Search Results (12)

Background: Most patients with testicular germ cell tumors (GCTs) are treated with cisplatin (CP)-based chemotherapy. However, some of them may develop CP resistance and therefore represent a clinical challenge. Cyclin-dependent kinase 5 (CDK5) is involved in chemotherapy resistance in different types of cancer. Here, we investigated the possible role of CDK5 and other CDKs targeted by dinaciclib in nonseminoma cell models (both CP-sensitive and CP-resistant), evaluating the potential of the CDK inhibitor dinaciclib as a single/combined agent for the treatment of advanced/metastatic testicular cancer (TC). Methods: The effects of dinaciclib and CP on sensitive and resistant NT2/D1 and NCCIT cell viability and proliferation were evaluated using MTT assays and direct count methods. Flow cytometry cell-cycle analysis was performed. The protein expression was assessed via Western blotting. The in vivo experiments were conducted in zebrafish embryos xenografted with TC cells. Results: Among all the CDKs analyzed, CDK5 protein expression was significantly higher in CP-resistant models. Dinaciclib reduced the cell viability and proliferation in each cell model, inducing changes in cell-cycle distribution. In drug combination experiments, dinaciclib enhances the CP effect both in vitro and in the zebrafish model. Conclusions: Dinaciclib, when combined with CP, could be useful for improving nonseminoma TC response to CP. Full article

Type II testicular germ cell tumors (TGCT) are the most frequently diagnosed solid malignancy in young men. Up to 15% of patients with metastatic non-seminomas show cisplatin resistance and a very poor survival rate due to lacking treatment options. Transcriptional cyclin-dependent kinases (CDK) have been shown to be effective targets in the treatment of different types of cancer. Here, we investigated the effects of the CDK inhibitors dinaciclib, flavopiridol, YKL-5-124, THZ1, NVP2, SY0351 and THZ531. An XTT viability assay revealed a strong cytotoxic impact of CDK7/12/13 inhibitor SY0351 and CDK9 inhibitor NVP2 on the TGCT wild-type cell lines (2102EP, NCCIT, TCam2) and the cisplatin-resistant cell lines (2102EP-R, NCCIT-R). The CDK7 inhibitor YKL-5-124 showed a strong impact on 2102EP, 2102EP-R, NCCIT and NCCIT-R cell lines, leaving the MPAF control cell line mostly unaffected. FACS-based analysis revealed mild effects on the cell cycle of 2102EP and TCam2 cells after SY0351, YKL-5-124 or NVP2 treatment. Molecular analysis showed a cell-line-specific response for SY0351 and NVP2 inhibition while YKL-5-124 induced similar molecular changes in 2102EP, TCam2 and MPAF cells. Thus, after TGCT subtype determination, CDK inhibitors might be a potential alternative for optimized and individualized therapy independent of chemotherapy sensitivity. Full article

Testicular cancer (TC) is a rare malignancy worldwide and is the most common malignancy in males aged 15–44 years. The Wnt/β-catenin signaling pathway mediates numerous essential cellular functions and has potentially important effects on tumorigenesis and cancer progression. The search for drugs to inhibit this pathway has identified a small molecule, PNU-74654, as an inhibitor of the β-catenin/TCF4 interaction. We evaluated the therapeutic role of PNU-74654 in two TC cell lines, NCCIT and NTERA2, by measuring cell viability, cell cycle transition and cell death. Potential pathways were evaluated by protein arrays and Western blots. PNU-74654 decreased cell viability and induced apoptosis of TC cells, with significant increases in the sub G1, Hoechst-stained, Annexin V-PI-positive rates. PNU-74654 treatment of both TC cell lines inhibited the TNFR1/IKB alpha/p65 pathway and the execution phase of apoptosis. Our findings demonstrate that PNU-74654 can induce apoptosis in TC cells through mechanisms involving the execution phase of apoptosis and inhibition of TNFR1/IKB alpha/p65 signaling. Therefore, small molecules such as PNU-74654 may identify potential new treatment strategies for TC. Full article

Germ cell tumors (GCTs) are considered to be highly curable; however, there are major differences in the outcomes related to histology and anatomical localization. GCTs originating from the testis are, overall, sensitive to platinum-based chemotherapy, whereas GCTs originating from the mediastinum show a worse response, which remains largely unexplained. Here, we address the differences among GCTs from two different anatomical locations (testicular versus mediastinal/extragonadal), with a specific focus on the role of the P53 pathway. It was recently shown that GCTs with TP53 mutations most often localize to the mediastinum. To elucidate the underlying mechanism, TP53 knock-out lines were generated in cisplatin-sensitive and -resistant clones of the representative 2102Ep cell line (wild-type TP53 testicular GCT) and NCCIT cell line (hemizygously mutated TP53, mutant TP53 mediastinal GCT). The full knock-out of TP53 in 2102Ep and resistant NCCIT resulted in an increase in cisplatin resistance, suggesting a contributing role for P53, even in NCCIT, in which P53 had been reported to be non-functional. In conclusion, these results suggest that TP53 mutations contribute to the cisplatin-resistant phenotype of mediastinal GCTs and, therefore, are a potential candidate for targeted treatment. This knowledge provides a novel model system to elucidate the underlying mechanism of clinical behavior and possible alternative treatment of the TP53 mutant and mediastinal GCTs. Full article

Embryonic cancer stem cells (CSCs) can differentiate into any cancer type. Targeting CSC using natural compounds is a good approach as it suppresses cancer recurrence with fewer adverse effects, and methylsulfonylmethane (MSM) is a sulfur-containing compound with well-known anticancer activities. This study determined the mechanistic aspects of the anticancer activity of MSM. We used Western blotting and real-time qPCR for molecular signaling studies and conducted flow cytometry for analyzing the processes in cells. Our results suggested an inhibition in the expression of CSC markers and Wnt/β-catenin signaling. MSM induced TRAIL-mediated extrinsic apoptosis in NCCIT and NTERA-2 cells rather than an intrinsic pathway. Inhibition of iron metabolism-dependent reactive oxygen species (ROS) generation takes part in TRAIL-mediated apoptosis induction by MSM. Suppressing iron metabolism by MSM also regulated p38/p53/ERK signaling and microRNA expressions, such as upregulating miR-130a and downregulating miR-221 and miR-222, which resulted in TRAIL induction and thereby extrinsic pathway of apoptosis. Hence, MSM could be a good candidate for neoadjuvant therapy by targeting CSCs by inhibiting iron metabolism. Full article

The majority of patients with testicular germ cell tumors (GCTs) can be cured with cisplatin-based chemotherapy. However, for a subset of patients present with cisplatin-refractory disease, which confers a poor prognosis, the treatment options are limited. Novel therapies are therefore urgently needed to improve outcomes in this challenging patient population. It has previously been shown that Wnt/β-catenin signaling is active in GCTs suggesting that its inhibitors LGK974 and PRI-724 may show promise in the management of cisplatin-refractory GCTs. We herein investigated whether LGK-974 and PRI-724 provide a treatment effect in cisplatin-resistant GCT cell lines. Taking a genoproteomic approach and utilizing xenograft models we found the increased level of β-catenin in 2 of 4 cisplatin-resistant (CisR) cell lines (TCam-2 CisR and NCCIT CisR) and the decreased level of β-catenin and cyclin D1 in cisplatin-resistant NTERA-2 CisR cell line. While the effect of treatment with LGK974 was limited or none, the NTERA-2 CisR exhibited the increased sensitivity to PRI-724 in comparison with parental cell line. Furthermore, the pro-apoptotic effect of PRI-724 was documented in all cell lines. Our data strongly suggests that a Wnt/β-catenin signaling is altered in cisplatin-resistant GCT cell lines and the inhibition with PRI-724 is effective in NTERA-2 CisR cells. Further evaluation of Wnt/β-catenin pathway inhibition in GCTs is therefore warranted. Full article

Less toxic treatment strategies for testicular germ cell tumor (TGCT) patients are needed, as overtreatment is a concern due to the long-term side effects of platin-based chemotherapy. Although clinical benefit from classical hypomethylating agents has to date been limited, TGCTs show an abnormal DNA methylome indicating the potential of treating TGCTs with hypomethylating drugs. We tested, for the first time in TGCT cell lines, a new synthetic flavonoid compound (MLo1302) from the 3-nitroflavanone family of DNA methyltransferase (DNMT) inhibitors. We show that MLo1302 reduces cell viability (including of cisplatin resistant cell line NCCIT-R), with IC₅₀s (inhibitory concentration 50) within the nanomolar range for NCCIT and NTERA-2 cells, and proved its cytotoxic effect. Exposure to MLo1302 reduced DNMT protein expression, similar to decitabine, and showed a partial effect in cell differentiation, reducing protein expression of pluripotency markers. RT² profiler expression array indicated several dysregulated targets, related to activation of apoptosis, differentiation, and cell cycle arrest. We validated these data by showing increased apoptosis, increased protein expression of cleaved caspase 8 and activated caspase 2, and reduced proliferation (BrdU assay), with increase in CDKN1A and decrease in MIB-1 expression. Therefore, synthetic drugs designed to target DNA methylation in cells may uncover effective treatments for TGCT patients. Full article

Novel treatment options are needed for testicular germ cell tumor (TGCT) patients, particularly important for those showing or developing cisplatin resistance, the major cause of cancer-related deaths. As TGCTs pathobiology is highly related to epigenetic (de)regulation, epidrugs are potentially effective therapies. Hence, we sought to explore, for the first time, the effect of the two most recently FDA-approved HDAC inhibitors (HDACis), belinostat and panobinostat, in (T)GCT cell lines including those resistant to cisplatin. In silico results were validated in 261 patient samples and differential expression of HDACs was also observed across cell lines. Belinostat and panobinostat reduced cell viability in both cisplatin-sensitive cells (NCCIT-P, 2102Ep-P, and NT2-P) and, importantly, also in matched cisplatin-resistant subclones (NCCIT-R, 2102Ep-R, and NT2-R), with IC50s in the low nanomolar range for all cell lines. Treatment of NCCIT-R with both drugs increased acetylation, induced cell cycle arrest, reduced proliferation, decreased Ki67 index, and increased p21, while increasing cell death by apoptosis, with upregulation of cleaved caspase 3. These findings support the effectiveness of HDACis for treating TGCT patients in general, including those developing cisplatin resistance. Future studies should explore them as single or combination agents. Full article

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. Full article

The emergence of cisplatin (CDDP) resistance is the main cause of treatment failure and death in patients with testicular germ cell tumors (TGCT), but its biologic background is poorly understood. To study the molecular basis of CDDP resistance in TGCT we prepared and sequenced CDDP-exposed TGCT cell lines as well as 31 primary patients’ samples. Long-term exposure to CDDP increased the CDDP resistance 10 times in the NCCIT cell line, while no major resistance was achieved in Tera-2. Development of CDDP resistance was accompanied by changes in the cell cycle (increase in G1 and decrease in S-fraction), increased number of acquired mutations, of which 3 were present within ATRX gene, as well as changes in gene expression pattern. Copy number variation analysis showed, apart from obligatory gain of 12p, several other large-scale gains (chr 1, 17, 20, 21) and losses (chr X), with additional more CNVs found in CDDP-resistant cells (e.g., further losses on chr 1, 4, 18, and gain on chr 8). In the patients’ samples, those who developed CDDP resistance and died of TGCT (2/31) showed high numbers of acquired aberrations, both SNPs and CNVs, and harbored mutations in genes potentially relevant to TGCT development (e.g., TRERF1, TFAP2C in one patient, MAP2K1 and NSD1 in another one). Among all primary tumor samples, the most commonly mutated gene was NSD1, affected in 9/31 patients. This gene encoding histone methyl transferase was also downregulated and identified among the 50 most differentially expressed genes in CDDP-resistant NCCIT cell line. Interestingly, 2/31 TGCT patients harbored mutations in the ATRX gene encoding a chromatin modifier that has been shown to have a critical function in sexual differentiation. Our research newly highlights its probable involvement also in testicular tumors. Both findings support the emerging role of altered epigenetic gene regulation in TGCT and CDDP resistance development. Full article

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. Full article

Despite high cure rates, about 20% of patients with advanced germ cell tumors (GCTs) fail cisplatin-based chemotherapy. High levels of DNA methylation have been identified in GCTs and linked to cisplatin resistance. Here, we examined the effects of DNA hypomethylating 5-azacitidine (5-aza) on two embryonal carcinoma cell lines (NCCIT, 2102Ep) and their cisplatin-resistant isogenic derivatives. Effects on cell viability and cisplatin sensitivity were assessed by the trypan blue exclusion method. Western blotting was used to examine induction of apoptosis 5-aza and results were validated by flow cytometry. Single agent treatment with 5-aza strongly impacted viability and induced apoptosis at low nanomolar concentrations, both in cisplatin-sensitive and -resistant cell lines. 5-aza exerted an immediate apoptotic response, followed by a prolonged inhibitory effect on cell viability and cell-cycle progression. Sequential treatment with 5-aza and cisplatin reduced cellular survival of the cisplatin-resistant sublines already at nanomolar concentrations, suggesting a partial restoration of cisplatin sensitivity by the compound. 5-aza demonstrated anti-tumor activity as a single agent at low nanomolar concentrations in GCT cells, irrespective of cisplatin-sensitivity. 5-aza may also have the potential at least to partially restore cisplatin-sensitivity in non-seminoma cells, supporting the hypothesis that combining DNA demethylating agents with cisplatin-based chemotherapy may be a valid therapeutic approach in patients with refractory GCTs. Full article