Halina Ostrowska

Lactacystin Inhibits Cathepsin A Activity in Melanoma Cell Lines

We describe the inhibitory effect of the proteasome inhibitor, lactacystin, on cathepsin A activity in murine melanoma cell lines. In vitro lactacystin metabolite, β-lactone, at a concentration of 1 µM, significantly suppressed cathepsin A activity in B78 melanoma cell lysates by about 50%. Exposure of three murine melanoma cell lines with different metastatic potential to lactacystin at a concentration of 5 µM for 6 h caused a significant reduction in the carboxypeptidase activity of this enzyme, while the inhibitory activity remained unchanged for at least 12 h. Other proteasome-specific inhibitors, e.g. epoxomicin and N-benzyloxycarbonyl-Ile-Glu(O-tert-Bu)-Ala-leucinal (PSI) at a concentration of 1 µM did not affect cathepsin A activity in melanoma cell line lysates. These data support our previous proposal that lactacystin is not a specific inhibitor of the proteasome. Since cathepsin A is also a tumor-associated enzyme, further research is needed to clarify its role and the significance of its inhibition by lactacystin in tumor biology.

Assessment of circulating proteasome chymotrypsin-like activity in plasma of patients with acute and chronic leukemias.

OBJECTIVE We evaluated whether the proteasomal chymotrypsin-like (ChT-L) activity is increased in plasma of patients with acute lymphoblastic (ALL), acute myeloblastic (AML) and chronic lymphocytic (CLL) leukemias. METHODS The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors (n=15) and ALL (n=15), AML (n=28) and CLL (n=22) patients. RESULTS The activity was significantly (P<0.001) higher in the plasma of ALL and AML patients at the diagnosis than in healthy subjects and decreased after therapy or remained unchanged or rose during relapse. By contrast, in CLL patients at the diagnosis, the activity did not differ significantly from the healthy controls. In each group, the activity positively correlated with the serum lactic dehydrogenase activity. CONCLUSIONS Plasma proteasome ChT-L activity can be a useful bio-marker for patients with acute leukemia at the blast stage.

Proteasome inhibitor prevents experimental arterial thrombosis in renovascular hypertensive rats

Recent studies indicate that highly selective proteasome inhibitors can be useful in prevention of some cardiovascular events. Here we demonstrate that proteasome inhibitor, Z-Ile-Glu (Ot-Bu) Ala-Leucinal (PSI), is active in the prevention of platelet-dependent arterial thrombosis induced in renovascular hypertensive rats (two-kidney, one clip Goldblatt model, and 2K1C, n=5). The administration of PSI intravenously at a single dose of 0.3 mg/kg before induction of arterial thrombosis markedly increased carotid final flow rate, as compared to control (vehicle) group (10.36 +/- 1.8 ml/min and 1.2 +/- 1.2 ml/min, respectively), significantly decreased the wet (1.23 +/- 0.23 mg and 4.1 +/- 0.94 mg, respectively), and dry (0.46 +/- 0.145 mg and 1.46 +/- 0.39, respectively) thrombus weight, and completely prevented arterial occlusion. Moreover, platelets from PSI - treated thrombotic 2K1C rats, showed in response to collagen a significant inhibition of aggregation in the whole blood (10.26 +/- 0.6 ohms vs. 15.51 +/- 0.91 ohms in the control group). In contrast, collagen-induced platelet aggregation was not inhibited in vitro, after pre-treatment of the blood with PSI at the concentration of 10 microM that effectively inhibited the 20S proteasome activity in platelets, indicating that ex vivo anti-aggregatory effect of PSI proceeds through an indirect mechanism not associated with suppression of 20S proteasome activity in platelets. In conclusion, our in vivo findings demonstrate that proteasome inhibitor, Z-Ile-Glu(Ot-Bu)Ala-Leucinal, prevents the development of arterial thrombosis in renovascular hypertensive rats and effectively suppresses platelet aggregation by an indirect mechanism. Thus the data provide a new insight into the potential role for the proteasome-dependent pathway in cardiovascular events.

Human platelet 20S proteasome: inhibition of its chymotrypsin-like activity and identification of the proteasome activator PA28. A preliminary report

Earlier studies have demonstrated that human platelets contain the 20S proteasome, and its protein activator. However, understanding the potential role of the proteasome in human platelets requires a detailed knowledge about its chymotryptic-like activity, a crucial one for protein degradation in all eukaryotic cells. In this communication we have shown that human platelet 20S proteasome exhibited chymotryptic-like activity towards succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin as substrate at a broad pH range, with optimum between pH 7.5-8.0 and 5.0-5.5. These two activities were markedly inhibited by a 10 μmol/l concentration of two structurally unrelated proteasome inhibitors: lactacystin/β-lactone or benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal, but not by ebelactone B, an inhibitor of lysosomal cathepsin A/deamidase. The chymotryptic-like activity of the 20S proteasome against succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin was also significantly inhibited in platelets, after exposure of platelet-rich plasma to 10 μmol/l lactacystin and benzyloxycarbonyl-Ile-Glu(O-tert.-butyl)-Ala-leucinal for up to 60 min. This indicates that these inhibitors can enter platelets and selectively inhibit 20S proteasome activity. We also demonstrated for the first time by Western blot analysis that human platelets contain a proteasome activator, PA28, which is known to play a key role in antigen processing by significant stimulation of the proteasomal chymotryptic-like activity. Since the platelet 20S proteasome was also present in a latent form, this suggests that its activity may be regulated in vivo in human platelets. All these results can therefore be beneficial in future studies on the role of the 20S proteasome in platelet biology.

S100A6 is transcriptionally regulated by β-catenin and interacts with a novel target, lamin A/C, in colorectal cancer cells.

In this paper we document an increased expression of S100A6, a calcium binding protein of the S100 family, and its co-localization with β-catenin in colorectal cancer tissues and in metastatic, SW620, versus non-metastatic, SW480, human colorectal cancer cell lines. Moreover, we show up-regulation of the S100A6 protein level in non-metastatic SW480 cells due to overexpression of β-catenin as well as the activation of the S100A6 gene promoter upon cell transfection with β-catenin and the TCF-Lef1 transcription factor. Since we found a high level of S100A6 in metastatic SW620 cells we searched for its interacting partners in the protein extract prepared from these cells. Using several methods we found that S100A6 interacts with lamin A/C, a protein known to be implicated in colon carcinogenesis. Our results reveal a novel and important network of relations and interactions between proteins potentially involved in colorectal cancer development and progression.

The ubiquitin-proteasome system: A novel target for anticancer and anti-inflammatory drug research

The ubiquitin-proteasome system is responsible for the degradation of most intracellular proteins, including those that control cell cycle progression, apoptosis, signal transduction and the NF-κB transcriptional pathway. Aberrations in the ubiquitin-proteasome system underlie the pathogenesis of many human diseases, so both the ubiquitin-conjugating system and the 20S proteasome are important targets for drug discovery. This article presents a few of the most important examples of the small molecule inhibitors and modulators targeting the ubiquitin-proteasome system, their mode of action, and their potential therapeutic relevance in the treatment of cancer and inflammatory-related diseases.

Increased plasma proteasome chymotrypsin-like activity in patients with advanced solid tumors

The chymotrypsin-like (ChT-L) activity is one of the key regulators of intracellular protein degradation. Elevated proteasome ChT-L activity has recently been reported in plasma of patients with leukemia and myelodysplastic syndrome and suggested to have a prognostic significance. The aim of the present study was to evaluate plasma proteasome ChT-L activity in patients with newly diagnosed solid tumors at early and advanced stages of the disease. The activity was assayed using the fluorogenic peptide substrate, Suc-Leu-Leu-Val-Tyr-AMC, in a cohort of 155 patients with early/advanced rectal (n = 43/29), gastric (n = 6/13), and breast (n = 37/27) cancer and compared with that in normal individuals (n = 55). The median plasma proteasome ChT-L activity was elevated by 20–32% in patients with advanced stage of rectal, gastric, and breast cancer compared with healthy donors. The difference turned out to be statistically significant (P < 0.001). By contrast, values in patients with early stage of malignancies were not significantly different from those observed in normal individuals. We also found that plasma proteasome activity correlated with serum carcinoembryonic antigen levels in the group of patients with rectal cancer (r = 0.433, P < 0.05). Elevated plasma proteasome ChT-L activity is indicative of advanced stage of rectal, gastric, and breast cancer. However, the activity does not seem to be a parameter with clinically relevant potential in terms of early detection of cancer in this subset of patients.

Ebelactone B, an inhibitor of extracellular cathepsin A-type enzyme, suppresses platelet aggregation ex vivo in renovascular hypertensive rats.

The present study was undertaken to investigate whether ebelactone B, an inhibitor of bradykinin and angiotensin I hydrolysis by serine carboxypeptidase Y-like enzymes, could influence platelet aggregation ex vivo in renovascular hypertensive rats (2-kidney, 1-clip Goldblatt model, 2K1C). We found that ebelactone B (5 mg/kg) administrated subcutaneously once a day for 5 days, 5 weeks after the development of hypertension, or a single dose of ebelactone B (0.5 mg/kg) injected intravenously into 2K1C hypertensive rats before the induction of arterial thrombosis, both markedly suppressed collagen-induced platelet aggregation in whole blood. In contrast, inhibition of collagen-induced platelet aggregation was not evident in vitro after pretreatment of the blood with ebelactone B, indicating that ex vivo the antiaggregatory action of this compound can proceed through an indirect mechanism. The injection of ebelactone B did not affect the mean blood pressure of 2K1C hypertensive rats but lowered an elevated extracellular serine carboxypeptidase cathepsin A-like activity. Thus, the data indicate that ebelactone B may be a promising antiaggregatory agent in renovascular hypertension and suggest that 1 of the possible mechanisms through which it exerts this effect may be related to the suppression of cathepsin A-like activity released locally during the development of renovascular hypertension.

Correlation between circulating proteasome activity, total protein and c-reactive protein levels following burn in children

AIM OF THE STUDY To characterize burn-induced changes following burn in children by analyzing circulating proteasome (c-proteasome) activity in the plasma in correlation with total protein and c-reactive protein levels in the plasma, and the severity of the burn. METHODS Fifty consecutive children scalded by hot water who were managed at the Department of Pediatric Surgery after primarily presenting with burns in 4-20% TBSA were included into the study. The children were aged 9 months up to 14 years (mean age 2.5±1 years). Patients were divided into groups according to the pediatric injury severity score used by American Burns Association. Plasma proteasome activity was assessed using Suc-Leu-Leu-Val-Tyr-AMC peptide substrate, 2-6 h, 12-16 h, 3 days, 5 days, and 7 days after injury. 20 healthy children consecutively admitted for planned inguinal hernia repair served as controls. RESULTS Statistically significant elevation of plasma c-proteasome activity was noted in all groups of burned children 12-16 h after the injury. We found a strong negative correlation of c-proteasome activity with total protein levels, and positive correlation with CRP levels 12-16 h after burn. We also found stronger correlation between c-proteasome activity and severity of burn, than CRP level and severity of burn 12-16 h, and 3 days after the burn. Correlations were statistically significant. CONCLUSIONS This study characterized circulating 20S proteasome activity levels after burn. C-proteasome activity elevate after burn and correlate negatively with plasma total protein level, thus plasma 20S proteasome activity could be additional biomarker of tissue damage in burn in pediatric population.

The comparison of C-proteasome activity in the plasma of children after burn injury, mild head injury and blunt abdominal trauma

PURPOSE We aimed to evaluate and compare the changes in circulating 20S proteasome activity in the plasma of children suffering from blunt abdominal trauma, thermal injury and mild head injury. PATIENTS AND METHODS The study population comprised 40 patients with burns, 35 children admitted due to mild head injury, and 30 children suffering from blunt abdominal trauma, who were admitted to Pediatric Surgery Department of Medical University of Bialystok Poland, between 2010 and 2014, and their parents gave informed consent, were included into the study. Patients were aged 9 months to 17 years (median=5.73±1.91y). The girls to boys ratio was nearly 1:2 (34 girls and 106 boys). Plasma proteasome activity was assessed using Suc-Leu-Leu-Val-Tyr-AMC peptide substrate, 2-6h, 12-16h, and 48h after the injury. 20 healthy children admitted for planned inguinal hernia repair served as controls. RESULTS In our series of patients, the C-proteasome activity was much higher 12-16h after burns, than after mild head injuries, or blunt abdominal injuries, and the difference was statistically significant (p<0.05). CONCLUSIONS Circulating 20S proteasome is probably released from damaged tissues in response to the injury and is a biomarker of tissue damage - more severe in the group of burnt patients in comparison to the patients with mild head injury and blunt abdominal trauma. Therefore detection of 20S proteasome may represent a novel marker of immunological activity and cellular degradation in trauma patients.