Serdar Karakurt

Transcriptome Sequencing Reveals PCAT5 as a Novel ERG-Regulated Long Noncoding RNA in Prostate Cancer.

Castration-resistant prostate cancers (CRPC) that arise after the failure of androgen-blocking therapies cause most of the deaths from prostate cancer, intensifying the need to fully understand CRPC pathophysiology. In this study, we characterized the transcriptomic differences between untreated prostate cancer and locally recurrent CRPC. Here, we report the identification of 145 previously unannotated intergenic long noncoding RNA transcripts (lncRNA) or isoforms that are associated with prostate cancer or CRPC. Of the one third of these transcripts that were specific for CRPC, we defined a novel lncRNA termed PCAT5 as a regulatory target for the transcription factor ERG, which is activated in approximately 50% of human prostate cancer. Genome-wide expression analysis of a PCAT5-positive prostate cancer after PCAT5 silencing highlighted alterations in cell proliferation pathways. Strikingly, an in vitro validation of these alterations revealed a complex integrated phenotype affecting cell growth, migration, invasion, colony-forming potential, and apoptosis. Our findings reveal a key molecular determinant of differences between prostate cancer and CRPC at the level of the transcriptome. Furthermore, they establish PCAT5 as a novel oncogenic lncRNA in ERG-positive prostate cancers, with implications for defining CRPC biomarkers and new therapeutic interventions.

A Comparative Study for the Evaluation of Two Doses of Ellagic Acid on Hepatic Drug Metabolizing and Antioxidant Enzymes in the Rat

The present study was designed to evaluate different doses of ellagic acid (EA) in vivo in rats for its potential to modulate hepatic phases I, II, and antioxidant enzymes. EA (10 or 30 mg/kg/day, intragastrically) was administered for 14 consecutive days, and activity, protein, and mRNA levels were determined. Although the cytochrome P450 (CYP) 2B and CYP2E enzyme activities were decreased significantly, the activities of all other enzymes were unchanged with the 10 mg/kg/day EA. In addition, western-blot and qRT-PCR results clearly corroborated the above enzyme expressions. On the other hand, while the NAD(P)H:quinone oxidoreductase 1 (NQO1), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST) activities were increased significantly, CYP1A, 2B, 2C, 2E, and 19 enzyme activities were reduced significantly with 30 mg/kg/day EA. In addition, CYP2B, 2C6, 2E1, and 19 protein and mRNA levels were substantially decreased by the 30 mg/kg/day dose of EA, but the CYP1A protein, and mRNA levels were not changed. CYP3A enzyme activity, protein and mRNA levels were not altered by neither 10 nor 30 mg/kg/day ellagic acid. These results indicate that EA exerts a dose-dependent impact on the metabolism of chemical carcinogens and drugs by affecting the enzymes involved in xenobiotics activation/detoxification and antioxidant pathways.

Tannic Acid Inhibits Proliferation, Migration, Invasion of Prostate Cancer and Modulates Drug Metabolizing and Antioxidant Enzymes.

The aim of this study was to investigate the effects of plant phenolic compound tannic acid (TA) on proliferative, metastatic, invasive properties of prostate cancer (PCa) cells; PC-3 and LNCaP, as well as drug metabolizing and antioxidant enzymes. Characterization of TA was done by using FT-IR and NMR. TA dose dependently inhibited the proliferation of PC-3 and LNCaP cells with IC50 values 35.3 μM and 29.1 μM, respectively. Wound healing assay showed that TA significantly inhibited (92.7%) migration of PCa cells (p<0.0001). In addition, TA was found to have anti-invasive potential on PC-3 cells and it inhibited (80.9%, p<0.0001) invasion of PC-3 cells into matrigel. Only 17.8% of PC-3 cells can form colony in the 0.7% agarose after treatment of cells with TA at the IC50 value concentration. Furthermore, flow cytometry analyses with Annexin V-APC and 7-AAD staining demonstrated that TA increases early apoptosis rate of PC-3 cells by 25.8% and LNCaP cells by 20.9%. Besides, Western blot and qRT-PCR analyses also demonstrated that TA regulates protein and mRNA expressions of CYP17A1, CYP3A4, CYP2B6, NQO1, GSTM1 and GSTP1 enzymes. The results obtained from this study show that TA might be a good candidate for combinational therapy and highly effective strategic molecule for reducing the occurrence of PCa.

Epilobium hirsutum alters xenobiotic metabolizing CYP1A1, CYP2E1, NQO1 and GPx activities, mRNA and protein levels in rats.

Abstract Context: Natural products have attracted increasing interests due to their use in flavoring, nutrition, cosmetics, pharmacy and medicine. Epilobium hirsutum L. (Onagraceae) is known for its analgesic, antimicrobial, and antiproliferative activity. CYP1A1 and CYP2E1, xenobiotic metabolizing enzymes, serve as a metabolic activation route yielding reactive metabolites that are eliminated by the action of NQO1 and glutathione peroxidase (GPx) enzymes. Objective: This study investigated in vivo effects of Epilobium hirsutum (EH) on CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA expressions in liver. Materials and methods: Male Wistar Albino rats were injected with EH at a dose of 37.5 mg/kg i.p. daily for 9 d. CYP2E1, CYP1A1, NQO1 and GPx activities, protein and mRNA levels were determined by enzyme assays, Western blotting and qPCR, respectively. Results: CYP1A1 associated ethoxyresorufin-O-deethylase activity of control and EH-treated animals were found as 6.54 ± 1.21 and 4.48 ± 1.67 nmol/min/mg, respectively. CYP2E1 associated aniline 4-hydroxylase of control and EH group were 0.537 ± 0.011 and 0.109 ± 0.01 nmol/min/mg, respectively. However, EH treatment increased the GPx and NQO1 activities from 0.069 ± 0.015 to 0.107 ± 0.026 nmol/min/mg and from 163.34 ± 92 to 588.3 ± 14 nmol/min/mg, respectively. Furthermore, protein and mRNA expression analysis revealed that CYP1A1 and CYP2E1 levels were decreased while those of NQO1 and GPx increased after EH treatment. Discussion and conclusion: Our current data suggest that the metabolism of xenobiotics, including drugs, may be altered due to changes in the expression and activity of these proteins by EH.

New water soluble Hg(2+) selective fluorescent calix[4]arenes: Synthesis and application in living cells imaging.

The present study demonstrates the synthesis of water-soluble fluorescent calix[4]arenes (6 and 7) and its application in living cell imaging for Hg2+ detection at a low level. The synthesized fluorescent ligands 6 and 7 were characterized by 1H NMR technique. The fluorescent study showed both water soluble ligands were Hg2+ selective and follow photo-induced electron transfer (PET) process. From the fluorimeter titration experiment detection limit was calculated as 1.14×10-5 and 3.42×10-5 for ligand 6 and 7, respectively. From the Benesi-Hildebrand plot binding constant values were evaluated as 666.7 and 733.3M-1 for 6 and 7, respectively. The interactions between ligands 6 and 7 and Hg2+ were also demonstrated in living cells, SW-620, using Fluorescent Cell Imager. While ligands 6 and 7 alone show fluorescent properties, they loss their action with the presence of Hg2+ in SW-620 cells.

Contribution of ellagic acid on the antioxidant potential of medicinal plant Epilobium hirsutum

abstract In the present study, the possible role of ellagic acid (EA) on antioxidant potential of Epilobium hirsutum (EH) in rat liver was investigated. Wistar rats were intraperitoneally treated with 37.5 mg/kg of EH and 10 mg/kg of EA for 9 days. Effects of EH and EA on antioxidant [glutathione peroxidase (GPx) and superoxide dismutases (SOD)] and Phase II [NADPH quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTs)] enzyme activities, as well as protein and mRNA expressions of those, were investigated. Polyphenolic content of EH was determined by LC-MS/MS analysis. EH and EA injection to rats resulted in a significant increase of NQO1 (3.6-fold and 4.7-fold), GPx (1.45-fold), and SOD (1.34-fold and 1.27-fold) enzyme activities, whereas total GST (46% and 57%) and its isoforms,and GST mu (57% and 72%), and GST theta (60% and 68%) activities were significantly decreased. Western-blot and qRT-PCR analysis showed that NQO1 and GPx protein and mRNA expressions were increased significantly (P < 0.0001), whereas GST mu and GST theta were significantly decreased (P < 0.0001).

Modulatory effects of rutin on the expression of cytochrome P450s and antioxidant enzymes in human hepatoma cells

Abstract Expression of a drug and xenobiotic metabolizing enzymes, cytochrome P450s (CYPs), and antioxidant enzymes can be modulated by various factors. The flavonoid rutin was investigated for its anti-carcinogen and protective effects as well as modulatory action on CYPs and phase II enzymes in human hepatocellular carcinoma cells. Rutin inhibited proliferation of HEPG2 cells in a dose-dependent manner with the IC50 value of 52.7 μmol L-1 and invasion of HEPG2 cells (21.6 %, p = 0.0018) and colony formation of those invaded cells (57.4 %, p < 0.0001). Rutin treatment also significantly increased early/late-stage apoptosis in HEPG2 cells (28.9 %, p < 0.001). Treatment by rutin significantly inhibited protein expressions of cytochrome P450-dependent CYP3A4 (75.3 %, p < 0.0001), elevated CYP1A1 enzymes (1.7-fold, p = 0.0084) and increased protein expressions of antioxidant and phase II reaction catalyzing enzymes, NQO1 (2.42-fold, p < 0.0001) and GSTP1 (2.03-fold, p < 0.0001). Besides, rutin treatment significantly inhibited mRNA expression of CYP3A4 (73.2 %, p=0.0014). Also, CYP1A1, NQO1 and GSTP1 mRNA expressions were significantly increased 2.77-fold (p = 0.029), 4.85- fold (p = 0.0051) and 9.84-fold (p < 0.0001), respectively.

Alteration of enzyme activities and kinetic properties of GST and NQO1 with naturally occurring phenolic compounds

Abstract Objective: Glutathione S-transferase (GST) and NAD(P)H:quinine oxidoreductase 1 (NQO1) are the enzymes important in cytoprotection and bioactivation of chemicals. This study has addressed effects of polyphenolic compounds; ellagic acid, quercetin, naringenin, resveratrol, rutin and hesperidin on rabbit liver GST and NQO1 enzyme activities. Methods: Cytosolic fractions were obtained from homogenized liver tissues of rabbit by differential centrifugation. After calculating IC50 values of individual enzymes for phenolic compounds, both Lineweaver-Burk and Dixon plots were drawn to determine the effect of phenolics on enzyme activity. Michaelis-Menten constant (Km), maximum velocity (Vmax), and inhibition constant (Ki) were calculated from Lineweaver-Burk and Dixon plots, respectively. Results: Resveratrol was found to be the most potent inhibitor for rabbit hepatic cytosolic GST activity with 75.9±2.06 μM IC50 value while naringenin was the least potent one with IC50 value of 260±1.92 μM. Hesperidin and quercetin were found to be the most and least potent inhibitors for NQO1 enzyme activity with IC50 values of 2.7±0.85 μM and 13.8±0.91 μM, respectively. Resveratrol and naringenin inhibited GST activity noncompetitively and mixed type with Ki of 6.2 μM and 245 μM, respectively; while both hesperidin with 0.64 μM Ki value and quercetin with 3.5 μM Ki value inhibited NQO1 activity in a competitive manner. Conclusion: These results indicate that phenolic compounds may modulate Phase II enzyme, GST and NQO1. Moreover, they can influence the metabolic activation of xenobiotic and toxic compounds metabolized by this enzyme. Ozet Amac: Glutatyon S-transferaz (GST) ve NAD(P)H:Kuinon Oksidoreduktaz 1 (NQO1) enzimleri kimyasal molekullerin biyoaktivasyonunda rol alırken diğer yandan da onların zararlı etkilerine karşı koruma gorevi gorurler. Bu calışma ile polifenolik bileşiklerden elajik asit, kuarsetin, naringenin, resveratrol, rutin ve hesperidinin tavşan karaciğer GST ve NQO1 enzim aktiviteleri uzerine etkileri amaclanmıştır. Metod: Homojenize tavsan karaciğer dokusundan, diferansiyel santrifugasyon ile sitozolik fraksiyonlar elde edilmiştir. Her bir enzime ait fenolik bileşikler icin IC50 değerleri hesaplandıktan sonra, fenoliklerin enzim aktiviteleri uzerine etkisini belirlemek icin Lineweaver-Burk ve Dixon grafikleri cizilmiştir. Bu grafiklerden Michaelis-Menten sabiti (Km), maksimum hız (Vmax) ve inhibisyon sabiti (Ki) hesaplanmıştır. Bulgular: Resveratrol, 75.9±2.06 μM IC50 değeri ile tavsan karaciğer sitozolik GST aktivitesi icin en guclu inhibitor iken, naringenin 260±1.92 μM IC50 değeri ile en az guclu inhi- bitor bulunmuştur. Hesperidin ve quersetin ise NQO1 enzimi icin sırasıyla 2.7±0.85 μM IC50 değeri ile en fazla ve 13.8±0.91 μM IC50 değeri ile en duşuk inhibitorler olarak bulunmuştur. Resveratrol ve naringenin GST aktivitesini sırasıyla 6.2 μM Ki değeriyle yarışmasız ve 245 μM Ki değeriyle karışık inhibisyon seklinde inhibe etmiştir. Ote yandan 0.64 μM Ki değeri ile hesperidin ve 3.5 μM Ki değeri ile quersetin NQO1 enzimini yarışmalı inhibisyon seklinde inhibe etmiştir. Sonuç: Bu bulgular gostermektedir ki fenolik bileşiklerin Faz II enzimlerinden GST ve NQO1’i module edebilir. Ayrıca, bu bileşikler bu enzimlerin metabolize ettiği ksenobiyotik ve toksik bileşiklerin metabolik aktivasyonunu etkileyebilir.

Abstract 148: Transcriptome sequencing reveals PCAT5 - new ERG-regulated non-coding transcript in prostate cancer

Background: Prostate cancer (PC) is the second most common cancer among men. Most PC-related deaths are due to invasive tumors that are treated with therapies inhibiting androgen production or androgen receptor (AR) activity. After an initial response, tumors invariably progress to castration resistant prostate cancers (CRPCs) for which no effective cure exists. Methods and Results: We used transcriptome sequencing to study fresh-frozen tissue specimens from 12 benign prostatic hyperplasias (BPHs), 28 PCs, and 13 CRPCs. Reference-based transcriptome assembly uncovered 145 previously unannotated intergenic PC and CRPC associated long non-coding transcripts (lncRNAs) or isoforms. One third of the transcripts were CRPC-specific. We showed that one of the novel transcripts, Prostate Cancer Associated Transcript 5 (PCAT5), expressed in half of the tumors, was likely regulated by ERG, the key transcription factor in ∼50% of prostate cancers. Genome-wide expression analysis of a PCAT5-positive prostate cancer cell line after PCAT5 knockdown suggested significant alterations in proliferation pathways. In vitro validation of the pathway alterations revealed concordantly dramatic effects in phenotype: stalling of cell growth, migration, invasion, and colony forming potential, and increase in the rate of apoptosis. Conclusions: We identified the key differences between PC and CRPC in transcriptome level, and validated the oncogenic potential of a novel lncRNA in ERG-positive prostate cancers, PCAT5. Our study presents a number of putative lncRNA biomarkers for CRPC, and opportunities for therapeutic intervention. Citation Format: Antti Ylipaa, Kati Kivinummi, Matti Annala, Annika Kohvakka, Leena Latonen, Mauro Scaravilli, Kimmo Kartasalo, Simo-Pekka Leppanen, Serdar Karakurt, Janne Seppala, Olli Yli-Harja, Teuvo L.J. Tammela, Wei Zhang, Tapio Visakorpi, Matti Nykter. Transcriptome sequencing reveals PCAT5 - new ERG-regulated non-coding transcript in prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 148. doi:10.1158/1538-7445.AM2015-148

Abstract 5217: Integrative sequencing reveals novel alterations in untreated and castration resistant prostate cancer.

Prostate cancer is the third most common source of male cancer deaths in developed countries. The standard of care for aggressive prostate cancer is androgen ablation, which prolongs survival until the tumor acquires a castration resistant phenotype. The molecular pathology underlying prostate cancer progression is not yet fully understood. We used integrative high throughput sequencing to study cancer-associated alterations in 53 prostate cancer related neoplasia at the DNA, RNA and epigenetic levels. The cohort included both hormone-naive and castration resistant prostate cancers, along with two neuroendocrine prostate cancers. We identified two new fusion genes, one of which associated with neuronal differentiation and castration resistance. We also identified a number of novel prostate cancer associated transcripts, including transcripts specific to castration resistant tumors. Based on ChIP-seq data from prostate cancer cell lines, many of the novel transcripts were regulated by known oncogenes such as ERG and AR. Methylation sequencing revealed a near-identical pattern of promoter hypermethylation in both hormone-naive and castration resistant tumors. Enrichment of hypermethylation was observed at EZH2 binding sites, supporting the role of EZH2 in the recruitment of DNA methyltransferase in prostate cancer. Promoter hypermethylation suppressed the expression of hundreds of genes, but a subset of genes characterized by promoter H3K27 trimethylation responded to hypermethylation with increased expression. Citation Format: Matti J. Annala, Kati K. Waltering, Antti Ylipaa, Kimmo Kartasalo, Kirsi Tuppurainen, Serdar Karakurt, Leena Latonen, Outi Saramaki, Simo-Pekka Leppanen, Janne Seppala, Hanna E. Rauhala, Teuvo LJ Tammela, Olli Yli-Harja, Wei Zhang, Tapio Visakorpi, Matti Nykter. Integrative sequencing reveals novel alterations in untreated and castration resistant prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5217. doi:10.1158/1538-7445.AM2013-5217