Technical challenges of working with extracellular vesicles

Extracellular Vesicles (EVs) are gaining interest as central players in liquid biopsies, with potential applications in diagnosis, prognosis and therapeutic guidance in most pathological conditions. These nanosized particles transmit signals determined by their protein, lipid, nucleic acid and sugar content, and the unique molecular pattern of EVs dictates the type of signal to be transmitted to recipient cells. However, their small sizes and the limited quantities that can usually be obtained from patient-derived samples pose a number of challenges to their isolation, study and characterization. These challenges and some possible options to overcome them are discussed in this review

MicroRNA-100 Acts as a Tumor Suppressor in Human Bladder Carcinoma 5637 Cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions.Univ São Paulo, Div Pediat Oncol, Dept Pediat, Fac Med Ribeirao Preto, BR-05508 São Paulo, BrazilUniv São Paulo, Dept Genet, BR-05508 São Paulo, BrazilSão Paulo State Univ UNESP, Dept Pathol, Fac Med Botucatu, São Paulo, BrazilSão Paulo State Univ UNESP, Dept Pathol, Fac Med Botucatu, São Paulo, Brazi

MicroRNA-100 acts as a tumor suppressor in human bladder carcinoma 5637 cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions.Department of Genetics Faculty of Medicine of Ribeirão Preto University of São PauloDivision of Pediatric Oncology Department of Pediatrics Faculty of Medicine of Ribeirão Preto University of São PauloDepartment of Pathology Faculty of Medicine of Botucatu São Paulo State University - UNESPDepartment of Pathology Faculty of Medicine of Botucatu São Paulo State University - UNES

MicroRNA-100 Acts as a Tumor Suppressor in Human Bladder Carcinoma 5637 Cells

Bladder carcinoma is one of the most common tumors in the world and, despite the therapy currently available, most of the patients relapse. Better understanding of the factors involved in disease pathogenesis would provide insights for the development of more effective strategies in treatment. Recently, differential miRNA expression profiles in bladder urothelial carcinomas identified miR-100 down-regulation and miR-708 up-regulation among the most common alterations, although the possible influence of these miRNAs in the control of basic mechanisms in bladder tumors has not been addressed. In this context, the present study aimed to evaluate the in vitro effects of miR-100 forced expression and miR-708 inhibition in the bladder carcinoma cell line 5637. Our results showed that overexpression of miR-100 significantly inhibited growth when compared to controls at both times tested (72 and 96 hours, p<0.01) with a maximum effect at 72 hours reducing proliferation in 29.6 %. Conversely, no effects on cell growth were observed after inhibition of miR-708. MiR-100 also reduced colony formation capacity of 5637 cells by 24.4%. No alterations in cell cycle progression or apoptosis induction were observed. The effects of miR-100 on growth and clonogenicity capacity in 5637 cells evince a possible role of this miRNA in bladder carcinoma pathogenesis. Further studies are necessary to corroborate our findings and examine the potential use of this microRNA in future therapeutic interventions

The Carbonic Anhydrase Inhibitor E7070 Sensitizes Glioblastoma Cells to Radio- and Chemotherapy and Reduces Tumor Growth

Glioblastomas (GBMs), the most common and lethal primary brain tumor, show inherent infiltrative nature and high molecular heterogeneity that make complete surgical resection unfeasible and unresponsive to conventional adjuvant therapy. Due to their fast growth rate even under hypoxic and acidic conditions, GBM cells can conserve the intracellular pH at physiological range by overexpressing membrane-bound carbonic anhydrases (CAs). The synthetic sulfonamide E7070 is a potent inhibitor of CAs that harbors putative anticancer properties; however, this drug has still not been tested in GBMs. The present study aimed to evaluate the effects of E7070 on CA9 and CA12 enzymes in GBM cells as well as in the tumor cell growth, migration, invasion, and resistance to radiotherapy and chemotherapy. We found that E7070 treatment significantly reduced tumor cell growth and increased radio- and chemotherapy efficacy against GBM cells under hypoxia. Our data suggests that E7070 has therapeutic potential as a radio-chemo-sensitizing in drug-resistant GBMs, representing an attractive strategy to improve the adjuvant therapy. We showed that CA9 and CA12 represent potentially valuable therapeutic targets that should be further investigated as useful diagnostic and prognostic biomarkers for GBM tailored therapy.Department of Paediatrics Ribeirão Preto Medical School University of São PauloDepartment of Neurosurgery Brigham and Women’s Hospital and Harvard Medical SchoolMolecular Oncology Research Center Barretos Cancer Hospital Pio XII Foundation, Rua Antenor Duarte Villela, 1331Department of Neuroscience and Physiology SUNY Upstate Medical UniversityDepartment of Human Genetics McGill UniversityDepartment of Genetics Ribeirão Preto Medical School University of São PauloDepartment of Pathology School of Medicine UNESP – Univ. Estadual PaulistaBarretos School of Health Sciences Dr. Paulo Prata – FACISBDepartment of Pathology and Forensic Medicine Ribeirão Preto Medical School University of São PauloDepartment of Surgery and Anatomy Ribeirão Preto Medical School University of São PauloDepartment of Radiation Oncology The Ohio State UniversityDepartment of Pathology School of Medicine UNESP – Univ. Estadual Paulist

Cytostatic in vitro Effects of DTCM-Glutarimide on Bladder Carcinoma Cells

Bladder cancer is a common malignancy worldwide. Despite the increased use of cisplatin-based combination therapy, the outcomes for patients with advanced disease remain poor. Recently, altered activation of the PI3K/Akt/mTOR pathway has been associated with reduced patient survival and advanced stage of bladder cancer, making its upstream or downstream components attractive targets for therapeutic intervention. In the present study, we showed that treatment with DTCM-glutaramide, a piperidine that targets PDK1, results in reduced proliferation, diminished cell migration and G1 arrest in 5637 and T24 bladder carcinoma cells. Conversely, no apoptosis, necrosis or autophagy were detected after treatment, suggesting that reduced cell numbers in vitro are a result of diminished proliferation rather than cell death. Furthermore previous exposure to 10 mu g/ml DTCM-glutarimide sensitized both cell lines to ionizing radiation. Although more studies are needed to corroborate our findings, our results indicate that PDK1 may be useful as a therapeutic target to prevent progression and abnormal tissue dissemination of urothelial carcinomas

<i>E2F1</i> and miRNA expression in a set of patient samples.

<p>A) <i>E2F1</i> expression in tumor relative to normal tissue was evaluated by qPCR; <i>PUM1</i> and <i>HMBS</i> expression were used for normalization. B) MIR205-5p and C) MIR136-5p tumor expression relative to normal expression was evaluated by qPCR; <i>RNU6b</i> expression was used for normalization. Red: patient #2 carries a somatic mutation at <i>E2F1</i>:MIR136-5p target site. Blue: patient #11 carries a germline alteration at <i>E2F1</i>:MIR205-5p target site. Dashed line represents the same expression value between normal and tumor tissues. Error bars indicate the SEM of experiments in triplicate.</p

List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.

<p>List of genes screened by next-generation sequencing for genetic alterations at miRNA predicted target sites.</p