Sepsis is a life-threatening clinical condition which is particularly serious among the elderly who experience considerably higher mortality rates compared to younger patients. Using a sterile endotoxemia model, we previously reported age-dependent mortality in conjunction with enhanced coagulation and insufficient levels of anti-coagulant factor activated protein C (aPC). The purpose of the present study was to further investigate the mechanisms for age-dependent coagulation and aPC insufficiency during experimental sepsis. Intra-abdominal sepsis was induced by cecal ligation and puncture (CLP) using 21 or 16 gauge (G) needles (double-puncture) on young (4-6 months old) and aged (20-25 months old) male C57BL/6 mice. Compared to young, aged mice showed significantly increased mortality (92% vs 28%), systemic inflammation, and coagulation in the lung and kidney after 21G CLP. Young mice with more severe CLP (16G) showed a mortality rate and inflammation equivalent to aged mice with 21G CLP; however, enhanced coagulation and kidney dysfunction were significant only in the aged. In young mice, increased levels of aPC after CLP was coupled with reduced levels of PC suggesting the conversion of PC to aPC; however, PC and aPC levels remained unchanged in aged mice, indicating a lack of PC to aPC conversion. Activation of fibrinolysis, determined by plasma D-dimer levels, was similar regardless of age or CLP severity and plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, showed severity-dependent induction independent of age. These results suggest that enhanced coagulation in aged mice during sepsis is due to dysfunction of the PC activation mechanism.
Expression of interleukin-6 (IL-6) upon acute inflammatory stress is significantly augmented by aging in adipose tissue, a major source of this cytokine. In the present study, we examined the mechanism of age-dependent IL-6 overproduction using visceral white adipose tissue from C57BL/6 mice. Upon treatment with lipopolysaccharide (LPS) in vitro, IL-6 was produced by adipose tissue explants, and secreted levels were significantly higher in cultures from aged (24 months) mice compared to young (4 months). Interleukin 1 beta (IL-1?) and tumor necrosis factor alpha (TNF?), two inducers of IL-6, were mainly produced by the lungs and spleen rather than adipose tissue in mice after LPS injection. Treatment of adipose explants with physiological levels of IL-1? induced significant age-dependent secretion of IL-6, while treatment with TNF? had little effect, demonstrating an augmented response of adipose tissues to IL-1? in the aged. In vitro experiments utilizing a neutralizing antibody against IL-1? and in vivo experiments utilizing IL-1-receptor-1 deficient mice, confirmed that IL-6 overproduction in the aged is regulated by autocrine/paracrine action of IL-1? which specifically occurs in aged adipose tissues. These findings indicate an elevated inflammatory potential of adipose tissue in the aged and a unique IL-1?-mediated mechanism for IL-6 overproduction, which may impact age-associated vulnerability to acute inflammatory diseases such as sepsis.
The intestinal mucosa undergoes a continual process of proliferation, differentiation, and apoptosis that is regulated by multiple signaling pathways. Previously, we have shown that the nuclear factor of activated T-cells 5 (NFAT5) is involved in the regulation of intestinal enterocyte differentiation. Here we show that treatment with sodium chloride (NaCl), which activates NFAT5 signaling, increased mTORC1 repressor regulated in development and DNA damage response 1 (REDD1) protein expression and inhibited mTOR signaling; these alterations were attenuated by knockdown of NFAT5. Knockdown of NFAT5 activated mammalian target of rapamycin (mTOR) signaling and significantly inhibited REDD1 mRNA expression and protein expression. Consistently, overexpression of NFAT5 increased REDD1 expression. In addition, knockdown of REDD1 activated mTOR and Notch signaling, whereas treatment with mTOR inhibitor rapamycin repressed Notch signaling and increased the expression of the goblet cell differentiation marker mucin 2 (MUC2). Moreover, knockdown of NFAT5 activated Notch signaling and decreased MUC2 expression, while overexpression of NFAT5 inhibited Notch signaling and increased MUC2 expression. Our results demonstrate a role for NFAT5 in the regulation of mTOR signaling in intestinal cells. Importantly, these data suggest that NFAT5 participates in the regulation of intestinal homeostasis via the suppression of mTORC1/Notch signaling pathway.
Wnt/?-catenin signaling plays a pivotal role in regulating cell growth and differentiation by activation of the ?-catenin/T-cell factor (TCF) complex and subsequent regulation of a set of target genes that have one or more TCF-binding elements (TBEs). Hyperactivation of this pathway has been implicated in numerous malignancies including human neuroendocrine tumors (NETs). Neurotensin (NT), an intestinal hormone, induces proliferation of several gastrointestinal (GI) cancers including cancers of the pancreas and colon. Here, we analyzed the human NT promoter in silico and found at least four consensus TBEs within the proximal promoter region. Using a combination of ChIP and luciferase reporter assays, we identified one TBE (located ?900 bp proximal from the transcription start site) that was immunoprecipitated efficiently by TCF4-targeting antibody; mutation of this site attenuated the responsiveness to ?-catenin. We also confirmed that the promoter activity and the mRNA and protein expression levels of NT were increased by various Wnt pathway activators and decreased by Wnt inhibitors in NET cell lines BON and QGP-1, which express and secrete NT. Similarly, the intracellular content and secretion of NT were induced by Wnt3a in these cells. Finally, inhibition of NT signaling suppressed cell proliferation and anchorage-independent growth and decreased expression levels of growth-related proteins in NET cells. Our results indicate that NT is a direct target of the Wnt/?-catenin pathway and may be a mediator for NET cell growth.
Upregulation of fatty acid synthase (FASN), a key enzyme of de novo lipogenesis, is associated with metastasis in colorectal cancer (CRC). However, the mechanisms of regulation are unknown. Since angiogenesis is crucial for metastasis, we investigated the role of FASN in the neovascularization of CRC. The effect of FASN on tumor vasculature was studied in orthotopic CRCs, the chick embryo chorioallantoic membrane (CAM) and Matrigel plug models using immunohistochemistry, immunofluorescent staining and confocal microscopy. Cell secretion was evaluated by ELISA and antibody arrays. Proliferation, migration and tubulogenesis of endothelial cells (ECs) were assessed in CRC-EC coculture models. In this study, we found that stable knockdown of FASN decreased microvessel density in HT29 and HCT116 orthotopic CRCs and resulted in 'normalization' of tumor vasculature in both orthotopic and CAM models. Furthermore, FASN regulated secretion of pro- and antiangiogenic factors, including vascular endothelial growth factor-A (VEGF-A). Mechanisms associated with the antiangiogenic activity noted with knockdown of FASN included: downregulation of VEGF(189), upregulation of antiangiogenic isoform VEGF(165b) and a decrease in expression and activity of matrix metalloproteinase-9. Furthermore, conditioned medium from FASN knockdown CRC cells inhibited activation of vascular endothelial growth factor receptor-2 and its downstream signaling and decreased proliferation, migration and tubulogenesis of ECs as compared with control medium. Together, these results suggest that cancer cell-associated FASN regulates tumor vasculature through alteration of the profile of secreted angiogenic factors and regulation of their bioavailability. Inhibition of FASN upstream of VEGF-A and other angiogenic pathways can be a novel therapeutic strategy to prevent or inhibit metastasis in CRC.
Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we report a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a "histone H4-mimic" GK-X-GK motif that is diacetylated by Tip60. The diacetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructing an activated Twist/BRD4/P-TEFb/RNA-Pol II complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.
The precise involvement of the PI3K/mTOR and RAS/MEK pathways in carcinoid tumors is not well defined. Therefore, the purpose of our study was to evaluate the role these pathways play in carcinoid cell proliferation, apoptosis, and secretion and to determine the effects of combined treatment on carcinoid tumor inhibition.
Combined resection of primary colorectal cancer and synchronous hepatic metastases has been shown to be safe and associated with acceptable oncologic outcomes in selected patients. The purpose of this study was to determine if selection criteria for combined resection could be identified using major morbidity or mortality as an avoidable outcome.
Activation of the Wnt/?-catenin pathway has been observed in at least 1/3 of hepatocellular carcinomas (HCC), and a significant number of these have mutations in the ?-catenin gene. Therefore, effective inhibition of this pathway could provide a novel method to treat HCC. The purposed of this study was to determine whether FH535, which was previously shown to block the ?-catenin pathway, could inhibit ?-catenin activation of target genes and inhibit proliferation of Liver Cancer Stem Cells (LCSC) and HCC cell lines. Using ?-catenin responsive reporter genes, our data indicates that FH535 can inhibit target gene activation by endogenous and exogenously expressed ?-catenin, including the constitutively active form of ?-catenin that contains a Serine37Alanine mutation. Our data also indicate that proliferation of LCSC and HCC lines is inhibited by FH535 in a dose-dependent manner, and that this correlates with a decrease in the percentage of cells in S phase. Finally, we also show that expression of two well-characterized targets of ?-catenin, Cyclin D1 and Survivin, is reduced by FH535. Taken together, this data indicates that FH535 has potential therapeutic value in treatment of liver cancer. Importantly, these results suggest that this therapy may be effective at several levels by targeting both HCC and LCSC.
Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.
The ingenious design of the bacterial virus phi29 DNA packaging nanomotor with an elegant and elaborate channel has inspired its application for single molecule detection of antigen/antibody interactions. The hub of this bacterial virus nanomotor is a truncated cone-shaped connector consisting of 12 protein subunits. These subunits form a ring with a central 3.6-nm channel acting as a path for dsDNA to enter during packaging and to exit during infection. The connector has been inserted into a lipid bilayer. Herein, we reengineered an Epithelial Cell Adhesion Molecule (EpCAM) peptide into the C-terminal of nanopore as a probe to specifically detect EpCAM antibody (Ab) in nanomolar concentration at the single molecule level. The binding of Abs sequentially to each peptide probe induced stepwise blocks in current. The distinctive current signatures enabled us to analyze the docking and undocking kinetics of Ab-probe interactions and determine the Kd. The signal of EpCAM antibody can be discriminated from the background events in the presence of nonspecific antibody or serum. Our results demonstrate the feasibility of generating a highly sensitive platform for detecting antibodies at extremely low concentrations in the presence of contaminants.
Human genome sequencing revealed that only ~1.5% of the DNA sequence coded for proteins. More and more evidence has uncovered that a substantial part of the 98.5% so-called "junk" DNAs actually code for noncoding RNAs. Two milestones, chemical drugs and protein drugs, have already appeared in the history of drug development, and it is expected that the third milestone in drug development will be RNA drugs or chemical drugs that target RNA. This review focuses on the development of RNA therapeutics for potential cancer treatment by applying RNA nanotechnology. A therapeutic RNA nanoparticle is unique in that its scaffold, ligand, and therapeutic component can all be composed of RNA. The special physicochemical properties lend to the delivery of siRNA, miRNA, ribozymes, or riboswitches; imaging using fluogenenic RNA; and targeting using RNA aptamers. With recent advances in solving the chemical, enzymatic, and thermodynamic stability issues, RNA nanoparticles have been found to be advantageous for in vivo applications due to their uniform nano-scale size, precise stoichiometry, polyvalent nature, low immunogenicity, low toxicity, and target specificity. In vivo animal studies have revealed that RNA nanoparticles can specifically target tumors with favorable pharmacokinetic and pharmacodynamic parameters without unwanted accumulation in normal organs. This review summarizes the key studies that have led to the detailed understanding of RNA nanoparticle formation as well as chemical and thermodynamic stability issue. The methods for RNA nanoparticle construction, and the current challenges in the clinical application of RNA nanotechnology, such as endosome trapping and production costs, are also discussed.
Small interfering RNA (siRNA) provides a highly selective method to target mutated pathways; however, its use is complicated by specific delivery to tumor cells. The aims of the present study were to develop a novel murine model of portal vein catheterization for the chronic delivery of therapeutic agents to liver metastases, determine the benefits of local delivery of siRNA to liver metastases, and determine the utility of epithelial cell adhesion molecule (EpCAM) as a selective target for siRNA delivery to colorectal cancer (CRC) metastases.
Phosphatidylinositol 4-phosphate 5-kinase type I ? (PIPKI?90) binds talin and localizes at focal adhesions (FAs). Phosphatidylinositol (4,5)-bisphosphate (PIP2) generated by PIPKI?90 is essential for FA formation and cell migration. On the other hand, PIPKI?90 and the ?-integrin tail compete for overlapping binding sites on talin. Enhanced PIPKI?90-talin interaction suppresses talin binding to the ?-integrin. It is unknown how PIPKI?90 is removed from the PIPKI?90-talin complex after on-site PIP2 production during cell migration. Here we show that PIPKI?90 is a substrate for HECTD1, an E3 ubiquitin ligase regulating cell migration. HECTD1 ubiquitinated PIPKI?90 at lysine 97 and resulted in PIPKI?90 degradation. Expression of the mutant PIPKI?90(K97R) enhanced PIP2 and PIP3 production, inhibited FA assembly and disassembly and inhibited cancer cell migration, invasion and metastasis. Interestingly, mutation at tryptophan 647 abolished the inhibition of PIPKI?90(K97R) on FA dynamics and partially rescued cancer cell migration and invasion. Thus, cycling PIPKI?90 ubiquitylation by HECTD1 and consequent degradation remove PIPKI?90 from talin after on-site PIP2 production, providing an essential regulatory mechanism for FA dynamics and cell migration.
PURPOSE: To develop novel hybrid paclitaxel (PTX) nanocrystals, in which bioactivatable (MMPSense® 750 FAST) and near infrared (Flamma Fluor® FPR-648) fluorophores are physically incorporated, and to evaluate their anticancer efficacy and diagnostic properties in breast cancer xenograft murine model. METHODS: The pure and hybrid paclitaxel nanocrystals were prepared by an anti-solvent method, and their physical properties were characterized. The tumor volume change and body weight change were evaluated to assess the treatment efficacy and toxicity. Bioimaging of treated mice was obtained non-invasively in vivo. RESULTS: The released MMPSense molecules from the hybrid nanocrystals were activated by matrix metalloproteinases (MMPs) in vivo, similarly to the free MMPSense, demonstrating its ability to monitor cancer progression. Concurrently, the entrapped FPR-648 was imaged at a different wavelength. Furthermore, when administered at 20 mg/kg, the nanocrystal formulations exerted comparable efficacy as Taxol®, but with decreased toxicity. CONCLUSIONS: Hybrid nanocrystals that physically integrated two fluorophores were successfully prepared from solution. Hybrid nanocrystals were shown not only exerting antitumor activity, but also demonstrating the potential of multi-modular bioimaging for diagnostics.
Paclitaxel (PTX) nanocrystals (200 nm) were produced by crystallization from a solution. Antitumor efficacy and toxicity were examined through a survival study in a human HT-29 colon cancer xenograft murine model. The antitumor activity of the nanocrystal treatments was comparable with that by the conventional solubilization formulation (Taxol®), but yielded less toxicity as indicated by the result of a survival study. Tritium-labeled PTX nanocrystals were further produced with a near infrared (NIR) fluorescent dye physically integrated in the crystal lattice. Biodistribution and tumor accumulation of the tritium-labeled PTX nanocrystals were determined immediately after intravenous administration and up to 48 h by scintillation counting. Whole-body optical imaging of animals was concurrently carried out; fluorescent intensities were also measured from excised tumors and major organs of euthanized animals. It was found that drug accumulation in the tumor was less than 1% of 20mg/kg intravenous dose. Qualitatively correlation was identified between the biodistribution determined by using tritium-labeled particles and that using optical imaging, but quantitative divergence existed. The divergent results suggest possible ways to improve the design of hybrid nanocrystals for cancer therapy and diagnosis. The study also raises questions of the general role of the enhanced permeability and retention (EPR) effect in tumor targeting and the effectiveness of bioimaging, specifically for theranostics, in tracking drug distribution and pharmacokinetics.
Deregulated Ras/Raf/mitogen-activated protein kinase and PI3 K/AKT/mTOR signaling pathways are significant in hepatocellular carcinoma proliferation (HCC). In this study we evaluated differences in the antiproliferative effect of dual PI3 K/Akt/mTOR and Ras/Raf/mitogen-activated protein kinase inhibition of non liver cancer stem cell lines (PLC and HuH7) and liver cancer stem cell (LCSC) lines (CD133, CD44, CD24, and aldehyde dehydrogenase 1-positive cells).
Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells in vitro and in vivo. Using a biotinylated FIDAS analogue, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC cell growth. A novel FIDAS agent delivered orally repressed CRC xenografts in athymic nude mice. These findings suggest that FIDAS analogues targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment.
Tolerance to physiological stress resulting from inflammatory disease decreases significantly with age. High mortality rates, increased cytokine production, and pronounced thrombosis are characteristic complications of aged mice with acute systemic inflammation induced by injection with lipopolysaccharide (LPS). As adipose tissue is now recognized as an important source of cytokines, we determined the effects of aging on visceral white adipose tissue gene expression during LPS-induced inflammation in male C57BL/6 mice. Microarray analysis revealed that the expression of 6025 genes was significantly changed by LPS; of those, the expression of 667 showed an age-associated difference. Age-associated differences were found in many genes belonging to the inflammatory response and blood clotting pathways. Genes for several procoagulant factors were upregulated by LPS; among these, tissue factor, thrombospondin-1, and plasminogen activator inhibitors-1 and -2, exhibited age-associated increases in expression which could potentially contribute to augmented thrombosis. Further analysis by qRT-PCR, histological examination, and cell fraction separation revealed that most inflammatory and coagulant-related gene expression changes occur in resident stromal cells rather than adipocytes or infiltrating cells. In addition, basal expression levels of 303 genes were altered by aging, including increased expression of component of Sp100-rs (Csprs). This study indicates that adipose tissue is a major organ expressing genes for multiple inflammatory and coagulant factors and that the expression of many of these is significantly altered by aging during acute inflammation. Data presented here provide a framework for future studies aimed at elucidating the impact of adipose tissue on age-associated complications during sepsis and systemic inflammation.
The epithelial-mesenchymal transition (EMT) enhances cancer invasiveness and confers tumor cells with cancer stem cell (CSC)-like characteristics. We show that the Snail-G9a-Dnmt1 complex, which is critical for E-cadherin promoter silencing, is also required for the promoter methylation of fructose-1,6-biphosphatase (FBP1) in basal-like breast cancer (BLBC). Loss of FBP1 induces glycolysis and results in increased glucose uptake, macromolecule biosynthesis, formation of tetrameric PKM2, and maintenance of ATP production under hypoxia. Loss of FBP1 also inhibits oxygen consumption and reactive oxygen species production by suppressing mitochondrial complex I activity; this metabolic reprogramming results in an increased CSC-like property and tumorigenicity by enhancing the interaction of ?-catenin with T-cell factor. Our study indicates that the loss of FBP1 is a critical oncogenic event in EMT and BLBC.
Carcinoid tumors are rare neuroendocrine tumors (NETs) that are increasing in incidence. Mutation and altered expression of Wnt/?-catenin signaling components have been described in many tumors but have not been well-studied in NETs. Here, we observed accumulation of ?-catenin in the cytoplasm and/or nucleus in 25% of clinical NET tissues. By mutational analysis, the mutations of ?-catenin (I35S) and APC (E1317Q, T1493T) were identified in NET cells and the tissues. Expression of representative Wnt inhibitors was absent or markedly decreased in BON, a human pancreatic carcinoid cell line; treatment with 5-aza-2-deoxycytidine (5-aza-CdR) increased expression levels of the Wnt inhibitors. Methylation analyses demonstrated that CpG islands of SFRP-1 and Axin-2 were methylated, whereas the promoters of DKK-1, DKK-3 and WIF-1 were unmethylated in four NET cells. Aberrant methylation of SFRP-1 was particularly observed in most of clinical NET tissues. In addition, the repression of these unmethylated genes was associated with histone H3 lysine 9 dimethylation (H3K9me2) in BON cells. Together, 5-aza-CdR treatment inhibited cell proliferation and decreased the protein levels of H3K9me2 and G9a. Moreover, a novel G9a inhibitor, UNC0638, suppressed BON cell proliferation through inhibition of Wnt/?-catenin pathway. Overexpression of the inhibitory genes, particularly SFRP-1 and WIF-1 in BON cells, resulted in suppression of anchorage-independent growth and inhibition of tumor growth in mice. Our findings suggest that aberrant Wnt/?-catenin signaling, through either mutations or epigenetic silencing of Wnt antagonists, contributes to the pathogenesis and growth of NETs and have important clinical implications for the prognosis and treatment of NETs.
LSD1 is a critical chromatin modulator that controls cellular pluripotency and differentiation through the demethylation of H3K4me1/2. Overexpression of LSD1 has been observed in many types of tumors and is correlated with its oncogenic effects in tumorigenesis. However, the mechanism leading to LSD1 upregulation in tumors remains unclear. Using an unbiased siRNA screening against all the human deubiquitinases, we identified USP28 as a bona fide deubiquitinase of LSD1. USP28 interacted with and stabilized LSD1 via deubiquitination. USP28 overexpression correlated with LSD1 upregulation in multiple cancer cell lines and breast tumor samples. Knockdown of USP28 resulted in LSD1 destabilization, leading to the suppression of cancer stem cell (CSC)-like characteristics in vitro and inhibition of tumorigenicity in vivo, which can be rescued by ectopic LSD1 expression. Our study reveals a critical mechanism underlying the epigenetic regulation by USP28 and provides another treatment approach against breast cancer.
Production of inflammatory cytokines by mesenteric adipose tissue (MAT) has been implicated in the pathogenesis of inflammatory bowel disease (IBD). Animal models of colitis have demonstrated inflammatory changes within MAT, but it is unclear if these changes occur in isolation or as part of a systemic adipose tissue response. It is also unknown what cell types are responsible for cytokine production within MAT. The present study was designed to determine whether cytokine production by MAT during experimental colitis is depot-specific, and also to identify the source of cytokine production within MAT.
B lymphoma Mo-MLV insertion region 1 (Bmi1) is a Polycomb Group (PcG) protein important in gene silencing. It is a component of Polycomb Repressive Complex 1 (PRC1), which is required to maintain the transcriptionally repressive state of many genes. Bmi1 was initially identified as an oncogene that regulates cell proliferation and transformation, and is important in hematopoiesis and the development of nervous systems. Recently, it was reported that Bmi1 is a potential marker for intestinal stem cells. Because Wnt signaling plays a key role in intestinal stem cells, we analyzed the effects of Wnt signaling on Bmi1 expression. We found that Wnt signaling indeed regulates the expression of Bmi1 in colon cancer cells. In addition, the expression of Bmi1 in human colon cancers is significantly associated with nuclear ?-catenin, a hallmark for the activated Wnt signaling. Krüppel-like factor 4 (KLF4) is a zinc finger protein highly expressed in the gut and skin. We recently found that KLF4 cross-talks with Wnt/?-catenin in regulating intestinal homeostasis. We demonstrated that KLF4 directly inhibits the expression of Bmi1 in colon cancer cells. We also found that Bmi1 regulates histone ubiquitination and is required for colon cancer proliferation in vitro and in vivo. Our findings further suggest that Bmi1 is an attractive target for cancer therapeutics.
Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation.
Colon cancer is the third most common cause of cancer and is the second leading cause of cancer deaths in the USA. Although inhibition of aldose reductase (AR) is known to prevent human colon cancer cell growth in nude mice xenografts, the role of AR in the regulation of cancer metastasis is not known. We now demonstrate the mechanisms by which AR regulates colon cancer metastasis in vitro and in vivo. Inhibition of AR prevented the epidermal growth factor (EGF) or fibroblast growth factor (FGF)-induced migration and invasion of human colon cancer (HT29; KM20) cells by >70% and also inhibited (>80%) the adhesion of the cancer cells to endothelial cells. Treatment of endothelial cells with AR inhibitors significantly (?85%) downregulated the EGF or FGF-induced expression of Inter-Cellular Adhesion Molecule-1, Vascular cell adhesion molecule-1 and vascular endothelial-cadherin. Furthermore, liver metastasis of green fluorescent protein-labeled KM20 cells injected into the spleen of athymic nude mice was significantly (>65%) prevented by AR inhibitor, fidarestat or ARsiRNA delivered systemically into the mice. Similar results were observed with HT29 cells. AR inhibition or ablation also prevented (70-90%) the increase in the levels of matrix metalloproteinase-2, cyclin D1, CD31, CD34 and the activation of nuclear factor-kappa-binding protein in metastatic liver. Thus, our results indicate that AR regulates cancer cell adhesion, invasion and migration events which initiate metastasis and therefore, AR inhibition could be a novel therapeutic approach for the prevention of colon cancer metastasis.
Carcinoid tumors are associated with the carcinoid syndrome, a set of symptoms resulting from the peptide and amine products, including serotonin, secreted from the cancer cells. The purpose of this study was to investigate the relationship between the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) inhibitor PTEN (phosphatase and tensin homolog deleted on chromosome ten) and serotonin synthesis and secretion in the carcinoid cancer cell line BON.
The mammalian target of rapamycin (mTOR) signaling exists in two complexes: mTORC1 and mTORC2. Neurotensin (NT), an intestinal hormone secreted by enteroendocrine (N) cells in the small bowel, has important physiological effects in the gastrointestinal tract. The human endocrine cell line BON abundantly expresses the NT gene and synthesizes and secretes NT in a manner analogous to that of N cells. Here, we demonstrate that the inhibition of mTORC1 by rapamycin (mTORC1 inhibitor), torin1 (both mTORC1 and mTORC2 inhibitor) or short hairpin RNA-mediated knockdown of mTOR, regulatory associated protein of mTOR (RAPTOR), and p70 S6 kinase (p70S6K) increased basal NT release via upregulating NT gene expression in BON cells. c-Jun activity was increased by rapamycin or torin1 or p70S6K knockdown. c-Jun overexpression dramatically increased NT promoter activity, which was blocked by PD98059, an mitogen-activated protein kinase kinase (MEK) inhibitor. Furthermore, overexpression of MEK1 or extracellular signal-regulated kinase 1 (ERK1) increased c-Jun expression and NT promoter activity. More importantly, PD98059 blocked rapamycin- or torin1-enhanced NT secretion. Consistently, rapamycin and torin1 also increased NT gene expression in Hep3B cells, a human hepatoma cell line that, similar to BON, expresses high levels of NT. Phosphorylation of c-Jun and ERK1/2 was also increased by rapamycin and torin1 in Hep3B cells. Finally, we showed activation of mTOR in BON cells treated with amino acids, high glucose, or serum and, concurrently, the attenuation of ERK1/2 and c-Jun phosphorylation and NT secretion. Together, mTORC1, as a nutrient sensor, negatively regulates NT secretion via the MEK/ERK/c-Jun signaling pathway. Our results identify a physiological link between mTORC1 and MEK/ERK signaling in controlling intestinal hormone gene expression and secretion.
Neurotensin is a 13-amino acid peptide found in the central nervous system central nervous system and the gastrointestinal tract. Since its initial discovery in 1973, neurotensin has been shown to play a role in a wide range of physiologic and pathologic processes throughout the body. Ongoing research efforts continue to clarify the role of neurotensin in various central nervous system and gastrointestinal processes, as well as how disruption of these normal mechanisms may lead to diseases ranging from schizophrenia to colorectal cancer. The goal of this review is to provide an overview of the most recent advances in the field of neurotensin research, in the context of what has been previously published.
Activation of phosphoinositide 3-kinase (PI3K)/Akt signaling is associated with growth and progression of colorectal cancer (CRC). We have previously shown that the mTOR kinase, a downstream effector of PI3K/Akt signaling, regulates tumorigenesis of CRC. However, the contribution of mTOR and its interaction partners toward regulating CRC progression and metastasis remains poorly understood. We found that increased expression of mTOR, Raptor, and Rictor mRNA was noted with advanced stages of CRC, suggesting that mTOR signaling may be associated with CRC progression and metastasis. mTOR, Raptor, and Rictor protein levels were also significantly elevated in primary CRCs (stage IV) and their matched distant metastases compared with normal colon. Inhibition of mTOR signaling, using rapamycin or stable inhibition of mTORC1 (Raptor) and mTORC2 (Rictor), attenuated migration and invasion of CRCs. Furthermore, knockdown of mTORC1 and mTORC2 induced a mesenchymal-epithelial transition (MET) and enhanced chemosensitivity of CRCs to oxaliplatin. We observed increased cell-cell contact and decreased actin cytoskeletal remodeling concomitant with decreased activation of the small GTPases, RhoA and Rac1, upon inhibition of both mTORC1 and mTORC2. Finally, establishment of CRC metastasis in vivo was completely abolished with targeted inhibition of mTORC1 and mTORC2 irrespective of the site of colonization. Our findings support a role for elevated mTORC1 and mTORC2 activity in regulating epithelial-mesenchymal transition (EMT), motility, and metastasis of CRCs via RhoA and Rac1 signaling. These findings provide the rationale for including mTOR kinase inhibitors, which inhibit both mTORC1 and mTORC2, as part of the therapeutic regimen for CRC patients.
Reactive oxygen species (ROS) are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC). Mitochondria as a major source of intracellular ROS and apoptotic signaling during oxidative stress in NEC have not been investigated. We sought to determine: (1) the effects of oxidative stress on intestinal mitochondrial apoptotic signaling, and (2) the role of growth factors in this process.
TNF-related apoptosis-inducing ligand (TRAIL; Apo2) has been shown to promote intestinal cell differentiation. Nuclear factor of activated T cells (NFAT) participates in the regulation of a variety of cellular processes, including differentiation. Here, we examined the role of NFAT in the regulation of TRAIL in human intestinal cells. Treatment with a combination of phorbol 12-myristate 13-acetate (PMA) plus the calcium ionophore A23187 (Io) increased NFAT activation and TRAIL expression; pretreatment with the calcineurin inhibitor cyclosporine A (CsA), an antagonist of NFAT signaling, diminished NFAT activation and TRAIL induction. In addition, knockdown of NFATc1, NFATc2, NFATc3, and NFATc4 blocked PMA/Io increased TRAIL protein expression. Expression of NFATc1 activated TRAIL promoter activity and increased TRAIL mRNA and protein expression. Deletion of NFAT binding sites from the TRAIL promoter did not significantly abrogate NFATc1-increased TRAIL promoter activity, suggesting an indirect regulation of TRAIL expression by NFAT activation. Knockdown of NFATc1 increased Sp1 transcription factor binding to the TRAIL promoter and, importantly, inhibition of Sp1, by chemical inhibition or RNA interference, increased TRAIL expression. These studies identify a novel mechanism for TRAIL regulation by which activation of NFATc1 increases TRAIL expression through negative regulation of Sp1 binding to the TRAIL promoter.
Colorectal cancer (CRC) is the second leading cause of cancer-related mortality in the United States. CRC is initiated by mutations of the tumor suppressor gene, adenomatous polyposis coli (APC), or ?-catenin gene. These mutations stabilize ?-catenin and constitutively activate Wnt/?-catenin target genes, such as c-Myc and cyclin D1, ultimately leading to cancer. Naturally occurring stilbene derivatives, resveratrol and pterostilbene, inhibit Wnt signaling and repress CRC cell proliferation but are ineffective at concentrations less than 10 ?M. To understand the structure--activity relationship within these stilbene derivatives and to develop more efficacious Wnt inhibitors than these natural products, we synthesized and evaluated a panel of fluorinated N,N-dialkylaminostilbenes. Among this panel, (E)-4-(2,6-difluorostyryl)-N,N-dimethylaniline (4r) inhibits Wnt signaling at nanomolar levels and inhibits the growth of human CRC cell xenografts in athymic nude mice at a dosage of 20 mg/kg. These fluorinated N,N-dialkylaminostilbenes appear to inhibit Wnt signaling downstream of ?-catenin, probably at the transcriptional level.
Aberrant Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study reports how sorafenib (a multi-kinase inhibitor) and PI-103 (a dual PI3K/mTOR inhibitor) alone and in combination inhibit the proliferation of the HCC cell line, Huh7.
The nuclear factor of activated T cell (NFAT) proteins are a family of transcription factors (NFATc1-c4) involved in the regulation of cell differentiation and adaptation. Previously we demonstrated that inhibition of phosphatidylinositol 3-kinase or overexpression of PTEN enhanced intestinal cell differentiation. Here we show that treatment of intestinal-derived cells with the differentiating agent sodium butyrate (NaBT) increased PTEN expression, NFAT binding activity, and NFAT mRNA expression, whereas pretreatment with the NFAT signaling inhibitor cyclosporine A (CsA) blocked NaBT-mediated PTEN induction. Moreover, knockdown of NFATc1 or NFATc4, but not NFATc2 or NFATc3, attenuated NaBT-induced PTEN expression. Knockdown of NFATc1 decreased PTEN expression and increased the phosphorylation levels of Akt and downstream targets Foxo1 and GSK-3?/?. Furthermore, overexpression of NFATc1 or the NFATc4 active mutant increased PTEN and p27(kip1) expression and decreased Akt phosphorylation. In addition, pretreatment with CsA blocked NaBT-mediated induction of intestinal alkaline phosphatase (IAP) activity and villin and p27(kip1) expression; knockdown of either NFATc1 or NFATc4 attenuated NaBT-induced IAP activity. We provide evidence showing that NFATc1 and NFATc4 are regulators of PTEN expression. Importantly, our results suggest that NFATc1 and NFATc4 regulation of intestinal cell differentiation may be through PTEN regulation.
Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine N cells in the distal small intestine. We have identified key regulatory elements in the promoter region that are involved in human NT/N (hNT/N) gene expression in the novel human endocrine cell line, BON, which resembles intestinal N cells in several important aspects including NT/N precursor protein processing, ratios of different NT/N mRNA isoforms, and high levels of constitutive expression of the NT/N gene. In this study, we demonstrated multiple cis-regulatory elements including a proximal region containing a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein (CREB)/ATF proteins (c-Jun, ATF-1, ATF-2, JunD, and CREB). Similar to the rat NT/N gene, this region is critical for constitutive hNT/N gene expression. Moreover, we identified a novel region that binds the orphan hormone receptor, NR2F2. We have demonstrated that the C terminus of NR2F2 strongly represses hNT/N transcription, whereas an N-terminal domain antagonizes this repressive effect. Regulation of NT/N expression by NR2F2 may have important consequences for lipid metabolism. We speculate that a complex interplay between the proximal CRE/AP-1-like motif and NR2F2 binding region exists to regulate hNT/N expression, which is critical for the high level of constitutive expression of NT/N in enteroendocrine cells. Finally, the BON cell line provides a unique model to characterize the factors regulating expression of the hNT/N gene and to better understand the mechanisms responsible for terminal differentiation of the N cell lineage in the gut.
Systemic inflammatory response syndrome (SIRS), a serious clinical condition characterized by whole-body inflammation, is particularly threatening for elderly patients, who suffer much higher mortality rates than the young. A major pathological consequence of SIRS is acute lung injury caused by neutrophil-mediated oxidative damage. Previously, we reported an increase in protein tyrosine nitration (a marker of oxidative/nitrosative damage) and a decrease in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in the lungs of young mice during endotoxemia-induced SIRS. Here we demonstrate that during endotoxemia, down-regulation of EC-SOD is significantly more profound and prolonged, whereas up-regulation of iNOS is augmented, in aged compared to young mice. Aged mice also showed 2.5-fold higher protein nitration levels, compared to young mice, with particularly strong nitration in the pulmonary vascular endothelium during SIRS. Additionally, by two-dimensional gel electrophoresis, Western blotting, and mass spectrometry, we identified proteins that show increased tyrosine nitration in age- and SIRS-dependent manners; these proteins (profilin-1, transgelin-2, LASP 1, tropomyosin, and myosin) include components of the actin cytoskeleton responsible for maintaining pulmonary vascular permeability. Reduced EC-SOD in combination with increased oxidative/nitrosative damage and altered cytoskeletal protein function due to tyrosine nitration may contribute to augmented lung injury in the aged with SIRS.
Neurotensin, a gut peptide, stimulates the growth of colorectal cancers that possess the high-affinity neurotensin receptor (NTR1). Sodium butyrate (NaBT) is a potent histone deacetylase inhibitor (HDACi) that induces growth arrest, differentiation, and apoptosis of colorectal cancers. Previously, we had shown that NaBT increases nuclear GSK-3beta expression and kinase activity; GSK-3beta functions as a negative regulator of extracellular signal-regulated kinase (ERK) signaling. The purpose of our current study was to determine: (a) whether HDACi alters NTR1 expression and function, and (b) the role of GSK-3beta/ERK in NTR1 regulation. Human colorectal cancers with NTR1 were treated with various HDACi, and NTR1 expression and function were assessed. Treatment with HDACi dramatically decreased endogenous NTR1 mRNA, protein, and promoter activity. Overexpression of GSK-3beta decreased NTR1 promoter activity (> 30%); inhibition of GSK-3beta increased NTR1 expression in colorectal cancer cells, indicating that GSK-3beta is a negative regulator of ERK and NTR1. Consistent with our previous findings, HDACi significantly decreased phosphorylated ERK while increasing GSK-3beta. Selective MAP/ERK kinase/ERK inhibitors suppressed NTR1 mRNA expression in a time- and dose-dependent fashion, and reduced NTR1 promoter activity by approximately 70%. Finally, pretreatment with NaBT prevented neurotensin-mediated cyclooxygenase-2 and c-myc expression and attenuated neurotensin-induced interleukin-8 expression. HDACi suppresses endogenous NTR1 expression and function in colorectal cancer cell lines; this effect is mediated, at least in part, through the GSK-3beta/ERK pathway. The downregulation of NTR1 in colorectal cancers may represent an important mechanism for the anticancer effects of HDACi.
The phosphatidylinositol 3-kinase (PI3K)/AKT pathway plays important roles in regulating cell motility. TSC2, a downstream target of AKT, is a central player in negatively controlling cell proliferation and protein translation through suppressing the activity of mTOR (mammalian target of rapamycin). However, the function of TSC2 in regulating cell migration remains unclear. Here, we show that TSC2 plays a critical role in the control of cell spreading, polarity, and migration. TSC2-deficient fibroblast cells were impaired in their ability to spread and alter actin cytoskeleton upon stimulation with insulin-like growth factor-1. Using scratch-induced polarization assay, we demonstrate that TSC2((-/-)) fibroblast cells polarized poorly toward the wound compared with wild-type cells. Similarly, knockdown of TSC2 expression in colon cancer cells resulted in a marked decrease in cell motility. Functionally, the activation of CDC42- and RAC1-GTPase was largely reduced in TSC2 knock-out fibroblast and TSC2 knockdown cancer cells. Furthermore, overexpression of an activating p110alpha mutant or short term rapamycin treatment rescued the cell polarization defect in TSC2((-/-)) fibroblast cells. Concurrently, the activation of CDC42 and RAC1 increased. The defect in cell migration and CDC42 and RAC1 activation was reversed by reintroducing TSC2 back into TSC2((-/-)) fibroblast cells. Taken together, we identified a novel role of TSC2 in controlling cell polarity and migration by regulating CDC42 and RAC1 activation.
We undertook focus groups, interviews, and an online survey with health care consumers as part of a recent project to assist purchasers in communicating more effectively about health care evidence and quality. Most of the consumers were ages 18-64; had health insurance through a current employer; and had taken part in making decisions about health insurance coverage for themselves, their spouse, or someone else. We found many of these consumers beliefs, values, and knowledge to be at odds with what policy makers prescribe as evidence-based health care. Few consumers understood terms such as "medical evidence" or "quality guidelines." Most believed that more care meant higher-quality, better care. The gaps in knowledge and misconceptions point to serious challenges in engaging consumers in evidence-based decision making.
An important step in carcinoma progression is loss of cell-cell adhesion leading to increased invasion and metastasis. We show here that the protein tyrosine phosphatase, PTP-PEST, is a critical regulator of cell-cell junction integrity and epithelial cell motility. Using colon carcinoma cells, we show that the expression level of PTP-PEST regulates cell motility. Either transient small interfering RNA or stable short hairpin RNA knockdown of PTP-PEST enhances haptotactic and chemotactic migration of KM12C colon carcinoma cells. Furthermore, KM12C cells with stably knocked down PTP-PEST exhibit a mesenchymal-like phenotype with prominent membrane ruffles and lamellae. In contrast, ectopic expression of PTP-PEST in KM20 or DLD-1 cells, which lack detectable endogenous PTP-PEST expression, suppresses haptotactic migration. Importantly, we find that PTP-PEST localizes in adherens junctions. Concomitant with enhanced motility, stable knockdown of PTP-PEST causes a disruption of cell-cell junctions. These effects are due to a defect in junctional assembly and not to a loss of E-cadherin expression. Adherens junction assembly is impaired following calcium switch in KM12C cells with stably knocked down PTP-PEST and is accompanied by an increase in the activity of Rac1 and a suppression of RhoA activity in response to cadherin engagement. Taken together, these results suggest that PTP-PEST functions as a suppressor of epithelial cell motility by controlling Rho GTPase activity and the assembly of adherens junctions.
Mechanisms responsible for crypt architectural distortion in chronic ulcerative colitis (CUC) are not well understood. Data indicate that serine/threonine protein kinase Akt (Akt) signaling cooperates with Wingless (Wnt) to activate beta-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-beta-catenin(552)). We investigated whether phosphoinositide 3-kinase (PI3K) is required for Akt-mediated activation of beta-catenin during intestinal inflammation.
The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway plays a critical role in the growth and progression of colorectal cancer (CRC). The purpose of our study was 2-fold: (1) to determine the expression levels of several key components of this pathway, including p85alpha, Akt1, Akt2, p-mTOR(Ser2448), and p-p70S6K(Thr389) in CRCs; and (2) to correlate the expression of these proteins with cancer stage and location (left versus right side).
The protein C (PC) pathway is an important anticoagulant mechanism that prevents thrombosis during the systemic inflammatory response. Thrombomodulin (TM), an endothelial cell membrane receptor, accelerates the conversion of PC to activated protein C (APC), which leads to the down-regulation of thrombin production and fibrin formation. Induction of acute endotoxemia in young and aged mice with a low dose of bacterial endotoxin lipopolysaccharide (LPS, 2.5 mg/kg) caused a high mortality rate in aged (80%) but not young (0%) mice. After injection with this dose of LPS, fibrin formation was significantly elevated only in aged mice, plasma APC levels were increased only in young mice, and TM expression was profoundly depressed in the aged. The increased thrombosis, suppressed APC level, and decreased TM expression were not observed in young mice receiving a higher dose of LPS (20 mg/kg), which resulted in a mortality rate (78%) equivalent to that seen in aged mice with the low-dose LPS. Mutant mice with reduced TM showed significantly less plasma APC and increased fibrin formation compared with wild-type mice after LPS. These results demonstrate that PC pathway activation is suppressed with aging and is partly responsible for age-associated thrombosis and high mortality during endotoxemia.
Epithelial-mesenchymal transition (EMT) is a transdifferentiation programme. The mechanism underlying the epigenetic regulation of EMT remains unclear. In this study, we identified that Snail1 interacted with histone lysine-specific demethylase 1 (LSD1). We demonstrated that the SNAG domain of Snail1 and the amine oxidase domain of LSD1 were required for their mutual interaction. Interestingly, the sequence of the SNAG domain is similar to that of the histone H3 tail, and the interaction of Snail1 with LSD1 can be blocked by LSD1 enzymatic inhibitors and a histone H3 peptide. We found that the formation of a Snail1-LSD1-CoREST ternary complex was critical for the stability and function of these proteins. The co-expression of these molecules was found in cancer cell lines and breast tumour specimens. Furthermore, we showed that the SNAG domain of Snail1 was critical for recruiting LSD1 to its target gene promoters and resulted in suppression of cell migration and invasion. Our study suggests that the SNAG domain of Snail1 resembles a histone H3-like structure and functions as a molecular hook for recruiting LSD1 to repress gene expression in metastasis.
RNA interference (RNAi) holds considerable promise as a novel therapeutic strategy to silence disease-causing genes not amenable to conventional therapeutics. Since it relies on small interfering RNAs (siRNAs), which are the mediators of RNAi-induced specific mRNA degradation, a major issue is the delivery of therapeutically active siRNAs into the target tissue. In vivo gene silencing with RNAi has been reported using both viral vector delivery and high-pressure, high-volume intravenous (i.v.) injection of synthetic siRNAs. For safety reasons, strategies based on viral vector delivery may be only of limited clinical use. The more desirable approach is to directly deliver active siRNAs. We describe the use of hydrodynamic administration as a technique to deliver naked siRNA constructs into experimental animals as a method of transient gene knockdown. This approach demonstrates that RNAi can be used to silence endogenous genes, involved in the cause of human diseases, with a clinically acceptable formulation and route of administration.
Notch signaling plays a crucial role in the development of colon cancer; targeting the Notch pathway may sensitize colon cancers to various adjuvant agents. The focus of our current study is to identify natural compounds that target Notch signaling and that might be beneficial for the prevention and treatment of colon cancer. Withaferin-A (WA) is a bioactive compound derived from Withania somnifera, which inhibits Notch-1 signaling and downregulates prosurvival pathways, such as Akt/NF-kappaB/Bcl-2, in three colon cancer cell lines (HCT-116, SW-480, and SW-620). In addition, WA downregulated the expression of mammalian target of rapamycin signaling components, pS6K and p4E-BP1, and activated c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cells. We also established the molecular link between Notch/Akt/mammalian target of rapamycin signaling by complementary approaches (i.e., overexpression of Notch-1 or inhibition of Notch-1 by small interfering RNA). Our results suggest that WA inhibits Notch-mediated prosurvival signaling, which facilitates c-Jun-NH(2)-kinase-mediated apoptosis in colon cancer cell lines. These results underscore the anticancer activity of WA, which exhibits potential for further development for targeted chemotherapy and/or chemoprevention strategies in the context of colon cancer.
The epithelial-mesenchymal transition is a critical early event in the invasion and metastasis of many types of cancer, including colorectal cancer (CRC). Chronic inflammation is an inducer of several cancer types and inflammatory cytokines have been implicated in tumor invasion.
The mammalian target of rapamycin (mTOR) kinase acts downstream of phosphoinositide 3-kinase/Akt to regulate cellular growth, metabolism, and cytoskeleton. Because approximately 60% of sporadic colorectal cancers (CRC) exhibit high levels of activated Akt, we determined whether downstream mTOR signaling pathway components are overexpressed and activated in CRCs.
Curcumin, a natural polyphenol product of the plant Curcuma longa, has been shown to inhibit the growth and progression of colorectal cancer; however, the anticancer mechanism of curcumin remains to be elucidated.
Toll-like receptors (TLRs) are involved in innate immunity. Overexpression of TLRs has been implicated in various types of cancer including colorectal cancer (CRC). The phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway is involved in CRC growth and progression. In this study, we determined whether TLR4 signaling and PI3K/Akt pathway activation occur in CRCs.
Carcinogenesis is a multifactorial process representing the accumulation of acquired and genetic defects, and it has become apparent that many gastrointestinal cancers originate from a state of chronic inflammation. Advances in the field of inflammation-induced carcinogenesis over the past several years have focused on the creation of agonists or antagonists of cytokines and pathways regulating proliferation and apoptosis, and include advances such as the discovery of pharmacological inhibitors of the histone deacetylase (HDAC) family of transcriptional co-repressors, which induce apoptosis of neoplastic cells; discovery of natural products, such as curcumin, which have been shown to regulate cytokine expression; and further investigation into the role of annexin A1, a downstream mediator of glucocorticoid action, in the regulation of inflammation, to name a few.
The purpose of our study was twofold: (1) to determine the incidence, patient and tumor characteristics, and outcome of patients with gastrointestinal carcinoid tumors using the Surveillance, Epidemiology and End Results (SEER) database, and (2) to delineate the expression pattern of growth factor receptors (GFRs) in carcinoid tumors.
Oxidative stress and inflammation may contribute to the disruption of the protective gut barrier through various mechanisms; mitochondrial dysfunction resulting from inflammatory and oxidative injury may potentially be a significant source of apoptosis during necrotizing enterocolitis (NEC). Tumor necrosis factor (TNF)-alpha is thought to generate reactive oxygen species (ROS) and activate the apoptosis signal-regulating kinase 1 (ASK1)-c-Jun N-terminal kinase (JNK)/p38 pathway. Hence, the focus of our study was to examine the effects of TNF-alpha/ROS on mitochondrial function, ASK1-JNK/p38 cascade activation in intestinal epithelial cells during NEC.
Phytochemicals are an important source of emerging preventive and therapeutic agents for cancer. Triptolide/PG490, an extract of the Chinese herb Tripterygium wilfordii Hook F, is a potent anti-inflammatory agent that also possesses anticancer activity. While its antiproliferative effects are well-established, the potential antimigratory effects of triptolide have not been characterized.
Increased mortality and overexpression of interleukin-6 (IL-6) during inflammatory stress are well-documented age-associated phenomena; however, the site of IL-6 overexpression is not entirely known. Here, we report that white adipose tissue is a major source of IL-6 in aged animals during lipopolysaccharide (LPS)-induced systemic inflammation. Among the various tissues examined, white adipose tissue from the epididymal fat pad (located in the abdominal cavity) expressed the highest level of IL-6 messenger RNA in both young and aged mice with a 5.5-fold higher level in the aged. Immunohistochemistry revealed that, within the adipose tissue, LPS-induced IL-6 expression is localized to both the adipocytes and stromal cells. Compared with age-matched wild-type mice, aged IL-6((-/-)) mice exhibited reduced mortality to LPS suggesting a deleterious effect of IL-6 overexpression in the aged. These results demonstrate that increased vulnerability to systemic inflammation with age is due in part, to augmented IL-6 production by the adipose tissue.
Carcinoid syndrome, characterized by flushing, diarrhea, and valvular heart disease, can occur following carcinoid tumor metastasis to the liver and systemic release of bioactive hormones into the systemic circulation. Treatment of this devastating disease is hampered by the lack of an in vivo model that recapitulates the clinical syndrome.
The protein kinase D (PKD) family of serine/threonine kinases, which can be activated by gastrointestinal hormones, consists of three distinct isoforms that modulate a variety of cellular processes including intracellular protein transport as well as constitutive and regulated secretion. Although isoform-specific functions have been identified in a variety of cell lines, the expression and function of PKD isoforms in normal, differentiated secretory tissues is unknown. Here, we demonstrate that PKD isoforms are differentially expressed in the exocrine and endocrine cells of the pancreas. Specifically, PKD3 is the predominant isoform expressed in exocrine cells of the mouse and human pancreas, whereas PKD1 and PKD2 are more abundantly expressed in the pancreatic islets. Within isolated mouse pancreatic acinar cells, PKD3 undergoes rapid membrane translocation, trans-activating phosphorylation, and kinase activation after gastrointestinal hormone or cholinergic stimulation. PKD phosphorylation in pancreatic acinar cells occurs viaaCa2+-independent, diacylglycerol- and protein kinase C-dependent mechanism. PKD phosphorylation can also be induced by physiologic concentrations of secretagogues and by in vivo stimulation of the pancreas. Furthermore, activation of PKD3 potentiates MEK/ERK/RSK (RSK, ribosomal S6 kinase) signaling and significantly enhances cholecystokinin-mediated pancreatic amylase secretion. These findings reveal a novel distinction between the exocrine and endocrine cells of the pancreas and further identify PKD3 as a signaling molecule that promotes hormone-stimulated amylase secretion.
Constitutive activation of the hedgehog pathway is implicated in the development of many human malignancies; hedgehog targets, PTCH1 and Gli1, are markers of hedgehog signaling activation and are expressed in most hedgehog-associated tumors. Protein kinase Cdelta (PKCdelta) generally slows proliferation and induces cell cycle arrest of various cell lines. In this study, we show that activated PKCdelta (wild-type PKCdelta stimulated by phorbol 12-myristate 13-acetate or constitutively active PKCdelta) decreased Gli-luciferase reporter activity in NIH/3T3 cells, as well as the endogenous hedgehog-responsive gene PTCH1. In human hepatoma (i.e. Hep3B) cells, wild-type PKCdelta and constitutively active PKCdelta decreased the expression levels of endogenous Gli1 and PTCH1. In contrast, PKCdelta siRNA increased the expression levels of these target genes. Silencing of PKCdelta by siRNA rescued the inhibition of cell growth by KAAD-cyclopamine, an antagonist of hedgehog signaling element Smoothened, suggesting that PKCdelta acts downstream of Smoothened. The biological relevance of our study is shown in hepatocellular carcinoma where we found that hepatocellular carcinoma with detectable hedgehog signaling had weak or no detectable expression of PKCdelta, whereas PKCdelta highly expressing tumors had no detectable hedgehog signaling. Our results demonstrate that PKCdelta alters hedgehog signaling by inhibition of Gli protein transcriptional activity. Furthermore, our findings suggest that, in certain cancers, PKCdelta plays a role as a negative regulator of tumorigenesis by regulating hedgehog signaling.
The increased motility and invasiveness of tumor cells are reminiscent of epithelial-mesenchymal transition (EMT), which occurs during embryonic development, wound healing, and metastasis. In this study, we found that Snail is stabilized by the inflammatory cytokine TNFalpha through the activation of the NF-kappaB pathway. We demonstrated that NF-kappaB is required for the induction of COP9 signalosome 2 (CSN2), which, in turn, blocks the ubiquitination and degradation of Snail. Furthermore, we showed that the expression of Snail correlated with the activation of NF-kappaB in cancer cell lines and metastatic tumor samples. Knockdown of Snail expression inhibited cell migration and invasion induced by inflammatory cytokines and suppressed inflammation-mediated breast cancer metastasis. Our study provides a plausible mechanism for inflammation-induced metastasis.
Down-regulation of E-cadherin plays an important role in epithelial-mesenchymal transition (EMT), which is critical in normal development and disease states such as tissue fibrosis and metastasis. Snail, a key transcription repressor of E-cadherin, is a labile protein with a short half-life and is regulated through phosphorylation, ubiquitination, and degradation. Previously, we showed that GSK-3beta phosphorylated two stretches of serine residues within the nuclear export signal and the destruction box of Snail, provoking its cytoplasmic export for ubiquitin-mediated proteasome degradation. However, the mechanism of Snail dephosphorylation and the identity of the Snail-specific phosphatase remain elusive. Using a functional genomic screening, we found that the small C-terminal domain phosphatase (SCP) is a specific phosphatase for Snail. SCP interacted and co-localized with Snail in the nucleus. We also found that SCP expression induced Snail dephosphorylation and stabilization in vitro and in vivo. However, a catalytically inactive mutant of SCP had no effect on Snail. Furthermore, we found that Snail stabilization induced by SCP enhanced snail activity in the suppression of E-cadherin and increased cell migration. Thus, our findings indicate that SCP functions as a Snail phosphatase to control its phosphorylation and stabilization, and our study provides novel insights for the regulation of Snail during EMT and cancer metastasis.
Necrotizing enterocolitis (NEC) remains a lethal condition for many premature infants. Peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor family, has been shown to play a protective role in cellular inflammatory responses; however, its role in NEC is not clearly defined. We sought to examine the expression of PPAR-gamma in the intestine using an ischemia-reperfusion (I/R) model of NEC, and to assess whether PPAR-gamma agonist treatment would ameliorate I/R-induced gut injury. Swiss-Webster mice were randomized to receive sham (control) or I/R injury to the gut induced by transient occlusion of superior mesenteric artery for 45 min with variable periods of reperfusion. I/R injury resulted in early induction of PPAR-gamma expression and activation of NF-kappaB in small intestine. Pretreatment with PPAR-gamma agonist, 15d-PGJ(2), attenuated intestinal NF-kappaB response and I/R-induced gut injury. Activation of PPAR-gamma demonstrated a protective effect on small bowel during I/R-induced gut injury.
Previously, we demonstrated that protein kinase D (PKD) plays a protective role during H(2)O(2)-induced intestinal cell death. Here, we sought to determine whether this effect is mediated by nuclear factor-kappaB (NF-kappaB) and mitogen-activated protein kinases (MAPKs). Treatment with H(2)O(2) activated NF-kappaB in RIE-1 cells; H(2)O(2) also induced the translocation of NF-kappaB p65 as well as phosphorylation of IkappaB-alpha. PKD1 siRNA inhibited H(2)O(2)-induced activation, translocation of NF-kappaB, and phosphorylation of IkappaB-alpha. We also found that overexpression of wild type PKD1 attenuated H(2)O(2)-induced phosphorylation of p38 MAPK and its upstream activator, MAPK kinase (MKK) 3/6, whereas the phosphorylation was increased by PKD1 siRNA or kinase-dead PKD1. Phosphorylation of neither extracellular signal-regulated kinases (ERK) 1/2 nor c-Jun N-terminal kinases (JNK) was altered by PKD1 plasmids or siRNA. Our findings suggest that PKD protects intestinal cells through up-regulation of NF-kappaB and down-regulation of p38 MAPK.
Neuroblastomas express increased levels of gastrin-releasing peptide receptor (GRP-R). However, the exact molecular mechanisms involved in GRP-R-mediated cell signaling in neuroblastoma growth and metastasis are unknown. Here, we report that focal adhesion kinase (FAK), as a critical downstream target of GRP-R, is an important regulator of neuroblastoma tumorigenicity. We found that FAK expression correlates with GRP-R expression in human neuroblastoma sections and cell lines. GRP-R overexpression in SK-N-SH cells increased FAK, integrin ?3 and ?1 expressions and cell migration. These cells demonstrated flatter cell morphology with broad lamellae, in which intense FAK expression was localized to the leading edges of lamellipodia. Interestingly, FAK activation was, in part, dependent on integrin ?3 and ?1 expression. Conversely, GRP-R silencing decreased FAK as well as Mycn levels in BE(2)-C cells, which displayed a denser cellular morphology. Importantly, rescue experiments in GRP-R silenced BE(2)-C cells showed FAK overexpression significantly enhanced cell viability and soft agar colony formation; similarly, FAK overexpression in SK-N-SH cells also resulted in increased cell growth. These effects were reversed in FAK silenced BE(2)-C cells in vitro as well as in vivo. Moreover, we evaluated the effect of FAK inhibition in vivo. FAK inhibitor (Y15) suppressed GRP-induced neuroblastoma growth and metastasis. Our results indicate that FAK is a critical downstream regulator of GRP-R, which mediates tumorigenesis and metastasis in neuroblastoma.
One of the advantages of nanotechnology is the feasibility to construct therapeutic particles carrying multiple therapeutics with defined structure and stoichiometry. The field of RNA nanotechnology is emerging. However, controlled assembly of stable RNA nanoparticles with multiple functionalities which retain their original role is challenging due to refolding after fusion. Herein, we report the construction of thermodynamically stable X-shaped RNA nanoparticles to carry four therapeutic RNA motifs by self-assembly of reengineered small RNA fragments. We proved that each arm of the four helices in the X-motif can harbor one siRNA, ribozyme, or aptamer without affecting the folding of the central pRNA-X core, and each daughter RNA molecule within the nanoparticle folds into their respective authentic structures and retains their biological and structural function independently. Gene silencing effects were progressively enhanced as the number of the siRNA in each pRNA-X nanoparticles gradually increased from one to two, three, and four. More importantly, systemic injection of ligand-containing nanoparticles into the tail-vein of mice revealed that the RNA nanoparticles remained intact and strongly bound to cancers without entering the liver, lung or any other organs or tissues, while remaining in cancer tissue for more than 8 h.
RNA interference has the potential to be more selective than small molecule inhibitors and can be used to target proteins, such as Ras, that are currently undruggable. The purpose of our study was to determine the optimal cotargeting strategy of the commonly mutated PI3K/AKT/mTOR and Ras pathways by a selective RNA interference approach in colorectal cancer cell lines possessing coexistent PIK3CA and KRAS mutations.
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.