Profiling and targeting of cellular bioenergetics: inhibition of pancreatic cancer cell proliferation.

BACKGROUND: Targeting both mitochondrial bioenergetics and glycolysis pathway is an effective way to inhibit proliferation of tumour cells, including those that are resistant to conventional chemotherapeutics. METHODS: In this study, using the Seahorse 96-well Extracellular Flux Analyzer, we mapped the two intrinsic cellular bioenergetic parameters, oxygen consumption rate and proton production rate in six different pancreatic cancer cell lines and determined their differential sensitivity to mitochondrial and glycolytic inhibitors. RESULTS: There exists a very close relationship among intracellular bioenergetic parameters, depletion of ATP and anti-proliferative effects (inhibition of colony-forming ability) in pancreatic cancer cells derived from different genetic backgrounds treated with the glycolytic inhibitor, 2-deoxyglucose (2-DG). The most glycolytic pancreatic cancer cell line was exquisitely sensitive to 2-DG, whereas the least glycolytic pancreatic cancer cell was resistant to 2-DG. However, when combined with metformin, inhibitor of mitochondrial respiration and activator of AMP-activated protein kinase, 2-DG synergistically enhanced ATP depletion and inhibited cell proliferation even in poorly glycolytic, 2-DG-resistant pancreatic cancer cell line. Furthermore, treatment with conventional chemotherapeutic drugs (e.g., gemcitabine and doxorubicin) or COX-2 inhibitor, celecoxib, sensitised the cells to 2-DG treatment. CONCLUSIONS: Detailed profiling of cellular bioenergetics can provide new insight into the design of therapeutic strategies for inhibiting pancreatic cancer cell metabolism and proliferation.

Epigenetic silencing of CXCL12 increases the metastatic potential of mammary carcinoma cells.

Expression of the chemokine receptor CXCR4 has been linked with increased metastasis and decreased clinical prognosis in breast cancer. The current paradigm dictates that CXCR4 fosters carcinoma cell metastasis along a chemotactic gradient to organs expressing the ligand CXCL12. The present study asked if alterations in autocrine CXCR4 signaling via dysregulation of CXCL12 in mammary carcinoma cells modulated their metastatic potential. While CXCR4 was consistently detected, expression of CXCL12 characteristic of human mammary epithelium was silenced by promoter hypermethylation in breast cancer cell lines and primary mammary tumors. Stable re-expression of functional CXCL12 in ligand null cells increased orthotopic primary tumor growth in the mammary fat-pad model of tumorigenesis. Those data parallel increased carcinoma cell proliferation measured in vitro with little-to-no-impact on apoptosis. Moreover, re-expression of autocrine CXCL12 markedly reduced metastatic lung invasion assessed using in vivo bioluminescence imaging following tail vein injection. Consistent with those data, decreased metastasis reflected diminished intracellular calcium signaling and chemotactic migration in response to exogenous CXCL12 independent of changes in CXCR4 expression. Together these data suggest that an elevated migratory signaling response to ectopic CXCL12 contributes to the metastatic potential of CXCR4-expressing mammary carcinoma cells, subsequent to epigenetic silencing of autocrine CXCL12.

Silencing of epithelial CXCL12 expression by DNA hypermethylation promotes colonic carcinoma metastasis.

Cellular metastasis is the most detrimental step in carcinoma disease progression, yet the mechanisms that regulate this process are poorly understood. CXCL12 and its receptor CXCR4 are co-expressed in several tissues and cell types throughout the body and play essential roles in development. Disruption of either gene causes embryonic lethality due to similar defects. Post-natally, CXCL12 signaling has a wide range of effects on CXCR4-expressing cells, including the directed migration of leukocytes, lymphocytes and hematopoietic stem cells. Recently, this signaling axis has also been described as an important regulator of directed carcinoma cell metastasis. We show herein that while CXCR4 expression remains consistent, constitutive colonic epithelial expression of CXCL12 is silenced by DNA hypermethylation in primary colorectal carcinomas as well as colorectal carcinoma-derived cell lines. Inhibition of DNA methyltransferase (Dnmt) enzymes with 5-aza-2'-deoxycytidine or genetic ablation of both Dnmt1 and Dnmt3b prevented promoter methylation and restored CXCL12 expression. Re-expression of functional, endogenous CXCL12 in colorectal carcinoma cells dramatically reduced metastatic tumor formation in mice, as well as foci formation in soft agar. Decreased metastasis was correlated with increased caspase activity in cells re-expressing CXCL12. These data constitute the unique observation that silencing CXCL12 within colonic carcinoma cells greatly enhances their metastatic potential.

Production of MDC/CCL22 by human intestinal epithelial cells.

The intestinal mucosa contains a subset of lymphocytes that produce Th2 cytokines, yet the signals responsible for the recruitment of these cells are poorly understood. Macrophage-derived chemokine (MDC/CCL22) is a recently described CC chemokine known to chemoattract the Th2 cytokine producing cells that express the receptor CCR4. The studies herein demonstrate the constitutive production of MDC/CCL22 in vivo by human colon epithelium and by epithelium of human intestinal xenografts. MDC/CCL22 mRNA expression and protein secretion was upregulated in colon epithelial cell lines in response to proinflammatory cytokines or infection with enteroinvasive bacteria. Inhibition of nuclear factor (NF)-kappaB activation abolished MDC/CCL22 expression in response to proinflammatory stimuli, demonstrating that MDC/CCL22 is a NF-kappaB target gene. In addition, tumor necrosis factor-alpha-induced MDC/CCL22 secretion was differentially modulated by Th1 and Th2 cytokines. Supernatants from the basal, but not apical, side of polarized epithelial cells induced a MDC/CCL22-dependent chemotaxis of CCR4-positive T cells. These studies demonstrate the constitutive and regulated production by intestinal epithelial cells of a chemokine known to function in the trafficking of T cells that produce anti-inflammatory cytokines.

Regulated MIP-3alpha/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity.

Human intestinal epithelial cells secrete an array of chemokines known to signal the trafficking of neutrophils and monocytes important in innate mucosal immunity. We hypothesized that intestinal epithelium may also have the capacity to play a role in signaling host adaptive immunity. The CC chemokine macrophage inflammatory protein (MIP)-3alpha/CCL20 is chemotactic for immature dendritic cells and CD45RO(+) T cells that are important components of the host adaptive immune system. In these studies, we demonstrate the widespread production and regulated expression of MIP-3alpha by human intestinal epithelium. Several intestinal epithelial cell lines were shown to constitutively express MIP-3alpha mRNA. Moreover, MIP-3alpha mRNA expression and protein production were upregulated by stimulation of intestinal epithelial cells with the proinflammatory cytokines tumor necrosis factor-alpha or interleukin-1alpha or in response to infection with the enteric bacterial pathogens Salmonella or enteroinvasive Escherichia coli. In addition, MIP-3alpha was shown to function as a nuclear factor-kappaB target gene. In vitro findings were paralleled in vivo by increased expression of MIP-3alpha in the epithelium of cytokine-stimulated or bacteria-infected human intestinal xenografts and in the epithelium of inflamed human colon. Mucosal T cells, other mucosal mononuclear cells, and intestinal epithelial cells expressed CCR6, the cognate receptor for MIP-3alpha. The constitutive and regulated expression of MIP-3alpha by human intestinal epithelium is consistent with a role for epithelial cell-produced MIP-3alpha in modulating mucosal adaptive immune responses.

Regulated production of interferon-inducible T-cell chemoattractants by human intestinal epithelial cells.

BACKGROUND & AIMS: Human intestinal epithelial cells inducibly express neutrophil and monocyte chemoattractants, yet little is known about the regulated production of T-cell chemoattractants by the intestinal epithelium. IP-10, Mig, and I-TAC are 3 CXC chemokines that are known to act as CD4(+) T-cell chemoattractants. METHODS: We studied constitutive chemokine expression in human colon, and defined the regulated expression of these chemokines by reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistology using cultured human intestinal epithelial cell lines and a novel adaptation of an in vivo human intestinal xenograft model. RESULTS: IP-10 and Mig were constitutively expressed by normal human colon epithelium, and their cognate receptor, CXCR3, was expressed by mucosal mononuclear cells. Interferon (IFN)-gamma stimulation increased mRNA expression and the polarized basolateral secretion of these chemokines by human colon epithelial cell lines; infection with enteroinvasive bacteria, or stimulation with the proinflammatory cytokines tumor necrosis factor alpha and interleukin 1alpha, strongly potentiated IFN-gamma-induced epithelial cell IP-10, Mig, and I-TAC production. Epithelial cell mRNA and protein expression of IP-10, Mig, and I-TAC were rapidly up-regulated in human intestinal xenografts in response to stimulation with IFN-gamma alone or in combination with IL-1. CONCLUSIONS: The constitutive and regulated production of the IFN-gamma-inducible chemokines IP-10, Mig, and I-TAC by human intestinal epithelium, and the expression of their cognate receptor, CXCR3, by mucosal mononuclear cells, suggest that the intestinal epithelium can play a role in modulating physiologic and pathologic T cell-mediated mucosal inflammation.

Analysis by high density cDNA arrays of altered gene expression in human intestinal epithelial cells in response to infection with the invasive enteric bacteria Salmonella.

Many clinically important enteric pathogens initiate disease by invading and passing through the intestinal epithelium, a process accompanied by increased epithelial expression of proinflammatory cytokines. To further define the role intestinal epithelial cells play in initiating and modulating the host response to infection with invasive bacteria, hybrid selection on high density cDNA arrays was used to characterize the mRNA expression profile of approximately 4,300 genes in human intestinal epithelial cells after infection with the prototypic invasive bacteria, Salmonella. Selected findings were further evaluated by reverse transcription-polymerase chain reaction, Northern blot analysis, and protein assays. Epithelial infection with Salmonella significantly up-regulated mRNA expression of a relatively small fraction of all genes tested. Of these, several cytokines (granulocyte colony-stimulating factor, inhibin A, Epstein-Barr virus-induced gene 3, interleukin-8, macrophage inflammatory protein-2alpha), kinases (TKT, Eck, HEK), transcription factors (interferon regulatory factor-1), and HLA class I were the most prominent. Furthermore, the transcription factor NF-kappaB is shown to be important for inducible mRNA expression for a broad group of genes tested. These findings expand the repertoire of known epithelial cell responses to infection with an invasive enteric pathogen. The results also show that evaluation of mRNA expression profiles by cDNA array analysis is a powerful approach to characterizing and understanding host-pathogen interactions.

Human intestinal epithelial cells express receptors for platelet-activating factor.

The intestinal epithelium produces and responds to cytokines and lipid mediators that play a key role in the induction and regulation of mucosal inflammation. The lipid mediator platelet-activating factor (PAF) can be produced and degraded by the human intestinal epithelium and is known to mediate a range of proinflammatory and other biological effects in the intestinal mucosa. In the studies herein, we assessed whether or not human intestinal epithelial cells express cell surface or intracellular PAF receptors (PAF-R), whether expression of these receptors can be regulated, and whether human intestinal epithelial cells respond to PAF. Several human colon epithelial cell lines (HT-29, Caco-2, T84, HCT-8, HCA-7, I407, and LS-174T) were shown by RT-PCR to constitutively express mRNA for PAF-R. In addition, PAF-R expression was demonstrated by immunoblot analysis and PAF-R was shown to be constitutively expressed on the cell surface of several of these cell lines, as assessed by flow cytometry. PAF-R expression by human colon epithelial cells was upregulated by stimulation with retinoic acid but not by stimulation with PAF, proinflammatory agonists (tumor necrosis factor-alpha, interleukin-1, interferon-gamma), or transforming growth factor-alpha. PAF-R on intestinal epithelial cells were functional, as PAF stimulation of the cells increased tyrosine phosphorylation of several cellular proteins, including proteins of 75 and 125 kDa, and this response was blocked by a PAF-R antagonist. Consistent with the findings using cell lines, PAF-R were also constitutively expressed by normal human colon and small intestinal epithelium in vivo, as shown by immunohistology. The constitutive and regulated expression of functional PAF-R by human intestinal epithelium suggests PAF produced by the intestinal epithelial cells or cells underlying the epithelium has autocrine or paracrine effects on intestinal epithelial cells.

Chemokine receptor expression by human intestinal epithelial cells.

BACKGROUND & AIMS: Intestinal epithelial cells produce an array of proinflammatory chemokines that can provide signals to mucosal immune and inflammatory cells. To determine if chemokines can also signal epithelial cells, we characterized the expression of chemokine receptors on human colon epithelial cells in vitro and in vivo. METHODS: Expression of chemokine receptor messenger RNAs (mRNAs) by the human colon epithelial cell lines HT-29, HT-29.18.C1, Caco-2, T84, HCA-7, and LS174T was assessed by reverse-transcription polymerase chain reaction. Chemokine receptors on intestinal epithelial cells in vitro were determined by flow cytometry, and expression in vivo was determined by immunostaining of human colon. Interleukin (IL)-8 and growth-related (GRO) alpha secretion were assayed by enzyme-linked immunosorbent assay. RESULTS: Human colon epithelial cells constitutively expressed mRNAs for an array of CC and CXC chemokine receptors, including CCR1-8 and CXCR4, but little if any CXCR1 or CXCR2. Further studies focused on CXCR4 and CCR5 because mRNA for those chemokine receptors was abundantly expressed by each of the colon epithelial cell lines, and these receptors were present on the cell surface. Analogous to their localization on polarized cell lines, CXCR4 and CCR5 had a predominant apical and, to a lesser extent, basolateral distribution on human enterocytes, as demonstrated by immunostaining of human colon. Human colon epithelial cells stimulated with stromal cell-derived factor 1alpha and macrophage inflammatory protein (MIP)- 1alpha or MIP-1beta, which are the chemokine ligands for CXCR4 or CCR5, up-regulated production of the CXC chemokines IL-8 and GROalpha. CONCLUSIONS: Human colon epithelial cells express chemokine receptors. Human colonocytes have the potential to serve as targets for chemokine signaling.

Mucosal immunity.

Advances have been made in understanding specialized aspects of acquired mucosal immunity and the importance of cross-talk between host innate and acquired immune responses in host mucosal defense. These advances include elucidation of a molecular basis for the coordinated trafficking and retention of lymphocytes in intestinal sites, new insights into the possible extrathymic origin of mucosal T cells in the intestine, and the description of ligands that may be the targets of intraepithelial T-cell recognition. The past year has witnessed the characterization of an array of regulated epithelial cell mediators and responses important for host mucosal defense against enteric microbial pathogens and firmly establishes epithelial cells as an integral component of the mucosal immune network. Animal models continued to further understanding of intestinal inflammatory disease, and studies on tissue transglutaminase have generated significant new insights into the immunopathogenesis of human celiac disease.

Hymenolepis diminuta fractions but not previous tapeworm infection stimulate intestinal myoelectric alterations in vivo in the rat.

Infection of rats with the enteric, lumen-dwelling tapeworm Hymenolepis diminuta causes electric changes in host intestinal smooth muscle and decreased luminal transit. The mechanisms that stimulate host intestinal alterations during this nontissue invasive infection may include the tapeworm's biomass, its diurnal migratory behavior, a host immune-mediated response, or direct parasite stimulation of host motor activity. In vivo intestinal myoelectric activity was monitored to evaluate the following: (1) that reinfection with H. diminuta is influenced by host immune regulation and (2) that administration of tapeworm fractions to never-before-infected rats initiates an alteration of enteric smooth muscle activity. To address the first hypothesis, we determined that altered intestinal myoelectric activity patterns were no different and did not occur earlier in a second infection with H. diminuta than in a primary infection. The lack of either a change in myoelectric pattern or an earlier onset of intestinal myoelectric changes indicates that tapeworm-induced myoelectric activity is not anamnestically stimulated by host immunomodulatory mechanisms. Consistent with the second hypothesis, administration of either H. diminuta carcass homogenate or tegument-enriched fractions directly into the intestinal lumen of tapeworm-naive rats initiated myoelectric patterns previously characteristic of chronic H. diminuta infection. Additionally, the appearance of characteristic nonmigrating myoelectric patterns in uninfected rats administered tapeworm fractions indicates that a substance from H. diminuta acts as the triggering signal molecule for intestinal myoelectric alterations. These findings also indicate that neither the tapeworm's biomass nor its diurnal movement is required for initiation of H. diminuta-altered myoelectric patterns. We have shown that H. diminuta possess a signal molecule(s) that alters host enteric electric activity, and we suggest that these alterations may play an important role in the symbiotic rat-tapeworm interrelationship.

Hymenolepsis diminuta: mucosal mastocytosis and intestinal smooth muscle hypertrophy occur in tapeworm-infected rats.

The mechanisms mediating motility changes during noninvasive tapeworm infection have not been characterized. In contrast, host intestinal motility changes during invasive nematode infection are mediated by mucosal mast cells (MMC). The purpose of this study was to examine and the correlate onset of myoelectric alterations 8 days after initial tapeworm infection with changes in intestinal morphology, MMC numbers, and MMC secretory activity. Segments of the small intestine, the tapeworms normal habitat, along with stomach, colon, and bladder were taken from tapeworm-infected and control rats. Tissues were fixed and stained to identify MMC and for morphologic measurement. Tapeworm-infected and uninfected rats with chronically implanted intestinal electrodes were treated with ketotifen, a mast cell stabilizer, and in vivo myoelectric activity monitored. In tapeworm-infected rats, the muscularis externa, on day 20 postinfection, and crypts of Lieberkuhn, on day 26 postinfection, from the entire small intestine appeared thickened or deeper, respectively. Increased muscularis thickness was due to smooth muscle hypertrophy in both the circular and the longitudinal muscle layers. Mucosal mastocytosis was first observed on day 26 postinfection and occurred only in the ileum of tapeworm-infected rats. Pharmacologic stabilization of mast cells with ketotifen did not prevent onset of enteric myoelectric alterations during tapeworm infection. Stomach, colon, and bladder MMC numbers and tissue dimensions were not different between Hymenolepis diminuta-infected rats and uninfected controls. Initiation of myoelectric alterations 8 days after infection precedes and may be a contributing factor to the onset of both smooth muscle hypertrophy and mucosal mastocytosis. Taken together, our data indicate that mast cells are not an initiating factor nor chronic stimulus maintaining intestinal myoelectric alterations during H. diminuta infection.

Human hepatocytes express an array of proinflammatory cytokines after agonist stimulation or bacterial invasion.

Inflammatory cells infiltrate the liver in response to microbial infection or hepatic injury. To assess the potential role hepatocytes may play in initiating or amplifying the acute inflammatory response in the liver, we used three human hepatocyte cell lines and primary human hepatocyte cultures to characterize the repertoire of cytokines that can be expressed and regulated in hepatocytes in response to agonist stimulation or bacterial infection. As reported herein, a proinflammatory cytokine gene program that includes C-X-C and C-C chemokines [interleukin-8(IL-8), growth related (GRO)-alpha, GRO-beta, GRO-gamma, epithelial neutrophil activating peptide-78 (ENA-78), and RANTES] and the cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage colony stimulating factor was upregulated in human hepatocytes after stimulation with IL-1 alpha or TNF-alpha or bacterial invasion. In contrast, expression of hematopoietic/ lymphoid growth factors by the same cells was either down-regulated (erythropoietin and stem cell factor) or unchanged (IL-7 and IL-15) in response to the identical stimuli. Hepatocytes did not express cytokines that often are associated with the regulation of antigen-specific immune responses (IL-2, IL-4, IL-5, IL-10, IL-12p40, IL-13, and interferon-gamma) or genes for several other proinflammatory cytokines [IL-1 alpha, IL-6, monocyte chemotactic protein-1 (MCP-1), and MCP-3] or hematopoietic growth factors (granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, IL-3, and IL-11). Together, these studies suggest that hepatocytes can both initiate and amplify acute inflammatory responses in the liver through the regulated expression and secretion of a specific array of proinflammatory cytokines.

Tapeworm infection decreases intestinal transit and enteric aerobic bacterial populations.

Intestinal myoelectric patterns in rats are altered after chronic luminal infection with the tapeworm Hymenolepis diminuta. This study evaluates whether these altered patterns were associated with changes in intestinal fluid transit and endogenous enteric microbe levels. Luminal transit, measured throughout the small intestine during the interdigestive state, was significantly decreased during tapeworm infection. Reduced transit was regional, occurring in the same location as that of the tapeworm and maximal myoelectric alterations. In other experimental systems, aerobic and anaerobic bacterial overgrowth is associated with decreased transit; however, reduced transit during tapeworm infection was unexpectedly associated with decreased numbers of aerobic bacteria, whereas anaerobic bacterial populations remained unchanged. The lack of overgrowing endogenous microflora suggests that overgrowth is not responsible for tapeworm-stimulated alterations in host myoelectric patterns. We speculate that a tapeworm secretion could be responsible for both transit and motility changes while delayed intestinal transit could prevent tapeworm expulsion, aid the tapeworms' migration, and contribute to the digestion and absorption of nutrients by hosts and/or parasites.

Praziquantel treatment normalizes intestinal myoelectric alterations associated with Hymenolepis diminuta-infected rats.

Hymenolepis diminuta-associated alterations in rat intestinal myoelectric patterns are abolished following therapeutic administration of the anthelmintic praziquantel (PZQ). Host intestinal smooth muscle myoelectric patterns, reflecting smooth muscle contractility and intestinal phasic motility, were recorded using in vivo serosal electrodes, surgically implanted on the duodenum, jejunum, and ileum. Repeated electromyographic recording from unrestrained and unanesthetized rats began 5 days after electrode implantation surgery. Three initial control recordings from each rat confirmed the appearance of normal intestinal myoelectric patterns, characterized by the interdigestive migrating myoelectric complex (MMC). All animals were subsequently infected with H. diminuta and myoelectric recordings beginning after day 8 postinfection confirmed the appearance of diminished frequency of the MMC and 2 nonmigrating myoelectric patterns, i.e., repetitive bursts of action potentials and sustained spike potentials. PZQ was used to remove the tapeworms from rats 12 days after Hymenolepis diminuta infection, as intestinal myoelectric changes become maximal at this time in tapeworm-infected rats. PZQ administered to uninfected rats at either of 2 dose levels did not affect host interdigestive myoelectric activity. After removal of the parasite with PZQ, electromyographic recordings indicated a return to normal uninfected electrical patterns within 24 hr of drug treatment. We have demonstrated that the presence of Hymenolepis diminuta is necessary to induce and maintain abnormal intestinal myoelectric patterns. The altered motor properties of tapeworm-infected rat intestine and the rapid reconversion to preinfection myoelectric patterns provides a new and unique model to examine the regulatory mechanisms of intestinal motility and its control by luminal parasites.

Intestinal myoelectric alterations in rats chronically infected with the tapeworm Hymenolepis diminuta.

This study determined that intestinal myoelectric activity was profoundly altered during a strictly luminal, chronic, tapeworm infection. Chronically implanted bipolar electrodes were attached to five sites on the serosal surface of the rat small intestine. One was placed on the duodenum, three on the jejunum, and the fifth on the ileum. Electromyographic recording in nonfasted unanesthetized animals was begun at day 5 postsurgery. All electromyographic recordings were analyzed for slow wave (SW) frequency, phase III frequency, duration of phase III, and percentage of SW with spike potentials. Three initial control recordings prior to infection confirmed the presence of normal interdigestive motility characterized by the three phases (I, II, III) of the migrating myoelectric complex (MMC). Two nonpropulsive myoelectric alterations were observed in infected animals: the repetitive bursts of action potentials (RBAP) and periods of sustained spike potentials (SSP). Myoelectric activity from infected animals indicated decreased cycling of the interdigestive MMC. RBAP and SSP were more prevalent in the distal small intestine corresponding to tapeworm location. The percent of spike potential activity indicated that there was a reversal in the spike potential gradient on the small intestine. The number of spike potentials was maximal in caudal and minimal in oral intestine. We propose that overall localized increases in myoelectric spike potential activity represent increased contractility and decreased propulsion triggered by the presence of the tapeworm. These motility changes were surprising, since the tapeworm Hymenolepis diminuta does not penetrate the intestinal mucosa. This interaction between parasite and host may prevent expulsion of the tapeworm from the small intestine.