Cancer‐associated fibroblasts contribute to cancer progression that is caused by epithelial–mesenchymal transition (EMT). Recently, mesenchymal stem cells (MSCs) were found to be the major candidate ...involved in the development of tumor‐promoting cancer stroma. Here we report that α‐smooth muscle actin‐positive myofibroblast‐like cells originating from MSCs contribute to inducing EMT in side population cells of pancreatic cancer. More importantly, MSC‐derived myofibroblasts function to maintain tumor‐initiating stem cell‐like characteristics, including augmenting expression levels of various stemness‐associated genes, enhancing sphere‐ forming activity, promoting tumor formation in a mouse xenograft model, and showing resistance to anticancer drugs. Furthermore, both γ‐secretase inhibitor and siRNA directed against Jagged‐1 attenuated MSC‐associated E‐cadherin suppression and sphere formation in pancreatic cancer side population cells. Thus, our results suggest that MSC‐derived myofibroblasts play important roles in regulating EMT and tumor‐initiating stem cell‐like properties of pancreatic cancer cells through an intermediating Notch signal.
Summary
Dendritic cells (DCs) are known as antigen‐presenting cells and play a central role in both innate and acquired immunity. Peripheral blood monocytes give rise to resident and recruited DCs in ...lymph nodes and non‐lymphoid tissues. The ligands of nuclear hormone receptors can modulate DC differentiation and so influence various biological functions of DCs. The role of bile acids (BAs) as signalling molecules has recently become apparent, but the functional role of BAs in DC differentiation has not yet been elucidated. We show that DCs derived from human peripheral blood monocytes cultured with a BA produce lower levels of interleukin‐12 (IL‐12) and tumour necrosis factor‐α in response to stimulation with commensal bacterial antigens. Stimulation through the nuclear receptor farnesoid X (FXR) did not affect the differentiation of DCs. However, DCs differentiated with the specific agonist for TGR5, a transmembrane BA receptor, showed an IL‐12 hypo‐producing phenotype. Expression of TGR5 could only be identified in monocytes and was rapidly down‐regulated during monocyte differentiation to DCs. Stimulation with 8‐bromoadenosine‐cyclic AMP (8‐Br‐cAMP), which acts downstream of TGR5 signalling, also promoted differentiation into IL‐12 hypo‐producing DCs. These results indicate that BAs induce the differentiation of IL‐12 hypo‐producing DCs from monocytes via the TGR5‐cAMP pathway.
This study aimed to evaluate the feasibility of and immune response to Wilms tumor gene 1 (WT1) peptide‐pulsed dendritic cell vaccination combined with gemcitabine (DCGEM) as a first‐line therapy ...among patients with advanced pancreatic cancer. Ten HLA‐A*2402 patients were treated with WT1 peptide‐pulsed DC vaccination (1 × 107 cells) on days 8 and 22 and gemcitabine (1000 mg/m2) on days 1, 8 and 15. Induction of a WT1‐specific immune response was evaluated using the delayed‐type hypersensitivity (DTH) skin test, interferon‐γ enzyme‐linked immunospot and HLA tetramer assays, along with assays for various immunological factors. DCGEM was well‐tolerated, and the relative dose intensity of gemcitabine was 87%. Disease control associated with a low neutrophil/lymphocyte ratio was observed in all three patients with DTH positivity; it was also correlated with a low percentage of granulocytic myeloid derived suppressor cells in the pretreatment peripheral blood (P = 0.017). Patients with liver metastases and high levels of inflammatory markers such as C‐reactive protein and interleukin‐8 (IL‐8) showed poor survival even though a WT1‐specific immune response was induced in them. WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions. This trial was registered with the University hospital Medical Information Network (UMIN) Clinical Trials Registry (http://www.umin.ac.jp/ctr/ number: UMIN‐000004855).
WT1 peptide‐pulsed DCGEM is feasible and effective for inducing anti‐tumor T‐cell responses. Our results support future investigations for pancreatic cancer patients with non‐liver metastases and favorable immunological conditions.
Aim
Liver macrophages play integral roles in both the progression and resolution of hepatic inflammation and fibrosis, comprising opposing functions that largely coincide with the activation state of ...nearby hepatic stellate cells (HSC). While cross‐talk between HSC and macrophages may be essential at various stages of inflammation and fibrogenesis, many facets of this interaction have yet to be thoroughly explored. Here, we examine the potential roles of HSC‐derived signaling molecules as mediators of liver macrophage differentiation.
Methods
Human peripheral blood mononuclear cells (PBMC) were differentiated to macrophages in the presence or absence of cultured HSC‐derived conditioned media. The phenotype of resulting macrophages was characterized by examination of cell surface marker expression, antigen‐presenting capabilities and cytokine secretion.
Results
Conditioned media from activated human HSC promoted the differentiation of a unique set of macrophages that differed in morphology and function from both classical (M1) and alternative (M2) macrophages, expressing increased levels of CD14 and CD16, as well as a distinct interleukin (IL)‐6high/IL‐10low/transforming growth factor (TGF)‐βhigh expression profile. These macrophages expressed high levels of CD206, CD209, CD80 and human leukocyte antigen DR, though no significant increases in antigen presentation were apparent. HSC‐derived macrophages exhibited specific activation of p38 mitogen‐activated protein kinase, and inhibition of this activation by p38 inhibitors during differentiation effectively reversed increases in IL‐6 and TGF‐β.
Conclusion
The present results suggest that HSC‐derived signaling molecules promote differentiation of liver macrophages with both pro‐inflammatory and profibrotic functions. Furthermore, these effects appear to be mediated, at least partially, in a p38‐dependent manner.
Background
Myeloid cell leukemia-1 (Mcl-1) is an anti-apoptotic protein that regulates apoptosis sensitivity in a variety of cell types. Here we evaluate the roles of Mcl-1 in chemotherapy-associated ...apoptosis in gastric cancer cells. In addition, our study examined whether Mcl-1 contributed to apoptosis resistance in so-called cancer stem cell (CSC)-like populations in gastric cancer.
Methods
Seven gastric cancer cell lines were used. The expression of Mcl-1 was assessed by either real-time polymerase chain reaction or Western blot analysis. Apoptosis was quantitated by morphological observation and caspase activity measurement. Adenovirus-mediated RNA interference (RNAi) technology was used to knockdown the expression of Mcl-1. The release of cytochrome
c
was evaluated by subcellular fractionation and immunoblot analysis. To identify and isolate the CSC-like populations, we used the CSC-associated cell surface marker CD44 and flow cytometry.
Results
Six out of the 7 gastric cancer cell lines overexpressed Mcl-1 protein. These Mcl-1-expressing cell lines were relatively resistant to chemotherapeutic agents such as 5-fluorouracil (5-FU) and cisplatin (CDDP). Depletion of Mcl-1 protein by RNAi technology effectively sensitized the cells to anticancer drug-induced mitochondrial cytochrome
c
release, caspase activation, and apoptosis. In addition, vast amounts of Mcl-1 mRNA were expressed in CD44-positive CSC-like cells. Mcl-1 suppression enhanced the apoptosis in CD44-positive cells to a level equivalent to that in CD44-negative cells, suggesting that Mcl-1 mediates chemotherapy resistance in CSC-like populations.
Conclusion
These results suggest that Mcl-1 mediates the resistance to apoptosis in gastric cancer cells by blocking the mitochondrial pathway of cell death. Mcl-1 depletion appears to be an attractive strategy to overcome chemotherapy resistance in gastric cancer cells.
DNA methylation plays a critical role in chromatin remodeling and gene expression. DNA methyltransferases (DNMTs) are hypothesized to mediate cellular DNA methylation status and gene expression ...during mammalian development and in malignant diseases. In this study, we examined the role of DNA methyltransferase 1 (DNMT1) and DNMT3b in cell proliferation and survival of hepatocellular carcinoma (HCC) cells. Gene silencing of both DNMT1 and DNMT3b by targeted siRNA knockdown reduces cell proliferation and sensitizes the cells to tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)‐mediated cell death. The proapoptotic protein caspase‐8 demonstrated promoter hypermethylation in HCC cells and was up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. In addition, death receptor TRAIL‐R2/DR5 (TRAIL receptor 2/death receptor 5) did not exhibit promoter hypermethylation in HCC cells but was also up‐regulated by knockdown of DNMT1 and DNMT3b both at mRNA and protein levels. Consistent with this observation, the combined transfection of DNMT1‐siRNA plus DNMT3b‐siRNA enhanced formation of the TRAIL‐death‐inducing signaling complex formation in HCC cells. In conclusion, our data suggest that DNA methylation of specific genomic regions maintained by DNMT1 and DNMT3b plays a critical role in survival of HCC cells, and a simultaneous knockdown of both DNMT1 and DNMT3b may be a novel anticancer strategy for the treatment of HCC. (Cancer Sci 2010)
Background/Aims: Hepatic steatosis sensitizes the liver to injury and inflammation by unclear mechanisms. Because Fas has been linked to liver injury and inflammation, Fas expression and ...sensitization to Fas signaling was examined in models of hepatic steatosis.
Methods: Mice were fed a carbohydrate diet while control animals received standard chow. Sensitization to Fas was examined following administration of Jo2 antibody. For the in vitro experiments, HepG2 cells were incubated with or without a mixture of long chain fatty acids (2:1 oleate:palmitate). Sensitization of the cells to Fas was examined using the CH11 antibody.
Results: Mice fed a high caloric diet developed hepatic steatosis, hyperlipidemia, insulin resistance, and hyperleptinemia, all features of the human syndrome. Fas expression in hepatocytes was increased as compared to lean animals and was coupled to cytotoxic signaling. Indeed, hepatocyte apoptosis, liver injury and chemokine generation were all accentuated in obese animals following administration of Jo-2, a Fas agonist. Hep G2 cells cultured in the presence of free fatty acids also developed ‘cellular steatosis’, upregulated Fas expression and were more sensitive to apoptosis by a Fas agonist.
Conclusions: Collectively, these data implicate Fas as a link between obesity associated fatty liver and increased susceptibility to liver damage.
Background and Aim: The efficacy of encircling abdominal compression devices in colonoscopies is inconsistent. We performed a meta-analysis of randomized controlled trials (RCTs) in which encircling ...abdominal compression devices were compared with control in colonoscopies.
We systematically searched RCTs published in the Cochrane Library, PubMed, and the Igaku-Chuo-Zasshi database. The data from the eligible RCTs were combined using the random-effects model. The weighted mean differences (WMDs), pooled odds ratios (ORs), and 95% confidence intervals (CIs) were calculated.
Five RCTs were included in this meta-analysis. Compared to the control group, encircling abdominal compression devices significantly reduced the caecal intubation time (WMD: -1.31, 95% CI: -2.40 to -0.23,
= 0.02). Compared to the control group, encircling abdominal compression devices significantly decreased the frequency of postural change (OR 0.30, 95% CI: 0.22 to 0.41,
< 0.00001). Compared to the control group, the use of encircling abdominal compression devices significantly reduced the need for abdominal compression (OR: 0.35, 95% CI: 0.17 to 0.70,
= 0.003).
Encircling abdominal compression devices in colonoscopies was found to reduce the caecal intubation time and the frequency of abdominal compression.
Hepatocyte apoptosis and stellate cell activation are both features of chronic liver diseases, but a relationship between these events has not been explored. In macrophages, engulfment of apoptotic ...bodies induces expression of transforming growth factor-β (TGF-β), a profibrogenic cytokine. We examined whether a similar response occurs in stellate cells. Fluorescently labeled hepatocyte apoptotic bodies were added to cultures of primary and immortalized human stellate cells. Stellate cells, but not hepatocytes, readily engulfed apoptotic bodies in a time-dependent manner as assessed by confocal microscopy. The activation of primary and immortalized human stellate cells after incubation with apoptotic bodies, as well as their fibrogenic activity, was indicated by an increase in α-smooth muscle actin (primary cells), TGF-β1, and collagen α1(I) mRNA (primary and immortalized cells). The profibrogenic response was dependent upon apoptotic body engulfment, because nocodazole, a microtubule-inhibiting agent, blocked both the engulfment and the increase of TGF-β1 and collagen α1(I) mRNA. As described in primary rodent stellate cells, up-regulation of collagen α1(I) mRNA was inhibited by a PI-3K inhibitor (LY294002) and a p38 mitogen-activated protein kinase inhibitor (SB203580) in LX-1 cells. In conclusion, these data support a model in which engulfment of hepatocyte apoptotic bodies by stellate cells leads to a fibrogenic response by eliciting a kinase-signaling pathway.
Background & Aims: Hepatocyte apoptosis and fibrosis are both features of liver injury. However, the potential mechanistic link between these 2 processes remains obscure. Our aim was to ascertain if ...Fas-mediated hepatocyte apoptosis promotes liver fibrogenesis during extrahepatic cholestasis. Methods: Wild-type and Fas-deficient lymphoproliferation (lpr) mice underwent bile duct ligation. Liver injury was assessed by quantitating hepatocyte apoptosis with the terminal deoxynucleotide transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and determining serum ALT values. mRNA expression was quantitated using real-time polymerase chain reaction technology. Liver fibrosis was assessed by digital image analysis of Sirius red–stained sections. Results: In 3-day bile duct ligated (BDL) animals, TUNEL-positive hepatocytes and serum ALT values were reduced in lpr versus wild-type animals. Likewise, hepatic mRNA transcripts for α-smooth muscle actin and platelet-derived growth factor receptor-β (initiation phase of stellate cell activation) and transforming growth factor β1 mRNA, collagen 1α, and tissue inhibitor of matrix metalloproteinases (perpetuation phase of stellate cell activation) were also reduced in 3-day BDL wild-type mice compared with lpr mice. Finally, in 3-week BDL mice, immunoreactivity for α-smooth muscle actin and Sirius red staining for collagen were significantly less in lpr compared with wild-type animals. Conclusion: Fas-mediated hepatocyte injury is mechanistically linked to liver fibrogenesis. These observations suggest that inhibition of Fas-mediated apoptosis may be a therapeutic antifibrogenic strategy in cholestatic liver diseases.
GASTROENTEROLOGY 2002;123:1323-1330
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