Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Conclusion: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients. (Hepatology 2014).
Core 3-derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional ?3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re-expressed core 3 synthase in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O-glycans induced altered expression of ?1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1? and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor-associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3-derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.
The presence of TNF-? in approximately 50% of surgically resected tumors suggests that the canonical NF-?B and the mTOR pathways are activated. Inhibitor of I?B kinase ? (IKK?) acts as the signaling node that regulates transcription via the p-I?B?/NF-?B axis and regulates translation via the mTOR/p-S6K/p-eIF4EBP axis. A kinome screen identified a quinoxaline urea analog 13-197 as an IKK? inhibitor. We hypothesized that targeting the NF-?B and mTOR pathways with 13-197 will be effective in malignancies driven by these pathways.
Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.
MUC1 is a transmembrane glycoprotein that modulates transcription via its cytoplasmic domain. We evaluated the capacity of MUC1 to regulate the global transcription of microRNAs in pancreatic cancer cells expressing MUC1. Results indicated that MUC1 regulated expression of at least 103 microRNAs. We evaluated further regulation of the microRNA transcript cluster miR-200c/141, which was among the most highly regulated microRNAs. We found that MUC1 directly interacted with ZEB1, a known transcriptional repressor of the miR-200c/141 cluster, at the promoter of miR-200c/141, and further reduced transcript production. These data indicate that signaling through MUC1 influences cancer progression by regulating transcription of microRNAs that are associated with the process of metastasis.
We describe the pathology of early pancreatic cancer and present an overview of known molecular alterations that occur in these lesions. There are three defined precursor lesions in current models of pancreatic cancer: pancreatic intraepithelial neoplasia (PanIN), mucinous cystic neoplasms (MCN), and intraductal papillary mucinous neoplasms (IPMN). Molecular alterations detected in these lesions include: telomeres, K-ras and downstream targets, p16/CDKN2A, p53, SMAD4/DPC4, microRNAs, mucins and their post-translational processing, inflammatory cytokines, CEACAM, and epigenetic alterations. We summarize previous analyses of these markers as diagnostic markers of disease, and suggest areas of future study.
Pancreatic ductal adenocarcinoma is a leading cause of cancer mortality with a dismal 2-5 % 5-year survival rate. Monotherapy with Gemcitabine has limited success, highlighting the need for additional therapies that enhance the efficacy of current treatments. We evaluated the combination of Gemcitabine and Rosiglitazone, an FDA-approved drug for the treatment of type II diabetes, in an immunocompetent transplantable mouse model of pancreatic cancer. Tumor progression, survival, and metastases were evaluated in immunocompetent mice with subcutaneous or orthotopic pancreatic tumors treated with Pioglitazone, Rosiglitazone, Gemcitabine, or combinations of these. We characterized the impact of high-dose Rosiglitazone and Gemcitabine therapy on immune suppressive mediators, including MDSC and T regulatory cells, and on modulation of peripheral and intra-tumoral T cell populations. Combinations of Rosiglitazone and Gemcitabine significantly reduced tumor progression and metastases, enhanced apoptosis, and significantly extended overall survival compared to Gemcitabine alone. Rosiglitazone altered tumor-associated immune suppressive mediators by limiting early MDSC accumulation and intra-tumoral T regulatory cells. Combination therapy with Rosiglitazone and Gemcitabine modulated T cell populations by enhancing circulating CD8(+) T cells and intra-tumoral CD4(+) and CD8(+) T cells while limiting T regulatory cells. The results suggest that Rosiglitazone, in combination with Gemcitabine, decreases immune suppressive mechanisms in immunocompetent animals and provides pre-clinical data in support of combining Rosiglitazone and Gemcitabine as a clinical therapy for pancreatic cancer.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.