RESUMO
OBJECTIVES: The liver's capability to completely regenerate after injury is a unique phenomenon in which cytokines are of particular interest. Here, we aimed to assess the release patterns and prognostic relevance of liver regeneration-related cytokines in the setting of living-donor liver transplant. MATERIALS AND METHODS: Eleven cytokines related to liver regeneration (hepatocyte growth factor, interleukin 6, insulin-like growth factor-1, tumor necrosis factor alpha, transforming growth factor beta, granulocyte colony-stimulating factor, stem cell factor, chemokine (C-X-C motif) ligand 12, angiogenin, fibroblast growth factor-2, and vascular endothelial growth factor) were compared in 13 living-donor liver transplant recipients and their corresponding donors before and daily (10 days) after transplant. Patients and donors were stratified by clinical outcomes (early graft loss within 4 weeks after transplant vs beneficial outcome). RESULTS: Most cytokines tested (especially tumor necrosis factor alpha and stem cell factor) were elevated in recipients versus donors. Many cytokines were also increased in recipients with graft loss (especially CXCL12) and in donors of recipients with beneficial outcomes (especially fibroblast growth factor 2). Fibroblast growth factor 2 levels were also correlated positively with serum gamma-glutamyltransferase, and higher preoperative concentrations in donors were associated with recipients having beneficial outcomes, indicating an improved regenerative capacity. In contrast, elevated CXCL12 levels in recipients before and after LDLT predicted graft loss and were linked to ongoing liver damage. CONCLUSIONS: In living-donor liver transplant, there are distinct differences between donors and recipients regarding the release of liver regeneration-related cytokines. Moreover, fibroblast growth factor 2 and CXCL12 may be of diagnostic value in a complementary way to describe or even predict the possible outcomes after transplant. These results may be of clinical interest not only for living-donor liver transplant but also for acute liver failure.
Assuntos
Citocinas/sangue , Regeneração Hepática , Transplante de Fígado/métodos , Doadores Vivos , Transplantados , Adulto , Idoso , Biomarcadores/sangue , Proliferação de Células , Quimiocina CXCL12/sangue , Feminino , Fator 2 de Crescimento de Fibroblastos/sangue , Hepatectomia , Humanos , Transplante de Fígado/efeitos adversos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Valor Preditivo dos Testes , Fatores de Risco , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Cholangiocyte senescence has been linked to primary sclerosing cholangitis (PSC). Persistent secretion of growth factors by senescent cholangiocytes leads to the activation of stromal fibroblasts (ASFs), which are drivers of fibrosis. The activated phenotype of ASFs is characterized by an increased sensitivity to apoptotic stimuli. Here, we examined the mechanisms of apoptotic priming in ASFs and explored a combined targeting strategy to deplete senescent cholangiocytes and ASFs from fibrotic tissue to ameliorate liver fibrosis. Using a coculture system, we determined that senescent cholangiocytes promoted quiescent mesenchymal cell activation in a platelet-derived growth factor (PDGF)-dependent manner. We also identified B-cell lymphoma-extra large (Bcl-xL) as a key survival factor in PDGF-activated human and mouse fibroblasts. Bcl-xL was also up-regulated in senescent cholangiocytes. In vitro, inhibition of Bcl-xL by the small molecule Bcl-2 homology domain 3 mimetic, A-1331852, or Bcl-xL-specific small interfering RNA induced apoptosis in PDGF-activated fibroblasts, but not in quiescent fibroblasts. Likewise, inhibition of Bcl-xL reduced the survival and increased apoptosis of senescent cholangiocytes, compared to nonsenescent cells. Treatment of multidrug resistance 2 gene knockout (Mdr2-/- ) mice with A-1331852 resulted in an 80% decrease in senescent cholangiocytes, a reduction of fibrosis-inducing growth factors and cytokines, decrease of α-smooth muscle actin-positive ASFs, and finally in a significant reduction of liver fibrosis. CONCLUSION: Bcl-xL is a key survival factor in ASFs as well as in senescent cholangiocytes. Treatment with the Bcl-xL-specific inhibitor, A-1331852, reduces liver fibrosis, possibly by a dual effect on activated fibroblasts and senescent cholangiocytes. This mechanism represents an attractive therapeutic strategy in biliary fibrosis. (Hepatology 2018;67:247-259).
Assuntos
Benzotiazóis/farmacologia , Ductos Biliares/citologia , Colangite Esclerosante/patologia , Fibroblastos/efeitos dos fármacos , Isoquinolinas/farmacologia , Fator de Crescimento Derivado de Plaquetas/efeitos dos fármacos , Animais , Biópsia por Agulha , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Colangite Esclerosante/tratamento farmacológico , Modelos Animais de Doenças , Resistência a Múltiplos Medicamentos , Fibroblastos/metabolismo , Fibroblastos/patologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Terapia de Alvo Molecular , Fator de Crescimento Derivado de Plaquetas/metabolismo , Distribuição Aleatória , Valores de ReferênciaRESUMO
AIM: To examine the influence on apoptotic mechanisms following inhibition of polo-like kinases as therapeutically approach for cholangiocellular cancer treatment. METHODS: As most cholangiocarcinomas are chemotherapy-resistant due to mechanisms preventing tumor cell death, we investigated the effect of Cisplatin on cholangiocellular carcinoma (CCA) cell lines KMCH-1 and Mz-Ch-1. Polo-like kinases (PLK) are important regulators of the cell cycle and their inhibition is discussed as a potential therapy while PLK inhibition can regulate apoptotic mediators. Here, cells were treated with PLK inhibitor BI6727 (Volasertib), Cisplatin, and in combination of both compounds. Cell viability was assessed by MTT; apoptosis was measured by DAPI staining and caspase-3/-7 assay. Western blot and qRT-PCR were used to measure expression levels of apoptosis-related molecules Bax and Bcl-2. RESULTS: The cell viability in the CCA cell lines KMCH-1 and Mz-Ch-1 was reduced in all treatment conditions compared to vehicle-treated cells. Co-treatment with BI6727 and cisplatin could even enhance the cytotoxic effect of cisplatin single treatment. Thus, co-treatment of cisplatin with BI6727 could slightly enhance the cytotoxic effect of the cisplatin in both cell lines whereas there was evidence of increased apoptosis induction solely in Mz-Ch-1 as compared to KMCH-1. Moreover, PLK inhibition decreases protein levels of Bcl-2; an effect that can be reversed by the proteasomal degradation inhibitor MG-132. In contrast, protein levels of Bax were not found to be altered by PLK inhibition. These findings indicate that cytotoxic effects of Cisplatin in Mz-Ch-1 cells can be enhanced by cotreatment with BI6727. CONCLUSION: In conclusion, BI6727 treatment can sensitize CCA cells to cisplatin-induced apoptosis with proteasomal Bcl-2 degradation as an additional pro-apoptotic effect.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/tratamento farmacológico , Proteínas de Ciclo Celular/antagonistas & inibidores , Colangiocarcinoma/tratamento farmacológico , Cisplatino/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Pteridinas/farmacologia , Neoplasias dos Ductos Biliares/enzimologia , Neoplasias dos Ductos Biliares/patologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Colangiocarcinoma/enzimologia , Colangiocarcinoma/patologia , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Quinase 1 Polo-LikeRESUMO
BACKGROUND & AIMS: Cholangiocarcinomas (CCA) paradoxically express the death ligand TRAIL and thus, are dependent on effective survival signals to circumvent apoptosis. Hedgehog signalling exerts major survival signals in CCA by regulating serine/threonine kinase polo-like kinase (PLK)2. We here aimed to examine the role of PLK1/2/3 expression for CCA tumour biology. METHODS: We employed CCA samples from 73 patients and human HUCCT-1/Mz-CHA1/KMCH-1 CCA cells. Immunohistochemistry for PLK1/2/3 was performed using tissue microarrays from representative tumour areas. RESULTS: PLK1/2/3-immunoreactive cancer cells were present in most of the CCA samples. However, only PLK1 and especially PLK3 were expressed in higher amounts within CCA cells as compared to normal liver. Given that fibroblast growth factor (FGF) can induce PLK3 expression and also is present in CCA, we examined the effect of FGF on PLK3 in vitro. Indeed, rhFGF rapidly increased PLK3 mRNA expression all three CCA cell lines giving an explanation for the abundant PLK3 presence in the tissue samples. Clinicopathologically, PLK3 expression was associated with decreased tumour cell migration and lymph/blood vessel infiltration whereas higher levels of PLK1 were correlated with larger tumour sizes. Moreover, strong PLK3 expression was associated with prolonged overall survival. CONCLUSIONS: The results suggest that PLK3 predominantly is expressed in CCA cells and that high PLK3 expression correlates with prolonged overall survival. These observations might have implications for prognosis prediction of human CCA as well as the potential therapeutic use of polo-like kinase inhibitors (i.e., PLK1/2 specifity).
Assuntos
Apoptose/genética , Neoplasias dos Ductos Biliares/mortalidade , Colangiocarcinoma/mortalidade , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais/genética , Idoso , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Feminino , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Supressoras de TumorRESUMO
BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide and therapeutic options are scarce. As they might represent future targets for cancer therapy, the expression of apoptosis-related genes in HCC is of particular interest. In this pilot study, we further examined apoptosis-related genes in human HCC and also focused on vitamin D signaling as this might be a regulator of HCC cell apoptosis. METHODS: We employed tumor tissue and serum samples from 62 HCC patients as well as 62 healthy controls for these studies. Tissue and serum specimens were analyzed by quantitative RT-PCR, immunohistochemistry and ELISA. RESULTS: In HCC patients the apoptosis marker M30 was found to be elevated and several pro-apoptotic (TRAIL, FasL and FasR) as well as anti-apoptotic genes (Mcl-1 and Bcl-2) were simultaneously upregulated in tumor tissue and especially tumor-surrounding tissue as compared to healthy control livers. Moreover, vitamin D serum levels were decreased in HCC patients whereas vitamin D receptor mRNA expression was increased in tumor tissue and tumor-surrounding tissue as compared to healthy livers. CONCLUSIONS: In human HCC, M30 serum levels are elevated indicating an increased cell turnover. Modulation of the vitamin D pathway might be a supportive, pro-apoptotic HCC therapy.
Assuntos
Anticorpos Monoclonais Murinos/sangue , Apoptose/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Vitamina D/metabolismo , Idoso , Biomarcadores/sangue , Carcinoma Hepatocelular/genética , Estudos de Casos e Controles , Feminino , Voluntários Saudáveis , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Receptor fas/sangueRESUMO
UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and thus rely on potent survival signals to circumvent cell death by TRAIL. Hedgehog (Hh) signaling is an important survival pathway in CCA. Herein, we further examine the mechanisms whereby Hh signaling mediates apoptosis resistance in CCA, revealing a pivotal role for the cell division regulating serine/threonine kinase polo-like kinase 2 (PLK2). We employed 50 human CCA samples (25 intrahepatic and 25 extrahepatic CCA) as well as human KMCH-1, Mz-CHA-1, and HUCCT-1 CCA cells for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. In human samples, polo-like kinase (PLK)1/2/3-immunoreactive cancer cells were present in the preponderance of intra- and extrahepatic CCA specimens. Inhibition of Hh signaling by cyclopamine reduced PLK2, but not PLK1 or PLK3, messenger RNA and protein expression in vehicle-treated and sonic Hh-treated CCA cells, confirming our previous microarray study. PLK2 regulation by Hh signaling appears to be direct, because the Hh transcription factors, glioma-associated oncogene 1 and 2, bind to the PLK2 promotor. Moreover, inhibition of PLK2 by the PLK inhibitor, BI 6727 (volasertib), or PLK2 knockdown was proapoptotic in CCA cells. BI 6727 administration or PLK2 knockdown decreased cellular protein levels of antiapoptotic myeloid cell leukemia 1 (Mcl-1), an effect reversed by the proteasome inhibitor, MG-132. Finally, BI 6727 administration reduced Mcl-1 protein expression in CCA cells, resulting in CCA cell apoptosis and tumor suppression in vivo. CONCLUSION: PLK2 appears to be an important mediator of Hh survival signaling. These results suggest PLK inhibitors to be of therapeutic value for treatment of human CCA.
Assuntos
Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/fisiopatologia , Proteínas Hedgehog/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Colangiocarcinoma/patologia , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Xenoenxertos , Humanos , Masculino , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , Pteridinas/farmacologia , Ratos , Ratos Endogâmicos F344 , Ligante Indutor de Apoptose Relacionado a TNF/fisiologiaRESUMO
Cancer-associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAFs are "activated" and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma as a desmoplastic tumor model, we investigated the sensitivity of liver CAFs to the cytotoxic drug navitoclax, a BH3 mimetic. Navitoclax induced apoptosis in CAF and in myofibroblastic human hepatic stellate cells but lacked similar effects in quiescent fibroblasts or cholangiocarcinoma cells. Unlike cholangiocarcinoma cells, neither CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAFs or cells treated with navitoclax both released the apoptogenic factors Smac and cytochrome c, suggesting that they are primed for cell death. Such death priming in CAFs appeared to be due, in part, to upregulation of the proapoptotic protein Bax. Short hairpin RNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors, and apoptotic cell death. In a syngeneic rat model of cholangiocarcinoma, navitoclax treatment triggered CAF apoptosis, diminishing expression of the desmoplastic extracellular matrix protein tenascin C, suppressing tumor outgrowth, and improving host survival. Together, our findings argue that navitoclax may be useful for destroying CAFs in the tumor microenvironment as a general strategy to attack solid tumors.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Colangiocarcinoma/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Miofibroblastos/efeitos dos fármacos , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular , Linhagem Celular Tumoral , Colangiocarcinoma/patologia , Humanos , Fígado/citologia , Metástase Neoplásica/terapia , Ratos , Carga Tumoral/efeitos dos fármacos , Microambiente Tumoral , Proteína X Associada a bcl-2/metabolismoRESUMO
UNLABELLED: It has been established that microRNA expression and function contribute to phenotypic features of malignant cells, including resistance to apoptosis. Although targets and functional roles for a number of microRNAs have been described in cholangiocarcinoma, many additional microRNAs dysregulated in this tumor have not been assigned functional roles. In this study, we identify elevated miR-25 expression in malignant cholangiocarcinoma cell lines as well as patient samples. In cultured cells, treatment with the Smoothened inhibitor, cyclopamine, reduced miR-25 expression, suggesting Hedgehog signaling stimulates miR-25 production. Functionally, miR-25 was shown to protect cells against TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Correspondingly, antagonism of miR-25 in culture sensitized cells to apoptotic death. Computational analysis identified the TRAIL Death Receptor-4 (DR4) as a potential novel miR-25 target, and this prediction was confirmed by immunoblot, cell staining, and reporter assays. CONCLUSION: These data implicate elevated miR-25 levels in the control of tumor cell apoptosis in cholangiocarcinoma. The identification of the novel miR-25 target DR4 provides a mechanism by which miR-25 contributes to evasion of TRAIL-induced cholangiocarcinoma apoptosis.
Assuntos
Apoptose , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Intra-Hepáticos/patologia , Colangiocarcinoma/metabolismo , MicroRNAs/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Humanos , Transdução de SinaisRESUMO
Isolated hepatocytes undergo lipoapoptosis, a feature of hepatic lipotoxicity, on treatment with saturated free fatty acids (FFA) such as palmitate (PA). However, it is unknown if palmitate is directly toxic to hepatocytes or if its toxicity is indirect via the generation of lipid metabolites such as lysophosphatidylcholine (LPC). PA-mediated hepatocyte lipoapoptosis is associated with endoplasmic reticulum (ER) stress, c-Jun NH(2)-terminal kinase (JNK) activation, and a JNK-dependent upregulation of the potent proapoptotic BH3-only protein PUMA (p53 upregulated modulator of apoptosis). Our aim was to determine which of these mechanisms of lipotoxicity are activated by PA-derived LPC. We employed Huh-7 cells and isolated murine and human primary hepatocytes. Intracellular LPC concentrations increase linearly as a function of the exogenous, extracellular PA, stearate, or LPC concentration. Incubation of Huh-7 cells or primary hepatocytes with LPC induced cell death by apoptosis in a concentration-dependent manner. Substituting LPC for PA resulted in caspase-dependent cell death that was accompanied by activating phosphorylation of JNK with c-Jun phosphorylation and an increase in PUMA expression. LPC also induced ER stress as manifest by eIF2α phosphorylation and CAAT/enhancer binding homologous protein (CHOP) induction. LPC cytotoxicity was attenuated by pharmacological inhibition of JNK or glycogen synthase kinase-3 (GSK-3). Similarly, short-hairpin RNA (shRNA)-targeted knockdown of CHOP protected Huh-7 cells against LPC-induced toxicity. The LPC-induced PUMA upregulation was prevented by JNK inhibition or shRNA-targeted knockdown of CHOP. Finally, genetic deficiency of PUMA rendered murine hepatocytes resistant to LPC-induced apoptosis. We concluded that LPC-induced lipoapoptosis is dependent on mechanisms largely indistinguishable from PA. These data suggest that FFA-mediated cytotoxicity is indirect via the generation of the toxic metabolite, LPC.
Assuntos
Apoptose/efeitos dos fármacos , Hepatócitos/metabolismo , Lisofosfatidilcolinas/farmacologia , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Lisofosfatidilcolinas/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Palmitatos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Estearatos/metabolismo , Fator de Transcrição CHOP/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Cholangiocarcinomas (CCAs) are highly desmoplastic neoplasms with a tumour microenvironment plentiful in myofibroblasts (MFBs). MFB-derived PDGF-BB survival signalling is a mediator of CCA cell resistance to apoptotic stimuli. This raises the concept that targeting PDGFR-ß, a cognate receptor of PDGF-BB, represents a potential strategy for the treatment of human CCA. AIMS: Herein, we examine a role for inhibiting PDGFR-ß in restoring CCA cell sensitivity to apoptotic stimuli in vitro and in vivo. METHODS: We employed human CCA samples from 41 patients (19 intrahepatic and 22 extrahepatic CCA samples), the human CCA cell lines KMCH-1 and HUCCT-1 as well as shPDGFR-ß-KMCH-1 and human myofibroblastic LX-2 cells for these studies. In vivo-experiments were conducted using a syngeneic rat orthotopic CCA model. RESULTS: Of several MFB-derived growth factors profiled, PDGF-BB and CTGF were most abundantly expressed; however, only PDGF-BB attenuated tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity. Co-culturing CCA cells with PDGF-BB-secreting MFBs significantly decreased TRAIL-induced CCA cell apoptosis when compared with monoculture conditions; this cytoprotective effect was abrogated in the presence of the tyrosine kinase inhibitors imatinib mesylate or linifanib, which inhibit PDGFR-ß. Consistent with these findings, MFB-imparted cytoprotection also was abolished when PDGFR-ß was knocked down as demonstrated in shPDGFR-ß-KMCH-1 cells. Finally, administration of imatinib mesylate increased CCA cell apoptosis and reduced tumour growth in a rodent in vivo-CCA model that mimics the human disease. CONCLUSIONS: Targeting PDGFR-ß sensitizes CCA cells to apoptotic stimuli and appears to be therapeutic in vivo.
Assuntos
Apoptose/fisiologia , Neoplasias dos Ductos Biliares/metabolismo , Ductos Biliares Extra-Hepáticos/metabolismo , Ductos Biliares Intra-Hepáticos/metabolismo , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/metabolismo , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Apoptose/efeitos dos fármacos , Becaplermina , Linhagem Celular Tumoral , Colangiocarcinoma/fisiopatologia , Primers do DNA/genética , Humanos , Immunoblotting , Imuno-Histoquímica , Queratina-7/metabolismo , Microscopia de Fluorescência , Miofibroblastos/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Proteínas Proto-Oncogênicas c-sis/metabolismo , Proteínas Proto-Oncogênicas c-sis/farmacologia , RNA Interferente Pequeno/metabolismo , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Ligante Indutor de Apoptose Relacionado a TNF/metabolismoRESUMO
Cell death by apoptosis is a prominent feature in a variety of liver diseases. It is likely that apoptosis is the initial cellular response to hepatocyte and biliary injury, which then leads to the initiation of cellular and cytokine cascades culminating in hepatocyte death with subsequent fibrosis and cirrhosis. This sequence of events is of paramount clinical importance. Recently, soluble forms of the major histocompatibility complex class I-related chains A and closely related B (MIC A and B) were reported to be increased in patients with a variety of liver diseases. MIC A and B are cell surface glycoproteins that function as indicators for cellular stress and thus activate circulating cytotoxic natural killer (NK) cells. The interaction between MIC A and B with their cognate receptor natural killer group 2 member D (NKG2D) culminates in enhanced liver cell death, which is mediated in part by apoptotic mechanisms. The present overview focuses on the role of the stress-induced NKG2D ligands MIC A and B in diverse liver diseases. Critical insights into these complex relations may help to promote rationally based therapies in liver diseases. Importantly, we hope that this overview will help to stimulate further studies into mechanisms by which stress ligands mediate cell death and its sequale.
Assuntos
Apoptose/fisiologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Hepatopatias/metabolismo , Modelos Biológicos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Estresse Fisiológico/fisiologia , Humanos , LigantesRESUMO
UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. CONCLUSIONS: MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.
Assuntos
Neoplasias dos Ductos Biliares/fisiopatologia , Ductos Biliares Intra-Hepáticos/fisiopatologia , Colangiocarcinoma/fisiopatologia , Proteínas Hedgehog/fisiologia , Proteínas Proto-Oncogênicas c-sis/fisiologia , Animais , Apoptose/efeitos dos fármacos , Becaplermina , Neoplasias dos Ductos Biliares/genética , Linhagem Celular Tumoral , Colangiocarcinoma/genética , Técnicas de Cocultura , Células Estreladas do Fígado/metabolismo , Humanos , Masculino , Ratos , Ratos Endogâmicos F344 , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptor Smoothened , Fatores de Transcrição/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de ZincoRESUMO
Nonalcoholic steatohepatitis is characterized by hepatic steatosis, elevated levels of circulating free fatty acids (FFA), endoplasmic reticulum (ER) stress, and hepatocyte lipoapoptosis. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor 5 (DR5) is significantly elevated in patients with nonalcoholic steatohepatitis, and steatotic hepatocytes demonstrate increased sensitivity to TRAIL-mediated cell death. Nonetheless, a role for TRAIL and/or DR5 in mediating lipoapoptotic pathways is unexplored. Here, we examined the contribution of DR5 death signaling to lipoapoptosis by free fatty acids. The toxic saturated free fatty acid palmitate induces an increase in DR5 mRNA and protein expression in Huh-7 human hepatoma cells leading to DR5 localization into lipid rafts, cell surface receptor clustering with subsequent recruitment of the initiator caspase-8, and ultimately cellular demise. Lipoapoptosis by palmitate was not inhibited by a soluble human recombinant DR5-Fc chimera protein suggesting that DR5 cytotoxic signaling is ligand-independent. Hepatocytes from murine TRAIL receptor knock-out mice (DR(-/-)) displayed reduced palmitate-mediated lipotoxicity. Likewise, knockdown of DR5 or caspase-8 expression by shRNA technology attenuated palmitate-induced Bax activation and apoptosis in Huh-7 cells, without altering induction of ER stress markers. Similar observations were verified in other cell models. Finally, knockdown of CHOP, an ER stress-mediated transcription factor, reduced DR5 up-regulation and DR5-mediated caspase-8 activation upon palmitate treatment. Collectively, these results suggest that ER stress-induced CHOP activation by palmitate transcriptionally up-regulates DR5, likely resulting in ligand-independent cytotoxic signaling by this death receptor.
Assuntos
Apoptose , Fígado Gorduroso/metabolismo , Hepatócitos/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Técnicas de Silenciamento de Genes , Hepatócitos/patologia , Humanos , Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/patologia , Camundongos , Camundongos Knockout , Ácido Palmítico/farmacologia , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Recent in vitro studies of cold-induced cell injury have revealed the detrimental effects of extracellular chloride on cold-stored isolated rat hepatocytes; however, its influence on endothelial cells is beneficial. To determine which of these effects is predominant in vivo, we tested both a chloride-poor variant of a new histidine-tryptophan-ketoglutarate (HTK)-based preservation solution and a chloride-containing variant in a rat liver transplantation model. The study, which was carried out in a blinded fashion with 7 or 8 rats per group, was divided into 2 parts: (1) a comparison of survival in 3 series under different conditions [different microsurgeons, rat strains, cold ischemia times (3, 12, and 24 hours), and warm ischemia times] and (2) an assessment of the microcirculation (30-90 minutes after reperfusion), laboratory data, bile production, and histology. In each of the survival experiments, a (strong) tendency toward prolonged survival was observed with the new chloride-containing solution (50% versus 12.5%, 75% versus 37.5%, and 100% versus 71.4% [chloride-containing vs. chloride-poor], overall P < 0.05). Additionally, the sinusoidal perfusion rates (83.9% ± 4.0% versus 69.2% ± 10.8%, P < 0.01) and the red blood cell velocities in sinusoids (147.7 ± 26.7 versus 115.5 ± 26.0 µm/second, P < 0.05) and in postsinusoidal venules (332.4 ± 87.3 versus 205.5 ± 53.5 µm/second, P < 0.01) were clearly higher with chloride. Moreover, the serum activities of liver enzymes were slightly reduced (not significantly), and bile production was significantly increased. These results suggest an overall beneficial effect of chloride in HTK-based liver preservation solutions.
Assuntos
Cloretos , Transplante de Fígado/métodos , Soluções para Preservação de Órgãos , Animais , Cloretos/farmacologia , Glucose/farmacologia , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Fígado/irrigação sanguínea , Fígado/citologia , Masculino , Manitol/farmacologia , Microcirculação , Modelos Animais , Soluções para Preservação de Órgãos/farmacologia , Cloreto de Potássio/farmacologia , Procaína/farmacologia , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Análise de SobrevidaRESUMO
TRAIL is a promising therapeutic agent for human malignancies. TRAIL often requires mitochondrial dysfunction, referred to as the Type II death receptor pathway, to promote cytotoxicity. However, numerous malignant cells are TRAIL resistant due to inhibition of this mitochondrial pathway. Using cholangiocarcinoma cells as a model of TRAIL resistance, we found that Hedgehog signaling blockade sensitized these cancer cells to TRAIL cytotoxicity independent of mitochondrial dysfunction, referred to as Type I death receptor signaling. This switch in TRAIL requirement from Type II to Type I death receptor signaling was demonstrated by the lack of functional dependence on Bid/Bim and Bax/Bak, proapoptotic components of the mitochondrial pathway. Hedgehog signaling modulated expression of X-linked inhibitor of apoptosis (XIAP), which serves to repress the Type I death receptor pathway. siRNA targeted knockdown of XIAP mimics sensitization to mitochondria-independent TRAIL killing achieved by Hedgehog inhibition. Regulation of XIAP expression by Hedgehog signaling is mediated by the glioma-associated oncogene 2 (GLI2), a downstream transcription factor of Hedgehog. In conclusion, these data provide additional mechanisms modulating cell death by TRAIL and suggest Hedgehog inhibition as a therapeutic approach for TRAIL-resistant neoplasms.
Assuntos
Proteínas Hedgehog/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas Hedgehog/genética , Humanos , Immunoblotting , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alcaloides de Veratrum/farmacologia , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genética , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismo , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
TNF-related apoptosis-inducing ligand (TRAIL) is a potential chemotherapeutic agent with high selectivity for malignant cells. Many tumors, however, are resistant to TRAIL cytotoxicity. Although cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and -2) are often over-expressed in cancers, their role in mediating TRAIL resistance remains unclear. Here, we demonstrate that TRAIL-induced apoptosis of liver cancer cells is associated with degradation of cIAP-1 and X-linked IAP (XIAP), whereas cIAP-2 remains unchanged. Lower concentrations of TRAIL causing minimal or no apoptosis do not alter cIAP-1 or XIAP protein levels. Silencing of cIAP-1 expression, but not XIAP or cIAP-2, as well as co-treatment with a second mitochondrial activator of caspases (SMAC) mimetic (which results in rapid depletion of cIAP-1), sensitizes the cells to TRAIL. TRAIL-induced loss of cIAP-1 and XIAP requires caspase activity. In particular, caspase 8 knockdown stabilizes both cIAP-1 and XIAP, while caspase 9 knockdown prevents XIAP, but not cIAP-1 degradation. Cell-free experiments confirmed cIAP-1 is a substrate for caspase 8, with likely multiple cleavage sites. These results suggest that TRAIL-mediated apoptosis proceeds through caspase 8-dependent degradation of cIAP-1. Targeted depletion of cIAP-1 by SMAC mimetics in conjunction with TRAIL may be beneficial for the treatment of human hepatobiliary malignancies.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 8/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/genética , Apoptose/fisiologia , Proteína 3 com Repetições IAP de Baculovírus , Caspase 8/genética , Caspase 8/fisiologia , Caspase 9/genética , Caspase 9/metabolismo , Inibidores de Caspase , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas Inibidoras de Apoptose/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/genéticaRESUMO
UNLABELLED: Cholangiocarcinoma (CCA) cells paradoxically express tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a death ligand that, failing to kill CCA cells, instead promotes their tumorigenicity and especially the metastatic behaviors of cell migration and invasion. Second mitochondria-derived activator of caspase (smac) mimetics are promising cancer therapeutic agents that enhance proapoptotic death receptor signaling by causing cellular degradation of inhibitor of apoptosis (IAP) proteins. Our aim was to examine the in vitro and in vivo effects of the smac mimetic JP1584 in CCA. Despite JP1584-mediated loss of cellular inhibitor of apoptosis-1 (cIAP-1) and cIAP-2, TRAIL failed to induce apoptosis in KMCH-1, TFK-1, and BDEneu CCA cells; a finding consistent with a downstream block in death signaling. Because cIAP-1 and cIAP-2 also promote nuclear factor kappa B (NF-kappaB) activation by the canonical pathway, the effect of JP1584 on this signaling pathway was examined. Treatment with JP1584 inhibited TRAIL-induced NF-kappaB activation as well as TRAIL-mediated up-regulation of the NF-kappaB target gene, matrix metalloproteinase 7 (MMP7). JP1584 also reduced TRAIL-mediated CCA cell migration and invasion in vitro. Finally, in a syngeneic rat orthotopic CCA model, JP1584 administration reduced MMP7 messenger RNA levels and extrahepatic metastases. CONCLUSION: : Although the smac mimetic JP1584 does not sensitize cells to apoptosis, it reduces TRAIL-induced CCA cell metastatic behavior. These data support the emerging concept that IAPs are prometastatic and represent targets for antimetastatic therapies.
Assuntos
Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/secundário , Ductos Biliares Intra-Hepáticos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Ligante Indutor de Apoptose Relacionado a TNF/efeitos dos fármacos , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Animais , Proteínas Reguladoras de Apoptose , Neoplasias dos Ductos Biliares/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Proteínas Mitocondriais , Invasividade Neoplásica , Ratos , Ratos Endogâmicos F344 , Células Tumorais CultivadasRESUMO
UNLABELLED: Stress-induced soluble major histocompatibility complex class I-related chains A/B (MIC A/B) are increased in chronic liver diseases and hepatocellular malignancy. We investigated the impact of these molecules on liver injury, apoptosis, and fibrosis in nonalcoholic steatohepatitis (NASH). Blood and liver tissue were obtained from 40 patients with NASH undergoing bariatric surgery for obesity. The control group consisted of 10 healthy individuals. We also investigated 10 patients with nonalcoholic fatty liver (NAFL). Polymerase chain reaction was used to measure messenger RNA (mRNA) transcripts of MIC A/B, natural killer cell receptor G2D (NKG2D), CD95/Fas, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-death receptor 5 (DR5). Apoptosis was quantified by way of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) (intrahepatic) and M30/M65 (systemic). Liver injury was assessed histopathologically and serologically (alanine aminotransferase/aspartate aminotransferase). Fibrosis was identified by Sirius red staining, quantitative morphometry, and alpha-smooth muscle actin and collagen 1alpha transcripts. Compared with controls, patients with NASH revealed significant increases in (1) NKG2D mRNA (13.1-fold) and MIC A/B mRNA (3.6-fold and 15.8-fold, respectively); (2) TRAIL-DR5 and CD95/Fas mRNA (2.7-fold and 3.6-fold, respectively); (3) TUNEL-positive hepatocytes (4.0-fold); and (4) M30 and M65 levels (4.6-fold and 3.4-fold, respectively). We found relevant correlations between MIC protein expression rates and NAS and fibrosis stages. In contrast, NKG2D and MIC A/B transcripts were attenuated in patients with NAFL compared with NASH. Histopathologically, NASH patients revealed increased NAS scores, an accumulation of natural killer cells, and 2.7-fold increased hepatic fibrosis by quantitative morphometry. CONCLUSION: Our findings suggest an important role for MIC A/B in liver injury. Therapeutic intervention aimed at reducing MIC A/B levels may beneficially affect the progression of NASH.
Assuntos
Fígado Gorduroso/patologia , Antígenos de Histocompatibilidade Classe I/biossíntese , Adulto , Apoptose , Fígado Gorduroso/imunologia , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/biossínteseRESUMO
AIM: To ascertain whether resveratrol affects the expression of free fatty acids (FFA)-induced profibrogenic genes, death receptors, and/or apoptosis-related molecules in human hepatic stellate cells, using the LX-2 cell line. METHODS: Cells were cultured in the presence of FFAs (2:1 oleate : palmitate) and subsequently treated with resveratrol. Gene expression rates were determined by quantitative real-time PCR. The 50% lethal dose (LD(50)) of resveratrol in the presence of FFAs was assessed with the MTT viability test. RESULTS: Compared to vehicle controls, incubation of LX-2 cells with 0.5 mM FFAs induced profibrogenic genes (alpha-SMA x 2.9; TGF-beta1 x 1.6; TIMP-1 x 1.4), death receptors (CD95/Fas x 3.8; TNFR-1 x 1.4), and anti-apoptotic molecules (Bcl-2 x 2.3; Mcl-1 x 1.3). Subsequent addition of 15 microM resveratrol (LD(50) = 23.2 microM) significantly (P < 0.05) upregulated further these genes (alpha-SMA x 6.5; TGF-beta1 x 1.9; TIMP-1 x 2.2; CD95/Fas x 13.1, TNFR-1 x 2.1; Bcl-2 x 3.6; Mcl-1 x 1.9). Importantly, this effect was only observed in the presence of FFAs. CONCLUSION: Resveratrol amplifies the profibrogenic activation of human hepatic LX-2 stellate cells. This finding raises the possibility that in obese patients with elevated FFAs reserveratrol could provoke hepatic fibrogenesis. In-vivo studies are necessary to further validate this conclusion.