RESUMO
Gene therapy has always been a promising therapeutic approach for Cystic Fibrosis (CF). However, numerous trials using DNA or viral vectors encoding the correct protein resulted in a general low efficacy. In the last years, chemically modified messenger RNA (cmRNA) has been proven to be a highly potent, pulmonary drug. Consequently, we first explored the expression, function and immunogenicity of human (h)CFTR encoded by cmRNAhCFTR in vitro and ex vivo, quantified the expression by flow cytometry, determined its function using a YFP based assay and checked the immune response in human whole blood. Similarly, we examined the function of cmRNAhCFTR in vivo after intratracheal (i.t.) or intravenous (i.v.) injection of the assembled cmRNAhCFTR together with Chitosan-coated PLGA (poly-D, L-lactide-co-glycolide 75:25 (Resomer RG 752 H)) nanoparticles (NPs) by FlexiVent. The amount of expression of human hCFTR encoded by cmRNAhCFTR was quantified by hCFTR ELISA, and cmRNAhCFTR values were assessed by RT-qPCR. Thereby, we observed a significant improvement of lung function, especially in regards to FEV0.1, suggesting NP-cmRNAhCFTR as promising therapeutic option for CF patients independent of their CFTR genotype.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Fibrose Cística/terapia , Terapia Genética/métodos , Pulmão/fisiopatologia , Animais , Linhagem Celular , Fibrose Cística/genética , Modelos Animais de Doenças , Humanos , Fluxo Expiratório Máximo/genética , Camundongos , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Cystic Fibrosis (CF) is the most common monogenic disease among people of Western European descent and caused by mutations in the CFTR gene. However, the disease severity is immensely variable even among patients with similar CFTR mutations due to the possible effect of 'modifier genes'. To identify genetic modifiers, we applied RNA-seq based transcriptomic analyses in CF patients with a mild and severe lung phenotype. Global gene expression and enrichment analyses revealed that genes of the type I interferon response and ribosomal stalk proteins are potential modifiers of CF related lung dysfunction. The results provide a new set of CF modifier genes with possible implications as new therapeutic targets for the treatment of CF.
Assuntos
Fibrose Cística/genética , Genótipo , Interferon Tipo I/genética , Proteínas Ribossômicas/genética , Transcriptoma , Adolescente , Adulto , Criança , Fibrose Cística/diagnóstico , Feminino , Humanos , Masculino , Mutação , Fenótipo , Índice de Gravidade de Doença , Adulto JovemRESUMO
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.
Assuntos
Proteína Morfogenética Óssea 2/genética , Regeneração Óssea/genética , Fator 2 de Diferenciação de Crescimento/genética , Células-Tronco Mesenquimais/citologia , Fosfatase Alcalina/genética , Animais , Humanos , Masculino , Osteogênese/genética , Polietilenoimina/química , RNA/administração & dosagem , Ratos Endogâmicos F344 , TransfecçãoRESUMO
BACKGROUND: The immunogenicity and limited stability of conventional messenger RNA (mRNA) has traditionally restricted its potential therapeutic use. In 1992, the first clinical application of mRNA was reported as a potential protein-replacement therapy; however, subsequent investigations have not been made for almost two decades. Recent developments, including increased stability, controlling immunogenicity, as well as utilization of mRNA encoding zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas9, have implicated modified mRNA as a very promising option for cancer immunotherapy, vaccines, protein expression replacement, and genome editing. This review aims to offer a summary of our present understanding of and improvements in mRNA-based drug technologies, along with a focus on the role in therapeutic options for pediatric respiratory diseases and hemoglobinopathies. CONCLUSIONS: This mini review summarizes the recent advances in modified mRNA-based therapy and its potential therapeutic effect in treating major pediatric diseases.
RESUMO
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.
Assuntos
Proteína Morfogenética Óssea 2/genética , Regeneração Óssea , Polietilenoimina/química , RNA/administração & dosagem , RNA/química , Fosfatase Alcalina/genética , Animais , Células da Medula Óssea/citologia , Sobrevivência Celular , Traumatismos Craniocerebrais/terapia , DNA/administração & dosagem , DNA/química , Terapia Genética , Humanos , Masculino , Camundongos Endogâmicos BALB C , Osteocalcina/genética , Plasmídeos , RNA/farmacologia , RNA/uso terapêutico , Ratos Endogâmicos F344 , Células Estromais/metabolismoAssuntos
Asma/terapia , Proteinose Alveolar Pulmonar/congênito , Proteína B Associada a Surfactante Pulmonar/deficiência , Edição de RNA/genética , RNA Mensageiro/uso terapêutico , Animais , Asma/genética , Quitosana/uso terapêutico , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Nanopartículas/uso terapêutico , Proteinose Alveolar Pulmonar/genética , Proteinose Alveolar Pulmonar/terapia , Proteína B Associada a Surfactante Pulmonar/genética , RNA Mensageiro/genéticaRESUMO
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 ß-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 ß-catenin inhibitor led to an additional reduction of cell viability. CONCLUSION: Sorafenib together with inhibitors of the ß-catenin pathway might be an effective tool in the treatment of pediatric HCC.
Assuntos
Carcinoma Hepatocelular/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Neoplasias Hepáticas/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Adulto , Animais , Western Blotting , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Criança , Transição Epitelial-Mesenquimal , Feminino , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Niacinamida/uso terapêutico , Sorafenibe , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/metabolismoRESUMO
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.
Assuntos
Compostos de Bifenilo/farmacologia , Carcinoma Hepatocelular/metabolismo , Movimento Celular/efeitos dos fármacos , Hepatoblastoma/metabolismo , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Actinas/química , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Carcinoma Hepatocelular/patologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Inibidores de Caspase/farmacologia , Adesão Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Ativação Enzimática , Imunofluorescência , Hepatoblastoma/patologia , Humanos , Indóis , Metástase Neoplásica , Oligopeptídeos/farmacologia , Piperazinas/farmacologia , Proteólise , Pseudópodes/química , Pirróis/farmacologia , Proteína cdc42 de Ligação ao GTP/metabolismoRESUMO
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<0.02). When using higher doses of CDDP (3 mg/kg) alone or in combination with ABT-737, dose-dependent toxicity was observed in this mouse strain. In conclusion, our results demonstrated the enhancement of chemotherapy efficacy by using modulators of apoptosis together with cytotoxic agents. Additive effects of ABT-737 may allow reduction in CDDP dosages with maintenance of antitumor activity. Sensitizing HB to apoptosis may also render resistant HB susceptible to established chemotherapy regimens.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Compostos de Bifenilo/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Nitrofenóis/farmacologia , Sulfonamidas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/efeitos adversos , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Cisplatino/efeitos adversos , Perfilação da Expressão Gênica , Hepatoblastoma/genética , Hepatoblastoma/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Nitrofenóis/administração & dosagem , Nitrofenóis/efeitos adversos , Piperazinas/administração & dosagem , Piperazinas/efeitos adversos , Piperazinas/farmacologia , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos , Redução de Peso/efeitos dos fármacosRESUMO
PURPOSE: 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. METHODS: The duplex drugs 2'-deoxy-5-fluorouridylyl-(3'-5')- 3'-C-ethynylcytidine (5-FdU(3'-5')ECyd) and 3'-C-ethynylcytidinylyl-(5'â1-O)-2-O-octadecyl-sn-glycerylyl-(3'-Οâ5')-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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Antimetabólitos Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Monofosfato de Citidina/análogos & derivados , Fluordesoxiuridilato/análogos & derivados , Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Oligonucleotídeos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Monofosfato de Citidina/administração & dosagem , Citometria de Fluxo/métodos , Fluordesoxiuridilato/administração & dosagem , Humanos , Células Tumorais CultivadasRESUMO
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.
Assuntos
Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Citotoxicidade Imunológica , Hepatoblastoma/patologia , Neoplasias Hepáticas/patologia , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Linfócitos T/imunologia , Anticorpos Biespecíficos/farmacologia , Antígenos de Neoplasias/imunologia , Complexo CD3/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular Tumoral , Sobrevivência Celular , Técnicas de Cocultura , Citotoxicidade Imunológica/efeitos dos fármacos , Difosfonatos/farmacologia , Molécula de Adesão da Célula Epitelial , Hepatoblastoma/imunologia , Inibidores de Histona Desacetilases/farmacologia , Humanos , Imidazóis/farmacologia , Neoplasias Hepáticas/imunologia , Ativação Linfocitária/efeitos dos fármacos , Microscopia de Fluorescência , Anticorpos de Cadeia Única/farmacologia , Esferoides Celulares , Linfócitos T/efeitos dos fármacos , Ácido ZoledrônicoRESUMO
PURPOSE: Hepatoblastoma (HB) has a poor prognosis in advanced stages. The aim of this study was to enhance effectiveness of chemotherapy with antineoplastic kinase inhibitors. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Assuntos
Hepatoblastoma/tratamento farmacológico , Neoplasias Hepáticas/tratamento farmacológico , Piperazinas/uso terapêutico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Aurora Quinases , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células Tumorais CultivadasRESUMO
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 < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment. CONCLUSION: Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.
Assuntos
Antineoplásicos/farmacologia , Benzenossulfonatos/farmacologia , Hepatoblastoma/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose/fisiologia , Benzenossulfonatos/uso terapêutico , Western Blotting , Caspase 3 , Linhagem Celular Tumoral , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Transcrição Forkhead/genética , Marcação In Situ das Extremidades Cortadas , Camundongos , Neovascularização Patológica/patologia , Niacinamida/análogos & derivados , Compostos de Fenilureia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/uso terapêutico , Sorafenibe , Sais de Tetrazólio , Tiazóis , Transplante Heterólogo , Transplantes , alfa-Fetoproteínas/metabolismoRESUMO
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.