Cellular and molecular mechanisms of nicotine's pro-angiogenesis activity and its potential impact on cancer

The present study examined the mechanisms of nicotine's effect on angiogenesis and its impact on tumor growth. Nicotine demonstrated significant (P<0.01) stimulation of the release of endothelial cell growth factor, basic fibroblast growth factor (b-FGF) but not vascular endothelial growth factor (VEGF). In a concentration-dependent manner, nicotine induced endothelial cell tube formation. Additionally, in the chick chorioallantoic membrane (CAM) model of angiogenesis, nicotine effectively induced the generation of new blood vessels (P<0.01), an effect that is mediated via b-FGF. The pro-angiogenesis effect of nicotine in the CAM model was maximally blocked by either anti-integrin alphavbeta3 or inhibitor of mitogen activated protein kinase (MAPK, ERK 1/2). In the CAM tumor implant model, nicotine doubled (P<0.01) the growth rate of breast, colon, and lung cancer. These data indicated that the pro-angiogenesis effect is mediated via b-FGF and induced through the nicotinic receptor, alphavbeta3 integrin, and MAPK.     

Cathepsin L inhibition suppresses drug resistance in vitro and in vivo: a putative mechanism

Cathepsin L is a lysosomal enzyme thought to play a key role in malignant transformation. Recent work from our laboratory has demonstrated that this enzyme may also regulate cancer cell resistance to chemotherapy. The present study was undertaken to define the relevance of targeting cathepsin L in the suppression of drug resistance in vitro and in vivo and also to understand the mechanism(s) of its action. In vitro experiments indicated that cancer cell adaptation to increased amounts of doxorubicin over time was prevented in the presence of a cathepsin L inhibitor, suggesting that inhibition of this enzyme not only reverses but also prevents the development of drug resistance. The combination of the cathepsin L inhibitor with doxorubicin also strongly suppressed the proliferation of drug-resistant tumors in nude mice. An investigation of the underlying mechanism(s) led to the finding that the active form of this enzyme shuttles between the cytoplasm and nucleus. As a result, its inhibition stabilizes and enhances the availability of cytoplasmic and nuclear protein drug targets including estrogen receptor-alpha, Bcr-Abl, topoisomerase-IIalpha, histone deacetylase 1, and the androgen receptor. In support of this, the cellular response to doxorubicin, tamoxifen, imatinib, trichostatin A, and flutamide increased in the presence of the cathepsin L inhibitor. Together, these findings provided evidence for the potential role of cathepsin L as a target to suppress cancer resistance to chemotherapy and uncovered a novel mechanism by which protease inhibition-mediated drug target stabilization may enhance cellular visibility and, thus, susceptibility to anticancer agents.     

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

Background

TRPA1 has been implicated in both chemo- and mechanosensation. Recent work demonstrates that inhibiting TRPA1 function reduces mechanical hypersensitivity produced by inflammation. Furthermore, a broad range of chemical irritants require functional TRPA1 to exert their effects. In this study we use the ex-vivo skin-nerve preparation to directly determine the contribution of TRPA1 to mechanical- and chemical-evoked responses at the level of the primary afferent terminal.

Results

Acute application of HC-030031, a selective TRPA1 antagonist, inhibited all formalin responses in rat C fibers but had no effect on TRPV1 function, assessed by capsaicin responsiveness. Genetic ablation experiments corroborated the pharmacological findings as C fibers from wild type mice responded to both formalin and capsaicin, but fibers from their TRPA1-deficient littermates responded only to capsaicin. HC-030031 markedly reduced the mechanically-evoked action potential firing in rat and wild type mouse C fibers, particularly at high-intensity forces, but had no effect on the mechanical responsiveness of Aδ fiber nociceptors. Furthermore, HC-030031 had no effect on mechanically-evoked firing in C fibers from TRPA1-deficient mice, indicating that HC-030031 inhibits mechanically-evoked firing via a TRPA1-dependent mechanism.

Conclusion

Our data show that acute pharmacological blockade of TRPA1 at the cutaneous receptive field inhibits formalin-evoked activation and markedly reduces mechanically-evoked action potential firing in C fibers. Thus, functional TRPA1 at sensory afferent terminals in skin is required for their responsiveness to both noxious chemical and mechanical stimuli.     

Synthesis and antiplatelet effects of an isoxazole series of glycoprotein IIb/IIIa antagonists

Despite the excellent in vitro potency of a series of benzamide glycoprotein IIb/IIIa antagonists, which have been reported previously, poor in vivo potency in the inhibition of platelet aggregation was observed when the most potent inhibitor XU057 was dosed intravenously to dogs. In this communication, we report that replacement of the benzamide in XU057 with an isoxazolecarboxamide resulted in significant improvement in in vivo potency. More importantly, the analogue XU065 showed an excellent oral antiplatelet effect in dogs.     

Antiangiogenesis efficacy of nitric oxide donors

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis‐mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis‐mediated processes using the NO donor, S‐nitroso N‐acetyl penicillamine (SNAP) and S‐nitroso N‐acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)‐stimulated tube formation and serum‐induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85–95% inhibition) of cytokine (FGF2)‐induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2‐induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 μg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases. J. Cell. Biochem. 80:104–114, 2000. © 2000 Wiley‐Liss, Inc.     

Implication of somatolactin in the regulation of sexual maturation and spawning of Mugil cephalus

Specific antibody for chum salmon somatolactin (SL) was used for immunocytochemical investigation of SL cell activity of Mugil cephalus during the gonadal cycle in both natural habitat and captivity. The SL-immunoreactive cells showed strong and specific immunoreactivity to antichum salmon SL. The number of SL-immunoreactive cells increased, as did the secretory and synthetic activity during sexual maturation and spawning in the natural habitat. The SL cells were rather small and moderately immunoreactive in immature fish; they were enlarged, their numbers increased, and they frequently showed more SL immunoreactivity during gonadal development. In addition, during late stages of maturation, small cell size with more or less SL immunoreactive cells were noted, indicating an active release of SL granules. Prespawning females tended to have more enlarged SL cells with stronger immunoreactivity than equivalent males. The SL cells showed an increase in the secretory activity during spawning as indicated by small size and weak immunoreactivity. The SL cells of M. cephalus reared in captivity showed high activity. This may be due to the low concentration of calcium in fresh water. The gradual stimulation of SL synthesis and release during sexual maturation and spawning of M. cephalus suggest that SL may be involved in the control of some steps of reproductive processes, such as steroidogenesis, calcium metabolism, and energy mobilization.     

Antiangiogenesis efficacy of nitric oxide donors

Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. Because nitric oxide (NO) is known to play a key role in various vascular diseases, the present study was undertaken to determine the role of NO in angiogenesis-mediated processes using the NO donor, S-nitroso N-acetyl penicillamine (SNAP) and S-nitroso N-acetyl glutathione (SNAG). The antiangiogenic efficacy of these NO donors was examined using in vivo and in vitro model systems. The in vitro studies demonstrated the ability of SNAP to inhibit cytokine fibroblast growth factor (FGF2)-stimulated tube formation and serum-induced cell proliferation. The inhibitory effect on cell proliferation by SNAP concentrations above the millimolar range was associated with significant shifts in the concentration of NO metabolites. Furthermore, using the mouse Matrigel implant model and the chick chorioallantoic membrane (CAM) models, SNAP demonstrated maximal inhibitory efficacy (85-95% inhibition) of cytokine (FGF2)-induced neovascularization in both in vivo models. SNAP and SNAG resulted in 85% inhibition of FGF2-induced neovascularization in the mouse Matrigel model when given at 5 mg/kg/day infusion in minipumps during 14 days and 87% inhibition of angiogenesis induced by FGF2 in the CAM when administered a single dose of 50 microg. Thus, NO donors might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or neovascular, ocular, and inflammatory diseases.     

Template-constrained cyclic peptide analogues of somatostatin: subtype-selective binding to somatostatin receptors and antiangiogenic activity

Beta-turns are a common secondary structure motif found in proteins that play a role in protein folding and stability and participate in molecular recognition interactions. Somatostatin, a peptide hormone possessing a variety of therapeutically-interesting biological activities, contains a beta-turn in its bioactive conformation. The beta-turn and biological activities of somatostatin have been succesfully mimicked in cyclic hexapeptide analogues. Two novel, structured, non-peptidic molecules were developed that are capable of holding the bioactive tetrapeptide sequence of somatostatin analogues in a beta-turn conformation, as measured by somatostatin receptor (SSTR) binding. Template-constrained cyclic peptides in which the ends of the -Tyr-D-Trp-Lys-Val-tetrapeptide were linked by scaffolds based on either an N,N'-dimethyl-N,N'-diphenylurea or a substituted biphenyl system (DJS631 and DJS811, respectively), bound selectively to mouse SSTR2B and rat and human SSTR5 with affinities as high as 1 nM. DJS811, at a dose of 3 mg/kg/day, was shown in a mouse Matrigel model to inhibit angiogenesis to a level of 79%. The development of structured turn scaffolds allows beta-turn sequences to be contained in the context of a compact structure, with less peptidic nature and potentially greater bioavailability than cyclic hexapeptides. These systems can be used to study the determinants of beta-turn formation, as well as to probe the importance of turn sequences occurring in molecular recognition interactions. The antiangiogenic activity of DJS811 suggests that it may have antitumor activity as well. In addition, because SSTR2 is overexpressed on many types of tumors, DJS631 and DJS811 may be useful in the development of agents for tumor imaging or the radiotherapy of cancer.     

Castration induced changes in dog prostate gland associated with diminished activin and activin receptor expression

This study was conducted to evaluate the effect of androgen ablation on dog prostate gland structure and the proliferation capacity of the prostatic cells and their association with the expression of Activin A and Activin RIIA receptor. The effect of androgen on the prostate gland was compared in intact and castrated dogs after one and two weeks. Specific primary antibodies were used to immunolocalize activin-A, activin receptor type II A and the proliferation marker (PCNA). The results showed that the glandular acini of the prostate gland of intact dogs are lined by tall columnar secretory cells and less abundant flattened basal cells and surrounded by a thin fibromuscular tissue. The cytoplasm of the glandular cells exhibited an intense immunoreaction for activin A and activin RIIA receptor while basal cells expressed PCNA. Castration induced a remarkable atrophy of the prostatic acini associated with a progressive loss of secretory epithelial cells, which showed a dramatic decrease to complete disappearance of Activin A and Activin RIIA receptor immunoreactions. The remaining cells of the atrophied acini continue to express PCNA and the inter-acinar fibromuscular tissue showed a remarkable increase in its mass and are induced to express PCNA. These results indicated that androgen is required for the survival of epithelial cells and to maintain growth-quiescent fibromuscular cells, while basal cell proliferation is androgen independent. The changes in the Activin A and Activin RIIA receptor localization and their association with the dynamic pattern of prostate gland regression after castration suggested that Activin A and Activin RIIA receptor expression are androgen dependent.