Joseph A. Spernyak

Genetically Altered Expression of Spermidine/Spermine N1-Acetyltransferase Affects Fat Metabolism in Mice via Acetyl-CoA

The acetylating enzyme, spermidine/spermine N1-acetyltransferase, participates in polyamine homeostasis by regulating polyamine export and catabolism. Previously, we reported that overexpression of the enzyme in cultured tumor cells and mice activates metabolic flux through the polyamine pathway and depletes the N1-acetyltransferase coenzyme and fatty acid precursor, acetyl-CoA. Here, we investigate this possibility in spermidine/spermine N1-acetyltransferase transgenic mice in which the enzyme is systemically overexpressed and in spermidine/spermine N1-acetyltransferase knock-out mice. Tissues of the former were characterized by increased N1-acetyltransferase activity, a marked elevation in tissue and urinary acetylated polyamines, a compensatory increase in polyamine biosynthetic enzyme activity, and an increase in metabolic flux through the polyamine pathway. These polyamine effects were accompanied by a decrease in white adipose acetyl- and malonyl-CoA pools, a major (20-fold) increase in glucose and palmitate oxidation, and a distinctly lean phenotype. In SSAT-ko mice, the opposite relationship between polyamine and fat metabolism was observed. In the absence of N1-acetylation of polyamines, there was a shift in urinary and tissue polyamines indicative of a decline in metabolic flux. This was accompanied by an increase in white adipose acetyl- and malonyl-CoA pools, a decrease in adipose palmitate and glucose oxidation, and an accumulation of body fat. The latter was further exaggerated under a high fat diet, where knock-out mice gained twice as much weight as wild-type mice. A model is proposed whereby the expression status of spermidine/spermine N1-acetyltransferase alters body fat accumulation by metabolically modulating tissue acetyl- and malonyl-CoA levels, thereby influencing fatty acid biosynthesis and oxidation.

Iron(II) PARACEST MRI Contrast Agents

The first examples of Fe(II) PARACEST magnetic resonance contrast agents are reported (PARACEST = paramagnetic chemical exchange saturation transfer). The iron(II) complexes contain a macrocyclic ligand, either 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane (L1) or 1,4,7-tris[(5-amino-6-methyl-2-pyridyl)methyl]-1,4,7-triazacyclononane (L2). The macrocycles bind Fe(II) in aqueous solution with formation constants of log K = 13.5 and 19.2, respectively, and maintain the Fe(II) state in the presence of air. These complexes each contain six exchangeable protons for CEST which are amide protons in [Fe(L1)](2+) or amino protons in [Fe(L2)](2+). The CEST peak for the [Fe(L1)](2+) amide protons is at 69 ppm downfield of the bulk water resonance whereas the CEST peak for the [Fe(L2)](2+) amine protons is at 6 ppm downfield of bulk water. CEST imaging using a MRI scanner shows that the CEST effect can be observed in solutions containing low millimolar concentrations of complex at neutral pH, 100 mM NaCl, 20 mM buffer at 25 °C or 37 °C.

Peroxiredoxin 1 Controls Prostate Cancer Growth through Toll-Like Receptor 4–Dependent Regulation of Tumor Vasculature

In recent years a number of studies have implicated chronic inflammation in prostate carcinogenesis. However, mitigating factors of inflammation in the prostate are virtually unknown. Toll-like receptor 4 (TLR4) activity is associated with inflammation and is correlated with progression risk in prostate cancer (CaP). TLR4 ligands include bacterial cell wall proteins, danger signaling proteins, and intracellular proteins such as heat shock proteins and peroxiredoxin 1 (Prx1). Here we show that Prx1 is overexpressed in human CaP specimens and that it regulates prostate tumor growth through TLR4-dependent regulation of prostate tumor vasculature. Inhibiting Prx1 expression in prostate tumor cells reduced tumor vascular formation and function. Furthermore, Prx1 inhibition reduced levels of angiogenic proteins such as VEGF within the tumor microenvironment. Lastly, Prx1-stimulated endothelial cell proliferation, migration, and differentiation in a TLR4- and VEGF-dependent manner. Taken together, these results implicate Prx1 as a tumor-derived inducer of inflammation, providing a mechanistic link between inflammation and TLR4 in prostate carcinogenesis. Our findings implicate Prx1 as a novel therapeutic target for CaP.

Mild Elevation of Body Temperature Reduces Tumor Interstitial Fluid Pressure and Hypoxia and Enhances Efficacy of Radiotherapy in Murine Tumor Models

Human and rodent solid tumors often exhibit elevated interstitial fluid pressure (IFP). This condition is recognized as a prognostic indicator for reduced responses to therapy and decreased disease-free survival rate. In the present study, we tested whether induction of a thermoregulatory-mediated increase in tissue blood flow, induced by exposure of mice to mild environmental heat stress, could influence IFP and other vascular parameters within tumors. Using several murine tumor models, we found that heating results in a sustained reduction in tumor IFP correlating with increased tumor vascular perfusion (measured by fluorescent imaging of perfused vessels, laser Doppler flowmetry, and MRI) as well as a sustained reduction in tumor hypoxia. Furthermore, when radiation therapy was administered 24 hours postheating, we observed a significant improvement in efficacy that may be a result of the sustained reduction in tumor hypoxia. These data suggest, for the first time, that environmental manipulation of normal vasomotor function is capable of achieving therapeutically beneficial changes in IFP and microvascular function in the tumor microenvironment.

The NiCEST Approach: Nickel(II) ParaCEST MRI Contrast Agents

Paramagnetic Ni(II) complexes are shown here to form paraCEST MRI contrast agents (paraCEST = paramagnetic chemical exchange saturation transfer; NiCEST = Ni(II) based CEST agents). Three azamacrocycles with amide pendent groups bind Ni(II) to form stable NiCEST contrast agents including 1,4,7-tris(carbamoylmethyl)-1,4,7-triazacyclononane (L1), 1,4,8,11-tetrakis(carbamoylmethyl)-1,4,8,11-tetraazacyclotetradecane (L2), and 7,13-bis(carbamoylmethyl)-1,4,10-trioxa-7,13-diazacyclopentadecane (L3). [Ni(L3)](2+), [Ni(L1)](2+), and [Ni(L2)](2+) have CEST peaks attributed to amide protons that are shifted 72, 76, and 76 ppm from the bulk water resonance, respectively. Both CEST MR images and CEST spectroscopy show that [Ni(L3)](2+) has the largest CEST effect in 100 mM NaCl, 20 mM HEPES pH 7.4 at 37 °C. This larger CEST effect is attributed to the sharper proton resonances of the complex which arise from a rigid structure and low relaxivity.

Tumor Vascular Response to Photodynamic Therapy and the Antivascular Agent 5,6-Dimethylxanthenone-4-Acetic Acid: Implications for Combination Therapy

Purpose: Photodynamic therapy (PDT) is a clinically approved treatment for a variety of solid malignancies. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent vascular targeting agent that has been shown to be effective against a variety of experimental rodent tumors and xenografts and is currently undergoing clinical evaluation. We have previously reported that the activity of PDT against transplanted mouse tumors is selectively enhanced by DMXAA. In the present study, we investigated the in vivo tumor vascular responses to the two treatments given alone and in combination. Experimental Design: Vascular responses to (i) four different PDT regimens using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) at two different fluences (128 and 48 J/cm2) and fluence rates (112 and 14 mW/cm2), (ii) 5-aminolevulinic acid (ALA)–sensitized PDT (135 J/cm2 at 75 mW/cm2), (iii) DMXAA at a high (30 mg/kg) and low dose (25 mg/kg), and (iv) the combination of HPPH-PDT (48 J/cm2 at 112 mW/cm2) and low-dose DMXAA were studied in BALB/c mice bearing Colon-26 tumors. Results: PDT-induced changes in vascular permeability, determined using noninvasive magnetic resonance imaging with a macromolecular contrast agent, were regimen dependent and did not predict tumor curability. However, a pattern of increasing (4 hours after treatment) and then decreasing (24 hours after) contrast agent concentrations in tumors, seen after high-dose DMXAA or the combination of PDT and low-dose DMXAA, was associated with long-term cure rates of >70%. This pattern was attributed to an initial increase in vessel permeability followed by substantial endothelial cell damage (CD31 immunohistochemistry) and loss of blood flow (fluorescein exclusion assay). Low dose–rate PDT, regardless of the delivered dose, increased the level of magnetic resonance contrast agent in peritumoral tissue, whereas treatment with either DMXAA alone, or PDT and DMXAA in combination resulted in a more selective tumor vascular response. Conclusions: The observed temporal and spatial differences in the response of tumor vessels to PDT and DMXAA treatments could provide valuable assistance in the optimization of scheduling when combining these therapies. The combination of PDT and DMXAA provides therapeutically synergistic and selective antitumor activity. Clinical evaluation of this combination is warranted.

A Redox‐Activated MRI Contrast Agent that Switches Between Paramagnetic and Diamagnetic States

The design of molecular switches for the production of responsive or “smart” imaging agents is a major challenge. Of particular interest are agents that respond to biological redox environment to map those disease states that involve redox imbalances.[1] Redox imbalances may be triggered in many ways, including very low oxygen pressure or hypoxia in the case of solid tumors.[2] Importantly, the development of probes to map oxygen levels and corresponding redox status of tumor tissue may guide the development of tumor-selective drugs.[3]

Light Delivery Over Extended Time Periods Enhances the Effectiveness of Photodynamic Therapy

Purpose: The rate of energy delivery is a principal factor determining the biological consequences of photodynamic therapy (PDT). In contrast to conventional high-irradiance treatments, recent preclinical and clinical studies have focused on low-irradiance schemes. The objective of this study was to investigate the relationship between irradiance, photosensitizer dose, and PDT dose with regard to treatment outcome and tumor oxygenation in a rat tumor model. Experimental Design: Using the photosensitizer HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide), a wide range of PDT doses that included clinically relevant photosensitizer concentrations was evaluated. Magnetic resonance imaging and oxygen tension measurements were done along with the Evans blue exclusion assay to assess vascular response, oxygenation status, and tumor necrosis. Results: In contrast to high-incident laser power (150 mW), low-power regimens (7 mW) yielded effective tumor destruction. This was largely independent of PDT dose (drug-light product), with up to 30-fold differences in photosensitizer dose and 15-fold differences in drug-light product. For all drug-light products, the duration of light treatment positively influenced tumor response. Regimens using treatment times of 120 to 240 min showed marked reduction in signal intensity in T2-weighted magnetic resonance images at both low (0.1 mg/kg) and high (3 mg/kg) drug doses compared with short-duration (6-11 min) regimens. Significantly greater reductions in pO2 were observed with extended exposures, which persisted after completion of treatment. Conclusions: These results confirm the benefit of prolonged light exposure, identify vascular response as a major contributor, and suggest that duration of light treatment (time) may be an important new treatment variable.

High Correlation of Whole-Body Red Fluorescent Protein Imaging and Magnetic Resonance Imaging on an Orthotopic Model of Pancreatic Cancer

We have developed genetically fluorescent orthotopic models of human pancreatic cancer. In these models, noninvasive fluorescent protein imaging (FPI) of internal primary tumors and metastatic deposits has been carried out. Whole-body tumor images are easily and inexpensively obtained using FPI, permitting both detection and quantification of tumor load. In this study, we simultaneously compared single mice with a highly fluorescent, red fluorescent protein-expressing orthotopic pancreatic cancer xenografts with both FPI and high-resolution magnetic resonance imaging (MRI). Images were acquired at multiple time points after tumor implantation in the pancreas. Indwelling pancreatic primary tumors and metastatic foci were detected by both FPI and MRI. Moreover, a strong correlation existed between images taken with these two technologies. FPI permitted rapid, high-throughput imaging without the need for either anesthesia or contrast agents. Both FPI and MRI enabled accurate imaging of tumor growth and metastasis, although MRI enabled tissue structure to be visualized as well. FPI has high resolution and is exceedingly rapid with instant image capture. We suggest a complimentary role for these two imaging modalities.

CoCEST: cobalt(II) amide-appended paraCEST MRI contrast agents

The first examples of air-stable Co(II) paraCEST MRI contrast agents are reported. Amide NH protons on the complexes give rise to CEST peaks that are shifted up to 112 ppm from the bulk water resonance. One complex has multiple CEST peaks that may be useful for ratiometric mapping of pH.