bluuebunny/crossref_metadata_2025 huggingface dataset:
Diaphragm muscle fiber function and structure in humans with hemidiaphragm paralysis
Authors: Welvaart W. N. , Paul M. A. , van Hees H. W. H. , Stienen G. J. M. , Niessen J. W. M. , de Man F. S. , Sieck G. C. , Vonk-Noordegraaf A. , Ottenheijm C. A. C.
Published: May 2011

Recent studies proposed that mechanical inactivity of the human diaphragm during mechanical ventilation rapidly causes diaphragm atrophy and weakness. However, conclusive evidence for the notion that diaphragm weakness is a direct consequence of mechanical inactivity is lacking. To study the effect of hemidiaphragm paralysis on diaphragm muscle fiber function and structure in humans, biopsies were obtained from the paralyzed hemidiaphragm in eight patients with hemidiaphragm paralysis. All patients had unilateral paralysis of known duration, caused by en bloc resection of the phrenic nerve with a tumor. Furthermore, diaphragm biopsies were obtained from three control subjects. The contractile performance of demembranated muscle fibers was determined, as well as fiber ultrastructure and morphology. Finally, expression of E3 ligases and proteasome activity was determined to evaluate activation of the ubiquitin-proteasome pathway. The force-generating capacity, as well as myofibrillar ultrastructure, of diaphragm muscle fibers was preserved up to 8 wk of paralysis. The cross-sectional area of slow fibers was reduced after 2 wk of paralysis; that of fast fibers was preserved up to 8 wk. The expression of the E3 ligases MAFbx and MuRF-1 and proteasome activity was not significantly upregulated in diaphragm fibers following paralysis, not even after 72 and 88 wk of paralysis, at which time marked atrophy of slow and fast diaphragm fibers had occurred. Diaphragm muscle fiber atrophy and weakness following hemidiaphragm paralysis develops slowly and takes months to occur.
Inhibition of neutrophil apoptosis by PAI-1
Authors: Zmijewski Jaroslaw W. , Bae Hong-Beom , Deshane Jessy S. , Peterson Cynthia B. , Chaplin David D. , Abraham Edward
Published: May 2011

Increased circulating and tissue levels of plasminogen activator inhibitor 1 (PAI-1) are often present in severe inflammatory states associated with neutrophil activation and accumulation and correlate with poor clinical outcome from many of these conditions. The mechanisms by which PAI-1 contributes to inflammation have not been fully delineated. In the present experiments, we found that addition of PAI-1 to neutrophil cultures diminished the rate of spontaneous and TNF-related apoptosis-inducing ligand-induced apoptotic cell death. The effects of PAI-1 on cell viability were associated with activation of antiapoptotic signaling pathways, including upregulation of PKB/Akt, Mcl-1, and Bcl-xL. Although urokinase-plasminogen activator receptor, lipoprotein receptor-related protein, and vitronectin are primary ligands for PAI-1, these molecules were not involved in mediating its antiapoptotic properties. In contrast, blocking pertussis toxin-sensitive G protein-coupled receptors and selective inhibition of phosphatidylinositide 3-kinase reversed the ability of PAI-1 to extend neutrophil viability. The antiapoptotic effects of PAI-1 were also evident under in vivo conditions during LPS-induced acute lung injury, where enhanced apoptosis was present among neutrophils accumulating in the lungs of PAI-1−/−compared with PAI-1+/+mice. These results demonstrate a novel antiapoptotic role for PAI-1 that may contribute to its participation in neutrophil-associated inflammatory responses.
Adenosine activation of A2Breceptor(s) is essential for stimulated epithelial ciliary motility and clearance
Authors: Allen-Gipson Diane S. , Blackburn Michael R. , Schneider Daniel J. , Zhang Hui , Bluitt DeAndre L. , Jarrell Justin C. , Yanov Daniel , Sisson Joseph H. , Wyatt Todd A.
Published: May 2011

Mucociliary clearance, vital to lung clearance, is dependent on cilia beat frequency (CBF), coordination of cilia, and the maintenance of periciliary fluid. Adenosine, the metabolic breakdown product of ATP, is an important modulator of ciliary motility. However, the contributions of specific adenosine receptors to key airway ciliary motility processes are unclear. We hypothesized that adenosine modulates ciliary motility via activation of its cell surface receptors (A1, A2A, A2B, or A3). To test this hypothesis, mouse tracheal rings (MTRs) excised from wild-type and adenosine receptor knockout mice (A1, A2A, A2B, or A3, respectively), and bovine ciliated bronchial epithelial cells (BBECs) were stimulated with known cilia activators, isoproterenol (ISO; 10 μM) and/or procaterol (10 μM), in the presence or absence of 5′-(N-ethylcarboxamido) adenosine (NECA), a nonselective adenosine receptor agonist [100 nM (A1, A2A, A3); 10 μM (A2B)], and CBF was measured. Cells and MTRs were also stimulated with NECA (100 nM or 10 μM) in the presence and absence of adenosine deaminase inhibitor, erythro-9- (2-hydroxy-3-nonyl) adenine hydrochloride (10 μM). Both ISO and procaterol stimulated CBF in untreated cells and/or MTRs from both wild-type and adenosine knockout mice by ∼3 Hz. Likewise, CBF significantly increased ∼2–3 Hz in BBECs and wild-type MTRs stimulated with NECA. MTRs from A1, A2A, and A3knockout mice stimulated with NECA also demonstrated an increase in CBF. However, NECA failed to stimulate CBF in MTRs from A2Bknockout mice. To confirm the mechanism by which adenosine modulates CBF, protein kinase activity assays were conducted. The data revealed that NECA-stimulated CBF is mediated by the activation of cAMP-dependent PKA. Collectively, these data indicate that purinergic stimulation of CBF requires A2Badenosine receptor activation, likely via a PKA-dependent pathway.
Bone marrow cells repair cigarette smoke-induced emphysema in rats
Authors: Huh Jin Won , Kim Sun-Yong , Lee Ji Hyun , Lee Jin-Seok , Van Ta Quang , Kim MiJung , Oh Yeon-Mok , Lee Yun-Song , Lee Sang-Do
Published: May 2011

The therapeutic potential of stem cells in chronic obstructive pulmonary disease is not well known although stem cell therapy is effective in models of other pulmonary diseases. We tested the capacities of bone marrow cells (BMCs), mesenchymal stem cells (MSCs), and conditioned media of MSCs (MSC-CM) to repair cigarette smoke-induced emphysema. Inbred female Lewis rats were exposed to cigarette smoke for 6 mo and then received BMCs, MSCs, or MSC-CM from male Lewis rats. For 2 mo after injection, the BMC treatment gradually alleviated the cigarette smoke-induced emphysema and restored the increased mean linear intercept. The BMC treatment significantly increased cell proliferation and the number of small pulmonary vessels, reduced apoptotic cell death, attenuated the mean pulmonary arterial pressure, and inhibited muscularization in small pulmonary vessels. However, only a few male donor cells were detected from 1 day to 1 mo after BMC administration. The MSCs and cell-free MSC-CM also induced the repair of emphysema and increased the number of small pulmonary vessels. Our data show that BMC, MSCs, and MSC-CM treatment repaired cigarette smoke-induced emphysema. The repair activity of these treatments is consistent with a paracrine effect rather than stem cell engraftment because most of the donor cells disappeared and because cell-free MSC-CM also induced the repair.
Sepiapterin improves angiogenesis of pulmonary artery endothelial cells with in utero pulmonary hypertension by recoupling endothelial nitric oxide synthase
Authors: Teng Ru-Jeng , Du Jianhai , Xu Hao , Bakhutashvili Ivane , Eis Annie , Shi Yang , Pritchard Kirkwood A. , Konduri Girija G.
Published: May 2011

Persistent pulmonary hypertension of the newborn (PPHN) is associated with decreased blood vessel density that contributes to increased pulmonary vascular resistance. Previous studies showed that uncoupled endothelial nitric oxide (NO) synthase (eNOS) activity and increased NADPH oxidase activity resulted in marked decreases in NO bioavailability and impaired angiogenesis in PPHN. In the present study, we hypothesize that loss of tetrahydrobiopterin (BH4), a critical cofactor for eNOS, induces uncoupled eNOS activity and impairs angiogenesis in PPHN. Pulmonary artery endothelial cells (PAEC) isolated from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were used to investigate the cellular mechanisms impairing angiogenesis in PPHN. Cellular mechanisms were examined with respect to BH4 levels, GTP-cyclohydrolase-1 (GCH-1) expression, eNOS dimer formation, and eNOS-heat shock protein 90 (hsp90) interactions under basal conditions and after sepiapterin (Sep) supplementation. Cellular levels of BH4, GCH-1 expression, and eNOS dimer formation were decreased in HTFL-PAEC compared with NFL-PAEC. Sep supplementation decreased apoptosis and increased in vitro angiogenesis in HTFL-PAEC and ex vivo pulmonary artery sprouting angiogenesis. Sep also increased cellular BH4 content, NO production, eNOS dimer formation, and eNOS-hsp90 association and decreased the superoxide formation in HTFL-PAEC. These data demonstrate that Sep improves NO production and angiogenic potential of HTFL-PAEC by recoupling eNOS activity. Increasing BH4 levels via Sep supplementation may be an important therapy for improving eNOS function and restoring angiogenesis in PPHN.
Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi
Authors: Martens Chelsea J. , Inglis Sarah K. , Valentine Vincent G. , Garrison Jennifer , Conner Gregory E. , Ballard Stephen T.
Published: May 2011

To better understand how airways produce thick airway mucus, nonvolatile solids were measured in liquid secreted by bronchi from normal pig, cystic fibrosis (CF) human, and non-CF human lungs. Bronchi were exposed to various secretagogues and anion secretion inhibitors to induce a range of liquid volume secretion rates. In all three groups, the relationship of solids concentration (percent nonvolatile solids) to liquid volume secretion rate was curvilinear, with higher solids concentration associated with lower rates of liquid volume secretion. In contrast, the secretion rates of solids mass and water mass as functions of liquid volume secretion rates exhibited positive linear correlations. The y-intercepts of the solids mass-liquid volume secretion relationships for all three groups were positive, thus accounting for the higher solids concentrations in airway liquid at low rates of secretion. Predictive models derived from the solids mass and water mass linear equations fit the experimental percent solids data for the three groups. The ratio of solids mass secretion to liquid volume secretion was 5.2 and 2.4 times higher for CF bronchi than for pig and non-CF bronchi, respectively. These results indicate that normal pig, non-CF human, and CF human bronchi produce a high-percent-solids mucus (>8%) at low rates of liquid volume secretion (≤1.0 μl·cm−2·h−1). However, CF bronchi produce mucus with twice the percent solids (∼8%) of pig or non-CF human bronchi at liquid volume secretion rates ≥4.0 μl·cm−2·h−1.
Endurance exercise training blunts the deleterious effect of high-fat feeding on whole body efficiency
Authors: Edwards Lindsay M. , Holloway Cameron J. , Murray Andrew J. , Knight Nicholas S. , Carter Emma E. , Kemp Graham J. , Thompson Campbell H. , Tyler Damian J. , Neubauer Stefan , Robbins Peter A. , Clarke Kieran
Published: June 2011

We recently showed that a week-long, high-fat diet reduced whole body exercise efficiency in sedentary men by >10% (Edwards LM, Murray AJ, Holloway CJ, Carter EE, Kemp GJ, Codreanu I, Brooker H, Tyler DJ, Robbins PA, Clarke K. FASEB J 25: 1088–1096, 2011). To test if a similar dietary regime would blunt whole body efficiency in endurance-trained men and, as a consequence, hinder aerobic exercise performance, 16 endurance-trained men were given a short-term, high-fat (70% kcal from fat) and a moderate carbohydrate (50% kcal from carbohydrate) diet, in random order. Efficiency was assessed during a standardized exercise task on a cycle ergometer, with aerobic performance assessed during a 1-h time trial and mitochondrial function later measured using 31P-magnetic resonance spectroscopy. The subjects then underwent a 2-wk wash-out period, before the study was repeated with the diets crossed over. Muscle biopsies, for mitochondrial protein analysis, were taken at the start of the study and on the 5th day of each diet. Plasma fatty acids were 60% higher on the high-fat diet compared with moderate carbohydrate diet ( P < 0.05). However, there was no change in whole body efficiency and no change in mitochondrial function. Endurance exercise performance was significantly reduced ( P < 0.01), most probably due to glycogen depletion. Neither diet led to changes in citrate synthase, ATP synthase, or mitochondrial uncoupling protein 3. We conclude that prior exercise training blunts the deleterious effect of short-term, high-fat feeding on whole body efficiency.
Potential Role of the Inflammasome-Derived Inflammatory Cytokines in Pulmonary Fibrosis
Authors: Biswas Rupa , Bunderson-Schelvan Melisa , Holian Andrij
Published: June 2011

Pulmonary fibrosis is a progressive, disabling disease with mortality rates that appear to be increasing in the western population, including the USA. There are over 140 known causes of pulmonary fibrosis as well as many unknown causes. Treatment options for this disease are limited due to poor understanding of the molecular mechanisms of the disease progression. However, recent progress in inflammasome research has greatly contributed to our understanding of its role in inflammation and fibrosis development. The inflammasome is a multiprotein complex that is an important component of both the innate and adaptive immune systems. Activation of proinflammatory cytokines following inflammasome assembly, such as IL-1βand IL-18, has been associated with development of PF. In addition, components of the inflammasome complex itself, such as the adaptor protein ASC have been associated with PF development. Recent evidence suggesting that the fibrotic process can be reversed via blockade of pathways associated with inflammasome activity may provide hope for future drug strategies. In this paper we will give an introduction to pulmonary fibrosis and its known causes. In addition, we will discuss the importance of the inflammasome in the development of pulmonary fibrosis as well as discuss potential future treatment options.
The Homological Kähler‐De Rham Differential Mechanism part I: Application in General Theory of Relativity
Authors: Mallios Anastasios , Zafiris Elias
Published: June 2011

The mechanism of differential geometric calculus is based on the fundamental notion of a connection on a module over a commutative and unital algebra of scalars
defined together with the associated de Rham complex. In this communication,
we demonstrate that the dynamical mechanism of physical fields can be formulated by purely algebraic means, in terms of the homological Kähler‐De Rham differential schema, constructed by connection inducing functors and their associated curvatures, independently of any background substratum. In this context, we show explicitly that the application of this mechanism in General Relativity, instantiating the case of gravitational dynamics, is
related with the absolute representability of the theory in the
field of real numbers, a byproduct of which is the fixed background
manifold construct of this theory. Furthermore, the background independence of the homological differential mechanism is of particular importance for the formulation of dynamics
in quantum theory, where the adherence to a fixed manifold substratum is
problematic due to singularities or other topological defects.
Optical Coherence Tomography in Multiple Sclerosis and Neuromyelitis Optica: An Update
Authors: Noval Susana , Contreras Inés , Muñoz Silvia , Oreja-Guevara Celia , Manzano Beatriz , Rebolleda Gema
Published: June 2011

Optical coherence tomography (OCT) uses light interference patterns to produce a cross-sectional image of the retina. It is capable of measuring the unmyelinated axons of the retinal ganglionar cells as they converge on the optic disc. In a disease like multiple sclerosis (MS), in which axonal loss has been identified as an important cause of sustained disability, it may prove an invaluable tool. OCT has demonstrated that axonal loss occurs after each episode of optic neuritis and that the degree of axonal loss is correlated to visual outcomes. Furthermore, axonal loss occurs in MS even in the absence of inflammatory episodes, and the degree of this loss is correlated with the duration of the disease process, with more thinning as the disease advances and in progressive forms. Thus, OCT retinal nerve fiber layer measurements may represent an objective outcome measure with which to evaluate the effect of treatment.
Statistical Convergence and Ideal Convergence of Sequences of Functions in 2‐Normed Spaces
Authors: Sarabadan Saeed , Talebi Sorayya
Published: June 2011

We present various kinds of statistical convergence and
ℐ‐convergence for sequences of functions with values in 2‐normed spaces and obtain
a criterion for ℐ‐convergence of sequences of functions in 2‐normed spaces. We also
define the notion of ℐ‐equistatistically convergence and study ℐ‐equi‐statistically convergence of sequences of functions.
A Systematic Review of the Current Role of Minimally Invasive Spine Surgery in the Management of Metastatic Spine Disease
Authors: Molina Camilo A. , Gokaslan Ziya L. , Sciubba Daniel M.
Published: June 2011

Although increasingly aggressive decompression and resection methods have resulted in improved outcomes for patients with metastatic spine disease, these aggressive surgeries are not feasible for patients with numerous comorbid conditions. Such patients stand to benefit from management via minimally invasive spine surgery (MIS), given its association with decreased perioperative morbidity. We performed a systematic review of literature with the goal of evaluating the clinical efficacy and safety of MIS in the setting of metastatic spine disease. Results suggest that MIS is an efficacious means of achieving neurological improvement and alleviating pain. In addition, data suggests that MIS offers decreased blood loss, operative time, and complication rates in comparison to standard open spine surgery. However, due to the paucity of studies and low class of available evidence, the ability to draw comprehensive conclusions is limited. Future investigations should be conducted comparing standard surgery versus MIS in a prospective fashion.
Pathogen Recognition by the Long Pentraxin PTX3
Authors: Moalli Federica , Jaillon Sebastien , Inforzato Antonio , Sironi Marina , Bottazzi Barbara , Mantovani Alberto , Garlanda Cecilia
Published: June 2011

Innate immunity represents the first line of defence against pathogens and plays key roles in activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules (PRMs) that recognise pathogen‐associated molecular patterns (PAMPs) and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. The long pentraxin PTX3 is a prototypic soluble PRM that is produced at sites of infection and inflammation by both somatic and immune cells. Gene targeting of this evolutionarily conserved protein has revealed a nonredundant role in resistance to selected pathogens. Moreover, PTX3 exerts important functions at the cross‐road between innate immunity, inflammation, and female fertility. Here, we review the studies on PTX3, with emphasis on pathogen recognition and cross‐talk with other components of the innate immune system.
Caveolin-1 Upregulation Mediates Suppression of Primary Breast Tumor Growth and Brain Metastases by Stat3 Inhibition
Authors: Chiu Wen-Tai , Lee Hsueh-Te , Huang Feng-Ju , Aldape Kenneth D. , Yao Jun , Steeg Patricia S. , Chou Cheng-Yang , Lu Zhimin , Xie Keping , Huang Suyun
Published: May 2011

Stat3 activation has been implicated as an important driver of brain metastasis in breast cancer, but the critical targets of Stat3 in this process are yet to be fully defined. In this study, we identified the lipid raft organizing protein Caveolin-1 (Cav-1) as a critical genetic target of Stat3 in this process. In human breast cancers, we found that activated Stat3 correlated with attenuation of Cav-1 in brain metastases relative to primary tumors. Cav-1 promoter activity and gene expression were increased by overexpressing an activated form of Stat3 but decreased by attenuation of Stat3 activity or expression. We identified putative Stat3-binding elements in the Cav-1 promoter and showed a direct repression of Cav-1 transcription by Stat3. Reciprocally, we showed that strategies to increase or decrease Cav-1 expression were sufficient to attenuate or promote breast cancer cell invasion. Furthermore, increased expression of Cav-1 phenocopied the effects of Stat3 activation in blocking primary tumor growth and abrogating formation of brain metastases. Collectively, our findings provide clinical and mechanistic evidence that Cav-1 is a critical target for suppression by Stat3 in driving invasion and metastasis of breast cancer cells. Cancer Res; 71(14); 4932–43. ©2011 AACR.
Sorafenib Enhances Pemetrexed Cytotoxicity through an Autophagy-Dependent Mechanism in Cancer Cells
Authors: Bareford M. Danielle , Park Margaret A. , Yacoub Adly , Hamed Hossein A. , Tang Yong , Cruickshanks Nichola , Eulitt Patrick , Hubbard Nisan , Tye Gary , Burow Matthew E. , Fisher Paul B. , Moran Richard G. , Nephew Kenneth P. , Grant Steven , Dent Paul
Published: May 2011

Pemetrexed (ALIMTA, Lilly) is a folate antimetabolite that has been approved by the U.S. Food and Drug Administration for the treatment of non–small cell lung cancer and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multikinase inhibitor sorafenib (Nexavar, Bayer), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K, and/or phosphorylated mTOR, in addition to class III receptor tyrosine kinases such as platelet-derived growth factor receptor beta and VEGF receptors, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors. Cancer Res; 71(14); 4955–67. ©2011 AACR.
Cancer Stem Cells and Chemosensitivity
Authors: Maugeri-Saccà Marcello , Vigneri Paolo , De Maria Ruggero
Published: May 2011

Cancer lethality is mainly due to the onset of distant metastases and refractoriness to chemotherapy. Thus, the development of molecular targeted agents that can restore or increase chemosensitivity will provide valuable therapeutic options for cancer patients. Growing evidence indicates that a cellular subpopulation with stem cell–like features, commonly referred to as cancer stem cells (CSCs), is critical for tumor generation and maintenance. Recent advances in stem cell biology are revealing that this cellular fraction shares many properties with normal adult stem cells and is able to propagate the parental tumor in animal models. CSCs seem to be protected against widely used chemotherapeutic agents by means of different mechanisms, such as a marked proficiency in DNA damage repair, high expression of ATP-binding cassette drug transporters, and activation of PI3K/AKT and Wnt pathways. Moreover, microenvironmental stimuli such as those involved in the epithelial-mesenchymal transition and hypoxia indirectly contribute to chemoresistance by inducing in cancer cells a stem-like phenotype. Understanding how CSCs overcome chemotherapy-induced death stimuli, and integrating such knowledge into clinical research methodology, has become a priority in the process of identifying innovative therapeutic strategies aimed at improving the outcome of cancer patients. Clin Cancer Res; 17(15); 4942–7. ©2011 AACR.
Impact of Clinical and Pathologic Features on Tumor-Infiltrating Lymphocyte Expansion from Surgically Excised Melanoma Metastases for Adoptive T-cell Therapy
Authors: Joseph Richard W. , Peddareddigari Vijay R. , Liu Ping , Miller Priscilla W. , Overwijk Willem W. , Bekele Nebiyou B. , Ross Merrick I. , Lee Jeffrey E. , Gershenwald Jeffrey E. , Lucci Anthony , Prieto Victor G. , McMannis John D. , Papadopoulos Nicholas , Kim Kevin , Homsi Jade , Bedikian Agop , Hwu Wen-Jen , Hwu Patrick , Radvanyi Laszlo G.
Published: June 2011

Purpose: Clinical trials on adoptive T-cell therapy (ACT) using expanded tumor-infiltrating lymphocytes (TIL) have shown response rates of over 50% in refractory melanoma. However, little is known how clinical and pathologic features impact TIL outgrowth isolated from metastatic melanoma tumors.

Experimental Design: We analyzed the impact of clinical and pathologic features on initial TIL outgrowth in 226 consecutive patients undergoing tumor resection. Successful initial TIL outgrowth was defined as ≥40 million viable lymphocytes harvested from all tumor fragments in a 5-week culture. To normalize for the different size of resected tumors and thus available tumor fragments, we divided the number of expanded TIL by the starting number of tumor fragments (TIL/fragment).

Results: Overall, initial TIL outgrowth was successful in 62% of patients, with patients ≤30 years of age (94%; P = 0.01) and female patients (71% vs. 57% for males; P = 0.04) having the highest rate of success. Systemic therapy 30 days before tumor harvest negatively impacted initial TIL outgrowth compared to patients who never received systemic therapy (47% vs. 71%, P = 0.02). Biochemotherapy within 0 to 60 days of tumor harvest negatively impacted the initial TIL outgrowth with a success rate of only 16% (P < 0.0001).

Conclusion: Parameters such as age, sex, and the type and timing of prior systemic therapy significantly affect the success rate of the initial TIL outgrowth from tumor fragments for ACT; these parameters may be helpful in selecting patients for melanoma ACT. Clin Cancer Res; 17(14); 4882–91. ©2011 AACR.
Brivanib, a Dual FGF/VEGF Inhibitor, Is Active Both First and Second Line against Mouse Pancreatic Neuroendocrine Tumors Developing Adaptive/Evasive Resistance to VEGF Inhibition
Authors: Allen Elizabeth , Walters Ian B. , Hanahan Douglas
Published: May 2011

Purpose: Preclinical trials of a mouse model of pancreatic neuroendocrine tumors (PNET) were conducted to determine whether dual FGF/VEGF pathway inhibition with brivanib can improve first-line efficacy in comparison with VEGF inhibitors lacking fibroblast growth factor (FGF)-inhibitory activity and to characterize second-line brivanib activity before and after the onset of evasive resistance to VEGF-selective therapy.

Experimental Design: An anti-VEGFR2 monoclonal antibody (DC101), an inhibitor of FGF signaling (FGF ligand trap), sorafenib, and brivanib were comparatively evaluated in first-line monotherapy in short and longer term fixed endpoint intervention trials in the RIP-Tag2 mouse model of PNET. Brivanib was also tested second line aiming to block adaptive resistance to selective VEGF therapies, assessing tumor growth, vascularity, hypoxia, invasion, and metastasis. The effects of initiating second-line brivanib therapy prior to or following overt relapse on sorafenib therapy were compared in overall survival trials to first-line therapies.

Results: Brivanib produced enduring tumor stasis and angiogenic blockade, both first and second line following the failure of DC101 or sorafenib. Overall survival was significantly extended by brivanib versus sorafenib, both first-line and when second-line therapy was initiated prior to sorafenib failure; second-line brivanib was less beneficial when initiated later, after the initiation of revascularization and incipient tumor progression.

Conclusions: Brivanib holds promise and deserves consideration for clinical evaluation as an antiangiogenic therapy, both in the context of impending failures of VEGF-selective therapy and in a first-line setting aiming to limit the adaptive response to VEGF inhibitors that results in evasive resistance. Clin Cancer Res; 17(16); 5299–310. ©2011 AACR.
FGFR-4 Arg388 Enhances Prostate Cancer Progression via Extracellular Signal–Related Kinase and Serum Response Factor Signaling
Authors: Yu Wendong , Feng Shu , Dakhova Olga , Creighton Chad J. , Cai Yi , Wang Jianghua , Li Rile , Frolov Anna , Ayala Gustavo , Ittmann Michael
Published: May 2011

Purpose: Increased expression of FGFR-4 and its ligands have been linked to lethal prostate cancer (PCa). Furthermore, a germ line polymorphism in the FGFR-4 gene, resulting in arginine at codon 388 (Arg388) instead of glycine (Gly388), is associated with aggressive disease. The FGFR-4 Arg388 variant results in increased receptor stability, sustained receptor activation, and increased motility and invasion compared with Gly388. However, the impact of sustained signaling on cellular signal transduction pathways is unknown.

Experimental Design: Expression microarray analysis of immortalized prostatic epithelial cells lines expressing FGFR-4 Arg388 or Gly388 was used to establish a gene signature associated with FGFR-4 Arg388 expression. Transient transfection of reporters and inhibitors was used to establish the pathways activated by FGFR-4 Arg388 expression. The impact of pathway knockdown in vitro and in an orthotopic model was assessed using inhibitors and/or short hairpin RNA (shRNA).

Results: Expression of the FGFR-4 Arg388 protein leads to increased activity of the extracellular signal–related kinase (ERK) pathway, increased activity of serum response factor (SRF) and AP1, and transcription of multiple genes that are correlated with aggressive clinical behavior in PCa. Increased expression of SRF is associated with biochemical recurrence in men undergoing radical prostatectomy. Consistent with these observations, knockdown of FGFR-4 Arg388 in PCa cells decreases proliferation and invasion in vitro and primary tumor growth and metastasis in vivo.

Conclusions: These studies define a signal transduction pathway downstream of FGFR-4 Arg388 that acts via ERK and SRF to promote PCa progression. Clin Cancer Res; 17(13); 4355–66. ©2011 AACR.
The Activity of Trabectedin As a Single Agent or in Combination with Everolimus for Clear Cell Carcinoma of the Ovary
Authors: Mabuchi Seiji , Hisamatsu Takeshi , Kawase Chiaki , Hayashi Masami , Sawada Kenjiro , Mimura Kazuya , Takahashi Kazuhiro , Takahashi Toshifumi , Kurachi Hirohisa , Kimura Tadashi
Published: May 2011

Purpose: The objective of this study was to evaluate the antitumor efficacy of trabectedin in clear cell carcinoma (CCC) of the ovary, which is regarded as an aggressive, chemoresistant, histologic subtype.

Experimental Design: Using 6 human ovarian cancer cell lines (3 CCC and 3 serous adenocarcinomas), the antitumor effects of trabectedin were examined in vitro, and we compared its activity according to histology. We next examined the antitumor activity of trabectedin in both cisplatin-resistant and paclitaxel-resistant CCC cells in vitro. Then, the in vivo effects of trabectedin were evaluated using mice inoculated with CCC cell lines. Using 2 pairs of trabectedin-sensitive parental and trabectedin-resistant CCC sublines, we investigated the role of mTOR in the mechanism of acquired resistance to trabectedin. Finally, we determined the effect of mTOR inhibition by everolimus on the antitumor efficacy of trabectedin in vitro and in vivo.

Results: Trabectedin showed significant antitumor activity toward chemosensitive and chemoresistant CCC cells in vitro. Mouse xenografts of CCC cells revealed that trabectedin significantly inhibits tumor growth. Greater activation of mTOR was observed in trabectedin-resistant CCC cells than in their respective parental cells. The continuous inhibition of mTOR significantly enhanced the therapeutic efficacy of trabectedin and prevented CCC cells from acquiring resistance to trabectedin.

Conclusion: Trabectedin is a promising agent for CCC as a first-line chemotherapy and as a second-line treatment of recurrent CCC that had previously been treated with cisplatin or paclitaxel. Moreover, trabectedin combined with everolimus may be more efficacious for the management of CCC. Clin Cancer Res; 17(13); 4462–73. ©2011 AACR.
Multiple Loci Modulate Opioid Therapy Response for Cancer Pain
Authors: Galvan Antonella , Skorpen Frank , Klepstad Pål , Knudsen Anne Kari , Fladvad Torill , Falvella Felicia S. , Pigni Alessandra , Brunelli Cinzia , Caraceni Augusto , Kaasa Stein , Dragani Tommaso A.
Published: May 2011

Purpose: Patients treated with opioid drugs for cancer pain experience different relief responses, raising the possibility that genetic factors play a role in opioid therapy outcome. In this study, we tested the hypothesis that genetic variations may control individual response to opioid drugs in cancer patients.

Experimental Design: We tested 1 million single-nucleotide polymorphisms (SNP) in European cancer patients, selected in a first series, for extremely poor (pain relief ≤40%; n = 145) or good (pain relief ≥90%; n = 293) responses to opioid therapy using a DNA-pooling approach. Candidate SNPs identified by SNP-array were genotyped in individual samples constituting DNA pools as well as in a second series of 570 patients.

Results: Association analysis in 1,008 cancer patients identified eight SNPs significantly associated with pain relief at a statistical threshold of P < 1.0 × 10−3, with rs12948783, upstream of the RHBDF2 gene, showing the best statistical association (P = 8.1 × 10−9). Functional annotation analysis of SNP-tagged genes suggested the involvement of genes acting on processes of the neurologic system.

Conclusion: Our results indicate that the identified SNP panel can modulate the response of cancer patients to opioid therapy and may provide a new tool for personalized therapy of cancer pain. Clin Cancer Res; 17(13); 4581–7. ©2011 AACR.
SKLB1002, a Novel Potent Inhibitor of VEGF Receptor 2 Signaling, Inhibits Angiogenesis and Tumor Growth In Vivo
Authors: Zhang Shuang , Cao Zhixing , Tian Hongwei , Shen Guobo , Ma Yongping , Xie Huanzhang , Liu Yalin , Zhao Chengjian , Deng Senyi , Yang Yang , Zheng Renlin , Li Weiwei , Zhang Na , Liu Shengyong , Wang Wei , Dai Lixia , Shi Shuai , Cheng Lin , Pan Youli , Feng Shan , Zhao Xia , Deng Hongxin , Yang Shengyong , Wei Yuquan
Published: May 2011

Purpose: VEGF receptor 2 (VEGFR2) inhibitors, as efficient antiangiogenesis agents, have been applied in the cancer treatment. However, currently most of these anticancer drugs suffer some adverse effects. Discovery of novel VEGFR2 inhibitors as anticancer drug candidates is still needed.

Experimental Design: In this investigation, we adopted a restricted de novo design method to design VEGFR2 inhibitors. We selected the most potent compound SKLB1002 and analyzed its inhibitory effects on human umbilical vein endothelial cells (HUVEC) in vitro. Tumor xenografts in zebrafish and athymic mice were used to examine the in vivo activity of SKLB1002.

Results: The use of the restricted de novo design method indeed led to a new potent VEGFR2 inhibitor, SKLB1002, which could significantly inhibit HUVEC proliferation, migration, invasion, and tube formation. Western blot analysis was conducted, which indicated that SKLB1002 inhibited VEGF-induced phosphorylation of VEGFR2 kinase and the downstream protein kinases including extracellular signal-regulated kinase, focal adhesion kinase, and Src. In vivo zebrafish model experiments showed that SKLB1002 remarkably blocked the formation of intersegmental vessels in zebrafish embryos. It was further found to inhibit a new microvasculature in zebrafish embryos induced by inoculated tumor cells. Finally, compared with the solvent control, administration of 100 mg/kg/d SKLB1002 reached more than 60% inhibition against human tumor xenografts in athymic mice. The antiangiogenic effect was indicated by CD31 immunohistochemical staining and alginate-encapsulated tumor cell assay.

Conclusions: Our findings suggest that SKLB1002 inhibits angiogenesis and may be a potential drug candidate in anticancer therapy. Clin Cancer Res; 17(13); 4439–50. ©2011 AACR.
Dual Blockade of EGFR and c-Met Abrogates Redundant Signaling and Proliferation in Head and Neck Carcinoma Cells
Authors: Xu Hai , Stabile Laura P. , Gubish Christopher T. , Gooding William E. , Grandis Jennifer R. , Siegfried Jill M.
Published: May 2011

Purpose: Head and neck squamous cell carcinoma (HNSCC) is usually fatal, and innovative approaches targeting growth pathways are necessary to effectively treat this disease. Both the epidermal growth factor receptor (EGFR) and the hepatocyte growth factor (HGF)/c-Met pathways are overexpressed in HNSCC and initiate similar downstream signaling pathways. c-Met may act in consort with EGFR and/or be activated as a compensatory pathway in the presence of EGFR blockade.

Experimental Design: Expression levels of EGFR and c-Met were determined by Western analysis in HNSCC cell lines and correlated with antitumor responses to inhibitors of these pathways.

Results: Combining the c-Met inhibitor PF2341066 with the EGFR inhibitor gefitinib abrogated HNSCC cell proliferation, invasion, and wound healing significantly more than inhibition of each pathway alone in HNSCC cell lines. When both HGF and the EGFR ligand, TGF-α, were present in vitro, P-AKT and P-MAPK expression were maximally inhibited by targeting both EGFR and c-Met pathways, suggesting that c-Met or EGFR can compensate when phosphorylation of the other receptor is inhibited. We also showed that TGF-α can induce phosphorylation of c-Met over sixfold by 8 hours in the absence of HGF, supporting a ligand-independent mechanism. Combined targeting of c-Met and EGFR resulted in an enhanced inhibition of tumor volumes accompanied by a decreased number of proliferating cells and increased apoptosis compared with single agent treatment in vivo.

Conclusions: Together, these results suggest that dual blockade of c-Met and EGFR may be a promising clinical therapeutic strategy for treating HNSCC. Clin Cancer Res; 17(13); 4425–38. ©2011 AACR.
NGAL Expression Is Elevated in Both Colorectal Adenoma–Carcinoma Sequence and Cancer Progression and Enhances Tumorigenesis in Xenograft Mouse Models
Authors: Sun Yan , Yokoi Kenji , Li Hui , Gao Jun , Hu Limei , Liu Ben , Chen Kexin , Hamilton Stanley R. , Fan Dominic , Sun Baocun , Zhang Wei
Published: May 2011

Purpose: There is growing evidence implicating that neutrophil gelatinase–associated lipocalin (NGAL) plays a role in the development and progression of cancers. However, the effect of NGAL in colorectal carcinoma (CRC) has not been clearly elucidated. In this study, we investigated the role of NGAL in the tumorigenesis and progression of CRC and evaluated the clinical value of NGAL expression.

Experimental Design: We examined NGAL expression in 526 colorectal tissue samples, including 53 sets of matched specimens (histologically normal mucosa, adenomas, and carcinomas) using immunohistochemical analysis. In CRCs, correlations between NGAL expression and clinicopathologic parameters were analyzed, and survival analysis was conducted. The role of NGAL was further tested using mouse xenograft models.

Results: NGAL expression was elevated during the colorectal adenoma–carcinoma sequence both among the 526 cases (rs = 0.66, P < 0.001) and in the 53 sets of matched specimens (rs = 0.60, P < 0.001). In CRCs, NGAL expression was associated with cancer stage (P = 0.041) and tumor recurrence in stage II patients (P = 0.037). Survival analysis revealed that NGAL expression was an independent prognostic factor for overall survival (HR = 1.84, P = 0.004) and for disease-free survival of stage II patients (HR = 5.88, P = 0.021). In mouse models, the xenografts in cecum and spleen were heavier and more numerous in the group injected with NGAL-overexpressing CRC cells (P < 0.05).

Conclusions: NGAL overexpression may promote the tumorigenesis and progression of CRC. Detecting NGAL expression in tumor tissues may be useful for evaluating prognosis of patients with CRC. Clin Cancer Res; 17(13); 4331–40. ©2011 AACR.
MicroRNA Profiling of Sporadic and Hereditary Medullary Thyroid Cancer Identifies Predictors of Nodal Metastasis, Prognosis, and Potential Therapeutic Targets
Authors: Abraham Deepak , Jackson Nicole , Gundara Justin S. , Zhao JingTing , Gill Anthony J. , Delbridge Leigh , Robinson Bruce G. , Sidhu Stan B.
Published: May 2011

Purpose: While the molecular basis of hereditary medullary thyroid cancer (HMTC) has been well defined, little is known about the molecular pathogenesis of sporadic medullary thyroid cancer (SMTC). In addition, microRNAs (miRNAs) have been shown to be important diagnostic and prognostic markers in cancer but have not been defined in MTC. Our aim was to study the miRNA profile of MTC to identify prognostic biomarkers and potential therapeutic targets.

Experimental Design: MiRNA microarray profiling was carried out in fresh frozen tissues from patients with SMTC (n = 12) and HMTC (n = 7). Differential expression of three miRNAs was confirmed in a validation cohort of SMTC and HMTC samples (n = 45) using quantitative reverse transcriptase-PCR and correlated with clinical outcomes. The functional role of a selected miRNA was investigated in vitro in the human medullary thyroid carcinoma cell line (TT cells) using cell proliferation assays and Western blotting analysis.

Results: MiRs-183 and 375 were overexpressed (P = 0.001; 0.031) and miR-9* was under-expressed (P = 0.011) in SMTC versus HMTC. Overexpression of miRs-183 and 375 in MTC predicted lateral lymph node metastases (P < 0.001; P = 0.001) and was associated with residual disease (P = 0.001; 0.003), distant metastases (P = 0.003; 0.001), and mortality (P = 0.01; 0.011). Knock down of miR-183 expression in the TT cell line induced a significant decrease in the viable cell count and upregulation of the protein LC3B, which is associated with autophagy.

Conclusions: Our data indicate that miRNAs play a pivotal role in the biology of MTC and represent an important class of prognostic biomarkers and therapeutic targets warranting further investigation. Clin Cancer Res; 17(14); 4772–81. ©2011 AACR.
Engineering the Brain Tumor Microenvironment Enhances the Efficacy of Dendritic Cell Vaccination: Implications for Clinical Trial Design
Authors: Mineharu Yohei , King Gwendalyn D. , Muhammad AKM G. , Bannykh Serguei , Kroeger Kurt M. , Liu Chunyan , Lowenstein Pedro R. , Castro Maria G.
Published: June 2011

Purpose: Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Clinical trials for GBM using dendritic cell (DC) vaccination resulted in antitumor immune responses. Herein, we tested the hypothesis that combining in situ (intratumoral) Ad-Flt3L/Ad-TK–mediated gene therapy with DC vaccination would increase therapeutic efficacy and antitumor immunity.

Experimental Design: We first assessed the immunogenicity of tumor lysates generated by Ad-TK (+GCV), temozolomide (TMZ), or freeze/thawing cycles (FTC) in a syngeneic brain tumor model. We also assessed phenotypic markers, cytokine release, and phagocytosis of bone marrow–derived DCs generated by fms-like tyrosine kinase 3 ligand (Flt3L) + IL-6 or by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4. Inhibition of tumor progression and production of anti-GBM antibodies was assessed following vaccination with (i) tumor cell lysates, (ii) DCs generated with either Flt3L/IL-6 or GM-CSF/IL-4 loaded with either Ad-TK/GCV-, TMZ-, or FTC-generated tumor lysates, or (iii) DCs in combination with in situ Ad-Flt3L/Ad-TK gene therapy.

Results: DCs loaded with tumor cell lysates generated with either Ad-TK/GCV or TMZ led to increased levels of phagocytosis, therapeutic efficacy, and humoral immune response. In situ immunogene therapy in combination with DC vaccination led to brain tumor regression and long-term survival in about 90% of animals, a significant increase when compared with either therapy alone.

Conclusions: Our results indicate that modifying the tumor microenvironment using intratumoral Ad-Flt3L/Ad-TK–mediated gene therapy potentiates therapeutic efficacy and antitumor immunity induced by DC vaccination. These data support novel phase I clinical trials to assess the safety and efficacy of this combined approach. Clin Cancer Res; 17(14); 4705–18. ©2011 AACR.
Restitution of Tumor Suppressor MicroRNAs Using a Systemic Nanovector Inhibits Pancreatic Cancer Growth in Mice
Authors: Pramanik Dipankar , Campbell Nathaniel R. , Karikari Collins , Chivukula Raghu , Kent Oliver A. , Mendell Joshua T. , Maitra Anirban
Published: May 2011

Mis-expression of microRNAs (miRNA) is widespread in human cancers, including in pancreatic cancer. Aberrations of miRNA include overexpression of oncogenic miRs (Onco-miRs) or downregulation of so-called tumor suppressor TSG-miRs. Restitution of TSG-miRs in cancer cells through systemic delivery is a promising avenue for pancreatic cancer therapy. We have synthesized a lipid-based nanoparticle for systemic delivery of miRNA expression vectors to cancer cells (nanovector). The plasmid DNA–complexed nanovector is approximately 100 nm in diameter and shows no apparent histopathologic or biochemical evidence of toxicity upon intravenous injection. Two miRNA candidates known to be downregulated in the majority of pancreatic cancers were selected for nanovector delivery: miR-34a, which is a component of the p53 transcriptional network and regulates cancer stem cell survival, and the miR-143/145 cluster, which together repress the expression of KRAS2 and its downstream effector Ras-responsive element binding protein-1 (RREB1). Systemic intravenous delivery with either miR-34a or miR-143/145 nanovectors inhibited the growth of MiaPaCa-2 subcutaneous xenografts (P < 0.01 for miR-34a; P < 0.05 for miR-143/145); the effects were even more pronounced in the orthotopic (intrapancreatic) setting (P < 0.0005 for either nanovector) when compared with vehicle or mock nanovector delivering an empty plasmid. Tumor growth inhibition was accompanied by increased apoptosis and decreased proliferation. The miRNA restitution was confirmed in treated xenografts by significant upregulation of the corresponding miRNA and significant decreases in specific miRNA targets (SIRT1, CD44 and aldehyde dehydrogenase for miR34a, and KRAS2 and RREB1 for miR-143/145). The nanovector is a platform with potential broad applicability in systemic miRNA delivery to cancer cells. Mol Cancer Ther; 10(8); 1470–80. ©2011 AACR.