The Intracellular and Secreted Sides of Osteopontin and Their Putative Physiopathological Roles.

Classically, osteopontin (OPN) has been described as a secreted glycophosprotein. Indeed, most data concerning its physiological and pathological roles are mainly related to the secreted OPN (sOPN). However, there are several instances in which intracellular OPN (iOPN) has been described, presenting some specific roles in distinct experimental models, such as in the immune system, cancer cells, and neurological disorders. We herein aimed to highlight and discuss some of these secreted and intracellular roles of OPN and their putative clinical and biological impacts. Moreover, by consolidating data from the OPN protein database, we also analyzed the occurrence of signal peptide (SP) sequences and putative subcellular localization, especially concerning currently known OPN splicing variants (OPN-SV). Comprehending the roles of OPN in its distinct cellular and tissue environments may provide data regarding the additional applications of this protein as biomarkers and targets for therapeutic purposes, besides further describing its pleiotropic roles.

LRP1B Expression as a Putative Predictor of Response to Pegylated Liposomal Doxorubicin Treatment in Ovarian Cancer.

BACKGROUND: Pegylated liposomal doxorubicin (PLD) is among the most active therapies for recurrent/progressive ovarian cancer (OC). Low-density lipoprotein receptor-related protein 1B (LRP1B) is one of the 10 most significantly deleted genes in human cancers. It mediates endocytosis of several factors from the cellular environment including liposomes. Although the LRP1B role in cancer has not been fully disclosed, its contribution to resistance to liposomal therapies has been hypothesized. This study aimed to evaluate the impact of LRP1B protein as a possible marker of response to PLD in patients with OC. METHODS: LRP1B expression and response to PLD were analyzed in OC cell lines by qRT-PCR and PrestoBlue viability assay, respectively. LRP1B protein expression was evaluated for the first time, in tumor samples from PLD-treated patients and controls (other chemotherapies) by immunohistochemistry. Association of LRP1B staining score (determined based on intensity and percentage of positively stained cells) with clinicopathological features, response to therapy and survival outcomes was evaluated. RESULTS: OC cells with increased expression of LRP1B were more sensitive to PLD. LRP1B staining score was associated with clinicopathological features, response to therapy, and survival outcomes. Higher LRP1B levels were associated with prolonged progression-free survival. This association was more evident in patients treated with PLD and in responders to PLD. CONCLUSION: Our results support a possible role of LRP1B as a predictor of response to PLD in patients with OC.

(3,3'-Methylene)bis-2-hydroxy-1,4-naphthoquinones induce cytotoxicity against DU145 and PC3 cancer cells by inhibiting cell viability and promoting cell cycle arrest.

We developed a novel method for the synthesis of bis-naphthoquinones (BNQ), which are hybrids of lawsone (2-hydroxy-1,4-naphthoquinone) and 3-hydroxy-juglone (3,5-dihydroxy-1,4-naphthoquinone). The anticancer activity of three synthesized compounds, named 4 (RC10), 5 (RCDFC), and 6 (RCDOH) was evaluated in vitro against two metastatic prostate cancer (PCa) cell lines, DU145 and PC3, using MTT assays. We found that 4 (RC10) and 5 (RCDFC) induced cytotoxicity against DU145 and PC3 cells. Flow cytometry analysis revealed that these two compounds promoted cell cycle arrest in G1/S and G2/M phases, increased Sub-G1 peak and induced inhibition in cell viability. We also showed that these effects are cell-type context dependent and more selective for these tested PCa cells than for HUVEC non-tumor cells. The two BNQ compounds 4 (RC10) and 5 (RCDFC) displayed promising anticancer activity against the two tested metastatic PCa cell lines, DU145 and PC3. Their effects are mainly associated with inhibition of cell viability, possibly through apoptotic cell death, besides altering the SubG1, G1/S and G2/M phases of cell cycle. 5 (RCDFC) compound was found to be more selective than 4 (RC10), when comparing their cytotoxic effects in relation to HUVEC non-tumoral cells. Future work should also test these compounds in combination with other chemotherapeutic drugs to evaluate their effects on further sensitizing drug-resistant metastatic PCa cells.

Loss of the p53 transactivation domain results in high amyloid aggregation of the Δ40p53 isoform in endometrial carcinoma cells.

Dysfunctional p53 formation and activity can result from aberrant expression and subcellular localization of distinct p53 isoforms or aggregates. Endometrial carcinoma (EC) is a cancer type in which p53 status is correlated with prognosis, and TP53 mutations are a frequent genetic modification. Here we aimed to evaluate the expression patterns of different p53 isoforms and their contributions to the formation and subcellular localization of p53 amyloid aggregates in both EC and endometrial nontumor cell lines. We found that full-length (fl) p53 and a truncated p53 isoform, Δ40p53, resulting from alternative splicing of exon 2 or alternative initiation of translation at ATG-40, are the predominantly expressed p53 variants in EC cells. However, Δ40p53 was the major p53 isoform in endometrial nontumor cells. Immunofluorescence assays revealed that Δ40p53 is mainly localized to cytoplasmic punctate structures of EC cells, resembling solid-phase structures similar to those found in neurodegenerative pathologies. Using light-scattering kinetics, CD, and transmission EM, we noted that the p53 N-terminal transactivation domain significantly reduces aggregation of the WT p53 DNA-binding domain, confirming the higher aggregation tendency of Δ40p53, which lacks this domain. This is the first report of cytoplasmic Δ40p53 in EC cells being a major component of amyloid aggregates. The differential aggregation properties of p53 isoforms in EC cells may open up new avenues in the development of therapeutic strategies that preferentially target specific p53 isoforms to prevent p53 amyloid aggregate formation.

Osteopontin-4 and Osteopontin-5 splice variants are expressed in several tumor cell lines.

Among osteopontin splice variants (OPN-SV), the expression profile of osteopontin-4 (OPN4) and osteopontin-5 (OPN5) has not been addressed in distinct cancer types. We herein aimed to investigate their expression in several cancer cell lines, besides comparing it in relation to the three previously described OPN-SV: OPNa, OPNb and OPNc. Total RNA from cancer cell lines, including prostate (PC3 and DU145), ovarian (A2780), breast (MCF-7 and MDA-MB-231), colorectal (Caco-2, HT-29 and HCT-116), thyroid (TT, TPC1 and 8505c) and lung (A549 and NCI-H460) was extracted, followed by cDNA synthesis. OPN-SV transcript analysis by RT-PCR or RT-qPCR were performed using OPN-SV specific oligonucleotides and gapdh and actin transcripts were used as housekeeping controls. OPN4 and OPN5 transcripts displayed co-expression in most tested cell lines. OPN4 was found expressed in similar or higher levels in relation to OPN5. Moreover, in most tested cell lines, OPN4 is also expressed in similar levels to OPNa or OPNb. The expression of OPN5 is also generally variable in relation to the other OPN-SV, but expressed in similar or higher levels in relation to OPNc, depending on each tested cell line. OPN4 and OPN5 seem to be co-expressed in several tumor types and OPN4 is one of the most overexpressed OPN-SV in distinct tumor cell lines. Once both OPN4 and OPN5 are differentially expressed and also evidence tumor-specific expression patterns, we hypothesize that similarly to the other OPN-SV, they also possibly contribute to key aspects of tumor progression, what should be further functionally investigated in distinct tumor models.

Molecular Aspects of Thyroid Calcification.

In thyroid cancer, calcification is mainly present in classical papillary thyroid carcinoma (PTC) and in medullary thyroid carcinoma (MTC), despite being described in benign lesions and in other subtypes of thyroid carcinomas. Thyroid calcifications are classified according to their diameter and location. At ultrasonography, microcalcifications appear as hyperechoic spots ≤ 1 mm in diameter and can be named as stromal calcification, bone formation, or psammoma bodies (PBs), whereas calcifications > 1 mm are macrocalcifications. The mechanism of their formation is still poorly understood. Microcalcifications are generally accepted as a reliable indicator of malignancy as they mostly represent PBs. In order to progress in terms of the understanding of the mechanisms behind calcification occurring in thyroid tumors in general, and in PTC in particular, we decided to use histopathology as the basis of the possible cellular and molecular mechanisms of calcification formation in thyroid cancer. We explored the involvement of molecules such as runt-related transcription factor-2 (Runx-2), osteonectin/secreted protein acidic and rich in cysteine (SPARC), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteopontin (OPN) in the formation of calcification. The present review offers a novel insight into the mechanisms underlying the development of calcification in thyroid cancer.

The LQB-223 Compound Modulates Antiapoptotic Proteins and Impairs Breast Cancer Cell Growth and Migration.

Drug resistance represents a major issue in treating breast cancer, despite the identification of novel therapeutic strategies, biomarkers, and subgroups. We have previously identified the LQB-223, 11a-N-Tosyl-5-deoxi-pterocarpan, as a promising compound in sensitizing doxorubicin-resistant breast cancer cells, with little toxicity to non-neoplastic cells. Here, we investigated the mechanisms underlying LQB-223 antitumor effects in 2D and 3D models of breast cancer. MCF-7 and MDA-MB-231 cells had migration and motility profile assessed by wound-healing and phagokinetic track motility assays, respectively. Cytotoxicity in 3D conformation was evaluated by measuring spheroid size and performing acid phosphatase and gelatin migration assays. Protein expression was analyzed by immunoblotting. Our results show that LQB-223, but not doxorubicin treatment, suppressed the migratory and motility capacity of breast cancer cells. In 3D conformation, LQB-223 remarkably decreased cell viability, as well as reduced 3D culture size and migration. Mechanistically, LQB-223-mediated anticancer effects involved decreased proteins levels of XIAP, c-IAP1, and Mcl-1 chemoresistance-related proteins, but not survivin. Survivin knockdown partially potentiated LQB-223-induced cytotoxicity. Additionally, cell treatment with LQB-223 resulted in changes in the mRNA levels of epithelial-mesenchymal transition markers, suggesting that it might modulate cell plasticity. Our data demonstrate that LQB-223 impairs 3D culture growth and migration in 2D and 3D models of breast cancer exhibiting different phenotypes.

OPNa Overexpression Is Associated with Matrix Calcification in Thyroid Cancer Cell Lines.

Osteopontin (OPN) spliced variants (OPN-SV: OPNa, OPNb, and OPNc) are aberrantly expressed in tumors and frequently associated with cancer progression. This holds true for papillary thyroid carcinoma (PTC), which is the most common type of thyroid cancer (TC). PTC often presents with desmoplasia and dystrophic calcification, including psammoma bodies (PB). This work aimed to investigate total OPN (tOPN) and OPN-SV expression and their association with the presence of PB in the PTC classical variants (cPTC), as well as the involvement of OPN-SV in matrix calcification of TC cell lines. We found that cPTC samples presenting PB showed higher OPN expression levels. In TC cell lines, OPNa overexpression promotes higher matrix calcification and collagen synthesis when compared to that of clones overexpressing OPNb or OPNc. In response to OPN knockdown, calcification was inhibited, paralleled with the downregulation of calcification markers. In conclusion, our data evidenced that OPN expression is associated with the presence of PB in cPTC samples. Among the OPN-SV, OPNa is the main contributor to matrix calcification in tested TC cells, providing clues to a better understanding on the biology and ethiopathogenesis of the calcification process in TC cells.

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.

PCA3 long noncoding RNA modulates the expression of key cancer-related genes in LNCaP prostate cancer cells.

Prostate cancer antigen 3 (PCA3) is a prostate-specific long noncoding RNA (lncRNA) involved in the control of prostate cancer (PCa) cell survival, through modulating androgen receptor (AR) signaling. To further comprehend the mechanisms by which PCA3 modulates LNCaP cell survival, we characterized the expression patterns of several cancer-related genes, including those involved in epithelial-mesenchymal transition (EMT) and AR cofactors in response to PCA3 silencing. We also aimed to develop a strategy to stably silence PCA3. Small interfering RNA (siRNA) or short hairpin RNA (shRNA) was used to knock down PCA3 in LNCaP cells. The expression of 84 cancer-related genes, as well as those coding for AR cofactors and EMT markers, was analyzed by quantitative real-time PCR (qRT-PCR). LNCaP-PCA3 silenced cells differentially expressed 16 of the 84 cancer genes tested, mainly those involved in gene expression control and cell signaling. PCA3 knockdown also induced the upregulation of several transcripts coding for AR cofactors and modulated the expression of EMT markers. LNCaP cells transduced with lentivirus vectors carrying an shRNA sequence targeting PCA3 stably downregulated PCA3 expression, causing a significant drop (60 %) in the proportion of LNCaP cells expressing the transgene. In conclusion, our data provide evidence that PCA3 silencing modulates the expression of key cancer-related genes, including those coding for AR cofactors and EMT markers. Transducing LNCaP cells with an shRNA sequence targeting PCA3 led to loss of viability of the cells, supporting the proposal of PCA3 knockdown as a putative therapeutic approach to inhibit PCa growth.

Changes in the transcriptional profile in response to overexpression of the osteopontin-c splice isoform in ovarian (OvCar-3) and prostate (PC-3) cancer cell lines.

BACKGROUND: Especially in human tumor cells, the osteopontin (OPN) primary transcript is subject to alternative splicing, generating three isoforms termed OPNa, OPNb and OPNc. We previously demonstrated that the OPNc splice variant activates several aspects of the progression of ovarian and prostate cancers. The goal of the present study was to develop cell line models to determine the impact of OPNc overexpression on main cancer signaling pathways and thus obtain insights into the mechanisms of OPNc pro-tumorigenic roles. METHODS: Human ovarian and prostate cancer cell lines, OvCar-3 and PC-3 cells, respectively, were stably transfected to overexpress OPNc. Transcriptomic profiling was performed on these cells and compared to controls, to identify OPNc overexpression-dependent changes in gene expression levels and pathways by qRT-PCR analyses. RESULTS: Among 84 genes tested by using a multiplex real-time PCR Cancer Pathway Array approach, 34 and 16, respectively, were differentially expressed between OvCar-3 and PC-3 OPNc-overexpressing cells in relation to control clones. Differentially expressed genes are included in all main hallmarks of cancer, and several interacting proteins have been identified using an interactome network analysis. Based on marked up-regulation of Vegfa transcript in response to OPNc overexpression, we partially validated the array data by demonstrating that conditioned medium (CM) secreted from OvCar-3 and PC-3 OPNc-overexpressing cells significantly induced endothelial cell adhesion, proliferation and migration, compared to CM secreted from control cells. CONCLUSIONS: Overall, the present study elucidated transcriptional changes of OvCar-3 and PC-3 cancer cell lines in response to OPNc overexpression, which provides an assessment for predicting the molecular mechanisms by which this splice variant promotes tumor progression features.

IKZF1 and BTG1 silencing reduces glucocorticoid response in B-cell precursor acute leukemia cell line.

INTRODUCTION: Secondary genetic alterations, which contribute to the dysregulation of cell cycle progression and lymphoid specialization, are frequently observed in B-cell precursor acute lymphoblastic leukemia (B-ALL). As IKZF1 and BTG1 deletions are associated with a worse outcome in B-ALL, this study aimed to address whether they synergistically promote glucocorticoid resistance. METHODS: Small interfering RNA was used to downregulate either IKZF1, or BTG1, or both genes in the 207 B-ALL cell line. Cell viability was investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and trypan blue exclusion assays. The expression levels of IKZF1, BTG1 and glucocorticoid-responsive genes (DUSP1, SGK1, FBXW7 and NR3C1) were evaluated by real time quantitative real time polymerase chain reaction (PCR). RESULTS: Isolated silencing of BTG1, IKZF1, or both genes in combination under dexamethasone treatment increased cell viability by 24%, 40% and 84%, respectively. Although BTG1 silencing did not alter the expression of glucocorticoid-responsive genes, IKZF1 knockdown decreased the transcript levels of DUSP1 (2.6-fold), SGK1 (1.8-fold), FBXW7 (2.2-fold) and NR3C1 (1.7-fold). The expression of glucocorticoid-responsive genes reached even lower levels (reducing 2.4-4 fold) when IKZF1 and BTG1 silencing occurred in combination. CONCLUSIONS: IKZF1 silencing impairs the transcription of glucocorticoid-responsive genes; this effect is enhanced by concomitant loss of BTG1. These results demonstrate the molecular mechanism by which the combination of both genetic deletions might contribute to higher relapse rates in B-ALL.

Expression analysis and in silico characterization of intronic long noncoding RNAs in renal cell carcinoma: emerging functional associations.

BACKGROUND: Intronic and intergenic long noncoding RNAs (lncRNAs) are emerging gene expression regulators. The molecular pathogenesis of renal cell carcinoma (RCC) is still poorly understood, and in particular, limited studies are available for intronic lncRNAs expressed in RCC. METHODS: Microarray experiments were performed with custom-designed arrays enriched with probes for lncRNAs mapping to intronic genomic regions. Samples from 18 primary RCC tumors and 11 nontumor adjacent matched tissues were analyzed. Meta-analyses were performed with microarray expression data from three additional human tissues (normal liver, prostate tumor and kidney nontumor samples), and with large-scale public data for epigenetic regulatory marks and for evolutionarily conserved sequences. RESULTS: A signature of 29 intronic lncRNAs differentially expressed between RCC and nontumor samples was obtained (false discovery rate (FDR) < 5%). A signature of 26 intronic lncRNAs significantly correlated with the RCC five-year patient survival outcome was identified (FDR < 5%, p-value ≤ 0.01). We identified 4303 intronic antisense lncRNAs expressed in RCC, of which 22% were significantly (p < 0.05) cis correlated with the expression of the mRNA in the same locus across RCC and three other human tissues. Gene Ontology (GO) analysis of those loci pointed to 'regulation of biological processes' as the main enriched category. A module map analysis of the protein-coding genes significantly (p < 0.05) trans correlated with the 20% most abundant lncRNAs, identified 51 enriched GO terms (p < 0.05). We determined that 60% of the expressed lncRNAs are evolutionarily conserved. At the genomic loci containing the intronic RCC-expressed lncRNAs, a strong association (p < 0.001) was found between their transcription start sites and genomic marks such as CpG islands, RNA Pol II binding and histones methylation and acetylation. CONCLUSION: Intronic antisense lncRNAs are widely expressed in RCC tumors. Some of them are significantly altered in RCC in comparison with nontumor samples. The majority of these lncRNAs is evolutionarily conserved and possibly modulated by epigenetic modifications. Our data suggest that these RCC lncRNAs may contribute to the complex network of regulatory RNAs playing a role in renal cell malignant transformation.

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.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia.

INTRODUCTION: This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. RESULTS: We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p = 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. CONCLUSION: The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

Total Osteopontin and Its Isoform OPN4 Are Differently Expressed in Respiratory Samples during Influenza A(H1N1)pdm09 Infection and Progression.

Influenza A virus (IAV) infection affects the human respiratory tract, causing an acute and highly contagious disease. Individuals with comorbidities and in the extremes of age are classified as risk groups for serious clinical outcomes. However, part of the severe infections and fatalities are observed among young healthy individuals. Noteworthy, influenza infections lack specific prognostic biomarkers that would predict the disease severity. Osteopontin (OPN) has been proposed as a biomarker in a few human malignancies and its differential modulation has been observed during viral infections. However, OPN expression levels in the primary site of IAV infection have not been previously investigated. Therefore, we evaluated the transcriptional expression patterns of total OPN (tOPN) and its splicing isoforms (OPNa, OPNb, OPNc, OPN4, and OPN5) in 176 respiratory secretion samples collected from human influenza A(H1N1)pdm09 cases and a group of 65 IAV-negative controls. IAV samples were differentially classified according to their disease severity. tOPN was more frequently detected in IAV samples (34.1%) when compared with the negative controls (18.5%) (p < 0.05), as well as in fatal (59.1%) versus non-fatal IAV samples (30.5%) (p < 0.01). OPN4 splice variant transcript was more prevalent in IAV cases (78.4%) than in the negative controls (66.1%) (p = 0.05) and in severe cases (85.7%) in relation to the non-severe ones (69.2%) (p < 0.01). OPN4 detection was also associated with severity symptoms such as dyspnea (p < 0.05), respiratory failure (p < 0.05), and oxygen saturation < 95% (p < 0.05). In addition, the OPN4 expression level was increased in the fatal cases of respiratory samples. Our data indicated that tOPN and OPN4 had a more pronounced expression pattern in IAV respiratory samples, pointing to the potential use of these molecules as biomarkers to evaluate disease outcomes.

Both osteopontin-c and osteopontin-b splicing isoforms exert pro-tumorigenic roles in prostate cancer cells.

BACKGROUND: Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN-SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays. METHODS: The expression levels of OPN-SI in prostate cell lines were analyzed by qRT-PCR. PC-3 was stably transfected with expression vectors containing OPNa, OPNb, and OPNc, as well as empty vector controls. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion, and soft agar colony formation. RESULTS: OPN-SI are overexpressed in PCa as compared to non-tumoral prostate cell lines. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of MMP-2, MMP-9, and VEGF. These isoforms also support sustained proliferative survival. We found that both OPNc and OPNb pro-tumorigenic roles are mainly mediated through PI3K signaling. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression. CONCLUSIONS: Our data provide evidence that both OPNc and OPNb splicing isoforms promote distinct aspects of PCa progression by inducing PI3K signaling. These data give support to strategies aiming to downregulate OPNc and OPNb expression as an approach to inhibit PCa progression.

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

BACKGROUND: PCA3 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. METHODS: LNCaP 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. RESULTS: LNCaP 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. CONCLUSIONS: Our 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.

Bovine Lactoferrin Induces Cell Death in Human Prostate Cancer Cells.

Bovine lactoferrin (bLf) is a multifunctional protein widely associated with anticancer activity. Prostate cancer is the second most frequent type of cancer worldwide. This study was aimed at evaluating the influence of bLf on cell viability, cell cycle progression, reactive oxygen species (ROS) production, and rate of apoptosis in the human prostate cancer cell line (DU-145). MTT assay and trypan blue exclusion were used to analyze cell viability. Morphological changes were analyzed through optical microscopy after 24 h and 48 h of bLf treatment. FITC-bLf internalization and cellular damage were observed within 24 h by confocal fluorescence microscopy. Cell cycle analyses were performed by flow cytometry and propidium iodide. For caspases 3/7 activation and reactive oxygen species production evaluation, cells were live-imaged using the high-throughput system Operetta. The cell viability assays demonstrated that bLf induces cell death and morphological changes after 24 h and 48 h of treatment compared to control on DU-145 cells. The bLf internalization was detected in DU-145 cells, G1-phase arrest of the cell cycle, caspase 3/7 activation, and increased oxidative stress on bLf-treated cells. Our data support that bLf has an important anticancer activity, thus offering new perspectives in preventing and treating prostate cancer.