ABSTRACT
BACKGROUND: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of ß-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). METHODS: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. RESULTS: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. CONCLUSIONS: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.
Subject(s)
Asparaginase/pharmacology , Asparaginase/therapeutic use , Carcinoma, Hepatocellular/drug therapy , Glutamate-Ammonia Ligase/antagonists & inhibitors , Glutamine , Liver Neoplasms/drug therapy , Tumor Burden/drug effects , beta Catenin/genetics , Animals , Antineoplastic Agents/therapeutic use , Asparagine/blood , Cadherins/analysis , Carcinoma, Hepatocellular/enzymology , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Enzyme Inhibitors/therapeutic use , Glutamate-Ammonia Ligase/genetics , Glutamate-Ammonia Ligase/metabolism , Glutamine/analysis , Glutamine/blood , Hep G2 Cells , Humans , Ki-67 Antigen/analysis , Liver Neoplasms/enzymology , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Male , Methionine Sulfoximine/therapeutic use , Mice , Mice, Nude , Mutation , Xenograft Model Antitumor Assays , beta Catenin/analysisABSTRACT
PURPOSE: The proto-oncogene beta-catenin is linked to an abnormal activation of the Wnt/beta-catenin-pathway and shows mutations in 50-90 % of hepatoblastoma (HB). Corresponding, the recently published murine orthotopic HB model differs from the former subcutaneous model by nuclear beta-catenin distribution. As the nuclear localization of beta-catenin is considered to reflect a more aggressive tumor growth, the influence of beta-catenin inhibition on cell viability and drug-efficiency in HB cells was analyzed. METHODS: Beta-catenin distribution in HB cells was analyzed by immunofluorescence. The influence of beta-catenin inhibitors Celecoxib, Etodolac, ICG001, and MET kinase inhibitor (SU11274) alone and in combination with cisplatin (CDDP) on HB cell lines (HuH6, HepT1) was evaluated by cell viability assays and BrdU incorporation. RESULTS: Celecoxib and ICG001 reduced dose-dependently HB cell viability and decreased nuclear beta-catenin in cultivated HB cells. Etodolac was without influence at concentrations up to 100 µM. Combinations of Celecoxib or ICG001 with MET kinase inhibitor or CDDP resulted in additive reduction of cell viability. CONCLUSION: Pharmaceutical beta-catenin inhibitors can modulate the nuclear localization of beta-catenin and reduce cell viability of HB cells in vitro. These promising effects might optimize the outcome of high-risk HB. The orthotopic HB model is a suitable basis for further in vivo studies.
Subject(s)
Antineoplastic Agents/pharmacology , Hepatoblastoma/drug therapy , Liver Neoplasms/drug therapy , beta Catenin/antagonists & inhibitors , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Celecoxib , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Cisplatin/pharmacology , Etodolac/pharmacology , Hepatoblastoma/metabolism , Hepatoblastoma/pathology , Humans , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Mas , Pyrazoles/pharmacology , Pyrimidinones/pharmacology , Sulfonamides/pharmacology , Tissue Distribution , Tumor Cells, Cultured , beta Catenin/metabolismABSTRACT
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<0.05). Levels of AFP were lower in both treated groups (P=0.08). Relative apoptotic areas were increased (P=0.003). Mean vascular density was the lowest in the sorafenib/CDDP group (P=0.02). CONCLUSION: The combination of sorafenib with cisplatin might be a promising treatment option for high risk or recurrent HB.
