alpha-chymotrypsin and Multiple-Myeloma

Topics: Acute Disease; Acute Kidney Injury; alpha 1-Antichymotrypsin; Blood Proteins; C-Reactive Protein; Chymotrypsin; Haptoglobins; Hemopexin; Humans; Immunodiffusion; Kidney Diseases; Kidney Neoplasms; Latex Fixation Tests; Multiple Myeloma; Nephelometry and Turbidimetry; Orosomucoid; Protease Inhibitors

Proteasome inhibitors (PIs) are highly active in multiple myeloma (MM) but resistance is commonly observed. All clinical stage PIs effectively inhibit chymotrypsin-like (CT-L) activity; one possible mechanism of resistance is compensatory hyperactivation of caspase-like (C-L) and trypsin-like (T-L) subunits, in response to CT-L blockade. Marizomib (MRZ), an irreversible PI that potently inhibits all three 20S proteasome subunits with a specificity distinct from other PIs, is currently in development for treatment of MM and malignant glioma. The pan-proteasome pharmacodynamic activity in packed whole blood and peripheral blood mononuclear cells was measured in two studies in patients with advanced solid tumours and haematological malignancies. Functional inhibition of all proteasome subunits was achieved with once- or twice-weekly MRZ dosing; 100% inhibition of CT-L was frequently achieved within one cycle at therapeutic doses. Concomitantly, C-L and T-L activities were either unaffected or increased, suggesting compensatory hyperactivation of these subunits. Importantly, this response was overcome by continued administration of MRZ, with robust inhibition of T-L and C-L (up to 80% and 50%, respectively) by the end of Cycle 2 and maintained thereafter. This enhanced proteasome inhibition was independent of tumour type and may underlie the clinical activity of MRZ in patients resistant to other PIs.

Topics: Caspases; Chymotrypsin; Enzyme Activation; Glioma; Humans; Lactones; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrroles; Trypsin

The ubiquitin-proteasome system is relevant in the pathobiology of many haematological malignancies, including multiple myeloma. The assessment of proteasome concentration and chymotrypsin-like (ChT-L) activity might constitute a new approach to diagnosis, prognosis and monitoring of anticancer treatment of patients with haematological malignancies and other diseases. The aim of our study was to determine which material, plasma or serum, is better for measuring chymotrypsin-like (ChT-L) activity and proteasome concentration. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in 70 plasma and serum samples drawn from 28 patients at different treatment stages for multiple myeloma (MM) and 31 healthy volunteers. Proteasome ChT-L activity and concentration in multiple myeloma patients were significantly higher in plasma compared to serum. In this group we observed significant and positive correlations both between the plasma and serum proteasome ChT-L activity and plasma and serum proteasome concentration. The higher values of proteasome concentration and ChT-L activity in plasma than in serum and their better correlations with parameters of tumour load and prognosis suggest that plasma constitutes a better biological material for measuring ChT-L activity and proteasome concentration than serum in multiple myeloma patients.

Topics: Adult; Aged; Aged, 80 and over; Chymotrypsin; Humans; Middle Aged; Multiple Myeloma; Prognosis; Proteasome Endopeptidase Complex

Although bortezomib is successfully used against multiple myeloma, the pharmacodynamics of proteasome inhibition and its association with efficacy or resistance is poorly understood. Using ultra performance liquid chromatography coupled to tandem mass spectrometry, site-specific luminogenic substrate assays and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assays, effects of bortezomib on cellular drug concentrations, chymotrypsin- , caspase- , and trypsin-like activities, and cytotoxic efficacy were evaluated in eight myeloma cell lines directly after 1 h of exposure and additionally after a 23-h washout phase. Bortezomib accumulated in myeloma cells by up to 100-fold and concentration-dependently inhibited the proteasomal activities with the chymotrypsin-like activity being the most sensitive. Whereas intracellular concentrations correlated with the inhibition of the chymotrypsin- and the caspase-like activities of the proteasome, the cytotoxic efficacy of bortezomib did not correlate with either intracellular concentrations or proteasomal inhibition. However, the ratio of concentrations measured directly after the exposure and after the washout phase (indicating drug disposition) correlated with efficacy, suggesting that the cell's ability to dispose bortezomib at least in part influences bortezomib's cytotoxicity. In conclusion, this data argues against a direct association of intracellular concentration or proteasomal inhibition with cytotoxic efficacy but advocates for an important role of cellular drug disposition. Moreover, this study underlines the pleiotropic mode of action of bortezomib going beyond proteasome inhibition.

Topics: Antineoplastic Agents; Bortezomib; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Liquid; Chymotrypsin; Dose-Response Relationship, Drug; Humans; Inhibitory Concentration 50; Multiple Myeloma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Serine Proteinase Inhibitors; Tandem Mass Spectrometry; Time Factors

Proteasome inhibition is associated with substantial antitumor effects in preclinical models of multiple myeloma (MM) as well as in patients. However, results of recent clinical trials to evaluate the effect of the proteasome inhibitor Bortezomib (Velcade(®), also called PS-341) in MM patients have shown limited activity when used as a single agent. This underscores the need to find new efficacious and less toxic proteasome inhibitors. Recently, carfilzomib was approved for the treatment of refractory/relapsed MM and several new agents have been introduced into the clinic, including marizomib and MLN9708, and trials investigating these second-generation proteasome inhibitors have demonstrated promising results. We have recently synthesized a novel proteasome inhibitor, BU-32, and tested its growth inhibitory effects in different human MM cells including RPMI8226, MM.1S, MM.1R, and U266. In this study, we evaluate the efficacy of the novel proteasome inhibitor BU-32 (NSC D750499) using an in vitro MM model. BU-32 exhibits strong cytotoxicity in a panel of MM cell lines--RPMI8226, MM1S, MM1R, and U266. In addition, we demonstrate by proteasome inhibition assay that BU-32 potently inhibits the chymotryptic- and caspase-like activities of the 26S proteasome. We further show from Annexin V-FITC binding studies that BU-32, like Bortezomib, induces apoptosis in a panel of MM cell lines but the effect is more pronounced with BU-32-treated cells. Invasion assay with the MM.1S cell line indicates that BU-32 inhibits the invasiveness of myeloma cells. Results from our studies using real-time PCR array analyses show that BU-32 effectively downregulates an array of angiogenesis and inflammatory markers. Our results suggest that BU-32 might be a potential chemotherapeutic agent with promising antitumor activity for the treatment of MM.

Topics: Animals; Apoptosis; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Proliferation; Chymotrypsin; Down-Regulation; Humans; Mice; Multiple Myeloma; Proteasome Inhibitors; Pyrazoles

The ubiquitin-proteasome pathway is implicated in the pathogenesis of many haematologic malignancies, including multiple myeloma. Under conditions of rapid cell turnover and growth rate, proteasomes are returned into circulation. The measurement of their levels or activity could offer a new approach to diagnosis, prognosis and monitoring of anticancer treatment in carcinoma patients. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in the plasma of 64 patients with a newly diagnosed multiple myeloma and 30 healthy volunteers. The values were found to be significantly higher in the studied patients and advanced disease stages compared to the control group, and decreased significant after chemotherapy. Both proteasome concentration and ChT-L activity correlated with adverse prognostic factors, such as lactate dehydrogenase and β2-macroglobulin. We also showed that proteasome concentration positively correlates with IL-6 level, as opposed to proteasome ChT-L activity. Of note, higher proteasome ChT-L activity, unlike the concentration, was proved to be an indicator of a shorter progression free survival, constituting thereby an important prognostic marker.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Chymotrypsin; Enzyme Assays; Female; Humans; Male; Middle Aged; Monitoring, Physiologic; Multiple Myeloma; Prognosis; Proteasome Endopeptidase Complex; Thalidomide

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much. These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.

Topics: Animals; B-Lymphocytes; Burkitt Lymphoma; Cattle; Chymotrypsin; Dose-Response Relationship, Drug; Flow Cytometry; Histones; Humans; Hybridomas; Immunoglobulin M; Multiple Myeloma; Protein Binding; Protein Conformation; Protein Transport; Thymus Gland; Trypsin; Tumor Cells, Cultured

To evaluate the impact of an additive therapy with an oral enzyme (OE) preparation given for more than 6 months additionally to standard combination chemotherapy (vincristine/melphalan/cyclophosphamide/prednisone (VMCP)- or methylprednisolone/ vincristine/CCNU/cyclophosphamide/melphalan (MOCCA)-regimen) in the primary treatment of patients with multiple myeloma stages I-III.. A cohort of 265 patients with multiple myeloma stages I-III was consecutively treated at our institution in two parallel groups (control group (n = 99): chemotherapy +/-OE for less than 6 months; OE-group (n = 166): chemotherapy + OE for more than 6 months). The median follow-up time in the stages I, II, and III for the OE-group was 61, 37, and 46.5 months, respectively; for the control group the respective values were 33, 51.5, and 31.5 months. The primary endpoint of the study was disease-specific survival. Secondary endpoints were response to therapy, duration of first response and side effects. The chosen method for evaluation was the technique of a retrolective cohort analysis with a concurrent control group. Survival analysis was performed by the Kaplan-Meier method and multivariate analysis was done with the Cox proportional hazards model.. Significantly higher overall response rates and longer duration of remissions were observed in the OE-group. Primary responders showed a longer mean survival time than non-responders. Additive therapy with OE given for more than 6 months decreased the hazard of death for patients at all stages of disease by approximately 60%. Observation time was not long enough to estimate the median survival for patients at stages I and II; for stage III patients it was 47 months in the control group versus 83 months for the patients treated with OE (P = 0.0014) which means a 3-year gain of survival time. Significant prognostic factors for survival, in the Cox regression analysis, were stage of disease and therapy with OE. The OE-therapy was generally well tolerated (3.6% of patients with mild to moderate gastrointestinal symptoms).. OEs represent a promising new additive therapy in multiple myeloma which will be further evaluated in a randomized phase III trial in the USA.

Topics: Administration, Oral; Adolescent; Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Chymotrypsin; Cohort Studies; Cyclophosphamide; Dexamethasone; Doxorubicin; Drug Combinations; Endopeptidases; Female; Humans; Lomustine; Male; Melphalan; Methylprednisolone; Middle Aged; Multiple Myeloma; Papain; Prednisone; Proportional Hazards Models; Retrospective Studies; Survival Rate; Trypsin; Vincristine

Topics: Animals; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Mice; Mice, Inbred BALB C; Mink; Multiple Myeloma; Neutralization Tests; Precipitin Tests; Recombination, Genetic; RNA Viruses; Species Specificity; Viral Envelope Proteins; Viral Proteins

The preceeding papers dealth with the purification and characterization of IgA immunoglobulin Tro and its H- and L-chains, the separation and characterization of the cyanogen bromide fragments and the isolation of the tryptic splitting products of the H-chain. In this paper the chymotryptic peptides of the H-chain are presented. All chymotryptic peptides were isolated and purified parallel to the isolation of tryptic peptides. On the basis of these overlapping splitting products the arrangement of the tryptic peptides in the protein could be determined. These and other splitting products listed in this paper contributed to the complete sequence determination of of the H-chain.

Topics: Amino Acid Sequence; Amino Acids; Chymotrypsin; Humans; Immunoglobulin A; Immunoglobulin Heavy Chains; Multiple Myeloma; Myeloma Proteins; Peptide Fragments

The human myeloma protein Boh (gamma 2, lambda) was isolated and completely reduced and aminoethylated. The light chain was obtained by chromatography on Sephadex G-100 in 4 M guanidine HC1. The amino-terminal sequence on the blocked light chain could be determined by automatic sequence degradation after PCAase treatment. Twenty-one peptides were isolated from a tryptic digest and 12 peptides from a chymotryptic digest. The sequence determination on these peptides was performed by automatic sequencing methods. The light chain of Boh protein belongs to the lambda II subgroup. Unique substitutions have been found at position 8 (Arg) and position 62 (Tyr). Furthermore, the Boh light chain has six cysteine residues, the additional (sixth) cysteine being adjacent to the invariable intrachain-S-S linking cysteine at position 91. Sequence comparison of lambda II proteins reveals a high degree of homology emphasizing the biologic significance of the hypervariable region sequences;

Topics: Amino Acid Sequence; Amino Acids; Ammonium Sulfate; Carboxypeptidases; Chromatography, DEAE-Cellulose; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Freeze Drying; Humans; Immunoglobulin Fragments; Immunoglobulin lambda-Chains; Multiple Myeloma; Myeloma Proteins; Peptide Chain Termination, Translational; Peptides; Protein Conformation; Pyrrolidonecarboxylic Acid; Sodium Dodecyl Sulfate; Subtilisins; Trypsin

Topics: Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Hemagglutination Inhibition Tests; Humans; Immune Sera; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin Fragments; Immunoglobulin G; Isoelectric Focusing; Methods; Multiple Myeloma; Trypsin

Topics: Amino Acid Sequence; Amino Acids; Bence Jones Protein; Chromatography, Ion Exchange; Chymotrypsin; Humans; Immunoglobulin Fragments; Multiple Myeloma; Peptide Fragments; Thermolysin; Trypsin

Topics: Alkylation; Amino Acid Sequence; Amino Acids; Chromatography, Gel; Chymotrypsin; Dansyl Compounds; Electrophoresis; Formates; Humans; Hydrogen-Ion Concentration; Immunoglobulin Fragments; Immunoglobulin G; Lysine; Multiple Myeloma; Myeloma Proteins; Oxidation-Reduction; Peptide Fragments; Serine; Thiocyanates; Tosyl Compounds; Trypsin; X-Ray Diffraction

Topics: Amino Acid Sequence; Amino Acids; Animals; Autoanalysis; Carbohydrates; Chromatography, Ion Exchange; Chymotrypsin; Complement Fixation Tests; Cyanogen Bromide; Guinea Pigs; Humans; Immunoglobulin Fc Fragments; Immunoglobulin Fragments; Immunoglobulin G; Latex; Mice; Microspheres; Multiple Myeloma; Myeloma Proteins; Peptide Fragments; Protein Conformation; Rabbits; Species Specificity; Subtilisins; Trypsin

Topics: Aluminum Silicates; Betamethasone; Blood Platelets; Blood Proteins; Cell Membrane; Chymotrypsin; Fibrinogen; gamma-Globulins; Humans; Immunochemistry; Immunoglobulin G; Immunoglobulins; Kaolin; Latex; Microspheres; Multiple Myeloma; Neoplasm Proteins; Platelet Adhesiveness

Topics: Alkylation; Amino Acid Sequence; Amino Acids; Carboxypeptidases; Chromatography, Gel; Chromatography, Ion Exchange; Chymotrypsin; Cold Temperature; Cryoglobulins; Cyanides; Humans; Immunoglobulin G; Immunoglobulin M; Immunoglobulins; Methods; Molecular Weight; Multiple Myeloma; Peptide Hydrolases; Peptides; Trypsin

Topics: Amino Acid Sequence; Animals; Bence Jones Protein; Chromatography, Ion Exchange; Chymotrypsin; Endopeptidases; Humans; Mice; Multiple Myeloma; Rabbits; Species Specificity