Effect of zoledronic acid on the doxycycline-induced decrease in tumour burden in a bone metastasis model of human breast cancer.

Bone is one of the most frequent sites for metastasis in breast cancer patients often resulting in significant clinical morbidity and mortality. Bisphosphonates are currently the standard of care for breast cancer patients with bone metastasis. We have shown previously that doxycycline, a member of the tetracycline family of antibiotics, reduces total tumour burden in an experimental bone metastasis mouse model of human breast cancer. In this study, we combined doxycycline treatment together with zoledronic acid, the most potent bisphosphonate. Drug administration started 3 days before the injection of the MDA-MB-231 cells. When mice were administered zoledronic acid alone, the total tumour burden decreased by 43% compared to placebo treatment. Administration of a combination of zoledronic acid and doxycycline resulted in a 74% decrease in total tumour burden compared to untreated mice. In doxycycline- and zoledronate-treated mice bone formation was significantly enhanced as determined by increased numbers of osteoblasts, osteoid surface and volume, whereas a decrease in bone resorption was also observed. Doxycycline greatly reduced tumour burden and could also compensate for the increased bone resorption. The addition of zoledronate to the regimen further decreased tumour burden, caused an extensive decrease in bone-associated soft tissue tumour burden (93%), and sustained the bone volume, which could result in a smaller fracture risk. Treatment with zoledronic acid in combination with doxycycline may be very beneficial for breast cancer patients at risk for osteolytic bone metastasis.

Malignant ascites fluid (MAF), including ovarian-cancer-associated MAF, contains angiostatin and other factor(s) which inhibit angiogenesis.

OBJECTIVE: The aim was to determine whether human malignant ascites fluid (MAF) associated with abdominal cancer, including ovarian cancer, contained factors which inhibit angiogenesis as well as others which stimulate this process. METHODS: MAF was collected from six patients, four with ovarian cancer, one with gastric cancer, and one with liver metastases. Using the chick chorioallantoic membrane (CAM) the effect of MAF on 7-day-old CAM capillaries was examined for 48 h. Vascular endothelial growth factor (VEGF) was evaluated by ELISA. Five samples of MAF were fractionated by lysine-Sepharose chromatography and the lysine-bound and -unbound fractions were eluted by epsilon-amino-n-hexanoic acid. Whole MAF, the lysine-bound and -unbound fractions, and human angiostatin were subjected to SDS-PAGE/Western blot analysis and immunostained after exposure to anti-human plasminogen. Human plasminogen was exposed to conditioned medium from ovarian epithelial cancer (HEY) cells and subjected to similar Western blot analysis. RESULTS: Despite containing VEGF, each MAF sample examined caused a loss of capillaries from the CAM; a similar response was seen using purified human angiostatin. Whole MAF and the lysine-bound fraction contained plasminogen (90 kDa) and a 55-kDa protein which migrated in a similar manner to human angiostatin on Western blot. Both the lysine-bound and -unbound fractions caused a loss of capillaries in the CAM. Human plasminogen exposed to conditioned medium from HEY cells yielded a fragment which was similar in size to angiostatin. CONCLUSIONS: MAF from patients with various clinical presentations contains angiostatin and VEGF as well as other factors which are capable of inhibiting angiogenesis.

Metabolism of rabbit angiostatin glycoforms I and II in rabbits: angiostatin-I leaves the intravascular space faster and appears to have greater anti-angiogenic activity than angiostatin-II.

Plasminogen (PLG) exists in the circulation as two glycoforms, I and II. Angiostatin (AST) is a polypeptide that has been cleaved from the kringle region of PLG and has strong anti-angiogenic properties. AST-I and AST-II, which consisted only of kringles 1 through 3, were prepared by the action of urokinase on purified rabbit PLG-I and PLG-II, respectively, in the presence of N-acetyl cysteine, followed by affinity chromatography on lysine-Sepharose. Purified AST-I and AST-II were tested for functional activity with a chick chorioallantoic membrane (CAM) model; when similar amounts were applied to a 6-day CAM, AST-I was substantially more effective than AST-II in decreasing vascular supply to the CAM over a 72-hour period; this activity correlated with a loss of capillaries, probably through apoptosis of endothelial cells. Radiolabeled AST-I and AST-II (iodine 125 and iodine 131) were co-injected intravenously into healthy rabbits to determine their clearances from plasma measured over 3 days. Over a dose range of 0.08 to 2.7 microg/kg, the fractional catabolic rate within the intravascular space (j(3)) indicated that AST-I was cleared 3-fold to 4-fold more rapidly than AST-II (P < .001). The catabolic half-life of AST-I (2.01 +/- 0.19 days) was significantly less than that of AST-II (2.62 +/- 0.20 days). The faster clearance of AST-I from the intravascular space was matched by its more rapid passage than AST-II to the extravascular space of various organs over 60 minutes in vivo. This property of AST-I as compared with AST-II may partially explain its greater anti-angiogenic potential. From the plasma concentrations of PLG-I and PLG-II and their relative behaviors toward rabbit VX-2 lung tumors in vivo, we predict that substantially greater quantities of AST-II than AST-I may be released into the extravascular space of tumors.

Function of cyclo-oxygenase-1 and cyclo-oxygenase-2 in the ductus arteriosus from foetal lamb: differential development and change by oxygen and endotoxin.

1. Prenatal patency of the ductus arteriosus is maintained mainly by prostaglandin(PG) E(2). Here we have examined the relative importance of cyclo-oxygenase-1 (COX1) and cyclo-oxygenase-2 (COX2) for PGE(2) formation in the foetal lamb ductus (0.65 gestation onwards). 2. Using fluorescence microscopy and immunogold staining, COX1 appeared more abundant than COX2 in endothelial and smooth muscle cells, and this difference was greater before-term. Inside muscle cells, COX1 and COX2 immunoreactivity was located primarily in the perinuclear region. Endotoxin, given to the lamb in utero (approximately 0.1 microg kg(-1)), caused COX2 upregulation, while an opposite effect with disappearance of the enzyme followed endotoxin treatment in vitro (100 ng ml(-1)). COX1 immunoreactivity remained virtually unchanged with either treatment; however, this isoform as well as any induced COX2 migrated towards the outer cytoplasm. 3. The COX2 inhibitor L-745,337 (1--10 microM) contracted the isolated ductus at term, the response being almost as high as that to indomethacin (dual COX1/COX2 inhibitor) over the same dose-range. Conversely, L-745,337 was relatively less effective in the premature. 4. Pretreatment of the premature in vivo with endotoxin enhanced the contraction of the ductus to L-745,337, while in vitro endotoxin had a variable effect. 5. The premature ductus exhibited a stronger contraction to L-745,337 following exposure to oxygen. On the other hand, the oxygen contraction, which is modest before-term, was enhanced by L-745,337. 6. We conclude that COX1 and COX2 develop unevenly in the ductus. While both enzymes contribute to PGE(2) formation at term, COX1 is the major isoform in the premature. COX2, however, may acquire greater importance before-term following physiological and pathophysiological stimuli.

Deletion of the endothelin-A-receptor suppresses oxygen-induced constriction but not postnatal closure of the ductus arteriosus.

Experiments were carried out in mutant 129/SvEv mice lacking the endothelin-A (ET(A))-receptor to determine whether endothelin-1 (ET-1), acting as a messenger for oxygen constriction, is responsible for closure of the ductus arteriosus at birth. The isolated ductus from ET(A) -/- fetuses, unlike that from ET(A) +/+ littermates, contracted marginally to oxygen and ET-1 but responded to a thromboxane analog. In vivo, reduction in ductus lumen was equally pronounced in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no such vessel narrowing was seen in hyperoxic ET(A), -/- fetuses, although it occurred in ET(A) +/+ littermates. Notwithstanding the uneven behaviour of the ductus in vitro and in vivo, no ET(A) genotype-related difference was noted in the morphology of the vessel on both light and electron microscopy. We conclude that ET-1 mediates the ductus constriction to oxygen. Without ET-1, however, the vessel still closes postnatally probably as a result of the withdrawal of the relaxing influence of prostaglandin E2 (PGE2).

Endothelin A receptor is necessary for O(2) constriction but not closure of ductus arteriosus.

In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O(2) mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ET(A) receptor -/-, +/-, and +/+ genotypes were used. Isolated DA from term ET(A) +/+ fetuses contracted to O(2) (5-95%) and a thromboxane A(2) analog (ONO-11113, 0.1 microM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1-100 nM). Indomethacin (2.8 microM) was also a constrictor. ET(A) -/- DA, unlike ET(A) +/+ DA, contracted marginally to O(2) and ET-1 but responded to ONO-11113. O(2) contraction was also reduced in ET(A) +/- DA. In vivo, DA constricted equally in tracheotomized ET(A) -/- and ET(A) +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ET(A) -/- fetuses in utero, although it occurred in ET(A) +/+ and +/- littermates. We conclude that ET-1 mediates the DA constrictor response to O(2). Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).

Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone.

1. We have previously shown that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction controlling the formation of endothelin-1 (ET-1). In the present study, we have examined whether CO is formed naturally in the vessel. 2. The CO-forming enzyme, haem oxygenase (HO), was identified in ductal tissue in its constitutive (HO-2) and inducible (HO-1) isoforms by Western immunoblotting and immunological staining procedures (both light and electron microscopy). HO-1 was localized to endothelial and muscle cells, while HO-2 was found only in muscle cells. Inside the muscle cells, HO-1 and HO-2 immunoreactivity was limited to the perinuclear region, and the Golgi apparatus in particular. However, upon exposure to endotoxin, HO-1 became more abundant, and both HO isoforms migrated towards the outer region of the cytoplasm close to the sarcolemma. 3. CO was formed enzymatically from added substrate (hemin, 50 microM) in the 10,000 g supernatant of the ductus and its formation was inhibited by zinc protoporphyrin IX (ZnPP, 200 microM). 4. ZnPP (10 microM) had no effect on the tone of the ductus under normal conditions (2.5 to 95% O2), but it contracted the endotoxin-treated ductus (at 2.5% O2). At the same concentration, ZnPP also tended to contract the hypoxic vessel (zero O2). 5. ZnPP (10 microM) curtailed the relaxant response of the oxygen (30%)/indomethacin (2.8 microM)-contracted ductus to bradykinin (35 nM), while it left the sodium nitroprusside (35 nM) relaxation unchanged. 6. We conclude that CO is formed in the ductus and may exert a relaxing influence when its synthesis is upregulated by an appropriate stimulus.

Carbon monoxide-induced relaxation of the ductus arteriosus in the lamb: evidence against the prime role of guanylyl cyclase.

1. We have previously found that carbon monoxide (CO) potently relaxes the lamb ductus arteriosus and have ascribed this response to inhibition of a cytochrome P450-based mono-oxygenase reaction which sustains contractile tone. Our proposal, however, has been questioned on the evidence of findings in other blood vessels implicating the guanylyl cyclase-based relaxing mechanism as the target for CO. To investigate this issue further, we have carried out experiments in the isolated ductus from near-term foetal lambs and have examined the effect of CO concomitantly on muscle tone and cyclic GMP content, both in the absence and presence of guanylyl inhibitors, or during exposure to monochromatic light at 450 nm. 2. CO (65 microM) reversed completely, or nearly completely, the tone developed by the vessel in the presence of oxygen (30%) and indomethacin (2.8 microM). Cyclic GMP content tended to increase with the relaxation, but the change did not reach significance. Sodium nitroprusside (SNP), a NO donor, mimicked CO in relaxing the ductus. Contrary to CO, however, SNP caused a marked accumulation of cyclic GMP with levels being positively correlated with the relaxation. 3. Methylene blue (10 microM) reduced marginally the CO relaxation, whilst LY-83583 (10 microM) had an obvious, albeit variable, inhibitory effect. Basal cyclic GMP content was lower in tissues treated with either compound and rose upon exposure to CO. However, the levels attained were still within the range of values for tissues prior to any treatment. Furthermore, the elevation in cyclic GMP was not related to the magnitude of the CO relaxation. 4. Illumination of the ductus with monochromatic light at 450 nm reversed the CO relaxation and any concomitant increase in cyclic GMP. In the absence of CO, light by itself had no effect. 5. Ductal preparation with only muscle behaved as the intact preparations in reacting to CO, both in the absence and presence of guanylyl cyclase inhibitors, or during illumination. 6. We conclude that the primary action of CO in the ductus arteriosus is not exerted on the guanylyl cyclase heme and that cyclic GMP may only have an accessory role in the relaxation to this agent. This finding reasserts the importance of a cytochrom P450-based mono-oxygenase reaction for generation of tone and as a target for CO in the ductus.

Inhibition of the contraction of the ductus arteriosus to oxygen by 1-aminobenzotriazole, a mechanism-based inactivator of cytochrome P450.

1. We have proposed that contractile tension of the ductus arteriosus is sustained by a cytochrome P450-linked mechanism acting as a limiting step in the synthesis of endothelin-1 (ET-1). In the present study, we have used the isolated ductus from near-term foetal lambs and guinea-pigs to investigate the effect on both muscle tone and ET-1 formation of 1-aminobenzotriazole (ABT), a suicide substrate for mono-oxygenase reactions. 2. ABT relaxed the lamb ductus at rest (2.5% O2) and during the oxygen contraction (15 to 95% O2). The effect was seen at 40 microM, and at 0.8 mM active tone was almost completely abolished. ABT (1 mM) also reversed the oxygen contraction in the guinea-pig ductus. 3. In the lamb ductus, the ABT response was not affected by removal of the endothelium or by treatment with 2.8 microM indomethacin (at 2.5% O2) and the ensuing contraction. 4. At both low and high concentration, ABT relaxed marginally, or not at all, the potassium-contracted (55 mM) ductus from either species. 5. ET-1 release from either the intact or endothelium-denuded lamb ductus tended to decrease in the presence of ABT (1 mM), whilst during the same treatment cyclic GMP content of the tissue remained unchanged. 6. We conclude that ABT relaxation is due to suppression of a contractile mechanism and not to activation of prostaglandin- and NO-mediated relaxing mechanisms. This contractile mechanism has a cytochrome P450-based mono-oxygenase reaction as a key component.

Post-activation potentiation and depression in the neocortex of the rat: II. Chronic preparations.

Although long-term potentiation (LTP) has been demonstrated in a number of subcortical sites in chronic preparations, there have been no demonstrations of LTP in the neocortex of chronic preparations. Even neocortical slice and acute preparations often require a drug-induced suppression of inhibition before LTP effects can be reliably induced. We have attempted to induce LTP in neocortical sites in 7 different experiments using chronically prepared adult rats. We were unable to obtain any evidence, even a trend, for the induction of LTP. The following manipulations were tested: (1) standard stimulation train parameters that have been shown to be highly effective in subcortical and hippocampal sites; (2) a 10-fold increase in the intra-train pulse durations; (3) variations in train pulse frequency (1 Hz to 300 Hz) and train duration (100 ms to 15 min); (4) co-activation of multiple inputs by stimulation of combinations of cortical sites or cortical and thalamic sites; (5) reduction of inhibition by administration of picrotoxin; 5) Housing of animals in an enriched environment; (6) utilization of the neocortical stimulation trains as a cue in a learning task; (7) application of pilocarpine to co-activate cholinergic systems. Although none of these manipulations produced LTP, the application of pilocarpine did facilitate the induction of a long-lasting depression effect. These findings contrast with the results obtained from anesthetized rats and from studies using brain slices, where LTP can be reliably induced. These results are discussed in light of other recent findings with respect to LTP and LTD effects.

Occurrence of endothelium-derived relaxing factor--nitric oxide in the lamb ductus arteriosus.

To determine whether the ductus arteriosus can form endothelium-derived relaxing factor--nitric oxide, we used isolated ductal strips from near-term fetal lamb and examined their response to bradykinin (a nitric oxide stimulator), L-arginine (a nitric oxide precursor), and agents interfering with the synthesis (N omega-nitro-L-arginine) and action (methylene blue) of nitric oxide. Bradykinin relaxed the indomethacin-contracted ductus dose dependently from a threshold of about 10(-10) M, and peak relaxation was greater at high (176-210 mmHg; 1 mmHg = 133.3 Pa) than low (15-25 mmHg) PO2. Bradykinin relaxation was nearly completely or completely abolished in endothelium-denuded preparations and, in its place, there was often a small contraction. Pretreatment with nitric oxide inhibitors also prevented, in part (methylene blue, 1 microM) or in full (N omega-nitro-L-arginine, 100 microM), the relaxant effect of bradykinin. Paradoxically, L-arginine (10 microM) had an inhibiting rather than an enhancing effect on the bradykinin relaxation. N omega-Nitro-L-arginine (100 microM) and methylene blue (1-100 microM) contracted by themselves the untreated ductus, and their action persisted after removal of the endothelium. These findings indicate the presence in the ductus arteriosus of a nitric oxide based relaxing mechanism, which may supplement prostaglandin E2 in keeping the vessel patent in the fetus. This mechanism may, on one hand, afford protection against nonsteroidal anti-inflammatory drugs in utero and may, on the other hand, complicate the management of prematures with persistent ductus and account for failures of the indomethacin therapy.

Evidence for an effector role of endothelin in closure of the ductus arteriosus at birth.

The ductus arteriosus is a special muscular shunt that in the fetus allows blood to bypass the unexpanded lungs. It closes rapidly after birth and this event is initiated by the physiologic rise in blood oxygen tension. Endothelin-1 has been proposed by us as a local mediator for oxygen after demonstrating that it is formed within the ductus and is a potent ductus constrictor. To confirm this possibility, we have now measured the release of endothelin-1 from the isolated ductus of near-term fetal lambs at different oxygen concentrations of the medium. In addition, using the same preparation, we have examined the effect on contractile tone of compounds interfering with the synthesis (phosphoramidon, 50 microM) and action (BQ123, 1 microM) of endothelin-1. We report that release of endothelin-1 from the ductus tends to increase with the oxygen concentration up to a value mimicking the neonatal condition. Phosphoramidon and, to a greater degree, BQ123 inhibit the contraction of the vessel to oxygen. These results implicate endothelin-1 as the effector agent for oxygen in the ductus and, by extension, assign to this peptide a critical role in the closure of the vessel at birth.

Effects of central and peripheral neuropeptide Y on sodium and water excretion in the rat.

Neuropeptide Y (NPY) is widely distributed throughout the central nervous system and in several sympathetically innervated tissues, including the kidney. Although many central and peripheral acting endogenous compounds alter renal function, the role of NPY is unknown. Accordingly, we examined the effects of intracerebroventricular (ICV) or intrarenal administration of NPY on sodium and water excretion in the barbiturate anesthetized rat. Sprague-Dawley rats were uninephrectomized 10 days prior to testing and, in rats undergoing ICV administration, cannulae were implanted 3 days prior to testing. For testing, rats were anesthetized (Nembutal) and the jugular vein, renal artery and ureter catheterized. The results showed that the intrarenal infusion of NPY at 1 microgram/kg/min increased sodium and water excretion relative to the saline control group without altering blood pressure or creatinine clearance. Similarly, ICV administration of NPY at 10 micrograms in a 5 microliters volume increased the excretion of sodium and water without altering blood pressure as compared to the artificial CSF group. These findings suggest that both central and peripheral NPY may contribute to the regulation of sodium and water excretion in the rat.