TCam-2 but not JKT-1 cells resemble seminoma in cell tradition. propose a signaling cascade including and as mediators of the romidepsin effects in GCC cells. (GCNIS) [1C3]. GCNIS cells are the result of a defective germ cell development, where a primordial germ cell (PGC) is usually thought to suffer from genetic aberrations leading to a developmental arrest [2, 4]. GCNIS cells eventually differentiate into the invasive type II germ cell cancers (GCCs), which are subdivided into seminomas and non-seminomas . Seminomas are highly much like GCNIS and PGCs regarding gene expression and histology . Contrarily, the stem cell populace of the non-seminomas, the embryonal carcinoma (EC) shows features of totipotency and is therefore able to differentiate into all three germ layers (teratomas) and extraembryonic tissues (yolk-sac tumors, choriocarcinomas). Familial predisposition, environmental parameters like exposure towards fertilizers, fine dust, endocrine disruptors and hormones are discussed as risk factors for development of GCCs . Additionally, presence of the testicular dysgenesis syndrome (cryptorchidism, azoospermia and testicular atrophy) increases the risk for GCC development [6, 7]. Generally, GCCs are treated by orchiectomy and depending on stage with chemo- or radiotherapy in addition. Early stage seminomas are very radiosensitive. Thus, stage I – IIb seminomas are treated by radiotherapy, whereas non-seminomas are treated with chemotherapy. More advanced stages of seminoma or patients that do not tolerate radiotherapy also receive TNFSF10 chemotherapy. Although most GCCs are sensitive towards a cisplatin-based therapy, approximately 20 – 50% of patients with metastatic disease cannot be cured by standard chemotherapy due to resistance mechanisms . Thus, there is a strong need for new therapeutic options to treat cisplatin-resistant disease. In this study, we treated GCC lines with the histone deacetylase inhibitor (HDI) romidepsin (ISTODAX, FK228, “type”:”entrez-nucleotide”,”attrs”:”text”:”FR901228″,”term_id”:”525229482″,”term_text”:”FR901228″FR901228) to sophisticated around the molecular mechanism and to address the question whether it is a therapeutic option for GCCs. RESULTS We reported previously that treatment of seminoma-like TCam-2 cells with romidepsin rapidly induced apoptosis . Based on this initial finding, we asked if romidepsin might also be harmful to other GCC cell lines. Thus, in this study we analyzed its molecular mode of action and and elaborated around the potential of romidepsin as a new therapeutic for GCCs. We utilized GCC cell lines and corresponding cisplatin-resistant subclones. The cell collection TCam-2 was used as a proxy for any seminoma, while the three cell lines 2102EP, NCCIT and NT2/D1 were derived from ECs and the two cell NKY 80 lines JAR and JEG-3 resemble a choriocarcinoma in culture [10C14]. As controls we included human main fibroblasts (MPAF, ARZ, EMF) and the Sertoli cell collection (FS1) . Romidepsin efficiently kills GCC cells and tumor model. To mimic GCCs, we xenografted 2102EP, 2102EP-R, NCCIT and NCCIT-R cells into the flank of nude mice and allowed tumors to grow for two weeks (-R = cisplatin-resistant subclone). Afterwards, we applied romidepsin (2 mg/kg) intravenously three times a week and monitored tumor growth for 10 days. Lately after 7 days, tumor sizes were significantly reduced in romidepsin treated mice compared to the control mice (Physique ?(Figure2).2). We confirmed induction of apoptosis by detection of PARP cleavage in romidepsin treated mice bearing 2102EP-R and NCCIT-R tumors (Supplementary Physique S1B). In conclusion, romidepsin efficiently kills tumor cells by inducing apoptosis. Open in a separate window Physique 2 Measurement of the tumor burden during treatment of xenografted 2102EP(-R) and NCCIT(-R) cells with 2.5 mg/kg romidepsin or the solvent for 10 daysInlay: photos of tumors of solvent (left) and romidepsin (right) treated mice after 10 days. n. s. = not significant, p-value > 0.05; asterisk = significant, p-value < 0.05. GCC cells mainly utilize HDAC1 for histone deacetylation Next, we were interested in alterations of molecular mechanisms induced by romidepsin in GCCs. HDIs like Romidepsin inhibit histone deacetylases (HDACs). Re-analyzing an expression microarray of GCC tissues published in a previous study  and a qRT-PCR analysis of GCC cell lines revealed that is highly expressed in all GCCs, GCC cell lines, human fibroblasts (ARZ, MPAF) and the Sertoli cell collection (FS1) (Supplementary Physique S1C, S1D). All other analyzed showed a lower expression compared to in all analyzed GCC samples (Supplementary Physique S1C, S1D), indicating that GCCs might mainly utilize HDAC1 for histone deacetylation. Romidepsin causes hyperacetylation of histones H3 and H4 Since inhibition of HDACs should lead to histone hyperacetylation, we analyzed NKY 80 the pan-acetylation status NKY 80 of histones 3 and 4 (pan-H3ac / -H4ac) 2 - 16h after 10 nM romidepsin treatment of GCC cell lines, fibroblasts and the Sertoli cell collection FS1. As shown by western blotting, within 2 - 16h after treatment H3 and H4 became hyperacetylated in all samples analyzed (Supplementary Physique S2A, S2B). In parallel, an ELISA-based.