RESUMO
In patients with hepatocellular carcinoma (HCC) receiving sorafenib, drug resistance is common. HCC develops in a microenvironment enriched with extracellular matrix proteins including laminin (Ln)-332, produced by hepatic stellate cells (HSCs). Ln-332 is the ligand of α3ß1 and α6ß4 integrins, differently expressed on the HCC cell surface, that deliver intracellular pathways. The aim of this study was to investigate the effect of Ln-332 on sorafenib's effectiveness. HCC cells were challenged with sorafenib in the presence of Ln-332 and of HSC conditioned medium (CM). Sorafenib impaired HCC cell proliferation and induced apoptosis. HSC-CM or Ln-332 inhibited sorafenib's effectiveness in HCC cells expressing both α3ß1 and α6ß4. Inhibiting α3 but not α6 integrin subunit using blocking antibodies or small interfering RNA abrogated the protection induced by Ln-332 and HSC-CM. Hep3B cells expressing α6ß4 but lacking the α3 integrin were insensitive to Ln-332 and HSC-CM protective effects. Hep3B α3-positive, but not wild-type and scramble transfected, cells acquired protection by sorafenib when plated on Ln-332-CM or HSCs. Sorafenib dephosphorylated focal adhesion kinase (FAK) and extracellular signal-regulated kinases 1/2, whereas Ln-332 and HSC-CM partially restored the pathways. Silencing FAK, but not extracellular signal-regulated kinases 1/2, abrogated the protection induced by Ln-332 and HSC-CM, suggesting a specific role for FAK. Sorafenib down-regulated total FAK, inducing its proteasomal degradation, while Ln-332 and HSC-CM promoted the escape of FAK from ubiquitination, probably inducing a preferential membrane localization. CONCLUSION: This study unveils a novel mechanism of sorafenib resistance depending on the α3ß1/Ln-332 axis and requiring FAK ubiquitination, providing new insights into personalizing therapy for patients with HCC. (Hepatology 2016;64:2103-2117).
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Células Estreladas do Fígado/fisiologia , Integrina alfa3/fisiologia , Laminina/fisiologia , Neoplasias Hepáticas/tratamento farmacológico , Niacinamida/análogos & derivados , Compostos de Fenilureia/uso terapêutico , Ubiquitinação , Humanos , Niacinamida/uso terapêutico , Sorafenibe , Células Tumorais CultivadasRESUMO
AIMS: In this study, we investigated whether pre-conditioning (PC) by electrical stimulation (EleS) induces cytoprotective effect on cardiac stem cells (CSCs) and determined its underlying molecular mechanisms. METHODS AND RESULTS: Sca-1(+) CSCs were isolated from male C57BL6 mice (12 weeks) hearts. PC of CSCs with EleS ((EleS)CSCs) was carried out for 3 h at 1.5 V followed by exposure to 300 µM H2O2 for 5 h. Cytoprotective effects and cell adhesion ability were significantly increased by EleS as evaluated by transferase-mediated dUTP nick-end labelling (TUNEL), lactate dehydrogenase (LDH) release assay, and adhesion assay. EleS increased phosphorylation of AKT, focal adhesion kinase (FAK), and glycogen synthase kinase (GSK3ß), as well as decreased caspase-3 cleavage. Interestingly, inhibition of AKT or FAK abolished the pro-survival effects of EleS. We found that connective tissue growth factor (Ctgf) was responsible for EleS-induced CSC survival and adhesion.The survival rate of (EleS)CSCs after transplantation in the infarcted myocardium was significantly increased together with improvement in cardiac function. Importantly, knockdown of Ctgf abolished EleS-induced cytoprotective effects and recovery of cardiac function. Furthermore, we identified miR-378 as a potential Ctgf regulator in (EleS)CSCs. CONCLUSION: EleS enhanced CSC survival in vitro and in vivo as well as functional recovery of the ischaemic heart through an AKT/FAK/CTGF signalling pathway. It is suggested that Ctgf and miR-378 are novel therapeutic targets for stem cell-based therapy.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/fisiologia , Terapia por Estimulação Elétrica , MicroRNAs/fisiologia , Isquemia Miocárdica/terapia , Miócitos Cardíacos/fisiologia , Transplante de Células-Tronco , Animais , Antígenos Ly/análise , Adesão Celular , Sobrevivência Celular , Citoproteção , Proteína-Tirosina Quinases de Adesão Focal/fisiologia , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/fisiopatologia , Estresse Oxidativo , Proteínas Proto-Oncogênicas c-akt/fisiologia , Células-Tronco/fisiologiaRESUMO
Different types of cancer take advantage of inflammatory components to improve their life-span in the organs. A sustenance of growth factors and cytokines (e.g. interleukin (IL)-1, tumor necrosis factor, IL-6, vascular endothelial growth factor) supports malignant cell progression and contributes to suppress the body immune defense. Strategies to modulate the host micro-environment offer new approaches for anti-cancer therapies. For these reasons new molecules with anti-tumor and anti-inflammatory features (e.g. trabectedin) are looked at with new eyes in the light of the crucial link between inflammation and cancer.