Enrique Castellón

Effect of Leuprolide and Cetrorelix on Cell Growth, Apoptosis, and GnRH Receptor Expression in Primary Cell Cultures from Human Prostate Carcinoma

Contradictory data have been reported regarding the effect of GnRH agonists and antagonists on cell growth and survival, using prostate cancer-derived cell lines expressing either endogenous or exogenous GnRH receptors. We addressed the issue studying the effect of leuprolide (agonist) and cetrorelix (antagonist) on cell growth, apoptosis and GnRH receptor expression using a primary cell coculture system. Also, binding characteristics of prostate GnRH receptor in this culture system are described. Epithelial and stromal cells were obtained from prostate adenocarcinoma samples and cocultured in a bicameral system. Expression of GnRH receptors was evaluated by semiquantitative RT-PCR (transcript level) and Western blot (protein level). Cell growth was estimated by MTT method and apoptosis by DNA fragmentation using COMET assay. Saturation and competition binding studies were carried out using 125I-GnRH as radioligand. GnRH receptors from cell cultures of prostate cancer exhibited a single class of binding sites with a Kd of 1.11 ± 0.28 nM and a Bmax of 2.81 ± 0.37 pmol/mg of membrane protein for GnRH. Leuprolide and cetrorelix showed no effect on GnRH receptor expression. Both analogues showed a significant reduction in cell growth rate and an increase in DNA-fragmented cell number. These effects were dependent on the analogue concentrations (from 5–20 ng/mL). Considering that the culture system used in this work represents more closely the in vivo conditions of tumor cells than metastatic derived cell lines, we conclude that GnRH analogues have a significant inhibitory effect on cell viability of cells expressing GnRH receptors. In addition, GnRH receptors expressed in tumor prostatic cells seem not discriminate between agonist and antagonist, both analogues activating these receptors. Also, leuprolide and cetrorelix treatments did not influence GnRH receptor expression in our culture system. These differences with pituitary receptors may be explained by differences in affinity, transduction mechanism and molecular context in prostatic tissue.

Platelets enhance tissue factor protein and metastasis initiating cell markers, and act as chemoattractants increasing the migration of ovarian cancer cells

BackgroundAn increase in circulating platelets, or thrombocytosis, is recognized as an independent risk factor of bad prognosis and metastasis in patients with ovarian cancer; however the complex role of platelets in tumor progression has not been fully elucidated. Platelet activation has been associated with an epithelial to mesenchymal transition (EMT), while Tissue Factor (TF) protein expression by cancer cells has been shown to correlate with hypercoagulable state and metastasis. The aim of this work was to determine the effect of platelet-cancer cell interaction on TF and “Metastasis Initiating Cell (MIC)” marker levels and migration in ovarian cancer cell lines and cancer cells isolated from the ascetic fluid of ovarian cancer patients.MethodsWith informed patient consent, ascitic fluid isolated ovarian cancer cells, cell lines and ovarian cancer spheres were co-cultivated with human platelets. TF, EMT and stem cell marker levels were determined by Western blotting, flow cytometry and RT-PCR. Cancer cell migration was determined by Boyden chambers and the scratch assay.ResultsThe co-culture of patient-derived ovarian cancer cells with platelets causes: 1) a phenotypic change in cancer cells, 2) chemoattraction and cancer cell migration, 3) induced MIC markers (EMT/stemness), 3) increased sphere formation and 4) increased TF protein levels and activity.ConclusionsWe present the first evidence that platelets act as chemoattractants to cancer cells. Furthermore, platelets promote the formation of ovarian cancer spheres that express MIC markers and the metastatic protein TF. Our results suggest that platelet-cancer cell interaction plays a role in the formation of metastatic foci.

Abstract 3990: Identification of microRNAs from exosomes of bulk and stem cells from prostate cancer by next generation sequencing

Cancer stem cells (CSCs) are related with cancer initiation, metastasis, resistance and recurrence, by regulating their environment in different ways, including the release of exosomes that modify surrounding and distant cells. Exosomes contain, among other molecules, miRNAs. By identifying the entire spectrum of miRNAs included in exosomes from prostate cancer (PCa) cells by next generation sequencing (NGS), is possible to identify miRNAs specifically released by CSCs, and those related with microenvironment regulation. We stablished adherent primary cell cultures (bulk cells) from PCa tissue from 5 patients. Later, from these cells we obtained CSCs enriched prostatospheres. From bulk and CSCs cultures we purified exosomes by precipitation (ExoQuick-TC, SBI) for miRNAs extraction (mirVana, Ambion). Quality of miRNAs was assessed by Bioanalizer (Agilent Technologies). NGS was performed in the Illumina platform (MiSeq, Illumina) according to manufacturer instructions. Bioinformatic analyses were carried out by Sistemas Genomicos. Overexpressed miRNAs were detected in plasma exosomes by TaqMan miRNA Assays (Lifetechnologies). We identified 1839 miRNAs in exosomes, and 223 species were differentially expressed between bulk and CSCs, but only 26 significantly. Nineteen of them were known species including 6 that were overexpressed in CSCs respect to bulk cells. These miRNAs target genes related with cell differentiation, matrix remodeling and genic expression. From all miRNA detected, 3 were highly overexpressed among all, and were detected in exosomes isolated from plasma of men with prostatic disease. Our study extensively characterizes the miRNA from exosomes of PCa cells. Exosomes contain high diversity of miRNAs, different in CSCs and bulk cells, suggesting differential regulation of their microenvironment. From these, CSCs exosome-related miRNAs could be released to bloodstream and play a role in PCa progression and metastasis (FONDECYT 11121525/1140417). Citation Format: Catherine A. Sanchez, Eliana Andahur, Rodrigo Valenzuela, Enrique Castellon, Christian Ramos, Juan Carlos Trivino. Identification of microRNAs from exosomes of bulk and stem cells from prostate cancer by next generation sequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3990. doi:10.1158/1538-7445.AM2015-3990