Matrix proteinase inhibition by AE-941, a multifunctional antiangiogenic compound.

BACKGROUND: Matrix metalloproteinases (MMPs) play an important role in tissue remodeling under normal physiological and pathological conditions and are thus attractive targets for both diagnostic and therapeutic purposes. Here, we examined the effect of AE-941, an orally bioavailable standardized extract made of cartilage that shows significant antiangiogenic and antimetastatic properties in vivo, on the activity of various members of the MMP family. MATERIALS AND METHODS: The effect of AE-941 on the activity of MMPs was assessed by fluorimetric assays and by substrate gel zymography. RESULTS: AE-941 markedly inhibits the gelatinolytic activity of MMP-2 and to a lesser extent those of MMP-1, MMP-7, MMP-9 and MMP-13. AE-941 also inhibited the elastinolytic activities of MMP-2 and MMP-9 as well as MMP-12 (metalloelastase), porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). Western blot analysis revealed the presence within AE-941 of immunoreactive TIMP-like proteins, suggesting that these proteins may be at least partly responsible for the observed MMP inhibition. CONCLUSIONS: Taken together, these results demonstrate that AE-941 contains TIMP-like proteins that could be responsible for the specific inhibition of MMPs. Given the recent studies suggesting the presence within this compound of specific inhibitor(s) of endothelial cell proliferation, AE-941 appears as a pleotropic agent able to interfere with several biochemical steps leading to angiogenesis and to other physiopathological conditions. Since AE-941 is currently under Phase III clinical investigations, these findings are also of considerable importance for our understanding of its anticancer properties.

NADPH oxidase activity is required for endothelial cell proliferation and migration.

NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor- or serum-induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor-induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.

Matrix metalloproteinases and their inhibitors in human pituitary tumors.

OBJECTIVE: To determine the expression of matrix metalloproteinases (MMP)-1, -2, and -3 and the tissue inhibitors of metalloproteinases (TIMP)-1, -2, and -3 in 12 tissue samples from normal pituitary glands and in 28 human pituitary tumors ranging from Grade 0 to Grade IV, and to establish a correlation between the level of expression of MMPs and TIMPs and the tumor grade. METHODS: The expression of MMPs and TIMPs was determined by Western blotting. MMP activity was detected by gelatin zymography. RESULTS: MMPs were expressed in the majority of tumors, and their levels of expression were unrelated to tumor grade or to their invasive phenotype. Some correlation was observed between MMP activity detected by zymography and tumor grade. TIMP-2 and TIMP-3 were poorly expressed in high-grade tumors and strongly expressed in normal pituitary glands and in the majority of low-grade tumors. CONCLUSION: No correlation could be established between the invasive potential of tumors and MMP-1, -2, and -3 expression levels. Some correlation was observed between MMP activity detected by zymography and tumor grade. A good inverse correlation was observed between TIMP-2 and TIMP-3 expression levels and tumor grade. These data suggest that monitoring the expression of TIMP-2 and TIMP-3 or gelatinolytic activity could be of prognostic value.

Expression of matrix metalloproteinases and their inhibitors in human brain tumors.

Sixty human brain tumors, classified according to the New World Health Organization (WHO) classification including, grade I schwannomas, meningiomas and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas, grade IV glioblastomas, grade III anaplastic oligodendrogliomas and grade IV glioblastomas and lung and melanoma metastases were analyzed for the expression of three matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs) and for MMP activity. Some correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. MMP-9 levels were found to be significantly higher in grade III anaplastic astrocytomas and anaplastic oligodendrogliomas than those in grade I schwannomas and meningiomas. Anaplastic astrocytomas and Grade IV glioblastomas expressed significantly higher levels MMP-12 than grade I meningiomas. All sixty tumors showed a similar pattern of activity in zymography, proMMP-9 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were especially low in tumors of grade II and grade III but significantly higher in tumors of grade I, particularly in schwannomas. Taken together, these data suggest that: 1) a balance between MMPs and TIMPs has an important role to play in human brain tumors; 2) TIMP expression may be valuable markers for tumor malignancy.

Expression of matrix metalloproteinases and their inhibitors in human brain tumors.

Sixty human brain tumors, including grade I meningiomas, schwannomas, and pilocytic astrocytomas, grade II astrocytomas, grade III anaplastic astrocytomas and oligodendrogliomas, and grade IV glioblastomas and lung and melanoma metastases were analyzed for expression of four matrix metalloproteinases (MMPs), two tissue inhibitors of MMPs (TIMPs), and MMP activity. No marked correlation was found between MMP expression and the degree of malignancy. Western blotting analysis revealed a more uniform pattern of distribution of MMP-2 (gelatinase A) than of MMP-9 (gelatinase B) and MMP-12 (metalloelastase) among tumors. All 60 tumors showed a similar pattern of activity in zymography, MMP-2 being the major species detected. Interestingly, TIMP-1 and TIMP-2 expression levels were low in tumors of grade III but significantly higher in tumors of grade I, particularly schwannomas. Altogether, these data suggest that: (1) the balance between MMP-2 and TIMP-2 is important in human brain tumors; and (2) TIMP expression may be a valuable marker for tumor malignancy.

Low molecular weight components but not dimeric HCG inhibit growth and down-regulate AP-1 transcription factor in Kaposi's sarcoma cells.

Kaposi's sarcoma, a sexually dimorphic disease inflicting high mortality in AIDS, remains at present without effective treatment. A recent report (Nature 375:64, 1995) showed that the placental glycoprotein hormone, human chorionic gonadotropin (HCG), and surprisingly its beta subunit, inhibit tumorigenicity and metastasis of Kaposi's sarcoma cells in mice xenografts. The anti-KS efficacy of a commercial HCG was subsequently demonstrated in clinical trials. Experimental data presented herein confirm that commercial HCG preparations (known to be about 25% pure) display significant inhibitory action in a dose-dependent manner. However, pure and biologically active HCG has no effect on Kaposi's sarcoma growth in culture. In fact, incubation of Kaposi's sarcoma cells with either one of four different well characterized preparations of pure HCG dimer or any of its two subunits did not alter cellular proliferation suggesting that a contaminant (or degradation product) may be the active agent. Commercial HCG preparations were subfractionated based on molecular size and each fraction was tested with respect to inhibition of KS cell growth, HCG radioreceptor binding and steroidogenic bioactivity. Results demonstrate that the anti-KS activity resides among low molecular weight components, and not in bona fide (macromolecular) HCG. Our study indicates that HCG activity and anti-KS action are separable. Interestingly, the active components in the crude HCG markedly down-regulate AP-1, a complex of transcription factors of the immediate-early response genes associated with cell growth. We conclude that, as yet unidentified molecules, present in the commercial HCG preparations, are responsible for the growth inhibitory effects presumably via the AP-1 signalling pathway.

Expression and cytokine regulation of glucocorticoid receptors in Kaposi's sarcoma.

Development of Kaposi's sarcoma (KS) after glucocorticoid therapy has been observed in a variety of clinical states including human immunodeficiency virus-1 infection and recent in vitro studies provided evidence for a direct stimulation effect of glucocorticoid hormones on KS cell proliferation. The importance of glucocorticoids in KS pathogenesis is further highlighted by the finding that glucocorticoids synergize with cytokines to promote acquired immune deficiency syndrome (AIDS)-associated KS (AIDS-KS) growth. Furthermore, cytokine effects were abrogated by the glucocorticoid antagonist RU-486. As glucocorticoid action is mediated through activation of their intracellular cognate receptors, we hypothesized that enhanced responsiveness of AIDS-KS cells to glucocorticoids may be due to elevated glucocorticoid receptor (GR) content. Indeed, high expression of GRs in AIDS-KS tumor biopsies was detected both at the level of mRNA and protein. Quantitative measurements of GRs in these specimens by a sensitive immunoassay showed that GR content was significantly elevated in the tumor tissue (4663 fmol/mg protein) compared with the uninvolved skin of the same patients (2777 fmol/mg protein), both of which were markedly above the normal skin of healthy donors (893 fmol/mg protein). Immunocytochemical analysis confirmed the presence of GRs in the cytoplasm and the nucleus of KS cells. Interestingly, four major KS cytokines, namely interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and oncostatin M, all of which are known autocrine growth factors for AIDS-KS cells, significantly increased the expression of functional GRs in cultured AIDS-KS cells. The latter result may explain, at least in part, the synergistic effect of glucocorticoid and oncostatin M on AIDS-KS cell proliferation. Thus, the high levels of GR expression in AIDS-KS and the up-regulation of GRs by KS-growth-promoting factors may confer enhanced and sustained sensitivity to the stimulatory effects of glucocorticoids. The data presented also provide molecular bases for therapeutic interventions targeting GRs in this disease.

The regulation of insulin-like growth factor-binding protein 1 messenger ribonucleic acid in cultured rat hepatocytes: the roles of glucagon and growth hormone.

In previous studies it was shown that bovine GH (bGH) suppressed and glucagon stimulated the level of 24- and 30- to 34-kilodalton insulin-like growth factor-binding proteins (IGFBPs) in the media of cultured rat hepatocytes. In the present study we have evaluated the regulation of IGFBP-1 gene expression in primary rat hepatocyte cultures. Glucagon produced a dose-dependent stimulation of hepatocyte IGFBP-1 messenger RNA (mRNA), attaining levels 2- to 6-fold greater than control at a glucagon concentration of 100 ng/ml. GH inhibited the accumulation of IGFBP-1 mRNA in a dose-dependent manner producing, 40-70% inhibition at 50 ng/ml. The effect of glucagon was comparable to and additive with dexamethasone (1 microM). The addition of 3-isobutyl-1-methylxanthine (100 microM) and (Bu)2cAMP (100 microM) augmented IGFBP-1 mRNA levels 5- to 6-fold. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (300 nM) was found to inhibit IGFBP-1 mRNA levels by 40-50%. The inhibitory effect of bGH on IGFBP-1 mRNA levels was abolished after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h, whereas glucagon's stimulatory effect was unaffected. The addition of staurosporine (500 nM) and H-7 (1 mM) abolished the inhibitory effect of GH but also significantly inhibited the stimulatory effect of glucagon, a result consistent with these agents acting on both protein kinase C (PKC) and PKA. In the presence of 10 micrograms/ml cycloheximide, IGFBP-1 gene expression was superinduced by bGH, whereas the effect of glucagon was uninfluenced. Thus the inhibitory action of GH involves, in part, the activation of PKC. Glucagon's stimulatory effect seems to involve the activation of PKA. The inhibitory effect of bGH on IGFBP-1 gene expression may require the continuing synthesis of one or more labile protein(s).

The augmentation of insulin-like growth factor-I messenger ribonucleic acid in cultured rat hepatocytes: activation of protein kinase-A and -C is necessary, but not sufficient.

In previous studies it was shown that bovine GH (bGH) and glucagon, when individually added to primary rat hepatocyte cultures, modestly stimulated IGF-I mRNA levels 1.8- to 2.5-fold, but when combined, synergized to stimulate IGF-I mRNA levels by 10- to 12-fold. In the present study we have explored further the mechanism of this effect in primary rat hepatocyte cultures. Like glucagon, the addition of 3-isobutyl-1-methylxanthine (100 microM) or (Bu)2cAMP (150 microM) augmented IGF-I mRNA levels 1.8- to 2.0-fold, but when combined with bGH (50 ng/ml), they augmented levels up to 12-fold. The half-life of IGF-I mRNA, determined by incubating hepatocytes with actinomycin-D was 12 h. Although bGH did not affect the decay rate, glucagon (100 ng/ml) or (Bu)2cAMP (100 microM) reduced the rate of loss by about 70%. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate minimally stimulated IGF-I mRNA levels 1.2- to 1.4-fold, but displayed no synergism when added with bGH, glucagon, or (Bu)2cAMP. The stimulatory effect of bGH plus glucagon was inhibited 80% after preincubation with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (10 microM) for 24 h. The addition of staurosporine, sphingosine, or H-7 [1-(5-isoquinolinyl sulfonyl)2-methyl piperazine] inhibited the stimulatory effect of bGH plus glucagon on hepatocyte IGF-I mRNA by 80%, 90%, and 85%, respectively. Preincubation with cycloheximide (10 micrograms/ml) blocked the synergistic effect of bGH plus either glucagon or (Bu)2cAMP by 65-80%. The effect of glucagon, mediated via the activation of adenylate cyclase, involves in part the posttranscriptional stabilization of IGF-I mRNA levels. The effect of GH, mediated in part by the activation of protein kinase-C, appears to be at the level of transcription. The synergistic augmentation of hepatocyte IGF-I mRNA levels by GH and glucagon involves the activation of PKA and PKC, but also appears to require the synthesis of one or more protein(s).

Octreotide inhibits insulin-like growth factor-I hepatic gene expression in the hypophysectomized rat: evidence for a direct and indirect mechanism of action.

Somatostatin and somatostatin analogs are known to interact with the GH-insulin-like growth factor (IGF)-I axis by inhibiting GH secretion and consequently hepatic IGF-I production. Indirect evidence suggests that octreotide, a somatostatin analog, reduces serum IGF-I levels relatively more than expected from GH reduction, implying a GH-independent pathway of action. To study the role of octreotide in the regulation of IGF-I production, independently of endogenous GH, we used the hypophysectomized (hypox) rat to measure hepatic IGF-I expression and also employed cultured rat hepatocytes to examine whether octreotide has any direct effect on the production of IGF-I. Forty male hypox Sprague-Dawley rats were randomized into 4 groups to receive daily injections for 3 days of either saline, human GH (hGH) (100 g), octreotide (100 g twice), or both hGH (100 g) and octreotide (100 g twice). GH stimulated serum IGF-I levels to 104 +/- 10 micrograms/liter as compared to saline (26 +/- 2 micrograms/liter). Octreotide alone had no effect, but combining octreotide and hGH significantly reduced the hGH-induced rise in the IGF-I levels (52 +/- 6 micrograms/liter). The relative expression of hepatic IGF-I in the rats treated with hGH increased by 4-fold compared to that in the saline-treated rats. Octreotide administered simultaneously with hGH potently blocked the hGH-induced IGF-I expression to control levels. In cultured hepatocytes, IGF-I mRNA levels maximally stimulated by combining bGH and glucagon were significantly inhibited in the presence of octreotide at low concentrations (0.3 and 3 ng/ml) by 25% and 45%, respectively. In contrast, high concentrations of octreotide (30 and 300 ng/ml) had no significant effect on IGF-I mRNA abundance. We conclude that: 1) octreotide inhibits IGF-I serum levels and hepatic gene expression in the hypox rat; and 2) octreotide can inhibit partially the direct effects of GH and glucagon on hepatic IGF-I production.

The differential regulation by glucagon and growth hormone of insulin-like growth factor (IGF)-I and IGF binding proteins in cultured rat hepatocytes.

The liver is a major site of production of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGF-BPs). GH decisively influences IGF-I production. To study the role of GH and glucagon in the regulation of IGF-I and IGF-BP production, we examined IGF-I and IGF-BPs secreted by primary rat hepatocytes cultured in a serum-free medium. Glucagon (1 x 10(-8) M) stimulated IGF-I secretion and IGF-BP secretion. Bovine GH (bGH, 300 ng/ml) stimulated IGF-I secretion but suppressed IGF-BP secretion. Combining bGH and glucagon significantly augmented IGF-I secretion above the level seen with each individual agent. The inhibitory effect of bGH on IGF-BP secretion was reversed by glucagon. The major species of IGF-BPs secreted by hepatocytes were found, on Western ligand blotting, to be 24K and 30-34K. All species of secreted IGF-BPs appeared to be comparably affected by glucagon, bGH, and their combination. Northern analysis of IGF-I mRNA revealed three transcripts of 0.7-1.1 kilobases (kb), 1.8 kb, and 7.0 kb. Glucagon stimulated IGF-I mRNA levels 1.8- to 2.0-fold, whereas bGH stimulated IGF-I mRNA levels 2.0- to 2.5-fold. When hepatocytes were incubated with glucagon and bGH for 6 h, IGF-I mRNA levels were augmented 10-fold. Glucagon, in the presence of 50 ng/ml bGH, had a dose-dependent effect on IGF-I mRNA accumulation from a 6-fold level of stimulation at 50 ng/ml of glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon to a 9-fold level of stimulation at 1000 ng/ml glucagon. This study has demonstrated that glucagon, as well as GH, has significant effects on the production of both IGF-I and IGF-BPs. Of particular interest was the marked augmentation of hepatic IGF-I messenger RNA levels and the reversal of the low levels of IGF-BP production seen on adding glucagon to bGH.

Renal tubular cells are potential targets for epidermal growth factor.

Epidermal growth factor (EGF) is a potent polypeptide mitogen with various receptor-mediated growth effects on cells from the skin, breast, and gastrointestinal tract. Recent studies indicate that EGF is produced in the kidney and is excreted in the urine, but the biological significance of renal EGF is uncertain. We demonstrate in vitro mitogenicity of EGF for LLC-PK1 cells, a tubular epithelial cell line derived from pig kidney cortex. Furthermore, when subconfluent monolayers of LLC-PK1 cells are exposed to EGF for 24 h, sodium-dependent phosphate transport is stimulated (209-410% of control). These cells possess EGF-specific high-affinity binding sites at their surface (Kd 300-700 pM) but cannot synthesize the growth factor. EGF binding sites are not a peculiarity of the LLC-PK1 cell line, since similar sites are present on MDCK cells (derived from dog kidney distal tubule or collecting duct), primary cultures of mouse proximal tubular cells, and freshly prepared membrane fractions from mouse kidney. Cortical basolateral membranes are highly enriched in EGF binding sites, whereas EGF binding by brush-border membrane fractions is minimal and is compatible with contamination.

Interaction of activating protein and surfactants with human liver hexosaminidase A and GM2 ganglioside.

The effects of surfactants on the human liver hexosaminidase A-catalysed hydrolysis of G(m2) ganglioside were assessed. Some non-ionic surfactants, including Triton X-100 and Cutscum, and some anionic surfactants, including sodium taurocholate, sodium dodecyl sulphate, phosphatidylinositol and N-dodecylsarcosinate, were able to replace the hexosaminidase A-activator protein [Hechtman (1977) Can. J. Biochem.55, 315-324; Hechtman & Leblanc (1977) Biochem. J.167, 693-701) and also stimulated the enzymic hydrolysis of substrate in the presence of saturating concentrations of activator. Other non-ionic surfactants, such as Tween 80, Brij 35 and Nonidet P40, and anionic surfactants, such as phosphatidylethanolamine, did not enhance enzymic hydrolysis of G(m2) ganglioside and inhibited hydrolysis in the presence of activator. The concentration of surfactants at which micelles form was determined by measurements of the minimum surface-tension values of reaction mixtures containing a series of concentrations of surfactant. In the case of Triton X-100, Cutscum, sodium taurocholate, N-dodecylsarcosinate and other surfactants the concentration range at which stimulation of enzymic activity occurs correlates well with the critical micellar concentration. None of the surfactants tested affected the rate of hexosaminidase A-catalysed hydrolysis of 4-methylumbelliferyl N-acetyl-beta-d-glucopyranoside. Both activator and surfactants that stimulate hydrolysis of G(m2) ganglioside decrease the K(m) for G(m2) ganglioside. Inhibitory surfactants are competitive with the activator protein. Evidence for a direct interaction between surfactants and G(m2) ganglioside was obtained by comparing gel-filtration profiles of (3)H-labelled G(M2) ganglioside in the presence and absence of surfactants. The results are discussed in terms of a model wherein a mixed micelle of surfactant or activator and G(M2) ganglioside is the preferred substrate for enzymic hydrolysis.