1 ABSTRACT Introduction and aim of the study Circulating tumor cells (CTCs) are a promising tool of identifying patients with castration- resistant prostate cancer (CRPC) who will benefit from often demanding cytotoxic therapy. The aim of this work was to evaluate the prognostic significance of CTC in docetaxel-treated CRPC patients. During the project, we also tested the various methods of CTC cultivation and studied their genetic profile as well as the genetic profile of histological specimen at the time of diagnosis. Patients and methods A total of 39 patients who met the CRPC criteria and were indicated for docetaxel chemotherapy were included in the prospective study. Blood collection for CTC analysis was done in all patients before chemotherapy and on the first day of the fourth or fifth cycle of docetaxel. In parallel, CTCs were cultivated. Isolation and detection of CTC was done using the AdnaTest system, which consists of immunomagnetic separation and subsequent detection of mRNA from the CTC lysate. The primary objective of the study was to evaluate the overall survival (OS) of patients. Survival analysis was performed using the Kaplan-Meier method of estimating the survival distribution function. The impact of individual factors was tested using the Log-rank test, the Wilcoxon test and the Cox...

Circulating tumor cells (CTCs) are a potential predictive surrogate marker for disease monitoring. Due to the sparse knowledge about their phenotype and its changes during cancer progression and treatment response, CTC isolation remains challenging. Here we focused on the mechanical characterization of circulating non-hematopoietic cells from breast cancer patients to evaluate its utility for CTC detection. For proof of premise, we used healthy peripheral blood mononuclear cells (PBMCs), human MDA-MB 231 breast cancer cells and human HL-60 leukemia cells to create a CTC model system. For translational experiments CD45 negative cells—possible CTCs—were isolated from blood samples of patients with mamma carcinoma. Cells were mechanically characterized in the optical stretcher (OS). Active and passive cell mechanical data were related with physiological descriptors by a random forest (RF) classifier to identify cell type specific properties. Cancer cells were well distinguishable from PBMC in cell line tests. Analysis of clinical samples revealed that in PBMC the elliptic deformation was significantly increased compared to non-hematopoietic cells. Interestingly, non-hematopoietic cells showed significantly higher shape restoration. Based on Kelvin–Voigt modeling, the RF algorithm revealed that elliptic deformation and shape restoration were crucial parameters and that the OS discriminated non-hematopoietic cells from PBMC with an accuracy of 0.69, a sensitivity of 0.74, and specificity of 0.63. The CD45 negative cell population in the blood of breast cancer patients is mechanically distinguishable from healthy PBMC. Together with cell morphology, the mechanical fingerprint might be an appropriate tool for marker-free CTC detection.

Despite significant improvements in medical sciences over the last decade, cancer still continues to be a major cause of death in humans throughout the world. Parallel to the efforts of understanding the intricacies of cancer biology, researchers are continuously striving to develop effective cancer detection and treatment strategies. Use of nanotechnology in the modern era opens up a wide range of possibilities for diagnostics, therapies and preventive measures for cancer management. Although, existing strategies of cancer detection and treatment, using nanoparticles, have been proven successful in case of cancer imaging and targeted drug deliveries, they are often limited by poor sensitivity, lack of specificity, complex sample preparation efforts and inherent toxicities associated with the nanoparticles, especially in case of in-vivo applications. Moreover, the detection of cancer is not necessarily integrated with treatment. X-rays have long been used in radiation therapy to kill cancer cells and also for imaging tumors inside the body using nanoparticles as contrast agents. However, X-rays, in combination with nanoparticles, can also be used for cancer diagnosis by detecting cancer biomarkers and circulating tumor cells. Moreover, the use of nanoparticles can also enhance the efficacy of X-ray radiation therapy for cancer treatment. This dissertation describes a novel in vitro technique for cancer detection and treatment using X-ray radiation and nanoparticles. Surfaces of synthesized metallic nanoparticles have been modified with appropriate ligands to specifically target cancer cells and biomarkers in vitro. Characteristic X-ray fluorescence signals from the X-ray irradiated nanoparticles are then used for detecting the presence of cancer. The method enables simultaneous detection of multiple iv cancer biomarkers allowing accurate diagnosis and early detection of cancer. Circulating tumor cells, which are the primary indicators of cancer metastasis, have also been detected where the use of magnetic nanoparticles allows enrichment of rare cancer cells prior to detection. The approach is unique in that it integrates cancer detection and treatment under one platform, since, X-rays have been shown to effectively kill cancer cells through radiation induced DNA damage. Due to high penetrating power of X-rays, the method has potential applications for in vivo detection and treatment of deeply buried cancers in humans. The effect of nanoparticle toxicity on multiple cell types has been investigated using conventional cytotoxicity assays for both unmodified nanoparticles as well as nanoparticles modified with a variety of surface coatings. Appropriate surface modifications have significantly reduced inherent toxicity of nanoparticles, providing possibilities for future clinical applications. To investigate cellular damages caused by X-ray radiation, an on-chip biodosimeter has been fabricated based on three dimensional microtissues which allows direct monitoring of responses to X-ray exposure for multiple mammalian cell types. Damage to tumor cells caused by X-rays is known to be significantly higher in presence of nanoparticles which act as radiosensitizers and enhance localized radiation doses. An analytical approach is used to investigate the various parameters that affect the radiosensitizing properties of the nanoparticles. The results can be used to increase the efficacy of nanoparticle aided X-ray radiation therapy for cancer treatment by appropriate choice of X-ray beam energy, nanoparticle size, material composition and location of nanoparticle with respect to the tumor cell nucleus.

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Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards ‘normal’ liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

<p>U radu je ispitan uticaj odabranih modifikovanih steroida na proliferaciju ćelija humanih malignih tumora i zdravih ćelija, a zatim njihov uticaj na modifikaciju ćelijskog ciklusa, indukovanje apoptoze i genetskih o&scaron;tećenja u tumorskim ćelijama najsenzitivnijim na ova jedinjenja (MDA-MB-231 ćelijska linija humanog adenokarcinoma dojke, ER-). Ispitana je<em> in vitro </em>citotoksičnost odabranih&nbsp; steroidnih derivata prema ćelijskim linijama MCF-7, MDA-MB-231, PC3, HeLa, HT-29, MRC-5, K562 i A549, i proučavan ćelijski mehanizam koji je u osnovi uočenog antiproliferativnog efekta svakog od ispitivanih derivata. Sva ispitivana jedinjenja indukuju apoptozu MDA-MB-231&nbsp; ćelija, na različite načine i sa različitim potencijalom. Genotoksikolo&scaron;ka studija upotpunjuje rezultate proučavanja efekta ispitivanih steroidnih jedinjenja na biolo&scaron;ke sisteme, ukazujući na izostanak genotoksičnosti ovih jedinjenja i njihov biomedicinski potencijal.</p> / <p>In this work the effect of selected modified steroids on proliferation of human&nbsp; malignant tumor and healthy cells, as well as their impact on the modification of cell cycle arrest, induction of apoptosis and genetic defects in tumor cells most sensitive&nbsp; to these compounds (MDA-MB-231 cell line of human breast adenocarcinoma, ER-) were examined. We examined the<em> in vitro </em>cytotoxicity of selected steroidal derivatives towards MCF-7, MDA-MB-231, PC-3, HeLa, HT-29, MRC-5, K562 and A549 cell lines, and studied the cellular mechanism that is underlying the antiproliferative activity expressed by these steroidal derivatives. All tested compounds induced apoptosis of&nbsp; MDA-MB-231 cells, in different ways and with different potential. Genotoxicological study complements the results of the study of the effect of tested steroidal compounds on biological systems, pointing out the lack of genotoxicity of these compounds and therefore their biomedical potential.</p>

O diferencial da cultura de células em 3-dimensões é permitir que as células explorem as 3-dimensões do espaço, aumentando assim as interações com o ambiente e entre as células. Em estudos relacionados à biologia do câncer de mama, vem ganhando espaço a utilização de esferóides para estudos que visam à compreensão da morfogênese do espaço luminal. Neste trabalho foi mostrado que as células MCF-7 reorganizam-se em estruturas tubulares e acinares. Em ambas as situações, a formação do lúmen veio acompanhada pelo estabelecimento de uma camada de células polarizadas, arranjo este muito semelhante ao encontrado em glândulas mamárias. Os resultados apresentados apontam para a existência de uma população de células na linhagem MCF-7 que não estão totalmente comprometidas ao fenótipo tumoral. Mantidos diferenciados, os esferóides de células MCF-7 apontam como um novo modelo para estudos relacionados à formação do lúmen, permitindo assim explorar o papel de diferentes vias como as relacionadas a apoptose, autofagia, diferenciação e sobrevivência celular. / As a particularity, a 3D cell culture permits cells to explore the three dimensions of the space thereby increasing cell-cell interactions, as well as interaction with the environment. In studies related to breast cancer biology, spheroids are becoming widely used in the aim to comprehend luminal space morphogenesis. We showed that MCF-7 cells reorganize themselves in tubular and acinar structures. In both situations, lumen formation was accompanied by the establishment of a layer of polarized cells, an arrangement that is very similar to that of breast glands. The presented results suggest the existence of an MCF cell line population not completely committed to the tumor phenotype. When maintained as differentiated, MCF-7 cell spheroids can be a new model for studies regarding lumen formation, thereby exploring the role of diiferent pathways, such as those related to cell apoptosis, autophagy, differentiation and survival.

A própolis, um produto natural derivado de resinas de plantas coletadas por abelhas, foi usada por milhares de anos na medicina tradicional pelo mundo inteiro. Neste estudo, investigamos o efeito de uma fração da própolis vermelha brasileira (BRVD), no crescimento das células de melanoma murino (B16F10), das linhagens hematológicas humanas (HL-60 e K562), e de fibroblastos humanos (MCR-5 e FP). Após a análise preliminar de várias frações da própolis BRV, encontramos que a Fração BRVD inibiu fortemente o crescimento das células de uma maneira dose-tempo dependente pela necrose. Os resultados mostraram que essa fração induz eficazmente um efeito citotóxico em todas as linhagens estudadas, com média da IC50 em torno de 30 g/mL em 24 h de exposição. Estes resultados sugerem que a atividade antitumor da fração BRVD ocorre com a indução de necrose e os compostos dessa fração podem ser úteis como um agente contra o câncer / Propolis, a natural product derived from plant resins collected by honeybees, has been used for thousands of years in traditional medicine all over the world. In this study we have investigated the effect of some fractions from red Brazilian propolis on the growth of murine melanoma cell ( B16/F10), human hematological cells ( HL-60 and K562), and human fibroblasts cells (MCR-5 and FP). We found that BRVD strongly inhibited the growth of the cells in a dose- and time-dependent through induction of necrosis. Our results showed that BRVD effectively induced a cytotoxic effect on all cell lines studied, with IC50 average about 30 g/mL for 24 h of exposition. These results suggest that the antitumor activity of BRVD from red Brazilian propolis occurs through the induction of necrosis and its compounds may be useful as a anticancer agent

Bcr-Abl é a tirosina quinase (TK) responsável por causar a Leucemia Mielóide Crônica (LMC). Os últimos estudos de follow-up mostram que apenas 50% dos pacientes tratados com a segunda geração de inibidores de TK atinge a remissão completa, o que significa que metade desses pacientes necessita de um algo melhor do que está disponível. Wiskott Aldrich Syndrome Protein (WASP) é um gene essencial para o bom desenvolvimento e função das células hematopoiéticas. Ante esse contexto, decidimos investigar se WASP poderia ter algum papel ou relevância na LMC. Em conclusão, Bcr-Abl suprime a expressão WASP por um mecanismo epigenético. A re-expressão de WASP torna as células mais suscetíveis à apoptose em resposta ao Imatinib. Sugerimos que a recuperação da expressão WASP deve ser discutida como estratégia para a terapia da LMC. / Bcr-Abl is the tyrosine kinase (TK) responsible for causing Chronic Myeloid Leukemia (CML). This fusion protein up- and down-regulates several genes and pathways, producing a strong resistance to apoptosis and a blockage of cell maturation in the hematopoietic compartment. The last follow-up studies provided that only 50% of the patients treated with second generation achieve complete remission, what means that one-half of these patients needs something better. Wiskott Aldrich Syndrome Protein (WASP) is an essential gene for the proper development and function of the hematopoietic cells. In the light of this background, we decided to investigate if WASP could have some role or relevance in the CML context. In conclusion, Bcr-Abl suppresses WASP expression by an epigenetic mechanism. The re-expression of WASP makes the CML cells more susceptible to apoptosis and contribute to respond to Imatinib. We suggest that recovery of WASP expression should be discussed as a new and additional strategy for CML therapy.