Extracellular Vesicles and Interleukins: Novel Frontiers in Diagnostic and Therapeutic for Cancer.

Tumor cells present many strategies for survival and dissemination in the tumor environment. Extracellular vesicles are a vital pathway used in crosstalk between tumor and non-malignant cells. They carry different types of molecules that, when internalized by target cells, can activate signaling pathways and molecular processes that will promote and disseminate neoplastic cells. Proteins, nucleic acids, and different cytokines, such as interleukins, are the main classes of molecules carried by extracellular vesicles and are being studied to understand the molecular mechanisms present in the tumor microenvironment. In particular, although poorly understood, the association between EVs and interleukins has revealed potential approaches to the diagnosis and therapeutics of several neoplasms.

Post-SELEX Optimization and Characterization of a Prostate Cancer Cell-Specific Aptamer for Diagnosis.

The RNA aptamer A4 binds specifically to tumor prostate cells. A4 was modified (mA4) by adding deoxyribonucleotides to its ends to remove the reactive 2' hydroxyl groups of RNA's sugar at the ends of the aptamer and to make it more stable to widespread RNase contamination in laboratories. Thus, mA4 would be more suitable to use in the clinical settings of prostate cancer (PCa). We aimed to characterize this optimized oligonucleotide to verify its potential as a diagnostic tool. The sequences and structures of A4 and mA4 were compared through in silico approaches to corroborate their similarity. Then, the degradation of mA4 was measured in appropriate media and human plasma for in vitro tests. In addition, the binding abilities of A4 to prostate cells were contrasted with those of mA4. The effects of mA4 were assessed on the viability, proliferation, and migration of human prostate cell lines RWPE-1 and PC-3 in three-dimensional (3D) cell cultures. mA4 showed configurational motifs similar to those of A4, displayed a half-life in plasma substantially higher than A4, and exhibited a comparable binding capacity to that of A4 and unaltered viability, proliferation, and migration of prostatic cells. Therefore, mA4 maintains the crucial 3D structures of A4 that would allow binding to its target, as suggested by in silico and binding analyses. mA4 may be a good PCa reporter as it does not change cellular parameters of prostate cells when incubated with it. Its additional deoxyribonucleotides make mA4 inherently more chemically stable than A4, avoiding its degradation and favoring its storage and handling for clinical applications. These characteristics support the potential of mA4 to be used in diagnostic systems for PCa.

Patents for the Physiological Quality in Seeds of Peach Rootstock Classified by Weight and Stored for Different Periods.

BACKGROUND: Among stone fruit, the peach (Prunus persica (L) Batsch) is one of the most widely grown species in Brazil, in both area cultivated and in production. OBJECTIVE: The aim of this study was to evaluate the physiological quality of heavy and light seeds of four cultivars of Prunus persica for two storage periods, from tests of electrical conductivity, germination, and an analysis of initial plantlets growth. METHODS: The Electrical Conductivity test (EC) was conducted in a Completely Randomised Design (CRD), in a 4 x 2 x 5 factorial scheme with five replications. The germination test was carried out in CRD, in a 4 x 2 factorial scheme with eight replications. The physiological quality of the seeds was determined at zero and twelve month's storage. For the growth analysis, the experimental design was in CRD, in a 4 x 2 factorial scheme with four replications. RESULTS: Under the conditions of the present study, it was found that the tests of germination and electrical conductivity were complementary in evaluating physiological quality in seeds of Prunus persica rootstock, suggesting that independent of the weight of the seeds, in 'Capdeboscq', 'Aldrighi', 'Okinawa' and 'Okinawa Roxo', there is a loss of quality and viability when the seeds are stored for a period of 12 months. CONCLUSION: Under the experimental conditions of the present study, it was concluded that storage for a period of 12 months in Recent patents is not rather recommendable for maintaining quality and viability in seeds of Prunus persica of the Capdeboscq, Aldrighi, Okinawa and Okinawa Roxo cultivars.

Patents for the Morphophysiological Quality of Seedlings and Grafted Peach Trees: Effects of Nutrient Solution and Substrates.

BACKGROUND: This study aimed to verify the influence of different substrates with and without addition of nutrient solution on the roostocks production of Capdeboscq cultivar and grafted peach plants of 'Chimarrita' scion. METHODS: In the first experiment, the height and stem diameter of the rootstocks were evaluated every two weeks, up to 90 days after transplanting (DAT). At 90 DAT, dry weight of shoots and roots, total dry mass and Dickson Quality Index were evaluated. In the second experiment, seedlings of 'Capdeboscq' were grafted with 'Chimarrita' scion. The growth of the scions and the percentage of living grafts were evaluated. At 146 DAT, the stem diameter of the scions, the SPAD index, the chlorophyll and nitrogen balance index were evaluated. DISCUSSION: The greatest mean values for the stem diameter of seedlings of the cv. Capdeboscq were obtained with the substrates T4 (5.53 mm); T2 (5.47 mm) and T1 (5.23 mm) with addition of nutrient solution, with seedlings reaching the plant standards according to the ordinance number 173 of May 27th of 1984, which recommends that rootstocks have to have a minimum stem diameter of 5.0 mm. Thus, the substrates which received the addition of nutrient solution, except the soil substrate, were adequate considering the rules that governing the peach tree production in Brazilian Nurserioes. CONCLUSION: The nutrient solution already avaliblabe for Recent patents is highly indicated to obtain seedlings of 'Capbdboscq' rootstocks with high mophophysiological quality in less time during nursery cycle of plant production. The largest stem diameter for 'Capdeboscq' was obtained with substrate 35% sand + 15% soil + 50% bovine manure (5.53 mm); 75% sand + 25% soil (5.47 mm) and 100% sand (5.23 mm) with addition of nutrient solution. The best morphophysiological characteristics of 'Chimarrita' plants grafted on 'Capdeboscq' seedlings was obtained with the use of substrates 75% sand + 25% soil and 35% sand + 15% soil + 50% bovine manure, whose plants reached a morphological standard for sale, in just four and a half months DAT, that is 56 days after grafting.

Biometric Characterization and Morphophysiological Quality of Peach Rootstock Seeds Using Images of their Seedling Vigor.

BACKGROUND: This study aimed to evaluate the efficiency of using the computerized imaging Seed Analysis System (SAS) in the biometric and morphophysiological characterization of seeds and the initial growth of seedlings from peach rootstocks. METHODS: The experimental design was completely randomized with five replicates of 20 seeds. The variables analyzed were the seed humidity content, length and width of seeds measured by SAS technology and manual measurements, mean germination time, germination percentage, radicle length and width, taproot length, length of the aerial part and taproot/aerial part ratio. RESULTS: The highest seed length, germination percentage (100%) and lower germination time (11.3), were obtained with the cv. Capdeboscq while, 'Tsukuba 1', 2' and 3' had intermediate seedlings length, varying from 1.55 to 1.65 cm with mean germination times between 14.5 and 18.0 days and average germination percentage of 96%. The computerized analysis of images is fast and efficient for biometric evaluations such as seed width and length, as well as initial growth of peach tree seedlings. The cvs Capdeboscq, Flordaguard and Tsukuba 2 presented greater radicle width, length and a mean taproot/aerial part ratio equal to 2, as well as higher number of adventitious roots, which indicated a strong positive correlation between radicle length, taproot length and initial seedling growth. CONCLUSION: The continuity of the research will certainly allow the development of reliable procedures for other species, besides allowing the identification of wider alternatives for the use of this system for the expansion of knowledge in the areas of physiology and evaluation of the physiological potential of seeds.

Comparative Assay of 2D and 3D Cell Culture Models: Proliferation, Gene Expression and Anticancer Drug Response.

BACKGROUND: In vitro tests allow establishing experimental variables. However, in vitro results cannot be extrapolated to in vivo tests. Considering that three-dimensional (3D) culture has been one of the best ways to portray the in vivo system of most cell types, it is possible to carry out assays with a great clinical relevance for the analysis of the screening, action and resistance of antitumor drugs. OBJECTIVE: Thus, the objective of the present study was to compare between 2D and 3D cell culture forms to conclude which is the most suitable model for preclinical in vitro drug testing. METHOD: We evaluated the proliferation, genetic expression and chemoresistance of prostate tumor cell lines, PC- 3, LNCaP and DU145. Prostate tumor cell lines PC-3, LNCaP and DU145 were treated with the antineoplastic drugs paclitaxel and docetaxel and evaluated with cytotoxicity, cell proliferation and gene expression assays in 2D and magnetic 3D bioprinting cultures. RESULTS: Lower cell proliferation rate, more resistance to paclitaxel and docetaxel and altered gene expression profile was shown in 3D cell culture comparing with its 2D counterpart. CONCLUSION: 3D cell culture exhibited a more similar behavior to in vivo systems, being a promising and more reliable tool for the development of new drugs.

Extracellular vesicles as drivers of epithelial-mesenchymal transition and carcinogenic characteristics in normal prostate cells.

There is increasing evidence that cancer dissemination and metastasis establishment may not only be due to the movement of tumor cells. Content of extracellular vesicles (EVs) secreted by tumor cells may also reflect the origin of these cells. Some molecules that constitute these EVs have already been used as targets for detection of specific tumors. However, to the best of our knowledge, EVs from biopsies and plasma have not yet been compared nor thoroughly investigated as triggers of malignant transformation and metastatic niche formation. To evaluate the role of EVs in the cellular microenvironment, we have treated the normal epithelial prostate cell lines, RWPE-1 and PNT-2, with a pool of EVs from biopsies of prostate primary tumors (bEVs), biopsies of benign prostate hyperplasia (hEVs), plasma of prostate cancer (PCa) patients (pEVs) or plasma of healthy individuals (pnEVs). Each of the four pools consisted of isolated EVs from several subjects, of which PCa patients were in different stages of cancer. Migration and proliferation profiles, cytokine release, and a panel of PCa-associated genes' expression of epithelial-mesenchymal transition in the cell lines were evaluated after 24 h incubation with EVs. When compared to the control groups, cells treated with the pool of EVs isolated from tumor biopsies and plasma of PCa patients showed greater migration and proliferation, significant alterations in gene expression, and high levels of IL-8, factors that are associated with cancer development. Specifically, isolated bEVs and pEVs may induce malignant features in non-tumor cells by activating several cellular events associated with cancer progression, suggesting that future PCa therapy may target multiple elements found in tumor-derived EVs.

3D Cell-SELEX: Development of RNA aptamers as molecular probes for PC-3 tumor cell line.

Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus being unable to predict the clinical outcome. Therefore, searching for new biomarkers aiming to improve diagnosis, prognosis and therapy is still required. In this study, we performed 3D Cell-SELEX against PC-3 prostate cancer cell line, a novel strategy to select specific nucleic acid ligands against spheroid cells in 3D cell culture. This original system combines Cell-SELEX, a process that exploits the cellular structure to generate specific ligands, and 3D cell culture, an approach that mimics the tissue microenvironment in vitro. In the first round of 3D Cell-SELEX, a negative selection against RWPE-1, non-tumor cell line, was performed to subtract non-tumor specific aptamers. The supernatant was used in eight additional rounds of selection, which were performed against PC-3 cell line. After nine selection cycles, eight PC-3 specific RNA aptamers were selected and sequenced. The aptamers presented sizes between 20 and 50 nucleotides-long, with low free energy (∆G<-13.6), which contributed for their spontaneous folding and high stability. Furthermore, our results showed the aptamer A4 as a specific ligand to prostate tumor cells, with dissociation constant in the nanomolar scale. Therefore, the novel 3D Cell-SELEX procedure improved the selection of PCa cell-surface ligands and the aptamer A4 has shown potential for the identification of prostate tumor cells, suggesting the application of this molecule in further screening assays for PCa.

Prostate-specific RNA aptamer: promising nucleic acid antibody-like cancer detection.

We described the selection of a novel nucleic acid antibody-like prostate cancer (PCa) that specifically binds to the single-stranded DNA molecule from a 277-nt fragment that may have been partially paired and bound to the PCA3 RNA conformational structure. PCA3-277 aptamer ligands were obtained, and the best binding molecule, named CG3, was synthesized for validation. Aiming to prove its diagnostic utility, we used an apta-qPCR assay with CG3-aptamer conjugated to magnetic beads to capture PCA3 transcripts, which were amplified 97-fold and 7-fold higher than conventional qPCR in blood and tissue, respectively. Histopathologic analysis of 161 prostate biopsies arranged in a TMA and marked with biotin-labeled CG3-aptamer showed moderate staining in both cytoplasm and nucleus of PCa samples; in contrast, benign prostatic hyperplasia (BPH) samples presented strong nuclear staining (78% of the cases). No staining was observed in stromal cells. In addition, using an apta-qPCR, we demonstrated that CG3-aptamer specifically recognizes the conformational PCA3-277 molecule and at least three other transcript variants, indicating that long non-coding RNA (lncRNA) is processed after transcription. We suggest that CG3-aptamer may be a useful PCa diagnostic tool. In addition, this molecule may be used in drug design and drug delivery for PCa therapy.

Evaluation of adverse effects in tamoxifen exposed healthy female dogs.

BACKGROUND: Mammary tumors are among the most frequent neoplasms in female dogs, but the strategies employed in animal treatment are limited. In human medicine, hormone manipulation is used in cancer therapy. Tamoxifen citrate is a selective inhibitor of oestrogen receptors and exerts a potent anti-oestrogen effect on the mammary gland. The aim of this study was to evaluate the adverse effects when exposing healthy female dogs to tamoxifen. METHODS: Tamoxifen was administered for 120 days at a dose of 0.5 or 0.8 mg/kg/day to either intact or spayed female dogs. The effects were assessed through clinical examination, haematology, serum biochemistry, ophthalmology and bone marrow aspirate examination. Ovariohysterectomy was performed and the uterus examined by histopathology. RESULTS: Vulva oedema and purulent vaginal discharge developed with 10 days of tamoxifen exposure in all groups. Pyometra was diagnosed after around 90 days of exposure in intact females with frequencies increasing during the following 30 days of exposure. Up to 50% of dogs within the groups developed retinitis but none of the dogs had signs of reduced visual acuity. The prevalence of retinitis in each group was similar after 120 days of exposure. Haematological, biochemical and bone marrow changes were not observed. Due to the high risk of developing pyometra after prolonged exposure to tamoxifen, only spayed animals should be given this medication. CONCLUSIONS: A dose of 0.8 mg tamoxifen/kg body weight/day is recommended when treating tamoxifen-responsive canine mammary tumors. Due to the high risk of developing pyometra, ovariohysterectomy is recommended.