Mechanistically, PLK1 overexpression enhances the activity of MAPK pathway, via upregulating RET expression in a kinase-dependent manner. Supporting our findings, PLK1 knockout in KP mice reduces RET gene expression, inhibits MAPK pathway activity, and strongly delays LADC development. Therefore, these data reveal that PLK1 functions as an oncogene in KP-driven LADC.</div><div> </div><div><br></div>

Whether to screen for prostate cancer in aging men is a topic that is fairly well researched, but recommendations are controversial, because the evidence supporting any recommendation is equivocal. The evidence clearly does not support routine screening of all average-risk men, but for men aged 55 to 69 years, either not routinely screening, or engaging each man in shared decision making for his individual preference on screening, is reasonable and consistent with the evidence. Many organizations, including the American Cancer Society, have not yet reassessed their guidelines, in response to the US Preventative Services Task Force revised guideline.

Jamaican males are a high-risk population for aggressive prostate cancer (PrCa) due to genetic influences, and identifying empirical data on treatments, which provide survival benefits is a prime challenge for clinicians who manage Jamaican PrCa patients. Thus, the purpose of this investigation was to elucidate treatment effects of brachytherapy and ERBT in the survival of a Jamaican PrCa cohort. Differences in survival outcomes of brachytherapy and ERBT treated Jamaican, and White U.S.-born PrCa patients with localized PrCa were compared. The mechanism of radiation programmed cell death in PrCa carcinogenesis explained in the oxidative stress theory, was the theoretical base for interpreting the research questions. A retrospective cohort design was used, and included survival analysis of secondary data from the Surveillance Epidemiology and End Results database. The sample size was 10,752 Jamaican and White U.S.-born prostate cancer patients diagnosed between 1992 and 2011. Kaplan-Meier and Cox proportional hazard regression models confirmed that brachytherapy resulted in enhanced survival benefits to the Jamaicans, HR 0.63, 95% CI [0.55, 0.73], p < .001, but ERBT did not, HR 1.6, 95% CI [1.38, 1.84] p < .001. Hence, brachytherapy may be an appropriate treatment option for Jamaican PrCa patients. Clinicians and health care planners can utilize the results for policy decisions aimed at increasing access to brachytherapy treatments to Jamaicans. Improving access to efficient PrCa treatments could reduce the morbidity and mortality rates of PrCa among Jamaicans, decrease years of potential life lost from PrCa, and enhance the life expectancy of the Jamaican male population.