Human Embryonal Carcinoma Cells Research Articles

Laccase-Treated Polystyrene Surfaces with Caffeic Acid, Dopamine, and L-3,4-Dihydroxyphenylalanine Substrates Facilitate the Proliferation of Melanocytes and Embryonal Carcinoma Cells NTERA-2.

This study presents the effects of treating polystyrene (PS) cell culture plastic with oxidoreductase enzyme laccase and the catechol substrates caffeic acid (CA), L-DOPA, and dopamine on the culturing of normal human epidermal melanocytes (NHEMs) and human embryonal carcinoma cells (NTERA-2). The laccase-substrate treatment improved PS hydrophilicity and roughness, increasing NHEM and NTERA-2 adherence, proliferation, and NHEM melanogenesis to a level comparable with conventional plasma treatment. Cell adherence dynamics and proliferation were evaluated. The NHEM endpoint function was quantified by measuring melanin content. PS surfaces treated with laccase and its substrates demonstrated the forming of polymer-like structures. The surface texture roughness gradient and the peak curvature were higher on PS treated with a combination of laccase and substrates than laccase alone. The number of adherent NHEM and NTERA-2 was significantly higher than on the untreated surface. The proliferation of NHEM and NTERA-2 correspondingly increased on treated surfaces. NHEM melanin content was enhanced 6-10-fold on treated surfaces. In summary, laccase- and laccase-substrate-modified PS possess improved PS surface chemistry/hydrophilicity and altered roughness compared to untreated and plasma-treated surfaces, facilitating cellular adherence, subsequent proliferation, and exertion of the melanotic phenotype. The presented technology is easy to apply and creates a promising custom-made, substrate-based, cell-type-specific platform for both 2D and 3D cell culture.

MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells.

Testicular cancer is fairly rare but can affect fertility in adult males. Leucine-rich repeats- and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker that mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 on testicular cancer development; however, the underlying mechanisms remain unclear. In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore the roles and regulatory effects of these molecules in NT2D1 cellular processes. A Dual-Glo luciferin-luciferase system was used to investigate LRWD1 transcriptional activity, and qRT-PCR and western blotting were used to determine gene and protein expression. The results suggested that miR-320a positively regulated LRWD1 and positively correlated with NT2D1 cell proliferation but negatively correlated with cell migration and invasion ability. In addition, the miRNA-ribonucleoprotein complex AGO2/FXR1 was shown to be essential in the mechanism by which miR-320a regulates LRWD1 mRNA expression. As miR-320a was required to regulate LRWD1 expression through the AGO2 and FXR1 complex, eEF2 and eLF4E were also found to be involved in miR-320a increasing LRWD1 expression. Furthermore, miR-320a and LRWD1 were responsive to oxidative stress, and NRF2 was affected by the presence of miR-320a in response to ROS stimulation. This is the first study showing the role of miR-320a in upregulating the testicular cancer-specific regulator LRWD1 and the importance of the AGO2/FXR1 complex in miR-320a-mediated upregulation of LRWD1 during testicular cancer progression.

A crucial stem cell plasticity regulation pathway: identification of key elements using the NCCIT human embryonic carcinoma cell line.

Upon removal of stemness factors, a small subpopulation of embryonic stem cells (ESCs) spontaneously extrudes the t-SNARE protein syntaxin-4, which upregulates the cell adhesion molecule P-cadherin and induces the onset of epithelial-mesenchymal transition (EMT)-like behaviors with loss of stemness in each cell. In this study, we identified a series of molecular elements responsible for this phenomenon using several small-molecule inhibitors and the human embryonic carcinoma cell line, NCCIT. We found that the syntaxin-4-triggered morphological changes and a decrease in stemness signatures were independently induced by the activation of Rho-associated kinase (ROCK) and the abrogation of PI3K/Akt signaling. We also found that the extracellular expression of syntaxin-4 inactivated focal adhesion kinase (FAK) in association with the augmented expression of P-cadherin, and comparable controls of either of these downstream elements of syntaxin-4 accelerated both ROCK-induced F-actin stress fiber formation and P13K/Akt-suppressed loss of stemness signatures. Cells expressing P-cadherin inactivated FAK but FAK inhibition did not affect P-cadherin expression, demonstrating a causal relationship between P-cadherin and FAK in the event of syntaxin-4 induction. These results reveal a novel signaling axis in stem cells and shed new light on the crucial elements for stem cell plasticity and the maintenance of stemness.

A Universal Strategy to Promote Secretion of G+/G- Bacterial Extracellular Vesicles and Its Application in Host Innate Immune Responses.

Both Gram-positive and Gram-negative bacteria release nanosized extracellular vesicles called membrane vesicles (MVs, 20-400 nm), which have great potential in various biomedical applications due to their abilities to deliver effector molecules and induce therapeutic responses. To fully utilize bacterial MVs for therapeutic purposes, regulated and enhanced production of MVs would be highly advantageous. In this study, we developed a universal method to enhance MV yields in both G+/G- bacteria through an autonomous controlled peptidoglycan hydrolase (PGase) expression system. A significant increase (9.37-fold) of MV concentration was observed in engineered E.coli Nissle 1917 compared to the wild-type. With the help of this autonomous system, for the first time we experimentally confirmed horizontal gene transfer and nutrient acquisition in a cocultured bacterial consortium. Furthermore, the engineered probiotic E.coli strains with high yield of MVs showed higher activation of the innate immune responses in human embryonic kidney 293T (HEK293T) and human colorectal carcinoma cells (HCT116), thereby demonstrating the great potential of engineering probiotics in immunology and further living therapeutics in humans.

Toxicity of Glycyl-l-Prolyl-l-Glutamate Pseudotripeptides: Cytotoxic, Oxidative, Genotoxic, and Embryotoxic Perspectives.

The tripeptide H-Gly-Pro-Glu-OH (GPE) and its analogs began to take much interest from scientists for developing effective novel molecules in the treatment of several disorders including Alzheimer's disease, Parkinson's disease, and stroke. The peptidomimetics of GPEs exerted significant biological properties involving anti-inflammatory, antiapoptotic, and anticancer properties. The assessments of their hematological toxicity potentials are critically required for their possible usage in further preclinical and clinical trials against a wide range of pathological conditions. However, there is so limited information on the safety profiling of GPE and its analogs on human blood tissue from cytotoxic, oxidative, and genotoxic perspectives. And, their embryotoxicity potentials were not investigated yet. Therefore, in this study, measurements of mitochondrial viability (using MTT assay) and lactate dehydrogenase (LDH) release as well as total antioxidant capacity (TAC) assays were performed on cultured human whole blood cells after treatment with GPE and its three novel peptidomimetics for 72 h. Sister chromatid exchange (SCE), micronucleus (MN), and 8-oxo-2-deoxyguanosine (8-OH-dG) assays were performed for determining the genotoxic damage potentials. In addition, the nuclear division index (NDI) was figured out for revealing their cytostatic potentials. Embryotoxicity assessments were performed on cultured human pluripotent NT2 embryonal carcinoma cells by MTT and LDH assays. The present results from cytotoxicity, oxidative, genotoxicity, and embryotoxicity testing clearly propounded that GPEs had good biosafety profiles and were trouble-free from the toxicological point of view. Noncytotoxic, antioxidative, nongenotoxic, noncytostatic, and nonembryotoxic features of GPE analogs are worthwhile exploring further and may exert high potentials for improving the development of novel disease-modifying agents.

PiRNAs Interact with Cold-Shock Domain-Containing RNA Binding Proteins and Regulate Neuronal Gene Expression During Differentiation.

piRNAs (PIWI-interacting RNAs) are a class of small non-coding RNAs (ncRNAs) abundantly expressed in germline cells and involved in suppressing the transposon activity. Interestingly, recent studies have found piRNA expression in the central nervous system (CNS), yet the underlying biological significance remains largely unknown. In this study, we investigated the expression and function of piRNAs during the retinoic acid (RA)-mediated neuronal differentiation in NT2 cells, a human embryonal carcinoma cell line. We identified a cohort of differentially expressed piRNAs by microarray. Two piRNAs, DQ582359 and DQ596268, were increasingly upregulated during the RA-induced differentiation and involved in regulating the expression of neuronal markers, MAP2 and TUBB3. Furthermore, these piRNAs were found to associate with cold-shock domain (CSD)-containing RNA binding proteins, DIS3, DIS3L2, and YB-1. Markedly, overexpression of these piRNAs further enhanced the protein levels of MAP2 and TUBB3, potentially by downregulating DIS3, DIS3L2, and YB-1. Hence, our study has identified a novel somatic function of piRNAs in regulating neuronal gene expression. The interaction of piRNA with some CSD-containing proteins can be further explored to enhance neuronal differentiation to treat neurodegenerative diseases.

MicroRNA 630 Represses NANOG Expression through Transcriptional and Post-Transcriptional Regulation in Human Embryonal Carcinoma Cells.

The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3′UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the NANOG 3′UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3′UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3′UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3′UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3′UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.

Polymeric Silver Nanoparticles: Potential for Folate-Targeted Delivery of Cisplatin In Vitro

Nanotechnology is a favorable avenue for improving therapeutic strategies, especially in cancer therapy. The harmful side effects of traditional cancer therapy impact dramatically on the patient’s quality of life. Cisplatin, a commonly used anticancer drug, is implicated in side effects such as neurotoxicity, nephrotoxicity and reduced blood cell count. Silver nanoparticles (AgNPs) have been investigated for their antibacterial effects and their anticancer activities to a lesser extent. Their capability as drug delivery vehicles has not been fully exploited, primarily due to their inconclusive cytotoxicity observed in healthy tissues. This study aimed to synthesize and characterize nanoparticles (NPs), consisting of Ag, chitosan (Cs) and folic acid (FA) (CsAg and FACsAg), loading them with cisplatin (C) (C-CsAg and C-FACsAg) and comparing their anticancer activities in the human embryonic kidney (HEK293), breast adenocarcinoma (MCF-7) and cervical carcinoma (HeLa) cells. All NPs and drug nanocomplexes were morphologically and physicochemically characterized, revealing NPs and nanocomplexes of favorable sizes ([Formula: see text][Formula: see text]nm), polydispersity and stability. The drug encapsulation efficiencies for C-CsAg and C-FACsAg were 50% and 72%, respectively, while drug release studies indicated that cisplatin release was pH dependent. The C-FACsAg nanocomplexes produced greater anticancer activity than C-CsAg. Folate receptor-mediated uptake was confirmed for the C-FACsAg nanocomplexes in the receptor-rich HeLa cells boding well for future in vivo research.

MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

You have accessJournal of UrologyPenile & Testicular Cancer I (MP40)1 Sep 2021MP40-04 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS, NCL1 AND NCD38, SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION Toshiki Etani, Taku Naiki, Nobuhiko Shimizu, Yusuke Noda, Takashi Nagai, Satoshi Nozaki, Keitaro Iida, Ryosuke Ando, Noriyasu Kawai, Satoru Takahashi, Takayoshi Suzuki, and Takahiro Yasui Toshiki EtaniToshiki Etani More articles by this author , Taku NaikiTaku Naiki More articles by this author , Nobuhiko ShimizuNobuhiko Shimizu More articles by this author , Yusuke NodaYusuke Noda More articles by this author , Takashi NagaiTakashi Nagai More articles by this author , Satoshi NozakiSatoshi Nozaki More articles by this author , Keitaro IidaKeitaro Iida More articles by this author , Ryosuke AndoRyosuke Ando More articles by this author , Noriyasu KawaiNoriyasu Kawai More articles by this author , Satoru TakahashiSatoru Takahashi More articles by this author , Takayoshi SuzukiTakayoshi Suzuki More articles by this author , and Takahiro YasuiTakahiro Yasui More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002055.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous and orthotopic tumors were induced by injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct 3/4 and SOX2 expression was decreased. In ChIP-qPCR analysis, LSD1 inhibition increased the methylation of promoter regions of SOX2 and Oct 3/4. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors. Source of Funding: Grants in Aid for Scientific Research (KAKENHI), Japan Society for the Promotion of Science (JSPS), Number 17K11152 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e715-e716 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Toshiki Etani More articles by this author Taku Naiki More articles by this author Nobuhiko Shimizu More articles by this author Yusuke Noda More articles by this author Takashi Nagai More articles by this author Satoshi Nozaki More articles by this author Keitaro Iida More articles by this author Ryosuke Ando More articles by this author Noriyasu Kawai More articles by this author Satoru Takahashi More articles by this author Takayoshi Suzuki More articles by this author Takahiro Yasui More articles by this author Expand All Advertisement Loading ...

In-vitro antioxidant and selective cytotoxicity of Garcinia cambogia and Garcinia indica leaf extracts on human kidney cancer cell line

Kidney cancer is one among the top 10 cancers. Renal cell carcinoma (RCC) is commonly known kidney cancer. Almost all clinically targeted drugs used in treating RCC have many aftereffects. To overcome this problem with herbal products, the present study is aimed to investigate the preliminary phytochemicals, antioxidants and selective cytotoxicity of aqueous leaves extract of Garcinia cambogia (GC)and Garcinia indica (GI)on(HEK-293) human embryonic kidney cells and (A498)human renal carcinoma cells. The phytochemical analyses were done using standard protocols. In-vitro antioxidant activity was carried out using DPPH, FRAP, and Phosphomolybdenum assay. Anticancer activity on A498 kidney cancer cell line was evaluated by MTT assay and the selectivity index was calculated. Preliminary phytochemical analysis of GC and GI leaf extract divulged the presence of various phyto-constituents. GI extract revealed higher phenolic content while flavonoid content was more in GC extract compared to alkaloids and saponins. Both plant extracts exhibited higher antioxidant capacities based on the test performed.GC and GIleaf extract wasselectively cytotoxic in-vitro to (A498) human renal carcinoma cells and can be safely used against kidney cancer at 500 µM (GC extract)and 300 µM (GI extract). Selective index for GC and GI extract was15.9 and 2.4respectively. The results indicate that GC and GI extracts are a favourable antioxidant and anti-cancer agents for A498 human renal carcinoma cells. However, further studies to isolate the bioactive compounds responsible for these activities are underway and to explore their molecular mechanism.

LRWD1 expression is regulated through DNA methylation in human testicular embryonal carcinoma cells

BackgroundSperm growth and maturation are correlated with the expression levels of Leucine-rich repeat and WD repeat-containing protein 1 (LRWD1), a widely expressed protein in the human testicles. The decrease in LRWD1 cellular level was linked to the reduction in cell growth and mitosis and the rise in cell microtubule atrophy rates. Since DNA methylation has a major regulatory role in gene expression, this study aimed at exploring the effect of the modulation of DNA methylation on LRWD1 expression levels.ResultsThe results revealed the presence of a CpG island up of 298 bps (− 253 ~ + 45) upon LRWD1 promoter in NT2/D1 cells. The hypermethylation of the LRWD1 promoter was linked to a reduction in the transcription activity in NT2/D1 cells, as indicated by luciferase reporter assay. The methylation activator, floxuridine, confirmed the decrease in the LRWD1 promoter transcriptional activity. On the other hand, 5-Aza-2′-deoxycytidine (5-Aza-dc, methylation inhibitor), significantly augmented LRWD1 promoter activity and the expression levels of mRNA and proteins. Furthermore, DNA methylation status of LRWD1 promoter in human sperm genomic DNA samples was analyzed. The results indicated that methylation of LRWD1 promoter was correlated to sperm activity.ConclusionsThus, the regulation of LRWD1 expression is correlated with the methylation status of LRWD1 promoter, which played a significant role in the modulation of spermatogenesis, sperm motility, and vitality. Based on these results, the methylation status of LRWD1 promoter may serve as a novel molecular diagnostic marker or a therapeutic target in males’ infertility.

Epithelium-specific ETS transcription factor-1 regulates NANOG expression and inhibits NANOG-induced proliferation of human embryonic carcinoma cells

The epithelium-specific ETS transcription factor-1 (ESE-1) plays multiple roles in pathogenesis and normal development of epithelial tissues. NANOG, a key mediator of stem cell self-renewal and pluripotency, is also expressed in various cancers and pluripotent cells. In this study, we investigated how ESE-1 influences NANOG expression and NANOG-induced proliferation in human germ cell-derived embryonic carcinoma NCCIT cells. Endogenous ESE-1 expression in NCCIT cells significantly increased during differentiation, whereas NANOG expression decreased. In addition, NANOG expression was downregulated by exogenous overexpression of ESE-1, and increased by shRNA-mediated knockdown of ESE-1. NANOG transcriptional activity was reduced by dose-dependent ESE-1 overexpression and a putative ESE-1 binding site (EBS) was mapped within conserved region 2. Site-directed mutagenesis of the putative EBS abrogated the repressive effect of ESE-1 on NANOG promoter activity. ESE-1 directly interacted with the putative EBS to regulate transcriptional activity of NANOG. Furthermore, NANOG-induced proliferation and colony formation of NCCIT cells were inhibited by ESE-1 overexpression and stimulated by ESE-1 shRNA-mediated knockdown. Altogether, our results suggest that ESE-1 exerts an anti-proliferative effect on NCCIT cells by acting as a novel transcriptional repressor of NANOG.

Expression and roles of system L amino acid transporters in human embryonal carcinoma cells.

Testicular germ cell tumors (TGCTs) are the most common malignant cancer in young men. Although TGCTs are generally responsive to platinum-based chemotherapy particularly cisplatin, acquired resistance in patients with metastasis still occurs resulting in poor prognosis. Specifically, differentiation of embryonal carcinoma (EC) cells, the stem cells of TGCTs, can lead to the reduction of cisplatin responsiveness. Therefore, novel therapeutic strategies for TGCTs are needed. System L amino acid transporters have been reported to be up-regulated and to play an important role in tumorigenesis. However, expression and role of system L amino acid transporters in TGCTs remain elusive. Expression of system L amino acid transporters was analyzed in TGCT samples from The Cancer Genome Atlas (TCGA). Expression of LAT1, LAT2, and 4F2hc was examined in human embryonal carcinoma cell line NTERA2. Roles of system L amino acid transporters on NTERA2 cell survival, cell proliferation, pluripotency, and cisplatin sensitivity were evaluated. Based upon TCGA datasets, we found that two isoforms of system L (LAT1 and LAT2) and their chaperone protein 4F2hc are highly expressed in EC samples compared with other groups. Treatment with the system L inhibitor BCH significantly suppressed leucine uptake into the pluripotent EC cell line NTERA2. The malignant phenotypes including cell viability, cell proliferation, and clonal ability were decreased following BCH treatment. Nonetheless, system L inhibition did not alter expression of stemness genes in NTERA2 cells. After NTERA2 differentiation, expressions of LAT1 and LAT2 were decreased. Finally, co-administration of BCH enhanced cisplatin sensitivity in both undifferentiated and differentiated cells. These effects were associated with the reduction in p70S6K phosphorylation. Taken together, these results shed light on the roles of system L amino acid transporters in TGCTs. Therefore, system L amino acid transporters could provide novel therapeutic targets for treatment against TGCTs.

Solubility and biological activity enhancement of the highly lipophilic viridicatins via interaction with cyclodextrins

Viridicatin and its derivatives were found to be the potent strong inhibitors of the tumor necrosis factor-induced replication of many serious diseases. Their extremely low solubility in aqueous medium at a physiological pH compromises bioavailability and leads to poor pharmacokinetic performance and low exposure. Since the pharmacologic activation represents a valuable strategy for treatment of major diseases, we first obtained water-soluble form of viridicatin and viridicatol and realized this by encapsulation in the cyclodextrin cavity. We also investigated self-assembling behavior of resulting complexes and showed that the cyclodextrin complex is more than four times more active against diseased cells than to healthy ones. Measurement of cytotoxic activity performed with normal human embryonic lung cells and human lung carcinoma cell cultures demonstrated that the functional group in the chemical structure of viridicatin derivatives also influences its biologic properties.

Role of PIWI-like 4 in modulating neuronal differentiation from human embryonal carcinoma cells

ABSTRACT PIWI homologs constitute a subclass of the Argonaute family. Traditionally, they have been shown to associate with a specific class of small RNAs, piRNAs, to suppress transposable elements and protect genomic integrity in germ cells. Recent studies imply that PIWI proteins may also exert important biological functions in somatic contexts, including the brain. However, their exact role in neural development remains unknown. Hence we investigated whether PIWI proteins are involved in neuronal differentiation. By using an established cell model for studying neurogenesis, NTera2/D1 (NT2) cells, we found that a particular PIWI homolog, PIWIL4 was increasingly upregulated throughout the course of all-trans retinoic acid (RA)-mediated neuronal differentiation. During this process, PIWIL4 knockdown led to partial recovery of embryonic stem cell markers, while suppressing RA-induced expression of neuronal markers. Consistently, PIWIL4 overexpression further elevated their expression levels. Furthermore, co-immunoprecipitation revealed an RA-induced interaction between PIWIL4 and the H3K27me3 demethylase UTX. Chromatin immunoprecipitation showed that this interaction could be essential for the removal of H3K27me3 from the promoters of RA-inducible genes. By a similar mechanism, PIWIL4 knockdown also suppressed the expression of PTN and NLGN3, two important neuronal factors secreted to regulate glioma activity. We further noted that the conditioned medium collected from PIWIL4-silenced NT2 cells significantly reduced the proliferation of glioma cells. Thus, our data suggest a novel somatic role of PIWIL4 in modulating the expression of neuronal genes that can be further characterized to promote neuronal differentiation and to modulate the activity of glioma cells.

MP11-03 NOVEL SELECTIVE LYSINE-SPECIFIC DEMETHYLASE 1 INHIBITORS SUPPRESS TESTICULAR TUMOR CELL PROLIFERATION

INTRODUCTION AND OBJECTIVE: Lysine-specific demethylase 1 (LSD1), the first histone demethylase to be discovered, is a novel target in prostate cancer therapy. NCL1 and NCD38, novel selective cell-active inhibitors of LSD1, were discovered at our university. We analyzed the efficacy of LSD1 inhibitors in germ cell tumors in vitro and in vivo. METHODS: Various tests were used to uncover mechanism of germ cell tumor growth and anticancer effects of NCL1 and NCD38. Cell viability was assessed using a WST-8 assay with standard vehicle, NCL1 or NCD38 in NTERA2 and Tera-1 cells (human testicular embryonal carcinoma cell lines). Flow cytometry and western blotting were performed to assess apoptosis and the cell cycle after treatment of NTERA2 cells with NCL1 or NCD38. Subcutaneous tumors were induced by subcutaneously injecting nude mice with NTERA2 cells. Thereafter, mice were injected intraperitoneally with vehicle, NCL1 or NCD38, twice a week, and their subsequent growth recorded. TUNEL staining was used to detect apoptosis in tumors. Finally, tissue arrays were prepared from tissue specimens from 35 patients with seminoma who underwent orchiectomy. RESULTS: The WST assay revealed a reduction in the number of viable cells in a dose-dependent manner after NCL1 or NCD38 treatment. In western blotting, NCL1 or NCD38 treatment induced caspase-dependent apoptosis, while Oct4 and SOX2 expression was decreased. In flow cytometry analysis, NCL1 or NCD38 significantly induced apoptosis in a dose-dependent manner. Subcutaneous tumor volumes and serum AFP levels were significantly lower in mice treated with NCL1 or NCD38 than in controls. TUNEL analysis showed that NCL1 and NCD38 treatment induced apoptosis in NTERA2 subcutaneous tumors. No adverse effect was observed in body weights, blood tests, and hematoxylin-eosin staining of various organs in mice. Tissue array analysis showed that LSD1 expression in human seminoma specimens was significantly higher than that in noncancerous specimens. CONCLUSIONS: Germ cell tumor growth was effectively suppressed with NCL1 or NCD38 treatment in vitro and in vivo without adverse events by regulating apoptosis and suppressing SOX2 and Oct4. These results indicate the potential of NCL1 and NCD38 as therapeutic agents for germ cell tumors.Source of Funding: Grant-in-Aid for Scientific Research 17K11152

Neuroprotective effects of boron nitride nanoparticles in the experimental Parkinson's disease model against MPP+ induced apoptosis.

Parkinson's disease (PD) is one of the most aggressive neurodegenerative diseases and characterized by the loss of dopamine-sensitive neurons in the substantia nigra region of the brain. There is no any definitive treatment to completely cure PD and existing treatments can only ease the symptoms of the disease. Boron nitride nanoparticles have been extensively studied in nano-biological studies and researches showed that it can be a promising candidate for PD treatment with its biologically active unique properties. In the present study, it was aimed to investigate ameliorative effects of hexagonal boron nitride nanoparticles (hBNs) against toxicity of 1-methyl-4-phenylpyridinium (MPP+) in experimental PD model. Experimental PD model was constituted by application of MPP+ to differentiated pluripotent human embryonal carcinoma cell (Ntera-2, NT-2) culture in wide range of concentrations (0.62 to 2mM). Neuroprotective activity of hBNs against MPP+ toxicity was determined by cell viability assays including MTT and LDH release. Oxidative alterations by hBNs application in PD cell culture model were investigated using total antioxidant capacity (TAC) and total oxidant status (TOS) tests. The impacts of hBNs and MPP+ on nuclear integrity were analyzed by Hoechst 33258 fluorescent staining method. Acetylcholinesterase (AChE) enzyme activities were determined by a colorimetric assay towards to hBNs treatment. Cell death mechanisms caused by hBNs and MPP+ exposure was investigated by flow cytometry analysis. Experimental results showed that application of hBNs increased cell viability in PD model against MPP+ application. TAS and TOS analysis were determined that antioxidant capacity elevated after hBNs applications while oxidant levels were reduced. Furthermore, flow cytometric analysis executed that MPP+ induced apoptosis was prevented significantly (p < 0.05) after application with hBNs. In a conclusion, the obtained results indicated that hBNs have a huge potential against MPP+ toxicity and can be used in PD treatment as novel neuroprotective agent and drug delivery system.

Tannic Acid Promotes TRAIL-Induced Extrinsic Apoptosis by Regulating Mitochondrial ROS in Human Embryonic Carcinoma Cells.

Human embryonic carcinoma (EC; NCCIT) cells have self-renewal ability and pluripotency. Cancer stem cell markers are highly expressed in NCCIT cells, imparting them with the pluripotent nature to differentiate into other cancer types, including breast cancer. As one of the main cancer stem cell pathways, Wnt/β-catenin is also overexpressed in NCCIT cells. Thus, inhibition of these pathways defines the ability of a drug to target cancer stem cells. Tannic acid (TA) is a natural polyphenol present in foods, fruits, and vegetables that has anti-cancer activity. Through Western blotting and PCR, we demonstrate that TA inhibits cancer stem cell markers and the Wnt/β-catenin signaling pathway in NCCIT cells and through a fluorescence-activated cell sorting analysis we demonstrated that TA induces sub-G1 cell cycle arrest and apoptosis. The mechanism underlying this is the induction of mitochondrial reactive oxygen species (ROS) (mROS), which then induce the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated extrinsic apoptosis pathway instead of intrinsic mitochondrial apoptosis pathway. Moreover, ribonucleic acid sequencing data with TA in NCCIT cells show an elevation in TRAIL-induced extrinsic apoptosis, which we confirm by Western blotting and real-time PCR. The induction of human TRAIL also proves that TA can induce extrinsic apoptosis in NCCIT cells by regulating mROS.

Vernolactone Promotes Apoptosis and Autophagy in Human Teratocarcinomal (NTERA-2) Cancer Stem-Like Cells.

Vernonia zeylanica, is a shrub endemic to Sri Lanka. V. zeylanica has been used in Sri Lankan traditional medicine for the treatment of various diseases and conditions. The present study was designed to determine antiproliferative, apoptotic, autophagic, and antioxidant effects of vernolactone, isolated from V. zeylanica, in human embryonal carcinoma cells (NTERA-2, a cancer stem cell model). Antiproliferative effects of vernolactone in NTERA-2 cells and human peripheral blood mononuclear cells (control cells) were evaluated using the Sulforhodamine B (SRB) assay and WST-1 antiproliferative assays, respectively. The antiproliferative effect of vernolactone was further investigated using the colony formation assay. Effects of vernolactone on apoptosis were investigated by phase contrast light microscopic and fluorescence microscopic analysis, caspase 3/7 expression, and real-time PCR of apoptosis-associated genes p53 and Survivin. The effect of vernolactone on NTERA-2 cell migration was monitored using the wound healing assay. Effects of vernolactone on the expression of autophagy-related genes (LC3, Beclin 1, PI3K, Akt, and mTOR) were evaluated using real-time PCR. 2,2-Diphenyl-1-2,2-diphenyl-picrylhydrazyl (DPPH) radical scavenging assay, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging, and ferric reducing antioxidant power (FRAP) assays were also carried out to evaluate the antioxidant activity of vernolactone. Overall results confirm that vernolactone can exert antiproliferative effects, induce apoptosis and autophagy, and decrease NTERA-2 cell migration in a dose- and time-dependent manner with a very small antioxidant property.

Disulfiram Overcomes Cisplatin Resistance in Human Embryonal Carcinoma Cells.

Cisplatin resistance in testicular germ cell tumors (TGCTs) is a clinical challenge. We investigated the underlying mechanisms associated with cancer stem cell (CSC) markers and modalities circumventing the chemoresistance. Chemoresistant models (designated as CisR) of human embryonal carcinoma cell lines NTERA-2 and NCCIT were derived and characterized using flow cytometry, gene expression, functional and protein arrays. Tumorigenicity was determined on immunodeficient mouse model. Disulfiram was used to examine chemosensitization of resistant cells. ALDH1A3 isoform expression was evaluated by immunohistochemistry in 216 patients’ tissue samples. Chemoresistant cells were significantly more resistant to cisplatin, carboplatin and oxaliplatin compared to parental cells. NTERA-2 CisR cells exhibited altered morphology and increased tumorigenicity. High ALDH1A3 expression and increased ALDH activity were detected in both refractory cell lines. Disulfiram in combination with cisplatin showed synergy for NTERA-2 CisR and NCCIT CisR cells and inhibited growth of NTERA-2 CisR xenografts. Significantly higher ALDH1A3 expression was detected in TGCTs patients’ tissue samples compared to normal testicular tissue. We characterized novel clinically relevant model of chemoresistant TGCTs, for the first time identified the ALDH1A3 as a therapeutic target in TGCTs and more importantly, showed that disulfiram represents a viable treatment option for refractory TGCTs.