Richard Mazurchuk

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.

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.

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.

Dynamic Imaging of Emerging Resistance during Cancer Therapy

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1omega) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2omega). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols.

A comparative study of manganese meso-sulfonatophenyl porphyrins: Contrast-enhancing agents for tumors

Four manganese meso-sulfonatophenyl porphyrins were prepared, characterized and investigated for their potential as tumor-specific MRI contrast-enhancing agents in mice bearing subcutaneous implants of a mammary carcinoma (SMT-F). The trisulfonated tetraphenyl porphyrin, MnTPPS3 presented the most favorable profile: bio-distribution, tumor concentration and tumor relaxivity, when compared at 24 hr postinjection. Imaging experiments revealed that a time-dependent delineation of tumor morphology occurs in response to MnTPPS3 that appears to correlate with necrotic regions of the tumor.

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.

Lack of microvessels in well-differentiated regions of human head and neck squamous cell carcinoma A253 associated with functional magnetic resonance imaging detectable hypoxia, limited drug delivery, and resistance to irinotecan therapy.

Purpose: Combination chemotherapy with irinotecan (CPT-11; 50 mg/kg/week × 4 intravenously), followed 24 hour later by 5-fluorouracil (50 mg/kg/week × 4 intravenously), results in 10 and 100% cure rates of animals bearing human head and neck squamous cell carcinoma xenografts A253 and FaDu, respectively. A253 consists of 30% well-differentiated and avascular and 70% poorly differentiated regions with low microvessel density (10/×400), whereas FaDu is uniformly poorly differentiated with higher microvessel density (19/×400). Studies were carried out for determining the role of well-differentiated and avascular regions in drug resistance in A253 and detection of such regions with noninvasive functional magnetic resonance (fMR) imaging. Experimental Design: Tumors were harvested for histopathologic evaluation and immunohistochemistry (CD31, CD34; differentiation marker: involucrin; hypoxia markers: carbonic anhydrase IX, pimonidazole; vascular endothelial factor (VEGF) and Ki67) immediately after fMR imaging following the 3rd dose of chemotherapy. High-performance liquid chromatography determination of intratumoral drug concentration of 7-ethyl-10-hydroxyl-camptothecin and autoradiography with 14C-labeled CPT-11 was done 2 hours after CPT-11 administration. Results: Although A253 xenografts showed three times higher concentration of 7-ethyl-10-hydroxyl-camptothecin, FaDu was more responsive to therapy. After therapy, A253 tumor consisted mostly (∼80%) of well-differentiated regions (positive for involucrin) lacking microvessels with a hypoxic rim (positive for carbonic anhydrase IX and pimonidazole) containing few proliferating (Ki67 positive) poorly differentiated cells. Autoradiography revealed that well-differentiated A253 tumor regions showed 5-fold lower 14C-labeled CPT-11 concentrations compared with poorly differentiated areas (P < 0.001). Blood oxygen level dependant fMR imaging was able to noninvasively distinguish the hypoxic and well-vascularized regions within the tumors. Conclusion: Avascular-differentiated regions in squamous cell carcinoma offer sanctuary to some hypoxic but viable tumor cells (carbonic anhydrase IX and Ki67 positive) that escape therapy because of limited drug delivery. This study provides direct evidence that because of a specific histologic structure, avascular, well-differentiated hypoxic regions in tumors exhibit low drug uptake and represent a unique form of drug resistance.

Functional magnetic resonance (fMR) imaging of a rat brain tumor model: implications for evaluation of tumor microvasculature and therapeutic response

Functional MR (fMR) imaging techniques based on blood oxygenation level dependent (BOLD) effects were developed and applied to a rat brain tumor model to evaluate the potential utility of the method for characterizing tumor growth and regression following treatment. Rats bearing 9L brain tumors in situ were imaged during inhalation of room air and after administration of 100% oxygen + acetazolamide (ACZ) injected 15 mg/kg intravenously. Pixel-to-pixel fMR maps of normalized signal intensity change from baseline values were calculated from T2 weighted spin echo (SE) images acquired pre- and post- oxygen + ACZ administration. Resultant fMR maps were then compared to gross histological sections obtained from corresponding anatomical regions. Regions containing viable tumor with increased cellular density and localized foci of necrotic tumor cells consistent with hypoxia were visualized in the fMR images as regions with decreased signal intensities, indicating diminished oxyhemoglobin concentration and blood flow as compared to normal brain. Histological regions having peritumor edema, caused by increased permeability of tumor vasculature, were visualized in the fMR images as areas with markedly increased signal intensities. These results suggest that fMR imaging techniques could be further developed for use as a non-invasive tool to assess changes in tumor oxygenation/hemodynamics, and to evaluate the pharmacologic effect of anti-neoplastic drugs.

The Vps33a gene regulates behavior and cerebellar Purkinje cell number

A mutation in the Vps33a gene causes Hermansky-Pudlak Syndrome (HPS)-like-symptoms in the buff (bf) mouse mutant. The encoded product, Vps33a, is a member of the Sec1 and Class C multi-protein complex that regulates vesicle trafficking to specialized lysosome-related organelles. As Sec1 signaling pathways have been implicated in pre-synaptic function, we examined brain size, cerebellar cell number and the behavioral phenotype of bf mutants. Standardized behavioral tests (SHIRPA protocols) demonstrated significant motor deficits (e.g., grip strength, righting reflex and touch escape) in bf mutants, worsening with age. Histological examination of brain revealed significant Purkinje cell loss that was confirmed with staining for calbindin, a calcium binding protein enriched in Purkinje cells. This pathologic finding was progressive, as older bf mutants (13-14 months) showed a greater attrition of neurons, with their cerebella appearing to be particularly reduced (approximately 30%) in size relative to those of age-matched-control cohorts. These studies suggest that loss of Purkinje neurons is the most obvious neurological atrophy in the bf mutant, a structural change that generates motor coordination deficits and impaired postural phenotypes. It is conceivable therefore that death of cerebellar cells may also be a clinical feature of HPS patients, a pathological event which has not been reported in the literature. In general, the bf mutant may be a potentially new and useful model for understanding Purkinje cell development and function.

Differential Pharmacodynamic Effects of Paclitaxel Formulations in an Intracranial Rat Brain Tumor Model

Nano- and microparticulate carriers can exert a beneficial impact on the pharmacodynamics of anticancer agents. To investigate the relationships between carrier and antitumor pharmacodynamics, paclitaxel incorporated in liposomes (L-pac) was compared with the clinical standard formulated in Cremophor-EL/ethanol (Cre-pac) in a rat model of advanced primary brain cancer. Three maximum-tolerated-dose regimens given by intravenous administration were investigated: 50 mg/kg on day 8 (d8) after implantation of 9L gliosarcoma tumors; 40 mg/kg on d8 and d15; 20 mg/kg on d8, d11, and d15. Body weight change and neutropenia were assessed as pharmacodynamic markers of toxicity. The pharmacodynamic markers of antitumor efficacy were increase in lifespan (ILS) and tumor volume progression, measured noninvasively by magnetic resonance imaging. At equivalent doses, neutropenia was similar for both formulations, but weight loss was more severe for Cre-pac. No regimen of Cre-pac extended survival, whereas L-pac at 40 mg/kg ×2 doses was well tolerated and mediated 26% ILS (p < 0.0002) compared with controls. L-pac at a lower cumulative dose (20 mg/kg ×3) was even more effective (40% ILS; p < 0.0001). In striking contrast, the identical regimen of Cre-pac was lethal. Development of a novel semimechanistic pharmacodynamic model permitted quantitative hypothesis testing with the tumor volume progression data, and suggested the existence of a transient treatment effect that was consistent with sensitization or “priming” of tumors by more frequent L-pac dosing schedules. Therefore, improved antitumor responses of carrier-based paclitaxel formulations can arise both from dose escalation, because of reduced toxicity, and from novel carrier-mediated alterations of antitumor pharmacodynamic effects.