Histone Deacetylase Inhibitors as Cognitive Enhancers and Modifiers of Mood and Behavior.

BACKGROUND: Epigenetic regulation of gene signalling is one of the fundamental molecular mechanisms for the generation and maintenance of cellular memory. Histone acetylation is a common epigenetic mechanism associated with increased gene transcription in the central nervous system (CNS). Stimulation of gene transcription by histone acetylation is important for the development of CNS-based long-term memory. Histone acetylation is a target for cognitive enhancement via the application of histone deacetylase (HDAC) inhibitors. The promising potential of HDAC inhibitors has been observed in the treatment of several neurodevelopmental and neurodegenerative diseases. OBJECTIVE: This study assessed the current state of HDAC inhibition as an approach to cognitive enhancement and treatment of neurodegenerative diseases. Our analysis provides insights into the mechanism of action of HDAC inhibitors, associated epigenetic priming, and describes the therapeutic success and potential complications after unsupervised use of the inhibitors. RESULTS AND CONCLUSION: Several chromatin-modifying enzymes play key roles in the regulation of cognitive processes. The importance of HDAC signaling in the brain is highlighted in this review. Recent advancements in the field of cognitive epigenetics are supported by the successful development of various HDAC inhibitors, demonstrating effective treatment of mood-associated disorders. The current review discusses the therapeutic potential of HDAC inhibition and observed complications after mood and cognitive enhancement therapies.

The crucial role of epigenetic regulation in breast cancer anti-estrogen resistance: Current findings and future perspectives.

Breast cancer (BC) cell de-sensitization to Tamoxifen (TAM) or other selective estrogen receptor (ER) modulators (SERM) is a complex process associated with BC heterogeneity and the transformation of ER signalling. The most influential resistance-related mechanisms include modifications in ER expression and gene regulation patterns. During TAM/SERM treatment, epigenetic mechanisms can effectively silence ER expression and facilitate the development of endocrine resistance. ER status is efficiently regulated by specific epigenetic tools including hypermethylation of CpG islands within ER promoters, increased histone deacetylase activity in the ER promoter, and/or translational repression by miRNAs. Over-methylation of the ER α gene (ESR1) promoter by DNA methyltransferases was associated with poor prognosis and indicated the development of resistance. Moreover, BC progression and spreading were marked by transformed chromatin remodelling, post-translational histone modifications, and expression of specific miRNAs and/or long non-coding RNAs. Therefore, targeted inhibition of histone acetyltransferases (e.g. MYST3), deacetylases (e.g. HDAC1), and/or demethylases (e.g. lysine-specific demethylase LSD1) was shown to recover and increase BC sensitivity to anti-estrogens. Indicated as a powerful molecular instrument, the administration of epigenetic drugs can regain ER expression along with the activation of tumour suppressor genes, which can in turn prevent selection of resistant cells and cancer stem cell survival. This review examines recent advances in the epigenetic regulation of endocrine drug resistance and evaluates novel anti-resistance strategies. Underlying molecular mechanisms of epigenetic regulation will be discussed, emphasising the utilization of epigenetic enzymes and their inhibitors to re-program irresponsive BCs.

Current insights into functions of phospholipase A2 receptor in normal and cancer cells: More questions than answers.

Lipid signaling network was proposed as a potential target for cancer prevention and treatment. Several recent studies revealed that phospholipid metabolising enzyme, phospholipase A2 (PLA2), is a critical regulator of cancer accelerating pathologies and apoptosis in several types of cancers. In addition to functioning as an enzyme, PLA2 can activate a phospholipase A2 receptor (PLA2R1) in plasma membrane. While the list of PLA2 targets extends to glucose homeostasis, intracellular energy balance, adipocyte development, and hepatic lipogenesis, the PLA2R1 downstream effectors are few and scarcely investigated. Among the most addressed PLA2R1 effects are regulation of pro-inflammatory signaling, autoimmunity, apoptosis, and senescence. Localized in glomeruli podocytes, the receptor can be identified by circulating anti-PLA2R1 autoantibodies leading to development of membranous nephropathy, a strong autoimmune inflammatory cascade. PLA2R1 was shown to induce activation of Janus-kinase 2 (JAK2) and estrogen-related receptor α (ERRα)-controlled mitochondrial proteins, as well as increasing the accumulation of reactive oxygen species, thus leading to apoptosis and senescence. These findings indicate the potential role of PLA2R1 as tumor suppressor. Epigenetic investigations addressed the role of DNA methylation, histone modifications, and specific microRNAs in the regulation of PLA2R1 expression. However, involvement of PLA2R1 in suppression of malignant growth and metastasis remains controversial. In this review, we summarize the recent findings that highlight the role of PLA2R1 in the regulation of carcinogenesis-related intracellular signaling.

Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR).

BACKGROUND: The analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. RESULTS: By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. CONCLUSIONS: We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.

Diverse effects of phospholipase A2 receptor expression on LNCaP and PC-3 prostate cancer cell growth in vitro and in vivo.

Physiological and pathophysiological functions of the phospholipase A2 receptor 1 (PLA2R1) are still not completely understood. To elucidate PLA2R1's function in prostate carcinoma, the receptor was ectopically overexpressed in LNCaP with silenced PLA2R1, and diminished in PC-3 cells with constitutively increased PLA2R1 expression relative to normal prostate epithelial cells. LNCaP cells were transfected to overexpress PLA2R1 (LNCaP-PLA2R1) and compared to control vector transfected cells (LNCaP-Ctrl). Alternatively, a CRISPR/Cas9-knockdown of PLA2R1 was achieved in PC-3 cells (PC-3 KD) and compared to the corresponding control-transfected cells (PC-3 Ctrl). The impact of PLA2R1 expression on proliferative and metastatic parameters was analysed in vitro. A pilot in vivo study addressed the effects of PLA2R1 in mice xenografted with transfected LNCaP and PC-3 cells. Cell viability/proliferation and motility were significantly increased in LNCaP-PLA2R1 and PC-3 Ctrl compared to LNCaP-Ctrl and PC-3 KD cells, respectively. However, levels of apoptosis, clonogenicity and cell invasion were reduced in LNCaP-PLA2R1 and PC-3 Ctrl cells. Gene expression analysis revealed an up-regulation of fibronectin 1 (FN1), TWIST homolog 1 (TWIST1), and cyclin-dependent kinase 6 (CDK6) in LNCaP-PLA2R1. In LNCaP xenografts, PLA2R1-dependent regulation of clonogenicity appeared to outweigh the receptor's pro-oncogenic properties, resulting in decreased tumour growth, supporting the tumour-suppressive role of PLA2R1. Alternatively, PC-3 Ctrl xenografts exhibited faster tumour growth compared to PC-3 KD cells, suggesting a pro-oncogenic effect of endogenous PLA2R1 expression. The differential growth-regulatory effects of PLA2R1 may be mediated by FN1, TWIST1, and CDK6 expression, although further investigation is required.

Identification of CpG Sites of SERPINA5 Promoter with Opposite Methylation Patterns in Benign and Malignant Prostate Cells.

BACKGROUND/AIM: To date there has been no investigation into the epigenetic regulation of the serine protease inhibitor SERPINA5 in prostate cancer, where lack of this gene was considered to facilitate invasive growth patterns. MATERIALS AND METHODS: Methylation degrees of eight CpG sites of SERPINA5 were analyzed in normal and malignant prostate cells using nucleotide sequencing, methylation-specific high resolution melting and digital droplet PCR techniques. RESULTS: The methylation degree of five CpG sites significantly correlated with lower SERPINA5 expression levels. In contrast, two CpG sites (at -19 bp and -14 bp from the transcription start site) were hypermethylated in normal epithelial prostate cells, benign hyperplasic cells and low-invasive malignant LNCaP cells, whereas in aggressive DU-145 and PC-3 cell lines, these sites were essentially unmethylated. CONCLUSION: Novel methylation patterns of two distinct CpG sites of the SERPINA5 promoter may be useful for differentiating benign from malignant prostate disease.

Epigenetic control of group V phospholipase A2 expression in human malignant cells.

Secreted phospholipases A2 (sPLA2) are suggested to play an important role in inflammation and tumorigenesis. Different mechanisms of epigenetic regulation are involved in the control of group IIA, III and X sPLA2s expression in cancer cells, but group V sPLA2 (GV-PLA2) in this respect has not been studied. Here, we demonstrate the role of epigenetic mechanisms in regulation of GV-PLA2 expression in different cell lines originating from leukaemia and solid cancers. In blood leukocytes from leukaemic patients, levels of GV-PLA2 transcripts were significantly lower in comparison to those from healthy individuals. Similarly, in DU-145 and PC-3 prostate and CAL-51 and MCF-7 mammary cancer cell lines, levels of GV-PLA2 transcripts were significantly lower in relation to those found in normal epithelial cells of prostate or mammary. By sequencing and methylation-specific high-resolution melting (MS-HRM) analyses of bisulphite-modified DNA, distinct CpG sites in the GV-PLA2 promoter region were identified that were differentially methylated in cancer cells in comparison to normal epithelial and endothelial cells. Spearman rank order analysis revealed a significant negative correlation between the methylation degree and the cellular expression of GV-PLA2 (r = -0.697; p = 0.01). The effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on GV-PLA2 transcription in the analysed cells confirmed the importance of DNA methylation and histone modification in the regulation of the GV-PLA2 gene expression in leukaemic, prostate and mammary cancer cell lines. The exposure of tumour cells to human recombinant GV-PLA2 resulted in a reduced colony forming activity of MCF-7, HepG2 and PC-3 cells, but not of DU-145 cells suggesting a cell-type-dependent effect of GV-PLA2 on cell growth. In conclusion, our results suggest that epigenetic mechanisms such as DNA methylation and histone modification play an important role in downregulation of GV-PLA2 expression in cancer cells.

Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

BACKGROUND: It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines. METHODS: Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system. RESULTS: Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2'-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, -181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, -154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression. CONCLUSIONS: Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, -154, and -302d, as well as hsa-miR-141, -181b, and -181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.

Silibinin down-regulates expression of secreted phospholipase A2 enzymes in cancer cells.

BACKGROUND: Silibinin, a naturally-occurring flavonoid produced by milk thistle, possesses antioxidant, anti-inflammatory and cancer-preventive activities. In the current study, we examined the effects of silibinin on the expression of secreted phospholipase A2 (sPLA2) enzymes, especially those of group IIA (hGIIA), which play a crucial role in inflammation and carcinogenesis. MATERIALS AND METHODS: The effects of silibinin on sPLA2 expressions in human HepG2 hepatoma and PC-3 prostate cancer cells were analyzed using quantitative reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay technique. RESULTS: Silibinin inhibited the expression of hGIIA in unstimulated and cytokine-primed HepG2 and PC-3 cells. The mRNA levels of sPLA2 of groups IB, III and V were also significantly decreased by silibinin. Analyses of transcription factor activation suggest that nuclear factor-κB, but not specificity protein 1 (SP1) is implicated in the silibinin-mediated down-regulation of hGIIA. CONCLUSION: Silibinin exhibits inhibitory effects on basal and cytokine-induced expression of sPLA2s in cancer cells and therefore, may have the potential to protect against up-regulation of hGIIA and other sPLA2 isoforms during inflammation and cancer.

Serum amyloid A, phospholipase A2-IIA and C-reactive protein as inflammatory biomarkers for prostate diseases.

INTRODUCTION: Serum amyloid A (SAA), secreted group IIA phospholipase A2 (sPLA2-IIA), and C-reactive protein (CRP) are acute-phase proteins whose serum concentrations increase not only during inflammatory disorders, but also in the course of malignant diseases. MATERIALS AND METHODS: In this study we analyzed serum levels of these inflammatory markers along with prostate-specific antigens (PSA) in patients with benign prostatic hyperplasia (BPH, n = 55), localized prostate cancers (PCa, n = 55), and metastatic prostate cancers (mPCa, n = 27) using immunological assays. RESULTS: We found that in comparison to healthy individuals (n = 55), patients with BPH, PCa and mPCa have elevated serum levels of SAA, sPLA2-IIA, and CRP, in addition to elevated levels of PSA. Significant differences with respect to inflammatory biomarkers were found between localized and metastatic PCa (p < 0.001), suggesting a prognostic value of these parameters. In addition, serum concentrations of SAA and sPLA2-IIA positively correlate with CRP in BPH patients (p < 0.05) and in patients with PCa and mPCa (p < 0.001), but not with PSA levels, Gleason score, or tumor stage, emphasizing a role of SAA and sPLA2-IIA as circulating biomarkers of inflammation rather than of neoplastic transformation. In contrast to PSA, which differed significantly between BPH and localized PCa patients (p < 0.01), such a difference was not found for SAA, sPLA2-IIA, and CRP. In order to elucidate whether the elevated levels of SAA and sPLA2-IIA can be caused by cancer cell-associated synthesis, in vitro studies were performed. These analyses demonstrated the expression of SAA and sPLA2-IIA in LNCaP and PC-3 prostate cell lines, which can be further upregulated by pro-inflammatory cytokines in a cell type-dependent manner. This might suggest that, in addition to the hepatic origin, SAA and sPLA2-IIA can also be synthesized and secreted by prostatic cancer tissue itself. CONCLUSION: The results of the present study emphasize the utility of SAA, sPLA2-IIA, and CRP as circulating biomarkers of inflammation during BPH development and PCa progression.

Plasma levels of phospholipase A2-IIA in patients with different types of malignancies: prognosis and association with inflammatory and coagulation biomarkers.

It is well-known that the plasma level of group IIA phospholipase A2 (sPLA2-IIA) is increased in patients with malignant diseases, but whether the up-regulated enzyme expression is directly related to tumorigenesis or a consequence of tumor-associated inflammation remains unresolved. In this study we analyzed circulating levels of sPLA2-IIA, C-reactive protein (CRP), fibrinogen, factor VIII (FVIII), von Willebrand factor (vWF), and antithrombin as biomarkers of inflammation and coagulation in patients with various types of malignancies. Underlying tumor entities were lung, esophageal, gastric, pancreatic, colorectal, head and neck, and hepatocellular carcinomas as well as multiple myeloma and non-Hodgkin's lymphoma. Plasma levels of sPLA2-IIA are shown to be markedly increased in all types of analysed malignancies in comparison to the normal range (22.8 ± 4.5 µg/L versus <1.9 µg/L). Levels of sPLA2-IIA correlate positively with CRP (p < 0.001), fibrinogen (p < 0.01), FVIII (p < 0.05), and vWF (p < 0.05) and negatively with antithrombin levels (p < 0.05). Kaplan-Meier analyses revealed a statistically prolonged survival time of patients with lower sPLA2-IIA concentrations (<4 µg/L) in comparison to those with elevated concentrations (>4 µg/L) of this enzyme. In conclusion, the study shows that the measurement of plasma sPLA2-IIA levels has prognostic values in patients with different types of malignancies. The association of sPLA2-IIA levels with CRP, fibrinogen, FVIII, and vWF levels supports the importance of inflammatory processes for the up-regulation of sPLA2-IIA during cancer progression.

Deregulated expression of urokinase and its inhibitor type 1 in prostate cancer cells: role of epigenetic mechanisms.

Plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) play a crucial role in cancer progression. In the present study we examined the regulation of PAI-1 and uPA expressions in normal prostate epithelial cells (PrEC) and the prostate cancer cell lines LNCaP, DU-145, and PC-3. The antigen and mRNA levels of PAI-1 were down-regulated in cancer cells, especially in LNCaP and DU-145. In the presence of proinflammatory cytokines, an increase of PAI-1 mRNA levels was observed in PrEC, LNCaP and PC-3, but not in DU-145 cells. Treatment with demethylating agent, 5-aza-2'-deoxycytidine increased the level of PAI-1 transcript in DU-145 cells and restored the inducing effect of cytokines on PAI-1 expression. An aberrant methylation of PAI-1 promoter in DU-145 and LNCaP cells was shown by methylation-sensitive high resolution melting (MS-HRM) analysis. PAI-1 methylation was also significantly increased in tumor samples (23.2±1.7%) in comparison to adjacent non-tumor tissue (6.0±0.8%). Furthermore, the expression of uPA was increased in high invasive cell lines DU-145 and PC-3 in comparison to PrEC and low invasive LNCaP cells. MS-HRM analysis revealed aberrant methylation of uPA promoter in LNCaP cells, but not in PrEC, DU-145 and PC-3 cells, as well as in normal and prostate cancer tissue samples. In conclusion, the study shows that PAI-1 and uPA expressions were changed in opposite directions in high invasive prostate cancer cell lines resulting in a strong decrease of PAI-1/uPA ratio, which may indicate a shift towards proteolytic activities. Methylation of the PAI-1 gene is suggested as one of the molecular mechanisms involved in the cancer-associated down-regulation of the PAI-1 expression.

Aberrant methylation of the M-type phospholipase A(2) receptor gene in leukemic cells.

BACKGROUND: The M-type phospholipase A2 receptor (PLA2R1) plays a crucial role in several signaling pathways and may act as tumor-suppressor. This study examined the expression and methylation of the PLA2R1 gene in Jurkat and U937 leukemic cell lines and its methylation in patients with myelodysplastic syndrome (MDS) or acute leukemia. METHODS: Sites of methylation of the PLA2R1 locus were identified by sequencing bisulfite-modified DNA fragments. Methylation specific-high resolution melting (MS-HRM) analysis was then carried out to quantify PLA2R1 methylation at 5'-CpG sites identified with differences in methylation between healthy control subjects and leukemic patients using sequencing of bisulfite-modified genomic DNA. RESULTS: Expression of PLA2R1 was found to be completely down-regulated in Jurkat and U937 cells, accompanied by complete methylation of PLA2R1 promoter and down-stream regions; PLA2R1 was re-expressed after exposure of cells to 5-aza-2'-deoxycytidine. MS-HRM analysis of the PLA2R1 locus in patients with different types of leukemia indicated an average methylation of 28.9% ± 17.8%, compared to less than 9% in control subjects. In MDS patients the extent of PLA2R1 methylation significantly increased with disease risk. Furthermore, measurements of PLA2R1 methylation appeared useful for predicting responsiveness to the methyltransferase inhibitor, azacitidine, as a pre-emptive treatment to avoid hematological relapse in patients with high-risk MDS or acute myeloid leukemia. CONCLUSIONS: The study shows for the first time that PLA2R1 gene sequences are a target of hypermethylation in leukemia, which may have pathophysiological relevance for disease evolution in MDS and leukemogenesis.

Regulation of thrombomodulin expression in prostate cancer cells.

In carcinomas the expression of thrombomodulin (TM) is inversely correlated with tumour progression and metastasis. In the present study a decreased TM expression in human prostate cancer cell lines, LNCaP, DU-145, and PC-3, in relation to normal prostate epithelial cells (PrEC) is shown. Sequencing and methylation-specific high resolution melting (MS-HRM) analyses of bisulphite-modified genomic DNA indicates a high degree of methylation in DU-145 cells and lesser degrees in PC-3 and LNCaP cells, whereas in PrEC the TM promoter is unmethylated. The expression of TM is negatively regulated by NF-κB- and GSK3-ß-dependent signalling pathways and positively regulated by retinoic acid and transcription factor Sp1 in PrEC, LNCaP and PC-3 cells, but not in DU-145 cells. However, exposure of DU-145 cells to the demethylating agent, 5-aza-2'deoxycytidine, restores the TM expression and its control by retinoic acid, NF-κB- and GSK3-ß-dependent signalling. In conclusion, the study establishes that in prostate cancer cell lines relative to PrEC the TM is down-regulated and that the TM promoter is hypermethylated, which seems to be responsible for the down-regulation and failed regulation of TM expression in DU-145 cells.

Serum levels of secreted group IIA phospholipase A(2) in benign prostatic hyperplasia and prostate cancer: a biomarker for inflammation or neoplasia?

Secreted group IIA phospholipase A(2) (sPLA(2)-IIA) is markedly up-regulated in human prostate cancer (PCa) specimens and in some PCa-derived cell lines, indicating an important role of this enzyme in tumourigenesis. In this study, we measured levels of sPLA(2)-IIA, C-reactive protein (CRP), and prostate-specific antigen (PSA) in serum samples obtained from patients with benign prostatic hyperplasia (BPH) and with PCa of different stages. We found that serum levels of sPLA(2)-IIA and CRP in BPH and PCa patients were significantly elevated compared to those of healthy individuals, but the concentrations of these inflammatory biomarkers did not differ between patients with BPH or PCa. Furthermore, serum levels of sPLA(2)-IIA correlated with concentrations of CRP, but not with PSA, Gleason grade or tumour stage. In conclusion, these findings suggest that cancer-related changes are not exclusive factors contributing to elevated serum sPLA(2)-IIA levels and emphasize the utility of sPLA(2)-IIA as a circulating marker of inflammation in patients with BPH and PCa.

Expression and shedding of endothelial protein C receptor in prostate cancer cells.

BACKGROUND: Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines. RESULTS: EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1ß or TNF-α result in a significant increase of sEPCR release. The effects of IL-1ß and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells. CONCLUSIONS: The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.

Reducing agents induce thrombomodulin shedding in human endothelial cells.

The level of thrombomodulin (TM) on cell surfaces reflects its biosynthesis, intracellular turnover, proteolytic cleavage, and release in soluble form (sTM). In the present study we examined the mechanisms mediating and regulating sTM release. Inducers of endothelial protein C receptor (EPCR) shedding, such as proinflammatory cytokines, phorbol ester, and ionomycin did not affect sTM release from human umbilical endothelial cells (HUVECs). In contrast, several natural and synthetic reducing compounds (i.e., glutathione, dihydrolipoic acid, homocysteine, N-acetyl-L-cysteine, dithiothreitol, and non-thiol cell-impermeable reductant, tris-(2-carboxyethyl)phosphine), but not oxidized glutathione or alpha-lipoic acid effectively up-regulated the release of sTM in endothelial cells. In addition, the direct activator of metalloproteases, 4-aminophenylmercuric acetate (APMA), was an effective inducer of TM shedding. Considerable inhibition of protein C activation was found with APMA, which is consistent with the effects of this agent on TM shedding. In addition to metalloproteases, serine proteases were shown by pharmacological inhibition studies to be involved in a similar degree in basal sTM release; however, serine proteases seem preferentially to be involved in thiol-induced TM proteolytic processing. From comparisons of non-thiol containing synthetic substrate with human recombinant TM it was demonstrated that disulfide bonds within TM are most likely modified by thiols making TM more susceptible to serine protease-mediated cleavage. In summary, the study shows that the extracellular redox state plays a crucial role in the regulation of TM shedding in HUVECs thereby offering new strategies to interfere with diminished activation of protein C during inflammatory diseases.

Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways.

The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), but not interferon-gamma and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1beta and TNF-alpha correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-l-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1beta and TNF-alpha, and downstream by MAP kinase signaling pathways and metalloproteinases.

Involvement of epigenetic mechanisms in the regulation of secreted phospholipase A2 expressions in Jurkat leukemia cells.

Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A(2) (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA(2) isozymes and the M-type receptor of sPLA(2) (sPLA(2)-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA(2)-IB, sPLA(2)-III, sPLA(2)-X, and sPLA(2)-R but not sPLA(2)-IIA and sPLA(2)-V. Transcription of sPLA(2)-IIA and sPLA(2)-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). Expression of sPLA(2)-IIA was further enhanced by additional exposure to interferon-gamma and blocked by inhibitors of specificity protein 1, nuclear factor kappaB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5'-CpG sites (-111 and -82) in the sPLA(2)-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA(2)-IIA and sPLA(2)-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation.

Differential expression of secretory phospholipases A2 in normal and malignant prostate cell lines: regulation by cytokines, cell signaling pathways, and epigenetic mechanisms.

Upregulation of group IIA phospholipase A(2) (sPLA(2)-IIA) correlates with prostate tumor progression suggesting prooncogenic properties of this protein. Here, we report data on expression of three different sPLA(2) isozymes (groups IIA, V, and X) in normal (PrEC) and malignant (DU-145, PC-3, and LNCaP) human prostate cell lines. All studied cell lines constitutively expressed sPLA(2)-X, whereas sPLA(2)-V transcripts were identified only in malignant cells. In contrast, no expression of sPLA(2)-IIA was found in PrEC and DU-145 cells, but it was constitutively expressed by IFN-gamma in LNCaP and PC-3 cells. Expression of sPLA(2)-IIA is upregulated in PC-3 and in PrEC cell in a signal transducer and activator of transcription-1-dependent manner, but not in LNCaP cell. Additional signaling pathways regulating sPLA(2)-IIA expression include cAMP/protein kinase A, p38 mitogen-activated protein kinase, protein kinase C, Rho-kinase, and mitogen-activated/extracellular response protein kinase / extracellular signal-regulated kinase. No deletions were revealed in the sPLA(2)-IIA gene from DU-145 cells lacking the expression of sPLA(2)-IIA. Reexpression of sPLA(2)-IIA was induced by 5-aza-2'-deoxycytidine demonstrating that DNA methylation is implicated in the regulation of sPLA(2)-II. Together, these data suggest that sPLA(2)-IIA and sPLA(2)-V, but not sPLA(2)-X, are differentially expressed in normal and malignant prostate cells under the control of proinflammatory cytokines; epigenetic mechanisms appear involved in the regulation of sPLA(2)-IIA expression, at least in DU-145 cells.