Diet as an epigenetic factor in inflammatory bowel disease.

Inflammatory bowel disease (IBD) has as a main characteristic the exacerbation of the immune system against enterocytes, compromising the individual's intestinal microbiota. This inflammatory cascade causes several nutritional deficiencies, which further compromise immunological functioning and, as a result, worsen the prognosis. This vicious cycle can be interrupted as the patient's dietary pattern meets their needs according to their clinical condition, acting directly on the inflammatory process of IBD through the interaction of food, intestinal microbiota, and epigenome. Specific nutritional intervention for IBD has a crucial role in preventing and managing disease activity. This review addresses epigenetic modifications through dietary compounds as a mechanism for modulating the intestinal microbiota of patients with IBD.

RNA Aptamer-functionalized Polymeric Nanoparticles in Targeted Delivery and Cancer Therapy: An up-to-date Review.

Cancer nanotechnology takes advantage of nanoparticles to diagnose and treat cancer. The use of natural and synthetic polymers for drug delivery has become increasingly popular. Polymeric nanoparticles (PNPs) can be loaded with chemotherapeutics, small chemicals, and/or biological therapeutics. Major problems in delivering such therapeutics to the desired targets are associated with the lack of specificity and the low capacity of PNPs to cross cell membranes, which seems to be even more difficult to overcome in multidrugresistant cancer cells with rigid lipid bilayers. Despite the progress of these nanocarrier delivery systems (NDSs), active targeting approaches to complement the enhanced permeability and retention (EPR) effect are necessary to improve their therapeutic efficiency and reduce systemic toxicity. For this, a targeting moiety is required to deliver the nanocarrier systems to a specific location. A strategy to overcome these limitations and raise the uptake of PNPs is the conjugation with RNA aptamers (RNApt) with specificity for cancer cells. The site-directed delivery of drugs is made by the functionalization of these specific ligands on the NDSs surface, thereby creating specificity for features of cancer cell membranes or an overexpressed target/receptor exposed to those cells. Despite the advances in the field, NDSs development and functionalization are still in their early stages and numerous challenges are expected to impact the technology. Thus, RNApt supplies a promising reply to the common problem related to drug delivery by NDSs. This review summarizes the current knowledge on the use of RNApt to generate functionalized PNPs for cancer therapy, discussing the most relevant studies in the area.

Cell-free DNA promotes malignant transformation in non-tumor cells.

Cell-free DNA is present in different biological fluids and when released by tumor cells may contribute to pro-tumor events such as malignant transformation of cells adjacent to the tumor and metastasis. Thus, this study analyzed the effect of tumor cell-free DNA, isolated from the blood of prostate cancer patients, on non-tumor prostate cell lines (RWPE-1 and PNT-2). To achieve this, we performed cell-free DNA quantification and characterization assays, evaluation of gene and miRNA expression profiling focused on cancer progression and EMT, and metabolomics by mass spectrometry and cellular migration. The results showed that tumor-free cell DNA was able to alter the gene expression of MMP9 and CD44, alter the expression profile of nine miRNAs, and increased the tryptophan consumption and cell migration rates in non-tumor cells. Therefore, tumor cell-free DNA was capable of altering the receptor cell phenotype, triggering events related to malignant transformation in these cells, and can thus be considered a potential target for cancer diagnosis and therapy.

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.

Characterization of oligonucleotide aptamers targeting the 5'-UTR from dengue virus.

AIM: The dengue virus is responsible for a high worldwide incidence of infections, aggravated by late diagnosis, and often confused with other tropical diseases. Results/methodology: Oligonucleotide aptamers binding to the 5'-UTR from dengue virus selected after eight rounds by systematic evolution of ligands by exponential enrichment technology were analyzed by dot-blot assay and in silico prediction of secondary structures, demonstrating the presence of stem-loops that may have the potential for interaction with the viral genome, which can lead to loss of their original conformation. CONCLUSION: This is the first description of RNA aptamers against functional RNA elements of the dengue virus genome with implications for disease control, which may have potential as tools in the future of antiviral therapies and for diagnostics.

Different Gene Therapy Strategies: A Overview for Prostate Cancer.

Gene therapy emerged as a mighty alternative for conventional treatment of multiple diseases. It has been defined as a product "that mediate their effects by transcription and/or translation of transferred genetic material and/or by integrating into the host genome and that are administered as nucleic acids, viruses, or genetically engineered microorganisms. The products may be used to modify cells in vivo or transferred to cells ex vivo prior to administration to the recipient". The first therapeutic gene therapy human trial was conducted in 1990 by Michael R. Blaese, and besides its potential, the technique suffered a major drawback after the tragical death of Jesse Gelsinger, caused by his immune response against the viral vector used in his treatment. To date, gene therapy has regained some popularity and more than 2000 clinical trials are ongoing, most of them related to the treatment or prevention of various types of cancer. Nevertheless, some types of cancer contain a rare population of stem-like cells, capable of differentiation into tumor cells, promoting the re-incidence of tumors. Those cells are generally more resilient to chemotherapy and radiotherapy and are related to tumor initiation, progression, recurrence and metastasis. The human prostate cancer (PCa) is highly heterogeneous and multifactorial, and even the markers are not precise enough to predict the clinical outcome. Furthermore, even though currently therapies can efficiently remove the tumors, the re-incidence rates are high. Gene therapy offers a handful of treatments that can halt oncogenes activation, promote the expression of suppressor genes or target cancer cells directly and induce apoptosis. Besides the risks involved, gene therapy can be of great help in the treatment of cancers and other diseases. This review aims to address the safety and potential of different gene therapy strategies used in the treatment of cancers.

Association of vitamin D receptor variants with clinical parameters in prostate cancer.

PURPOSE: Prostate Cancer (PCa) is one of the most common cancers in men and its early detection can provide a high chance of cure. The detection of Vitamin D Receptor (VDR) gene polymorphisms may be useful as a molecular indicator of clinical outcome, once VDR is implicated in a wide variety of biological processes including modulation of the immune response and inhibition of cancer cell growth, angiogenesis and metastasis. In this study we explored the Single Nucleotide Polymorphisms (SNPs) FokI, BsmI, ApaI and TaqI, to evaluate the susceptibility locus for PCa and verify its correlation with clinical parameters. METHODS: VDR polymorphisms were detected by PCR followed by Restriction Fragment Length Polymorphism (PCR-RFLP). DNA samples were extracted from peripheral blood of 342 patients: 132 PCa, 41 Benign Prostatic Hyperplasia and 169 young healthy volunteers. RESULTS: Statistical analysis showed a noteworthy correlation among SNPs and clinical pathological features. CC genotype (TaqI) was correlated with the age at diagnosis (>58 years old), and GG (BsmI) was associated to lower Prostate-Specific Antigen (PSA) levels (<10 ng/mL). Moreover, when PCa patients were subgrouped, G allele (BsmI) significantly increased the estimated chance for PSA < 10 ng/mL, and GG/GG genotype (BsmI/ApaI) provided a 9.75 fold increased chance of patients with PCa to present lower PSA levels. CONCLUSIONS: The polymorphisms of VDR gene showed a genotype-phenotype association and presented new correlations with different parameters as age and PSA levels.

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.

Dynamic dialog between cytokeratin 18 and annexin A1 in breast cancer: a transcriptional disequilibrium.

Cytokeratins (CKs) constitute the cytoskeletal network and are regulated by post-translational modifications, acting not only as a mechanical support, but also in cell signaling and regulatory processes. Signaling is mediated by CK-associated proteins, such as Annexin A1 (ANXA1), a ligand of the CK18/CK8 complex. ANXA1 has a pivotal role in cellular and immunological responses, and together with CK18 have been implicated in several processes related to malignant transformation in breast cancer (BC). Our aim was to demonstrate how their interaction might be linked to BC development. We investigated transcript levels, protein expression and distribution for both targets in breast tissues of 92 patients (42 BCs and 50 benign diseases) using qPCR and immunohistochemistry, respectively. ANXA1 and CK18 mRNAs were inversely correlated, and their ratio in each TNM stage significantly differentiated BC from benign diseases (OR=5.62). These differences did not mirror tissue protein levels, but a significant dichotomous protein distribution in tumor tissues was observed, differing from the expected co-localization observed during cell homeostasis. The disequilibrium of transcriptional levels between ANXA1/CK18 and alterations in their tissue distribution are present either in initial events or tumor progression, which suggest a critical event in BC. The broken dialog between ANXA1 and CK18 in normal breast tissues may play a critical role in BC development, and together may be used as combined targets for BC diagnostics.

The interaction of AGT and NOS3 gene polymorphisms with conventional risk factors increases predisposition to hypertension.

Renin-angiotensin and kallikrein-kinin systems are interconnected, regulating blood pressure homeostasis. We have demonstrated the interactions among polymorphisms of the angiotensinogen (AGT) and endothelial nitric oxide synthase (NOS3) genes and conventional risk factors affecting the hypertension occurrence. Individuals were recruited (n=192) and classified into hypertensive (HG; n=140) and normotensive (NG; n=52) groups. The genotypic distribution of the Met235Thr (AGT) and Glu298Asp (NOS3) polymorphisms demonstrated that both are independent risk factors of hypertension (p=0.02 and p=0.008, respectively). The concomitant presence of these polymorphisms in the HG group was significantly different (p=0.001) from the NG. Both gene polymorphisms presented an additive effect for the unfavourable alleles T and A, respectively, and 95% of the double mutant homozygotes were classified into the HG. Specific interactions among certain conventional factors and the presence of at least one unfavourable allele presented significant odds towards hypertension. Blood pressure homeostasis was affected by genetic polymorphisms conditioned by the T and A alleles of the AGT and NOS3 genes, respectively, which acted independently. However, their interaction with smoking, sedentariness, age and total cholesterol may have increased the predisposition to hypertension, which may explain most of the hypertension cases.

Prostate cancer antigen 3 (PCA3) RNA detection in blood and tissue samples for prostate cancer diagnosis.

BACKGROUND: The non-coding prostate cancer antigen 3 (PCA3) RNA is currently the most specific biomarker for prostate cancer (PCa) diagnosis. Although its clinical value has been validated in a urine assay after intensive prostatic massage, few studies have been conducted to establish its diagnostic value in the peripheral blood (PBL). The aim of the present study was to examine the PCA3 expression in blood as a diagnostic tool, and to provide an additional strategy to improve PCa diagnosis. METHODS: PCA3 transcripts were detected by RT-PCR in PBL and prostatic tissues from patients. PBL sampling also included a group of young healthy volunteers. The relationship between the PCA3 RNA detection and clinical characteristics was analyzed. RESULTS: PCA3 detection in blood presented 94% specificity and 32% sensitivity, and its combined detection in tissues significantly improved diagnostic parameters. However, PCA3 RNA detection in blood was also associated with PSA levels ≥10 ng/mL, and their combination provided a sensitivity of 60% and specificity of 93%. CONCLUSIONS: Detection of the PCA3 RNA in patients' blood is an efficient tool for PCa diagnosis because it allows a routine collection procedure, which is also supported by the ongoing screening marker, prostate-specific antigen (PSA). We propose its combined use with PSA levels ≥10 ng/mL, which improves accuracy, and prevents overdiagnosis and overtreatment.

Gene expression profile in the peripheral blood of patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: Prostate cancer consists of multifactorial and multifocal events, generating differential gene expression in tumor cells. METHODS: The molecular profile of 14 gene expression was analyzed through cDNA array in blood samples of patients with prostate cancer (PCa) and benign prostatic hyperplasia (BPH). RESULTS: Messenger RNA from patient's blood showed significant differences between PCa and BPH groups only for the NOS3 gene, with an occurrence chance for PCa5.8-fold higher than BPH disease. CONCLUSION: The NOS3 gene expression in the patient's blood may be used as a putative biomarker for prostate cancer.

The -786T>C promoter polymorphism of the NOS3 gene is associated with prostate cancer progression.

BACKGROUND: There is no biological or epidemiological data on the association between NOS3 promoter polymorphisms and prostate cancer. The polymorphisms in the promoter region of NOS3 gene may be responsible for variations in the plasma NO, which may promote cancer progression by providing a selective growth advantage to tumor cells by angiogenic stimulus and by direct DNA damage. METHODS: This study aimed evaluating the NOS3 promoter polymorphisms by PCR-SSCP and sequencing, associating genotypes and haplotypes with NOS3 expression levels through semi-quantitative RT-PCR, and with PCA3 mRNA detection, a specific tumor biomarker, in the peripheral blood of pre-surgical samples from 177 patients; 83 PCa and 94 BPH. RESULTS: Three novel SNPs were identified -764A>G, -714G>T and -649G>A in the NOS3 gene promoter region, which together with the -786T>C generated four haplotypes (N, T, C, A). NOS3 gene expression levels were affected by the -786T>C polymorphism, and there was a 2-fold increase in NOS3 levels favored by the incorporation of each C allele. NOS3 levels higher than 80% of the constitutive gene expression level (B2M) presented a 4-fold increase in PCa occurrence. CONCLUSION: The -786T>C polymorphism was the most important promoter alteration of the NOS3 gene that may affect the PCa progression, but not its occurrence, and the incorporation of the C allele is associated with increased levels of NOS3 transcripts. The NOS3 transcript levels presented a bimodal behavior in tumor development and may be used as a biomarker together with the PCA3 marker for molecular staging of the prostate cancer.

Transforming growth factor-beta 1 gene polymorphisms and expression in the blood of prostate cancer patients.

The transforming growth factor beta 1 (TGF-beta1) is a multifunctional cytokine with several regulatory activities in tumor cells affecting growth, differentiation, and function. Alterations in gene expression, secretion, and regulation of TGF-beta1 may lead to a favorable environment for tumor development by angiogenesis stimulation and immune system suppression. We evaluated the influence of the TGFB1 polymorphisms by ARMS-PCR, Leu10Pro, and Arg25Pro, on prostate cancer (PCa) and benign prostatic hyperplasia (BPH). We assessed TGFB1 polymorphisms and their relation to mRNA levels (semi-quantitative RT-PCR) in blood samples as well as the implications in disease occurrence and progression. Peripheral blood samples from 175 patients were analyzed as to 92 BPH and 83 PCa. Samples obtained from 132 healthy males were used as negative controls. PCa patients with a Gleason score greater than 7 presented a higher frequency of the C allele (Leu10Pro). This allele was associated with a higher risk of developing PCa and BPH compared to the population (2.6 and 3.6 times higher, respectively). Patients with TGFB1 transcript levels equal to or more than 70% higher than control levels presented a 5.34 and 2.14-fold higher risk of having PCa and BPH, respectively, relative to the population. No association was detected between polymorphisms and mRNA levels. The C allele of the Leu10Pro polymorphism may predispose men to a more rapid cancer progression. Additionally, higher mRNA levels in the peripheral blood of PCa patients suggest that tumor cells may be disseminated in the circulation and could be used as a biomarker for extra-capsular invasion.

The endothelial nitric oxide synthase Glu-298-Asp polymorphism and its mRNA expression in the peripheral blood of patients with prostate cancer and benign prostatic hyperplasia.

BACKGROUND: The endothelial nitric oxide synthase (ecNOS) has an important role in vascular development and in the carcinogenesis process of prostate cancer (PCa). The nitric oxide (NO) production may promote cancer progression by providing a selective growth advantage to tumor cells, by angiogenic stimulus and by direct DNA damage. METHODS: The present study aimed at evaluating the ecNOS Glu-298-Asp polymorphism by the PCR-RFLP technique, associating genotypes with gene expression levels and the tumor biomarker, Prostate Cancer Antigen (DD3), through semi-quantitative RT-PCR. Pre-surgical peripheral blood samples from 160 patients were analyzed: 84 PCa, 11 prostate intraepithelial neoplasia (PIN) and 65 benign prostatic hyperplasia (BPH). RESULTS: The GG and GT Glu-298-Asp genotypes were associated with positive DD3 expression in the peripheral blood, presenting a 3.32-fold higher risk of PCa occurrence. There was no association between genotypes and ecNOS mRNA expression levels; however, the presence of the G allele is closely related to the hematogenous dissemination event of tumoral cells, as evidenced by the DD3 positivity. The higher G allele frequency among pT3 and pT4 staged PCa patients suggests that this would be associated with advanced phenotypes of the disease and may also be contributing to higher NO levels, causing cancer progression. CONCLUSIONS: The G allele may have a secondary influence on the prostate cancer predisposition, but an essential role on the event of tumor cells hematogenous dissemination, probably due to the angiogenic stimulus.