benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Acute-Disease

ST1926 is an atypical retinoid and a promising anti-tumour agent with selective apoptotic activity on the leukaemic blast. The anti-tumour activity of the compound has been associated with its capacity to induce DNA double stranded breaks. Target profiling by affinity chromatography coupled to mass spectrometry led to the identification of histone H2A.Z as a protein capable of binding ST1926 specifically. The result was confirmed by studies involving Surface Plasmon Resonance (SPR). This indicates that H2A.Z is a primary target of ST1926 and links the perturbations of the histone pathway observed by microarray analysis to the DNA damage and apoptotic responses caused by the atypical retinoid. Comparison of the whole-genome gene-expression profiles of the ST1926-sensitive NB4 and the ST1926-resistant NB4.437r cell lines demonstrated differential expression of numerous genes. Network analysis of the data indicated enrichment of the cellular pathways controlling cAMP (cyclic adenosine-monophosphate)-dependent signal transduction, proteasome-dependent protein degradation and nuclear histones in NB4.437r cells. Pharmacological inhibition of cAMP-dependent protein kinase A with H89 partially reverted resistance of NB4.437r cells to ST1926. Conversely, inhibition of the proteasome with MG132 or bortezomib blocked the apoptotic response afforded by ST1926 in the NB4 cell line. This last effect was associated with a dramatic reduction in the DNA damage caused by the atypical retinoid. The results corroborate the idea that DNA damage is an important determinant of ST1926 apoptotic activity. More importantly, they demonstrate a proactive role of the proteasome in the DNA damaging and ensuing apoptotic response observed upon the challenge of acute myeloid leukaemia cells with ST1926.

Topics: Acute Disease; Adamantane; Apoptosis; Blotting, Western; Cell Line, Tumor; Cell Survival; Cinnamates; Cyclic AMP-Dependent Protein Kinases; Drug Resistance, Neoplasm; Enzyme Inhibitors; Gene Expression Profiling; Gene Expression Regulation, Leukemic; Histones; Humans; Isoquinolines; Leukemia, Myeloid; Leupeptins; Oligonucleotide Array Sequence Analysis; Proteasome Endopeptidase Complex; Protein Binding; Signal Transduction; Sulfonamides; Surface Plasmon Resonance

To observe the effect of proteasome inhibitor MG-132 on severe acute pancreatitis (SAP) and associated lung injury of rats.. Male adult SD rats were randomly divided into SAP group, sham-operation group, and MG-132 treatment group. A model of SAP was established by injection of 5% sodium taurocholate into the biliary-pancreatic duct of rats. The MG-132 group was pretreated with 10 mg/kg MG-132 intraperitoneally (ip) 30 min before the induction of pancreatitis. The changes in serum amylase, myeloperoxidase (MPO) activity of pancreatic and pulmonary tissue were measured. The TNF-alpha level in pancreatic cytosolic fractions was assayed with an enzyme-linked immunosorbent assay (ELISA) kit. Meanwhile, the pathological changes in both pancreatic and pulmonary tissues were also observed.. MG-132 significantly decreased serum amylase, pancreatic weight/body ratio, pancreatic TNF-alpha level, pancreatic and pulmonary MPO activity (P < 0.05). Histopathological examinations revealed that pancreatic and pulmonary samples from rats pretreated with MG-132 demonstrated milder edema, cellular damage, and inflammatory activity (P < 0.05).. The proteasome inhibitor MG-132 shows a protective effect on severe acute pancreatitis and associated lung injury of rats.

Topics: Acute Disease; Amylases; Animals; Cysteine Proteinase Inhibitors; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Leupeptins; Lung; Lung Diseases; Male; Organ Size; Pancreas; Pancreatitis; Peroxidase; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Taurocholic Acid; Tumor Necrosis Factor-alpha

To establish the therapeutic potential of proteasome inhibition, we examined the therapeutic effects of MG132 (Z-Leu-Leu-Leu-aldehyde) in an experimental model of acute pancreatitis.. Pancreatitis was induced in rats by two hourly intraperitoneal (ip) injections of cholecystokinin octapeptide (CCK; 2 x 100 microg/kg) and the proteasome inhibitor MG132 (10 mg/kg ip) was administered 30 min after the second CCK injection. Animals were sacrificed 4 h after the first injection of CCK.. Administering the proteasome inhibitor MG132 (at a dose of 10 mg/kg, ip) 90 min after the onset of pancreatic inflammation induced the expression of cell-protective 72 kDa heat shock protein (HSP72) and decreased DNA-binding of nuclear factor-kappaB (NF-kappaB). Furthermore MG132 treatment resulted in milder inflammatory response and cellular damage, as revealed by improved laboratory and histological parameters of pancreatitis and associated oxidative stress.. Our findings suggest that proteasome inhibition might be beneficial not only for the prevention, but also for the therapy of acute pancreatitis.

Topics: Acute Disease; Animals; Body Weight; Cholecystokinin; Cysteine Proteinase Inhibitors; Cytokines; HSP72 Heat-Shock Proteins; Leupeptins; Male; NF-kappa B; Organ Size; Oxidative Stress; Pancreas; Pancreatitis; Peroxidase; Proteasome Inhibitors; Rats; Rats, Wistar

To observe the protective effect of the proteasome inhibitor MG-132 in rats with severe acute pancreatitis (SAP) and the associated lung injury.. In rat models of the SAP established with injection of 5% sodium taurocholate into the biliary-pancreatic duct, the changes of the serum amylase and myeloperoxidase (MPO) activity in the pancreatic and lung tissues were evaluated. The pathological changes of the pancreatic and lung tissues were also observed.. MG-132 significantly decreased serum amylase, pancreatic weight/body weight ratio, and pancreatic and pulmonary myeloperoxidase activity (P<0.05). Histopathological examinations revealed milder edema, cellular damage, and inflammation in the pancreatic and lung tissues of rats pretreated with the peptide (P<0.05).. MG-132 ameliorates SAP and the associated lung injury in rats.

Topics: Acute Disease; Amylases; Animals; Cysteine Proteinase Inhibitors; Leupeptins; Lung; Lung Injury; Pancreas; Pancreatitis; Peroxidase; Rats; Rats, Sprague-Dawley

The cell-permeant MG132 tripeptide (Z-Leu-Leu-Leu-aldehyde) is a peptide aldehyde proteasome inhibitor that also inhibits other proteases, including calpains and cathepsins. By blocking the proteasome, this tripeptide has been shown to induce the expression of cell-protective heat shock proteins (HSPs) in vitro. Effects of MG132 were studied in an in vivo model of acute pancreatitis. Pancreatitis was induced in male Wistar rats by injecting 2 x 100 microug/kg cholecystokinin octapeptide intraperitoneally (ip) at an interval of 1 h. Pretreating the animals with 10 mg/kg MG132 ip before the induction of pancreatitis significantly inhibited IkappaB degradation and subsequent activation of nuclear factor-kappaB (NF-kappaB). MG132 also increased HSP72 expression. Induction of HSP72 and inhibition of NF-kappaB improved parameters of acute pancreatitis. Thus MG132 significantly decreased serum amylase, pancreatic weight/body weight ratio, pancreatic myeloperoxidase activity, proinflammatory cytokine concentrations, and the expression of pancreatitis-associated protein. Parameters of oxidative stress (GSH, MDA, SOD, etc.) were improved in both the serum and the pancreas. Histopathological examinations revealed that pancreatic specimens of animals pretreated with the peptide demonstrated milder edema, cellular damage, and inflammatory activity. Our findings show that simultaneous inhibition of calpains, cathepsins, and the proteasome with MG132 prevents the onset of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Body Weight; Cysteine Proteinase Inhibitors; Cytokines; HSP72 Heat-Shock Proteins; Leupeptins; Lung; Male; NF-kappa B; Oxidative Stress; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Proteasome Inhibitors; Rats; Rats, Wistar; Sincalide

The human herpes virus 8 (HHV8)-encoded viral FLICE (Fas-associating protein with death domain-like interleukin-1-converting enzyme) inhibitory protein (vFLIP) is believed to protect cells against death receptor-mediated apoptosis. In the present study we demonstrate that expression of HHV8 vFLIP in a growth factor-dependent TF-1 leukemia cell line protects against growth factor withdrawal-induced apoptosis. Unlike vector-expressing cells, those expressing HHV8 vFLIP maintain their mitochondrial membrane potential upon withdrawal from growth factor and also exhibit a block in the activation of caspases. The protective effect of HHV8 vFLIP is associated with its ability to activate the nuclear factor-kappa B (NF-kappaB) pathway and is missing in the vFLIP encoded by equine herpes virus 2 that lacks this activity. Inhibition of the NF-kappaB pathway by IkappaB superrepressor, lactacystin, MG132, arsenic trioxide, and phenylarsine oxide reverse the protection against growth factor withdrawal-induced apoptosis conferred by HHV8 vFLIP. HHV8 vFLIP up-regulates the expression of Bcl-x(L), an antiapoptotic member of the Bcl2 family, which is a known target of the NF-kappaB pathway. Collectively, the above results suggest that HHV8 vFLIP-induced NF-kappaB activation may contribute to cellular transformation seen in association with HHV8 infection by preventing the apoptosis of cells destined to die because of growth factor deprivation.

Topics: Acetylcysteine; Acute Disease; Apoptosis; Arsenic Trioxide; Arsenicals; bcl-X Protein; Cell Transformation, Viral; Enzyme Activation; Gene Expression Regulation, Viral; Granulocyte-Macrophage Colony-Stimulating Factor; Herpesvirus 8, Human; Humans; I-kappa B Proteins; Leukemia, Myeloid; Leupeptins; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Neoplasm Proteins; NF-kappa B; Oxides; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Recombinant Proteins; Rhadinovirus; Transcription, Genetic; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha; Viral Proteins

Acute myelogenous leukemia (AML) is typically a disease of stem progenitor cell origin. Interestingly, the leukemic stem cell (LSC) shares many characteristics with normal hematopoietic stem cells (HSCs) including the ability to self-renew and a predominantly G(0) cell-cycle status. Thus, although conventional chemotherapy regimens often ablate actively cycling leukemic blast cells, the primitive LSC population is likely to be drug-resistant. Moreover, given the quiescent nature of LSCs, current drugs may not effectively distinguish between malignant stem cells and normal HSCs. Nonetheless, based on recent studies of LSC molecular biology, we hypothesized that certain unique properties of leukemic cells could be exploited to induce apoptosis in the LSC population while sparing normal stem cells. In this report we describe a strategy using treatment of primary AML cells with the proteasome inhibitor carbobenzoxyl-l-leucyl-l-leucyl-l-leucinal (MG-132) and the anthracycline idarubicin. Comparison of normal and leukemic specimens using in vitro culture and in vivo xenotransplantation assays shows that the combination of these two agents induces rapid and extensive apoptosis of the LSC population while leaving normal HSCs viable. Molecular genetic studies using a dominant-negative allele of inhibitor of nuclear factor kappaB (IkappaBalpha) demonstrate that inhibition of nuclear factor kappaB (NF-kappaB) contributes to apoptosis induction. In addition, gene-expression analyses suggest that activation of p53-regulated genes are also involved in LSC apoptosis. Collectively, these findings demonstrate that malignant stem cells can be preferentially targeted for ablation. Further, the data begin to elucidate the molecular mechanisms that underlie LSC-specific apoptosis and suggest new directions for AML therapy.

Topics: Acute Disease; Alleles; Animals; Antibiotics, Antineoplastic; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Gene Expression Regulation, Leukemic; Graft Survival; Hematopoietic Stem Cells; Humans; I-kappa B Proteins; Idarubicin; Leukemia, Myeloid; Leukocytes; Leupeptins; Mice; Mice, Inbred NOD; Mice, SCID; Multienzyme Complexes; Neoplasm Proteins; Neoplasm Transplantation; Neoplastic Stem Cells; NF-kappa B; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Tumor Suppressor Protein p53

The 26S proteasome is a non-lysosomal multicatalytic protease complex for degrading intracellular proteins by ATP/ubiquitin-dependent proteolysis. Tightly ordered proteasomal degradation of proteins critical for cell cycle control implies a role of the proteasome in maintaining cell proliferation and cell survival. In this study, we demonstrate that cell-permeable proteasome inhibitors, lactacystin, benzyloxycarbonyl(Z)-leucyl-leucyl-leucinal (ZLLLal; MG-132) and 4-hydroxy-5-iodo-3-nitrophenylacetyl-leucyl-leucyl-leucine vinyl sulfone (NLVS), induce apoptosis abundantly in p53-defective leukemic cell lines CCRF-CEM, U937 and K562 as well as in myelogenic and lymphatic leukemic cells obtained from adult individuals with relapsed acute leukemias. Leukemic cell apoptosis induced by the proteasome inhibitors was dependent on activation of caspase-3 and related caspase family proteases, because caspase-3 inhibitor N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartal (Ac-DEVD-cho) and, more effectively, the general caspase-inhibitor N-benzyloxycarbonyl-L-valyl-L-alanyl-L-aspartate fluoromethylketone (Z-VAD-fmk) were capable of blocking apoptosis induced by lactacystin, ZLLLal or NLVS. Induction of apoptosis by lactacystin or ZLLLal was accompanied by cell cycle arrest at G2/M phase and by accumulation and stabilization of cyclin-dependent kinase inhibitor p21WAF1/Cip and tumor suppressor protein p53. A role of p53 in mediating apoptosis or induction of p21WAF1/Cip1 was ruled out since CCRF-CEM and U937 cells express non-functional mutant p53, and K562 cells lack expression of p53. Viability and hematopoietic outgrowth of human CD34+ progenitor cells treated with lactacystin were slightly reduced, whereas treatment of CD34 + cells with ZLLLal or the cytostatic drugs doxorubicin and gemcitabine resulted in markedly reduced viability and hematopoietic outgrowth. These results demonstrate a basic role of the proteasome in maintaining survival of human leukemic cells, and may define cell-permeable proteasome inhibitors as potently anti-leukemic agents which exhibit a moderate hematopoietic toxicity in vitro.

Topics: Acetylcysteine; Acute Disease; Adult; Antigens, CD34; Apoptosis; Caspase 3; Caspases; Cell Culture Techniques; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Flow Cytometry; G2 Phase; Hematopoietic Stem Cells; Humans; K562 Cells; Leukemia; Leupeptins; Mitosis; Multienzyme Complexes; Neoplasm Proteins; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells