Search

There exists a dire need for improved therapeutics to achieve predictable bone regeneration. Gene therapy using non-viral vectors that are safe and efficient at transfecting target cells is a promising approach to overcoming the drawbacks of protein delivery of growth factors. Here, we investigated the transfection efficiency, cytotoxicity, osteogenic potential and in vivo bone regenerative capacity of chemically modified ribonucleic acid (cmRNA) (encoding BMP-2) complexed with polyethylenimine (PEI) and made comparisons with PEI complexed with conventional plasmid DNA (encoding BMP-2). The polyplexes were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficiency using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these polyplexes was validated by determining the expression of bone-specific genes, osteocalcin and alkaline phosphatase as well as through the detection of bone matrix deposition. Using a calvarial bone defect model in rats, it was shown that PEI-cmRNA (encoding BMP-2)-activated matrices promoted significantly enhanced bone regeneration compared to PEI-plasmid DNA (BMP-2)-activated matrices. Our proof of concept study suggests that scaffolds loaded with non-viral vectors harboring cmRNA encoding osteogenic proteins may be a powerful tool for stimulating bone regeneration with significant potential for clinical translation.

Background Treatment outcome of children with pediatric hepatocellular carcinoma (pHCC) is poor. Therefore, we evaluated the tyrosine kinase inhibitor sorafenib in a model of pHCC. Methods Cell viability after treatment with sorafenib was evaluated in HC-AFW1 cells (pHCC) using MTT assay and compared to an adult HCC (aHCC) and two hepatoblastoma (HB) cell lines. ERK, pERK, E-cadherin, and vimentin expression were investigated using Western Blot. Sorafenib (60 mg/kg) was administered orally to NOD.Cg-Prkdcscid-IL2rgtmWjl/Sz mice bearing subcutaneous HC-AFW1-derived tumors. Tumor progression, viability, and vascularization were monitored by tumor volume, AFP levels, and CD31 immunostaining, respectively. Sensitization to sorafenib was evaluated using the β-catenin inhibitor ICG001. Results Sorafenib reduced cell viability in HC-AFW1 (IC50: 8 µM), comparable to HB cells, however less pronounced in aHCC cells (IC50: 23 µM). Sorafenib inhibited ERK signaling in both, HC-AFW1 cells and -xenografts. In vivo, sorafenib treatment only led to a moderate tumor growth inhibition, although significant reduction of vascularization and tumor growth kinetics was observed. Long-term treatment with sorafenib decreased E-cadherin, but showed no induction of vimentin expression. Combining sorafenib with a β-catenin inhibitor led to an additional reduction of cell viability. Conclusion Sorafenib together with inhibitors of the β-catenin pathway might be an effective tool in the treatment of pediatric HCC.

Employing cost-effective biomaterials to deliver chemically modified ribonucleic acid (cmRNA) in a controlled manner addresses the high cost, safety concerns, and lower transfection efficiency that exist with protein and gene therapeutic approaches. By eliminating the need for nuclear entry, cmRNA therapeutics can potentially overcome the lower transfection efficiencies associated with non-viral gene delivery systems. Here, we investigated the osteogenic potential of cmRNA-encoding BMP-9, in comparison to cmRNA-encoding BMP-2. Polyethylenimine (PEI) was used as a vector to increase in vitro transfection efficacy. Complexes of PEI-cmRNA (encoding BMP-2 or BMP-9) were fabricated at an amine (N) to phosphate (P) ratio of 10 and characterized for transfection efficacy in vitro using human bone marrow stromal cells (BMSCs). The osteogenic potential of BMSCs treated with these complexes was determined by evaluating the expression of bone-specific genes as well as through the detection of bone matrix deposition. It was found that alkaline phosphatase (ALP) expression 3 days post transfection in the group treated with BMP-9-cmRNA was significantly higher than that in the group that received BMP-2-cmRNA treatment. Alizarin red staining and atomic absorption spectroscopy demonstrated enhanced osteogenic differentiation as evidenced by increased bone matrix production by the BMSCs treated with BMP-9-cmRNA when compared to cells treated with BMP-2-cmRNA. In vivo studies showed increased bone formation in calvarial defects treated with the BMP-9-cmRNA and BMP-2-cmRNA collagen scaffolds when compared to empty defects. The connectivity density of the regenerated bone was higher (2-fold-higher) in the group that received BMP-9-cmRNA compared to BMP-2-cmRNA. Together, these findings suggest that cmRNA-activated matrix encoding osteogenic molecules can provide a powerful strategy for bone regeneration with significant clinical translational potential.

Advanced stages of tumour and development of metastases are the two major problems in treating liver tumours such as hepatoblastoma (HB) and hepatocellular carcinoma (HCC), in paediatric patients. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We analysed the effect of apoptosis modulators, BH3 mimetics, on mechanisms of dissemination such as adhesion or migration of HB and HCC cells. BH3 mimetics such as ABT-737 and obatoclax can reduce cell migration in a scratch assay as well as adhesion of HB and HCC cells to matrigel. Immunofluorescence staining of F-actin demonstrated that development of lamellipodia, which are important for migration, decreased. BH3 mimetics increase the level of activated caspases 3 and 7 in HUH6 cells. This results in the degradation of GTPase Cdc42, which can be determined by western blot analysis. A pan-caspase inhibitor can block the migration and degradation of Rho-GTPase. In summary, our study showed that BH3 mimetics not only enhance drug sensitivity but also may prevent metastasis by inhibiting HB and HCC cell motility.

Genome editing using site-specific endonucleases (SSE) holds great potential to repair disease-causing genes. In this study we offer a promising approach to deliver SSEs as nuclease-encoded, chemically modified mRNA (nec-mRNA) together with a repair template to correct the lung disease Surfactant Protein-B (SP-B) deficiency while simultaneously reducing SSE-induced off-target effects. To assess the potential of mRNA-encoded nucleases to create site specific double-strand breaks (DSBs), we evaluated a panel of 15 transcription activator-like effector nucleases (TALENs) and 36 Zink-Finger nucleases (ZFNs) for non-homologous end-joining (NHEJ) and also tested for the integration of a promoter template by homology-directed repair (HDR) in vitro . In addition we compared different mRNA modifications to assess immunoreactions against nec-mRNA. Finally, we co-administered intratracheally nec-mRNA with the repair template to SP-B deficient mice and evaluated the survival and promoter integration efficacy. Compared to plasmid-DNA, delivery of mRNA-encoded nucleases resulted in more frequent induction of DSBs ( P P in vitro . While being only transiently expressed, incorporation of modified nucleosides into nec-mRNA and complexation into nanoparticles (NPs) increased mRNA expression levels ( P in vivo . Also, no immune activation was observed. Importantly, delivery of nec-mRNA-NP to SP-B deficient mice lead to prolonged survival rates compared with matched control groups ( P In conclusion, we have shown that delivery of nec-mRNA-NP together with a repair template results in successful, site-specific genome editing in vivo .

Duplex drugs are promising anticancer agents. After in vivo cleavage into active nucleoside analogues, they exert their anti-tumour activity with reduced toxicity and side effects. Here we evaluated the impact of two duplex drugs on the viability of hepatoblastoma (HB) cells lines and their toxicity against human fibroblasts.The duplex drugs 2'-deoxy-5-fluorouridylyl-(3'-5')- 3'-C-ethynylcytidine (5-FdU(3'-5')ECyd) and 3'-C-ethynylcytidinylyl-(5'→1-O)-2-O-octadecyl-sn-glycerylyl-(3'-Ο→5')-2'-deoxy-5-fluorouridine (ECyd-lipid-5-FdU) were analysed in two HB cell lines (HUH6, HepT1) and fibroblasts by MTT assay. The treatment potential was compared to the single substances 2'-deoxy-5-fluorourindine (5-FdU), 3'-C-ethynylycytidine (ECyd) and an equimolar mixture of both. Cell cycle analyses were performed using flow cytometry after 7-AAD staining.Both duplex drugs achieve a potent cytotoxic effect at low μM concentrations, which was more pronounced than the mixture of ECyd + 5-FdU. Further, both substances exert toxicity on fibroblasts of tumour samples, with less toxicity in foreskin fibroblasts cultures. Cell cycle analyses revealed a shift towards apoptotic cells for both drugs in HB cells.5-FdU(3'-5')ECyd and ECyd-lipid-5-FdU exert a highly potent anti-tumoural effect on HB cells and might therefore be a treatment option in HB. Pharmacological formulations of both duplex drugs have to be evaluated in vivo to reduce possible side effects.

The response of standard-risk hepatoblastoma (HB) to neoadjuvant cisplatin (CDDP) chemotherapy is excellent; however, in high-risk HB, drug resistance remains a major challenge. Alternative therapeutic strategies may consider combining cytotoxic drugs with apoptosis sensitizers as this has shown additive effects in various types of malignancies. Analysis of published expression databases have revealed an anti-apoptosis state in HB samples. Herein, we evaluated the synergistic effects of ABT-737 as a modulator of apoptosis in combination with CDDP in HB. To this end, clonogenic assays were performed with HepT1 and HUH6 HB cells to evaluate the synergistic effects of CDDP and ABT-737. Combination treatment with CDDP and ABT-737 reduced the clonogenicity of HB cells more than 5-fold compared to treatment with CDDP alone. Furthermore, the HUH6 mixed-type HB cells showed higher sensitivity to CDDP and combination treatment compared to the HepT1 embryonal-type cells. Subcutaneous HUH6 tumors in NOD/LtSz-scid IL2Rγnull mice were treated with CDDP (1.25 and 3 mg/kg body weight, n=6), ABT-737 (100 mg/kg, n=5) and the combination of both agents (n=5). Combined treatment led to a significantly reduced tumor growth compared to CDDP treatment alone (p

Background Chemoresistance and advanced tumour stage at time of diagnosis are the major reasons for poor treatment results in hepatoblastoma (HB) and paediatric hepatocellular carcinoma (HCC). Positive results with transplantation of liver and bone marrow revealed the impact of the immune system on the treatment of liver malignancies. Aim Cytotoxic-immune-cells-like natural killer (NK) and T cells are major player in the defence against developing tumours. This study aimed to specifically analyse the ability of ex-vivo expanded γδ T cells to recognise and lyse HB and HCC cell lines in coculture assays. Methods Cell viability after treatment with γδ T cells was evaluated with two HB (HUH6 and HepT1) and one HCC cell line (HC-AFW1) using a MTT-based cytotoxicity assay. The binding of T cells to target cells was monitored using immunofluorescence microscopy. Results Incubation of hepatic tumour cell lines with γδ T cells led to a significant decrease in tumour cell viability. This was enhanced by zoledronic acid and histone deacetylase inhibitors. MT110, an EpCAM/CD3-bispecific BiTE antibody could bluntly enhance tumour cell lysis close to completion. γδ T cells efficiently interacted with HB and HCC cells in a spheroid culture model. Conclusion Bispecific antibodies such as MT110 might be used to intensify the antitumoural effect of γδ T cells in context of adoptive immune cell transfer. Optimised immunotherapeutic strategies might therefore improve the outcome of high risk hepatoblastoma and hepatocellular carcinoma.

Zink-Finger- bzw. transcription activator-like effector-Nukleasen (ZFNs/TALENs) sind vielversprechende Werkzeuge im Zuge der Reparatur genomischer Mutationen, indem diese den Mechanismus der Homologen Rekombination begünstigen. In Studien zur Hämophilie konnte bereits eine Nuklease-vermittelte Genkorrektur unter Verwendung von AAV-Vektoren demonstriert werden. Dabei kodierten diese sowohl für die Doppelstrangbruch- (DSB) induzierenden ZFN-Nukleasen, als auch den Einbau des Reparatur-Templates durch den Reparaturmechanismus der Homologen Rekombination (HDR, engl. homology-directed repair). Während der Einsatz von stabil exprimierenden AAV-Vektoren zwar als sicher angesehen werden kann, ist eine transiente Expression von Nukleasen ausreichend, um genomische Modifikationen zu erzeugen. Demzufolge wäre die zeitlich begrenzte Expression von ZFNs ein entscheidender Vorteil, um potentielle Nuklease-bedingte Nebeneffekte zu reduzieren. In den letzten Jahren hat sich als Alternative zu traditionellen, viralen gentherapeutischen Vektoren, modifizierte messenger RNA (mRNA) als vielversprechend herausgestellt. Dessen Vorteile liegen sowohl in einer transienten Proteinexpression, bei gleichzeitiger Vermeidung genomischer Integration, als auch in dessen chemischen Modifizierungen, die eine stabile Expression und das Ausbleiben von Immunantworten begünstigen. Eine solche Co-Transfektion mRNA-kodierender Nukleasen in einem gentherapeutischen Kontext macht mRNA daher zu einem idealen Vehikel. In dieser Studie wurden solche optimierten mRNA-kodierenden Nukleasen verwendet, um die erfolgreiche Genkorrektur am murinen Modell der SP-B Defizienz zu demonstrieren. Für die Genkorrektur des SP-B Lokus wurde zunächst ein Panel aus in silico generierten ZFNs und TALENs hergestellt. Der dual-luciferase single-strand Assay sowie der T7-Endonuklease I -Assay verifizierten für TALEN-1 (T1) und ZFN-3 (Z3) eine hohe Schnitt-Effizienz ex vivo, wodurch bis zu 39 % der Allele durch das nicht-homologe end-joining (NHEJ) repariert wurden. Im Gegensatz zu Plasmid-DNA (pDNA) stieg dabei die Induktion an DSBs signifikant an (P < 0,05). Die Kombination mit einem Reparatur-Template, welches eine NheI-Restriktionsschnittstelle in den SP-B Lokus einfügte, konnte ebenfalls ein signifikanter Anstieg in der HDR für mRNA verzeichnet werden (P < 0,05). Da Z3 effizienter in der DSB-Induktion sowie der HDR war, wurde diese für alle folgenden Experimente verwendet. Zur Optimierung der Z3-Expression in vivo wurden zwei Modifikationen (Ψ(1.0)/m5C(1.0) und s2U(0.25)/m5C(0.25)) mit und ohne Komplexierung in mit Chitosan-beschichteten poly(D,L-Laktid-Co-Glycolid) Nanopartikeln (NPs) getestet. Dabei ergaben sich für markierte s2U(0.25)/m5C(0.25) Z3 mRNA (im Weiteren als Z3 nec-mRNA bezeichnet) mit NPs die höchsten Expressionslevel- nach intratrachealer (i.t.) Applikation in die Lunge. Über einen IFN-α ELISA wurde zusätzlich ein signifikanter immunstimulatorischer Effekt der Z3 nec-mRNA ausgeschlossen. Die sequenzspezifische HDR wurde anschließend ex vivo über die Co-Applikation eines CAG-Promoter enthaltenden AAV- Donor-Templates (AAV6-Donor) zusammen mit AAV-kodierender Z3 (Z3 AAV) oder Z3 nec-mRNA erfolgreich getestet. Diese gentherapeutische Manipulation zeigte sich ebenfalls in Gen-korrigierten, transgenen SP-B Mäusen mit signifikant höheren Überlebensraten als die der Mock-behandelten Gruppen (P < 0,001). Korrigierte SP-B Mäuse zeigten dabei eine normale Lungenfunktion im Vergleich zu Positivkontrollen. Ebenfalls wurde im Vergleich zu PBS-behandelten Mäusen kein signifikanter Anstieg in der IL-12 Produktion beobachtet. Sequenzspezifische DSB-Induktion sowie HDR bestätigten die Ausprägung eines normalen Phänotyps, wobei zwischen Z3 AAV und Z3 nec-mRNA keine Unterschiede zu erkennen waren. Die immunhistochemischen Analysen bestätigten zusätzlich die zeitlich limitierte Expression der Z3 nec-mRNA im Vergleich zu Z3 AAV. Die hier erhaltenen Ergebnisse bestätigen eindrucksvoll, dass die Applikation von nec-mRNA in Kombination eines AAV-Donors zur erfolgreichen Genkorrektur des transgenen SP-B Lokus führt und somit die konstitutive SP-B Expression sowie eine normale Lungenfunktion nach Absetzen von Doxycyclin erlaubt. Verglichen mit AAV, zeigte Z3 nec-mRNA eine zeitlich limitierte Nuklease-Expression, wodurch mögliche Nebeneffekte im Gegensatz zu dauerhaft aktiven AAV-Nukleasen reduziert werden können. Zusätzlich macht die Abwesenheit einer Immunantwort nec-mRNA zu einem idealen Vehikel für mehrmalige Applikationen hinsichtlich einer dauerhaften Genkorrektur der Lunge. Zusammengefasst besitzt nec-mRNA damit ein enormes Potential im Zuge klinisch relevanter, gentherapeutischer Strategien für die Behandlung erblich-bedingter Lungenerkrankungen sowie weiterer monogenetischer Defekte.

Background:Sorafenib has recently been shown to reduce tumour growth in hepatoblastoma (HB) xenografts. The effect of a combined administration with cytostatic agents was now investigated.Methods:Cell viability after treatment with sorafenib and different cytostatic agents was evaluated in two HB cell lines (HUH6 and HepT1) using MTT assay. ERK signalling was investigated by western blot, NOXA expression by rt-PCR, and formation of DNA adducts using immunocytology. NMRI mice bearing subcutaneous HUH6-derived tumours were treated with sorafenib alone or in combination with cisplatin. Tumour progression, viability, apoptosis, and vascularisation were monitored by tumour volume, AFP levels, TUNEL assay, and CD31 immunostaining, respectively.Results:The combination of sorafenib and cisplatin led to a remarkable decrease in cell viability. The cisplatin-induced enhanced ERK1/2 activation, but not NOXA expression and the formation of DNA adducts was partly abrogated by sorafenib. In HB xenografts, both, sorafenib and alternated application of sorafenib and cisplatin significantly reduced tumour growth (P

Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors.Viability was monitored in HB cells (HUH6, HepT1) in monolayer and spheroid cultures treated with kinase inhibitors VX-680, Wee1-InhibitorII, and SU11274 alone or in combination with cisplatin (CDDP) using MTT assays. Apoptosis was revealed by Caspase-3 assay. Western blot and immunohistochemical analyses were performed to determine histone H3 phosphorylation.Among the kinase inhibitors strongest anti-proliferative effect on HB cells was documented for VX-680. HUH6 cells responded more sensitively to the Aurora kinase inhibitor as HepT1 cells (IC(50) 8 and 16.6 μM, respectively). While VX-680 and CDDP showed no additive effects, the combination of VX-680 and histone deacetylase inhibitor SAHA had a synergistic effect on the proliferation of HUH6 cells. The inhibition with VX-680 led to reduced histone H3 phosphorylation, to an increase of apoptotic cells, and to morphological changes such as vacuolization and swelling of the cells and nuclei.The data provide evidence that VX-680 might improve treatment results in HB with increased Aurora kinase activity by inhibiting cell proliferation and induction of apoptosis.

Background Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients Aims This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours. Methods Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence. Results Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P

Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.

OBJECTIVES: The clinical value of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of pancreatic lesions is uncertain in patients with normal parenchyma and chronic pancreatitis. The aim of this study was to analyze the diagnostic yield and influence of EUS-FNA on the clinical management of patients with pancreatic lesions, in the presence (CP) or absence (NP) of chronic pancreatitis. METHODS: A total of 207 consecutive patients with NP (n = 133) and CP (n = 74) were examined using linear array echo endoscopes for the procedure and 22-gauge needles. RESULTS: Adequate specimens were obtained from 200 lesions. A correct final diagnosis was established at histology (n = 108), bacteriology (n = 9), and clinical follow-up (n = 83). Cytology gave 17 false-negative EUS-FNA results (overall sensitivity: 85%). In patients with NP, 60 solid adenocarcinomas were detected, 32 other malignancies, and 38 benign lesions, with 11 false-negative results (sensitivity: 89%). In patients with CP, only seven of 13 malignancies (all solid adenocarcinomas) were identified using FNA (sensitivity: 54%). Overall, malignancy was identified in 116 patients, 32 of whom (27%) had lesions other than primary solid adenocarcinomas. Management was altered in 25 of these patients, which changed the surgical approach in 21%. EUS-FNA influenced the therapeutic approach in 44% of the total patient group. CONCLUSIONS: EUS-FNA was especially useful in patients with a focal pancreatic lesion with normal parenchyma. Its sensitivity in patients with CP was unacceptably low, and resection of the tumor using standard surgical techniques was still usually required to confirm the correct diagnosis. Diagnostic EUS-FNA influenced clinical management in nearly half of patients.

Background: Hepatoblastoma (HB) represents the most common malignant liver tumour in childhood. For patients with high risk or recurrent HB treatment results remain still poor due to a developing multidrug resistance during chemotherapy. Therefore new therapy strategies have to be evaluated. The primary objective was to investigate the combined therapy of cytotoxic agents with sorafenib. Sorafenib has been shown to reduce tumour progression and angiogenesis. In order to investigate a treatment close to a clinical setting the effect of the combined administration of cytotoxic agents with sorafenib on HB cell lines and xenotransplanted HB tumours was determined. Methods: Cell viability of two HB cell lines (HUH6 and HepT1) was evaluated in using MTT assays after treatment with sorafenib and different cytostatic agents (cisplatin (CDDP), doxorubicin, irinotecan, topotecan). ERK signalling was investigated by Western blot analysis. NMRI mice (nu/nu) bearing subcutaneous HUH6 derived tumours were treated with Sorafenib orally (30 mg/kg, on days 4-13 and 17-24) alone or in combination with CDDP i.p. (3 mg/kg, on days 1-3 and 14-16). Tumor progression and viability were monitored by tumour volume and AFP levels. Apoptosis was assessed by TUNEL assay. Angiogenesis was determined by CD31 staining and mean vascular density (MVD) was calculated. Results: In vitro the combination of sorafenib with CDDP showed a remarkable decrease in cell viability compared to the other agents tested. However with increasing concentrations of CDDP this additive effect disappeared. CDDP alone showed an enhanced phosphorylation of ERK1/2, which is a common pathway for apoptosis induction through NOXA. CDDP in combination with sorafenib led to an overall reduced phosphorylation level of ERK1/2, which also resulted in a low HB cell viability. Since CDDP and sorafenib have an opposing effect on phosphorylation of ERK1/2, we avoid the concomitant administration of both drugs in vivo. In HB xenografts both sorafenib alone or in alternating combination with CDDP reduced tumour growth compared to the control untreated group (p < 0.05). The combination treatment showed a higher and earlier reduction of tumor volume than sorafenib alone. In addition AFP levels did not significantly change in the treatment groups. Apoptotic areas as determined by TUNEL analysis increased significantly after administration of sorafenib or sorafenib and CDDP (p < 0.08). Furthermore CD31 staining showed a significant inhibition of angiogenesis as revealed by determination of the MVD in both treatment groups (p < 0.02). Discussion: Taken together the combination therapy of Sorafenib and CDDP led to an enhanced decrease in cell viability as well as in tumour progression and angiogenesis than sorafenib alone. Therefore a combined therapy might be a promising option in the treatment of high risk or recurrent hepatoblastoma. 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 4337. doi:1538-7445.AM2012-4337