Endorepellin in vivo: targeting the tumor vasculature and retarding cancer growth and metabolism.

BACKGROUND: The antiangiogenic approach to controlling cancer requires a better understanding of angiogenesis and the discovery of new compounds that modulate this key biological process. Here we investigated the role of endorepellin, an angiostatic protein fragment that is derived from the C-terminus of perlecan, a heparan sulfate proteoglycan, in controlling tumor angiogenesis in vivo. METHODS: We administered human recombinant endorepellin systemically to mice bearing orthotopic squamous carcinoma xenografts or syngeneic Lewis lung carcinoma tumors. We monitored tumor growth, angiogenesis, metabolism, hypoxia, and mitotic index by using quantitative immunohistochemistry and positron emission tomography scan imaging. In addition, we determined the localization of injected endorepellin using near-infrared labeling and immunohistochemistry of frozen tumor sections. Finally, we isolated tumor-derived endothelial cells and tested whether endorepellin could interact with these cells and disrupt in vitro capillary morphogenesis. All statistical tests were two-sided. RESULTS: Endorepellin specifically targeted the tumor vasculature as determined by immunohistochemical analysis and accumulated in the tumor perivascular zones where it persisted for several days as discrete deposits. This led to inhibition of tumor angiogenesis (as measured by decreased CD31-positive cells, mean control = 1902 CD31-positive pixels, mean endorepellin treated = 343.9, difference between means = 1558, 95% confidence interval [CI] = 1296 to 1820, P<.001), enhanced tumor hypoxia, and a statistically significant decrease in tumor metabolism and mitotic index (as measured by decreased Ki67-positive cells, mean control Ki67 pixels = 5970, mean endorepellin-treated Ki67 pixels = 3644, difference between means = 2326, 95% CI = 1904 to 2749, P<.001) compared to untreated controls. Endorepellin was actively internalized by tumor-derived endothelial cells causing a redistribution of alpha2beta1 integrin such that both proteins colocalized to punctate deposits in the perivascular region. Endorepellin treatment inhibited in vitro capillary morphogenesis of both normal and tumor-derived endothelia. CONCLUSIONS: Our results provide support for the hypothesis that endorepellin is an effective antitumor vasculature agent that could be used as a therapeutic modality to combat cancer.

Localization and characterization of glutathione-s-transferase isozymes alpha, mu, and pi within the mouse vomeronasal organ.

The nasal cavity of vertebrates contains a variety of xenobiotic metabolizing enzymes that possess a broad range of substrate specificity ranging from metabolism of drugs, carcinogens, and steroid hormones, to dietary components and environmental pollutants. This investigation sought to localize the cellular expression and distribution of glutathione-s-transferase (GST) alpha, mu, and pi detoxifying enzymes, and to study GST activity toward different substrates in the mouse vomeronasal organ (VNO). Immunohistochemistry was used to identify GST alpha, mu and pi in the non-sensory and sensory layer of the VNO. Western blot analysis of cytosolic proteins revealed a qualitatively higher enzyme expression of GST alpha and mu in the main olfactory tissue (OE) in comparison to VNO tissue, whereas the GST pi isozyme was equally expressed in both. Total GST metabolism of 1-chloro-2, 4-dinitrobenzene (CDNB) revealed a higher activity level in the OE when compared to the VNO. In contrast, thin-layer chromatographic analysis of GST conjugation of the odorant, trans-2-hexenal (t-hex) (10 mM) showed more conjugate formed per unit protein in the VNO than the OE. The analysis of GST expression and enzyme activity within the VNO parallels the reported localization of phase I metabolizing enzymes and suggests that GST isozymes play independent roles that characterize multiple processes within VNO chemosensitivity.

Characterization of the mouse olfactory glutathione S-transferases during the acute phase response.

The acute phase response (APR) has been shown to alter expression and activity of biotransformation enzymes, such as the phase I cytochromes p450 and phase II glutathione S-transferases (GSTs). The cytochromes p450 and GSTs are expressed abundantly and colocalized to non-neuronal cells of the olfactory mucosa. Previous studies indicate that olfactory cytochromes p450 expression and activity is altered during periods of localized inflammation and infection. Little is understood, however, about the influence of the APR on olfactory GST enzymes. This study investigated effects of the APR on olfactory GST isozymes expression and activity in mouse olfactory mucosa after 24-hr treatment with the acute phase inducer, polyinosinic: polycytidylic acid (polyIC). Western blot analysis using antibodies directed against specific GST isoforms alpha (A1-1), micro (M1-1), and pi (P1-1) demonstrated that their expression was unaltered by polyIC treatment. In contrast, olfactory p450 2E1 expression was significantly decreased. Enzymatic activity of the olfactory GSTs toward the general substrate, 1-chloro-2,4-dinitrobenzene (CDNB) was unchanged during the APR. Analysis of olfactory glutathione content during the APR showed that it was also unaffected by polyIC. The insensitivity of these olfactory GST isoforms during the APR may play a significant role toward limiting the impact of infection and inflammation on the olfactory system.