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1.  Grape Seed Extract Efficacy against Azoxymethane-induced Colon Tumorigenesis in A/J Mice: Interlinking miRNA with Cytokine Signaling and Inflammation 
Colorectal cancer (CRC) is the second leading cause of cancer-associated deaths, suggesting that additional strategies are needed to prevent/control this malignancy. Since CRC growth and progression involve a large window (10-15 years), chemopreventive intervention could be a practical/translational strategy. Azoxymethane (AOM)-induced colon tumorigenesis in mice resembles human CRC in terms of progression of ACF to polyps, adenoma and carcinomas, and associated molecular mechanisms. Accordingly, herein we investigated grape seed extract (GSE) efficacy against AOM-induced colon tumorigenesis in A/J mice. GSE was fed in diet at 0.25% or 0.5% (w/w) dose starting two-weeks after last AOM injection for 18 or 28 weeks. Our results showed that GSE feeding significantly decreases colon tumor multiplicity and overall tumor size. In biomarker analysis, GSE showed significant anti-proliferative and pro-apoptotic activities. Detailed mechanistic studies highlighted that GSE strongly modulates cytokines/interleukins and miRNA expression profiles as well as miRNA processing machinery associated with alterations in NF-κB, β-catenin and MAPK signaling. Additional studies using immunohistochemical analyses found that indeed GSE inhibits NF-κB activation and decreases the expression of its downstream targets (COX-2, iNOS, VEGF) related to inflammatory signaling, down-regulates β-catenin signaling and decreases its target gene C-myc, and reduces phosphorylated ERK1/2 levels. Together, these finding suggested that inflammation, proliferation and apoptosis are targeted by GSE to prevent CRC. In summary, this study for the first time shows alterations in the expression of miRNAs and cytokines by GSE in its efficacy against AOM-induced colon tumorigenesis in A/J mouse sporadic CRC model, supporting its translational potential in CRC chemoprevention.
doi:10.1158/1940-6207.CAPR-13-0053
PMCID: PMC3701740  PMID: 23639480
colorectal cancer; chemoprevention; grape seed extract; inflammation; apoptosis
2.  Energy deprivation by silibinin in colorectal cancer cells 
Autophagy  2013;9(5):697-713.
Small molecules with the potential to initiate different types of programmed cell death could be useful ‘adjunct therapy’ where current anticancer modalities fail to generate significant activity due to a defective apoptotic machinery or resistance of cancer cells to the specific death mechanism induced by that treatment. The current study identified silibinin, for the first time, as one such natural agent, having dual efficacy against colorectal cancer (CRC) cells. First, silibinin rapidly induced oxidative stress in CRC SW480 cells due to reactive oxygen species (ROS) generation with a concomitant dissipation of mitchondrial potential (ΔΨm) and cytochrome c release leading to mild apoptosis as a biological effect. However, with increased exposure to silibinin, cytoplasmic vacuolization intensified within the cells followed by sequestration of the organelles, which inhibits the further release of cytochrome c. Interestingly, this decrease in apoptotic response correlated with increased autophagic events as evidenced by tracking the dynamics of LC3-II within the cells. Mechanistic studies revealed that silibinin strongly inhibited PIK3CA-AKT–MTOR but activated MAP2K1/2-MAPK1/3 pathways for its biological effects. Corroborating these effects, endoplasmic reticulum stress was generated and glucose uptake inhibition as well as energy restriction were induced by silibinin, thus, mimicking starvation-like conditions. Further, the cellular damage to tumor cells by silibinin was severe and irreparable due to sustained interference in essential cellular processes such as mitochondrial metabolism, phospholipid and protein synthesis, suggesting that silibinin harbors a deadly ‘double-edged sword’ against CRC cells thereby further advocating its clinical effectiveness against this malignancy.
doi:10.4161/auto.23960
PMCID: PMC3669180  PMID: 23445752
colorectal cancer; silibinin; autophagy; oxidative stress; energy restrictions
3.  Assessment of the In vivo Antitumor Effects of ENMD-2076, a Novel Multitargeted Kinase Inhibitor, against Primary and Cell Line–Derived Human Colorectal Cancer Xenograft Models 
Purpose
This in vivo study was designed to investigate the efficacy of ENMD-2076, a small-molecule kinase inhibitor with activity against the Aurora kinases A and B, and several other tyrosine kinases linked to cancer, including vascular endothelial growth factor receptor 2, cKit, and fibroblast growth factor receptor 1, against murine xenograft models of human colorectal cancer (CRC).
Experimental Design
HT-29 CRC cell line xenografts were treated with either vehicle or ENMD-2076 (100 or 200 mg/kg) orally daily for 28 days. Tumor growth inhibition, dynamic contrast-enhanced magnetic resonance imaging, and 18FDG-positron emission tomography were conducted to assess the anti-proliferative, antiangiogenic, and antimetabolic responses, respectively. Effects on proliferation were also analyzed by immunohistochemical methods. Additionally, three patient-derived xenografts from primary and metastatic sites were treated with ENMD-2076 (100 mg/kg) and assessed for tumor growth inhibition.
Results
In the HT-29 xenograft model, ENMD-2076 induced initial tumor growth inhibition followed by regression. Treatment was associated with significant tumor blanching, indicating a loss of vascularity and substantial reductions in tumor vascular permeability and perfusion as measured by dynamic contrast-enhanced magnetic resonance imaging. Positron emission tomography scanning showed significant decreases in 18FDG uptake at days 3 and 21 of treatment, which was associated with a marked reduction in proliferation as assessed by Ki-67. All three of the patient-derived xenografts tested were sensitive to treatment with ENMD 2076 as measured by tumor growth inhibition.
Conclusions
ENMD-2076 showed robust antitumor activity against cell line and patient-derived xenograft models of CRC that is detectable by functional imaging, supporting clinical investigation of this agent in CRC.
doi:10.1158/1078-0432.CCR-10-0325
PMCID: PMC3928713  PMID: 20406842
4.  Translational imaging endpoints to predict treatment response to novel targeted anticancer agents 
Response Evaluation Criteria in Solid Tumors (RECIST) and World Health Organization (WHO) Criteria have been traditionally used for the evaluation of therapeutic response to chemotherapeutic treatment regimens. They determine anatomic criteria for patients response to anti-cancer therapy based on morphological measurements of each target lesion. While this assessment is justified for cytotoxic (chemotherapeutic) drugs, it is now recognized that morphological imaging protocols are poorly suited to the evaluation of the efficacy of novel signal transduction inhibitors (STIs) which exhibit cytostatic rather than cytotoxic properties. New imaging technologies are now designed to evaluate, in a functional manner, modifications in tumor metabolic activity, cellularity, vascularization before a reduction in tumor volume can be detected. Introduction of physiological imaging end-points, derived from dynamic contrast-enhanced (DCE) imaging protocols – including magnetic resonance imaging (MRI), computed tomography (CT) and ultrasound (US) - allow for early assessment of disruption in tumor perfusion and permeability for targeted anti-angiogenic agents. Diffusion-weighted MRI (DWI) provides another physiological imaging end-point since tumor necrosis and cellularity are seen early in response to anti-angiogenic treatment. Changes in glucose and phospholipid turnover, based on metabolic MRI and positron emission tomography (PET), provide reliable markers for therapeutic response to novel receptor-targeting agents. Finally, novel molecular imaging techniques of protein and gene expression have been developed in animal models followed by a successful human application for gene therapy-based protocols.
doi:10.1016/j.drup.2011.04.004
PMCID: PMC3159818  PMID: 21640633
functional imaging; magnetic resonance imaging; positron emission tomography; signal transduction inhibitors; contrast enhanced imaging; personalized medicine
5.  First-in-Human Phase I Trial of Two Schedules of OSI-930, a Novel Multikinase Inhibitor, Incorporating Translational Proof-of-Mechanism Studies 
Purpose
OSI-930 is a novel, potent, oral small-molecule receptor tyrosine kinase inhibitor, predominantly against VEGF receptors (VEGFR), c-Kit, and platelet-derived growth factor receptors. A phase I trial was undertaken to determine safety, maximum-tolerated dose (MTD), pharmacokinetics, pharmacodynamics, and antitumor activity of OSI-930 in patients with advanced solid tumors.
Experimental Design
OSI-930 was administered once or twice a day using a modified accelerated titration design. Pharmacokinetics and plasma soluble VEGFR2 (sVEGFR2) studies were undertaken. Dynamic contrast-enhanced MRI (DCE-MRI) and 2[18F]fluoro-2-deoxy-D-glucose-positron emission tomography (FDG-PET) MTD expansion cohorts were conducted.
Results
Fifty-eight patients received OSI-930 in 2 schedules; once a day schedule: 12 patients at doses up to 1,600 mg without reaching MTD; twice a day schedule: 46 patients at 400 mg (n = 7), 500 mg (n = 31), and 600 mg (n =8). Dose-limiting toxicities were observed at 600 mg twice a day (n =3): G3 rash (n =2) and G4 γ-glutamyltransferase, establishing the MTD at 500 mg twice a day. Common G1–2 toxicities included fatigue, diarrhea, nausea, and rash. Antitumor responses were seen in 2 patients with advanced ovarian cancer [Response Evaluation Criteria in Solid Tumors (RECIST) partial response (PR) (n = 1); GCIG CA125 response (n = 1)]. Eleven of 19 heavily pretreated imatinib-resistant patients with gastrointestinal stromal tumors achieved RECIST stable disease (median duration: 126 days), with FDG-PET scans showing PRs in 4 of 9 patients. OSI-930 exposure increased with dose; substantial decreases in sVEGFR levels were observed with OSI-930 twice a day doses ≥400 mg, while DCE-MRI responses were shown in 4 of 6 patients.
Conclusions
OSI-930 is safe and well tolerated, with pharmacokinetic–pharmacodynamic data supporting proof-of-mechanism with clinically relevant antitumor activity.
doi:10.1158/1078-0432.CCR-12-2258
PMCID: PMC3880798  PMID: 23403628
6.  Inositol Hexaphosphate Inhibits Tumor Growth, Vascularity, and Metabolism in TRAMP Mice: A Multiparametric Magnetic Resonance Study 
Herein, employing anatomical and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), we evaluated non-invasively, the in vivo, chemopreventive efficacy of inositol hexaphosphate (IP6), a major constituent of high fiber diets, against prostate tumor growth and progression in transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Male TRAMP mice, beginning 4 weeks of age, were fed with 1, 2 or 4% (w/v) IP6 in drinking water or only drinking water till 28 weeks of age and monitored using MRI over the course of study. Longitudinal assessment of prostate volumes by conventional MRI and tumor vascularity by gadolinium-based DCE-MRI showed a profound reduction in tumor size partly due to anti-angiogenic effects by IP6 treatment. As potential mechanisms of IP6 efficacy, decrease in the expression of glucose transporter GLUT-4 protein together with an increase in levels of phospho-AMP-activated kinase (AMPKTh172) were observed in prostate tissues of mice from IP6 fed-groups, suggesting that IP6 is interfering with the metabolic events occurring in TRAMP prostate. Investigative metabolomics study utilizing quantitative high-resolution 1H-NMR on prostate tissue extracts showed that IP6 significantly decreased glucose metabolism and membrane phospholipid synthesis, in addition to causing an increase in myo-inositol levels in the prostate. Together, these findings show that oral IP6 supplement blocks PCa growth and angiogenesis in TRAMP model in conjunction with metabolic events involved in tumor sustenance. This results in energy deprivation within the tumor, suggesting a practical and translational potential of IP6 treatment in suppressing growth and progression of prostate cancer in humans.
doi:10.1158/1940-6207.CAPR-12-0387
PMCID: PMC3662486  PMID: 23213071
TRAMP; prostate cancer; inositol hexaphosphate; GLUT-4; Magnetic Resonance Imaging
7.  Preclinical Activity of the Rational Combination of Selumetinib (AZD6244) in Combination with Vorinostat in KRAS-Mutant Colorectal Cancer Models 
Purpose
Despite the availability of several active combination regimens for advanced colorectal cancer (CRC), the 5-year survival rate remains poor at less than 10%,supporting the development of novel therapeutic approaches. In this study, we focused on the preclinical assessment of a rationally based combination against KRAS-mutated CRC by testing the combination of the MEK inhibitor, selumetinib, and vorinostat, a histone deacetylase (HDAC) inhibitor.
Experimental Design
Transcriptional profiling and gene set enrichment analysis (baseline and post-treatment) of CRC cell lines provided the rationale for the combination. The activity of selumetinib and vorinostat against the KRAS-mutant SW620 and SW480 CRC cell lines was studied in vitro and in vivo. The effects of this combination on tumor phenotype were assessed using monolayer and 3-dimensional cultures, flow cytometry, apoptosis, and cell migration. In vivo, tumor growth inhibition, 18F-fluoro-deoxy-glucose positron emission tomography (FDG-PET), and proton nuclear magnetic resonance were carried out to evaluate the growth inhibitory and metabolic responses, respectively, in CRC xenografts.
Results
In vitro, treatment with selumetinib and vorinostat resulted in a synergistic inhibition of proliferation and spheroid formation in both CRC cell lines. This inhibition was associated with an increase in apoptosis, cell-cycle arrest in G1, and reduced cellular migration and VEGF-A secretion. In vivo, the combination resulted in additive tumor growth inhibition. The metabolic response to selumetinib and vorinostat consisted of significant inhibition of membrane phospholipids; no significant changes in glucose uptake or metabolism were observed in any of the treatment groups.
Conclusion
These data indicate that the rationally based combination of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, selumetinib, with the HDAC inhibitor vorinostat results in synergistic antiproliferative activity against KRAS-mutant CRC cell lines in vitro. In vivo, the combination showed additive effects that were associated with metabolic changes in phospholipid turnover, but not on FDG-PET, indicating that the former is a more sensitive endpoint of the combination effects.
doi:10.1158/1078-0432.CCR-11-1507
PMCID: PMC3876277  PMID: 22173548
8.  Simultaneous Activation of Kras and Inactivation of p53 Induces Soft Tissue Sarcoma and Bladder Urothelial Hyperplasia 
PLoS ONE  2013;8(9):e74809.
The development of the Cre recombinase-controlled (Cre/LoxP) technique allows the manipulation of specific tumorigenic genes, temporarily and spatially. Our original intention of this study was to investigate the role of Kras and p53 in the development of urinary bladder cancer. First, to validate the effect of intravesical delivery on Cre recombination (Adeno-Cre), we examined activity and expression of β-galactosidase in the bladder of control ROSA transgenic mice. The results confirmed specific recombination as evidenced by β-galactosidase activity in the bladder urothelium of these mice. Then, we administered the same adenovirus into the bladder of double transgenic KrasLSLG12D/+. p53fl/fl mice. The virus solution was held in place by a distal urethral retention suture for 2 hours. To our surprise, there was a rapid development of a spindle-cell tumor with sarcoma characteristics near the suture site, within the pelvic area but outside the urinary track. Since we did not see any detectable β-galactosidase in the area outside of the bladder in the validating (control) experiment, we interpreted that this sarcoma formation was likely due to transduction by Adeno-Cre in the soft tissue of the suture site. To avoid the loss of skin integrity associated with the retention suture, we transitioned to an alternative technique without suture to retain the Adeno-Cre into the bladder cavity. Interestingly, although multiple Adeno-Cre treatments were applied, only urothelial hyperplasia but not carcinogenesis was observed in the subsequent experiments of up to 6 months. In conclusion, we observed that the simultaneous inactivation of p53 and activation of Kras induces quick formation of spindle-cell sarcoma in the soft tissues adjacent to the bladder but slow formation of urothelial hyperplasia inside the bladder. These results strongly suggest that the effect of oncogene regulation to produce either hyperplasia or carcinogenesis greatly depends on the tissue type.
doi:10.1371/journal.pone.0074809
PMCID: PMC3776760  PMID: 24058630
9.  Induction of the Unfolded Protein Response Drives Enhanced Metabolism and Chemoresistance in Glioma Cells 
PLoS ONE  2013;8(8):e73267.
The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.
doi:10.1371/journal.pone.0073267
PMCID: PMC3748289  PMID: 24039668
10.  Early Detection of Graft Failure Using the Blood Metabolic Profile of a Liver Recipient 
Transplantation  2007;83(4):517-521.
In this case report we describe the blood metabolic profile (“metabolomics”) by nuclear magnetic resonance (NMR) spectroscopy and principle component analysis (PCA) from a patient who underwent two consecutive liver transplantations. The first graft from a living-related donor failed and was followed by a second successful transplant from a deceased donor. Using quantitative high-resolution 1H-NMR spectroscopy, 48 endogenous metabolites were analyzed in whole blood samples at baseline and different time points after each transplantation. From 48 analyzed metabolites, six metabolites were identified by PCA as metabolic markers consistent with a non-functional liver after first transplantation. Importantly, this distinctive metabolic profile was present as early as two hours after first transplant surgery when no other variable or conventional laboratory tests indicated poor graft function. This article reports the potential usefulness of quantitative 1H-NMR based metabolomics to diagnose early graft dysfunction in liver transplantation.
doi:10.1097/01.tp.0000251649.01148.f8
PMCID: PMC2709529  PMID: 17318087
Primary graft dysfunction; Living-related liver transplantation; Metabolomics; 1H-NMR spectroscopy
11.  Left Ventricular Assist Device Effects on Metabolic Substrates in the Failing Heart 
PLoS ONE  2013;8(4):e60292.
Background
Heart failure patients have inadequate nutritional intake and alterations in metabolism contributing to an overall energy depleted state. Left ventricular assist device (LVAD) support is a common and successful intervention in patients with end-stage heart failure. LVAD support leads to alterations in cardiac output, functional status, neurohormonal activity and transcriptional profiles but the effects of LVADs on myocardial metabolism are unknown. This study set out to measure cardiac metabolites in non-failing hearts, failing hearts, and hearts post-LVAD support.
Methods
The study population consisted of 8 non-ischemic failing (at LVAD implant) and 8 post-LVAD hearts, plus 8 non-failing hearts obtained from the tissue bank at the University of Colorado. NMR spectroscopy was utilized to evaluate differences in myocardial energy substrates. Paired and non-paired t-tests were used to determine differences between the appropriate groups.
Results
Glucose and lactate values both decreased from non-failing to failing hearts and increased again significantly in the (paired) post-LVAD hearts. Glutamine, alanine, and aromatic amino acids decreased from non-failing to failing hearts and did not change significantly post-LVAD. Total creatine and succinate decreased from non-failing to failing hearts and did not change significantly post-LVAD.
Discussion
Measured metabolites related to glucose metabolism are diminished in failing hearts, but recovered their values post-LVAD. This differed from the amino acid levels, which decreased in heart failure but did not recover following LVAD. Creatine and the citric acid cycle intermediate succinate followed a similar pattern as the amino acid levels.
doi:10.1371/journal.pone.0060292
PMCID: PMC3613395  PMID: 23560088
12.  Utilization of Quantitative In Vivo Pharmacology Approaches to Assess Combination Effects of Everolimus and Irinotecan in Mouse Xenograft Models of Colorectal Cancer 
PLoS ONE  2013;8(3):e58089.
Purpose
The PI3K/AKT/mTOR pathway is frequently dysregulated in cancers and inhibition of mTOR has demonstrated the ability to modulate pro-survival pathways. As such, we sought to determine the ability of the mTOR inhibitor everolimus to potentiate the antitumor effects of irinotecan in colorectal cancer (CRC).
Experimental Design
The combinatorial effects of everolimus and irinotecan were evaluated in vitro and in vivo in CRC cell lines harboring commonly found mutations in PIK3CA, KRAS and/or BRAF. Pharmacokinetically-directed dosing protocols of everolimus and irinotecan were established and used to assess the in vivo antitumor effects of the agents. At the end of treatment, 3–6 tumors per treatment arm were harvested for biomarker analysis by NMR metabolomics.
Results
Everolimus and irinotecan/SN38 demonstrated synergistic anti-proliferative effects in multiple CRC cell lines in vitro. Combination effects of everolimus and irinotecan were determined in CRC xenograft models using clinically-relevant dosing protocols. Everolimus demonstrated significant tumor growth inhibition alone and when combined with irinotecan in HT29 and HCT116 tumor xenografts. Metabolomic analysis showed that HT29 tumors were more metabolically responsive than HCT116 tumors. Everolimus caused a decrease in glycolysis in both tumor types whilst irinotecan treatment resulted in a profound accumulation of lipids in HT29 tumors indicating a cytotoxic effect.
Conclusions
Quantitative analysis of tumor growth and metabolomic data showed that the combination of everolimus and irinotecan was more beneficial in the BRAF/PIK3CA mutant HT29 tumor xenografts, which had an additive effect, than the KRAS/PIK3CA mutant HCT116 tumor xenografts, which had a less than additive effect.
doi:10.1371/journal.pone.0058089
PMCID: PMC3592886  PMID: 23520486
13.  The Emerging Field of Quantitative Blood Metabolomics for Biomarker Discovery in Critical Illnesses 
Metabolomics, a science of systems biology, is the global assessment of endogenous metabolites within a biologic system and represents a “snapshot” reading of gene function, enzyme activity, and the physiological landscape. Metabolite detection, either individual or grouped as a metabolomic profile, is usually performed in cells, tissues, or biofluids by either nuclear magnetic resonance spectroscopy or mass spectrometry followed by sophisticated multivariate data analysis. Because loss of metabolic homeostasis is common in critical illness, the metabolome could have many applications, including biomarker and drug target identification. Metabolomics could also significantly advance our understanding of the complex pathophysiology of acute illnesses, such as sepsis and acute lung injury/acute respiratory distress syndrome. Despite this potential, the clinical community is largely unfamiliar with the field of metabolomics, including the methodologies involved, technical challenges, and, most importantly, clinical uses. Although there is evidence of successful preclinical applications, the clinical usefulness and application of metabolomics in critical illness is just beginning to emerge, the advancement of which hinges on linking metabolite data to known and validated clinically relevant indices. In addition, other important aspects, such as patient selection, sample collection, and processing, as well as the needed multivariate data analysis, have to be taken into consideration before this innovative approach to biomarker discovery can become a reliable tool in the intensive care unit. The purpose of this review is to begin to familiarize clinicians with the field of metabolomics and its application for biomarker discovery in critical illnesses such as sepsis.
doi:10.1164/rccm.201103-0474CI
PMCID: PMC3208597  PMID: 21680948
sepsis;; acute lung injury;; pneumonia;; trauma
14.  Detection of glomerular complement C3 fragments by magnetic resonance imaging in murine lupus nephritis 
Kidney International  2011;81(2):152-159.
One of the challenges of treating patients with glomerulonephritis (GN) is to accurately assess disease activity. We recently developed a magnetic resonance imaging (MRI)-based method of detecting glomerular C3, and hypothesized that this agent could be used to monitor the severity of GN. In the current study we used this imaging method to track the progression of renal disease in the MRL/lpr mouse model of lupus nephritis (LN). The targeting agent is comprised of superparamagnetic iron oxide (SPIO) nanoparticles conjugated to complement receptor type 2 (CR2-targeted SPIO). Glomerular C3b/iC3b/C3d deposition in progressively aging MRL/lpr and control mice was monitored with quantitative immunofluorescence or with CR2-targeted SPIO and T2-weighted MRI. Immunofluorescence showed that glomerular C3b/iC3b increased with disease activity. This finding was replicated with the T2-weighted MRI: T2-relaxation times decreased (as SPIO reduce T2-relaxation times) with disease activity in the cortex and medullas of MRL/lpr mice, but not of control mice. Our findings demonstrate that an MRI contrast agent targeted to glomerular C3b/iC3b/C3d can be used to non-invasively monitor disease activity in GN. Further, therapeutic complement-inhibitors have recently been used in patients with renal disease, and this method could identify patients likely to benefit from complement inhibition.
doi:10.1038/ki.2011.332
PMCID: PMC3375901  PMID: 21956190
CR2-targeted iron oxide nanoparticles; T2-relaxation time; glomerulonephritis
15.  Silibinin Prevents Lung Tumorigenesis in Wild-type but not in iNOS−/− Mice: Potential of Real-time Micro-CT in Lung Cancer Chemoprevention Studies 
Purpose
Sustained nitric oxide (NO) generation positively correlates with lung cancer development and progression. Herein, we genetically confirmed this role of iNOS and evaluated the chemopreventive efficacy of silibinin in carcinogen-treated B6/129 wild-type (WT) and iNOS−/− mice.
Experimental Design
Male B6/129-Nos2tm1Lau (iNOS −/−) and B6/129PF2 WT mice were injected i.p. with 1mg/g body weight urethane once weekly for 7 consecutive weeks, followed by silibinin gavage (742mg/kg body weight) for 5 days/week for 18 weeks.
Results
Quantification of micro-CT data in real-time showed that silibinin significantly decreases urethane-induced tumor number and size in WT mice, consistent with measurements made ex vivo at study termination. Genetic ablation of iNOS decreased urethane-induced tumor multiplicity by 87% (P<0.001) compared to WT mice. Silibinin decreased tumor multiplicity by 71% (P<0.01) in WT mice, but did not show any such considerable effect in iNOS−/− mice. Tumors from WT mice expressed more iNOS (P<0.01) but almost similar eNOS and nNOS than those in silibinin-treated mice. In these tumors, silibinin moderately (P<0.01) inhibited cell proliferation but strongly (P<0.01) reduced the number of newly formed nestin-positive microvessels. Silibinin decreased VEGFR2 level, and STAT3 and NF-κB activation in tumors.
Conclusions
The lack of effect of silibinin in iNOS−/− mice suggests that silibinin exerts most of its chemopreventive and angiopreventive effects through its inhibition of iNOS expression in lung tumors. Our results support iNOS as a potential target for controlling lung cancer, and demonstrate the value of real-time non-invasive micro-CT imaging modality for evaluating the efficacy of lung cancer chemopreventive agents.
doi:10.1158/1078-0432.CCR-10-2290
PMCID: PMC3066181  PMID: 21148748
Chemoprevention; lung cancer; micro-CT; silibinin; iNOS
16.  Distinct patterns of kidney and liver cyst growth in pkd2(WS25/−) mice 
Nephrology Dialysis Transplantation  2010;25(11):3496-3504.
Background. Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease that results in the development of cystic kidneys and liver. Pkd2(WS25/−) mice are a key genetic mouse model of human ADPKD that recapitulate the ‘molecular recessive’ nature of human ADPKD. Providing the foundation for future long-term studies, the present work documents distinct patterns of long-term cyst growth in the kidneys and liver of male and female pkd2(WS25/−) mice.
Methods. Gravimetric measurements documented the progression of kidney and liver growth in male and female pkd2(WS25/−) mice over 12 months. A fast imaging with steady-state precision–magnetic resonance imaging (FISP-MRI) technique to measure kidney and liver organ and cyst volumes was optimized and validated. Longitudinal FISP-MRI analyses of changes in cyst volumes were performed in pkd2(WS25/−) mice over 15 months.
Results. Male and female pkd2(WS25/−) mice had significant increases in kidney weights after 4 months of age. The progression of kidney growth was minimal after 4 months of age. Liver cyst growth in male pkd2(WS25/−) mice was minimal after 4 months of age but showed an accelerated rate of growth after 8 months of age. Female pkd2(WS25/−) mice also showed accelerated growth but this was delayed in time when compared with male pkd2(WS25/−) mice.
Conclusions. Pkd2(WS25/−) mice are a genetic mouse model that recapitulates the early phenotypic characteristics of human ADPKD kidney cystogenesis. Male pkd2(WS25/−) mice consistently display a late progression in liver growth that is seen in clinically impacted livers of human ADPKD patients.
doi:10.1093/ndt/gfq195
PMCID: PMC2980992  PMID: 20388629
Autosomal dominant polycystic kidney disease (ADPKD); Fast imaging with steady-state precision (FISP); Magnetic resonance imaging (MRI)
17.  IMMUNOSUPPRESSANT NEUROTOXICITY IN RAT BRAIN MODELS: OXIDATIVE STRESS AND CELLULAR METABOLISM 
Chemical research in toxicology  2010;23(3):608-619.
Co-administration of the calcineurin inhibitor cyclosporine (CsA) and the mTOR inhibitors sirolimus (SRL) or everolimus (RAD) increases efficacy of immunosuppression after organ transplantation. Neurotoxicity of CsA is a major clinical problem. Our goal was to assess the effects of CsA, SRL and RAD on the brain cell metabolism.
The studies included the comparison of immunosuppressant-mediated effects on glucose metabolism, energy production and reactive oxygen species (ROS) formation in perfused rat brain slices, primary rat astrocytes and C6-glioma cells.
In brain slices and astrocytes, CsA inhibited Krebs cycle metabolism, while activating anaerobic glycolysis most likely to compensate for the inhibition of mitochondrial energy production. SRL and RAD inhibited cytosolic glycolysis, but did not cause changes in mitochondrial energy production. CsA+SRL inhibited Krebs cycle and glycolysis, thus reducing the ability of the cell to compensate for the negative effects of CsA on mitochondrial nucleoside triphosphate synthesis. In contrast to SRL at the concentrations tested, RAD reduced the CsA-induced ROS formation and antagonized CsA-induced effects on glucose and energy metabolism. Surprisingly, in C6 cells, SRL and RAD exposure resulted in high ROS concentrations without significant impairment of cell metabolism.
Our results suggested that SRL enhances CsA-induced ROS formation and negative metabolic effects in brain cells, while RAD seems to antagonize the CsA effects. However, the three models showed different metabolic responses when challenged with the study drugs. In contrast to SRL, RAD enhances ROS formation in C6 glioma cells, but has only minor effects on normal rat brain tissue.
doi:10.1021/tx900351q
PMCID: PMC2841482  PMID: 20148532
reactive oxygen species; brain metabolism; cyclosporine; sirolimus; everolimus; immunosuppressant; nuclear magnetic resonance spectroscopy (NMR)
18.  Development and validation of a sensitive assay for the quantification of imatinib using LC/LC-MS/MS in human whole blood and cell culture 
Biomedical chromatography : BMC  2009;23(12):1251-1258.
We developed and validated a semi-automated LC/LC-MS/MS assay for the quantification of imatinib in human whole blood and leukemia cells. After protein precipitation, samples were injected into the HPLC system and trapped onto the enrichment column (flow 5 mL/min); extracts were back-flushed onto the analytical column. Ion transitions [M + H]+ of imatinib (m/z = 494.3 → 394.3) and its internal standard trazodone (372.5 → 176.3) were monitored. The range of reliable response was 0.03–75 ng/mL. The inter-day precisions were: 8.4% (0.03 ng/mL), 7.2% (0.1 ng/mL), 6.5% (1 ng/mL), 8.2% (10 ng/mL) and 4.3% (75 ng/mL) with no interference from ion suppression. Autosampler stability was 24 hs and samples were stable over three freeze–thaw cycles. This semi-automated method is simple with only one manual step, uses a commercially available internal standard, and has proven to be robust in larger studies.
doi:10.1002/bmc.1247
PMCID: PMC3044679  PMID: 19517424
imatinib; gleevec; liquid chromatography; sample enrichment; tandem mass spectrometry; LC/LC-MS/MS
19.  Phase I Pharmacologic and Biologic Study of Ramucirumab (IMC-1121B), a Fully Human Immunoglobulin G1 Monoclonal Antibody Targeting the Vascular Endothelial Growth Factor Receptor-2 
Journal of Clinical Oncology  2010;28(5):780-787.
Purpose
To evaluate the safety, maximum-tolerated dose (MTD), pharmacokinetics (PKs), pharmacodynamics, and preliminary anticancer activity of ramucirumab (IMC-1121B), a fully human immunoglobulin G1 monoclonal antibody targeting the vascular endothelial growth factor receptor (VEGFR)-2.
Patients and Methods
Patients with advanced solid malignancies were treated once weekly with escalating doses of ramucirumab. Blood was sampled for PK studies throughout treatment. The effects of ramucirumab on circulating vascular endothelial growth factor-A (VEGF-A), soluble VEGFR-1 and VEGFR-2, tumor perfusion, and vascularity using dynamic contrast-enhanced magnetic resonance imaging were assessed.
Results
Thirty-seven patients were treated with 2 to 16 mg/kg of ramucirumab. After one patient each developed dose-limiting hypertension and deep venous thrombosis at 16 mg/kg, the next lower dose (13 mg/kg) was considered the MTD. Nausea, vomiting, headache, fatigue, and proteinuria were also noted. Four (15%) of 27 patients with measurable disease had a partial response (PR), and 11 (30%) of 37 patients had either a PR or stable disease lasting at least 6 months. PKs were characterized by dose-dependent elimination and nonlinear exposure consistent with saturable clearance. Mean trough concentrations exceeded biologically relevant target levels throughout treatment at all dose levels. Serum VEGF-A increased 1.5 to 3.5 times above pretreatment values and remained in this range throughout treatment at all dose levels. Tumor perfusion and vascularity decreased in 69% of evaluable patients.
Conclusion
Objective antitumor activity and antiangiogenic effects were observed over a wide range of dose levels, suggesting that ramucirumab may have a favorable therapeutic index in treating malignancies amenable to VEGFR-2 inhibition.
doi:10.1200/JCO.2009.23.7537
PMCID: PMC2834394  PMID: 20048182
20.  Human placental metabolic adaptation to chronic hypoxia, high altitude: hypoxic preconditioning 
We have previously demonstrated placentas from laboring deliveries at high altitude have lower binding of hypoxia-inducible transcription factor (HIF) to DNA than those from low altitude. It has recently been reported that labor causes oxidative stress in placentas, likely due to ischemic hypoxic insult. We hypothesized that placentas of high-altitude residents acquired resistance, in the course of their development, to oxidative stress during labor. Full-thickness placental tissue biopsies were collected from laboring vaginal and nonlaboring cesarean-section term (37–41 wk) deliveries from healthy pregnancies at sea level and at 3,100 m. After freezing in liquid nitrogen within 5 min of delivery, we quantified hydrophilic and lipid metabolites using 31P and 1H NMR metabolomics. Metabolic markers of oxidative stress, increased glycolysis, and free amino acids were present in placentas following labor at sea level, but not at 3,100 m. In contrast, at 3,100 m, the placentas were characterized by the presence of concentrations of stored energy potential (phosphocreatine), antioxidants, and low free amino acid concentrations. Placentas from pregnancies at sea level subjected to labor display evidence of oxidative stress. However, laboring placentas at 3,100 m have little or no oxidative stress at the time of delivery, suggesting greater resistance to ischemia-reperfusion. We postulate that hypoxic preconditioning might occur in placentas that develop at high altitude.
doi:10.1152/ajpregu.00383.2009
PMCID: PMC2806207  PMID: 19864339
oxidative stress; labor; pregnancy; antioxidants; protein metabolism
21.  Silibinin Feeding Alters the Metabolic Profile in TRAMP Prostatic Tumors: 1H-NMRS-Based Metabolomics Study 
Cancer research  2009;69(9):3731-3735.
Herein, we evaluated for the first time silibinin efficacy on prostate cancer (PCa) metabolism in transgenic adenocarcinoma of the mouse prostate (TRAMP) model utilizing quantitative high-resolution proton nuclear magnetic resonance spectroscopy (1H-NMRS) metabolomics. Prostate tissues were from mice fed control or silibinin diet for 20 weeks. Comparative metabolic profiling indicated that antitumor effect of silibinin is accompanied by alteration in metabolic profile of TRAMP prostatic tumors as indicated by 6 fold (P=0.016) increase in glucose content and 48% (P=0.015) reduction in lactate levels. Increase in citrate utilization by prostate tissue also reversed with silibinin, as indicated by 3-fold (P=0.01) increase in citrate levels in silibinin-fed group. Also, 61% and 50% (P<0.01) decrease in cholesterol and phosphatidylcholine levels, respectively, was observed with silibinin. These results corroborate our earlier findings regarding PCa chemopreventive potential of silibinin in TRAMP model and warrant additional metabolic profiling in other silibinin-fed PCa tumor model tissues. This will help identify specific metabolic biomarkers altered during silibinin treatment, which when detected in clinical biopsies or non-invasive MRS studies could help monitor silibinin effectiveness against PCa malignancy.
doi:10.1158/0008-5472.CAN-09-0096
PMCID: PMC2679526  PMID: 19366793
22.  Use of nuclear magnetic resonance-based metabolomics in detecting drug resistance in cancer 
Biomarkers in medicine  2009;3(3):289-306.
Cancer cells possess a highly unique metabolic phenotype, which is characterized by high glucose uptake, increased glycolytic activity, decreased mitochondrial activity, low bioenergetic and increased phospholipid turnover. These metabolic hallmarks can be readily assessed by metabolic technologies – either in vitro or in vivo – to monitor responsiveness and resistance to novel targeted drugs, where specific inhibition of cell proliferation (cytostatic effect) occurs rather than direct induction of cell death (cytotoxicity). Using modern analytical technologies in combination with statistical approaches, ‘metabolomics’, a global metabolic profile on patient samples can be established and validated for responders and nonresponders, providing additional metabolic end points. Discovered metabolic end points should be translated into noninvasive metabolic imaging protocols.
doi:10.2217/BMM.09.15
PMCID: PMC2756767  PMID: 20160999
anticancer treatment; cancer biomarkers; choline metabolism; quantitative metabolomics; signal transduction inhibitor; Warburg effect
23.  Clinical Applications of Metabolomics in Oncology: A Review 
Metabolomics, an omic science in systems biology, is the global quantitative assessment of endogenous metabolites within a biological system. Either individually or grouped as a metabolomic profile, detection of metabolites is carried out in cells, tissues, or biofluids by either nuclear magnetic resonance spectroscopy or mass spectrometry. There is potential for the metabolome to have a multitude of uses in oncology, including the early detection and diagnosis of cancer and as both a predictive and pharmacodynamic marker of drug effect. Despite this, there is lack of knowledge in the oncology community regarding metabolomics and confusion about its methodologic processes, technical challenges, and clinical applications. Metabolomics, when used as a translational research tool, can provide a link between the laboratory and clinic, particularly because metabolic and molecular imaging technologies, such as positron emission tomography and magnetic resonance spectroscopic imaging, enable the discrimination of metabolic markers noninvasively in vivo. Here, we review the current and potential applications of metabolomics, focusing on its use as a biomarker for cancer diagnosis, prognosis, and therapeutic evaluation.
doi:10.1158/1078-0432.CCR-08-1059
PMCID: PMC2676437  PMID: 19147747
24.  Activated Akt1 accelerates MMTV-c-ErbB2 mammary tumourigenesis in mice without activation of ErbB3 
Introduction
ErbB2, a member of the epidermal growth factor receptor (EGFR) family, is overexpressed in 20% to 30% of human breast cancer cases and forms oncogenic signalling complexes when dimerised to ErbB3 or other EGFR family members.
Methods
We crossed mouse mammary tumour virus (MMTV)-myr-Akt1 transgenic mice (which express constitutively active Akt1 in the mammary gland) with MMTV-c-ErbB2 transgenic mice to evaluate the role of Akt1 activation in ErbB2-induced mammary carcinoma using immunoblot analysis, magnetic resonance spectroscopy and histological analyses.
Results
Bitransgenic MMTV-c-ErbB2, MMTV-myr-Akt1 mice develop mammary tumours twice as fast as MMTV-c-ErbB2 mice. The bitransgenic tumours were less organised, had more mitotic figures and fewer apoptotic cells. However, many bitransgenic tumours displayed areas of extensive necrosis compared with tumours from MMTV-c-ErbB2 mice. The two tumour types demonstrate dramatically different expression and activation of EGFR family members, as well as different metabolic profiles. c-ErbB2 tumours demonstrate overexpression of EGFR, ErbB2, ErbB3 and ErbB4, and activation/phosphorylation of both ErbB2 and ErbB3, underscoring the importance of the entire EGFR family in ErbB2-induced tumourigenesis. Tumours from bitransgenic mice overexpress the myr-Akt1 and ErbB2 transgenes, but there was dramatically less overexpression and phosphorylation of ErbB3, diminished phosphorylation of ErbB2, decreased level of EGFR protein and undetectable ErbB4 protein. There was also an observable attenuation in a subset of tyrosine-phosphorylated secondary signalling molecules in the bitransgenic tumours compared with c-ErbB2 tumours, but Erk was activated/phosphorylated in both tumour types. Finally, the bitransgenic tumours were metabolically more active as indicated by increased glucose transporter 1 (GLUT1) expression, elevated lactate production and decreased intracellular glucose (suggesting increased glycolysis).
Conclusion
Expression of activated Akt1 in MMTV-c-ErbB2 mice accelerates tumourigenesis with a reduced requirement for signalling through the EGFR family, as well as a reduced requirement for a subset of downstream signaling molecules with a metabolic shift in the tumours from bitransgenic mice. The reduction in signalling downstream of ErbB2 when Akt is activated suggest a possible mechanism by which tumour cells can become resistant to ErbB2-targeted therapies, necessitating therapies that target oncogenic signalling events downstream of ErbB2.
doi:10.1186/bcr2132
PMCID: PMC2575543  PMID: 18700973
25.  N-(3-Hydroxyhexanoyl)-l-Homoserine Lactone Is the Biologically Relevant Quormone That Regulates the phz Operon of Pseudomonas chlororaphis Strain 30-84▿  
Applied and Environmental Microbiology  2007;73(22):7443-7455.
Phenazine production by Pseudomonas fluorescens 2-79 and P. chlororaphis isolates 30-84 and PCL1391 is regulated by quorum sensing through the activator PhzR and acyl-homoserine lactones (acyl-HSLs) synthesized by PhzI. PhzI from P. fluorescens 2-79 produces five acyl-HSLs that include four 3-hydroxy species. Of these, N-(3-hydroxyhexanoyl)-HSL is the biologically relevant ligand for PhzR. The quorum-sensing systems of P. chlororaphis strains 30-84 and PCL1391 have been reported to produce and respond to N-(hexanoyl)-HSL. These differences were of interest since PhzI and PhzR of strain 2-79 share almost 90% sequence identity with orthologs from strains 30-84 and PCL1391. In this study, as assessed by thin-layer chromatography, the three strains produce almost identical complements of acyl-HSLs. The major species produced by P. chlororaphis 30-84 were identified by mass spectrometry as 3-OH-acyl-HSLs with chain lengths of 6, 8, and 10 carbons. Heterologous bacteria expressing cloned phzI from strain 30-84 produced the four 3-OH acyl-HSLs in amounts similar to those seen for the wild type. Strain 30-84, but not strain 2-79, also produced N-(butanoyl)-HSL. A second acyl-HSL synthase of strain 30-84, CsaI, is responsible for the synthesis of this short-chain signal. Strain 30-84 accumulated N-(3-OH-hexanoyl)-HSL to the highest levels, more than 100-fold greater than that of N-(hexanoyl)-HSL. In titration assays, PhzR30-84 responded to both N-(3-OH-hexanoyl)- and N-(hexanoyl)-HSL with equal sensitivities. However, only the 3-OH-hexanoyl signal is produced by strain 30-84 at levels high enough to activate PhzR. We conclude that strains 2-79, 30-84, and PCL1391 use N-(3-OH-hexanoyl)-HSL to activate PhzR.
doi:10.1128/AEM.01354-07
PMCID: PMC2168216  PMID: 17921283

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