Etel Rodrigues Pereira Gimba

PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling

BackgroundPCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling.MethodsLNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR.ResultsLNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions.ConclusionsOur findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control.

Human osteopontin splicing isoforms: Known roles, potential clinical applications and activated signaling pathways

Human osteopontin is subject to alternative splicing, which generates three isoforms, termed OPNa, OPNb and OPNc. These variants show specific expression and roles in different cell contexts. We present an overview of current knowledge of the expression profile of human OPN splicing isoforms (OPN-SIs), their tissue-specific roles, and the pathways mediating their functional properties in different pathophysiological conditions. We also describe their putative application as biomarkers, and their potential use as therapeutic targets by using antibodies, oligonucleotides or siRNA molecules. This synthesis provides new clues for a better understanding of human OPN splice variants, their roles in normal and pathological conditions, and their possible clinical applications.

Influence of Lycopene on Cell Viability, Cell Cycle, and Apoptosis of Human Prostate Cancer and Benign Hyperplastic Cells

Prostate cancer is the most common malignancy in men and the second leading cause of cancer-related mortality in men of the Western world. Lycopene has received attention because of its expcted potential to prevent cancer. In the present study, we evaluated the influence of lycopene on cell viability, cell cycle, and apoptosis of human prostate cancer cells and benign prostate hyperplastic cells. Using MTT assay, we observed a decrease of cell viability in all cancer cell lines after treatment with lycopene, which decreased the percentage of cells in G0/G1 phase and increased in S and G2/M phases after 96 h of treatment in metastatic prostate cancer cell lineages. Flow citometry analysis of cell cycle revealed lycopene promoted cell cycle arrest in G0/G1 phase after 48 and 96 h of treatment in a primary cancer cell line. Using real time PCR assay, lycopene also induced apoptosis in prostate cancer cells with altered gene expression of Bax and Bcl-2. No effect was observed in benign prostate hyperplasia cells. These results suggest an effect of lycopene on activity of human prostate cancer cells.

Osteopontin-a splice variant is overexpressed in papillary thyroid carcinoma and modulates invasive behavior

Osteopontin (OPN) is a matricellular protein overexpressed in cancer cells and modulates tumorigenesis and metastasis, including in thyroid cancer (TC). The contribution of each OPN splice variant (OPN-SV), named OPNa, OPNb and OPNc, in TC is currently unknown. This study evaluates the expression of total OPN (tOPN) and OPN-SV in TC tissues and cell lines, their correlation with clinicopathological, molecular features and their functional roles. We showed that tOPN and OPNa are overexpressed in classic papillary thyroid carcinoma (cPTC) in relation to adjacent thyroid, adenoma and follicular variant of papillary thyroid carcinoma (fvPTC) tissues. In cPTC, OPNa overexpression is associated with larger tumor size, vascular invasion, extrathyroid extension and BRAFV600E mutation. We found that TC cell lines overexpressing OPNa exhibited increased proliferation, migration, motility and in vivo invasion. Conditioned medium secreted from cells overexpressing OPNa induce MMP2 and MMP9 metalloproteinases activity. In summary, we described the expression pattern of OPN-SV in cPTC samples and the key role of OPNa expression on activating TC tumor progression features. Our findings highlight OPNa variant as TC biomarker, besides being a putative target for cPTC therapeutic approaches.

Expression analysis of thrombospondin 2 in prostate cancer and benign prostatic hyperplasia.

Thrombospondin 2 (TSP2) is a protein with important roles in different tumor types, mainly related to tumor inhibition. However, there are limiting data regarding TSP2 in prostate cancer (PCa) and benign prostatic hyperplasia (BPH). We aimed to investigate TSP2 transcript and protein expression in tumoral and non-tumoral prostate tissues and cell lines, and its implications for PCa diagnosis and progression. TSP2 transcript expression was evaluated by real time PCR in PCa and BPH tissue samples and in tumoral and non-tumoral cell lines. TSP2 protein expression analysis was conducted by immunohistochemistry in a tissue microarray (TMA) containing PCa and BPH tissue samples. TSP2 transcript was down-regulated in PCa tissue samples and cell lines, when compared to BPH and non-tumoral samples (P<0.01). Receiver Operating Curve (ROC) analysis demonstrated that TSP2 transcript levels can better distinguish PCa from BPH tissue samples (P<0.01) than serum PSA levels (P=0.299). TSP2 protein expression has been observed in the cytoplasm of both PCa and BPH epithelial and stromal compartments. TSP2 stromal staining scores were significantly lower in PCa than in BPH tissues (P<0.01), while similar TSP2 epithelial staining patterns were observed in both diseases. Notably, the TSP2 epithelial staining score was significantly correlated to vascular invasion and biochemical recurrence in PCa tissue samples (P<0.05). Our data indicate that TSP2 is down-regulated at PCa tissues and cell lines, especially at stroma compartment, which could be related to PCa progression. TSP2 levels could potentially be applied for differential PCa and BPH diagnosis.

Osteopontin splice variants expression is involved on docetaxel resistance in PC3 prostate cancer cells.

Osteopontin (OPN) is a phosphoprotein that activates several aspects of tumor progression. Alternative splicing of the OPN primary transcript generates three splicing isoforms, OPNa, OPNb and OPNc. In this report, we investigated some cellular mechanisms by which OPN splice variants could mediate PC3 prostate cancer (PCa) cell survival and growth in response to docetaxel (DXT)-induced cell death. Cell survival before and after DXT treatment was analyzed by phase-contrast microscopy and crystal-violet staining assays. Quantitative real-time PCR and immunocytochemical staining assays were used to evaluate the putative involvement of epithelial-mesenchymal transition (EMT) and OPN isoforms on mediating PC3 cell survival. Upon DXT treatment, PC3 cells overexpressing OPNb or OPNc isoforms showed higher cell densities, compared to cells overexpressing OPNa and controls. Notably, cells overexpressing OPNb or OPNc isoforms showed a downregulated pattern of EMT epithelial cell markers, while mesenchymal markers were mostly upregulated in these experimental conditions. We concluded that OPNc or OPNb overexpression in PC3 cells can mediate resistance and cell survival features in response to DXT-induced cell death. Our data also provide evidence the EMT program could be one of the molecular mechanisms mediating survival in OPNb- or OPNc-overexpressing cells in response to DXT treatment. These data could further contribute to a better understanding of the mechanisms by which PCa cells acquire resistance to DXT treatment.

Lycopene Extracts from Different Tomato-Based Food Products Induce Apoptosis in Cultured Human Primary Prostate Cancer Cells and Regulate TP53, Bax and Bcl-2 Transcript Expression

Carotenoids are the main tomato components, especially lycopene. Lycopene is more bioavailable in tomato processed products than in raw tomatos, since formation of lycopene cis-isomers during food processing and storage may increase its biological activity. In the current study, we evaluated the influence of lycopene extracts (5 mg / mL) from different tomato-based food products (paste, sauce, extract and ketchup) on cell viability and apoptosis on primary human prostate cancer cells (PCa cels) for 96h. Using MTT assay, we observed a significant decrease on primary PCa cell viability upon treatment with lycopene extracted from either 4 tomato-based food products. Flow cytometeric analysis revealed that lycopene from tomato extract and tomato sauce promoted up to fifty-fold increase on the proportion of apoptotic cells, when compared to the control group. Using real time PCR assay, we found that lycopene promoted an upregulation of TP53 and Bax transcript expression and also downregulation of Bcl-2 expression in PCa cells. In conclusion, our data demostrate that cis-lycopene promoted a significant inhibition on primary PCa cell viability, as well as an increase on their apoptotic rates, evidencing that cis-lycopene contained in tomato sauce and extract cain mainly modulate of primary human prostate cancer cell survival.

Chemosensitizing effects of metformin on cisplatin- and paclitaxel-resistant ovarian cancer cell lines

BACKGROUND Epithelial ovarian cancer (EOC) remains the most lethal gynecologic malignancy. Primary cytoreductive surgery with adjuvant taxane-platinum chemotherapy is the standard treatment to fight ovarian cancer, however, their side effects are severe, and chemoresistance emerges at high rates. Therefore, EOC clinic urges for novel treatment strategies to reverse chemoresistance and to improve the survival rates. Metformin has been shown to act in synergy with certain anti-cancer agents, overcoming chemoresistance in various types of tumors. This paper aims to investigate the use of metformin as a new treatment option for cisplatin- and paclitaxel-resistant ovarian cancer. METHODS The effects of metformin alone or in combination with conventional drugs on resistant EOC cell lines were investigated using the MTT assay for cell proliferation; Flow Cytometry analysis for cell cycle and the mRNA expression was analyzed using the real-time PCR technique. RESULTS We found that metformin exhibited antiproliferative effects in paclitaxel-resistant A2780-PR, and in cisplatin-resistant ACRP cell lines. The combined therapy containing conventional drugs and metformin improved the effect of the treatment in cell proliferation rate, especially in the resistant cells. We found that metformin, in clinical relevant doses, could significantly reduce the mRNA expression of inflammatory cytokines and NF-κB signaling pathway. CONCLUSIONS Taken together, our observations suggest that metformin inhibits the inflammatory pathway induced by paclitaxel and cisplatin treatment. Furthermore, metformin in combination with paclitaxel or cisplatin improved the sensitivity in drug-resistant ovarian cancer cells. Therefore, metformin may be beneficial treatment strategy, particularly in patients with tumors refractory to platinum and taxanes.

Osteopontin and their roles in hematological malignancies: Splice variants on the new avenues

Osteopontin (OPN) is a protein expressed in several tissues, including bone marrow, in which it performs distinct roles, such as modulating hematopoietic stem cell niche and bone remodeling. Most data in hematological malignancies (HMs) refers to total OPN (tOPN), comprehending the sum of distinct OPN splicing isoforms (OPN-SI), while reports describing the expression and roles of each OPN-SI are scarce. This review aims to summarize tOPN roles in HMs and provide evidence that OPN-SIs can also modulate specific functions in HMs biology. We summarize that upregulated tOPN can modulate HMs (leukemia, lymphoma and myeloma) progression, inducing cell adhesion, invasion, angiogenesis, cell differentiation and extramedullary and/or central nervous system infiltration. Based on this expression pattern, tOPN has been pointed out as a biomarker in those HMs, thus providing potential targets for therapeutic approaches. Our group found that OPN-SIs are expressed in childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cell lines (unpublished data), providing early evidence that OPN-SIs are also expressed in BCP-ALL. Further studies should investigate whether these OPN-SIs can differently modulate HMs biology and their putative application as auxiliary biomarkers for HMs.

Osteopontin-c isoform levels are associated with SR and hnRNP differential expression in ovarian cancer cell lines

Osteopontin-c splicing isoform activates ovarian cancer progression features. Imbalanced expression of splicing factors from serine/arginine -rich and heterogeneous ribonucleoproteins families has been correlated with the generation of oncogenic splicing isoforms. Our goal was to investigate whether there is any association between the transcriptional patterns of these splicing factors in ovarian cells and osteopontin-c expression levels. We also aimed to investigate the occurrence of these splicing factors binding sites inside osteopontin exon 4 and adjacent introns. To test associations between osteopontin-c and splicing factors expression patterns, we used an in vitro model in which OVCAR-3 cells overexpressing osteopontin-c (OVCAR-3/OPNc++) presented higher transcriptional levels of osteopontin-c than two other ovarian carcinoma cells (TOV-112D, SKOV-3) and ovarian non-tumoral cell lines (IOSE 364 and IOSE 385). The transcriptional levels of osteopontin-c, serine/arginine-rich, and hnRNP factors were evaluated using real-time polymerase chain reaction. Human Splice Finder software was used to search for putative splicing factor binding sites in osteopontin genomic regions. OVCAR-3/OPNc++ cells presented higher transcriptional levels of hnRNP than serine/arginine-rich when compared to TOV-112D, SKOV-3, and IOSE cells. TOV-112D and SKOV-3 cells also overexpressed hnRNP in relation to serine/arginine-rich transcripts. Putative binding sites for these splicing factors have been predicted on osteopontin exon 4 and their upstream and downstream intronic regions. Our data showed that higher osteopontin-c expression levels are associated with a predominance of hnRNP in relation to serine/arginine-rich transcripts and that osteopontin exon 4 and adjacent intronic sequences contain predicted binding sites for some of these tested splicing factors. In conclusion, differential expression of these splicing factors in ovarian cancer cells could be one of the putative mechanisms leading to aberrant splicing of the osteopontin primary transcript. Future work, aiming to control ovarian cancer progression by downregulating osteopontin-c levels, could include strategies that also regulate heterogeneous ribonucleoproteins and serine/arginine-rich expression levels in order to modulate osteopontin splicing.