Free radicals and ethanol-induced cytotoxicity in neural crest cells

Associations between ethanol-induced cranial neural crest cell (NCC) damage in mammalian embryos and subsequent malformations as observed in human fetal alcohol syndrome have previously been illustrated. The vulnerability of NCCs to this teratogen may result, at least in part, from their sensitivity to free radical damage. To examine relationships between free radical generation and NCC cytotoxicity, primary culture of mouse NCCs was used. NCC viability was determined in both dose- and time-response studies involving ethanol exposure. After 48 hr of culture, cell viability was significantly diminished at all doses tested (i.e., 50, 100, 150, and 200 mM ethanol). At 100 mM ethanol (a dosage that is teratogenic in vivo and in whole embryo culture), cell viability decreased to approximately 50% of control values over the first 12 hr of culture, and decreased further, to approximately 20% by 48 hr. Using nitroblue tetrazolium as a probe, it was observed that exposure of NCCs to ethanol stimulated the production of superoxide anion radicals. Co-treatment of the ethanol-exposed NCCs with free radical scavengers including 300 units/ml of superoxide dismutase, catalase (500 units/ml), or alpha-tocopherol (300 microM) significantly improved NCC viability. These results suggest that the ethanol-induced NCC injury is mediated, at least in part, through the generation of free radicals. To test this hypothesis further, NCCs were exposed in culture to xanthine/xanthine oxidase. Exogenous free radicals generated by the xanthine/xanthine oxidase system resulted in reduced NCC viability, the severity of which increased in a time and enzyme concentration-related manner. Superoxide dismutase (300 units/ml) and catalase (500 units/ml) significantly reduced the effects of the xanthine/xanthine oxidase-generated free radicals on NCC viability. The similarity between the susceptibility of NCCs to ethanol and their susceptibility to exogenous free radicals in concert with the free radical scavenger-mediated amelioration of ethanol and exogenous free radical-induced NCC death strongly suggest that free radicals play a significant role in ethanol-induced NCC death.     

Inhibition of tumour cell invasion by protease inhibitors: correlation with the protease profile

The invasive potential of eight established human tumour cell lines of different origin has been studied in the Matrigel assay. Between 25% and 70% of the cells migrated through the Matrigel layer within 24 h, indicating that invasiveness varies with the cell type. Semiquantitative measurements of the proteases MMP-2 and MMP-9, and cathepsins B and L were performed in these cell lines and the cell culture media. High invasive potential was found in those cell lines expressing high levels of cathepsins B and L or matrix metal proteases (MMP), either alone or in combination. Overexpression of one of these enzymes is enough to explain a high invasive potential of a cell line. Selective protease inhibitors at 10 nM concentration in the culture medium were used to inhibit the migration of tumour cells in the Matrigel assay. The MMP inhibitor Batimastat reduced the invasive potential of all cell lines studied independently of the MMP expression. The effect of cysteine protease inhibitors was strongly correlated with the protease profile of the tumour cell line. Our findings support the hypothesis of a very complex activation cascade of matrix-degrading proteolytic enzymes and they underline the need to analyse the protease profile of any tumour before beginning an antiproteolytic tumour treatment.     

Chemopreventive agents: protease inhibitors

Certain protease inhibitors, called the anticarcinogenic protease inhibitors in this review, are capable of preventing carcinogenesis in a wide variety of in vivo and in vitro model systems. The anticarcinogenic protease inhibitors are extremely potent agents with the ability to prevent cancer, with some unique characteristics as anticarcinogenic agents. The anticarcinogenic protease inhibitors have the ability to irreversibly suppress the carcinogenic process. They do not have to be continuously present to suppress carcinogenesis. They can be effective when applied in both in vivo and in vitro carcinogenesis assay systems at long time periods after carcinogen exposure, and are effective as anticarcinogenic agents at extremely low molar concentrations. While several different types of protease inhibitors can prevent the carcinogenic process, the most potent of the anticarcinogenic protease inhibitors on a molar basis are those with the ability to inhibit chymotrypsin or chymotrypsin-like proteases. The soybean-derived protease inhibitor, Bowman-Birk inhibitor (BBI), is a potent chymotrypsin inhibitor that has been extensively studied for its ability to prevent carcinogenesis in many different model systems. Much of this review is focused on the characteristics of BBI as the anticarcinogenic protease inhibitor, as this is the protease inhibitor that has risen to the human trial stage as a human cancer chemopreventive agent. Part of this review hypothesizes that the Bowman-Birk family of protease inhibitors plays a role in plants similar to that of alpha1-antichymotrypsin in people. Both BBI and alpha1-antichymotrypsin are potent inhibitors of chymotrypsin and chymotrypsin-like enzymes, are highly anti-inflammatory, and are thought to play important roles in the defense of their respective organisms. It is believed that BBI will be shown to play a major role in the prevention and/or treatment of several different diseases, in addition to cancer.     

Cerebral vasospasm and free radicals

The literature implicating free radical reactions in the genesis of cerebral vasospasm following aneurysmal subarachnoid hemorrhage is reviewed. While this condition has features of a prototypical free radical-mediated disease and a plausible theory can be outlined, data to support the theory are limited. An association of lipid peroxidation with vasospasm has been observed, but more sophisticated techniques for detection of free radicals and for detection of free radical damage to arterial wall proteins and nucleic acids have not been used. There are conflicting reports about efficacy of various antioxidant treatments for vasospasm. In these studies, concomitant experiments have usually not confirmed that the treatments have decreased free radicals or lipid peroxides in cerebrospinal fluid. Because smooth muscle contraction is involved in vasospasm, it would be interesting to investigate the actions of free radicals on smooth muscle cells using, for example, isometric tension recordings and patch clamp techniques. Studies of cardiac myocytes indicate that free radicals alter conductances through potassium and calcium channels and through the sodium-calcium exchanger and may result in elevations in intracellular calcium. Few studies have been performed on cerebral smooth muscle cells. In one study, exposure of cerebrovascular smooth muscle cells to free radicals resulted in increased outward currents, decreased membrane resistance, cell contraction, appearance of membrane blebs, and cell death. In summary, more investigations using better experimental techniques are required before free radicals and reactions induced by them can be said with certainty to be the primary cause of vasospasm.     

Optimal utilization of cryopreserved human semen for assisted reproduction: recovery and maintenance of sperm motility and viability

PURPOSE: Our purpose was to evaluate sperm motility and viability and the maintenance of these parameters in already cryopreserved semen samples following repeated freezing/thawing cycles. METHODS: Human spermatozoa were subjected to five cycles of cryopreservation/thawing. Recovery of sperm motility and viability and the proportion of viable nonmotile sperm were determined up to 6 hr after thaw. RESULTS: Sperm motilities (prefreeze motility, 70.1%; n = 9 samples) after each of five freeze/thaw cycles were 24.4, 8.0, 3.5, 1.5 and 1.8%. The recovery of sperm viability was higher than that of motility after each cycle: 39.1, 25.3, 22.6, 17.8, and 16.5%. Recoveries of motility and viability were improved if the thawed samples were left in the original cryopreservation medium prior to refreezing vs. if a washing/ resuspension step was included. The recovery of sperm motility in the first thawing cycle was indicative of the expected motile sperm recovery in the second thawing cycle. CONCLUSIONS: Cryopreserved semen that is intended to be reused in future assisted reproduction treatments should be thawed only once and aliquoted in the original freezing medium before refreezing. The recovery of sperm motility and viability in the second thawing cycle, thus the applicability of the sample in conventional in vitro fertilization or intracytoplasmic sperm injection may be anticipated in > 90% of the samples. In view of intracytoplasmic sperm injection it is important that sperm viability is maintained better than motility; after the first, second, and third thawing cycles the ratios of motile:nonmotile viable sperm were 1:1, 1:4, and 1:7, respectively.     

Purification and characterization of a novel isoform of mast cell tryptase from rat tongue

Rat mast cell tryptase was purified to homogeneity from rat tongue by a series of standard chromatographic procedures. Since the enzyme gave band corresponding to molecular mass of 32-35 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis and exhibited a molecular mass of 135 kDa on gel filtration, it was presumed to be a noncovalently associated tetramer. The N-terminal amino acid sequence of 50 residues of the enzyme showed the highest degree of homology with the same region in mouse mast cell protease 7 (92%), and less homology to those of tryptases from man and dog, and peritoneal cells of rats and Mongolian gerbils. The inhibitor specificity of rat tongue tryptase was similar to that of rat peritoneal mast cell tryptase free from trypstatin: it was inhibited by alpha 1-antitrypsin, Kunitz-type soybean trypsin inhibitor and Bowman-Birk soybean trypsin inhibitor, but these inhibitors do not inhibit the tryptases from rat skin, human lung, and dog mast cells. Judging from these results, together with other enzymatic properties, the enzyme may be a novel isoform of tryptase in rat tongue. Analysis by differential staining with peroxidase-labeled lectins of the enzyme suggested that it has tri- and/or tetraantennary complex-type oligosaccharides containing a relatively high amount of sialic acid. The immunohistochemical distribution of this enzyme indicated that the reactive antigen was specific in connective tissue but not in mucosal mast cells.     

The skin, free radicals, and oxidative stress

Oxidative stress involves the adverse effects of oxygen and other free radicals on living tissue. An overview of the various types of free radicals generated in the body, the basic chemistry of free radicals, and how they arise is presented. The importance of understanding the action of free radicals on specific target tissues in the skin and how this affects the physiology of the skin in relation to treatment is covered.     

Exposure to lysosomotropic amines and protease inhibitors retard corneal endothelial cell migration along the natural basement membrane during wound repair

Regulation of cell migration along the natural basement membrane during wound repair in the organ culture corneal endothelium was investigated using various lysosomotropic amines and protease inhibitors. Following a circular transcorneal freeze injury, cells within the area die and expose the underlying basement membrane (Descemet's membrane). During normal wound repair, cells traverse this expanse and repopulate the region by approximately 48 h postinjury. During this time, acid phosphatase histochemistry revealed distinct alterations in the lysosomal population of cells that were adjacent to, and migrated into, the wound region. To explore whether relationships may exist between changes in the lysosome population and cell migration, injured endothelia were organ cultured in the presence of either methylamine or chloroquine, two lysosomotropic amines. Methylamine significantly retarded cell translocation (85%) into the injury zone when compared to nontreated controls. In comparison, chloroquine was less effective in restricting injury-induced cell migration and propylamine, also a lysosomotropic amine, had no influence on the repair process. In addition, two serine/thio protease inhibitors, leupeptin and antipain, were both able to impede cell translocation during wound repair by 85 and 52%, respectively, whereas soybean trypsin inhibitor, a serine protease inhibitor, exhibited no inhibitory effect on the repair process. Similarly, incubating injured tissues in either 1,10-phenanthroline or phosphoramidon, both metalloproteinase inhibitors, did not prevent endothelial cell movement nor wound repair. Results indicate that corneal endothelial cell migration along the natural basement membrane is dependent on protease function. Although the precise nature of the proteases involved has yet to be ascertained, results indicate that lysosomal enzymes may have a distinct role in corneal endothelial cell movement along the natural basement membrane during wound repair.     

Treatment with protease inhibitors associated with peripheral insulin resistance and impaired oral glucose tolerance in HIV-1-infected patients

BACKGROUND: The use of protease inhibitors in the treatment of HIV-1 infection is associated with the new onset of diabetes mellitus, hyperlipidaemia and lipodystrophy. It is unclear whether these findings are coincidental or whether they reflect a causative effect of protease inhibitors. OBJECTIVE: To evaluate the effect of treatment with protease inhibitors on insulin sensitivity, oral glucose tolerance and serum lipids in HIV-infected patients in order to determine whether treatment with protease inhibitors can cause peripheral insulin resistance. DESIGN: Cross-sectional controlled study in HIV-infected patients treated with protease inhibitors to assess insulin sensitivity, oral glucose tolerance and changes in serum lipids. METHODS: Sixty-seven patients treated with protease inhibitors, 13 therapy-naive patients and 18 HIV-negative control subjects were tested for insulin sensitivity (intravenous insulin tolerance test). In a subgroup of 24 treated patients, oral glucose tolerance was determined. Serum lipids prior to and under treatment with protease inhibitors were compared. RESULTS: Patients on protease inhibitors had a significantly decreased insulin sensitivity when compared with therapy-naive patients (median, 75 and 156 micromol/l/min, respectively; P < 0.001). All treated patients with impaired (n=4) or diabetic (n=9) oral glucose tolerance, and four out of 11 patients with normal glucose tolerance showed peripheral insulin resistance; all therapy-naive patients had normal insulin sensitivity. Treatment with protease inhibitors led to a significant increase in total triglycerides and cholesterol in the 67 treated patients (median increase, 113 and 37 mg/ml, respectively). CONCLUSION: Treatment with protease inhibitors is associated with peripheral insulin resistance, leading to impaired or diabetic oral glucose tolerance in some of the patients, and with hyperlipidaemia. Overall, there is a large variation in the severity and clinical presentation of protease inhibitor-associated metabolic side-effects.     

Heterocycle-containing bisphosphonates cause apoptosis and inhibit bone resorption by preventing protein prenylation: evidence from structure-activity relationships in J774 macrophages

Recent evidence suggests that bisphosphonates (BPs) may inhibit bone resorption by mechanisms that lead to osteoclast apoptosis. We have previously shown that BPs also reduce cell viability and induce apoptosis in the macrophage-like cell line J774. To determine whether BPs inhibit osteoclast-mediated bone resorption and affect J774 macrophages by the same molecular mechanism, we examined the potency to reduce J774 cell viability of pairs of nitrogen-containing BPs that differ slightly in the structure of the heterocycle-containing side chain but that differ markedly in antiresorptive potency. In all cases, the most potent antiresorptive BP of each pair also caused the greatest loss of J774 viability, while the less potent antiresorptive BPs were also less potent at reducing J774 cell viability. Similarly, the bisphosphinate, phosphonoalkylphosphinate and monophosphonate analogs of BPs (in which one or both phosphonate groups are modified, giving rise to much less potent or inactive antiresorptive agents) were much less potent or inactive at reducing J774 cell viability. Thus, the structure-activity relationships of BPs for inhibiting bone resorption match those for causing loss of cell viability in J774 cells, indicating that BPs inhibit osteoclast-mediated bone resorption and reduce J774 macrophage viability by the same molecular mechanism. Loss of J774 cell viability after treatment with BPs was associated with a parallel increase in apoptotic cell death. We have recently proposed that nitrogen-containing BPs reduce cell viability and cause J774 apoptosis as a consequence of inhibition of enzymes of the mevalonate pathway and hence loss of prenylated proteins. In this study, the BPs that were potent inducers of J774 apoptosis and potent antiresorptive agents were also found to be effective inhibitors of protein prenylation in J774 macrophages, whereas the less potent BP analogs did not inhibit protein prenylation. This provides strong evidence that BPs with a heterocyclic, nitrogen-containing side chain, such as risedronate, inhibit osteoclast-mediated bone resorption and induce J774 apoptosis by preventing protein prenylation.     

Antibody selection for immunohistochemical survey of equine tissue

A membrane-bound protease induced by sulfur mustard in cultured normal human epidermal keratinocytes (NHEK) was purified and partially characterized. Maximum enzyme stimulation occurred at 16 hr after normal human epidermal keratinocytes were exposed to 300 microM sulfur mustard. Purification to homogeneity of the protease was accomplished by Triton X-100 solubilization, ultracentrifugation, and dialysis, followed by ion-exchange chromatography through DEAE-cellulose and finally hydrophobic column chromatography through phenyl Sepharose. Analysis of the purified enzyme by SDS-PAGE revealed a single polypeptide at the 80 kDa region. Further investigation of biochemical properties showed that a synthetic serine-specific Chromozym TRY peptide and the physiological protein laminin were good substrates for this enzyme. Moreover, this enzyme was inhibited mostly by the serine-protease inhibitors leupeptin and di-isopropyl fluorophosphate and not by the cysteine protease inhibitor E-64 or the metalloprotease inhibitor 1,10-phenanthroline (Component H, CH), indicating the serine protease nature of this enzyme. This enzyme had a pH optimum in the range of 7.0 to 8.0. Amino acid sequencing of the purified enzyme revealed that this enzyme belongs to the endopeptidase family (serine protease), and is homologous with a mammalian-type bacterial serine endopeptidase that can preferentially cleave K-X, including K-P. These results suggest that serine-protease stimulation may be one of the mechanisms of mustard-induced skin blister formation, and that some specific serine-protease inhibitors may be useful for the treatment of this sulfur mustard toxicity.     

Protease inhibitors in patients with HIV disease. Clinically important pharmacokinetic considerations

Since its introduction in 1987, zidovudine monotherapy has been the treatment of choice for patients with HIV infection. Unfortunately it has been established that the beneficial effects of zidovudine are not sustained due to the development of resistant viral strains. This has led to the strategy of combination therapy, and in 1995 treatment with zidovudine plus didanosine, or zidovudine plus zalcitabine, was demonstrated to be more effective than zidovudine monotherapy in preventing disease progression and reducing mortality in patients with HIV disease. Recent work demonstrates an even greater antiviral effect from triple therapy with 2 nucleosides, zidovudine plus zalcitabine with the addition of saquinavir, a new protease inhibitor drug. The HIV protease enzyme is responsible for the post-translational processing of gag and gag-pol polyprotein precursors, and its inhibition by drugs such as saquinavir, ritonavir, indinavir and VX-478 results in the production of non-infectious virions. As resistance may also develop to the protease inhibitors they may be used in combination, and future strategies may well include quadruple therapy with 2 nucleoside analogues plus 2 protease inhibitors. Administration of protease inhibitors alone or in combination with other drugs does raise a number of important pharmacokinetic issues for patients with HIV disease. Some protease inhibitors (e.g. saquinavir) have kinetic profiles characterised by reduced absorption and a high first pass effect, resulting in poor bioavailability which may be improved by administrating with food. Physiological factors including achlorhydria, malabsorption and hepatic dysfunction may influence the bioavailability of protease inhibitors in HIV disease. Protease inhibitors are very highly bound to plasma proteins (> 98%), predominantly to alpha 1-acid glycoprotein. This may influence their antiviral activity in vitro and may also predispose to plasma protein displacement interactions. Such interactions are usually only of clinical relevance if the metabolism of the displaced drug is also inhibited. This is precisely the situation likely to pertain to the protease inhibitors, as ritonavir may displace other protease inhibitor drugs, such as saquinavir, from plasma proteins and inhibit their metabolism. Protease inhibitors are extensively metabolised by the cytochrome P450 (CYP) enzymes present in the liver and small intestine. In vitro studies suggest that the most influential CYP isoenzyme involved in the metabolism of the protease inhibitors is CYP3A, with the isoforms CYP2C9 and CYP2D6 also contributing. Ritonavir has an elimination half-life (t1/2 beta) of 3 hours, indinavir 2 hours and saquinavir between 7 and 12 hours. Renal elimination is not significant, with less than 5% of ritonavir and saquinavir excreted in the unchanged form. As patients with HIV disease are likely to be taking multiple prolonged drug regimens this may lead to drug interactions as a result of enzyme induction or inhibition. Recognised enzyme inducers of CYP3A, which are likely to be prescribed for patients with HIV disease, include rifampicin (rifampin) [treatment of pulmonary tuberculosis], rifabutin (treatment and prophylaxis of Mycobacterium avium complex), phenobarbital (phenobarbitone), phenytoin and carbamazepine (treatment of seizures secondary to cerebral toxoplasmosis or cerebral lymphoma). These drugs may reduce the plasma concentrations of the protease inhibitors and reduce their antiviral efficacy. If coadministered drugs are substrates for a common CYP enzyme, the elimination of one or both drugs may be impaired. Drugs which are metabolised by CYP3A and are likely to be used in the treatment of patients with HIV disease include the azole antifungals, macrolide antibiotics and dapsone; therefore, protease inhibitors may interact with these drugs. (ABSTRACT TRUNCATED)     

GA strategy for variable selection in QSAR studies: GA-based PLS analysis of calcium channel antagonists

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.     

Evaluation of methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as reagents in flow cytometry of hematopoietic progenitor cells

BACKGROUND: Flow cytometry is used to quantitate CD34+ hematopoietic progenitor cells for transplantation. The present study evaluates methods for preparing and thawing cryopreserved CD34+ and CD34- cell lines for use as flow cytometry reagents. STUDY DESIGN AND METHODS: The human myeloid leukemic cell lines KG 1 a (CD34+) and K562 (CD34-) were grown in culture under standard conditions and then prepared on ficoll gradients of different densities to determine which gave the component that was most reproducible. After ficoll preparation, the cells were frozen in standard cryopreservation media and four methods of thawing were examined. Determination of the method that gave the cell component that was most reproducible was based on viability, percentage of cell recovery, and maintenance of CD34 antigenicity status. RESULTS: Ficoll gradient preparation improved the ease of flow cytometry analysis when original viability was low, and it produced a more uniform cell population. However, it resulted in significant cell loss for both cell lines. While the cell recovery for K562 cells was not significantly different with any of the densities of ficoll, recovery was significantly better for KG 1 a cells with ficoll at a specific gravity of 1.077. Of the thawing methods examined, all three that involved a rapid thaw at 37 degrees C were statistically equivalent to each other and were better than thawing at 4 degrees C. CONCLUSION: With a standardized method of preparing cell lines as reagents for quality control purposes, data comparison among cell processing laboratories may more readily be initiated. Such cell lines could also be useful as a teaching tool for flow cytometry and in proficiency testing.     

Loss of heterozygosity on chromosome 16 in sporadic Wilms'' tumour

Activated cell-mediated immunity, associated for example with HIV infection, is accompanied by elevated concentrations of neopterin and 7,8-dihydroneopterin. Recent data have indicated a role of neopterin derivatives in virus activation and apoptotic cell death, processes likely to involve the action of oxygen free radicals. Because T cell death in AIDS is likely to involve the Fas/Fas ligand system and the action of oxygen free radicals and 7,8-dihydroneopterin, we compared the kinetics and sensitivity of apoptotic cell death of human leukemic Jurkat T cells to that of treatments with 7,8-dihydroneopterin, anti-Fas, and H2O2. Upon incubation with 5 mM 7,8-dihydroneopterin and 50 microM hydrogen peroxide over a period of 24 hr, bimodal kinetics were observed with peaks at 5.5 hr (7,8-dihydroneopterin, 13.1%; H2O2, 11.4%) and at 24 hr (7,8-dihydroneopterin, 11.2%; H2O2, 13.2%). In contrast, anti-Fas (20 ng/mL)-induced apoptosis increased steadily over time, peaking at 11 hr (43.2%). Interestingly, anti-Fas-induced apoptosis was suppressed upon co-incubation with 7,8-dihydroneopterin and H2O2 by 62% and 68%, respectively. We also compared the sensitivity to drug treatments of apoptosis induced by 7,8-dihydroneopterin, anti-Fas antibodies, and H2O2. 7,8-Dihydroneopterin-mediated, and similarly anti-Fas- and H2O2-mediated, apoptosis was not inhibited by a broad range of pharmacological inhibitors, such as actinomycin D, cycloheximide, cyclosporin A, and various protein kinase inhibitors. On the contrary, inhibitors with antioxidant abilities, such as pyrrolidinedithiocarbamate, significantly blocked 7,8-dihydroneopterin-, H2O2- as well as anti-Fas-mediated apoptosis. These results imply that 7,8-dihydroneopterin-, H2O2-, and anti-Fas-mediated cell death might involve related redox sensitive signal transduction pathways.     

Verapamil inhibits tumor protease production, local invasion and metastasis development in murine carcinoma cells

The invasion and metastasis process involves degradation of the extracellular matrix mediated by tumor- and host-produced proteolytic enzymes. The main enzymes involved in this process are urokinase-type plasminogen activator (uPA) and the matrix metalloproteinases (MMPs). Calcium is a main co-factor in the signaling pathways that regulate cell proliferation and protease production. We have studied here the effect of verapamil, a calcium channel blocker widely used to treat hypertensive diseases, on local tumor growth, spontaneous and experimental metastasis development, tumor-associated protease production and circulating MMP activity in tumor-bearing mice. BALB/c mice treated for 45 days with verapamil showed no toxic effects. Oral administration of verapamil to mice injected with F311 tumor cells, either pre-treated or not with verapamil, showed a significant decrease of local tumor invasion and both spontaneous and experimental metastasis development (51.3% inhibition of metastasis in both cases, p < 0.01). uPA and MMP-9 production by tumor cells in vitro was significantly inhibited by verapamil in a dose-dependent manner, showing a long-term inhibition after removal of the drug. Verapamil also exhibited a marked cytostatic effect on F311 cell proliferation in vitro. In addition, circulating MMP activity, usually enhanced in tumor-bearing mice, diminished significantly with all verapamil treatments. Our results suggest that modulation of the calcium-dependent signaling pathways that regulate tumor- or host-dependent production of proteases and tumor cell proliferation could contribute to the inhibition of metastasis development. Finally, we describe the inhibitory effects of a commonly used hypotensor in humans, verapamil, on the invasive and metastatic capacity of mammary tumor cells.     

Effect of dietary phytochemicals on cancer development (review)

Vegetables, fruits, and whole grains contain a wide variety of phytochemicals that have the potential to modulate cancer development. There are many biologically plausible reasons why consumption of plant foods might slow or prevent the appearance of cancer. These include the presence in plant foods of such potentially anticarcinogenic substances as carotenoids, chlorophyll, flavonoids, indole, isothiocyanate, polyphenolic compounds, protease inhibitors, sulfides, and terpens. The specific mechanisms of action of most phytochemicals in cancer prevention are not yet clear but appear to be varied. Considering the large number and variety of dietary phytochemicals, their interactive effects on cancer risk may be extremely difficult to assess. Phytochemicals can inhibit carcinogenesis by inhibiting phase I enzymes, and induction of phase II enzymes, scavenge DNA reactive agents, suppress the abnormal proliferation of early, preneoplastic lesions, and inhibit certain properties of the cancer cell.     

Pathogenesis of HIV-1-protease inhibitor-associated peripheral lipodystrophy, hyperlipidaemia, and insulin resistance

HIV-1 protease-inhibitor treatments are associated with a syndrome of peripheral lipodystrophy, central adiposity, breast hypertrophy in women, hyperlipidaemia, and insulin resistance. The catalytic region of HIV-1 protease, to which protease inhibitors bind, has approximately 60% homology to regions within two proteins that regulate lipid metabolism: cytoplasmic retinoic-acid binding protein type 1 (CRABP-1) and low density lipoprotein-receptor-related protein (LRP). We hypothesise that protease inhibitors inhibit CRABP-1-modified, and cytochrome P450 3A-mediated synthesis of cis-9-retinoic acid, a key activator of the retinoid X receptor; and peroxisome proliferator activated receptor type gamma (PPAR-gamma) heterodimer, an adipocyte receptor that regulates peripheral adipocyte differentiation and apoptosis. Protease-inhibitor binding to LRP would impair hepatic chylomicron uptake and triglyceride clearance by the endothelial LRP-lipoprotein lipase complex. The resulting hyperlipidaemia contributes to central fat deposition (and in the breasts in the presence of oestrogen), insulin resistance, and, in susceptible individuals, type 2 diabetes. Understanding the syndrome's pathogenesis should lead to treatment strategies and to the design of protease inhibitors that do not cause this syndrome.