A Novel Detection Method of Breast Cancer through a Simple Panel of Biomarkers.

Circulating tumor cells (CTCs) have been identified as responsible for the spread of tumors to other organs of the body. In this sense, the development of sensitive and specific assays for their detection is important to reduce the number of deaths due to metastases. Here, we assessed whether the detection of CTCs in peripheral blood can serve in the construction of a panel of diagnosis and monitoring treatments of breast cancer (BC), focusing on the expression of markers of epithelial-mesenchymal transition. Through analyzing the blood from women without breast alterations (control), women with benign alterations, women with breast cancer without chemotherapy, and women with breast cancer with chemotherapy, we identified the best markers by transcriptional levels and determined three profiles of CTCs (mesenchymal, intermediate, and epithelial) by flow cytometry which, combined, can be used for diagnosis and therapy monitoring with sensitivity and specificity between 80% and 100%. Therefore, we have developed a method for detecting breast cancer based on the analysis of CTC profiles by epithelial-mesenchymal transition markers which, combined, can be used for the diagnosis and monitoring of therapy.

New Insights into the Role of Polybromo-1 in Prostate Cancer.

The human protein Polybromo-1 (PBMR1/BAF180) is a component of the SWI/SNF chromatin-remodeling complex that has been reported to be deregulated in tumors. However, its role in prostate cancer (PCa) is largely unknown. In this study, we described the PBRM1 transcriptional levels and the protein expression/localization in tissues of PCa patients and in prostatic cell lines. Increased PBRM1 mRNA levels were found in PCa samples, when compared to benign disease, and were correlated with higher Gleason score. We also verified that only the nuclear localization of PBRM1 protein is correlated with a more aggressive disease and high Prostate-Specific Antigen (PSA) levels in tissue microarrays. Intriguing expression patterns of mRNA and protein were identified in the cell lines. Although PBRM1 protein was restricted to the nuclei, in tumor cell lines in non-neoplastic cells, it was also present in vesicular-like structures that were dispersed within the cytoplasm. We knocked-down PBRM1 in the castration-resistant PCa (CRPC) cell line PC-3 and we verified that PBRM1 promotes the expression of several markers of aggressiveness, including EpCAM, TGF-ß, and N-Cadherin. Therefore, our data supported the hypothesis that PBRM1 displays a pivotal role in the promotion and maintenance of the malignant behavior of PCa, especially in CRPC.

Ethanolic Extracts from Azadirachta indica Leaves Modulate Transcriptional Levels of Hormone Receptor Variant in Breast Cancer Cell Lines.

Breast Cancer (BC) encompasses numerous entities with different biological and behavioral characteristics, favored by tumor molecular complexity. Azadirachta indica (neem) presents phenolic compounds, indicating its potential as an antineoplastic compound. The present study aimed to evaluate the cellular response of MCF10, MCF7, and MDA-MB-231 breast cell lines to ethanolic extracts of neem leaves (EENL) obtained by dichloromethane (DCM) and ethyl acetate (EA) solvent. Extracts' antiproliferative activities were evaluated against MCF 10A, MCF7, and MDA-MB-231 for 24 and 48 h using MTT assay. ESR1, ESR2, AR, AR-V1, AR-V4, and AR-V7 transcripts were quantified through qPCR for 0.03125 µg/mL of DCM and 1.0 µg/mL for EA for 48 h. The EENL was tested on Drosophila melanogaster as a sole treatment and then also together with doxorubicin. Antiproliferative effect on tumor cell lines without affecting MCF 10A were 1.0 µg/mL (P < 0.001) for EA, and 0.03125 µg/mL (P < 0.0001) for DCM, both after 48 h. Transcriptional levels of AR-V7 increased after treatment. In vivo assays demonstrated that EENL induced fewer tumors at a higher concentration with doxorubicin (DXR). The behavior of AR-V7 in the MDA-MB-231 tumor lineage indicates new pathways involved in tumor biology and this may have therapeutic value for cancer.

Altered Leukocyte Sphingolipid Pathway in Breast Cancer.

Sphingolipid metabolism pathway is essential in membrane homeostasis, and its dysfunction has been associated with favorable tumor microenvironment, disease progression, and chemotherapy resistance. Its major components have key functions on survival and proliferation, with opposing effects. We have profiled the components of the sphingolipid pathway on leukocytes of breast cancer (BC) patients undergoing chemotherapy treatment and without, including the five sphingosine 1-phosphate (S1P) receptors, the major functional genes, and cytokines, in order to better understand the S1P signaling in the immune cells of these patients. To the best of our knowledge, this is the first characterization of the sphingolipid pathway in whole blood of BC patients. Skewed gene profiles favoring high SPHK1 expression toward S1P production during BC development was observed, which was reversed by chemotherapy treatment, and reached similar levels to those found in healthy donors. Such levels were also correlated with high levels of TNF-α. Our data revealed an important role of the sphingolipid pathway in immune cells in BC with skewed signaling of S1P receptors, which favored cancer development even under chemotherapy, and may probably be a trigger of cancer resistance. Thus, these molecules must be considered as a target pathway for combined BC therapeutics.

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.

Arrabidaea chica chloroform extract modulates estrogen and androgen receptors on luminal breast cancer cells.

BACKGROUND: Breast Cancer (BC) is the most common cancer in women worldwide and, although 70% of patients are responsive to selective Estrogen Receptor (ER) modulators such as Tamoxifen (Tam), patients' survival is comprised by resistance to endocrine therapy. Brazilian flora, especially the Amazon biome, is one of the richest global sources of native species with potentially bioactive compounds. Arrabidaea chica is a plant native to the Amazon that has been used in the treatment of different diseases. However, its action on BC remains unclear. METHODS: Herein the biological effects of the chloroform extract of A. chica (CEAC) were evaluated on BC cells and in in vivo model. After confirmation of CEAC antioxidant capacity, cells were treated with CEAC and Tam, alone and with CEAC+Tam. The cell viability was evaluated by MTT and hormone receptor transcripts levels were assessed (ESR1, ESR2 and AR). Finally, anticarcinogenicity of CEAC was recorded in Drosophila melanogaster through Epithelial Tumor Test (ETT). RESULTS: The study confirmed the antioxidant activity of CEAC. CEAC was selective for MCF-7, downregulating ESR2 and AR transcripts and upregulating ESR2 expression. The modulatory effects of CEAC on ERs did not differ between cells treated with Tam and with CEAC+Tam. Interestingly, previous treatment with CEAC, followed by treatment with Tam promoted a significant decrease in cell viability. The extract also presented anticarcinogenic effect in in vivo assay. CONCLUSION: The bioassays on breast tumor cells demonstrated the antiproliferative activity of the extract, which modulated the expression of hormone receptors and sensitized luminal tumor cells to Tam. These results suggest that CEAC could be a complementary treatment for BC.

Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) Spectroscopy Analysis of Saliva for Breast Cancer Diagnosis.

Saliva biomarkers using reagent-free biophotonic technology have not been investigated as a strategy for early detection of breast cancer (BC). The attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy has been proposed as a promising tool for disease diagnosis. However, its utilization in cancer is still incipient, and currently saliva has not been used for BC screening. We have applied ATR-FTIR onto saliva from patients with breast cancer, benign breast disease, and healthy matched controls to investigate its potential use in BC diagnosis. Several salivary vibrational modes have been identified in original and second-derivative spectra. The absorbance levels at wavenumber 1041 cm-1 were significantly higher (p < 0.05) in saliva of breast cancer patients compared with those of benign patients, and the ROC curve analysis of this peak showed a reasonable accuracy to discriminate breast cancer from benign and control patients. The 1433-1302.9 cm-1 band area was significantly higher (p < 0.05) in saliva of breast cancer patients than in control and benign patients. This salivary ATR-FTIR spectral area was prevalidated as a potential diagnostic biomarker of BC. This spectral biomarker was able to discriminate human BC from controls with sensitivity and specificity of 90% and 80%, respectively. Besides, it was able to differentiate BC from benign disease with sensitivity and specificity of 90% and 70%, respectively. Briefly, for the first time, saliva analysis by ATR-FTIR spectroscopy has demonstrated the potential use of salivary spectral biomarkers (1041 cm-1 and 1433-1302.9 cm-1) as a novel alternative for noninvasive BC diagnosis, which could be used for screening purposes.

Overweight Women with Breast Cancer on Chemotherapy Have More Unfavorable Inflammatory and Oxidative Stress Profiles.

Chronic inflammation and redox imbalance are strongly influenced by diet and nutritional status, and both are risk factors for tumor development. This prospective study aimed to explore the associations between inflammatory and antioxidant markers and nutritional status in women with breast cancer undergoing chemotherapy. The women were evaluated at three times: T0, after the infusion of the first cycle; T1, after infusion of the intermediate cycle; and T2, after the infusion of the last chemotherapy cycle. The consumption of antioxidant nutrients and the Total Dietary Antioxidant Capacity reduced between T0 and T2 and the Dietary Inflammatory Index scores increased throughout the chemotherapy. Blood samples taken at the end of the chemotherapy showed lower levels of glutathione reductase and reduced glutathione, with greater quantification of the transcripts for Interleukin-6 and Tumor Necrosis Factor α. It should be emphasized that the Total Dietary Antioxidant Capacity is lower and the Dietary Inflammatory Index is higher in the group of overweight patients at the end of the follow-up, besides showing lower levels of the redox status, especially the plasma levels of glutathione reductase (p = 0.039). In addition, trends towards higher transcriptional levels of cytokines in peripheral blood were observed more often in overweight women than in non-overweight women. In this study of 55 women with breast cancer, nine (16%) with metastases, diet became more pro-inflammatory with fewer antioxidants during the chemotherapy. Briefly, we have shown that chemotherapy is critical for high-risk overweight women due to their reduced intake of antioxidant nutrients, generating greater inflammatory and oxidative stress profiles, suggesting the adoption of healthier dietary practices by women with breast cancer throughout their chemotherapy.

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.

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.

A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers.

The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.

Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

Combined analysis of multiple mRNA markers by RT-PCR assay for prostate cancer diagnosis.

OBJECTIVES: To develop a semi-quantitative method for prostate cancer diagnosis and to validate this technique in clinical protocols with the use of multiplex RT-PCR assays for five different biomarkers associated with carcinogenesis, including the PCA3 gene. DESIGN AND METHODS: AR, SRD5A2, KLK2, PSMA, and PCA3 transcripts were analyzed by multiplex RT-PCR assay in 73 prostatic tissue samples from patients with prostate cancer (PCa) and benign hyperplasia (BPH). RESULTS: Significant differences were observed between cancerous and hypertrophic tissues in the relative expression of these genes. AR, KLK2, PSMA, and PCA3 genes displayed increased transcriptional levels in the cancer specimens; on the other hand, SRD5A2 mRNA levels were higher in the BPH samples. CONCLUSIONS: Our results suggest that the most promising marker for PCa diagnosis was positive PCA3 detection associated with serum PSA levels, which showed 28-fold higher chances for cancer occurrence, with 92% specificity and 94% positive predictive value.