The combined treatment of transplantable solid mammary carcinoma in Wistar rats by use of photodynamic therapy and cysteine proteinase inhibitor.

Numerous studies have shown that lysosomal proteinases play an important role in carcinogenesis. The enzymatic activity of tumor-associated proteases is counter-balanced by specific inhibitors. Photodynamic therapy (PDT) is a technique which involves photoexcitation of sensitizing drugs retained in neoplastic tissue that is subsequently destroyed. Intraperitoneal injections of hematoporphyrin derivative (HpD) were given at a dose of 20 mg/kg in rats transplanted with mammary carcinoma. A halogen lamp was used 24 hours later at 630 +/- 20 nm and total dose--200 J/sq.cm. Cysteine proteinase inhibitor (CPI) was dissolved in saline and injected subcutaneously in doses of 50 mg and 200 mg per animal. The effectiveness of the treatment was evaluated with regard to survival time and tumor response and to depth of necrosis. In several cases tumors completely disappeared following HpD-PDT + CPI. The number of complete tumor responses was higher when PDT + 200 mg of CPI was used, i.e. 6 out of 10 rats. Promising results have also been obtained with regard to survival time of treated animals and to induction of tumor necrosis. We may presume that a combination of PDT and proteinase inhibitors could be a useful tool in further anticancer studies and, hopefully, in anticancer therapy.

Activity of cysteine protease inhibitors in human brain tumors.

BACKGROUND: Cysteine proteases (mainly cathepsins B and L) are thought to play an important role in the progress of cancer, including brain tumors. Together with other proteases, they hydrolyze the extracellular matrix and basement membrane proteins, thus enabling the tumor to grow and spread. Therefore cysteine protease inhibitors are regarded as protective factors, able to prevent tumor growth and dissemination. MATERIAL AND METHODS: In this study, the activity of cysteine protease inhibitors (CPIs) was investigated in material derived from patients with brain tumors (astrocytoma and meningioma). The activity of CPIs was measured as antipapain activity in tissue homogenates, cerebrospinal fluid, and serum, with N-benzoyl-DL-arginine-2-naphthylamide hydrochloride (BANA) as a substrate, according to Barret's method. RESULTS: Tumorous tissues showed higher activity of cysteine protease inhibitors than control tissues, but this difference proved to be statistically insignificant. The activity of CPIs was lower in cerebrospinal fluid and serum from patients with brain tumors. CONCLUSIONS: The activity of CPIs measured in brain tumor tissue cannot be taken as a marker of any type of tumor, whereas CPI activity in cerebrospinal fluid and serum may be considered a marker of meningioma. In meningioma patients the level of CPIs may be too low to prevent the host tissues from the growing tumor.

Role of cysteine endopeptidases in cancerogenesis.

Role of cysteine endopeptidases in cancerogenesis steps: neoplastic transformation, invasion and metastasis is reviewed and discussed. Positive correlation between tumor invasiveness, as well as its metastatic potential and secretion of cysteine endopeptidase (particularly cathepsins B and L) has been documented well in literature. Based on our recent results we postulate that serum endopeptidase-like activity could be used as a marker of cancer aggressiveness in diagnostic procedures in oncology. We also propose that the cysteine endopeptidase inhibitor levels (total, active and latent) could be useful factors for recognising the activation of the organism self-defence mechanisms against cancer. In addition, our idea of use of urinary cysteine peptidase inhibitors (UCPI) as potential anticancer agents is presented and discussed.

Cysteine peptidase inhibitors and activator(s) in urine of patients with colorectal cancer.

The total activity of cysteine peptidase inhibitors and activator(s) was determined in the samples of urine received from colorectal cancer patients. Patients with peptic ulcer and healthy volunteers agreed to be a control group. The studies revealed a marked difference between the values of the determined parameters for the patients with colorectal cancer and those for the control group. Determination of cysteine peptidase inhibitors in patient's urine is proposed as a new diagnostic procedure.

Decrease in vivo of cysteine endopeptidases in blood of patients with tumor of the larynx.

Since cysteine endopeptidase (cathepsins B and L) have been proposed to be implicated in tumor malignancy, we have attempted to decrease these in vivo. Large amounts of urine cysteine peptidase inhibitors (UCPI) are present in the urine of patients. Our results indicate protective effects of a UCIP preparation against human serum cysteine endopeptidases.

Serum cathepsin B-like activity as a potential marker of laryngeal carcinoma.

Early detection and reliable monitoring of treatment constitute one of the most challenging problems in oncology. For this reason, we studied the potential value of serum cathepsin B-like activity as a possible marker of laryngeal carcinoma. The results indicate that serial analyses of this parameter have predictive value in the assessment of surgical treatment of laryngeal carcinoma. Activity of the enzyme reveals a progressive increment which could be associated with increased severity of the neoplasm present. The serum level of the cathepsin B-like activity remained close to zero in the control group of healthy volunteers and patients with inflammatory diseases.

Method of purification of thiol proteinase inhibitors from human urine.

Urinary thiol proteinase inhibitors (UTPI) had been isolated from their complex with papain. Almost 90% of these inhibitors, previously inactivated by native or immobilized papain, were recovered after heating the complex to 80 degrees C at pH 2.0 for 20 min. Two protein inhibitors with molecular weights of 76,000 (UTPI-76) and about 45,000 (UTPI-45) were isolated from urine of patients with malignant colorectal tumor. UTPI-fraction consisted of about 40% of UTPI-76 (probably light form of kininogen) and of 60% of UTPI-45 fraction. The inhibitors differed in affinity to Concanavalin A. No electrophoretic mobility changes of both UTPI, before and after isolation by described affinity chromatography were observed.

Critical carboxyl group(s) in Na+-dependent cotransporters of the intestinal brush-border membrane.

We have previously provided functional evidence for a role of carboxyl group(s) in the mechanism of coupling of Na+ and D-glucose fluxes by the small-intestinal cotransporter(s) (Kessler, M. and Semenza, G. (1983) J. Membrane Biol. 76, 27-56). We present here a study on the inactivation of the Na+-dependent transport systems, but not of the Na+-independent ones, in the small-intestinal brush-border membrane, by hydrophobic carbodiimides. Although marginal or insignificant protection by the substrates or by Na+ was observed, the parallelism between Na+-dependence and inactivation by these carbodiimides strongly indicates the role of carboxyl group(s) previously indicated. Contrary to the carboxyl group identified by Turner [1986) J. Biol. Chem. 261, 1041-1047) in the sugar binding site of the renal Na+/D-glucose cotransporter, the carboxyl group(s) studied here probably occur elsewhere in the cotransporter molecule.

Phenylalkylsulfonyl derivatives as covalent inhibitors of penicillin amidase.

It was demonstrated that phenylmethanesulfonyl fluoride-a very potent inhibitor of penicillin amidase from Escherichia coli-binds covalently to the enzyme in molar ratio 1:1. The chloride, the azide and the N-hydroxysuccinimide ester of phenylmethanesulfonic acid are also very strong inactivators of the amidase. Weaker inhibition was noted with para-substituted phenylmethanesulfonyl chlorides and with phenylethanesulfonyl and alkylsulfonyl chlorides. The inactivated amidase could be reactivated by incubation either with 6-amino-penicillanic acid or with proteins from E. coli extract. Benzyl isocyanate is also a potent covalent inhibitor of the amidase but inactivated amidase could be not reactivated in this way. It was demonstrated that representatives of all inactivator types bind to one active site of the amidase. Interdependence between inactivation rate and stability of some sulfonyl inhibitors was observed. No inhibition was noted the amide, the hydrazide and the methyl ester of phenylmethanesulfonic acid.

Molecular forms of bovine gamma-glutamyltransferase and their enzyme immunoassay.

Light form of bovine kidney gamma-glutamyl transferase was isolated from heavy form of the enzyme after digestion with bromelain. Its apparent molecular weight was 95,000 and in SDS solution it dissociated into two non-identical subunits with molecular weights 26,000 and 69,000. No substantial differences between both forms in activation, kinetic parameters and inhibition with anthglutin and its isomers were noted. Using enzyme immunoassay it was possible to determine one enzyme form in the presence of the other. This was applied for studies of gamma-glutamyltransferase forms in cow serum and colostrum.

A new fluorimetric method for the determination of gamma-glutamyltransferase activity in blood serum.

Using 7-(gamma-L-glutamyl)-4-methyl-coumarylamide as the fluorogenic substrate a simple and sensitive method for the assay of transferase activity of gamma-glutamyl-transferase in human blood serum and some other biological fluids is described. The substrate was synthesized with a good yield by the King and Kidd method. Close correlation and good agreement was noted between activities measured by the fluorimetric method and by the old colorimetric one in which gamma-L-glutamyl-p-nitroanilide was used.

Properties of penicillin amidase immobilized by copolymerization with acrylamide.

An enzyme preparation in a spherical granule form was obtained by copolymerization of penicillin amidase (EC 3.5.1.11) (previously modified with maleic anhydride) and acrylamide via a crosslinking agent. As compared with the native enzyme, immobilized amidase is more resistant to heating, has a lower affinity to benzylpenicillin, and is less inhibited by phenylacetate. Its substrate specificity and optimum pH remain unchanged.