NTERA-2 Cells Research Articles

Human Ntera2 cells as a predictive in vitro test system for developmental neurotoxicity

Developmental neurotoxicity (DNT) of environmental chemicals is a serious threat to human health. Current DNT testing guidelines propose investigations in rodents, which require large numbers of animals. With regard to the "3Rs" (reduction, replacement, and refinement) of animal testing, alternative testing strategies are needed in order to refine and reduce animal experiments and allow faster and less expensive screening. The goal of this study was to establish components for a human cell-based test system to assess DNT potential of chemicals at an early stage of brain development. A human neural precursor cell line should be tested for suitability for semi-automated high-throughput DNT screening. We established assays suitable for detecting disturbances in two basic processes of brain development in 96-well scale: neuronal differentiation and migration using the human Ntera2 (NT2) cell line. We assessed the effects of four test compounds with well-established DNT potential in comparison with three compounds without specific DNT potential. We found that human NT2 cell cultures treated with the morphogen, retinoic acid, imitate neuronal differentiation, and migration in vitro. The developmental neurotoxicants methylmercury chloride, sodium arsenite, sodium valproate, and methylazoxymethanol significantly reduced the expression of the neuronal marker β-tubulin type III and decreased the migration distance in developing NT2 cells. Both endpoints, differentiation and migration, can be read out directly in a standard fluorescence plate reader, enabling high-throughput screening. We conclude that NT2 cell tests are likely to become valuable components of a human cell-based modular in vitro DNT test systems.

Cyclic AMP and c-KIT Signaling in Familial Testicular Germ Cell Tumor Predisposition

Familial testicular germ cell tumors (FTGCTs) are hypothesized to result from the combined interaction of multiple low-penetrance genes. We reported inactivating germline mutations of the cAMP-binding phosphodiesterase 11A (PDE11A) as modifiers of FTGCT risk. Recent genome-wide association studies have identified single-nucleotide polymorphisms in the KITLG gene, the ligand for the cKIT tyrosine kinase receptor, as strong modifiers of susceptibility to both familial and sporadic testicular germ cell tumors. We studied 94 patients with FTGCTs and 50 at-risk male relatives from 63 unrelated kindreds, in whom the PDE11A gene had been sequenced by investigating the association between KITLG genome-wide association study single-nucleotide polymorphisms rs3782179 and rs4474514 and FTGCT risk in these patients and in 692 controls. We also examined cAMP and c-KIT signaling in testicular tissues and cell lines and extended the studies to 2 sporadic cases, one with a PDE11A defect and one without, as a comparison. We found a higher frequency of the KITLG risk alleles in FTGCT patients who also had a PDE11A sequence variant, compared with those with a wild-type PDE11A sequence. In NTERA-2 and Tcam-2 cells transfected with the mutated forms of PDE11A (R52T, F258Y, Y727C, R804H, V820M, R867G, and M878V), cAMP levels were significantly higher, and the relative phosphodiesterase activity was lower than in the wild-type cells. KITLG expression was consistently increased in the presence of PDE11A-inactivating defects, both at the RNA and protein levels, in familial testicular germ cell tumors. The 2 sporadic cases that were studied, one with a PDE11A defect and another without, agreed with the data in FTGTCT and in the cell lines. Patients with FTGCT and PDE11A defects also carry KITLG risk alleles more frequently. There may be an interaction between cAMP and c-KIT signaling in predisposition to testicular germ cell tumors.

Curcumin inhibits AP-2γ-induced apoptosis in the human malignant testicular germ cells in vitro

To investigate the effects of curcumin on proliferation and apoptosis in testicular cancer cells in vitro and to investigate its molecular mechanisms of action. NTera-2 human malignant testicular germ cell line and F9 mouse teratocarcinoma stem cell line were used. The anti-proliferative effect was examined using MTT and colony formation assays. Hoechst 33258 staining, TUNEL and Annexin V-FITC/PI staining assays were used to analyze cell apoptosis. Protein expression was examined with Western blot analysis and immunocytochemical staining. Curcumin (5, 10 and 15 μmol/L) inhibited the viability of NTera-2 cells in dose- and time-dependent manners. Curcumin significantly inhibited the colony formation in both NTera-2 and F9 cells. Curcumin dose-dependently induced apoptosis of NTera-2 cells by reducing FasL expression and Bcl-2-to-Bax ratio, and activating caspase-9, -8 and -3. Furthermore, curcumin dose-dependently reduced the expression of AP transcription factor AP-2γ in NTera-2 cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Curcumin inhibited ErbB2 expression, and decreased the phosphorylation of Akt and ERK in NTera-2 cells. Curcumin induces apoptosis and inhibits proliferation in NTera-2 cells via the inhibition of AP-2γ-mediated downstream cell survival signaling pathways.

Assessment of tissue distribution and subcellular localization of miR-302 and miR-21 by means of in situ hybridization technique

MicroRNAs (miRNAs) are a group of short non-coding RNAs implicated in numerous fundamental cellular processes, and their disregulations have been linked to several pathologic conditions, mainly cancers. Determining tissue distribution of miRNAs is a prerequisite for understanding their exact functions during development, tissue homeostasis and abnormality. In situ hybridization is a powerful technique to delineate the sub-cellular localization and tissue distribution patterns of mRNAs as well as miRNAs. Due to the important role of miRNAs in tumorigenesis, we optimized an ISH technique for detection of two well-known miRNAs (miR-302 and miR-21) in formalin-fixed paraffin-embedded (FFPE) tumor samples along with a pluripotent embryonal carcinoma cell line, NTERA-2 (NT2). After fixation of cells on slides/sectioning of FFPE blocks, proteinase K digestion, probe concentration, antibody development and light sensitive color reaction were optimized for both the FFPE samples and cell line. Signals for U6 snRNA, as an internal control, were detected in the nuclei of the cells. MiR-21 and miR-302 expression was detected in the cytoplasm of FFPE samples of seminoma carcinoma and in NT2 cell line, respectively. In this study, we optimized ISH for miRNA detection in FFPE samples and NT2 cell line.

The targeting and functions of miRNA-383 are mediated by FMRP during spermatogenesis

Our previous studies have shown that microRNA-383 (miR-383) expression is downregulated in the testes of infertile men with maturation arrest (MA). Abnormal testicular miR-383 expression may potentiate the connections between male infertility and testicular germ cell tumors. However, the mechanisms underlying the targeting and functions of miR-383 during spermatogenesis remain unknown. In this study, we found that fragile X mental retardation protein (FMRP) was associated with 88 miRNAs in mouse testis including miR-383. Knockdown of FMRP in NTERA-2 (NT2) (testicular embryonal carcinoma) cells enhanced miR-383-induced suppression of cell proliferation by decreasing the interaction between FMRP and miR-383, and then affecting miR-383 binding to the 3′-untranslated region of its target genes, including interferon regulatory factor-1 (IRF1) and Cyclin D1 both in vivo and in vitro. On the other hand, FMRP levels were also downregulated by overexpression of miR-383 in NT2 cells and GC1 (spermatogonia germ cell line). miR-383 targeted to Cyclin D1 directly, and then inhibited its downstream effectors, including phosphorylated pRb and E2F1, which ultimately resulted in decreased FMRP expression. Reduced miR-383 expression, dysregulated cyclin-dependent kinase 4 expression (one of the downstream genes of miR-383) and increased DNA damage were also observed in the testes of Fmr1 knockout mice and of MA patients with a downregulation of FMRP. A potential feedback loop between FMRP and miR-383 during spermatogenesis is proposed, and FMRP acts as a negative regulator of miR-383 functions. Our data also indicate that dysregulation of the FMRP–miR-383 pathway may partially contribute to human spermatogenic failure with MA.

A human pluripotent carcinoma stem cell-based model for in vitro developmental neurotoxicity testing: Effects of methylmercury, lead and aluminum evaluated by gene expression studies

The major advantage of the neuronal cell culture models derived from human stem cells is their ability to replicate the crucial stages of neurodevelopment such as the commitment of human stem cells to the neuronal lineage and their subsequent stages of differentiation into neuronal and glial-like cell. In these studies we used mixed neuronal/glial culture derived from the NTERA-2 (NT-2) cell line, which has been established from human pluripotent testicular embryonal carcinoma cells. After characterization of the different stages of cell differentiation into neuronal- and glial-like phenotype toxicity studies were performed to evaluate whether this model would be suitable for developmental neurotoxicity studies. The cells were exposed during the differentiation process to non-cytotoxic concentrations of methylmercury chloride, lead chloride and aluminum nitrate for two weeks. The toxicity was then evaluated by measuring the mRNA levels of cell specific markers (neuronal and glial). The results obtained suggest that lead chloride and aluminum nitrate at low concentrations were toxic primarily to astrocytes and at the higher concentrations it also induced neurotoxicity. In contrast, MetHgCl was toxic for both cell types, neuronal and glial, as mRNA specific for astrocytes and neuronal markers were affected. The results obtained suggest that a neuronal mixed culture derived from human NT2 precursor cells is a suitable model for developmental neurotoxicity studies and gene expression could be used as a sensitive endpoint for initial screening of potential neurotoxic compounds.

Regulation of human Cripto‐1 expression by nuclear receptors and DNA promoter methylation in human embryonal and breast cancer cells

Human Cripto-1 (CR-1) plays an important role in regulating embryonic development while also regulating various stages of tumor progression. However, mechanisms that regulate CR-1 expression during embryogenesis and tumorigenesis are still not well defined. In the present study, we investigated the effects of two nuclear receptors, liver receptor homolog (LRH)-1 and germ cell nuclear factor receptor (GCNF) and epigenetic modifications on CR-1 gene expression in NTERA-2 human embryonal carcinoma cells and in breast cancer cells. CR-1 expression in NTERA-2 cells was positively regulated by LRH-1 through direct binding to a DR0 element within the CR-1 promoter, while GCNF strongly suppressed CR-1 expression in these cells. In addition, the CR-1 promoter was unmethylated in NTERA-2 cells, while T47D, ZR75-1, and MCF7 breast cancer cells showed high levels of CR-1 promoter methylation and low CR-1 mRNA and protein expression. Treatment of breast cancer cells with a demethylating agent and histone deacetylase inhibitors reduced methylation of the CR-1 promoter and reactivated CR-1 mRNA and protein expression in these cells, promoting migration and invasion of breast cancer cells. Analysis of a breast cancer tissue array revealed that CR-1 was highly expressed in the majority of human breast tumors, suggesting that CR-1 expression in breast cancer cell lines might not be representative of in vivo expression. Collectively, these findings offer some insight into the transcriptional regulation of CR-1 gene expression and its critical role in the pathogenesis of human cancer.

Blocking Nogo Receptor 1 Promotes Functional Regeneration after Spinal Cord Injury

An insult to the mammalian spinal cord often results in persistent functional deficits. Proteins in CNS myelin are important determinants of this situation as they inhibit neurite growth. Among those proteins Nogo-A, oligodendrocyte myelin glycoprotein (OMgp), and myelin-associated glycoprotein (MAG) all bind to the neuronal Nogo-66 receptor (NgR1) and thereby block neuronal regeneration after injury. Neutralizing the interaction between the inhibitory ligands and NgR1 may alleviate the inhibition and therefore result in increased recovery after injury. Thus, antibodies neutralizing ligand/receptor interaction might have therapeutic value. From a set of 300 monoclonal anti-NgR1 antibodies one anti-NgR1 ligand blocking antibody (mAb50) was selected for in vivo studies. mAb50 binds with high affinity (below 100 pM) to human and rat NgR1, competes for binding of a ligand peptide (derived from Nogo-A: Nogo66) to the isolated NgR1 protein and cellular NgR1, and reduces the inhibitory effects of Nogo66 on neurite outgrowth in differentiated human NTera2 cells and rat dorsal root ganglion neurons. In a rat spinal cord hemisection model mAb50 enhanced functional locomotor recovery. These data suggest that neutralization of NgR1 activity with an antibody may be a valuable strategy to neutralize neurite outgrowth inhibition in the mammalian CNS. Thus anti-NgR1 antibodies are a potential approach for the treatment of spinal cord injury and even neurodegenerative diseases.

Effects of the neurotoxic thionophosphate pesticide chlorpyrifos on differentiating alternative models

Studies by researchers worldwide have revealed that, even in industrialised nations, people, infants and the aged in particular, are even more exposed to neurotoxic drugs as a consequence of the increased quantity of pesticide residues in food. This phenomenon, as underlined by The Worldwatch Institute (2006), is linked to the exponential increase in the use of these toxic compounds over the last 40years, up from 0.49kg per hectare in 1961 to 2kg in 2004, with the result that these substances are found in the daily diet.Many studies have demonstrated how the assumption of pesticides in the neonatal period and early infancy can alter the development and function of the nervous, immune, endocrine and reproductive apparatuses. Moreover, the unequivocal relationship between brain tumours, infant leukemia and pesticides are well recognised.On the basis of the above information, the effects of the neurotoxic thionophosphate pesticide chlorpyrifos (CPF) have been tested, considering biomarkers of toxicity and toxicity endpoint, on the biological models Dictyostelium discoideum, Paracentrotus lividus, and NTera2 Cells, as they are compatible with the 3Rs strategy (Reduction, Replacement, and Refinement in animal experiments). Our results have revealed that developing organisms are particularly sensitive to the toxic effects of CPF.

Vitamin D Metabolism and Effects on Pluripotency Genes and Cell Differentiation in Testicular Germ Cell Tumors In Vitro and In Vivo

Testicular germ cell tumors (TGCTs) are classified as either seminomas or nonseminomas. Both tumors originate from carcinoma in situ (CIS) cells, which are derived from transformed fetal gonocytes. CIS, seminoma, and the undifferentiated embryonal carcinoma (EC) retain an embryonic phenotype and express pluripotency factors (NANOG/OCT4). Vitamin D (VD) is metabolized in the testes, and here, we examined VD metabolism in TGCT differentiation and pluripotency regulation. We established that the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human fetal germ cells, CIS, and invasive TGCTs. VD metabolism diminished markedly during the malignant transformation from CIS to EC but was reestablished in differentiated components of nonseminomas, distinguished by coexpression of mesodermal markers and loss of OCT4. Subsequent in vitro studies confirmed that 1,25(OH)2D3 (active VD) downregulated NANOG and OCT4 through genomic VDR activation in EC-derived NTera2 cells and, to a lesser extent, in seminoma-derived TCam-2 cells, and up-regulated brachyury, SNAI1, osteocalcin, osteopontin, and fibroblast growth factor 23. To test for a possible therapeutic effect in vivo, NTera2 cells were xenografted into nude mice and treated with 1,25(OH)2D3, which induced down-regulation of pluripotency factors but caused no significant reduction of tumor growth. During NTera2 tumor formation, down-regulation of VDR was observed, resulting in limited responsiveness to cholecalciferol and 1,25(OH)2D3 treatment in vivo. These novel findings show that VD metabolism is involved in the mesodermal transition during differentiation of cancer cells with embryonic stem cell characteristics, which points to a function for VD during early embryonic development and possibly in the pathogenesis of TGCTs.

Effects of N-acetyl-l-cysteine on bleomycin induced oxidative stress in malignant testicular germ cell tumors

Testicular cancer is a very common cancer in males aged 15-44 years. Bleomycin is used in chemotherapy regimens in the treatment of patients having testicular germ-cell tumor. Bleomycin generates oxygen radicals, induces oxidative cleavage of DNA strand and induces apoptosis in cancer cells. There is no study in the literature investigating effects of N-Acetyl-L-Cysteine (NAC) on bleomycin-induced oxidative stress in testicular germ cell tumors. For this reason, we studied effects of NAC on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testis cancer cells incubated with bleomycin and compared the results with H(2)O(2) which directly produces oxidative stress. We determined protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide levels and total antioxidant capacity in both testicular cancer cells. Bleomycin and H(2)O(2) significantly increased 8-isoprostane, TBARS, protein carbonyl and lipid hydroperoxide levels in NTera-2 and NCCIT cells. Bleomycin and H(2)O(2) significantly decreased antioxidant capacity and GSH levels in both cell lines. Co-incubation with NAC significantly decreased lipid hydroperoxide, 8-isoprostane, protein carbonyl content and TBARS levels increased by bleomycin and H(2)O(2). NAC enhanced GSH levels and antioxidant capacity in the NTera-2 and NCCIT cells. It can be concluded that NAC diminishes oxidative stress in human testicular cancer cells induced by bleomycin and H(2)O(2).

Transcription Factors Sp1 and p73 Control the Expression of the Proapoptotic Protein NOXA in the Response of Testicular Embryonal Carcinoma Cells to Cisplatin

Testicular germ cell tumors (TGCTs) are highly responsive to and curable by cisplatin-based chemotherapy even in advanced stages. We have studied the molecular mechanisms involved in the induction of apoptosis in response to cisplatin, and found that proapoptotic Noxa is transcriptionally up-regulated following cisplatin exposure, even in the absence of p53, in NTERA2 cisplatin-sensitive cells but not in 1411HP-resistant cells. Blockade of Noxa reduced the apoptotic response of embryonal carcinoma (EC) NTERA2 cells to cisplatin. A detailed analysis of the Noxa promoter revealed that p73 and Sp1-like factors, Sp1 and KLF6, played key roles in the transcriptional control of this gene. Overexpression of TAp73 induced Noxa whereas the dominant negative isoform ΔNp73, reduced the levels of Noxa after cisplatin exposure in NTERA2 and 2102EP. Interestingly, down-regulation of Sp1 increased Noxa expression in response to cisplatin. However, blockade of KLF6 decreased cisplatin-induced up-regulation of Noxa in EC cell lines. In addition, tissue microarray analyses of TGCTs revealed that expression of Noxa correlates with good clinical prognosis in patients with embryonal carcinoma. Thus, our data show the transcriptional network that regulates Noxa in EC cells, which is key for their apoptotic response to cisplatin-based chemotherapy, and propose Noxa as a predictive factor of therapeutic response.

Effects of curcumin on bleomycin-induced oxidative stress in malignant testicular germ cell tumors

Bleomycin is commonly used in the treatment of testicular cancer. Bleomycin generates oxygen radicals, induces the oxidative cleavage of DNA strands and induces cancer cell apoptosis. Curcumin (diferuloylmethane) is a potent antioxidant and chief component of the spice turmeric. No study investigating the effects of curcumin on intrinsic and bleomycin-induced oxidative stress in testicular germ cell tumors has been reported in the literature. For this reason, the present study aimed to examine the effects of curcumin on oxidative stress produced in wild-type NTera-2 and p53-mutant NCCIT testicular cancer cells incubated with bleomycin and the results were compared with cells treated with H2O2 which directly produces oxidative stress. The protein carbonyl content, thiobarbituric acid reactive substances (TBARS), glutathione (GSH), 8-isoprostane, lipid hydroperoxide (LPO) levels and total antioxidant capacity in the two testicular cancer cell lines were determined. Results showed that bleomycin and H2O2 significantly increased protein carbonyl, TBARS, 8-isoprostane and LPO levels in the NTera-2 and NCCIT cell lines. Bleomycin and H2O2 significantly decreased the antioxidant capacity and GSH levels in NTera-2 cells. Curcumin significantly decreased LPO, 8-isoprostane and protein carbonyl content, and TBARS levels increased in cells treated with bleomycin and H2O2. Curcumin enhanced GSH levels and the antioxidant capacity of NTera-2 cells. In conclusion, curcumin inhibits bleomycin and H2O2-induced oxidative stress in human testicular cancer cells.

Tumourigenic characteristics of embryonal carcinoma cells as a model for studying tumour progression of human embryonic stem cells

The objective of this study was to compare tumourigenic characteristics of human embryonic stem cells (HESCs) and embryonal carcinoma cells (ECCs) to identify a robust and simple model for studying certain aspects of cell transformation and tumourigenesis, in tumour progression of HESCs. SSEA-3 positive ECCs (NTERA-2) cells were identified and compared to HESCs (ch HES-20) in terms of pluripotency and differentiation capacity, growth characteristics, gene expression profiles and signalling pathways. Our results showed that NTERA-2 cells shared similarities in expression markers of pluripotency to ch HES-20 cells. However, NTERA-2 cells also expressed some markers of differentiation and had a tendency to differentiate towards ectodermal endpoints. We identified NTERA-2 cells with higher S-phase fraction in cell cycle distribution, anti-apoptosis markers and robust self-renewal ability, compared to ch HES-20 cells. Microarray analysis and real-time PCR results showed that some oncogenes were up-regulated and tumour-suppression genes were down-regulated, whereas pluripotency-related genes were up-regulated and differentiation-related genes were down-regulated, and that Wnt and Notch signalling pathways were activated during progression from ES cells to EC cells. Tumourigenic characteristics of ECCs may provide a valuable insight into possible tumour progression of HESCs.

Cisplatin resistance induced in germ cell tumour cells is due to reduced susceptibility towards cell death but not to altered DNA damage induction or repair

To identify factors involved in cisplatin (CDDP) resistance of germ cell tumours (GCTs), we exposed NTERA-2 cells, and the platinum-adapted subline NTERA-2R to CDDP and compared their response. While both cell lines showed comparable proliferation, NTERA-2R cells were clearly more resistant to the drug than the parental NTERA-2 cell line. Interestingly, the two lines showed identical extent of DNA adduct formation and elimination, indicating that neither changes in CDDP uptake, nor altered drug efflux, DNA binding, or repair caused the difference in resistance. Similarly, no difference occurred in the time-course of γH2AX formation, which was not linked to 53BP1 accumulation. In contrast, NTERA-2R cells showed a more pronounced dose-dependent S phase delay, a transient G2/M-block, and subsequent release into immediate cell death. We thus conclude that the enhanced resistance against CDDP is linked to reduced susceptibility to cell death rather than to an altered DNA adduct formation or adduct removal.

Stromal factors SDF1α, sFRP1, and VEGFD induce dopaminergic neuron differentiation of human pluripotent stem cells

Human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons hold potential for treating Parkinson's disease (PD) through cell replacement therapy. Generation of DA neurons from hESCs has been achieved by coculture with the stromal cell line PA6, a source of stromal cell-derived inducing activity (SDIA). However, the factors produced by stromal cells that result in SDIA are largely undefined. We previously reported that medium conditioned by PA6 cells can generate functional DA neurons from NTera2 human embryonal carcinoma stem cells. Here we show that PA6-conditioned medium can induce DA neuronal differentiation in both NTera2 cells and the hESC I6 cell line. To identify the factor(s) responsible for SDIA, we used large-scale microarray analysis of gene expression combined with mass spectrometric analysis of PA6-conditioned medium (CM). The candidate factors, hepatocyte growth factor (HGF), stromal cell-derived factor-1 α (SDF1α), secreted frizzled-related protein 1 (sFRP1), and vascular endothelial growth factor D (VEGFD) were identified, and their concentrations in PA6 CM were established by immunoaffinity capillary electrophoresis. Upon addition of SDF1α, sFRP1, and VEGFD to the culture medium, we observed an increase in the number of cells expressing tyrosine hydroxylase (a marker for DA neurons) and βIII-tubulin (a marker for immature neurons) in both the NTera2 and I6 cell lines. These results indicate that SDF1α, sFRP1, and VEGFD are major components of SDIA and suggest the potential use of these defined factors to elicit DA differentiation of pluripotent human stem cells for therapeutic intervention in PD.

Abstract 4910: Apoptotic effects of curcumin and bleomycin alone or in combination on malignant testicular germ cells

Abstract Aims: Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols in testicular cancer, but side effects are common. Bleomycin increases oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in several types of malignancies. However, no study has been carried out so far to elucidate its possible anticancer activity and interaction with Bleomycin in testicular cancer cells. Methods: In this study, we investigated the effects of Curcumin and Bleomycin alone or in combination on apoptosis-signalling-pathways. We determined the cytotoxic effect of Bleomycin and Curcumin on NTera-2 (NT2) cells using 3-(4,5) dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and measured apoptosis signalling pathway markers such as Caspase-3, Caspase-8, Caspase-9 activities and Bcl-2, Bax and Cyt-c levels in NT2 cells incubated with Bleomycin in the presence or absence of Curcumin. Results: Median lethal dose (LD50) of Bleomycin on NT2 cell viability as 400 μg/ml after an incubation of 24 h and LD50 of Curcumin as 20 μM. Treatment with Bleomycin (400 μg/ml) and Curcumin (20 μM) alone for 24 h caused 3.2-fold and 3-fold increase in caspase-3 activity, 3.4-fold and 2-fold increase in caspase-8 activity, 2.9-fold and 2.8 fold increase in caspase-9 activities respectively. These treatments alone also caused 2-fold increase in Bax and 2-fold increase in cytoplasmic Cyt-c levels while leading 2-fold decrease in Bcl-2 levels. Combination of Curcumin with Bleomycin induced Caspase-3,-8,-9 activities more than their separate effects in NT2 cells. Specifically combination of Curcumin (20 μM) with Bleomycin (400 μg/ml) induced 4.2-fold increase in Caspase-3, 4.3-fold increase in caspase-8 activity, 4-fold increase in caspase-9 activity, 3-fold increase in cytoplasmic Cyt-c levels and 3-fold decrease in Bcl-2 levels.Conclusion: Bleomycin has proven to be an essential component of the cisplatin-based chemotherapy regimens, used in the treatment of testicular cancer. However, the use of Bleomycin is limited because of Bleomycin-induced pneumonitis, a complication occurring in approximately 10% of patients treated with Bleomycin and fatal in approximately 10% of the cases. Our observations suggest that the effects of Bleomycin on apoptosis-signalling-pathways were improved by Curcumin. Co-administration of Bleomycin with Curcumin could be a new strategy to use less dosage of Bleomycin and prevent patients from Bleomycin-induced side effects. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4910. doi:1538-7445.AM2012-4910

Abstract 3325: The role of MyD88 in chemoresistance, differentiation and hypoxia in cancer stem cells

Abstract Ovarian cancer is the leading cause of gynaecological cancer death worldwide. This high mortality is caused in large part by development of chemoresistant recurrent disease. In primary tumours differentiation status of the tumour is considered a key prognostic factor however in recurrent disease this is no longer the case, suggesting that in recurrent tumours the differentiation status of the tumour has no bearing on the response to chemotherapy. The origins of recurrent disease may be explained by the cancer stem cell (CSC) theory. CSCs are a minority population of cancer cells with stem like properties including enhanced proliferation. The adaptor molecule myeloid differentiation-primary response gene (88) (MyD88), is a key constituent of the toll like receptor 4 (TLR4) pathway. In normal circumstances TLR pathways mediate the body's response to pathogens; however MyD88 has been recently suggested as a possible marker of cancer stemness in ovarian cancer. This work investigates the involvement of MyD88 in the CSC response to chemotherapy, differentiation stimulus and hypoxia treatment. Cell line experiments were carried out in two CSC lines: NTera2 and 2102Ep. NTera2 cells readily differentiate in response to retinoic acid (RA) treatment while 2102Ep cells are nullipotent and resist differentiation via RA. NTera2 cells are more chemosensitive than 2102Ep cells. Cells were incubated in the presence of both retinoic acid and a platinum based chemotherapy drug, both in isolation and in various combinations. RNA was isolated from treated cells, cDNA was synthesised and interrogated for TLR4/MyD88 expression via Q-PCR. To assess whether MyD88 was necessary and/or sufficient for the normal cell response, MyD88 was knocked down in cells via siRNA transfection, and overexpressed via insertion of an overexpression plasmid. The effect of these alterations on the cell lines response to chemotherapy, differentiation stimulus and hypoxia was then measured. The downstream effect of ablation of the MyD88 signal was also assessed using an Affymetrix array, and the chemokine and cytokine expression profile of both cell lines in all treatments was assessed using Quantibody arrays from RayBiotech. MyD88 expression was shown to be affected by chemotherapy, differentiation stimulus and hypoxic treatments. Furthermore when cells were pre-treated with differentiation stimulus or chemotherapy, their normal responses were altered. So far, our work has shown that ablation of the MyD88 signal in nullipotent 2102Ep cells removes their ability to resist differentiation via RA. To close this mechanism we have also shown that overexpression of MyD88 in pluripotent NTera2 cells allows them to resist differentiation via RA. Functional analysis of the role of MyD88 in chemotherapy and hypoxia resistance is currently being assessed. This and the mechanisms involved will be detailed, along with the chemokine/cytokine profile of treated cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3325. doi:1538-7445.AM2012-3325

Abstract 4187: A novel genome-wide assay identifies chromatin domains involved in stem cell behavior

Abstract The activity of DNA regulatory elements are often controlled by chromatin structure. Active regulatory elements are located within “open” or “accessible” chromatin. If a regulatory element is in a “closed” or “inaccessible” chromatin environment it is functionally silenced. We find that when permeabilized cells are treated with nuclease in situ, small DNA fragments, corresponding to accessible chromatin, can be readily isolated. These small DNA fragments, when analyzed by next-generation sequencing, provide a global map of active genomic regulatory domains. We used this technology to identify chromatin domains involved in stem cell behavior using NTera2 cells as a model system. By comparing differentiated and undifferentiated NTera2 cells we identified more than 500 genomic regions that are accessible only in undifferentiated cells. We then used these results, together with ENCODE data analyzing human embryonic stem cells and human cancer cells, to identify genomic regulatory regions that are active only in stem cells. Importantly, many of these regions are proximal to genes that are associated with cell “stemness” (NANOG and LIN28 are examples). In addition, several uncharacterized genes that have RNA expression characteristics similar to NANOG and LIN28 were identified. Interestingly, several stem-cell specific active regulatory domains were found adjacent to previously uncharacterized, stem-cell specific non-coding RNA transcript regions. These findings demonstrate that this approach can serve as a new discovery tool to identify chromatin regions that control stem cell behavior. In addition, chromatin regions associated with other biological or pathological states could also be identified using this approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4187. doi:1538-7445.AM2012-4187

Synergistic anticancer activity of curcumin and bleomycin: An in�vitro study using human malignant testicular germ cells

Testicular cancer is the most common cancer among young men of reproductive age. Bleomycin is a frequently used drug for the treatment of several malignancies and is part of the chemotherapy protocols used for testicular cancer; however, side-effects are common. Bleomycin causes an increase in oxidative stress which has been shown to induce apoptosis in cancer cells. Curcumin (diferuloylmethane), an active component of the spice turmeric, has been demonstrated to induce apoptosis in a number of malignancies. However, to date no study has been carried out to elucidate its anticancer activity and interaction with bleomycin in testicular cancer cells. In this study, we investigated and compared the effects of curcumin, bleomycin and hydrogen peroxide (H2O2) on apoptotic signaling pathways. Curcumin (20 µM), bleomycin (400 µg/ml) and H2O2 (400 µM) incubation for 24 h decreased the viability of NTera-2 cells, and increased caspase-3, -8 and -9 activities, Bax and cytoplasmic cytochrome c levels and decreased Bcl-2 levels. The concurrent use of curcumin with bleomycin induced caspase-3, -8 and -9 activities to a greater extent in NTera-2 cells than the use of each drug alone. Our observations suggest that the effects of curcumin and bleomycin on apoptotic signaling pathways are synergistic. Therefore, we propose to use curcumin together with bleomycin to decrease its therapeutic dose and, therefore, its side-effects.