This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein β1, β1i, β2, β2i, β5, β5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of β1i,β2, β5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.
Apoptosis ; drug effects ; Cell Line, Tumor ; Diterpenes ; pharmacology ; Humans ; Multiple Myeloma ; pathology ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; pharmacology

Ubiquitin-proteasome pathway (UPP) is one of the ways utilized for selective degradation of many proteins in cells, and the 20S proteasome takes the functional machinery where hydrolysis of targeted proteins takes place. Based on existing peptide inhibitors, a series of novel tripeptidic tetrazoles have been designed, synthesized, and the structures have been confirmed with 1H NMR, MS and elemental analysis. Among them, three compounds (6b, 6d and 6h) showed inhibitory activities of ChT-L of 20S proteasome.
Biological Assay ; Drug Design ; Molecular Structure ; Oligopeptides ; chemical synthesis ; chemistry ; pharmacology ; Proteasome Endopeptidase Complex ; chemistry ; Proteasome Inhibitors ; chemical synthesis ; chemistry ; pharmacology ; Tetrazoles ; chemical synthesis ; chemistry ; pharmacology

Cell Line, Tumor ; Enzyme Inhibitors ; pharmacology ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Proteasome Endopeptidase Complex ; drug effects

The aim of the study was to explore the synergistic effect of the proteasome inhibitor bortezomib (bor) and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) on apoptosis of T lymphoma cell lines Jurkat and Hut78, and on the formation of aggresome. Jurkat and Hut78 cells were treated with bor (10 nmol/L) or bor (10 nmol/L) combined with SAHA (2 micromol/L) respectively. Cell growth inhibition was estimated by trypan blue dye exclusion test. Cell morphology was evaluated by light microscopy with Wright's staining of cytocentrifuge preparations. Cell apoptosis was analyzed by flow cytometry. Ultrastructure of cell apoptosis and aggresome were observed by transmission electron microscopy. The results showed that proliferation of both Jurkat and Hut78 cells was significantly inhibited in the bor+SAHA group, as compared with the control group and the bor alone group. Flow cytometric analysis confirmed that the percentage of apoptosis in Jurkat and Hut78 cells in the bor+SAHA group (41.8+/-4.7% and 72.7+/-11.7% respectively) was remarkably higher than those in the control group (3.6+/-1.3% and 7.0+/-1.9% respectively) and the bor alone group (6.3+/-2.3% and 18.7+/-9.2% respectively) (p<0.01). Ultrastructure examination revealed that typical aggresomes in cells could be observed in bor alone group. The combination of bor and SAHA diminished both the amount and density of aggresomes, or even eliminated them, accompanied by the increased rate of apoptosis. It is concluded that proteasome inhibitor combined with histone deacetylase inhibitor synergically induces T lymphoma cell apoptosis. Bortezomib stimulates the formation of aggresome, while SAHA destroys this aggresome structure, which may be one of the mechanisms underlying the enhancement of bortezomib-induced apoptosis.
Apoptosis ; drug effects ; Boronic Acids ; pharmacology ; Bortezomib ; Drug Synergism ; Histone Deacetylase Inhibitors ; pharmacology ; Humans ; Jurkat Cells ; Lymphoma, T-Cell ; drug therapy ; Proteasome Inhibitors ; Pyrazines ; pharmacology

Amyloid beta (Aβ) precursor protein (APP) is a key protein in the pathogenesis of Alzheimer's disease (AD). Both APP and its paralogue APLP1 (amyloid beta precursor-like protein 1) have multiple functions in cell adhesion and proliferation. Previously it was thought that autophagy is a novel beta-amyloid peptide (Aβ)-generating pathway activated in AD. However, the protein proteolysis of APLP1 is still largely unknown. The present study shows that APLP1 is rapidly degraded in neuronal cells in response to stresses, such as proteasome inhibition. Activation of the endoplasmic reticulum (ER) stress by proteasome inhibitors induces autophagy, causing reduction of mature APLP1/APP. Blocking autophagy or JNK stress kinase rescues the protein expression for both APP and APLP1. Therefore, our results suggest that APP/APLP1 is degraded through autophagy and the APLP1 proteolysis is mainly mediated by autophagy-lysosome pathway.
Amyloid beta-Protein Precursor ; genetics ; metabolism ; Animals ; Autophagy ; Cell Line ; Endoplasmic Reticulum ; metabolism ; JNK Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Leupeptins ; pharmacology ; Mice ; Neurons ; cytology ; metabolism ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; Protein Stability ; Rats

Hypoxia-inducible factor 1alpha (HIF-1alpha), which transactivates a variety of hypoxia-induced genes, is rapidly degraded under nomoxia through the hydroxylation-ubiquitination-proteasome pathway. In this study, we addressed how HIF-1alpha is stabilized by proteasome inhibitors. The ubiquitin pool was rapidly reduced after proteasome inhibition, followed by the accumulation of non-ubiquitinated HIF-1alpha. The poly-ubiquitination of HIF-1alpha was resumed by restoration of free ubiquitin, which suggests that the HIF-1alpha stabilization under proteasome inhibition is attributed to depletion of the free ubiquitin pool. Ni2+ and Zn2+ also stabilized HIF-1alpha with depletion of the free ubiquitin pool and these effects of metal ions were attenuated by restoration of free ubiquitin. Ni2+ and Zn2+ may disturb the recycling of free ubiquitin, as MG132 does. Based on these results, the state of the ubiquitin pool seems to be another critical factor determining the cellular level of HIF-1alpha.
Cell Hypoxia/physiology ; Cell Line, Tumor ; HCT116 Cells ; HEK293 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/biosynthesis/*metabolism ; Leupeptins/pharmacology ; Nickel/chemistry ; Proteasome Endopeptidase Complex/*metabolism ; Proteasome Inhibitors/*pharmacology ; Ubiquitin/*metabolism ; Ubiquitination/*physiology ; Up-Regulation ; Zinc/chemistry

Multiple myeloma (MM) is a common plasma cell malignancy, accounting for the second largest hematological malignancy. Proteasome inhibitors represented by bortezomib (BTZ) have been the main treatment for patients with newly diagnosed and relapsed or refractory myeloma in nearly two decades. Although BTZ has improved the prognosis of MM patients, MM remains incurable in most patients, mainly because MM cells become resistant to BTZ. This review is to better understand the mechanism of MM resistance to BTZ and explore possible new therapeutic strategies.
Humans ; Bortezomib/therapeutic use* ; Multiple Myeloma/pathology* ; Proteasome Inhibitors/pharmacology* ; Prognosis ; Plasma Cells/pathology* ; Drug Resistance, Neoplasm ; Antineoplastic Agents/pharmacology* ; Cell Line, Tumor

Gene medicine based on recombinant adeno-associated virus (rAAV) vector has rapidly become the prior-choose reagent for gene therapy, since it had been shown that the rAAV was able to stably express many genes in vivo without detectable side-effect. However, recent findings of CTL immune responses to AAV capsid in a clinical trial highlighted a new issue regarding safety that previously was not identified in animal studies. Obviously it is so important to understand the interaction of rAAV with the immune system in details for the safety and success of rAAV gene medicine. In this review we evaluate several current hypotheses aiming to explain the cellular immunotoxicity, also analysis the current findings including the presentation kinetics of the capsid antigen and the activation of CTL. Focusing on the key steps of the immune response several solutions are proposed, including immunosuppression, optimization of vector and improvement of purity, in order to insure clinical safety and efficacy of rAAV.
Animals ; Capsid ; immunology ; Dependovirus ; genetics ; Genetic Therapy ; Genetic Vectors ; adverse effects ; immunology ; Humans ; Immune Tolerance ; Immunity, Cellular ; Immunosuppressive Agents ; pharmacology ; Proteasome Endopeptidase Complex ; metabolism ; Proteasome Inhibitors ; Recombinant Proteins ; adverse effects ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVETo investigate whether proteasome inhibitor MG-132 induces apoptosis of human erythroleukemia cell line K562 and possible mechanisms.

METHODSK562 cells were incubated with RPMI 1640 and exposed to 0, 1, 5, 10, 15 micromol/L of MG-132 for 24 hrs, respectively. The apoptosis of cells were detected by fluorescence microscope, DNA fragments and flow cytometry. The NF-kappaB mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). Expression of NF-kappaB and caspase-3 was semiquantitatively analyzed with SABC techniques. Caspase-3 activities were measured with a colorimetric method.

RESULTSThe growth of K562 cells was inhibited and the apoptosis of the cells increased after MG-132 treatment in a dose-dependent manner. After 24 hrs of 15 micromol/L MG-132 treatment, the percentage of apoptotic cells (26.5+/-0.6%) increased significantly when compared with the untreated controls (1.2+/-0.1%) (P<0.01). MG-132 treatment decreased the mRNA and protein expression of NF-kappaB, and increased the protein expression of caspase-3.

CONCLUSIONSMG-132 can induce apoptosis of human erythroleukemia cell line K562 through the down-regulation of NF-kappaB expression and up-regulation of caspase-3 expression.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cysteine Proteinase Inhibitors ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; K562 Cells ; Leupeptins ; pharmacology ; NF-kappa B ; Proteasome Inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: To explore the mechanism of proteasome inhibitor MG132 in improving osteoporosis. METHODS: Total of 32 female SD rats, weighing 220 to 250 g and 8 weeks old, were selected. They were randomly divided into 4 groups(n=8). Rats of group A and group B were cut off ovaris on both sides to make model of osteoporosis, and then they were given proteasome inhibitors MG132 and dimethyl sufoxide (DMSO) respectively. Group C was a sham group and rats were given MG132. Group D was a normal group and rats were given MG132 too. The rats were killed in batches at 6 and 12 weeks after administration, and the femoral neck tissues were obtained. Relevant data were analyzed, such as pathomorphological observation, micro-CT analysis, detection of 20S proteasome activity in tissues, and expression of Wnt and β-catenin. RESULTS: Morphological observation showed that the trabecular were slightly thinner, reticulated, and occasionally interrupted in group A, while the trabecular were obviously thinner and discontinuous in group B. And the trabecular were intact and arranged reticulated in group C and D. The analysis results of bone mineral density(BMD), bone surface(BS), bone volume/total volume(BV/TV) and trabecular thickness(Tb.Th) showed that group B was worse than other groups in all parameters at different time points(P<0.05), and group A was worse than group C and group D in BS(P<0.05), there was no significant difference in all parameters between group C and group D. RFU value of 20S proteasome in group B was significantly higher than that in other groups(P<0.05). According to the results of Western blot, the gray values of Wnt protein and β-catenin protein in group A were significantly higher than those in other groups (P<0.05). CONCLUSION MG-132, a ubiquitin proteasome inhibitor, can regulate Wnt/β-catenin signaling pathway by inhibiting the degradation of β-catenin protein, and delaying the occurrence and development of osteoporosis.
Animals ; Bone Density ; Female ; Leupeptins ; Osteoporosis/drug therapy* ; Proteasome Inhibitors/pharmacology* ; Rats ; Rats, Sprague-Dawley ; Wnt Signaling Pathway ; beta Catenin/metabolism*