The role of statins in the differentiation and function of bone cells.

BACKGROUND: Statins are 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors blocking cholesterol biosynthesis in hepatic cells, thereby causing an increase in low-density lipoprotein (LDL) receptors resulting in enhanced uptake and clearance of atherogenic LDL-cholesterol (LDL-C) from the blood. Accordingly, statins decrease the risk of developing atherosclerosis and its acute complications, such as acute myocardial infarction and ischaemic stroke. Besides the LDL-C-lowering impact, statins also have other so-called pleiotropic effects. Among them, the ability to modulate differentiation and function of bone cells and exert direct effects on osteosynthesis factors. Specifically, earlier studies have shown that statins cause in vitro and in vivo osteogenic differentiation. DESIGN: The most relevant papers on the bone-related 'pleiotropic' effects of statins were selected following literature search in databases and were reveiwed. RESULTS: Statins increase the expression of many mediators involved in bone metabolism including bone morphogenetic protein-2 (BMP-2), glucocorticoids, transforming growth factor-beta (TGF-ß), alkaline phosphatase (ALP), type I collagen and collagenase-1. As a result, they enhance bone formation and improve bone mineral density by modulating osteoblast and osteoclast differentiation. CONCLUSION: This review summarizes the literature exploring bone-related 'pleiotropic' effects of statins and suggests an anabolic role in the bone tissue for this drug class. Accordingly, current knowledge encourages further clinical trials to assess the therapeutic potential of statins in the treatment of bone disorders, such as arthritis and osteoporosis.

Allantoin may modulate aging impairments, symptoms and cancers.

Allantoin increases in different stress conditions and environment such as physical activity, amniotic fluids and oxidative stress. So, we inspired to explore the role of allantoin as a metabolic by-product in health improvement and protection using irradiation as simulator for oxidative stress. Allantoin was injected i.p. (100 mg/kg) in senile male rats in irradiated and non-irradiated groups in comparison to sham operated group. The studied parameters were superoxide dismutase, Glutathione reductase, Glutathione, total antioxidant capacity, collagenase, urea, creatine kinase, alanine transaminase, aspartate aminotransferase, triglycerides, total cholesterol, and HDL and LDL cholesterol. Allantoin in vitro antitumor activity was MTT assayed for some age dependent cancers. Allantoin showed improvement in all in vivo studied oxidative stress parameters. Allantoin showed an increase in lipogenesis was recorded as a hepatic energy targeting muscles. Allantoin improves aging process indicated by its collagenase inhibitory effect. Allantoin showed cytotoxicity against prostate, colon, intestinal ovarian and breast cancers and weak inhibitory against larynx cancer. Allantoin may be the possible mysterious key factor involved in health and aging improvement and cancer protection in stress conditions such as physically activity and radiation hazards.

Inhibitory activity of Podospermum canum and its active components on collagenase, elastase and hyaluronidase enzymes.

Present study is aimed to investigate in vitro inhibitory effects of the extract prepared from the aerial parts of Podospermum canum (syn: Scorzonera cana var. jacquiniana) (Asteraceae) on hyaluronidase, collagenase, and elastase enzymes using a bioassay-guided fractionation. Inhibitory effects of the extract, sub-extracts, fractions obtained by column chromatography, and isolated compounds on collagenase, elastase, and hyaluronidase were performed by using in vitro enzyme inhibitory assays based on spectrophotometric evaluation. The methanolic extract obtained from P. canum exhibited strong inhibitory activities on elastase and collagenase while the insignificant activity was observed on hyaluronidase. Through bioactivity-guided fractionation, the ethyl acetate and remaining water sub-extracts obtained from the methanolic extract displayed significant inhibitory activities on collagenase and elastase, while petroleum ether and chloroform extracts did not show any inhibitory activity. Eleven known compounds: arbutin, 6́-O-caffeoylarbutin, cichoriin, 3,5-dicaffeoylquinic acid methyl ester, apigenin 7-O-ß-glucoside, luteolin 7-O-ß-glucoside, apigenin 7-O-ß-rutinoside, isoorientin, orientin, vitexin, procatechuic acid, and new compound 4-hydroxy-benzoic acid 4-(6-O-α-rhamnopyranosyl-ß-glucopyranosyl) benzyl ester have been obtained from ethyl acetate sub-extract. Results of the present study have revealed that apigenin 7-O-ß-glucoside, luteolin 7-O-ß-glucoside, apigenin 7-O-ß-rutinoside, and isoorientin showed potent enzyme inhibitory activities. However, methanolic extract of P. canum displayed a greater inhibitory activity than fractions and isolated compounds both on collagenase and elastase.

Absolute Configuration of Mycosporine-Like Amino Acids, Their Wound Healing Properties and In Vitro Anti-Aging Effects.

Mycosporine-like amino acids (MAAs) are water-soluble metabolites, reported to exhibit strong UV-absorbing properties. They have been found in a wide range of marine organisms, especially those that are exposed to extreme levels of sunlight, to protect them against solar radiation. In the present study, the absolute configuration of 14 mycosporine-like-amino acids was determined by combining the results of electronic circular dichroism (ECD) experiments and that of advanced Marfey's method using LC-MS. The crystal structure of a shinorine hydrate was determined from single crystal X-ray diffraction data and its absolute configuration was established from anomalous-dispersion effects. Furthermore, the anti-aging and wound-healing properties of these metabolites were evaluated in three different assays namely the inhibition of collagenase, inhibition of advanced glycation end products (AGEs) and wound healing assay (scratch assay).

Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases.

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.

In Vitro Chondroprotective Potential of Extracts Obtained from Various Phyllantus Species.

Phyllanthus amarus has been proven to exhibit chondroprotection. Regarding the morphological similarities among Phyllanthus species, we were attracted to evaluate the chondroprotective potential of Phyllanthus species including P. amarus obtained from Chiang Mai and Phuket, Phyllanthus urinaria L., Phyllanthus urinaria subsp. chamaepeuce, Phyllanthus debilis, and Phyllanthus airy-shawii using interleukin-1ß-induced degradation of cartilage explants. The ethanolic extracts of the plants were evaluated for major lignans, phyllanthin, and hypophyllanthin by HPLC and further measurements of the total contents of flavonoids and phenolic compounds along with the assays for antioxidant and anti-collagenase activities. The interleukin-1ß-induced cartilage explant degradation was performed with/without the extracts at concentrations of 50-250 µg/mL. After 4-14 days of incubation, the medium was assayed for the level of sulfated glycosaminoglycans while the explants were measured for the remaining content of uronic acid. Proteoglycan intensity in the explants was determined by safranin O staining. Diacerein, the antiarthritic agent, was used as the positive control. Although the two major lignans were found in P. amarus from Chiang Mai, P. amarus from Phuket, and P. urinaria L. extracts, similar chondroprotective activities were observed in all Phyllanthus extracts. Total phenolic content and total flavonoid content of the extracts showed a correlation with antioxidation, whereas the total phenolic content correlated with anti-collagenase activity. Among the six extracts, P. airy-shawii showed the greatest antioxidant and collagenase inhibitory activities. The results revealed that chondroprotective activities of all of the extracts of Phyllanthus species might result from an additive or synergistic influence of some constituents of these plants, which could be considered for antiarthritic purposes.

Thai plants with high antioxidant levels, free radical scavenging activity, anti-tyrosinase and anti-collagenase activity.

BACKGROUND: Ultraviolet radiation from sunlight induces overproduction of reactive oxygen species (ROS) resulting in skin photoaging and hyperpigmentation disorders. Novel whitening and anti-wrinkle compounds from natural products have recently become of increasing interest. The purpose of this study was to find products that reduce ROS in 14 Thai plant extracts. METHODS: To determine total phenolic and flavonoid content, antioxidant activity, anti-tyrosinase activity and anti-collagenase activity, we compared extracts of 14 Thai plants prepared using different solvents (petroleum ether, dichloromethane and ethanol). Antioxidant activities were determined by DPPH and ABTS assays. RESULTS: Total phenolic content of the 14 Thai plants extracts was found at the highest levels in ethanol followed by dichloromethane and petroleum ether extracts, respectively, while flavonoid content was normally found in the dichloromethane fraction. Scavenging activity ranged from 7 to 99% scavenging as assessed by DPPH and ABTS assays. The ethanol leaf extract of Ardisia elliptica Thunb. had the highest phenolic content, antioxidant activity and collagenase inhibition, while Cassia alata (L.) Roxb. extract had the richest flavonoid content. Interestingly, three plants extracts, which were the ethanolic fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) Roxb., had high antioxidant content and activity, and significantly inhibited both tyrosinase and collagenase. CONCLUSION: Our finding show that the ethanol fractions of Annona squamosa L., Ardisia elliptica Thunb. and Senna alata (L.) Roxb. show promise as potential ingredients for cosmetic products such as anti-wrinkle agents and skin whitening products.

The effects of tacrolimus on colonic anastomotic healing in rats.

AIM: The aim of this experimental study is to investigate the effects of tacrolimus on colonic anastomotic healing after subcutaneous administration. MATERIALS AND METHODS: Forty Albino-Wistar male rats were divided into two groups, with two equal subgroups each. They all underwent colonic resection followed by a single-layer, inverted colon anastomosis and were injected subcutaneously with either 1 ml of 0.9% NaCl solution or tacrolimus (0.1 mg/kg body weight) depending on their group. Half of the rats were sacrificed on the fourth postoperative day, while the remaining half were sacrificed on the eighth postoperative day. Macroscopical and histological assessment was performed, while anastomotic bursting pressures and the tissue concentrations in hydroxyproline and collagenase I were evaluated. RESULTS: On the fourth postoperative day, the bursting pressures (217.00 ± 11.12, p < 0.001), the fibroblast activity (2.80 ± 0.42, p = 0.022), the neoangiogenesis (2.10 ± 0.32, p = 0.007) and the tissue hydroxyproline concentration (254.23 ± 67.10, p = 0.001) were significantly higher in the tacrolimus-treated animals. Furthermore, tacrolimus significantly decreased the inflammatory cell infiltration (1.50 ± 0.53, p < 0.001) and the tissue collagenase I concentration (4.16 ± 0.76, p = 0.002). On the eighth day, the bursting pressure (264.00 ± 32.61, p < 0.001) and the hydroxyproline tissue concentration (331.04 ± 55.56, p = 0.002) were significantly higher in the tacrolimus subgroups. The inflammatory cell infiltration (1.20 ± 0.42, p < 0.001) and the collagenase I concentration (1.61 ± 0.83, p < 0.001) were significantly lower. In addition, the adhesion formation score was significantly lower (1.20 ± 0.92, p = 0.065). CONCLUSION: Tacrolimus, when injected subcutaneously, promotes healing of colonic anastomoses in rats. It impairs not only inflammatory response but also collagen degradation, resulting to increased anastomotic strength on the fourth as well as on the eighth postoperative day.

Sublethal doses of surfactants induce premature senescence in normal human skin cells.

We evaluated the cytotoxicity of surfactants in human cells. Synthetic surfactants showed different cytotoxicity levels depending on their structures. The cytotoxicity of commercial washing products was determined mainly by the contents of surfactants. All of them induced premature senescence in normal cells, but not in tumor-derived or immortalized cells, under sublethal conditions. Residual surfactants might be a risk factor for skin aging.

The effects of iloprost on colonic anastomotic healing in rats.

PURPOSE: The purpose of this experimental study is to investigate the effects of iloprost on colonic anastomotic healing in rats, after intraperitoneal administration. METHODS: Forty male Albino-Wistar rats were randomized into two groups of twenty animals each. They all underwent colonic resection followed by an inverted anastomosis. The rats of Group A (control) received 3 ml of NaCl intraperitoneally, while those of Group B (iloprost) received iloprost (2 µg/kg body weight), immediately postoperatively and daily until killed. Each group was further divided into two equal subgroups, depending on the day of killing. The animals of subgroups 1 were killed on the fourth postoperative day, while those of subgroups 2 on the eighth. Macroscopical and histological assessments were performed. Besides, anastomotic bursting pressures and the tissue concentrations in hydroxyproline and collagenase I were also evaluated. RESULTS: No anastomotic dehiscence was noted. The mean bursting pressure was higher in the iloprost group compared with the control group, but a significant difference was revealed only on the fourth postoperative day. Furthermore, iloprost significantly increased the new vessel formation on the fourth, as well as on the eighth postoperative day. CONCLUSION: Iloprost enhances the early phase of colonic anastomotic healing in rats.

Herbal Infusions as a Valuable Functional Food.

Herbal infusions are an underestimated and easy to intake a source of biologically active natural compounds (polyphenols), which, in the dissolved form, are more easily absorbed. Therefore, this study aimed to assess the potential of herbal infusions as a functional food to reduce postprandial hyperglycemia (inhibition of α-amylase and α-glucosidase) and to reduce the effects of increased blood glucose level (antioxidant effect-DPPH, CUPRAC, and Fe2+ chelating assays, as well as anti-inflammatory activity-inhibition of collagenase). We showed that polyphenols are present in the examined aqueous herbal infusions (including chlorogenic and gallic acids). Subsequently, our research has shown that herbal infusions containing cinnamon bark, mulberry leaves, and blackberry fruits most strongly inhibit glucose release from complex carbohydrates, and that all herbal infusions can, to different degrees, reduce the effects of elevated blood sugar. In conclusion, infusions prepared from herbal blends could be recommended to prevent type II diabetes.

Effect of lanthanides on Porphyromonas gingivalis proteases.

Host and bacterial proteases play a vital role in periodontitis. Inhibitors of these proteases are necessary for control of this disease. The purpose of this study was to evaluate the effect of lanthanides on proteins from Porphyromonas gingivalis, a major pathogen in periodontitis. Benzoyl-L-Arg-p-nitroanilide (BAPNA); H-Gly-Pro-pNA x HCl and gelatin were used to evaluate the activity of P. gingivalis proteins in the presence of lanthanides. Proteins extracted from cell surfaces and culture media of P. gingivalis were assessed for activity in the presence of different lanthanides by BAPNA assay. Only gadolinium chloride was used for H-Gly-Pro-pNA x HCl assay and gelatin-zymography. Concentration-dependent reduction of absorbance was observed in the presence of lanthanides with BAPNA and a similar observation was made with gadolinium chloride using H-Gly-Pro-pNa. Collagenolytic activity in cell surface extracts and culture media-precipitated proteins was absent in the presence of gadolinium chloride. These results suggest that the lanthanide gadolinium can be a potential inhibitor of P. gingivalis proteases.

Effect of smoking, abstention, and nicotine patch on epidermal healing and collagenase in skin transudate.

Delayed wound healing may explain postoperative tissue and wound dehiscence in smokers, but the effects of smoking and smoking cessation on the cellular mechanisms remain unclear. Suction blisters were raised in 48 smokers and 30 never smokers. The fluid was retrieved and the epidermal roof was excised. Transepidermal water loss (TEWL) was measured after 2, 4, and 7 days. Then, the smokers were randomized to continuous smoking or abstinence with a transdermal nicotine patch or a placebo by concealed allocation. The sequence was repeated after 4, 8, and 12 weeks in all smokers and abstainers and in 6 never smokers. Matrix metalloproteinase (MMP)-8 and MMP-1 levels in suction blister fluid were assessed by an enzyme-linked immunosorbent assay. Random-effects models for repeated measurements were applied and p< or =0.05 was considered significant. One week after wounding the TEWL was 17.20 (14.47-19.92) g/cm(2) hour (mean, 95% CI) in smokers and 13.89 (9.46-18.33) in never smokers (p<0.01). In abstinent smokers TEWL was 18.95 (15.20-22.70)(p<0.01, when compared with smokers). In smokers, MMP-8 was 36.4 (24.3-48.5) ng/mL (mean, 95% CI) and 15.2 (1.4-30.2) ng/mL in never smokers (p<0.01). Abstinent smokers' MMP-8 level was 21.2 ng/mL (6.6-43.0) (p=0.02, when compared with smokers). MMP-1 was unaffected by smoking and abstention. Transdermal nicotine patch did not affect any parameter. We conclude that smoking attenuates epidermal healing and may enhance extracellular matrix degradation. Three months of abstinence from smoking does not restore epidermal healing, whereas 4 weeks of abstinence normalizes suction blister MMP-8 levels. These findings suggest sustained impaired wound healing in smokers and potential reversibility of extracellular matrix degradation.

Subantimicrobial-dose doxycycline modulates gingival crevicular fluid biomarkers of periodontitis in postmenopausal osteopenic women.

BACKGROUND: We recently demonstrated that a 2-year subantimicrobial-dose doxycycline (SDD) regimen (double-masked, placebo-controlled clinical trial) in postmenopausal (PM) women exhibiting mild systemic bone loss (osteopenia) and local bone loss (periodontitis) reduced the progression of periodontal attachment loss (intent-to-treat analysis) and the severity of gingival inflammation and alveolar bone loss (subgroups) without producing antibiotic side effects. We now describe SDD effects on biomarkers of collagen degradation and bone resorption in the gingival crevicular fluid (GCF) of the same vulnerable subjects. METHODS: GCF was collected from SDD- and placebo-treated PM subjects (n=64 each) at the baseline and 1- and 2-year appointments; the volume was determined; and the samples were analyzed for collagenase activity (using a synthetic peptide as substrate), relative levels of three genetically distinct collagenases (Western blot), a type-1 collagen breakdown product/bone resorption marker (a carboxyterminal telopeptide cross-link fragment of type I collagen [ICTP]; radioimmunoassay), and interleukin-1beta (enzyme-linked immunosorbent assay). Statistical analyses were performed using generalized estimating equations; primary analyses were intent-to-treat. RESULTS: Collagenase activity was significantly reduced by SDD treatment relative to placebo based on intent-to-treat (P=0.01). ICTP showed a similar pattern of change during SDD treatment, and GCF collagenase activity and ICTP were positively correlated at all time periods (P<0.001). Matrix metalloproteinase (MMP)-8 accounted for approximately 80% of total collagenase in GCF, with much less MMP-1 and -13, and SDD reduced the odds of elevated MMP-8 by 60% compared to placebo (P=0.006). CONCLUSION: These observations support the therapeutic potential of long-term SDD therapy to reduce periodontal collagen breakdown and alveolar bone resorption in PM women; effects on serum biomarkers of systemic bone loss in these subjects are being analyzed.

Inhibition of Shedding of Low-Density Lipoprotein Receptor-Related Protein 1 Reverses Cartilage Matrix Degradation in Osteoarthritis.

OBJECTIVE: The aggrecanase ADAMTS-5 and the collagenase matrix metalloproteinase 13 (MMP-13) are constitutively secreted by chondrocytes in normal cartilage, but rapidly endocytosed via the cell surface endocytic receptor low-density lipoprotein receptor-related protein 1 (LRP-1) and subsequently degraded. This endocytic system is impaired in osteoarthritic (OA) cartilage due to increased ectodomain shedding of LRP-1. The aim of this study was to identify the LRP-1 sheddase(s) in human cartilage and to test whether inhibition of LRP-1 shedding prevents cartilage degradation in OA. METHODS: Cell-associated LRP-1 and soluble LRP-1 (sLRP-1) released from human cartilage explants and chondrocytes were measured by Western blot analysis. LRP-1 sheddases were identified by proteinase inhibitor profiling and gene silencing with small interfering RNAs. Specific monoclonal antibodies were used to selectively inhibit the sheddases. Degradation of aggrecan and collagen in human OA cartilage was measured by Western blot analysis using an antibody against an aggrecan neoepitope and a hydroxyproline assay, respectively. RESULTS: Shedding of LRP-1 was increased in OA cartilage compared with normal tissue. Shed sLRP-1 bound to ADAMTS-5 and MMP-13 and prevented their endocytosis without interfering with their proteolytic activities. Two membrane-bound metalloproteinases, ADAM-17 and MMP-14, were identified as the LRP-1 sheddases in cartilage. Inhibition of their activities restored the endocytic capacity of chondrocytes and reduced degradation of aggrecan and collagen in OA cartilage. CONCLUSION: Shedding of LRP-1 is a key link to OA progression. Local inhibition of LRP-1 sheddase activities of ADAM-17 and MMP-14 is a unique way to reverse matrix degradation in OA cartilage and could be effective as a therapeutic approach.

Peptides of type II collagen can induce the cleavage of type II collagen and aggrecan in articular cartilage.

The objective of this study was to determine whether a fragment(s) of type II collagen can induce cartilage degradation. Fragments generated by cyanogen bromide (CB) cleavage of purified bovine type II collagen were separated by HPLC. These fragments together with selected overlapping synthetic peptides were first analysed for their capacity to induce cleavage of type II collagen by collagenases in chondrocyte and explant cultures of healthy adult bovine articular cartilage. Collagen cleavage was measured by immunoassay and degradation of proteoglycan (mainly aggrecan) was determined by analysis of cleavage products of core protein by Western blotting. Gene expression of matrix metalloproteinases MMP-13 and MMP-1 was measured using Real-time PCR. Induction of denaturation of type II collagen in situ in cartilage matrix with exposure of the CB domain was identified with a polyclonal and monoclonal antibodies that only react with this domain in denatured but not native type II collagen. As well as the mixture of CB fragments and peptide CB12, a single synthetic peptide CB12-II (residues 195-218), but not synthetic peptide CB12-IV (residues 231-254), potently and consistently induced in explant cultures at 10 microM and 25 microM, in a time, cell and dose dependent manner, collagenase-induced cleavage of type II collagen accompanied by upregulation of MMP-13 expression but not MMP-1. In isolated chondrocyte cultures CB12-II induced very limited upregulation of MMP-13 as well as MMP-1 expression. Although this was accompanied by concomitant induction of cleavage of type II collagen by collagenases, this was not associated by aggrecan cleavage. Peptide CB12-IV, which had no effect on collagen cleavage, clearly induced aggrecanase specific cleavage of the core protein of this proteoglycan. Thus these events involving matrix molecule cleavage can importantly occur independently of each other, contrary to popular belief. Denaturation of type II collagen with exposure of the CB12-II domain was also shown to be much increased in osteoarthritic human cartilage compared to non-arthritic cartilage. These observations reveal that peptides of type II collagen, to which there is increased exposure in osteoarthritic cartilage, can when present in sufficient concentration induce cleavage of type II collagen (CB12-II) and aggrecan (CB12-IV) accompanied by increased expression of collagenases. Such increased concentrations of denatured collagen are present in adult and osteoarthritic cartilages and the exposure of chondrocytes to the sequences they encode, either in soluble or more likely insoluble form, may therefore play a role in the excessive resorption of matrix molecules that is seen in arthritis and development.

Hydrogen peroxide-mediated downregulation of matrix metalloprotease-2 in indomethacin-induced acute gastric ulceration is blocked by melatonin and other antioxidants.

Gastric mucosal damage is directly associated with extracellular matrix degradation in which matrix metalloproteinases (MMPs) play a crucial role. Remodeling of connective tissues and loss of tissue integrity due to the action of MMPs are reported in several inflammatory diseases, including gastric ulcer. Indomethacin-induced gastric ulceration involves the generation of reactive oxygen species (ROS) and a reduction in MMP-2 transcription and translation. Our aim was to identify the mechanism for suppression of MMP-2 activity by ROS during acute ulceration and further to examine the possible actions of antioxidants, especially melatonin, during healing. Melatonin (N-acetyl-5-methoxytryptamine) blocked hydroxyl radical and nitrite anion generation, protein oxidation, mucosal cell disruption, and MMP-2 downregulation. In addition, suppression of MMP-2 activity by H2O2 in a dose- and time-dependent manner in vitro is blocked by melatonin, omeprazole, and curcumin. We observed that melatonin and other antioxidants (e.g., curcumin and omeprazole) offered gastroprotection in vivo by upregulation of suppressed MMP-2 expression and activity at the level of secretion and synthesis. Moreover, antioxidants reversed the suppression of MMP-2 expression by upregulation of MT1-MMP and downregulation of TIMP-2. Hence, we hypothesize that antioxidants exerted protection against H2O2-mediated inactivation and downregulation of MMP-2 expression during onset of indomethacin-induced ulceration.

Effect of matrix metalloproteinase inhibitor batimastat on breast cancer regrowth and metastasis in athymic mice.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in the invasion and metastasis of human cancers by mediating the degradation of extracellular matrix components. Therefore, these enzymes constitute promising targets in the development of anticancer therapies. Batimastat ([(4-N-hydroxyamino)-2R-isobutyl-3S-(thienyl-thiomethyl)succinyl]-L- phenyl-alanine-N-methylamide) is one of a new class of agents designed to inhibit MMP activity. PURPOSE: We asked whether batimastat, given as adjuvant therapy after primary tumor resection, could inhibit local-regional tumor regrowth and the formation of lung metastases in a human breast cancer xenograft model. We also explored possible effects of batimastat on breast cancer cell viability and on the accumulation of specific messenger RNAs (mRNAs). METHODS: Human MDA-MB-435 breast cancer cells were treated in vitro for 6 days with batimastat at concentrations ranging from 0.1 to 10.0 microM, and then viable cell counts were performed. The activity of collagenases, directly associated with cultured MDA-MB-435 cells or released into their culture fluids, was assessed by gelatin zymography after 1 and 3 days of batimastat treatment (drug range, 0.2-2.0 microM). Athymic nude mice were given daily intraperitoneal injections of batimastat (30 mg/kg body weight) after resection of MDA-MB-435 primary tumors grown in their mammary fat pads; the volumes of tumor regrowths and the numbers and volumes of lung metastases were calculated; neovascularization in the regrowths was assessed by immunohistochemical analysis with an antibody directed against CD31, an endothelial cell antigen. The effect of batimastat treatment on the accumulation of mRNAs encoding specific MMPs and the tissue inhibitor of metalloproteinases-2 (TIMP-2) in cultured cells, primary tumors, and tumor regrowths was measured by RNA dot blotting and hybridization with complementary probes. Linear regression analysis, Student's t tests, and chi-squared analysis were used to evaluate the data. RESULTS: The viability of cultured MDA-MB-435 cells was not affected by treatment with batimastat; however, measured activities for the 72-kd and 92-kd collagenases released by these cells were reduced after batimastat treatment. Intraperitoneal injection of batimastat significantly inhibited the local-regional regrowth of resected MDA-MB-435 tumors in athymic nude mice (in comparison with control mice, P = .035), and it reduced the incidence (P < .05), number (P = .0001), and total volume (P = .0001) of lung metastases. Batimastat treatment did not affect cellular levels of MMP or TIMP-2 mRNAs. CONCLUSION: Batimastat inhibits human breast cancer regrowth and metastasis in a nude mouse xenograft model. Potential mechanisms for batimastat's inhibitory activity do not include direct cell toxicity or alteration of MMP or TIMP mRNA levels.

Liarozole and 13-cis-retinoic acid anti-prostatic tumor activity.

Liarozole fumarate (R85,246), a novel benzimidazole derivative, reduced s.c. and bone metastasis tumor growth by the androgen-independent PC-3ML-B2 human prostatic carcinoma clone in SCID mice. The drug inhibited cell invasion of Matrigel in Boyden chamber chemotactic assays and the secretion of type IV collagenase. In vitro, liarozole failed to inhibit cell proliferation and cell attachment to various substrates (Matrigel, laminin, type IV collagen, and fibronectin). In vivo, the drug also blocked type IV collagenase production in established s.c. tumors. Liarozole has been postulated by others (R. De Coster, W. Wouters, R. Van Ginckel, D. End, et al. J. Steroid Biochem. Mol. Biol., 43: 197-201, 1992) to inhibit retinoic acid catabolism. Our data indicate that liarozole treatment can increase the tumor retinoic acid levels in vivo. Studies of retinoic acid revealed that the drug independently reduced tumor growth in vivo and inhibited cell invasion of Matrigel and the secretion of collagenase IV. Surprisingly, liarozole and retinoic acid failed to exhibit measurable synergistic activity both in vitro and in vivo. Taken together these data suggest that liarozole might inhibit retinoic acid catabolism in vivo and consequently have significant therapeutic value as an anti-prostatic tumor agent.

Retinyl methyl ether down-regulates activator protein 1 transcriptional activation in breast cancer cells.

Retinyl methyl ether (RME) is known to prevent the development of mammary cancer. However, the mechanism by which RME exerts its anticancer effect is presently unclear. The diverse biological functions of retinoids, the vitamin A derivatives, are mainly mediated by their nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs are ligand-dependent transcriptional factors that either activate gene transcription through their binding to retinoic acid response elements or repress transactivation of genes containing the activator protein 1 (AP-1) binding site. Previous studies demonstrated that RME can modulate transcriptional activity of retinoid receptors on retinoic acid response elements, suggesting that regulation of retinoid receptor activity may mediate the anticancer effect of RME. In this study, we present evidence that RME can down-regulate AP-1 activity induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, insulin, growth factors, and the nuclear proto-oncogenes c-Jun and c-Fos. Transient transfection assays demonstrate that inhibition of AP-1 activity occurs on the human collagenase promoter containing an AP-1 binding site or the thymidine kinase promoter linked with an AP-1 binding site. In HeLa cells, the inhibition is observed when RAR-alpha and/or RXR-alpha but not RAR-beta or RAR-gamma expression vectors are cotransfected, whereas the endogenous retinoid receptors in breast cancer cells T-47D and ZR-75-1 were sufficient to confer the inhibition by RME. Furthermore, using gel retardation assay, we show that 12-O-tetradecanoylphorbol-13-acetate- and epidermal growth factor-induced AP-1 binding activity in breast cancer cells is inhibited by RME. These results suggest that one of the mechanisms by which RME prevents cancer development may be due to the repression of AP-1-responsive genes.