alpha-chymotrypsin and Carcinoma--Hepatocellular

The proteasomal system is a promising target for cancer treatment. Quercetin (Que), a flavonoid compound with antitumor ability, displays the inhibitory effect on proteasome activity. However, the underlying molecular mechanisms are ill defined. The present study found that Que treatment significantly reduced the chymotrypsin-like protease activity of proteasome whereas the trypsin- and caspase-like protease activities remained unchanged in HepG2 cancer cells, along with activation of p38 MAPK and JNK and reduction of ERK1/2 phosphorylation. Que-reduced proteasome activity could not be reverted by inhibition of p38 MAPK and JNK signaling pathway. In addition, MEK1 overexpression or knockdown upregulated or downregulated the chymotrypsin-like protease activity of proteasome, respectively. Both Que and MEK1/ERK1/2 inhibitor attenuated the expression levels of proteasome β subunits. These results indicate that Que-induced suppression of MEK1/ERK1/2 signaling and subsequent reduction of proteasome β subunits is responsible for its inhibitory impacts on proteasome activity.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Cell Survival; Chymotrypsin; Enzyme Activation; Hep G2 Cells; Humans; Liver Neoplasms; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Proteasome Endopeptidase Complex; Quercetin

A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 degrees C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC(50) of 10.7+/-4.2 microM. It inhibited HIV-1 reverse transcriptase with an IC(50) of 8+/-1.5 microM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability.

Topics: Acacia; Adsorption; Amino Acid Sequence; Antineoplastic Agents, Phytogenic; Breast Neoplasms; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Chromatography; Chymotrypsin; Dimerization; Dose-Response Relationship, Drug; Female; Fungi; Hep G2 Cells; HIV Reverse Transcriptase; Humans; Hydrogen-Ion Concentration; Inhibitory Concentration 50; Molecular Structure; Phytotherapy; Plant Proteins; Reverse Transcriptase Inhibitors; Seeds; Sepharose; Sequence Homology; Temperature

The proteasome is a major cellular proteinase. Its activity is modulated by cellular oxidants. Hepatitis C core protein and ethanol exposure both cause enhanced oxidant generation. The aim was to investigate whether core protein, by its ability to generate oxidants, alters proteasome activity and whether these alterations are further affected by ethanol exposure.. These interactions were examined in Huh-7 cell lines that expressed inducible HCV core protein and/or constitutive cytochrome P450 2E1 (CYP2E1) and as purified components in a cell-free system. Chymotrypsin-like proteasome activity was measured fluorometrically.. Proteasome activity in core-positive 191-20 cells was 20% higher than that in core-negative cells and was enhanced 3-fold in CYP2E1-expressing L14 cells. Exposure of core-positive cells to glutathione ethyl ester, catalase, or the CYP2E1 inhibitor diallyl sulfide partially reversed the elevation of proteasome activity in core-positive cells, whereas ethanol exposure suppressed proteasome activity. The results indicate that proteasome activity was up-regulated by low levels of core-induced oxidative stress but down-regulated by high levels of ethanol-elicited stress. These findings were partially mimicked in a cell-free system. Addition of core protein enhanced the peptidase activity of purified 20S proteasome containing the proteasome activator PA28 and was further potentiated by addition of liver mitochondrial and/or microsome fractions. However, proteasome activation was significantly attenuated when fractions were obtained from ethanol-fed animals.. HCV core protein interacts with PA28, mitochondrial, and endoplasmic reticulum proteins to cause low levels of oxidant stress and proteasome activation, which is dampened during ethanol metabolism when oxidant generation is higher.

Topics: Allyl Compounds; Carcinoma, Hepatocellular; Catalase; Cell Line, Tumor; Chymotrypsin; Cytochrome P-450 CYP2E1; Cytochrome P-450 CYP2E1 Inhibitors; Enzyme Activation; Enzyme Inhibitors; Ethanol; Glutathione; Humans; Liver Neoplasms; Microsomes, Liver; Mitochondria, Liver; Oxidants; Oxidative Stress; Proteasome Endopeptidase Complex; Proteins; Sulfides; tert-Butylhydroperoxide; Transfection; Viral Core Proteins

Conformational diseases are caused by a structural rearrangement within a protein that results in aberrant intermolecular linkage and tissue deposition. This is typified by the polymers that form with the Z deficiency variant of alpha 1-antitrypsin (Glu-342 --> Lys). These polymers are retained within hepatocytes to form inclusions that are associated with hepatitis, cirrhosis, and hepatocellular carcinoma. We have assessed a surface hydrophobic cavity in alpha1-antitrypsin as a potential target for rational drug design in order to prevent polymer formation and the associated liver disease. The introduction of either Thr-114 --> Phe or Gly-117 --> Phe on strand 2 of beta-sheet A within this cavity significantly raised the melting temperature and retarded polymer formation. Conversely, Leu-100 --> Phe on helix D accelerated polymer formation, but this effect was abrogated by the addition of Thr-114 --> Phe. None of these mutations affected the inhibitory activity of alpha 1-antitrypsin. The importance of these observations was underscored by the finding that the Thr-114 --> Phe mutation reduced polymer formation and increased the secretion of Z alpha 1-antitrypsin from a Xenopus oocyte expression system. Moreover cysteine mutants within the hydrophobic pocket were able to bind a range of fluorophores illustrating the accessibility of the cavity to external agents. These results demonstrate the importance of this cavity as a site for drug design to ameliorate polymerization and prevent the associated conformational disease.

Topics: alpha 1-Antitrypsin; Animals; Binding Sites; Carcinoma, Hepatocellular; Chymotrypsin; Crystallography, X-Ray; Cysteine; Fibrosis; Glycine; Hepatitis; Hepatocytes; Hydrogen-Ion Concentration; Models, Molecular; Mutation; Oocytes; Phenylalanine; Polymers; Potassium Chloride; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Temperature; Threonine; Time Factors; Xenopus

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Samuel W. French and R. J. Mayer. The presentations were (1) The ubiquitin-proteasome 26s pathway in liver cell protein turnover: Effect of alcohol and drugs, by Samuel W. French and F. Bardag-Gorce; (2) The role of CYP2E1 phosphorylation and degradation pathway in the induction of the enzyme, by Magnus Ingelman-Sundberg; (3) Role of proteasome in the proteolysis of oxidized proteins in experimental chronic alcoholism, by Helen Rouach; (4) Alcohol, proteolysis and liver cancer, by R. J. Mayer; (5) Effect of ethanol feeding on the ATP-ubiquitin-proteasome pathway in the liver cell, by F. Bardag-Gorce; (6) Novel mechanisms and targets for intracellular transport of CYP2E1, by E. Neve; and (7) Gankyrin, an oncoprotein commonly over expressed in hepatoma, by H. Higashitsuji.

Topics: Animals; Carcinoma, Hepatocellular; Central Nervous System Depressants; Chymotrypsin; Cytochrome P-450 CYP2E1; Ethanol; Hepatocytes; Humans; Liver; Liver Neoplasms; Mice; Peptide Hydrolases; Proteasome Endopeptidase Complex; Rats; Ubiquitins

Hepatocellular carcinoma with osteoclast-like giant cells (hepatic giant cell carcinoma [HGCC]) is a rare entity, with only three cases reported. The tumor is histologically similar to giant cell tumor (GCT) of bone, and the origin of the multinucleated giant cells and mononuclear stromal cells has not been determined. The purpose of this report is to present a case of this rare tumor and compare its ultrastructural and immunohistochemical features with those of a conventional GCT of bone. Histologically, the HGCC consists of sheets of osteoclast-like giant cells with a background of mononuclear cells. The giant cells lack the pleomorphism seen in hepatocellular carcinomas with anaplastic giant cells. At the light microscopic level, most of this tumor was nearly identical to a GCT of bone, but several microscopic fields (less than 5% of the tumor) had the histologic appearance of a "usual" hepatocellular carcinoma. The hepatic tumor was negative for HAM 56, epithelial cytokeratins, muramidase, and alpha-1-antitrypsin, with only focal positivity for chymotrypsin in mononuclear and giant cells. The GCT was strongly positive for alpha-1-antitrypsin and chymotrypsin in both the mononuclear and giant cells and showed focal, weak staining for AE1 and AE3 in the mononuclear stromal cells. Ultrastructurally, both mononuclear and giant cells of the HGCC showed features typical of hepatocellular carcinoma. Although the patient presented in this report died, the pattern of growth was different from most hepatocellular carcinomas. The overall histologic features of this tumor are distinctive and appear to justify separating this variant from other types of hepatocellular carcinoma.

Topics: Adult; Biomarkers, Tumor; Bone Neoplasms; Carcinoma; Carcinoma, Hepatocellular; Cell Nucleus; Chymotrypsin; Cytoplasm; Female; Humans; Immunohistochemistry; Liver Neoplasms; Microscopy, Electron; Osteoclasts; Protease Inhibitors

Hepatoid adenocarcinomas of the stomach are gastric carcinomas with both adenocarcinomatous and hepatocellular differentiations. They usually produce large amounts of alpha-fetoprotein (AFP) with a Concanavalin A-binding property of hepatic type. In this study, these carcinomas occurred in older persons, with the antrum being a common site. Observed grossly, growth of the tumors was nodular and massive. Prognosis was poor because of frequent liver metastases. In the cytoplasms of tumor cells, various serum proteins were identified, including AFP, alpha-1 antitrypsin (AAT), alpha-1 antichymotrypsin (ACT), albumin, and prealbumin. Localizations of ferritin, prothrombin, and transferrin were demonstrated with less frequency. Adenocarcinomatous foci were composed of well-differentiated, intestinal-type epithelial cells and often contained carcinoembryonic antigen. These adenocarcinomatous and hepatoid areas were often intermingled with each other. There were extensive venous involvements by tumor cells. The poor prognosis of the tumors may be attributed to these involvements as well as to production of AFP and presence of AAT/ACT, which have immunosuppressive and protease-inhibitory properties, respectively.

Topics: Adenocarcinoma; Adult; Aged; Albumins; alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; alpha-Fetoproteins; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Cell Differentiation; Cell Nucleus; Chymotrypsin; Cytoplasm; Female; Histocytochemistry; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Middle Aged; Prealbumin; Prognosis; Staining and Labeling; Stomach Neoplasms

Plasmodium berghei sporozoites were observed to react with human hepatoma (HepG2) target cells which had been fixed with methanol, formaldehyde, or glutaraldehyde. The reaction consisted of attachment of sporozoites to the fixed target cells and the release of circumsporozoite protein which bound to target cell areas adjacent to the attachment sites. Treatment of fixed target cells with 0.1 N H2SO4 at 80 C, neuraminidases, neuraminidase plus galactose oxidase or inclusion of transferrin, orosomucoid, their asialo forms, or various monosaccharides in the incubation medium had no significant effect on target cell reactivity with sporozoites. Fixed cells oxidized with periodate or cells extracted with methanol or chloroform-methanol were reactive but lost activity if allowed to air dry after treatment. Treatment with papain or chymotrypsin at levels producing heavy cell structure damage caused a major loss of activity.

Topics: Carcinoma, Hepatocellular; Cells, Cultured; Chymotrypsin; Formaldehyde; Galactose Oxidase; Glutaral; Host-Parasite Interactions; Liver Neoplasms; Malaria; Methanol; Neuraminidase; Papain; Plasmodium berghei; Transferrin

Twenty-four cases of hepatoblastoma, 14 cases of hepatocellular carcinoma and three cases of malignant mesenchymoma out of a total of 54 primary liver tumours were studied by light microscopy and immunohistochemistry. A remarkable finding in one case of hepatoblastoma and one case of hepatocellular carcinoma was a sarcoid-like reaction in the tumour tissue. Three cases of hepatoblastoma presented a macrotrabecular pattern. Among hepatocellular carcinomas, three cases corresponded to the fibrolamellar variant. By immunohistochemistry, the proportion of cases with positive staining for alpha 1-fetoprotein was higher in hepatoblastoma than in hepatocellular carcinoma. HBs-antigen could be demonstrated in non-neoplastic liver cells in two cases of hepatocellular carcinoma, but not in the tumour cells. No strong correlation between histological pattern and prognosis could be established in hepatoblastoma. However, there was a tendency to more aggressive biological behavior in cases with pronounced mitotic activity. The number of mitoses in hepatoblastoma varied widely. As in previous studies, patients with the fibrolamellar variant of hepatocellular carcinoma fared better than those with the classical type of this tumour. Prognosis in malignant mesenchymoma was not as poor as suggested from previous studies.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; alpha-Fetoproteins; Carcinoma, Hepatocellular; Child; Child, Preschool; Chorionic Gonadotropin; Chymotrypsin; Female; Hepatitis B Antigens; Humans; Immunoenzyme Techniques; Liver Neoplasms; Male; Mesenchymoma; Neurotensin

Radioiodinated, native and denatured bovine serum albumin (albumin) beta-lactoglobulin and cytochrome c were introduced into hepatoma tissue culture cells by erythrocyte-ghost-mediated microinjection, and their rates of degradation were compared. Denatured albumin was degraded at 20% of the rate of undenatured albumin, denatured beta-lactoglobulin was degraded three times faster than undenatured beta-lactoglobulin, while denatured and undenatured cytochrome c were degraded at the same rate. Thus, denaturation does not affect the rates of intracellular breakdown of microinjected proteins in a simple predictable way. Exhaustive methylation did not inhibit the degradation of denatured beta-lactoglobulin or albumin, indicating that, like their undenatured counterparts, they are not degraded via the ubiquitin pathway. In reticulocyte lysates, in the presence of ATP, denatured albumin and beta-lactoglobulin were broken down at slightly slower rates than the parent proteins. Exhaustive methylation of both denatured and undenatured proteins completely abolished their ATP-dependent breakdown. This inhibition is consistent with the hypothesis that free -NH2 groups are required for the attachment of ubiquitin prior to degradation in this system. Removal of an ammonium sulfate fraction from reticulocyte lysates produces a proteolytic system markedly different from the whole lysate [Speiser, S. & Etlinger, J. D. (1983) Proc. Natl Acad. Sci. USA 80, 3577-3580]. In this system both denatured and undenatured albumin and beta-lactoglobulin were degraded essentially independently of ATP. Methylation only slightly decreased the breakdown of denatured proteins, suggesting that they are not degraded via the ubiquitin pathway. A possible explanation of these results is that removal of the ammonium sulfate fraction unmasks an ATP-independent proteolytic system unrelated to the ubiquitin pathway.

Topics: Carcinoma, Hepatocellular; Cell-Free System; Cells, Cultured; Chymotrypsin; Cytochrome c Group; Lactoglobulins; Liver Neoplasms; Methylation; Protein Denaturation; Reticulocytes; Serum Albumin; Trypsin

We studied, by electrophoretic techniques, the physiochemical properties of 4 glycoproteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, alpha 1-acid glycoprotein and transferrin synthesized by three different human hepatoma cell lines. A common feature was the export of glycoproteins with retarded electrophoretic mobility, indicating incomplete sialylation, and a predominance of atypical, highly branched carbohydrate chains. The abnormal glycosylation pattern may be specific for malignant transformation of hepatocytes and possibly related to the intracellular accumulation of some of these proteins in malignant cells.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Cell Line; Chymotrypsin; Glycoproteins; Humans; Immunoassay; Immunoelectrophoresis, Two-Dimensional; Liver Neoplasms; Neoplasm Proteins; Orosomucoid; Transferrin

Alpha-1-antitrypsin (A1AT) has been shown to be a useful tumor marker for hepatocellular carcinoma (HCC) and some other neoplasms. No studies of alpha-1-antichymotrypsin (A1AChy) in HCC have been reported. A comparative study of A1AT and A1AChy in HCC was undertaken. While 19/33 HCC were positive for A1AT, all 33 HCC contained immunoreactive A1AChy. Cells showing positive immunoreaction for both A1AT and A1AChy were more numerous in moderately differentiated HCC than in well differentiated or poorly differentiated HCC. Although the pattern of staining with both antisera was similar, in cases showing positive staining for both antisera, A1AChy-positive cells were more numerous than A1AT-positive cells. The results suggest a useful role for A1AChy as a marker of HCC.

Topics: alpha 1-Antichymotrypsin; alpha 1-Antitrypsin; Carcinoma, Hepatocellular; Cell Transformation, Neoplastic; Chymotrypsin; Histocytochemistry; Humans; Hyperplasia; Immunoenzyme Techniques; Liver; Liver Cirrhosis; Liver Neoplasms

The serum levels of alpha-1-antichymotrypsin (ACT) were studied in 168 patients with various liver diseases and cancers in conjunction with other liver function tests, serum sialic acid, AFP and CEA. The ACT levels in acute viral hepatitis and chronic hepatitis were not significantly altered compared with the normal level (220 +/- 40 microgram/ml), although the level was slightly increased or decreased temporarily during the acute phase of the former. In liver cirrhosis, the mean level was significantly lower than the normal in spite of the absence of signs of hepatic decompensation (168 +/- 51 microgram/ml, p less than 0.001). In contrast to cirrhosis, the levels were increased to various extents in 65% of cases with hepatoma, in spite of the association of liver cirrhosis in the majority of them. Much higher levels were observed in all cases of metastatic liver cancers and cancers of the pancreas and the biliary tract. The elevations were observed even in cases without the increase of AFP or CEA. Both in cirrhosis and cancers, ACT levels were not correlated with any of serum bilirubin and serum enzyme activities, but were positively correlated with the levels of plasma fibrinogen and serum sialic acid. The measurement of serum ACT level can be taken advantage of for the diagnosis and monitoring of liver cirrhosis and liver cancers, particularly of hepatoma without AFP elevation.

Topics: alpha 1-Antichymotrypsin; alpha-Fetoproteins; Blood Sedimentation; Carcinoembryonic Antigen; Carcinoma, Hepatocellular; Chymotrypsin; Fibrinogen; Humans; Liver Cirrhosis; Liver Diseases; Liver Neoplasms; Sialic Acids

Topics: Animals; BCG Vaccine; Carcinoma, Hepatocellular; Cell Wall; Chymotrypsin; Hydrochloric Acid; Injections; Liver Neoplasms; Lymphatic Metastasis; Male; Mice; Mycobacterium; Mycobacterium bovis; Mycobacterium tuberculosis; Neoplasm Transplantation; Neoplasms, Experimental; Nitrosamines; Nocardia asteroides; Pronase; Propionibacterium acnes; Skin Neoplasms; Sodium Hydroxide; Transplantation, Homologous; Trypsin

Topics: Amino Acids; Animals; Bromine; Carcinoma, Hepatocellular; Cattle; Cell Nucleus; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Female; Genes, Regulator; Histones; Liver; Liver Neoplasms; Liver Regeneration; Lysine; Peptides; Rabbits; Rats; Species Specificity; Succinimides; Thymus Gland; Uterus

Topics: Adenosine; Biological Transport; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Chloromercuribenzoates; Chromatography, Paper; Chymotrypsin; Cytidine; Dipyridamole; Ethanol; Guanosine; Hot Temperature; Inosine; Kinetics; Liver Neoplasms; Neoplasms, Experimental; Neuraminidase; Nucleic Acids; Nucleosides; Phospholipases; Phosphotransferases; Thymidine; Tritium; Trypsin; Uridine

Histones F2al extracted from normal and neoplastic cells possess similar amino acid compositions. Tryptic and chymotryptic peptides of the F2al histones have identical chromato-electrophoretic R(F) values. It is concluded that histones F2al from various sources have similar overall structures. The observed differences in the ratios of in-N-monomethyl- and di-in-N-methyl-lysine in the histones from normal and neoplastic cells may be of significance with respect to gene regulation.

Topics: Amino Acids; Animals; Carcinoma, Hepatocellular; Cattle; Cell Line; Chromatography; Chromatography, Gel; Chymotrypsin; Cyanides; Electrophoresis, Disc; Histones; Humans; Leukemia, Lymphoid; Liver Neoplasms; Lymphoma, Non-Hodgkin; Neoplasms, Experimental; Thymus Gland; Trypsin

Topics: Amino Acid Sequence; Arginine; Carcinoma, Hepatocellular; Chymotrypsin; Glycine; Histones; Liver Neoplasms; Neoplasm Proteins; Pepsin A