Darshini Kuruppu

Epidermal Growth Factor Gene Functional Polymorphism and the Risk of Hepatocellular Carcinoma in Patients With Cirrhosis

CONTEXT Overexpression of epidermal growth factor (EGF) in the liver induces transformation to hepatocellular carcinoma in animal models. Polymorphisms in the EGF gene modulate EGF levels. OBJECTIVE To assess the relationship among human EGF gene single-nucleotide polymorphism, EGF expression, and risk of hepatocellular carcinoma. DESIGN, SETTING, AND PARTICIPANTS Molecular mechanisms linking the 61*G allele polymorphism to EGF expression were examined in human hepatocellular carcinoma cell lines and human liver tissue. A case-control study involving 207 patients with cirrhosis was conducted at the Massachusetts General Hospital (1999-2006) and a validation case-control study involving 121 patients with cirrhosis was conducted at Hôpital Paul Brousse (1993-2006). Restriction fragment-length polymorphism was used to determine the EGF gene polymorphism genotype. Logistic regression analysis was used to assess the association between the EGF polymorphism and hepatocellular carcinoma risk. MAIN OUTCOME MEASURES Mechanisms by which the EGF gene polymorphism modulates EGF levels and associations among EGF gene polymorphism, EGF levels, and hepatocellular carcinoma. RESULTS Transcripts from the EGF 61*G allele exhibited more than a 2-fold longer half-life than those from the 61*A allele, and EGF secretion was 2.3-fold higher in G/G hepatocellular carcinoma cell lines than A/A cell lines. Serum EGF levels were 1.8-fold higher in G/G patients than A/A patients, and liver EGF levels were 2.4-fold higher in G/G patients than A/A patients. Among the 207 patients with cirrhosis in the Massachusetts study population, 59 also had hepatocellular carcinoma. Analysis of the distribution of allelic frequencies revealed that there was a 4-fold odds of hepatocellular carcinoma in G/G patients compared with A/A patients in the Massachusetts study population (odds ratio, 4.0; 95% confidence interval [CI], 1.6-9.6; P = .002). Logistic regression analysis demonstrated that the number of copies of G was significantly associated with hepatocellular carcinoma after adjusting for age, sex, race, etiology, and severity of cirrhosis (G/G or A/G vs A/A; hazard ratio, 3.49; 95% CI, 1.29-9.44; P = .01). The significant association was validated in the French patients with alcoholic cirrhosis and hepatocellular carcinoma. CONCLUSION The EGF gene polymorphism genotype is associated with risk for development of hepatocellular carcinoma in liver cirrhosis through modulation of EGF levels.

Characterization of an animal model of hepatic metastasis

The experimental study of possible therapies for control of the growth of liver metastases requires the availability of a model which is technically feasible and appears to exhibit growth characteristics similar to human tumours. We report on the development of an intrasplenic injection model of liver metastases, and describe the histology, growth pattern and blood flow demonstrated by light microscopy, stereology and laser Doppler flowmetry. The hepatic metastases were induced in mice by intrasplenic injection of dimethylhydrazine (DMH) induced primary colonic carcinoma cells (106 cells in 1 mL). The growth and development of metastases was studied over a period of 3 weeks at predetermined time points. Tumour cells were visible in the hepatic sinusoids by day 7 by light microscopy. Macroscopically visible tumours with a diameter of 0.18 ± 0.02 cm (mean ± s.d.) were seen by day 10. By this time the tumours had derived a blood supply from the hepatic sinusoids adjacent to the tumour periphery. With further vascularization the tumours reached a diameter of 0.96 ± 0.50 cm by day 22. Metastatic growth was quantitated by stereological analysis of tumour volume in relation to non‐diseased hepatic tissue. Normal mouse liver had a mean volume of 1.13 ± 0.14 cm3. Tumour growth occurred in three phases. During the initial slow phase the volume of metastases increased from 0.03 ± 0.02 cm3 at day 10 to 0.22 ± 0.24 cm3 by day 16. Rapid tumour growth, occurring over the next 3 days, constituted the intermediate phase with metastatic volume reaching 1.21 ± 0.74 cm3 by day 19 (P= 0.0003 compared with day 16). This growth was followed by a plateau phase when the metastatic volume was 1.40 ± 0.55 cm3 at day 22. The volume of total liver and of tumour necrosis followed a similar growth pattern. A necrotic tumour volume of 0.004 ± 0.006 cm3 first seen on day 10 increased to 0.05 ± 0.06 cm3 by day 16, and to 0.25 ± 0.20 cm3 by day 22 (P=0.0022 compared with day 16). The blood flow in metastases measured by laser Doppler flowmetry was lower compared to the non‐diseased liver. Tumour blood flow, expressed as a percentage of normal liver blood flow, was 63.31 ± 26.28% at day 10 and diminished to 27.91 ± 8.99% by day 22, with an increase in tumour size and age. The decrease in flow was significant between days 13 and 16 (P= 0.0015). This intrasplenic mouse model of metastases is reproducible and should prove useful in the study of treatment of hepatic metastases.

A short duration of high-fat diet induces insulin resistance and predisposes to adverse left ventricular remodeling after pressure overload

Insulin resistance is an increasingly prevalent condition in humans that frequently clusters with disorders characterized by left ventricular (LV) pressure overload, such as systemic hypertension. To investigate the impact of insulin resistance on LV remodeling and functional response to pressure overload, C57BL6 male mice were fed a high-fat (HFD) or a standard diet (SD) for 9 days and then underwent transverse aortic constriction (TAC). LV size and function were assessed in SD- and HFD-fed mice using serial echocardiography before and 7, 21, and 28 days after TAC. Serial echocardiography was also performed on nonoperated SD- and HFD-fed mice over a period of 6 wk. LV perfusion was assessed before and 7 and 28 days after TAC. Nine days of HFD induced systemic and myocardial insulin resistance (assessed by myocardial 18F-fluorodeoxyglucose uptake), and myocardial perfusion response to acetylcholine was impaired. High-fat feeding for 28 days did not change LV size and function in nonbanded mice; however, TAC induced greater hypertrophy, more marked LV systolic and diastolic dysfunction, and decreased survival in HFD-fed compared with SD-fed mice. Compared with SD-fed mice, myocardial perfusion reserve was decreased 7 days after TAC, and capillary density was decreased 28 days after TAC in HFD-fed mice. A short duration of HFD induces insulin resistance in mice. These metabolic changes are accompanied by increased LV remodeling and dysfunction after TAC, highlighting the impact of insulin resistance in the development of pressure-overload-induced heart failure.

Variable Inhibition of Thrombospondin 1 against Liver and Lung Metastases through Differential Activation of Metalloproteinase ADAMTS1

Metastasis relies on angiogenesis for tumor expansion. Tumor angiogenesis is restrained by a variety of endogenous inhibitors, including thrombospondin 1 (TSP1). The principal antiangiogenic activity of TSP1 resides in a domain containing three TSP1 repeats (3TSR), and TSP1 cleavage is regulated, in part, by the metalloproteinase ADAMTS1. In this study, we examined the role of TSP1 and ADAMTS1 in controlling metastatic disease in the liver and lung. TSP1 overexpression inhibited metastatic growth of colon or renal carcinoma cells in liver but not lung. Metastatic melanoma in liver grew more rapidly in Tsp1-null mice compared with controls, whereas in lung grew similarly in Tsp1-null mice or controls. Recombinant TSP1 was cleaved more efficiently in lysates from liver than lung. ADAMTS1 inhibition by neutralizing antibody, small interfering RNA, or genetic deletion abrogated cleavage activity. To confirm that lack of cleavage of TSP1 ablated its antiangiogenic function in the lung, we generated colon cancer cells stably secreting only the 3TSR domain and found that they inhibited formation of both liver and lung metastases. Collectively, our results indicate that the antiangiogenic activity of TSP1 is differentially regulated by ADAMTS1 in the liver and lung, emphasizing the concept that regulation of angiogenesis is varied in different tissue environments.

Enhanced Binding of Metabotropic Glutamate Receptor Type 5 (mGluR5) PET Tracers in the Brain of Parkinsonian Primates

The interplay between dopamine and glutamate in the basal ganglia regulates critical aspects of motor learning and behavior. Metabotropic glutamate receptors (mGluR) are increasingly regarded as key modulators of neuroadaptation in these circuits, in normal and disease conditions. Using PET, we demonstrate a significant upregulation of mGluR type 5 in the striatum of MPTP-lesioned, parkinsonian primates, providing the basis for therapeutic exploration of mGluR5 antagonists in Parkinson disease.

Microvascular Architecture of Hepatic Metastases in a Mouse Model

Development of effective treatment for hepatic metastases can be initiated by a better understanding of tumour vasculature and blood supply. This study was designed to characterise the microvascular architecture of hepatic metastases and observe the source of contributory blood supply from the host. Metastases were induced in mice by an intrasplenic injection of colon carcinoma cells (106 cells/ml.) Vascularization of tumours was studied over a three week period by scanning electron microscopy of microvascular corrosion casts. Metastatic liver involvement was observed initially within a week post induction, as areas approximately 100 μm in diameter not perfused by the casting resin. On histology these spaces corresponded to tumour cell aggregates. The following weeks highlighted the angiogenesis phase of these tumours as they received a vascular supply from adjacent hepatic sinusoids. Direct sinusoidal supply of metastases was maintained throughout tumour growth. At the tumour periphery most sinusoids were compressed to form a sheath demarcating the tumour from the hepatic vasculature. No direct supply from the hepatic artery or the portal vein was observed. Dilated vessels termed vascular lakes dominated the complex microvascular architecture of the tumours, most tapering as they traversed towards the periphery. Four vascular branching patterns could be identified as true loops, bifurcations and trifurcations, spirals and capillary networks. The most significant observation in this study was the direct sinusoidal supply of metastases, together with the vascular lakes and the peripheral sinusoidal sheaths of the tumour microculature.

Cerebellar neurons possess a vesicular compartment structurally and functionally similar to Glut4-storage vesicles from peripheral insulin-sensitive tissues

The insulin-sensitive isoform of the glucose transporting protein, Glut4, is expressed in fat as well as in skeletal and cardiac muscle and is responsible for the effect of insulin on blood glucose clearance. Recent studies have revealed that Glut4 is also expressed in the brain, although the intracellular compartmentalization and regulation of Glut4 in neurons remains unknown. Using sucrose gradient centrifugation, immunoadsorption and immunofluorescence staining, we have shown that Glut4 in the cerebellum is localized in intracellular vesicles that have the sedimentation coefficient, the buoyant density, and the protein composition similar to the insulin-responsive Glut4-storage vesicles from fat and skeletal muscle cells. In cultured cerebellar neurons, insulin stimulates glucose uptake and causes translocation of Glut4 to the cell surface. Using 18FDG (18fluoro-2-deoxyglucose) positron emission tomography, we found that physical exercise acutely increases glucose uptake in the cerebellum in vivo. Prolonged physical exercise increases expression of the Glut4 protein in the cerebellum. Our results suggest that neurons have a novel type of translocation-competent vesicular compartment which is regulated by insulin and physical exercise similar to Glut4-storage vesicles in peripheral insulin target tissues.

Viral oncolysis by herpes simplex virus and other viruses

The use of viruses to destroy tumors, also known as viral oncolysis, dates back to the early 1900s. Although the mechanism of cancer cell lysis was unknown in the early years of development, advances in tumor biology, molecular biology, and virology have been critical for numerous advances that have brought the field to where it is today. Oncolytic viruses have been developed based on innate and engineered properties to preferentially target tumor cells. Engineered properties include alterations in endogenous gene expression and introduction of foreign genes. Methods to non-invasively monitor sites of viral replication is required for preclinical and clinical studies. Positron emission tomography (PET) can be used for this purpose. This review focuses on commonly used oncolytic viruses, their selection for oncolytic therapy, the design of HSV-1 viral mutants, and monitoring their replication by PET.

Positron Emission Tomography of Herpes Simplex Virus 1 Oncolysis

Viral oncolysis, the destruction of cancer cells by replicating viruses, is under clinical investigation for cancer therapy. Lytic viral replication in cancer cells both destroys the cells and liberates progeny virion to infect adjacent cancer cells. The safety and efficacy of this approach are dependent on selective and robust viral replication in cancer cells rather than in normal cells. Methods to detect and quantify viral replication in tissues have relied on organ sampling for molecular analyses. Preclinical and clinical studies of viral oncolysis will benefit significantly from development of a noninvasive method to repetitively measure viral replication. We have shown that positron emission tomography (PET) allows for in vivo detection of herpes simplex virus (HSV)-1 replication in tumor cells using 9-(4-[(18)F]-fluoro-3-[hydroxymethyl]butyl)guanine ([(18)F]FHBG) as the substrate for HSV thymidine kinase (HSV-TK). As expected, phosphorylated [(18)F]FHBG is initially trapped within HSV-1-infected tumor cells and is detectable as early as 2 h following virus administration. MicroPET images reveal that [(18)F]FHBG accumulation in HSV-1-infected tumors peaks at 6 h. However, despite progressive accumulation of HSV-1 titers and HSV-TK protein in the tumor as viral oncolysis proceeds, tumor cell degradation resulting from viral oncolysis increases over time, which limits intracellular retention of [(18)F]FHBG. These observations have important consequences with regard to strategies to use [(18)F]FHBG PET for monitoring sites of HSV-TK expression during viral oncolysis.

Metformin—an Adjunct Antineoplastic Therapy—Divergently Modulates Tumor Metabolism and Proliferation, Interfering with Early Response Prediction by 18F-FDG PET Imaging

Over the last several years, epidemiologic data have suggested that the antidiabetes drug metformin (MET), an adenosine monophosphate–activated protein kinase (AMPK) activator, improves progression-free survival of patients with multiple cancers; more than 30 clinical trials are under way to confirm this finding. We postulated that the role of AMPK as a central cellular energy sensor would result in opposite effects on glucose uptake and proliferation, suggesting different roles for 18F-FDG and 3′-deoxy-3′-18F-fluorothymidine (18F-FLT) in assessing its effectiveness as an antineoplastic agent. Methods: Colon cancer cell lines HT29 (human) and MC26 (murine) were treated for 24 or 72 h with a range of MET concentrations (0–10 mM). Western blotting was used to study the activation of AMPK after MET treatment. Glucose uptake and cell proliferation were measured by cell retention studies with either 18F-FDG or 18F-FLT. EdU (ethynyl deoxyuridine, a thymidine analog) and annexin-propidium iodide flow cytometry was performed to determine cell cycle S-phase and apoptotic changes. In vivo 18F-FDG and 18F-FLT PET images were acquired before and 24 h after MET treatment of HT29 tumor–bearing mice. Results: After 24 h of MET incubation, phosphorylated AMPK levels increased severalfold in both cell lines, whereas total AMPK levels remained unchanged. In cell retention studies, 18F-FDG uptake increased but 18F-FLT retention decreased significantly in both cell lines. The numbers of HT29 and MC26 cells in the S phase decreased 36% and 33%, respectively, after MET therapy. Apoptosis increased 10.5-fold and 5.8-fold in HT29 and MC26 cells, respectively, after 72 h of incubation with MET. PET imaging revealed increased 18F-FDG uptake (mean ± SEM standardized uptake values were 0.71 ± 0.03 before and 1.29 ± 0.11 after MET therapy) (P < 0.05) and decreased 18F-FLT uptake (mean ± SEM standardized uptake values were 1.18 ± 0.05 before and 0.89 ± 0.01 after MET therapy) (P < 0.05) in HT29 tumor–bearing mice. Conclusion: MET, through activation of the AMPK pathway, produces a dose-dependent increase in tumor glucose uptake while decreasing cell proliferation in human and murine colon cancer cells. Thus, changes in 18F-FDG uptake after MET treatment may be misleading. 18F-FLT imaging is a promising alternative that correlates with the tumor response.