Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Decreased activity of cathepsins L+B and decreased invasive ability of PC3 prostate cancer cells

Cancer metastasis involves multiple factors, one of which is the production and secretion of matrix degrading proteases by the cancer cells. Many metastasizing cancer cells secrete the lysosomal proteases, cathepsins L and B, which implicates them in the metastatic process. Cathepsins L and B are regulated by endogenous cysteine proteinase inhibitors (CPI) known as cystatins. An imbalance between cathepsin L and/or B and cystatin expression/activity may be a characteristic of the metastatic phenotype. To determine whether cystatins can attenuate the invasive ability of PC3 prostate cancer cells, cells were transfected with a cDNA coding for chicken cystatin. Expression of chicken cystatin mRNA was determined by PCR analysis. Total cysteine proteinase inhibitory activity, cathepsins L+B activity, and invasion through a Matrigel® matrix were assessed. Stably transfected cells expressed the chicken cystatin mRNA and exhibited a significant decrease in secreted cathepsin L+B activity and a small increase in secreted cysteine proteinase inhibitor activity. The ability of cystatin transfected cells to invade the reconstituted basement membrane, Matrigel®, was attenuated compared to nontransfected cells or cells transfected with vector alone. We have demonstrated that the cysteine proteinases cathepsins L and B participate in the invasive ability of the PC3 prostate cancer cell line, and we discuss here the potential of using cysteine proteinase inhibitors such as the cystatins as anti-metastatic agents.     

Novel roles of protease inhibitors in infection and inflammation

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. alpha(1)-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.     

Effects of proteinase inhibitors on preimplantation embryos in the rat

Proteinase inhibitors of microbial origin were injected into the uterine horns of mated rats at 14:00 h on Day 5 of pregnancy (spermatozoa in vaginal smear = Day 1), and 5 or 6 h later the embryos were flushed from the horns and examined. Chymostatin and alpha-MAPI, inhibitors of chymotrypsin-like serine proteinase and thiol proteinases, as well as thiolstatin, an inhibitor of thiol proteinases, significantly inhibited embryo growth. The inhibitory activity of alpha-MAPI on embryonic growth was distinctly greater than that of thiolstatin, although the ID50 values of the two inhibitors to papain are similar. Antipain and leupeptin which are inhibitors of trypsin-like and thiol proteinases, and talopeptin, an inhibitor of metal proteinases, significantly interrupted the removal of the zona pellucida from expanding blastocysts. These results suggest that (1) a chymotrypsin-like proteinase seems to be important to the growth of the embryo, (2) a thiol proteinase may participate in embryonic growth, and (3) a trypsin-like proteinase and a metal proteinase are likely to participate in zonalysis.     

Molecular imaging of proteolytic activity in cancer

The early detection of both primary tumors and metastatic disease continue to be significant challenges in the diagnosis and staging of cancer. The growing recognition of the role of proteinases and proteolytic cascades in both the growth and metastasis of tumors has led to the development not only of therapeutic strategies using proteinase inhibitors, but also of methods to detect and image tumors in vivo via tumor-associated proteolytic activities. These imaging strategies derive from the enhanced sensitivity afforded by amplification that can be obtained by enzymatic processing to increase the efficacy of imaging "contrast agents" coupled with the inherent substrate specificity and selectivity of proteinases. This review describes key proteinases important in cancer progression, the strategies that have been devised to detect and image proteolytic activity in vivo, and the potential for this kind of functional imaging to serve as a marker for targeted therapy. The intent is to draw attention to the developing methods of molecular imaging to facilitate not only cancer diagnosis, but also for devising strategies for individualized targeted therapy and non-invasive monitoring of therapeutic efficacy.     

Effect of various proteinase inhibitors on ovulation of explanted hamster ovaries

Hamster ovaries explanted at 21:00-24:00 h on the day of pro-oestrus were incubated with microbial proteinase inhibitors until 10:30 h on the next morning and the ovulatory blocking effect of these inhibitors was examined. Amongst 11 proteinase inhibitors examined, talopeptin, a specific inhibitor for metallo-proteinases, and alpha-MAPI, a specific inhibitor for serine and thiol proteinases, were the strongest blockers. These 2 inhibitors exhibited a chronological discrepancy in their blocking effect on ovulation. S-SI, plasminostreptin, elastatinal, antipain and chymostatin, which are inhibitors for serine proteinases, partly but significantly inhibited ovulation. The results suggest that, in addition to a metallo-proteinase reported previously, a proteinase which is sensitive to alpha-MAPI is essential for the ovulatory process, and that serine proteinase(s) also participate in ovulation of the hamster ovary.     

Effect of cysteine proteinase inhibitors on murine B16 melanoma cell invasion in vitro

Various types of proteinases are implicated in the malignant progression of human and animal tumors. Proteinase inhibitors may therefore be useful as therapeutic agents in anti-invasive and anti-metastatic treatment. The aims of this study were (1) to estimate the relative importance of proteinases in B16 cell invasion in vitro using synthetic, class-specific proteinase inhibitors and (2) to assess the inhibitory effect of some naturally occurring cysteine proteinase inhibitors. Serine proteinase inhibitor reduced invasiveness by up to 24%, whereas inhibition of aspartic proteinases reduced invasion by 11%. Synthetic inhibitors of cysteine proteinases markedly impaired invasion: cathepsin B inhibitors, particularly Ca-074Me, inhibited invasion from 20-40%, whereas cathepsin L inhibitor Clik 148 reduced invasion by 11%. The potato cysteine proteinase inhibitor PCPI 8.7 inhibited invasion by 21%, whereas another potato inhibitor, PCPI 6.6, and the mushroom cysteine proteinase inhibitor clitocypin had no effects. As the inhibitors that inhibited cathepsin B were in general more efficient at impairing the invasiveness, we conclude that of the two cysteine proteinases, cathepsin B plays a more important role than cathepsin L in murine melanoma cell invasion.     

Structural basis of the endoproteinase-protein inhibitor interaction

Proteolytic enzymes are potentially hazardous to their protein environment, so that their activity must be carefully controlled. Living organisms use protein inhibitors as a major tool to regulate the proteolytic activity of proteinases. Most of the inhibitors for which 3D structures are available are directed towards serine proteinases, interacting with the active sites in a 'canonical' i.e. substrate-like manner via an exposed reactive site loop of conserved conformation. More recently, some non-canonically binding serine proteinase inhibitors directed against coagulation factors, in particular thrombin, a few cysteine proteinase inhibitors inhibitory towards papain-like proteinases, and three zinc endopeptidase inhibitors directed against metzincins and thermolysin have been characterised in the free and complexed state, displaying novel mechanisms of inhibition with their target proteinases. These different interaction modes are presented and briefly discussed with respect to the different strategies applied by nature.     

Compensatory proteolytic responses to dietary proteinase inhibitors in the red flour beetle, Tribolium castaneum (Coleoptera: Tenebrionidae)

Increasing levels of inhibitors that target cysteine and/or serine proteinases were fed to Tribolium castaneum larvae, and the properties of digestive proteinases were compared in vitro. Cysteine proteinases were the major digestive proteinase class in control larvae, and serine proteinase activity was minor. Dietary serine proteinase inhibitors had minimal effects on either the developmental time or proteolytic activity of T. castaneum larvae. However, when larvae ingested cysteine proteinase inhibitors, there was a dramatic shift from primarily cysteine proteinases to serine proteinases in the proteinase profile of the midgut. Moreover, a combination of cysteine and serine proteinase inhibitors in the diet prevented this shift from cysteine proteinase-based digestion to serine proteinase-based digestion, and there was a corresponding substantial retardation in growth. These data suggest that the synergistic inhibitory effect of a combination of cysteine and serine proteinase inhibitors in the diet of T. castaneum larvae on midgut proteolytic activity and beetle developmental time is achieved through the prevention of the adaptive proteolytic response to overcome the activity of either type of inhibitor.     

Proteinases and proteinase inhibitors as modulators of animal cell growth.

1. Three distinct lines of evidence indicate that proteinases are involved in the growth of cultured animal cells. 2. Endogenous growth-related proteinases have been identified, and exogenous proteinases can also stimulate cell proliferation, probably by different mechanisms. In some cases, higher concentrations of proteinases are cytotoxic. 3. Proteinase inhibitors, not surprisingly, inhibit cell growth, but can also be mitogenic at sub-inhibitory concentrations. 4. There must, therefore, be at least three major cellular processes in which proteinases or proteinase inhibitors can operate to exert a direct effect on cell proliferation. 5. Details of one action of an exogenous proteinase, typified by thrombin and the thrombin receptor, are becoming clear at the molecular level, but thrombin probably activates at least two intracellular signalling systems, as well as acting as a growth inhibitor in some situations. 6. Much remains to be investigated in other examples.     

Proteinases involved in matrix turnover during cartilage and bone breakdown

The joint is a discrete unit that consists of cartilage, bone, tendon and ligaments. These tissues are all composed of an extracellular matrix made of collagens, proteoglycans and specialised glycoproteins that are actively synthesised, precisely assembled and subsequently degraded by the resident connective tissue cells. A balance is maintained between matrix synthesis and degradation in healthy adult tissues. Different classes of proteinases play a part in connective tissue turnover in which active proteinases can cleave matrix protein during resorption, although the proteinase that predominates varies between different tissues and diseases. The metalloproteinases are potent enzymes that, once activated, degrade connective tissue and are inhibited by tissue inhibitors of metalloproteinases (TIMPs); the balance between active matrix metalloproteinases and TIMPs determines, in many tissues, the extent of extracellular matrix degradation. The serine proteinases are involved in the initiation of activation cascades and some, such as elastase, can directly degrade the matrix. Cysteine proteinases are responsible for the breakdown of collagen in bone following the removal of the osteoid layer and the attachment of osteoclasts to the exposed bone surface. Various growth factors increase the synthesis of matrix and proteinase inhibitors, whereas cytokines (alone or in combination) can inhibit matrix synthesis and stimulate proteinase production and matrix destruction.     

Characterization of midgut proteinase activities of white grubs: Lepidiota noxia,Lepidiota negatoria,and Antitrogus consanguineus (scarabaeidae,melolonthini)

The proteinases in the midguts of three scarab white grub species, Lepidiota noxia, L. negatoria, and Antitrogus consanguineus, were investigated to classify the proteinases present and to determine the most effective proteinase inhibitor for potential use as an insect control agent. pH activity profiles indicated the presence of serine proteinases and the absence of cysteine proteinases. This was confirmed by the lack of inhibition by specific cysteine proteinase inhibitors. Trypsin, chymotrypsin, elastase, and leucine aminopeptidase activities were detected by using specific synthetic substrates. A screen of 32 proteinase inhibitors produced 9 inhibitors of trypsin, chymotrypsin, and elastase which reduced proteolytic activity by greater than 75%. © 1995 Wiley-Liss, Inc.     

Inhibition of Helicoverpa armigera gut pro‐proteinase activation in response to synthetic protease inhibitors

Protease inhibitors play an important role in host plant defence against herbivores. However, insects have the ability to elevate the production of proteinases or resort to production of a diverse array of proteinases to offset the effect of proteinase inhibitors. Therefore, we studied the inhibition of pro‐proteinase(s) activation in the midgut of the polyphagous pest Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in response to protease inhibitors to develop appropriate strategies for the control of this pest. Gelatin coating present on X‐ray film was used as a substrate to detect electrophoretically separated pro‐proteinases and proteinases of H. armigera gut extract on native‐ and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. Six activated pro‐proteinase bands were detected in H. armigera gut lumen, which were partially purified and characterized using substrate assays. Activated H. armigera midgut pro‐proteinase(s) showed activity maxima at pH 8 and 10, and exhibited optimal activity at 40 °C. The activation of H. armigera gut pro‐proteinase isoforms was observed in the fraction eluted on benzamidine‐sepharose 4B column. Purification and substrate assay studies revealed that 23–70 kDa polypeptides were likely the trypsin/chymotrypsin‐like pro‐proteinases. Larvae of H. armigera fed on a cocktail of synthetic inhibitors (antipain, aprotinin, leupeptin, and pefabloc) showed maximum activation of pro‐proteinases compared with the larvae fed on individual inhibitors. The implications of these results for developing plants expressing proteinase inhibitors for conferring resistance to H. armigera are discussed.     

Human skin chymotryptic proteinase. Isolation and relation to cathepsin g and rat mast cell proteinase I

A chymotrypsin-like proteinase was purified 2400-fold from human skin. The procedure involves extraction of the proteinase from skin in 2 M KCl, precipitation with protamine chloride, fractionation by gel filtration chromatography, and fractionation by chromatography using a CH-Sepharose-D-tryptophan methyl ester affinity column. The properties of this proteinase were compared to the rat mast cell proteinase I and human cathepsin G. Differences were observed in the rates at which the proteinases were inhibited by diisopropyl fluorophosphate, the sensitivity of the proteinases to protein proteolytic inhibitors, the relative hydrolytic rates of the proteinases for a series of substrates, and the kinetic constants of the proteinases for synthetic substrates. The human skin proteinase did not react with antiserum to the rat skin proteinase and did not elute in the same position as the rat skin proteinase on gel filtration columns. These data demonstrate that the human skin proteinase is distinct from the other proteinases. Extracts of involved skin from patients with cutaneous mastocytosis had 15-fold higher levels of chymotryptic activity than extracts of uninvolved skin or skin from normal controls. The enzymatic properties of the material extracted from the biopsied skin were similar to those of the proteinase from normal skin, suggesting that the human skin chymotrypsin-like proteinase is a mast cell constituent.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

The role of conformational change in serpin structure and function

Serpins are members of a family of structurally related protein inhibitors of serine proteinases, with molecular masses between 40 and 100kDa. In contrast to other, simpler, proteinase inhibitors, they may interact with proteinases as inhibitors, as substrates, or as both. They undergo conformational interconversions upon complex formation with proteinase, upon binding of some members to heparin, upon proteolytic cleavage at the reactive center, and under mild denaturing conditions. These conformational changes appear to be critical in determining the properties of the serpin. The structures and stabilities of these various forms may differ significantly. Although the detailed structural changes required for inhibition of proteinase have yet to be worked out, it is clear that the serpin does undergo a major conformational change. This is in contrast to other, simpler, families of protein inhibitors of serine proteinases, which bind in a substrate-like or product-like manner. Proteolytic cleavage of the serpin can result in a much more stable protein with new biological properties such as chemo-attractant behaviour. These structural transformations in serpins provide opportunities for regulation of the activity and properties of the inhibitor and are likely be important in vivo, where serpins are involved in blood coagulation, fibrinolysis, complement activation and inflammation.     

Proteinase,Lipase and Amylase Activities in the Small Intestine of Pigs Suffering from Colienterotoxaemia

The activities of proteinases, lipases, amylases and the activities of proteinase inhibitors, as well as the numbers of Escherichia coli in the contents from the small intestine were examined for pigs suffering from colienterotoxaemia and for healthy pigs. Enzyme activities were determined using an agar diffusion test. Compared with healthy animals the activities of proteinases and amylases in diseased animals were reduced while lipases showed increased activity. In pathologically changed contents showing large numbers of E. coli, proteinases could not be demonstrated; however, proteinase inhibitors were found in these contents. In healthy animals, proteinase inhibitors were not demonstrated in ingesta-con-taining contents. In diseased animals, E. coli were found in large numbers in all parts of the small intestine. In healthy animals, E. coli was demonstrated especially in the posterior part of the small intestine and regularly in small numbers. The possible influence of digestive enzymes, especially proteinases and their inhibitors, on enterotoxins from E. coli is discussed.     

Regulation of proteolytic enzyme activities in muscles practice and hypothesis.

Two weeks of immobilization in shortened state caused 50% decrease in muscle mass and an increase in lysosomal proteinase activities. In this study causes of elevated protease activities in muscle are studied. Many factors could play a role in these elevated proteinase activities. We have found that redox state, ATP content, fuel supply and glucocorticoid receptor number were important in this period. Testosterone, insulin or proteinase inhibitors were not proved to play role in elevated proteinase activities. These practical results are explained by the results achieved in other types of cells. We conclude that changes in redox potential and/or oxygen free radical content of muscle elements can cause a post-translational covalent modification of cysteine proteinases and -SH dependent metalloproteinases, leading thereby to their activation. Lysosomal cysteine proteinases can activate procathepsin D that can damage lysosomal cysteine proteinase inhibitors and in another path it activates procathepsin B, L and H reversely. This feed-back regulation and the activation of cysteine proteinases by metalloproteinases might accelerate the proteinase activities in skeletal muscles.     

Purification and identification of two serine class proteinases from dog mast biochemically and immunologically similar to human proteinases tryptase and chymase

Serine class proteinases with trypsin-like and chymotrypsin-like specificity were purified from dog mastocytoma tissue. An antiserum was produced against the chymotrypsin-like proteinase. The antiserum reacted with mast cells in skin sections prepared from normal dogs consistent with the proteinase being a mast cell constituent. The antiserum also cross-reacted with the major chymotrypsin-like proteinase isolated from normal dog skin and partially cross-reacted with human skin chymase. No cross-reaction was detected with rat chymase. The trypsin-like proteinase from dog mastocytoma tissue was similar to tryptase isolated from human skin. It had a similar subunit structure, was not inhibited by many protein proteolytic enzyme inhibitors, bound to heparin, and reacted strongly with antiserum against human tryptase. Antiserum against human tryptase also reacted with mast cells in skin sections prepared from normal dog skin. No immunocytochemical labeling of rat skin mast cells was observed with anti-human tryptase. These studies establish the presence of a trypsin-like and chymotrypsin-like proteinase in dog skin mast cells and provide immunological evidence which suggests that both proteinases are more closely related to human than rat mast cell proteinases. These immunological and biochemical relationships are important when comparing the roles of these proteinases in different animals.     

Digestive proteinases in Lasioderma serricorne (Coleoptera: Anobiidae)

The cigarette beetle, Lasioderma serricorne (Fabricius), is a common pest of stored foods. A study of digestive proteinases in L. serricorne was performed to identify potential targets for proteinaceous biopesticides, such as proteinase inhibitors. Optimal casein hydrolysis by luminal proteinases of L. serricorne was in pH 8.5-9.0 buffers, although the pH of luminal contents was slightly acidic. Results from substrate and inhibitor analyses indicated that the primary digestive proteinases were serine proteinases. The most effective inhibitors of caseinolytic hydrolysis were from soybean (both Bowman Birk and Kunitz), with some inhibition by chymostatin, N-tosyl-L-phenylalanine chloromethyl ketone, and leupeptin. Casein zymogram analysis identified at least eight proteolytic activities. Activity blot analyses indicated one major proteinase activity that hydrolysed the trypsin substrate N-alpha-benzoyl-L-arginine rho-nitroanilide, and three major proteinase activities that hydrolysed the chymotrypsin substrate N-succinyl ala-ala-pro-phe rho-nitroanilide. The absence of cysteine, aspartic, and metallo proteinases in L. serricorne digestion was evidenced by the lack of activation by thiol reagents, alkaline pH optima, and the results from class-specific proteinase inhibitors. The data suggest that protein digestion in L. serricorne is primarily dependent on trypsin- and chymotrypsin-like proteinases.