Ray R. Zhang

MicroRNA-217 Promotes Ethanol-induced Fat Accumulation in Hepatocytes by Down-regulating SIRT1

Background: To investigate the role of miR-217 in mediating ethanol action in the liver. Results: miR-217 promotes ethanol-induced fat accumulation through down-regulating SIRT1 in vitro and in vivo. Conclusion: miR-217 is a specific target of ethanol action in the liver and contributes to development of alcoholic fatty liver. Significance: miR-217 may present as a potential therapeutic target for treating human alcoholic fatty liver disease. Ethanol-mediated inhibition of hepatic sirtuin 1 (SIRT1) plays a crucial role in the pathogenesis of alcoholic fatty liver disease. Here, we investigated the underlying mechanisms of this inhibition by identifying a new hepatic target of ethanol action, microRNA-217 (miR-217). The role of miR-217 in the regulation of the effects of ethanol was investigated in cultured mouse AML-12 hepatocytes and in the livers of chronically ethanol-fed mice. In AML-12 hepatocytes and in mouse livers, chronic ethanol exposure drastically and specifically induced miR-217 levels and caused excess fat accumulation. Further studies revealed that overexpression of miR-217 in AML-12 cells promoted ethanol-mediated impairments of SIRT1 and SIRT1-regulated genes encoding lipogenic or fatty acid oxidation enzymes. More importantly, miR-217 impairs functions of lipin-1, a vital lipid regulator, in hepatocytes. Taken together, our novel findings suggest that miR-217 is a specific target of ethanol action in the liver and may present as a potential therapeutic target for treating human alcoholic fatty liver disease.

Regulation of hepatic lipin‐1 by ethanol: Role of AMP‐activated protein kinase/sterol regulatory element‐binding protein 1 signaling in mice

Lipin‐1 is a protein that exhibits dual functions as a phosphatidic acid phosphohydrolase enzyme in the triglyceride synthesis pathways and a transcriptional coregulator. Our previous studies have shown that ethanol causes fatty liver by activation of sterol regulatory element‐binding protein 1 (SREBP‐1) and inhibition of hepatic AMP‐activated protein kinase (AMPK) in mice. Here, we tested the hypothesis that AMPK‐SREBP‐1 signaling may be involved in ethanol‐mediated up‐regulation of lipin‐1 gene expression. The effects of ethanol on lipin‐1 were investigated in cultured hepatic cells and in the livers of chronic ethanol‐fed mice. Ethanol exposure robustly induced activity of a mouse lipin‐1 promoter, promoted cytoplasmic localization of lipin‐1, and caused excess lipid accumulation, both in cultured hepatic cells and in mouse livers. Mechanistic studies showed that ethanol‐mediated induction of lipin‐1 gene expression was inhibited by a known activator of AMPK or overexpression of a constitutively active form of AMPK. Importantly, overexpression of the processed nuclear form of SREBP‐1c abolished the ability of 5‐aminoimidazole‐4‐carboxamide ribonucleoside to suppress ethanol‐mediated induction of lipin‐1 gene‐expression level. Chromatin immunoprecipitation assays further revealed that ethanol exposure significantly increased the association of acetylated histone H3 at lysine 9 with the SRE‐containing region in the promoter of the lipin‐1 gene. Conclusion: In conclusion, ethanol‐induced up‐regulation of lipin‐1 gene expression is mediated through inhibition of AMPK and activation of SREBP‐1. (Hepatology 2012)

Volumetric modulated arc planning for lung stereotactic body radiotherapy using conventional and unflattened photon beams: a dosimetric comparison with 3D technique

PurposeFrequently, three-dimensional (3D) conformal beams are used in lung cancer stereotactic body radiotherapy (SBRT). Recently, volumetric modulated arc therapy (VMAT) was introduced as a new treatment modality. VMAT techniques shorten delivery time, reducing the possibility of intrafraction target motion. However dose distributions can be quite different from standard 3D therapy. This study quantifies those differences, with focus on VMAT plans using unflattened photon beams.MethodsA total of 15 lung cancer patients previously treated with 3D or VMAT SBRT were randomly selected. For each patient, non-coplanar 3D, coplanar and non-coplanar VMAT and flattening filter free VMAT (FFF-VMAT) plans were generated to meet the same objectives with 50 Gy covering 95% of the PTV. Two dynamic arcs were used in each VMAT plan. The couch was set at ± 5° to the 0° straight position for the two non-coplanar arcs. Pinnacle version 9.0 (Philips Radiation Oncology, Fitchburg WI) treatment planning system with VMAT capabilities was used. We analyzed the conformity index (CI), which is the ratio of the total volume receiving at least the prescription dose to the target volume receiving at least the prescription dose; the conformity number (CN) which is the ratio of the target coverage to CI; and the gradient index (GI) which is the ratio of the volume of 50% of the prescription isodose to the volume of the prescription isodose; as well as the V20, V5, and mean lung dose (MLD). Paired non-parametric analysis of variance tests with post-tests were performed to examine the statistical significance of the differences of the dosimetric indices.ResultsDosimetric indices CI, CN and MLD all show statistically significant improvement for all studied VMAT techniques compared with 3D plans (p < 0.05). V5 and V20 show statistically significant improvement for the FFF-VMAT plans compared with 3D (p < 0.001). GI is improved for the FFF-VMAT and the non-coplanar VMAT plans (p < 0.01 and p < 0.05 respectively) while the coplanar VMAT plans do not show significant difference compared to 3D plans. Dose to the target is typically more homogeneous in FFF-VMAT plans. FFF-VMAT plans require more monitor units than 3D or non-coplanar VMAT ones.ConclusionBesides the advantage of faster delivery times, VMAT plans demonstrated better conformity to target, sharper dose fall-off in normal tissues and lower dose to normal lung than the 3D plans for lung SBRT. More monitor units are often required for FFF-VMAT plans.

Beyond the margins: real-time detection of cancer using targeted fluorophores

Over the past two decades, synergistic innovations in imaging technology have resulted in a revolution in which a range of biomedical applications are now benefiting from fluorescence imaging. Specifically, advances in fluorophore chemistry and imaging hardware, and the identification of targetable biomarkers have now positioned intraoperative fluorescence as a highly specific real-time detection modality for surgeons in oncology. In particular, the deeper tissue penetration and limited autofluorescence of near-infrared (NIR) fluorescence imaging improves the translational potential of this modality over visible-light fluorescence imaging. Rapid developments in fluorophores with improved characteristics, detection instrumentation, and targeting strategies led to the clinical testing in the early 2010s of the first targeted NIR fluorophores for intraoperative cancer detection. The foundations for the advances that underline this technology continue to be nurtured by the multidisciplinary collaboration of chemists, biologists, engineers, and clinicians. In this Review, we highlight the latest developments in NIR fluorophores, cancer-targeting strategies, and detection instrumentation for intraoperative cancer detection, and consider the unique challenges associated with their effective application in clinical settings.

Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice

Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis.

Fluorescent cancer-selective alkylphosphocholine analogs for intraoperative glioma detection

BACKGROUND 5-Aminolevulinic acid (5-ALA)-induced tumor fluorescence aids brain tumor resections but is not approved for routine use in the United States. We developed and describe testing of 2 novel fluorescent, cancer-selective alkylphosphocholine analogs, CLR1501 (green) and CLR1502 (near infrared), in a proof-of-principle study for fluorescence-guided glioma surgery. OBJECTIVE To demonstrate that CLR1501 and CLR1502 are cancer cell-selective fluorescence agents in glioblastoma models and to compare tumor-to-normal brain (T:N) fluorescence ratios with 5-ALA. METHODS CLR1501, CLR1502, and 5-ALA were administered to mice with magnetic resonance imaging-verified orthotopic U251 glioblastoma multiforme- and glioblastoma stem cell-derived xenografts. Harvested brains were imaged with confocal microscopy (CLR1501), the IVIS Spectrum imaging system (CLR1501, CLR1502, and 5-ALA), or the Fluobeam near-infrared fluorescence imaging system (CLR1502). Imaging and quantitative analysis of T:N fluorescence ratios were performed. RESULTS Excitation/emission peaks are 500/517 nm for CLR1501 and 760/778 nm for CLR1502. The observed T:N ratio for CLR1502 (9.28±1.08) was significantly higher (P<.01) than for CLR1501 (3.51±0.44 on confocal imaging; 7.23±1.63 on IVIS imaging) and 5-ALA (4.81±0.92). Near-infrared Fluobeam CLR1502 imaging in a mouse xenograft model demonstrated high- contrast tumor visualization compatible with surgical applications. CONCLUSION CLR1501 (green) and CLR1502 (near infrared) are novel tumor-selective fluorescent agents for discriminating tumor from normal brain. CLR1501 exhibits a tumor-to-brain fluorescence ratio similar to that of 5-ALA, whereas CLR1502 has a superior tumor-to-brain fluorescence ratio. This study demonstrates the potential use of CLR1501 and CLR1502 in fluorescence-guided tumor surgery.

Analysis of Cancer-Targeting Alkylphosphocholine Analogue Permeability Characteristics Using a Human Induced Pluripotent Stem Cell Blood-Brain Barrier Model.

Cancer-targeting alkylphosphocholine (APC) analogues are being clinically developed for diagnostic imaging, intraoperative visualization, and therapeutic applications. These APC analogues derived from chemically synthesized phospholipid ethers were identified and optimized for cancer-targeting specificity using extensive structure-activity studies. While they strongly label human brain cancers associated with disrupted blood-brain barriers (BBB), APC permeability across intact BBB remains unknown. Three of our APC analogues, CLR1404 (PET radiotracer), CLR1501 (green fluorescence), and CLR1502 (near-infrared fluorescence), were tested for permeability across a BBB model composed of human induced pluripotent stem cell-derived brain microvascular endothelial cells (iPSC-derived BMECs). This in vitro BBB system has reproducibly consistent high barrier integrity marked by high transendothelial electrical resistance (TEER > 1500 Ω-cm(2)) and functional expression of drug efflux transporters. The radioiodinated and fluorescent APC analogues demonstrated fairly low permeability across the iPSC-BMEC (35 ± 5.7 (CLR1404), 54 ± 3.2 (CLR1501), and 26 ± 4.9 (CLR1502) × 10(-5) cm/min) compared with BBB-impermeable sucrose (13 ± 2.5) and BBB-permeable diazepam (170 ± 29). Only the fluorescent APC analogues (CLR1501, CLR1502) underwent BCRP and MRP polarized drug efflux transport in the brain-to-blood direction of the BBB model, and this efflux can be specifically blocked with pharmacological inhibition. None of the tested APC analogues appeared to undergo substantial P-gp transport. Limited permeability of the APC analogues across an intact BBB into normal brain likely contributes to the high tumor to background ratios observed in initial human trials. Moreover, addition of fluorescent moieties to APCs resulted in greater BMEC efflux via MRP and BCRP, and may affect fluorescence-guided applications. Overall, the characterization of APC analogue permeability across human BBB is significant for advancing future brain tumor-targeted applications of these agents.

Mutational analysis reveals the origin and therapy-driven evolution of recurrent glioma.

U niversity of California, San Francisco researchers recently reported in Science the mutational profiling of 23 initially low-grade gliomas (LGGs) and associated recurrent tumors and profiled a subset of recurrent tumors in temozolomide-treated patients. Three key findings with potentially important clinical implications for LGG management were demonstrated: (1) LGGs and paired recurrent tumors are highly divergent and often only share a few early mutations, thus partly explaining their differential therapeutic responses; (2) mutant isocitrate dehydrogenase-1 (IDH1) may be critical for LGG formation and is a potential therapeutic target; and (3) temozolomide therapy may contribute to malignant transformation and affect clinical outcome. Johnson et al determined the genetic profiles of LGGs and associated recurrences. Mutations that are shared or exclusive to the initial tumor or recurrences were characterized for each of 23 initial tumors and recurrences found up to 11 years later, and tumor phylogenies were mapped via evolutionary analyses. Overall, the paired tumors shared a significant percentage of mutations exclusive to the initial tumors in 43% of cases. These findings suggest that gliomas and recurrent tumors share early tumorigenic mutational origins but diverge afterward in tumorigenesis. LGG sequencing also revealed that an IDH1 mutation was present and remained unchanged in all paired tumors, highlighting IDH1 as a potentially critical LGG driver mutation. IDH1 mutants produce 2-hydroxyglutarate R-enantiomer, an interesting tumor metabolism product that inhibits histone enzymes and alters gene expression. Recent work highlights a possible LGG-selective therapeutic opportunity because inhibitors of IDH-1 mutant activity selectively reduce tumor growth rate and stimulate glioma differentiation. This work also reported the effects of temozolomide therapy on mutational profiles of recurrent gliomas, especially given that temozolomide use in LGG therapy is controversial. Mutational profiles of paired tumors in 10 temozolomide-treated patients were determined. Recurrent tumors from 6 of the 10 patients exhibited hypermutated phenotypes after temozolomide therapy, carryingmanymoremutations per million base pairs compared with their initial tumors. The hypermutated state is likely caused by the propensity of temozolomide tomutate and compromise DNA mismatch repair pathways. Additionally, the authors characterized the unique hypermutated signature and found significant association with high-grade glioma signaling pathways such as retinoblastoma and protein kinase B–mammalian target of rapamycin signaling (Figure, B). These results suggest that temozolomide therapy may contribute to malignant transformation of LGGs, and further studies are needed to determine whether this alters clinical outcomes.

Is wax equivalent to tissue in electron conformal therapy planning? A Monte Carlo study of material approximation introduced dose difference

With CT‐based Monte Carlo (MC) dose calculations, material composition is often assigned based on the standard Hounsfield unit ranges. This is known as the density threshold method. In bolus electron conformal therapy (BolusECT), the bolus material, machineable wax, would be assigned as soft tissue and the electron density is assumed equivalent to soft tissue based on its Hounsfield unit. This study investigates the dose errors introduced by this material assignment. BEAMnrc was used to simulate electron beams from a Trilogy accelerator. SPRRZnrc was used to calculate stopping power ratios (SPR) of tissue to wax, SPRwaxtissue, and tissue to water, SPRwatertissue, for 6, 9, 12, 15, and 18 MeV electron beams, of which 12 and 15 MeV beams are the most commonly used energies in BolusECT. DOSXYZnrc was applied in dose distribution calculations in a tissue phantom with either flat wax slabs of various thicknesses or a wedge‐shaped bolus on top. Dose distribution for two clinical cases, a chest wall and a head and neck, were compared with the bolus material treated as wax or tissue. The SPRwaxtissue values for 12 and 15 MeV beams are between 0.935 and 0.945, while the SPRwatertissue values are between 0.990 and 0.991. For a 12 MeV beam, the dose in tissue immediately under the bolus is overestimated by 2.5% for a 3 cm bolus thickness if the wax bolus is treated as tissue. For 15 MeV beams, the error is 1.4%. However, in both clinical cases the differences in the PTV DVH is negligible. Due to stopping power differences, dose differences of up to 2.5% are observed in MC simulations if the bolus material is misassigned as tissue in BolusECT dose calculations. However, for boluses thinner than 2 cm that are more likely encountered in practice, the error is within clinical tolerance. PACS numbers: 87.55.km, 87.56.ng

364 Creation of a Dual-Labeled Cancer-Targeting Alkylphosphocholine Analog for Dual Modality Quantitative Positron Emission Tomography and Intraoperative Tumor Visualization.

INTRODUCTION Near-infrared fluorescence (NIRF) imaging is a promising noninvasive, real-time, sensitive, high-resolution modality for cancer detection due to high tissue penetrance and low background autofluorescence. However, accurate fluorescence quantitation is problematic due to photon scattering by tissues, photon absorption by pigments and water, and static quenching of close-proximity fluorophores. CLR1502 is a NIRF-labeled cancer-specific alkylphosphocholine analog with selective uptake and retention in over 60 preclinical cancer models (including glioblastoma cancer stem cell and brain met models), and approaching clinical trials. To address many of NIRF limitations, we synthesized Iodo-1502, a novel dual-labeled (DL) positron emission tomography (PET) NIRF agent by adding radiolabeled iodine to CLR1502, enabling simultaneous whole-body cancer staging and intraoperative tumor illumination with a single agent. Initial preclinical in vivo studies of Iodo-1502 will be presented. METHODS Standard organic synthetic chemistry and biology methods. Multiple human cancer lines were implanted for tumor xenografts in athymic nude mice. Once tumors reached 3 mm diameter, IV injection of dual-labeled Iodo-1502 and animal imaging up to 120 hours with IVIS Spectrum (excitation = 745 nm, emission = 800 nm) or small-animal Siemens Inveon Hybrid PET/CT scanner was done. Tumors were then removed for similar ex vivo quantitative IVIS and PET studies. RESULTS Like the NIRF CLR1502 predecessor, the new dual-labeled Iodo-1502 showed striking tumor selectivity and retention for at least 6 days postinjection in xenograft models. Moreover, ex vivo quantitation showed excellent correlation of fluorescence and PET imaging. CONCLUSION This new agent overcomes many NIRF limitations, and enables broad-spectrum cancer-specific intraoperative tumor margin illumination and whole-body quantitative PET imaging.