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.
...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.
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.
Detailed profiling of cellular bioenergetics can provide new insight into the design of therapeutic strategies for inhibiting pancreatic cancer cell metabolism and proliferation.
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.
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.
Model organism research is essential for discovering the mechanisms of human diseases by defining biologically meaningful gene to disease relationships. The Rat Genome Database (RGD, (
...https://rgd.mcw.edu
)) is a cross-species knowledgebase and the premier online resource for rat genetic and physiologic data. This rich resource is enhanced by the inclusion and integration of comparative data for human and mouse, as well as other human disease models including chinchilla, dog, bonobo, pig, 13-lined ground squirrel, green monkey, and naked mole-rat. Functional information has been added to records via the assignment of annotations based on sequence similarity to human, rat, and mouse genes. RGD has also imported well-supported cross-species data from external resources. To enable use of these data, RGD has developed a robust infrastructure of standardized ontologies, data formats, and disease- and species-centric portals, complemented with a suite of innovative tools for discovery and analysis. Using examples of single-gene and polygenic human diseases, we illustrate how data from multiple species can help to identify or confirm a gene as involved in a disease and to identify model organisms that can be studied to understand the pathophysiology of a gene or pathway. The ultimate aim of this report is to demonstrate the utility of RGD not only as the core resource for the rat research community but also as a source of bioinformatic tools to support a wider audience, empowering the search for appropriate models for human afflictions.
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) α 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 1α and macrophage inflammatory protein (MIP)- 1α or MIP-1β, which are the chemokine ligands for CXCR4 or CCR5, up-regulated production of the CXC chemokines IL-8 and GROα. Conclusions: Human colon epithelial cells express chemokine receptors. Human colonocytes have the potential to serve as targets for chemokine signaling.
Angiogenesis plays a critical role in metastasis and tumor growth. Human tumors, including colorectal adenocarcinoma, secrete
angiogenic factors, inducing proliferation and chemotaxis of ...microvascular endothelial cells, eventually leading to tumor
neovascularization. The chemokine interleukin 8 (IL-8; CXCL8) exerts potent angiogenic properties on endothelial cells through
interaction with its cognate receptors CXCR1 and CXCR2. As CXCR1 and CXCR2 expression is differentially regulated in tissue-specific
endothelial cells and effects of IL-8 on intestinal endothelial cells are not defined, we characterized the potential IL-8-induced
angiogenic mechanisms in primary cultures of human intestinal microvascular endothelial cells (HIMEC) and IL-8 receptor expression
in human intestinal microvessels. CXCR1 and CXCR2 expression on HIMEC were defined using reverse transcriptase-PCR, immunohistochemistry,
flow cytometry, and Western blot analysis. IL-8-induced downstream signaling events were assessed using immunoblot analysis
and immunofluorescence. The angiogenic effects of IL-8 on HIMEC were determined using proliferation and chemotaxis assays.
HIMEC responded to IL-8 with rapid stress fiber assembly, chemotaxis, enhanced proliferation, and phosphorylation of extracellular
signal-regulated protein kinase 1/2 (ERK 1/2). HIMEC express CXCR2, but not CXCR1. Neutralizing antibodies to CXCR2 diminished
IL-8-induced chemotaxis and stress fiber assembly. Specific inhibitors of ERK 1/2 and phosphoinositide 3-kinase abrogated
endothelial tube formation and IL-8-induced chemotaxis in HIMEC. IL-8 elicits angiogenic responses in microvascular endothelial
cells isolated from human intestine by engaging CXCR2. We confirmed tissue expression of CXCR2 in human intestinal microvessels.
Supported by the notion that malignant colonic epithelial cells overexpress IL-8, CXCR2 blockade may be a novel target for
anti-angiogenic therapy in colorectal adenocarcinoma.
1 The John Rankin Laboratory of Pulmonary Medicine,
Department of Preventive Medicine, University of Wisconsin School of
Medicine, Madison 53705-2368; 2 Department of Comparative
Biosciences, ...School of Veterinary Medicine, University of
Wisconsin, Madison, Wisconsin 53706-1102; and 3 Division
of Physiology, Department of Medicine, and White Mountain Research
Station, University of California, San Diego, La Jolla, California
92093-0623
We
tested the hypothesis that unanesthetized rats exhibit ventilatory
long-term facilitation (LTF) after intermittent, but not continuous,
hypoxia. Minute ventilation ( E ) and carbon dioxide production ( CO 2 ) were measured in
unanesthetized, unrestrained male Sprague-Dawley rats via barometric
plethysmography before, during, and after exposure to continuous or
intermittent hypoxia. Hypoxia was either isocapnic inspired
O 2 fraction (F I O 2 ) = 0.08-0.09 and inspired CO 2 fraction
(F I CO 2 ) = 0.04 or poikilocapnic
(F I O 2 = 0.11 and
F I CO 2 = 0.00). Sixty minutes after
intermittent hypoxia, E or
E / CO 2 was
significantly greater than baseline in both isocapnic and poikilocapnic
conditions. In contrast, 60 min after continuous hypoxia,
E and
E / CO 2 were not
significantly different from baseline values. These data demonstrate
ventilatory LTF after intermittent hypoxia in unanesthetized rats.
Ventilatory LTF appeared similar in its magnitude (after accounting for
CO 2 feedback), time course, and dependence on intermittent
hypoxia to phrenic LTF previously observed in anesthetized,
vagotomized, paralyzed rats.
ventilation; plasticity; intermittent hypoxia; hypoxia; respiratory
control
Department of Medicine, University of California, San Diego, La
Jolla, California 92093-0623
Chronic exposure
to hypoxia results in a time-dependent increase in ventilation called
ventilatory ...acclimatization to hypoxia. Increased
O 2 sensitivity of arterial
chemoreceptors contributes to ventilatory acclimatization to hypoxia,
but other mechanisms have also been hypothesized. We designed this
experiment to determine whether central nervous system processing of
peripheral chemoreceptor input is affected by chronic hypoxic exposure.
The carotid sinus nerve was stimulated supramaximally at different
frequencies (0.5-20 Hz, 0.2-ms duration) during recording of
phrenic nerve activity in two groups of anesthetized, ventilated,
vagotomized rats. In the chronically hypoxic group (7 days at 80 Torr
inspired P O 2 ), phrenic burst
frequency (f R , bursts/min) was
significantly higher than in the normoxic control group with carotid
sinus nerve stimulation frequencies >5 Hz. In the chronically hypoxic
group, peak amplitude of integrated phrenic nerve activity
( Phr, percent baseline) or change in Phr was
significantly greater at stimulation frequencies between 5 and 17 Hz,
and minute phrenic activity ( Phr × f R ) was significantly greater at
stimulation frequencies >5 Hz. These experiments show that chronic
hypoxia facilitates the translation of arterial chemoreceptor afferent
input to ventilatory efferent output through a mechanism in the central
nervous system.
hypoxic ventilatory response; acclimatization; central nervous
system
Department of Physiology, Medical College of Wisconsin and Zablocki Veterans Affairs Medical Center, Milwaukee, Wisconsin
Submitted 29 March 2006
; accepted in final form 15 June 2006
Ventilatory ...sensitivity to CO 2 in awake adult Brown Norway (BN) rats is 5075% lower than in adult Sprague-Dawley (SD) and salt-sensitive Dahl S (SS) rats. The purpose of the present study was to test the hypothesis that this difference would be apparent during the development of CO 2 sensitivity. Four litters of each strain were divided into four groups such that rats were exposed to 7% inspired CO 2 for 5 min in a plethysmograph every third day from postnatal day (P) 0 to P21 and again on P29 and P30. From P0 to P14, CO 2 exposure increased pulmonary ventilation ( E ) by 2550% in the BN and SD strains and between 25 to over 200% in the SS strain. In all strains beginning around P15, the response to CO 2 increased progressively reaching a peak at P1921 when E during hypercapnia was 175225% above eucapnia. There were minimal changes in CO 2 sensitivity between P21 and P30, and at both ages there were minimal between-strain differences. At P30, the response to CO 2 in the SS and SD strains was near the adult response, but the response in the BN rats was 100% greater at P30 than in adults. We conclude that 1 ) CO 2 -sensing mechanisms, and/or mechanisms downstream from the chemoreceptors, change dramatically at the age in rats when other physiological systems are also maturing ( P15), and 2 ) there is a high degree of age-dependent plasticity in CO 2 sensitivity in rats, which differs between strains.
neural control of breathing; consomics; hypercapnia
Address for reprint requests and other correspondence: H. V. Forster, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: bforster{at}mcw.edu )
The Rat Genome Database (RGD, http://rgd.mcw.edu) was developed to provide a core resource for rat researchers combining genetic, genomic, pathway, phenotype and strain information with a focus on ...disease. RGD users are provided with access to structured and curated data from the molecular level through to the level of the whole organism, including the variations associated with disease phenotypes. To fully support use of the rat as a translational model for biological systems and human disease, RGD continues to curate these datasets while enhancing and developing tools to allow efficient and effective access to the data in a variety of formats including linear genome viewers, pathway diagrams and biological ontologies. To support pathophysiological analysis of data, RGD Disease Portals provide an entryway to integrated gene, QTL and strain data specific to a particular disease. In addition to tool and content development and maintenance, RGD promotes rat research and provides user education by creating and disseminating tutorials on the curated datasets, submission processes, and tools available at RGD. By curating, storing, integrating, visualizing and promoting rat data, RGD ensures that the investment made into rat genomics and genetics can be leveraged by all interested investigators.
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