Several analogues of somatostatin were examined in the Mia PaCa-2 human pancreatic cancer cell line for their ability to promote tyrosine phosphatase activity affecting the receptors for the epidermal growth factor. The inhibition of growth of the Mia PaCa-2 cells in culture was also evaluated to determine the mechanism of action of somatostatin analogues and their relative effectiveness in inhibiting cancer growth. Of the analogues tested D-Phe-Cys-Tyr-D-Trp-LysVal-Cys-Trp-NH2 (RC-160) caused the greatest stimulation of tyrosine phosphatase activity. Analogue D-Phe-Cys-Tyr-DTrp-Lys-Val-Cys-Thr-NH2 (RC-121) had less effect but was more potent than somatostatin-14. Analogue D-Phe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr(ol) (SMS 201-995) produced no significant dephosphorylation. The analogues displayed the same order of activity in assays on growth inhibition of Mia PaCa-2 cells in cultures. Analogue (SMS-201-995) caused virtually no tyrosine phosphatase stimulation or growth inhibition in this cancer cell line, although it possesses a much higher antisecretory activity than somatostatin-14 in normal tissues. These observations indicate that somatostatin and some of its analogues can act as growth inhibitors in cancer cells through the activation of tyrosine phosphatase. These data reinforce the view that somatostatin analogue RC-160 and related compounds could be used for treatment of pancreatic cancer.
Abstract-Hypertriglyceridemia may contribute to the development of atherosclerosis by increasing expression of cell adhesion molecules (CAMs). Although the cellular expression of CAMs is difficult to assess clinically, soluble forms of CAMs (sCAMs) are present in the circulation and may serve as markers for CAMs. In this study, we examined the association between sCAMs and other risk factors occurring with hypertriglyceridemia, the effect of triglyceride reduction on sCAM levels, and the role of soluble vascular cell adhesion molecule-1 (sVCAM-1) in monocyte adhesion in vitro. Compared with normal control subjects (nϭ20), patients with hypertriglyceridemia and low HDL (nϭ39) had significantly increased levels of soluble intercellular adhesion molecule-1 (sICAM-1) (316Ϯ28.8 versus 225Ϯ16.6 ng/mL), sVCAM-1 (743Ϯ52.2 versus 522Ϯ43.6 ng/mL), and soluble E-selectin (83Ϯ5.9 versus 49Ϯ3.6 ng/mL). ANCOVA showed that the higher sCAM levels in patients occurred independently of diabetes mellitus and other risk factors. In 27 patients who received purified n-3 fatty acid (Omacor) 4 g/d for Ն7 months, triglyceride level was reduced by 47Ϯ4.6%, sICAM-1 level was reduced by 9Ϯ3.4% (Pϭ.02), and soluble E-selectin level was reduced by 16Ϯ3.2% (PϽ.0001), with the greatest reduction in diabetic patients. These results support previous in vitro data showing that disorders in triglyceride and HDL metabolism influence CAM expression and treatment with fish oils may alter vascular cell activation. In a parallel-plate flow chamber, recombinant sVCAM-1 at the concentration seen in patients significantly inhibited adhesion of monocytes to interleukin-
Heparan sulfate was isolated from the cell surface, cell pellet, and culture medium of exponentially growing as well as postconfluent bovine aortic smooth muscle cells (SMCs). After chromatography on DEAE-Sephadex and Sepharose 4B, the various mucopolysaccharides were examined for their ability to cause growth inhibition in a SMC bioassay. The heparan sulfate isolated from the surface of postconfluent SMCs possessed approximately eight times the antiproliferative potency per cell of the heparan sulfate obtained from the surface of exponentially growing SMCs. Heparan sulfate isolated from other fractions of exponentially growing or postconfluent SMCs possesses little growth inhibitory activity. The difference in the antiproliferative activities of heparan sulfate obtained from the surface of SMCs in the two growth states could not be attributed to the synthesis of a greater mass of mucopolysaccharide by postconfluent SMCs. Indeed, heparan sulfate isolated from the surface of the postconfluent SMCs exhibits a specific antiproliferative activity which is 13-fold greater than mucopolysaccharide obtained from the surface of exponentially growing SMCs and more than 40-fold greater than commercially available heparin. In addition, exponentially growing SMCs did not exhibit an enhanced ability to degrade the complex carbohydrate. Furthermore, other investigations indicate that the small amount of growth inhibitory activity intrinsic to heparan sulfate isolated from the surface of exponentially growing SMCs is due to residual, biologically active, mucopolysaccharide produced by the primary postconfluent SMCs from which the exponentially growing SMCs were derived. These studies suggest that bovine aortic SMCs are capable of controlling their own growth by the synthesis of a specific form of heparan sulfate with antiproliferative potency.Most connective tissues contain glycosaminoglycans as major components of their extracellular matrix. These high molecular weight, negatively charged sulfated mucopolysaccharides have been implicated in determining certain general overall properties of tissues such as hydration, elasticity, and permeability (1). It has also been suggested that glycosaminoglycans that are known to be associated with the surface of the cell could be involved in basic cellular functions, such as adhesion and motility (2, 3). However, little is known about whether specific structural elements on the glycosaminoglycans are responsible for the biologic properties of these components.Several investigators have demonstrated that the mucopolysaccharide, heparin, must satisfy unique structure-function relationships to interact with its protein co-factor, antithrombin, to alter allosterically the conformation of this protease inhibitor, and thereby accelerate neutralization of coagulation system enzymes (4). Our laboratory has recently shown that anticoagulantly active heparan sulfate molecules are present on the surface of endothelial cells and are, in part, responsible for maintaining the nonthrombogenic p...
Human cathepsin G is a serine proteinase with chymotrypsin-like specificity found in both polymorphonuclear leukocytes (neutrophils) and the U937 leukemic cell line. Utilizing RNA from the latter, we have constructed a cDNA library in lambda gt11 and isolated a clone which apparently codes for the complete amino acid sequence of this enzyme. Analysis of the sequence reveals homology with rat mast cell proteinase II (47%) but a greater degree of identity (56%) with a product of activated mouse cytotoxic T lymphocytes. The close relationship between the three proteins indicates similarities in substrate specificity and in biosynthesis which we predict involves removal of a two amino acid activation peptide during or just before packaging into their respective storage granules.
The potential of a given amount of heparin to inhibit smooth muscle cell (SMC) proliferation can be increased more than 13 fold if quiescent cultures are pretreated with this mucopolysaccharide for 48 h. The large increase in antiproliferative activity was attributable to a 74% inhibition of the first cell cycle traverse of SMC after serum addition. If the mucopolysaccharide was added to SMC coincident with serum, the initial cell cycle traverse was only suppressed by 27%. In both heparin pretreated and nonpretreated SMC cultures, 48 to 72 h elapsed before substantial inhibition was observed. The inhibitory effects of heparin were reversible and inversely proportional to the starting cell density of the cultures. The effects of known heparin binding proteins on the inhibitory capability of heparin were examined. Neither platelet-derived growth factor (PDGF), low density lipoprotein (LDL), nor platelet factor 4 (PF4) were able to reduce the antiproliferative effects. Heparin retained full biological activity in medium containing serum depleted of all heparin binding proteins by heparin-Sepharose chromatography. These results indicate that heparin does not inhibit growth by preventing serum mitogens or nutrients from interacting with SMC. Rather, our data suggest that heparin is slowly internalized by SMC following binding to specific, non-PF4 dissociable sites. Heparin may accumulate intracellularly and block a crucial point in the proliferative machinery of SMC.
The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.
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