bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Regulation of T-Type Calcium Channels by S-Nitrosothiols in the Rat Thalamus
Authors: Joksovic Pavle M. , Doctor Allan , Gaston Benjamin , Todorovic Slobodan M.
Published: February 2007

Although T-type Ca2+ channels in the reticular thalamic nucleus (nRT) have a central function in tuning neuronal excitability and are implicated in sensory processing, sleep, and epilepsy, the mechanisms involved in their regulation are poorly understood. Here we recorded T-type Ca2+ currents from intact nRT neurons in brain slices from young rats and investigated the mechanisms of T-type channel modulation by S-nitrosothiols (SNOs). We found that extracellular application of S-nitrosoglutathione (GSNO), S-nitrosocysteine (CSNO) and S-nitroso- N-acetyl-penicillamin (SNAP) rapidly and reversibly reduced T-type currents. The effects of SNOs are strongly stereoselective at physiological concentrations: L-CSNO was fourfold more effective in inhibiting T-type current than was D-CSNO. The effects of GSNO were abolished if cells had been treated with free hemoglobin or N-ethylmaleimide, an irreversible alkylating agent but not by 8-bromoguanosine-3′,5′-cyclomonophosphate sodium salt, a membrane-permeant cGMP analogue or 1H-(1,2,4) oxadiazolo (4,3-a) quinoxalin-1-one, a specific soluble guanylyl cyclase inhibitor. In addition, bath applications of GSNO inhibited T-type currents in nucleated outside-out patches and whole cell recordings to a similar extent, with minimal effect on cell-attached recordings, suggesting a direct effect of GSNO on putative extracellular thiol residues on T-type channels. Biophysical studies indicate that GSNO decreased the availability of T-type channels at physiological potentials by modifying gating and stabilizing inactive states of the channels. In current-clamp experiments, GSNO diminished the amplitude of low-threshold calcium spikes and frequency of spike firing with minimal effects on the passive membrane properties. Collectively, the results indicate that SNOs may be a class of endogenous agents that control the functional states of the thalamus.
Identification and Dynamics of Spontaneous Burst Initiation Zones in Unidimensional Neuronal Cultures
Authors: Feinerman Ofer , Segal Menahem , Moses Elisha
Published: February 2007

Spontaneous activity is typical of in vitro neural networks, often in the form of large population bursts. The origins of this activity are attributed to intrinsically bursting neurons and to noisy backgrounds as well as to recurrent network connections. Spontaneous activity is often observed to emanate from localized sources or initiation zones, propagating from there to excite large populations of neurons. In this study, we use unidimensional cultures to overcome experimental difficulties in identifying initiation zones in vivo and in dissociated two-dimensional cultures. We found that spontaneous activity in these cultures is initiated exclusively in localized zones that are characterized by high neuronal density but also by recurrent and inhibitory network connections. We demonstrate that initiation zones compete in driving network activity in a winner-takes-most scenario.
Control of Hand Impedance Under Static Conditions and During Reaching Movement
Authors: Darainy Mohammad , Towhidkhah Farzad , Ostry David J.
Published: February 2007

It is known that humans can modify the impedance of the musculoskeletal periphery, but the extent of this modification is uncertain. Previous studies on impedance control under static conditions indicate a limited ability to modify impedance, whereas studies of impedance control during reaching in unstable environments suggest a greater range of impedance modification. As a first step in accounting for this difference, we quantified the extent to which stiffness changes from posture to movement even when there are no destabilizing forces. Hand stiffness was estimated under static conditions and at the same position during both longitudinal (near to far) and lateral movements using a position-servo technique. A new method was developed to predict the hand “reference” trajectory for purposes of estimating stiffness. For movements in a longitudinal direction, there was considerable counterclockwise rotation of the hand stiffness ellipse relative to stiffness under static conditions. In contrast, a small counterclockwise rotation was observed during lateral movement. In the modeling studies, even when we used the same modeled cocontraction level during posture and movement, we found that there was a substantial difference in the orientation of the stiffness ellipse, comparable with that observed empirically. Indeed, the main determinant of the orientation of the ellipse in our modeling studies was the movement direction and the muscle activation associated with movement. Changes in the cocontraction level and the balance of cocontraction had smaller effects. Thus even when there is no environmental instability, the orientation of stiffness ellipse changes during movement in a manner that varies with movement direction.
Limits of Linear Rate Coding of Dynamic Stimuli by Electroreceptor Afferents
Authors: Gussin Daniel , Benda Jan , Maler Leonard
Published: February 2007

We estimated the frequency-intensity (f-I) curves of P-unit electroreceptors using 4-Hz random amplitude modulations (RAMs) and using the covariance method (50-Hz RAMs). Both methods showed that P units are linear encoders of stimulus amplitude with additive noise; the gain of the f-I curve was, on average, 0.32 and 2.38 spikes·s−1·μV−1for the low- and high-frequency cutoffs, respectively. There were two sources of apparent noise in the encoding process: the first was the variability of baseline P-unit discharge and the second was the variation of receptor discharge due to variability of the stimulus slope independent of its intensity. The covariance method showed that a linear combination of eigenvectors representing the time-weighted stimulus intensity (E1) and its derivative (E2) could account for, on average, 92% of the total response variability; E1 by itself accounted for 76% of the variability. The low gain of the low-frequency f-I curve implies that detection of small (1 μV) signals would require integration over many receptors (∼1,200) and time (200 ms); even then, signals that elicit behavioral responses could not be detected using rate coding with the estimated gain and noise levels. Weak signals at the limit of behavioral thresholds could be detected if the animal were able to extract E1 from the population of responding P units; we propose a tentative mechanism for this operation although there is no evidence as to whether it is actually implemented in the nervous system of these fish.
Multiple Saccades Are More Automatic Than Single Saccades
Authors: van Donkelaar Paul , Saavedra Sandy , Woollacott Marjorie
Published: February 2007

In this paper, we demonstrate that when a peripheral object is foveated by a sequence of multiple saccades, the initial saccade in the sequence is initiated markedly faster than a single accurate saccade to the same object. We suggest that multiple saccades represent a more automatic form of oculomotor planning that may be the result of a reduced influence from the cerebral cortex. To test this, we compared single and multiple saccade characteristics across development. We find that in contrast to the reduction in the latency of single saccades that is observed across development, the latency of initial saccades in multiple saccade sequences is remarkably stable across all age groups. Moreover, the longer the latency of this initial saccade, the more accurate it is, suggesting that there is a relation between the degree of procrastination and the accuracy of the response. Finally, the frequency with which multiple saccades occurred within each age group was positively correlated with the tendency to generate erroneous saccades during a fixation control task. Taken together, the present data suggest that multiple saccades are generated in a more automatic manner than single saccades.
Decompensation of Cardiac Hypertrophy: Cellular Mechanisms and Novel Therapeutic Targets
Authors: Diwan Abhinav , Dorn Gerald W.
Published: February 2007

Cardiac hypertrophy leads to heart failure, and both conditions can ultimately prove lethal. Here, traditional and novel mechanisms relating hypertrophy and heart failure are described at the physiological, cellular, and molecular levels. The rational application of these mechanistic considerations to therapeutics targeting hypertrophy and heart failure is discussed.
PKA, Germ Cells, and Fertility
Authors: Burton Kimberly A. , McKnight G. Stanley
Published: February 2007

Temporal and spatial regulation of PKA activity are essential for vigorous sperm motility and for the resumption of meiosis in oocytes, two events required for successful fertilization. Genetic mutations in mice that affect PKA signaling in germ cells lead to infertility and illustrate the importance of this pathway in mammalian reproduction.
Intracellular pH Sensors: Design Principles and Functional Significance
Authors: Srivastava Jyoti , Barber Diane L. , Jacobson Matthew P.
Published: February 2007

Changes in intracellular pH regulate many cell behaviors, including proliferation, migration, and transformation. However, our understanding of how physiological changes in pH affect protein conformations and macromolecular assemblies is limited. We present design principles, current modeling predictions, and examples of pH sensors or proteins that have activities or ligand-binding affinities that are regulated by changes in intracellular pH.
Epigenetic Inactivation of the Chromosomal Stability Control Genes BRCA1, BRCA2, and XRCC5 in Non–Small Cell Lung Cancer
Authors: Lee Ming-Ni , Tseng Ruo-Chia , Hsu Han-Shui , Chen Jia-Yang , Tzao Ching , Ho William L. , Wang Yi-Ching
Published: February 2007

Purpose: Lung cancer cells frequently exhibit marked chromosome instability. We postulated that alterations of the double-strand break repair genes (BRCA1, BRCA2, and XRCC5) might be involved in lung cancer.

Patients and Methods: We examined the loss of protein and mRNA expression and the 5′CpG hypermethylation and allelic imbalance of the BRCA1, BRCA2, and XRCC5 genes in 98 non–small cell lung cancer (NSCLC) samples. Anchorage-dependent growth after reexpression of these genes was examined in a lung cancer cell line that originally lacked BRCA1 and BRCA2 expression.

Results: The data indicated that low protein expression of BRCA1 and BRCA2 was frequent in lung adenocarcinomas (42-44%), whereas low XRCC5 protein expression was more prevalent among squamous cell carcinoma (32%). In addition, low BRCA1 expression was significantly associated with low RB expression, especially in lung adenocarcinoma. Concurrent alterations in XRCC5 and p53 were the most frequent profiles in smoking patients. Importantly, low mRNA and protein expressions of BRCA1, BRCA2, and XRCC5 were significantly associated with their promoter hypermethylation. 5-Aza-2′-deoxycytidine treatment of NSCLC cells showed demethylation and reexpression of the BRCA1 and BRCA2 genes and reduced anchorage-independent growth.

Conclusions: Our retrospective study provides compelling evidence that low mRNA and protein expression in the BRCA1/BRCA2 and XRCC5 genes occur in lung adenocarcinoma and squamous cell carcinoma, respectively, and that promoter hypermethylation is the predominant mechanism in deregulation of these genes. Alteration of the double-strand break repair pathway, perhaps by interacting with p53 and RB deregulation, is important in the pathogenesis of a subset of NSCLC.
Celecoxib and Mucosal Protection: Translation from an Animal Model to a Phase I Clinical Trial of Celecoxib, Irinotecan, and 5-Fluorouracil
Authors: Javle Milind M. , Cao Shousong , Durrani Farukh A. , Pendyala Lakshmi , Lawrence David D. , Smith Patrick F. , Creaven Patrick J. , Noel Diane C. , Iyer Renuka V. , Rustum Youcef M.
Published: February 2007

Purpose: Chemotherapy-induced diarrhea occurs secondary to mucosal inflammation and may be cyclooxygenase-2 mediated. Cyclooxygenase-2 inhibitors may ameliorate chemotherapy-induced mucosal toxicity and enhance its antitumor effect. We investigated this hypothesis in the Ward colorectal cancer rat model and in a phase I clinical study.

Experimental Design: In the Ward rat model, irinotecan was given daily × 3 or weekly × 4 with or without celecoxib. In the phase I clinical study, we planned to escalate the dose of irinotecan in the FOLFIRI regimen (irinotecan, 5-fluorouracil, and leucovorin) with a fixed dose of celecoxib. Irinotecan was escalated in four dose levels: 180, 200, 220, and 260 mg/m2. Celecoxib was administered as 400 mg, twice daily starting on day 2 of cycle 1. Pharmacokinetics of irinotecan, SN-38, and SN-38G were obtained on days 1 and 14. A standard 3 + 3 dose escalation scheme was used. Plasma concentrations of irinotecan, SN-38, and SN-38G were measured using high-pressure liquid chromatography.

Results: Celecoxib ameliorated diarrhea, weight loss, and lethality and resulted in synergistic antitumor effect in the rat model. Twelve patients with advanced cancers were enrolled and evaluable for dose-limiting toxicity (DLT). Diarrhea was the cause for discontinuation in one. Grade 2 and 3 diarrhea occurred in three and two patients, respectively. One patient had DLT at dose level 2 (grade 3 diarrhea). Two had a DLT at DL3 (G3 emesis and myocardial infarct). Celecoxib had limited influence on the pharmacokinetics of irinotecan in this data set.

Conclusions: Maximum tolerated dose of irinotecan in FOLFIRI schedule with celecoxib is 200 mg/m2.
Vascular Endothelial Growth Factor Gene Polymorphisms Are Associated with Prognosis in Ovarian Cancer
Authors: Hefler Lukas A. , Mustea Alexander , Könsgen Dominique , Concin Nicole , Tanner Berno , Strick Reiner , Heinze Georg , Grimm Christoph , Schuster Eva , Tempfer Clemens , Reinthaller Alexander , Zeillinger Robert
Published: February 2007

Purpose: Vascular endothelial growth factor (VEGF), an important regulator of angiogenesis and vascular permeability, is involved in various steps of ovarian carcinogenesis. Gene polymorphisms within the gene encoding VEGF were shown to be independently associated with an adverse outcome in various malignancies. No data are available for ovarian cancer.

Experimental Design: In the present multicenter study, we examined three common polymorphisms within the VEGF gene (−634G/C, −1154G/A, and −2578C/A) known to be associated with an increased VEGF production in 563 Caucasian patients with ovarian cancer from Austria and Germany using pyrosequencing. Results were correlated with clinical data.

Results: The three investigated polymorphisms did not correlate with any of the investigated clinicopathologic variables. In univariate and multivariate models, no significant correlations between any polymorphism and patients' overall survival were ascertained. Simultaneous carriage of the three homozygous genotypes (i.e., VEGF −634C/C, VEGF −1154G/G, VEGF −2578C/C) known to be associated with increased VEGF expression in an individual patient, however, was independently associated with a shortened overall survival (hazard ratio, 2.1; 95% confidence interval, 1.1-3.9; P = 0.02).

Conclusions: We present the first data on VEGF gene polymorphisms in ovarian cancer. Simultaneous carriage of the three investigated homozygous genotypes was shown to be an independent adverse prognosticator of overall survival.
Suberoylanilide Hydroxamic Acid, a Histone Deacetylase Inhibitor: Effects on Gene Expression and Growth of Glioma Cells In vitro and In vivo
Authors: Yin Dong , Ong John M. , Hu Jinwei , Desmond Julian C. , Kawamata Norihiko , Konda Bindu M. , Black Keith L. , Koeffler H. Phillip
Published: February 2007

Purpose: Histone acetylation is one of the main mechanisms involved in regulation of gene expression. During carcinogenesis, tumor-suppressor genes can be silenced by aberrant histone deacetylation. This epigenetic modification has become an important target for tumor therapy. The histone deacetylation inhibitor, suberoylanilide hydroxamic acid (SAHA), can induce growth arrest in transformed cells. The aim of this study is to examine the effects of SAHA on gene expression and growth of glioblastoma multiforme (GBM) cells in vitro and in vivo.

Experimental Design: The effect of SAHA on growth of GBM cell lines and explants was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Changes of the cell cycle and relative gene expression were detected by fluorescence-activated cell sorting, real-time reverse transcription-PCR, and Western blotting. After glioma cells were implanted in the brains of mice, the ability of SAHA to decrease tumor growth was studied.

Results: Proliferation of GBM cell lines and explants were inhibited in vitro by SAHA (ED50, 2 × 10−6 to 2 × 10−5 mol/L, 5 days). SAHA exposure of human U87 and T98G glioma cell lines, DA66 and JM94 GBM explants, as well as a murine GL26 GBM cell line resulted in an increased accumulation of cells in G2-M of the cell cycle. Many proapoptotic, antiproliferative genes increased in their expression (DR5, TNFα, p21WAF1, p27KIP1), and many antiapoptotic, progrowth genes decreased in their levels (CDK2, CDK4, cyclin D1, cyclin D2) as measured by real-time reverse transcription-PCR and/or Western blot after these GBM cells were cultured with SAHA (2.5 × 10−6 mol/L, 1 day). Chromatin immunoprecipitation assay found that acetylation of histone 3 on the p21WAF1 promoter was markedly increased by SAHA. In vivo murine experiments suggested that SAHA (10 mg/kg, i.v., or 100 mg/kg, i.p.) could cross the blood-brain barrier as shown by prominent increased levels of acetyl-H3 and acetyl-H4 in the brain tissue. Furthermore, the drug significantly (P < 0.05) inhibited the proliferation of the GL26 glioma cells growing in the brains of mice and increased their survival.

Conclusions: Taken together, SAHA can slow the growth of GBM in vitro and intracranially in vivo. SAHA may be a welcome addition for the treatment of this devastating disease.
Chimmitecan, a Novel 9-Substituted Camptothecin, with Improved Anticancer Pharmacologic ProfilesIn vitroandIn vivo
Authors: Huang Min , Gao Heyong , Chen Yi , Zhu Hong , Cai Yujun , Zhang Xiongwen , Miao Zehong , Jiang Hualiang , Zhang Jian , Shen Hongwu , Lin Liping , Lu Wei , Ding Jian
Published: February 2007

Purpose: This study aimed to evaluate antitumor activities and pharmacologic profiles of chimmitecan, a novel 9-small-alkyl–substituted lipophilic camptothecin, in comparison with irinotecan (CPT-11) and topotecan.

Experimental Design: The in vitro cytotoxities of chimmitecan in human tumor cell lines and multidrug resistance (MDR) cells were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and sulforhodamin B assays. DNA relaxation, cleavage assays, and cellular band depletion assay were combined to delineate its effects on topoisomerase I. DNA damage, cell cycle arrest, and apoptosis were assessed using comet assay, flow cytometry, and DNA ladder analysis, respectively. The in vivo antitumor activities were measured in nude mice bearing human tumor xenografts.

Results: Chimmitecan displayed more potent cytotoxicity than SN38 and topotecan. Neither a cross-resistance to chimmitecan in MDR cells nor an influence of human serum albumin in its cytotoxity was observed. Chimmitecan exhibited comparable effects on topoisomerase I compared with the reference drugs, including inhibiting topoisomerase I catalytic activity and trapping and stabilizing covalent topoisomerase I-DNA complexes. Furthermore, nanomolar levels of chimmitecan caused impressive DNA damage, G2-M phase arrest, and apoptosis in human leukemia HL60 cells. I.v. administration of chimmitecan inhibited the growth of HCT-116, MDA-MB-435, BEL-7402, and A549 human carcinoma xenografts in nude mice, with greater potency than CPT-11 against the latter two tumors models. Chimmitecan presented potent efficacy in A549 tumor model when given orally.

Conclusions: Chimmitecan is a potent inhibitor of topoisomerase I and displays outstanding activity in vitro and in vivo. The substitution at the 9-position benefits chimmitecan a salient anti-MDR activity, stability in human serum albumin, improved solubility, and oral availability, which might favorably promise its therapeutic potential in clinical settings.
α1-Antitrypsin Precursor in Gastric Juice Is a Novel Biomarker for Gastric Cancer and Ulcer
Authors: Hsu Ping-I , Chen Chung-Hsuan , Hsieh Chieu-Shia , Chang Wei-Chao , Lai Kwok-Hung , Lo Gin-Ho , Hsu Ping-Ning , Tsay Feng-Woei , Chen Yu-Shan , Hsiao Michael , Chen Hui-Chun , Lu Pei-Jung
Published: February 2007

Purpose: To search for novel disease-specific markers in gastric juice by investigating the protein concentrations and components in gastric juice from patients with various gastroduodenal diseases.

Experimental Design: Protein concentrations and pH values in fasting gastric juice were examined in 120 healthy subjects and 39 gastric ulcer, 38 duodenal ulcer, and 31 gastric cancer patients. The protein components in gastric juice were studied by two-dimensional PAGE and mass spectrometric analysis.

Results: Protein concentrations in gastric juice of patients with gastric ulcers and gastric cancer were significantly higher than those in healthy subjects (1.06 and 2.61 mg/mL versus 0.48 mg/mL; P = 0.001 and P < 0.001, respectively), and duodenal ulcer patients had lower gastric juice protein concentrations compared with healthy subjects (0.26 versus 0.48 mg/mL; P < 0.05). Gastric hypoacidity and advanced age were independent factors affecting the protein concentrations in gastric juice with odds ratios of 32.9 (95% confidence interval, 11.8-90.9) and 3.2 (95% confidence interval, 1.3-8.3), respectively. Each electrophoresis images of gastric juice could be classified into one of three patterns: basic band, specific band, or nonspecific band. The frequencies of specific band pattern in healthy subjects, gastric ulcer, duodenal ulcer, and gastric cancer patients were 6%, 42%, 6%, and 93%, respectively. Proteomic analysis revealed that α1-antitrypsin precursor was the principal peptide in the specific band.

Conclusions: α1-antitrypsin precursor in gastric juice is a novel biomarker for gastric cancer and ulcer. A noninvasive method to obtain gastric juice followed by proteomic analysis may serve as a new tool to screen for gastric malignancies.
NF-Y–Dependent Cyclin B2 Expression in Colorectal Adenocarcinoma
Authors: Park Seon-Hwa , Yu Gyung-Ran , Kim Woo-Ho , Moon Woo-Sung , Kim Jong-Hun , Kim Dae-Ghon
Published: February 2007

Purpose: Cyclin B2, a G2-M cyclin, is overexpressed in colorectal adenocarcinomas compared with the normal mucosa. This study examined the level of cyclin B2 overexpression according to the histologic findings and investigated the mechanism(s) and clinical implications of cyclin B2 overexpression in colorectal adenocarcinomas.

Experimental Design: The immunoreactivity of the polyclonal antibodies to cyclin B2 was determined in colorectal cancer cells. The transcriptional regulation of cyclin B2 by NF-Y was analyzed using an in vitro transfection assay and an in vivo chromatin immunoprecipitation assay. The proliferative activity of the colorectal cancer cells in relation to cyclin B2 overexpression was further examined.

Results: The cytoplasmic distribution of cyclin B2 immunoreactivity was positive in 42 of 65 (64.6%) cases of colorectal adenocarcinoma, and the level was similar regardless of the histologic type. A dominant-negative form of NF-YA effectively inhibited the cyclin B2 promoter activity, and NF-Y was found to bind three conserved CCAAT boxes in the cyclin B2 promoter in colorectal adenocarcinoma cells. Tumor cells with a higher functional cyclin B2 activity grew faster than those with a lower activity. Furthermore, there was a correlation between the cells showing immunoreactivity to cyclin B2 and those containing the proliferating cell nuclear antigen, a G1-S cyclin, which is also downstream of NF-Y in colorectal adenocarcinoma cells.

Conclusions: Cyclin B2 seems to be a molecular marker of a colorectal adenocarcinoma and that its up-regulation and coordinate expression of the other cell cycle–related genes by NF-Y might contribute to tumor cell proliferation by accelerating cell cycle progression.
Class III β-Tubulin Expression and Benefit from Adjuvant Cisplatin/Vinorelbine Chemotherapy in Operable Non–Small Cell Lung Cancer: Analysis of NCIC JBR.10
Authors: Sève Pascal , Lai Raymond , Ding Keyue , Winton Timothy , Butts Charles , Mackey John , Dumontet Charles , Dabbagh Laith , Aviel-Ronen Sarit , Seymour Lesley , Whitehead Marlo , Tsao Ming-Sound , Shepherd Frances A. , Reiman Tony
Published: February 2007

Purpose: High class III β-tubulin (bTubIII) expression in advanced non–small cell lung cancer is known to correlate with reduced response rates and inferior survival with anti-microtubule agents. JBR.10 showed a 12% and 15% improvement in 5-year recurrence-free survival (RFS) and overall survival (OS), respectively, with the addition of cisplatin and vinorelbine following resection of stage IB-II non–small cell lung cancer. We sought to determine the effect of bTubIII on patient outcome and benefit from adjuvant chemotherapy in the JBR.10 trial.

Experimental Design: We did a semiquantitative immunohistochemical assay for bTubIII on primary tumor tissue available from 265 of the 482 patients in JBR.10. Tumors were classified as bTubIII “low” or “high” using a validated method. We examined the prognostic effect of bTubIII in patients treated with or without chemotherapy and the survival benefit from chemotherapy in low versus high bTubIII subgroups.

Results: High bTubIII expression was associated with poorer RFS and OS in patients treated with surgery alone but not in patients treated with adjuvant chemotherapy. The RFS and OS benefits of adjuvant chemotherapy were greater in high versus low tubulin expressors. However, with Cox regression, the interaction between bTubIII status and chemotherapy treatment in predicting RFS or OS did not reach statistical significance.

Conclusions: Chemotherapy seemed to overcome the negative prognostic effect of high bTubIII expression. Greater benefit from adjuvant chemotherapy was seen in patients with high bTubIII expression. This is contrary to what has been seen in the setting of advanced disease; possible reasons for this difference are being explored.
Flaxseed and Its Lignans Inhibit Estradiol-Induced Growth, Angiogenesis, and Secretion of Vascular Endothelial Growth Factor in Human Breast Cancer Xenografts In vivo
Authors: Bergman Jungeström Malin , Thompson Lilian U. , Dabrosin Charlotta
Published: February 2007

Purpose: Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, which is crucial in cancer progression. We have previously shown that estradiol (E2) increases VEGF in breast cancer. Phytoestrogens are potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. The main phytoestrogens in Western diet are lignans, and flaxseed is a rich source of the mammalian lignans enterodiol and enterolactone.

Experimental Design: In the present study, ovariectomized mice were treated with continuous release of E2. MCF-7 tumors were established and mice were fed with basal diet or 10% flaxseed, and two groups that were fed basal diet received daily injections with enterodiol or enterolactone (15 mg/kg body weight).

Results: We show that flaxseed, enterodiol, and enterolactone counteracted E2-induced growth and angiogenesis in solid tumors. Extracellular VEGF in vivo, sampled using microdialysis, in all intervention groups was significantly decreased compared with tumors in the basal diet group. Our in vivo findings were confirmed in vitro. By adding enterodiol or enterolactone, E2-induced VEGF secretion in MCF-7 cells decreased significantly without agonistic effects. The increased VEGF secretion by E2 in MCF-7 cells increased the expression of VEGF receptor-2 in umbilical vein endothelial cells, suggesting a proangiogenic effect by E2 by two different mechanisms, both of which were inhibited by the addition of lignans.

Conclusions: Our results suggest that flaxseed and its lignans have potent antiestrogenic effects on estrogen receptor–positive breast cancer and may prove to be beneficial in breast cancer prevention strategies in the future.
Proteomic Signature Corresponding to the Response to Gefitinib (Iressa, ZD1839), an Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor in Lung Adenocarcinoma
Authors: Okano Tetsuya , Kondo Tadashi , Fujii Kiyonaga , Nishimura Toshihide , Takano Toshimi , Ohe Yuichiro , Tsuta Koji , Matsuno Yoshihiro , Gemma Akihiko , Kato Harbumi , Kudoh Shoji , Hirohashi Setsuo
Published: February 2007

Purpose: We aimed to identify candidate proteins for tumor markers to predict the response to gefitinib treatment.

Experimental Design: We did two-dimensional difference gel electrophoresis to create the protein expression profile of lung adenocarcinoma tissues from patients who showed a different response to gefitinib treatment. We used a support vector machine algorithm to select the proteins that best distinguished 31 responders from 16 nonresponders. The prediction performance of the selected spots was validated by an external sample set, including six responders and eight nonresponders. The results were validated using specific antibodies.

Results: We selected nine proteins that distinguish responders from nonresponders. The predictive performance of the nine proteins was validated examining an additional six responders and eight nonresponders, resulting in positive and negative predictive values of 100% (six of six) and 87.5% (seven of eight), respectively. The differential expression of one of the nine proteins, heart-type fatty acid–binding protein, was successfully validated by ELISA. We also identified 12 proteins as a signature to distinguish tumors based on their epidermal growth factor receptor gene mutation status.

Conclusions: Study of these proteins may contribute to the development of personalized therapy for lung cancer patients.
Detection of Circulating Tumor Cells in Peripheral Blood of Patients with Metastatic Breast Cancer: A Validation Study of the CellSearch System
Authors: Riethdorf Sabine , Fritsche Herbert , Müller Volkmar , Rau Thomas , Schindlbeck Christian , Rack Brigitte , Janni Wolfgang , Coith Cornelia , Beck Katrin , Jänicke Fritz , Jackson Summer , Gornet Terrie , Cristofanilli Massimo , Pantel Klaus
Published: February 2007

Purpose: The CellSearch system (Veridex, Warren, NJ) is designed to enrich and enumerate circulating tumor cells (CTCs) from peripheral blood. Here, we validated the analytic performance of this system for clinical use in patients with metastatic breast cancer.

Experimental Design: This prospective multicenter study conducted at three independent laboratories involved samples from 92 patients with metastatic breast cancer. Intra- and inter-assay variability using controls containing defined numbers of cells (average, 50 and 1,000, respectively), cell stability based on varying storage and shipment conditions, recovery precision from samples spiked with 4 to 12 tumor cells, inter-instrument variability, and positivity of samples from metastatic breast cancer patients were tested.

Results: Intra- and inter-assay precision for two sites were high: All eight positive controls analyzed in the same run and >95% of the run to run control values (n = 299) were within the specified ranges. Recovery rate of spiked samples averaged between 80% and 82%. CTCs were detected in ∼70% of metastatic breast cancer patients. CTC values of identical samples processed either immediately after blood drawing or after storage for 24, 48, or 72 h at room temperature or at 4°C did not differ significantly. Shipment of samples had no influence on CTC values. When analyzing identical samples in different centers, inter-instrument accordance was high.

Conclusions: The CellSearch system enables the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory. Blood samples should be shipped at room temperature and CTC counts are stable for at least 72 h.
CD8+ T cells Reactive to Survivin Antigen in Patients with Multiple Myeloma
Authors: Grube Matthias , Moritz Stephanie , Obermann Ellen C. , Rezvani Katayoun , Mackensen Andreas , Andreesen Reinhard , Holler Ernst
Published: February 2007

Purpose: Survivin is a member of the inhibitors of apoptosis family and is overexpressed in different types of malignancies. Cytotoxic T cells recognizing survivin epitopes can be elicited in vitro and by vaccination in patients with leukemia, breast cancer, and melanoma. We did this study to investigate whether survivin-specific CD8+ T cells occur in patients with multiple myeloma.

Experimental Design: An HLA-A2.1–binding survivin peptide was used to detect peptide-specific T cells by a quantitative real-time PCR to measure antigen-specific IFN-γ mRNA expression in 23 patients with myeloma and 21 healthy volunteers. T cells producing IFN-γ in response to survivin were further analyzed for expression of CD45RA and CCR7 to determine phenotypic characterization. Additional immunohistochemical analyses of survivin antigen expression in bone marrow specimens of patients was done.

Results: T cells recognizing HLA-A2.1–binding survivin peptide were detected in 9 of 23 patients and in 1 of 21 healthy volunteers. Survivin-reactive T cells were identified as terminally differentiated effector T cells (CD8+, CD45RA+, and CCR7−). Positive survivin expression of myeloma cells in bone marrow specimens was shown in 7 of 11 patients.

Conclusion: We provide, for the first time, evidence of T cell reactivity against survivin antigen in patients with multiple myeloma. Our data suggest the immunogenicity of survivin antigen in multiple myeloma and that immunotherapeutic strategies using survivin as a target antigen might be an option for patients with this disease.
Celecoxib Prevents Neuroblastoma Tumor Development and Potentiates the Effect of Chemotherapeutic Drugs In vitro and In vivo
Authors: Ponthan Frida , Wickström Malin , Gleissman Helena , Fuskevåg Ole M. , Segerström Lova , Sveinbjörnsson Baldur , Redfern Christopher P.F. , Eksborg Staffan , Kogner Per , Johnsen John I.
Published: February 2007

Purpose: Neuroblastoma is the most common and deadly solid tumor of childhood. Cyclooxygenase-2 is expressed in clinical neuroblastoma tumors and cell lines and inhibitors of this enzyme induce apoptosis in human neuroblastoma cells in vitro and in neuroblastoma xenografts in vivo. We hypothesized that the cyclooxygenase-2–specific inhibitor celecoxib could enhance the cytotoxic effect of chemotherapeutic drugs currently used in neuroblastoma treatment. Furthermore, we investigated if prophylactic treatment with celecoxib could prevent neuroblastoma tumor development in vivo.

Experimental Design: Neuroblastoma cell cytotoxicity of chemotherapeutic drugs in combination with celecoxib was examined. In vivo, athymic rats carrying established SH-SY5Y xenografts were treated with celecoxib in combination with irinotecan, doxorubicin or etoposide, or with either drug alone. For prevention studies, rats received celecoxib in the diet, 250 to 2,500 ppm, from the time of tumor cell injection.

Results: Celecoxib induced a synergistic or an additive cytotoxic effect in combination with doxorubicin, etoposide, irinotecan or vincristine in vitro. In vivo, treatment with celecoxib in combination with irinotecan or doxorubicin induced a significant growth inhibition of established neuroblastoma tumors. Rats receiving celecoxib in the diet showed a distinct dose-dependent delay in tumor development compared with untreated rats. Plasma levels of celecoxib were comparable with levels obtainable in humans.

Conclusions: Celecoxib potentiates the antitumor effect of chemotherapeutic drugs currently used in neuroblastoma treatment, which argues for clinical trials combining these drugs. Celecoxib could also be a potential drug for treatment of minimal residual disease.
Clinical Pharmacodynamic Effects of the Growth Hormone Receptor Antagonist Pegvisomant: Implications for Cancer Therapy
Authors: Yin Donghua , Vreeland Franzanne , Schaaf Larry J. , Millham Robert , Duncan Barbara A. , Sharma Amarnath
Published: February 2007

Purpose: The present study evaluated and compared the efficacy of pegvisomant and octreotide in blocking the growth hormone (GH) axis in humans based on pharmacodynamic biomarkers associated with the GH axis. The study also evaluated the safety of pegvisomant given at high s.c. doses for 14 days.

Experimental Design: Eighty healthy subjects were enrolled in five cohorts: cohorts 1 to 3, s.c. pegvisomant at 40, 60, or 80 mg once daily × 14 days (n = 18 per cohort); cohort 4, s.c. octreotide at 200 μg thrice daily × 14 days (n = 18); and cohort 5, untreated control (n = 8). Serial blood samples were collected to measure plasma concentrations of total insulin-like growth factor type I (IGF-I), free IGF-I, IGF-II, IGF-binding protein 3 (IGFBP-3), and GH in all subjects and serum pegvisomant concentrations in subjects of cohorts 1 to 3. All subjects receiving treatment were monitored for adverse events (AE).

Results: After s.c. dosing of pegvisomant once daily for 14 days, the mean maximum suppression values of total IGF-I were 57%, 60%, and 62%, at 40, 60, and 80 mg dose levels, respectively. The maximum suppression was achieved ∼7 days after the last dose and was sustained for ∼21 days. Pegvisomant also led to a sustained reduction in free IGF-I, IGFBP-3, and IGF-II concentrations by up to 33%, 46%, and 35%, respectively, and an increase in GH levels. In comparison, octreotide resulted in a considerably weaker inhibition of total IGF-I and IGFBP-3 for a much shorter duration, and no inhibition of IGF-II. AEs in pegvisomant-treated subjects were generally either grade 1 or 2. The most frequent treatment-related AEs included injection site reactions, headache, and fatigue.

Conclusions: Pegvisomant at well-tolerated s.c. doses was considerably more efficacious than octreotide in suppressing the GH axis, resulting in substantial and sustained inhibition of circulating IGF-I, IGF-II, and IGFBP-3 concentrations. These results provide evidence in favor of further testing the hypothesis that pegvisomant, through blocking the GH receptor–mediated signal transduction pathways, could be effective in treating tumors that may be GH, IGF-I, and/or IGF-II dependent, such as breast and colorectal cancer.
A Pilot Study of CTLA-4 Blockade after Cancer Vaccine Failure in Patients with Advanced Malignancy
Authors: O'Mahony Deirdre , Morris John C. , Quinn Cate , Gao Wendy , Wilson Wyndham H. , Gause Barry , Pittaluga Stefania , Neelapu Sattva , Brown Margaret , Fleisher Thomas A. , Gulley James L. , Schlom Jeffrey , Nussenblatt Robert , Albert Paul , Davis Thomas A. , Lowy Israel , Petrus Mike , Waldmann Thomas A. , Janik John E.
Published: February 2007

Purpose: Eleven patients with progressive advanced malignancy after administration of a cancer vaccine received a fully human anti-CTLA-4 monoclonal antibody (ipilimumab). The primary end point was to determine drug toxicity. Tumor response, tumor-specific CD8+ T-cell immune responses, and modulation of CD4+ CD25+ FoxP3+ regulatory T-cell (Treg) numbers were secondary end points.

Experimental Design: Three patients with colon cancer, four with non–Hodgkin's lymphoma, and four with prostate cancer were treated. The first dose was given at 3 mg/kg and subsequent doses were administered monthly at 1.5 mg/kg for a total of four cycles.

Results: Tumor regression was observed in two patients with lymphoma; one of which obtained a partial response of 14-month duration. Ipilimumab was well tolerated with predominantly grade 1/2 toxicities. One drug-related grade 3 toxicity was observed. One patient died within 30 days of treatment due to progressive colon cancer. No increase in vaccine-specific T-cell responses was observed after therapy. Tregs as detected by expression of CD4+CD25 +CD62L + declined at early time points but rebounded to levels at or above baseline values at the time of the next infusion.

Conclusions: Ipilimumab treatment depressed Treg numbers at early time points in the treatment cycle but was not accompanied by an increase in vaccine-specific CD8+ T-cell responses in these patients previously treated with a variety of investigational anticancer vaccines. A partial response was observed in one patient with follicular lymphoma. A phase I/II trial evaluating ipilimumab in patients with follicular lymphoma is currently ongoing.
Reactive Oxygen Species: A Breath of Life or Death?
Authors: Fruehauf John P. , Meyskens Frank L.
Published: February 2007

New insights into cancer cell–specific biological pathways are urgently needed to promote development of rationally targeted therapeutics. Reactive oxygen species (ROS) and their role in cancer cell response to growth factor signaling and hypoxia are emerging as verdant areas of exploration on the road to discovering cancer's Achilles heel. One of the distinguishing and near-universal hallmarks of cancer growth is hypoxia. Unregulated cellular proliferation leads to formation of cellular masses that extend beyond the resting vasculature, resulting in oxygen and nutrient deprivation. The resulting hypoxia triggers a number of critical adaptations that enable cancer cell survival, including apoptosis suppression, altered glucose metabolism, and an angiogenic phenotype. Ironically, recent investigations suggest that oxygen depletion stimulates mitochondria to elaborate increased ROS, with subsequent activation of signaling pathways, such as hypoxia inducible factor 1α, that promote cancer cell survival and tumor growth. Because mitochondria are key organelles involved in chemotherapy-induced apoptosis induction, the relationship between mitochondria, ROS signaling, and activation of survival pathways under hypoxic conditions has been the subject of increased study. Insights into mechanisms involved in ROS signaling may offer novel avenues to facilitate discovery of cancer-specific therapies. Preclinical and clinical evaluation of agents that modify ROS signaling in cancer offers a novel avenue for intervention. This review will cover recent work in ROS-mediated signaling in cancer cells and its potential as a target for developmental therapeutics.
Somatic TP53 Mutations Are Relatively Rare among Adrenocortical Cancers with the Frequent 17p13 Loss of Heterozygosity
Authors: Libè Rossella , Groussin Lionel , Tissier Frédérique , Elie Caroline , René-Corail Fernande , Fratticci Amato , Jullian Eric , Beck-Peccoz Paolo , Bertagna Xavier , Gicquel Christine , Bertherat Jérôme
Published: February 2007

Purpose: Allelic losses [loss of heterozygosity (LOH)] at the 17p13 locus are frequent (85%) in adrenocortical cancers. The tumor suppressor gene TP53 is located at 17p13. The aim of the study was to determine the frequency of TP53 somatic inactivating mutations in adrenocortical tumors with 17p13 LOH and their clinico-biological correlations.

Experimental Design: TP53 somatic mutations, intragenic LOH (VNTR1 marker), and p53 overexpression were studied in 36 adrenocortical tumors with 17p13 LOH determined by Southern blot.

Results: TP53 mutations were detected in 33% of the tumors, and VNTR1 LOH was present in 44% of the cases and did not always correlate with the presence of a TP53 mutation. Only the TP53-mutant tumors exhibit a strong nuclear immunoreactivity. TP53-mutant tumors were significantly larger than wild-type TP53 tumors (median tumor weight: 640 versus 185 g; P = 0.02), were associated with a more advanced stage of tumor progression (MacFarlane stage IV; P = 0.01), and had a shorter disease-free survival (P = 0.03).

Conclusions: The finding that only a minority of adrenocortical tumors with 17p13 LOH had either a VNTR1 LOH or a TP53 mutation indicates that TP53 might not be the only or major tumor suppressor gene at 17p13 involved in adrenocortical cancer progression. We suggest that a genetic instability of the 17p13 region, occurring early in adrenocortical cancer development, involves various genes located in this region. TP53 might be only one of them, and its alteration by the occurrence of inactivating mutation is associated with the development of more aggressive tumors.
Epidermal Growth Factor Receptor Protein Expression and Gene Amplification in Small Cell Carcinoma of the Urinary Bladder
Authors: Wang Xiaoyan , Zhang Shaobo , MacLennan Gregory T. , Eble John N. , Lopez-Beltran Antonio , Yang Ximing J. , Pan Chong-Xian , Zhou Honghong , Montironi Rodolfo , Cheng Liang
Published: February 2007

Purpose: Small cell carcinoma of the urinary bladder is a highly aggressive malignancy with an average life expectancy of only a few months. Epidermal growth factor receptor (EGFR) has been implicated in the pathogenesis and progression of many malignancies. This study was done to investigate EGFR protein expression and gene amplification in a large series of small cell carcinomas of the urinary bladder.

Experimental Design: Fifty-two cases of urinary bladder small cell carcinoma were included in this study. Immunostaining for EGFR was done on paraffin-embedded tissue sections, and gene amplification for EGFR was done by fluorescence in situ hybridization. EGFR expression was correlated with clinicopathologic characteristics and clinical outcome.

Results: All 52 patients, except 1, had advanced disease (T2 or above) at presentation. Immunohistochemically, positive EGFR expression was observed in 14 of 52 (27%) cases. No EGFR gene amplification was observed in any of 52 cases by fluorescence in situ hybridization. Forty cases had polysomy and the remaining 12 cases displayed disomy. No correlation between EGFR protein expression and gene amplification was shown. There was no correlation between EGFR expression and clinicopathologic characteristics.

Conclusions: EGFR is expressed in a subset of urinary bladder small cell carcinomas; however, expression of EGFR does not correlate with clinicopathologic variables. At the molecular level, EGFR overexpression in small cell carcinoma of the urinary bladder does not seem to be caused by gene amplification. The expression of EGFR raises the possibility that EGFR may be a potential therapeutic target in the treatment of this malignancy.