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.
Cholangiocarcinoma (CCA) cells paradoxically express the death 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. Co-culturing CCA cells with myofibroblastic human primary HSCs or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing PDGF-BB-antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling as it was abolished by the smoothened (the transducer of Hh signaling) inhibitor cyclopamine. PDGF-BB induced PKA-dependent trafficking of smoothened to the plasma membrane resulting in GLI2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide mRNA expression analysis identified 67 target genes to be commonly up- (50 genes) or downregulated (17 genes) by both SHH 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 Myofibroblast-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 cholangiocarcinoma.
Cancer associated fibroblasts (CAF) are abundant in the stroma of desmoplastic cancers where they promote tumor progression. CAF are `activated' and as such may be uniquely susceptible to apoptosis. Using cholangiocarcinoma (CCA) as a desmoplastic tumor model, we investigated the sensitivity of liver CAF 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 CCA cells. Unlike CCA cells, niether CAF nor quiescent fibroblasts expressed Mcl-1, a known resistance factor for navitoclax cytotoxicity. Explaining this paradox, we found that mitochondria isolated from CAF 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 CAF appeared to be due in part to upregulation of the pro-apoptotic protein Bax. shRNA-mediated attenuation of Bax repressed navitoclax-mediated mitochondrial dysfunction, release of apoptogenic factors and apoptotic cell death. In a syngeneic rat model of CCA, 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 CAF in the tumor microenvironment as a general strategy to attack solid tumors.
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. Obesity and insulin resistance, cardinal features of the metabolic syndrome, are associated with enhanced lipolysis in adipose tissue (1, 2). This excessive lipolysis results in increased serum concentrations of free fatty acids (FFA), 2 augmenting their delivery to non-adipose tissues such as liver, heart, and pancreatic -cells (2). Inundation of these tissues with FFA overwhelms fatty acid oxidative pathways with toxic cellular consequences. An advanced response to toxic FFA is cellular demise by apoptosis, termed lipoapoptosis (3). Hepatocyte lipotoxicity is particularly germane to injury in the liver, where it contributes to the syndrome of nonalcoholic fatty liver disease (NAFLD) (4). For example, the magnitude of hepatocyte lipoapoptosis correlates with hepatic disease severity (4). Thus, the mechanisms initiating lipoapoptosis are of biomedical interest and human health relevance. Lipotoxicity is likely multifactorial. Sustained endoplasmic reticulum (ER) stress (5-7), c-Jun N-terminal kinase (JNK) activation (8, 9), and oxidative stress (10) have all been implicated in lipotoxicity. Despite the fact that death receptors are potent mediators of cytotoxicity (11), especially in hepatic diseases, their contribution to lipotoxicity is incompletely defined. Fas, the prototype death receptor, has been implicated in adipocyte toxicity and inflammation (12), and its hepatic expression is increased in NAFLD (4). Tumor necrosis factor-␣ (TNF-␣) and TNF receptor-1 (TNFR-1) have also been implicated in hepatic steatosis (13)(14)(15). In contrast, the role of TNF-related apoptosis-inducing ligand (TRAIL) and its cognate death receptors (DR)-4 and -5 in lipotoxicity is understudied. Yet, increasing data implicate a critical role for DR5 in lipotoxicity. For example, DR5 expression is increased in steatotic hepatocytes and sensitizes hepatocytes to exogenous TRAIL cytotoxicity (16). Thus, the role of DR5 in lipoapoptosis merits further investigation.In this study, we explored the potential contribution of DR5 death signaling during lipoapoptosis by saturated FFA. The results implicate TRAIL death signaling by DR...
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