bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in insulin sensitivity during leptin replacement therapy in leptin-deficient patients
Authors: Paz-Filho Gilberto , Esposito Karin , Hurwitz Barry , Sharma Anil , Dong Chuanhui , Andreev Victor , Delibasi Tuncay , Erol Halil , Ayala Alejandro , Wong Ma-Li , Licinio Julio
Published: October 2008

Leptin replacement rescues the phenotype of morbid obesity and hypogonadism in leptin-deficient adults. However, leptin's effects on insulin resistance are not well understood. Our objective was to evaluate the effects of leptin on insulin resistance. Three leptin-deficient adults (male, 32 yr old, BMI 23.5 kg/m2; female, 42 yr old, BMI 25.1 kg/m2; female, 46 yr old, BMI 31.7 kg/m2) with a missense mutation of the leptin gene were evaluated during treatment with recombinant methionyl human leptin (r-metHuLeptin). Insulin resistance was determined by euglycemic hyperinsulinemic clamps and by oral glucose tolerance tests (OGTTs), whereas patients were on r-metHuLeptin and after treatment was interrupted for 2–4 wk in the 4th, 5th, and 6th years of treatment. At baseline, all patients had normal insulin levels, C-peptide, and homeostatic model assessment of insulin resistance index, except for one female diagnosed with type 2 diabetes. The glucose infusion rate was significantly lower with r-metHuLeptin (12.03 ± 3.27 vs. 8.16 ± 2.77 mg·kg−1·min−1, P = 0.0016) but did not differ in the 4th, 5th, and 6th years of treatment when all results were analyzed by a mixed model [ F( 1 , 4 ) = 0.57 and P = 0.5951]. The female patient with type 2 diabetes became euglycemic after treatment with r-metHuLeptin and subsequent weight loss. The OGTT suggested that two patients showed decreased insulin resistance while off treatment. During an off-leptin OGTT, one of the patients developed a moderate hypoglycemic reaction attributed to increased posthepatic insulin delivery and sensitivity. We conclude that, in leptin-deficient adults, the interruption of r-metHuLeptin decreases insulin resistance in the context of rapid weight gain. Our results suggest that hyperleptinemia may contribute to mediate the increased insulin resistance of obesity.
Effects of acute acidemia on the fetal cardiovascular defense to acute hypoxemia
Authors: Thakor A. S. , Giussani D. A.
Published: October 2008

In complicated pregnancy, fetal hypoxemia rarely occurs in isolation but is often accompanied by fetal acidemia. There is growing clinical concern about the combined effects of fetal hypoxemia and fetal acidemia on neonatal outcome. However, the effects on the fetal defense responses to acute hypoxemia during fetal acidemia are not well understood. This study tested the hypothesis that fetal acidemia affects the fetal defense responses to acute hypoxemia. The hypothesis was tested by investigating, in the late-gestation sheep fetus surgically prepared for long-term recording, the in vivo effects of acute fetal acidemia on 1) the fetal cardiovascular responses to acute hypoxemia and 2) the neural and endocrine mechanisms mediating these responses. Under general anesthesia, five sheep fetuses at 0.8 gestation were instrumented with catheters and Transonic flow probes around the femoral and umbilical arteries. After 5 days, animals were subjected to an acute hypoxemia protocol during intravenous infusion of saline or treatment with acidified saline. Treatment with acidified saline reduced fetal basal pH from 7.35 ± 0.01 to 7.29 ± 0.01 but did not alter basal cardiovascular variables, blood glucose, or plasma concentrations of catecholamines, ACTH, and cortisol. During hypoxemia, treatment with acidified saline increased the magnitude of the fetal bradycardia and femoral vasoconstriction and concomitantly increased chemoreflex function and enhanced the increments in plasma concentrations of catecholamines, ACTH, and cortisol. Acidemia also reversed the increase in umbilical vascular conductance during hypoxemia to vasoconstriction. In conclusion, the data support our hypothesis and show that acute acidemia markedly alters fetal hemodynamic, metabolic, and endocrine responses to acute hypoxemia.
Sensor Network‐Based Localization for Continuous Tracking Applications: Implementation and Performance Evaluation
Authors: Denegri Livio , Zappatore Sandro , Davoli Franco
Published: October 2008

The increasing interest in systems able to provide users with
immersive services (e.g., domotics, context‐aware applications,
and immersive distance learning tools) has encouraged
the development of cheap and effective platforms aimed at
tracking objects and people within a certain space. In this
context, wireless sensor networks (WSNs) can play a very
important role, since specialized sensors can be fruitfully
exploited in order to generate/receive signals by means of
which the WSN can derive the position of nodes joined to
the objects to be tracked. The paper presents an original localization platform that exploits
a single‐hop WSN, based on a Microchip MCU and
a Cypress RF device, to track its moving nodes. Specifically,
the nodes of the network are divided into three sets:
the first set consists of anchor nodes that, according to the
commands from the sink (the central node of the WSN),
generate ultrasonic pulses. These pulses are received by
the second set of (moving) nodes, which estimate the pulse time trip and communicate it to the sink. Finally, the last
set is constituted by general purpose nodes that collect any
kind of data from the surrounding field. The sink gathers
all the data, computes the position of moving nodes, and transfers information to external users on the Internet. The algorithms adopted to manage the network and to localize
moving nodes are discussed. A working prototype
based upon the hardware platform, software, and protocol
described in this paper has been deployed and tested, and
some results are shown. Simulation results of the localization
system are presented to show system scalability.
Review of Available Data for Validation of Nuresim Two‐Phase CFD Software Applied to CHF Investigations
Authors: Bestion D. , Anglart H. , Caraghiaur D. , Péturaud P. , Smith B. , Andreani M. , Niceno B. , Krepper E. , Lucas D. , Moretti F. , Galassi M. C. , Macek J. , Vyskocil L. , Koncar B. , Hazi G.
Published: October 2008

The NURESIM Project of the 6th European Framework Program initiated the development of a new‐generation common European Standard Software Platform for nuclear reactor simulation. The thermal‐hydraulic subproject aims at improving the understanding and the predictive capabilities of the simulation tools for key two‐phase flow thermal‐hydraulic processes such as the critical heat flux (CHF). As part of a multi‐scale analysis of reactor thermal‐hydraulics, a two‐phase CFD tool is developed to allow zooming on local processes. Current industrial methods for CHF mainly use the sub‐channel analysis and empirical CHF correlations based on large scale experiments having the real geometry of a reactor assembly. Two‐phase CFD is used here for understanding some boiling flow processes, for helping new fuel assembly design, and for developing better CHF predictions in both PWR and BWR. This paper presents a review of experimental data which can be used for validation of the two‐phase CFD application to CHF investigations. The phenomenology of DNB and Dry‐Out are detailed identifying all basic flow processes which require a specific modeling in CFD tool. The resulting modeling program of work is given and the current state‐of‐the‐art of the modeling within the NURESIM project is presented.
Euler‐Euler Large Eddy Simulation of a Square Cross‐Sectional Bubble Column Using the Neptune_CFD Code
Authors: Ničeno B. , Boucker M. , Smith B. L.
Published: October 2008

In this work, we report on Euler‐Euler large eddy simulation (EELES) of dispersed bubbly flow in a square cross‐sectional bubble column. Simulations are performed using the Neptune_CFD package, and results processed using the SALOME platform. The motivation to undertake this study is to check our implementation of the Smagorinsky subgrid‐scale (SGS) model into NEPTUNE_CFD. We outline all the physical models used, and we present instantaneous realizations of velocity and void fraction fields in order to illustrate the structure of the turbulence field, and long‐time averaged results, to compare with analogous simulations performed using the CFX‐4 code and experimental data. The same physical models and constants have been used in both the CFX‐4 and NEPTUNE_CFD codes, except the SGS model, which is Smagorinsky in case of NEPTUNE_CFD and a one‐equation model in CFX‐4. The results obtained with EELES compare reasonably well with experiment, meaning in particular that the implementations have been successful. Some perspectives on the further use of EELES are also given.
A Second‐Order Turbulence Model Based on a Reynolds Stress Approach for Two‐Phase Flow—Part I: Adiabatic Cases
Authors: Mimouni S. , Archambeau F. , Boucker M. , Laviéville J. , Morel C.
Published: October 2008

In our work in 2008, we evaluated the aptitude of the code Neptune_CFD to reproduce the incidence of a structure topped by vanes on a boiling layer, within the framework of the Neptune project. The objective was to reproduce the main effects of the spacer grids. The turbulence of the liquid phase was modeled by a first‐order K‐ε model. We show in this paper that this model is unable to describe the turbulence of
rotating flows, in accordance with the theory. The objective of this paper is to improve the turbulence
modeling of the liquid phase by a second turbulence model based on a Rij‐ε approach. Results obtained on typical single‐phase cases highlight the improvement of the prediction for all computed values. We tested the turbulence model Rij‐ε implemented in the code versus typical adiabatic two‐phase flow experiments. We check that the simulations with the Reynolds stress transport model (RSTM) give satisfactory results in a simple geometry as compared to a K‐ε model: this point is crucial before calculating rod bundle geometries where the K‐ε model may fail.
A Novel Dietary Flavonoid Fisetin Inhibits Androgen Receptor Signaling and Tumor Growth in Athymic Nude Mice
Authors: Khan Naghma , Asim Mohammad , Afaq Farrukh , Abu Zaid Mohammad , Mukhtar Hasan
Published: October 2008

Androgen receptor (AR)–mediated signaling plays an important role in the development and progression of prostate cancer (PCa). Hormonal therapies, mainly with combinations of antiandrogens and androgen deprivation, are the mainstay treatment for advanced disease. However, emergence of androgen resistance largely due to inefficient antihormone action limits their therapeutic usefulness. Here, we report that fisetin, a novel dietary flavonoid, acts as a novel AR ligand by competing with the high-affinity androgen to interact with the ligand binding domain of AR. We show that this physical interaction results in substantial decrease in AR stability and decrease in amino-terminal/carboxyl-terminal (N-C) interaction of AR. This results in blunting of AR-mediated transactivation of target genes including prostate-specific antigen (PSA). In addition, treatment of LNCaP cells with fisetin decreased AR protein levels, in part, by decreasing its promoter activity and by accelerating its degradation. Fisetin also synergized with Casodex in inducing apoptosis in LNCaP cells. Treatment with fisetin in athymic nude mice implanted with AR-positive CWR22Rυ1 human PCa cells resulted in inhibition of tumor growth and reduction in serum PSA levels. These data identify fisetin as an inhibitor of AR signaling axis and suggest that it could be a useful chemopreventive and chemotherapeutic agent to delay progression of PCa. [Cancer Res 2008;68(20):8555–63]
Direct Presentation of a Melanocyte-Associated Antigen in Peripheral Lymph Nodes Induces Cytotoxic CD8+ T Cells
Authors: Schuler Prisca , Contassot Emmanuel , Irla Magali , Hugues Stéphanie , Preynat-Seauve Olivier , Beermann Friederich , Donda Alena , French L.E. , Huard Bertrand
Published: October 2008

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8+ T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8+ T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8+ T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8+ T cells, independently of bone marrow–derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer. [Cancer Res 2008;68(20):8410–8]
MicroRNA-Mediated Inhibition of Prostate-Derived Ets Factor Messenger RNA Translation Affects Prostate-Derived Ets Factor Regulatory Networks in Human Breast Cancer
Authors: Findlay Victoria J. , Turner David P. , Moussa Omar , Watson Dennis K.
Published: October 2008

Prostate-derived Ets factor (PDEF) is an ETS transcription factor expressed in normal tissues with high epithelial cell content and noninvasive breast cancer cells. A putative tumor suppressor PDEF protein expression is often lost during progression to a more invasive phenotype. Interestingly, PDEF mRNA has been found to be retained or even overexpressed in the absence of protein; however, the mechanisms for this remain to be elucidated. This study identifies two microRNAs (miRNA) that directly act on and repress PDEF mRNA translation, leading to the loss of PDEF protein expression and the gain of phenotypes associated with invasive cells. In addition, we show that these miRNAs are elevated in human breast tumor samples. Together, these data describe a mechanism of regulation that explains, for the first time, the lack of correlation between PDEF mRNA and protein levels, providing insight into the underexplored role of posttranscriptional regulation and how this contributes to dysregulated protein expression in cancer. These observations have critical implications for therapeutically targeting miRNAs that contribute to cancer progression. [Cancer Res 2008;68(20):8499–506]
A Subset of Host B Lymphocytes Controls Melanoma Metastasis through a Melanoma Cell Adhesion Molecule/MUC18-Dependent Interaction: Evidence from Mice and Humans
Authors: Staquicini Fernanda I. , Tandle Anita , Libutti Steven K. , Sun Jessica , Zigler Maya , Bar-Eli Menashe , Aliperti Fabiana , Pérez Elizabeth C. , Gershenwald Jeffrey E. , Mariano Mario , Pasqualini Renata , Arap Wadih , Lopes José Daniel
Published: October 2008

Host immunity affects tumor metastasis but the corresponding cellular and molecular mechanisms are not entirely clear. Here, we show that a subset of B lymphocytes (termed B-1 population), but not other lymphocytes, has prometastatic effects on melanoma cells in vivo through a direct heterotypic cell-cell interaction. In the classic B16 mouse melanoma model, one mechanism underlying this phenomenon is a specific up-regulation and subsequent homophilic interaction mediated by the cell surface glycoprotein MUC18 (also known as melanoma cell adhesion molecule). Presence of B-1 lymphocytes in a panel of tumor samples from melanoma patients directly correlates with MUC18 expression in melanoma cells, indicating that the same protein interaction exists in humans. These results suggest a new but as yet unrecognized functional role for host B-1 lymphocytes in tumor metastasis and establish a biochemical basis for such observations. Our findings support the counterintuitive central hypothesis in which a primitive layer of the immune system actually contributes to tumor progression and metastasis in a mouse model and in melanoma patients. Given that monoclonal antibodies against MUC18 are in preclinical development but the reason for their antitumor activity is not well understood, these translational results are relevant in the setting of human melanoma and perhaps of other cancers. [Cancer Res 2008;68(20):8419–28]
Netrin-1 Induces Apoptosis in Human Cervical Tumor Cells via the TAp73α Tumor Suppressor
Authors: Roperch Jean-Pierre , El Ouadrani Karima , Hendrix Ann , Emami Shahin , De Wever Olivier , Melino Gerry , Gespach Christian
Published: October 2008

Netrins and their receptors deleted in colon cancer (DCC), neogenin, UNC5, and integrins are involved in axon guidance, epithelial morphogenesis, vascular pattering, cancer cell survival, invasion, tumor angiogenesis, and metastasis. Here, we considered the possible contribution of the p53-related apoptosis mediators p63 and p73 in the mechanisms underlying the antagonism between netrin-1 and DCC at the cell death control. We have showed that ectopic expression and external addition of netrin-1 in HeLa and HEK-293 cells with inactive p53 lead to impaired cell viability and induction of apoptosis. These responses were associated with up-regulation of the proapoptotic protein TAp73α, decreased Bcl-2/Bax ratio, and caspase-3 cleavage, with no change in protein levels of the antiapoptotic NH2-terminal–truncated ΔNp73α isoform, p73 adapter Yap-1 and p73 E3 ubiquitin ligase Itch, and p63, as well as the transcripts encoding p63, TAp73α, and ΔNp73α. However, the proteasome inhibitor MG132 potentiated, while DCC counteracted, netrin-1–induced TAp73α. Consistently, netrin-1 expression correlated with stabilization of the TAp73α protein and lower levels of TAp73α ubiquitination that was conversely enhanced by DCC, in a netrin-dependent manner. Our data indicate that netrin-1 selectively up-regulates TAp73α by preventing its ubiquitination and degradation. Targeted repression of p73α by shRNA reversed TAp73α and the apoptosis induced by netrin-1, and exacerbated the growth of HeLa tumor xenografts. Apoptosis induced by cisplatin was markedly enhanced in netrin-1 or DCC-expressing cells. Collectively, our data reveal that the transcriptionally active TAp73α tumor suppressor is implicated in the apoptosis induced by netrin-1 in a p53-independent and DCC/ubiquitin-proteasome dependent manner. [Cancer Res 2008;68(20):8231–9]
Sperm-Associated Antigen 9 Is Associated With Tumor Growth, Migration, and Invasion in Renal Cell Carcinoma
Authors: Garg Manoj , Kanojia Deepika , Khosla Aashima , Dudha Namrata , Sati Satish , Chaurasiya Dipak , Jagadish Nirmala , Seth Amlesh , Kumar Rajive , Gupta Samir , Gupta Anju , Lohiya Nirmal Kumar , Suri Anil
Published: October 2008

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiation and chemotherapy. Current therapies for RCC patients are inefficient due to the lack of diagnostic and therapeutic markers. Our recent studies have suggested an association of sperm-associated antigen 9 (SPAG9) with ovarian carcinomas. In the present study, we investigated the clinical relevance of SPAG9 in RCC patients. RT-PCR analysis showed expression of SPAG9 transcript in RCC tissues and RCC cell lines. In situ RNA hybridization and immunohistochemistry analyses confirmed the expression of SPAG9 in 88% of cancer patients, suggesting that SPAG9 participates in renal cancer. In addition, immunoblotting and ELISA analyses revealed a humoral immune response against SPAG9 in the sera of RCC patients but not in healthy individuals. Consistent with the clinical findings, knockdown of SPAG9 expression in RCC cells with specific siRNA significantly reduced cell growth and colony formation. Using in vitro wound healing and Matrigel invasion assays, we found that cell migration and invasive ability were also significantly inhibited. Furthermore, in vivo xenograft studies in nude mice revealed that administration of a SPAG9 siRNA plasmid significantly inhibited tumor growth. In conclusion, SPAG9 expression is associated with clinicopathologic features of tumors, suggesting that SPAG9 could contribute to the early spread of cancer. These results indicate that SPAG9 may have a role in tumor development and metastasis and thus could serve as a novel target for early detection and treatment of RCC. [Cancer Res 2008;68(20):8240–8]
MicroRNA-7, a Homeobox D10 Target, Inhibits p21-Activated Kinase 1 and Regulates Its Functions
Authors: Reddy Sirigiri Divijendra Natha , Ohshiro Kazufumi , Rayala Suresh K. , Kumar Rakesh
Published: October 2008

MicroRNAs are noncoding RNAs that inhibit the expression of their targets in a sequence-specific manner and play crucial roles during oncogenesis. Here we show that microRNA-7 (miR-7) inhibits p21-activated kinase 1 (Pak1) expression, a widely up-regulated signaling kinase in multiple human cancers, by targeting the 3′-untranslated region (UTR) of Pak1 mRNA. We noticed an inverse correlation between the levels of endogenous miR-7 and Pak1 expression in human cancer cells. We discovered that endogenous miR-7 expression is positively regulated by a homeodomain transcription factor, HoxD10, the loss of which leads to an increased invasiveness. HoxD10 directly interacts with the miR-7 chromatin. Accordingly, the levels of Pak1 protein are progressively up-regulated whereas those of miR-7 and its upstream activator HoxD10 are progressively down-regulated in a cellular model of breast cancer progression from low to highly invasive phenotypes. Furthermore, HoxD10 expression in highly invasive breast cancer cells resulted in an increased miR-7 expression but reduced Pak1 3′-UTR-luciferase activity and reduced Pak1 protein. Finally, we show that miR-7 introduction inhibits the motility, invasiveness, anchorage-independent growth, and tumorigenic potential of highly invasive breast cancer cells. Collectively, these findings establish for the first time that Pak1 is a target of miR-7 and that HoxD10 plays a regulatory role in modifying the expression of miR-7 and, consequently, the functions of the miR-7-Pak1 pathway in human cancer cells. [Cancer Res 2008;68(20):8195–200]
Human epidermal growth factor receptor (HER-1:HER-3) Fc-mediated heterodimer has broad antiproliferative activityin vitroand in human tumor xenografts
Authors: Sarup Jay , Jin Pei , Turin Lisa , Bai Xiaomei , Beryt Malgorzata , Brdlik Cathleen , Higaki Jeffrey N. , Jorgensen Brett , Lau Francis W. , Lindley Peter , Liu Jim , Ni Irene , Rozzelle James , Kumari Rajendra , Watson Susan A. , Zhang Juan , Shepard H. Michael
Published: October 2008

All four members of the human epidermal growth factor (EGF) receptor (HER) family are implicated in human cancers. Although efficacious in a subset of patients, resistance to single-targeted anti-HER therapy [i.e., cetuximab (Erbitux) and trastuzumab (Herceptin)] is often associated with coexpression of other HER family members. This may be overcome by a HER ligand binding molecule that sequesters multiple EGF-like ligands, preventing ligand-dependent receptor activation. Toward this end, we have combined the HER-1/EGFR and HER-3 ligand binding domains, dimerized with fusion of an Fc fragment of human IgG1. This resulted in a mixture of HER-1/Fc homodimer (HFD100), HER-3/Fc homodimer (HFD300), and HER-1/Fc:HER-3/Fc heterodimer (RB200), also termed Hermodulins. The purified first-generation RB200 bound EGF and neuregulin 1 (NRG1)-β1 ligands, determined by cross-linking and direct binding studies. The binding affinity for both was ∼10 nmol/L by dissociation-enhanced lanthanide fluorescence immunoassay using europium (Eu)-labeled ligands. Competition studies with RB200 using Eu-EGF or Eu-NRG1-β1 revealed that RB200 bound HER-1 ligands, including transforming growth factor-α and heparin-binding EGF, and HER-3 ligands NRG1-α and NRG1-β3. RB200 inhibited EGF- and NRG1-β1–stimulated tyrosine phosphorylation of HER family proteins, proliferation of a diverse range of tumor cells in monolayer cell growth assays, tumor cell proliferation as a single agent and in synergy with tyrosine kinase inhibitors, lysophosphatidic acid–stimulated cell proliferation, and tumor growth in two human tumor xenograft nude mouse models. Taken together, the data reveal that RB200 has the potential to sequester multiple HER ligands and interfere with signaling by HER-1, HER-2, and HER-3. [Mol Cancer Ther 2008;7(10):3223–36]
Preclinical overview of sorafenib, a multikinase inhibitor that targets both Raf and VEGF and PDGF receptor tyrosine kinase signaling
Authors: Wilhelm Scott M. , Adnane Lila , Newell Philippa , Villanueva Augusto , Llovet Josep M. , Lynch Mark
Published: October 2008

Although patients with advanced refractory solid tumors have poor prognosis, the clinical development of targeted protein kinase inhibitors offers hope for the future treatment of many cancers. In vivo and in vitro studies have shown that the oral multikinase inhibitor, sorafenib, inhibits tumor growth and disrupts tumor microvasculature through antiproliferative, antiangiogenic, and/or proapoptotic effects. Sorafenib has shown antitumor activity in phase II/III trials involving patients with advanced renal cell carcinoma and hepatocellular carcinoma. The multiple molecular targets of sorafenib (the serine/threonine kinase Raf and receptor tyrosine kinases) may explain its broad preclinical and clinical activity. This review highlights the antitumor activity of sorafenib across a variety of tumor types, including renal cell, hepatocellular, breast, and colorectal carcinomas in the preclinical setting. In particular, preclinical evidence that supports the different mechanisms of action of sorafenib is discussed. [Mol Cancer Ther 2008;7(10):3129–40]
Cisplatin abrogates the geldanamycin-induced heat shock response
Authors: McCollum Andrea K. , Lukasiewicz Kara B. , TenEyck Cynthia J. , Lingle Wilma L. , Toft David O. , Erlichman Charles
Published: October 2008

Benzoquinone ansamycin antibiotics such as geldanamycin (GA) bind to the NH2-terminal ATP-binding domain of heat shock protein (Hsp) 90 and inhibit its chaperone functions. Despite in vitro and in vivo studies indicating promising antitumor activity, derivatives of GA, including 17-allylaminogeldanamycin (17-AAG), have shown little clinical efficacy as single agents. Thus, combination studies of 17-AAG and several cancer chemotherapeutics, including cisplatin (CDDP), have begun. In colony-forming assays, the combination of CDDP and GA or 17-AAG was synergistic and caused increased apoptosis compared with each agent alone. One measurable response that results from treatment with Hsp90-targeted agents is the induction of a heat shock factor-1 (HSF-1) heat shock response. Treatment with GA + CDDP revealed that CDDP suppresses up-regulation of HSF-1 transcription, causing decreased levels of stress-inducible proteins such as Hsp27 and Hsp70. However, CDDP treatment did not prevent trimerization and nuclear localization of HSF-1 but inhibited DNA binding of HSF-1 as shown by chromatin immunoprecipitation. Melphalan, but not camptothecin, caused similar inhibition of GA-induced HSF-1–mediated Hsp70 up-regulation. 3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt cell survival assays revealed that deletion of Hsp70 caused increased sensitivity to GA (Hsp70+/+ IC50 = 63.7 ± 14.9 nmol/L and Hsp70−/− IC50 = 4.3 ± 2.9 nmol/L), which confirmed that a stress response plays a critical role in decreasing GA sensitivity. Our results suggest that the synergy of GA + CDDP is due, in part, to CDDP-mediated abrogation of the heat shock response through inhibition of HSF-1 activity. Clinical modulation of the HSF-1–mediated heat shock response may enhance the efficacy of Hsp90-directed therapy. [Mol Cancer Ther 2008;7(10):3256–64]
Stat3 contributes to resistance toward BCR-ABL inhibitors in a bone marrow microenvironment model of drug resistance
Authors: Bewry Nadine N. , Nair Rajesh R. , Emmons Michael F. , Boulware David , Pinilla-Ibarz Javier , Hazlehurst Lori A.
Published: October 2008

Imatinib mesylate is a potent, molecularly targeted therapy against the oncogenic tyrosine kinase BCR-ABL. Although imatinib mesylate has considerable efficacy against chronic myeloid leukemia (CML), advanced-stage CML patients frequently become refractory to this agent. The bone marrow is the predominant microenvironment of CML and is a rich source of both soluble factors and extracellular matrices, which may influence drug response. To address the influence of the bone marrow microenvironment on imatinib mesylate sensitivity, we used an in vitro bone marrow stroma model. Our data show culturing K562 cells, in bone marrow stroma-derived conditioned medium (CM), is sufficient to cause resistance to BCR-ABL inhibitors. Drug resistance correlated with increased pTyrStat3, whereas no increases in pTyrStat5 was noted. Moreover, resistance was associated with increased levels of the Stat3 target genes Bcl-xl, Mcl-1, and survivin. Finally, reducing Stat3 levels with small interfering RNA sensitized K562 cells cultured in CM to imatinib mesylate-induced cell death. Importantly, Stat3 dependency was specific for cells grown in CM, as reducing Stat3 levels in regular growth conditions had no effect on imatinib mesylate sensitivity. Together, these data support a novel mechanism of BCR-ABL-independent imatinib mesylate resistance and provides preclinical rationale for using Stat3-inhibitors to increase the efficacy of imatinib mesylate within the context of the bone marrow microenvironment. [Mol Cancer Ther 2008;7(10):3169–75]
Role of histone deacetylase inhibitor-induced reactive oxygen species and DNA damage in LAQ-824/fludarabine antileukemic interactions
Authors: Rosato Roberto R. , Almenara Jorge A. , Maggio Sonia C. , Coe Stefanie , Atadja Peter , Dent Paul , Grant Steven
Published: October 2008

The role of reactive oxygen species (ROS) production on DNA damage and potentiation of fludarabine lethality by the histone deacetylase inhibitor (HDACI) LAQ-824 was investigated in human leukemia cells. Preexposure (24 h) of U937, HL-60, Jurkat, or K562 cells to LAQ-824 (40 nmol/L) followed by fludarabine (0.4 μmol/L) dramatically potentiated apoptosis (≥75%). LAQ-824 triggered an early ROS peak (30 min-3 h), which declined by 6 h, following LAQ-824-induced manganese superoxide dismutase 2 (Mn-SOD2) upregulation. LAQ-824/fludarabine lethality was significantly diminished by either ROS scavengers N-acetylcysteine or manganese (III) tetrakis (4-benzoic acid) porphyrin or ectopic Mn-SOD2 expression and conversely increased by Mn-SOD2 antisense knockdown. During this interval, LAQ-824 induced early (4-8 h) increases in γ-H2AX, which persisted (48 h) secondary to LAQ-824-mediated inhibition of DNA repair (e.g., down-regulation of Ku86 and Rad50, increased Ku70 acetylation, diminished Ku70 and Ku86 DNA-binding activity, and down-regulated DNA repair genes BRCA1, CHEK1, and RAD51). Addition of fludarabine further potentiated DNA damage, which was incompatible with cell survival, and triggered multiple proapoptotic signals including activation of nuclear caspase-2 and release of histone H1.2 into the cytoplasm. The latter event induced activation of Bak and culminated in pronounced mitochondrial injury and apoptosis. These findings provide a mechanistic basis for understanding the role of early HDACI-induced ROS generation and modulation of DNA repair processes in potentiation of nucleoside analogue-mediated DNA damage and lethality in leukemia. Moreover, they show for the first time the link between HDACI-mediated ROS generation and the recently reported DNA damage observed in cells exposed to these agents. [Mol Cancer Ther 2008;7(9):3285–97]
FOXO3a mediates the cytotoxic effects of cisplatin in colon cancer cells
Authors: Fernández de Mattos Silvia , Villalonga Priam , Clardy Jon , Lam Eric W-F.
Published: October 2008

Cisplatin is a conventional chemotherapeutic agent that binds covalently to purine DNA bases and mediates cellular apoptosis. A better understanding of the downstream cellular targets of cisplatin will provide information on its mechanism of action and help to understand the mechanism of drug resistance. In this study, we have investigated the effects of cisplatin in a panel of colon carcinoma cell lines and the involvement of the phosphoinositide-3-kinase/forkhead/winged helix box class O (FOXO) pathway in cisplatin action and resistance. Cisplatin-sensitive and cisplatin-resistant cell lines have been characterized in cell viability, flow cytometry, and clonogenic assays. The main components of the phosphoinositide-3-kinase/protein kinase B pathway, particularly FOXO3a, have been analyzed in sensitive and resistant cells on cisplatin treatment. Interestingly, in sensitive cells, cisplatin induces FOXO3a dephosphorylation and nuclear translocation, and expression of its target genes, whereas in resistant cells the effect of cisplatin on FOXO3a is incomplete. Consistent with this, protein kinase B/FOXO signaling axis modulators triciribine and psammaplysene A sensitize the resistant HT29 cells to cisplatin treatment. Critically, knockdown of FOXO3a expression using small interfering RNA rescues sensitive SW620 cells from cisplatin-induced short- and long-term cell death. Together, our findings suggest that FOXO3a is a relevant mediator of the cytotoxic effects of cisplatin in colon cancer cells. [Mol Cancer Ther 2008;7(10):3237–46]
Heparin-binding epidermal growth factor–like growth factor promotes transcoelomic metastasis in ovarian cancer through epithelial-mesenchymal transition
Authors: Yagi Hiroshi , Yotsumoto Fusanori , Miyamoto Shingo
Published: October 2008

Heparin-binding epidermal growth factor–like growth factor (HB-EGF) is involved in several biological processes including cell adhesion, invasion, and angiogenesis. HB-EGF also plays a pivotal role in the progression of ovarian cancer. To investigate the significance of HB-EGF in peritoneal dissemination, we examined the roles of HB-EGF in cell adhesion, invasion, and angiogenesis in ovarian cancer. Through the suppression of focal adhesion kinase and EGF receptor activation, cell adhesive properties mediated by integrin β1 were diminished by the inhibition of HB-EGF expression. The reduction of HB-EGF expression attenuated the chemotactic invasive ability and the expression of matrix metalloprotease (MMP)-2 and vascular endothelial growth factor (VEGF), leading to the inhibition of cell invasion and angiogenesis. Suppression of the Snail family, which regulates the epithelial-mesenchymal transition, blocked the cell adhesion properties on extracellular matrices, the chemotactic invasive ability, and the expression of MMP9 and VEGF through the reduction of HB-EGF expression. The volume of tumor burden in the peritoneal cavity was dependent on the expression of HB-EGF. According to these results, HB-EGF contributes to cell adhesion, invasion, and angiogenesis, which are integral to transcoelomic metastasis in ovarian cancer. CRM197, an inhibitor of HB-EGF, resulted in a significant decrease of tumor burden in peritoneal dissemination, accompanied with a reduction in both cellular spreading, when assayed on an extracellular matrix, and invasive ability, when assayed in a chemotaxis chamber, as well as decreased expression of MMP9 and VEGF. Thus, HB-EGF is a mutual validating target in the peritoneal dissemination of ovarian cancer, and CRM197 may be useful as a anticancer agent for advanced ovarian cancer. [Mol Cancer Ther 2008;7(9):3441–51]
Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions
Authors: Liaw Tracy Y.E. , Salam Noeris K. , McKay Matthew J. , Cunningham Anne M. , Hibbs David E. , Kavallaris Maria
Published: October 2008

2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 μmol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G2-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I β-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on β-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions. [Mol Cancer Ther 2008;7(9):3150–9]
Structural basis for nuclear receptor corepressor recruitment by antagonist-liganded androgen receptor
Authors: Hodgson Myles C. , Shen Howard C. , Hollenberg Anthony N. , Balk Steven P.
Published: October 2008

Androgen receptor (AR) recruitment of transcriptional corepressors NCoR and SMRT can be enhanced by antagonists such as mifepristone. This study shows that enhanced NCoR binding to the mifepristone-liganded AR is mediated by the NCoR COOH-terminal N1 CoRNR box and that this selectivity is due to charged residues unique to the COOH-terminal CoRNR boxes of NCoR and SMRT. Significantly, these residues are on a helical face adjacent to oppositely charged residues in helix 4 of the AR ligand-binding domain. Mutagenesis of these AR residues in helix 4, as well as mutation of lysine 720 in helix 3 (predicted to interact with the CoRNR box), markedly impaired AR recruitment of NCoR, indicating that N1 CoRNR box binding is being stabilized by these ionic interactions in the AR ligand-binding domain coactivator/corepressor binding site. Finally, results using a helix 12–deleted AR indicate that mifepristone induces allosteric changes in addition to helix 12 displacement that are critical for NCoR binding. These findings show that AR antagonists can enhance corepressor recruitment by stabilizing a distinct antagonist conformation of the AR coactivator/corepressor binding site and support the development of additional antagonists that may be able to further enhance AR recruitment of corepressors. [Mol Cancer Ther 2008;7(10):3187–94]
Identification of small-molecule inhibitors of autotaxin that inhibit melanoma cell migration and invasion
Authors: Saunders Lauren P. , Ouellette Amy , Bandle Russ , Chang William Chozen , Zhou Hongwen , Misra Raj N. , De La Cruz Enrique M. , Braddock Demetrios T.
Published: October 2008

Autotaxin (ATX) is a prometastatic enzyme initially isolated from the conditioned medium of human melanoma cells that stimulates a myriad of biological activities, including angiogenesis and the promotion of cell growth, survival, and differentiation through the production of lysophosphatidic acid (LPA). ATX increases the aggressiveness and invasiveness of transformed cells, and ATX levels directly correlate with tumor stage and grade in several human malignancies. To study the role of ATX in the pathogenesis of malignant melanoma, we developed antibodies and small-molecule inhibitors against recombinant human protein. Immunohistochemistry of paraffin-embedded human tissue shows that ATX levels are markedly increased in human primary and metastatic melanoma relative to benign nevi. Chemical screens identified several small-molecule inhibitors with binding constants ranging from nanomolar to low micromolar. Cell migration and invasion assays with melanoma cell lines show that ATX markedly stimulates melanoma cell migration and invasion, an effect suppressed by ATX inhibitors. The migratory phenotype can be rescued by the addition of the enzymatic product of ATX, LPA, confirming that the observed inhibition is linked to suppression of LPA production by ATX. Chemical analogues of the inhibitors show structure-activity relationships important for ATX inhibition and indicate pathways for their optimization. These studies suggest that ATX is an approachable molecular target for the rational design of chemotherapeutic agents directed against malignant melanoma. [Mol Cancer Ther 2008;7(10):3352–62]
Therapeutic efficacy of soluble receptor activator of nuclear factor-κB-Fc delivered by nonviral gene transfer in a mouse model of osteolytic osteosarcoma
Authors: Lamoureux François , Picarda Gaëlle , Rousseau Julie , Gourden Clothilde , Battaglia Séverine , Charrier Céline , Pitard Bruno , Heymann Dominique , Rédini Françoise
Published: October 2008

Osteosarcoma is the most frequent primary bone tumor that develops mainly during youth, the median age of diagnosis being 18 years. Despite improvement in osteosarcoma treatment, survival rate is only 30% after 5 years for patients with pulmonary metastases at diagnosis. This warrants exploration of new therapeutic options. The anti-bone resorption molecule receptor activator of NF-κB (RANK) is very promising, as it may block the vicious cycle between bone resorption and tumor proliferation that takes place during tumor development in bone site. The cDNA encoding murine RANK-Fc (mRANK-Fc) was administered by gene transfer using an amphiphilic polymer in a mouse model of osteolytic osteosarcoma. Clinical and bone microarchitecture variables were assessed by radiography and micro-CT analyses. In vitro experiments were designed to determine the mechanism of action of RANK-Fc on tumor cell proliferation (XTT assays), apoptosis (caspase activation), cell cycle distribution (fluorescence-activated cell sorting analysis), or gene expression (reverse transcription-PCR). RANK-Fc was effective in preventing the formation of osteolytic lesions associated with osteosarcoma development and in reducing the tumor incidence, the local tumor growth, and the lung metastases dissemination leading to a 3.9-fold augmentation of mice survival 28 days after implantation. On the contrary, mRANK-Fc did not prevent the development of nonosseous tumor nodules, suggesting that bone environment is necessary for mRANK-Fc therapeutic efficacy. Furthermore, mRANK-Fc has no direct activity on osteosarcoma cells in vitro. mRANK-Fc exerts an indirect inhibitory effect on osteosarcoma progression through inhibition of bone resorption. [Mol Cancer Ther 2008;7(10):3389–98]
Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments
Authors: Gao Jie , Kou Geng , Chen Huaiwen , Wang Hao , Li Bohua , Lu Ying , Zhang Dapeng , Wang Shuhui , Hou Sheng , Qian Weizhu , Dai Jianxin , Zhao Jian , Zhong Yanqiang , Guo Yajun
Published: October 2008

We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), a recombinant immunotoxin in which a single-chain antibody derived from mouse SM5-1 monoclonal antibody is genetically fused to PE38KDEL-I. In comparison with the SMFv-PE38KDEL wild-type, Mut-I showed improved therapeutic efficacy and reduced toxicity. To overcome the problems associated with the immune response to the Pseudomonas exotoxin A (PE) component of Mut-I, we have constructed PE38KDEL-I-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with F(ab′) fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S). PE-NP-S specifically bound to SM5-1 binding protein-expressing hepatocellular carcinoma cell lines and was then internalized by these cells, resulting in significant cytotoxic effect. In SM5-1 binding protein-overexpressing tumor xenograft model, administration of PE-NP-S significantly inhibited tumor development and induced tumor regression. Moreover, PE-NP-S was shown to be much weaker in inducing vascular leakage syndrome in mice than Mut-I. The LD50 of PE-NP-S was about 4-fold higher than that of Mut-I. Remarkably, PE-NP-S was of low immunogenicity in development of anti-PE neutralizing antibodies in vivo and was less susceptible to inactivation by anti-PE neutralizing antibodies compared with Mut-I. In conclusion, the resultant PE-NP-S possessed increased cancer therapeutic efficacy and had reduced nonspecific toxicity and immunogenicity, suggesting that it is a potential candidate in cancer therapy. [Mol Cancer Ther 2008;7(10):3399–407]
EBP1, an ErbB3-binding protein, is decreased in prostate cancer and implicated in hormone resistance
Authors: Zhang Yuexing , Linn Douglas , Liu Zhenqiu , Melamed Jonathan , Tavora Fabio , Young Charles Y. , Burger Angelika M. , Hamburger Anne W.
Published: October 2008

Aberrant activation of the androgen receptor (AR) by the ErbB2/ErbB3 heterodimer contributes to the development of hormone resistance in prostate cancer. EBP1, an ErbB3-binding protein, acts as an AR corepressor. As EBP1 is decreased in preclinical models of hormone-refractory prostate cancer, we studied the expression of EBP1 in human prostate cancer. We found that the expression of the EBP1 gene was significantly decreased in prostate cancer tissues compared with benign prostate at both mRNA and protein levels. Restoration of EBP1 expression in the hormone-refractory LNCaP C81 cell line led to an amelioration of the androgen-independent phenotype based on established biological criteria and a reduction in the expression of a cohort of AR target genes. The ability of the ErbB3 ligand heregulin (HRG) to stimulate growth and AKT phosphorylation of hormone-refractory prostate cancer cells was abolished. Abrogation of EBP1 expression by short hairpin RNA in hormone-dependent LNCaP cells, which undergo apoptosis in response to HRG, resulted in HRG-stimulated cell growth. Restoration of EBP1 expression decreased the tumorigenicity of C81 xenografts in female mice, whereas elimination of EBP1 expression enhanced the ability of LNCaP cells to grow in female mice. Our data support a role for EBP1 in the development of hormone-refractory prostate cancer via inhibition of both AR- and HRG-stimulated growth and present a novel strategy for treating androgen-refractory prostate cancer. [Mol Cancer Ther 2008;7(10):3176–86]