OBJECTIVE: The study was performed to establish the purification processes of both 26S and 20S proteasomes, also to investigate the inhibitory properties and patterns of two different proteasome inhibitors on the isolated proteasomes. METHODS: The 26S and 20S proteasomes were purified respectively using liquid chromatographies and glycerol density gradient fractionation. The inhibitory patterns and kinetics of two different proteasome inhibitors were investigated using purified 26S and 20S proteasomes. RESULTS: The purity of the isolated proteasomes were determined by their biochemical properties and electrophoretic patterns. 3-nitro-4-hydroxy-5-indophenylacetyl-leucyl-leucyl-leucyl-vinylsulfone (Nip-L₃-VS) inhibited exclusively the chymotrypsin-like peptidase activities of the 26S and 20S proteasomes. On the other hand, dansyl-phenylyl-leucyl-boronic acid (DFLB) inhibited chymotrpsin-like, trypsin-like, and caspase-like peptidase activities of both proteasomes with different sensitivity. CONCLUSION: The proposed purification method provides efficient separation and isolation of the 26S and 20S proteasomes. Nip-L₃-VS and DFLB were shown to have different inhibitory effects and kinetics on the peptidase activities of the isolated proteasomes. These studies are suggested to be applied to the researches on proteasome inhibitors as therapeutic reagents for many related diseases.
Bortezomib ; Chromatography ; Glycerol ; Hand ; Indicators and Reagents ; Kinetics ; Methods ; Proteasome Endopeptidase Complex ; Proteasome Inhibitors

Bortezomib is a proteasome inhibitor used for the treatment of patients with multiple myeloma. Recently, several case reports about acute pancreatitis caused by Bortezomib were published in the international literature. But Bortezomib induced pancreatitis case was not reported in Korea. Herein, we report a case of acute pancreatitis caused by Bortezomib therapy in a 76-year-old female with multiple myeloma. On three months after the first administration of Bortezomib, the patient visited the hospital with symptoms of acute pancreatitis. The common etiological factors for acute pancreatitis were all excluded. Then the patient was diagnosed as Bortezomib-induced pancreatitis. After cessation of Bortezomib, she showed clinical and laboratory improvement.
Aged ; Female ; Humans ; Korea ; Multiple Myeloma* ; Pancreatitis* ; Proteasome Inhibitors ; Bortezomib

Boronic Acids ; Bortezomib ; Humans ; Liver Diseases ; Proteasome Inhibitors ; Pyrazines

PURPOSE: One of possible mechanisms of the antineoplastic effect by nonsteroidal anti-inflammatory drugs (NSAIDs) is an induction of apoptosis. The NSAIDs-induced apoptosis appears to be caspase- and mitochondria-dependent. The ubiquitin-proteasome system, which is a fundamental non- lysosomal tool that cells use to process or degrade a variety of short-lived proteins, is known to be involved in apoptosis and to be located upstream of mitochondrial changes and caspase activation. The present study was conducted to explore the potential role of proteasome pathway in NSAIDs-induced apoptosis. METHODS: We employed sulindac as a NSAID, and the lactacystin as a proteasome inhibitor to investigate the extent of the apoptosis in colon cancer cell line, HT-29 cells. The proteasome activity and the amount of apoptosis were quantified after cells were treated with 1 mM sulindac, 1micrometer lactacystin or both. RESULTS: Sulindac treatment caused apoptosis of the HT-29 cells in a time-dependent manner with resultant changes in nuclear morphology. Western blots also showed caspase-3 activation and PARP cleavage after sulindac treatment. Not only single treatment with lactacystin decreased proteasome activity, but co-treatment with sulindac enhanced decrease in proteasome activity further (P<0.01). Treatment with lactacystin only did not induce apoptosis. However, lactacystin augmented the induction of sulindac-induced apoptosis (P<0.01). This synergistic effect was also proven by Western blot analyses, where co-treatment augmented the caspase-3 activation and PARP degradation. CONCLUSIONS: The combination treatment of sulindac with a proteasome inhibitor lactacystin is suggested to be a very effective strategy for the induction of cancer cell apoptosis. Elucidation of the mechanism underlying the regression of colon cancers by combination of sulindac and lactacystin seems to be an immediate challenge in the near future.
Apoptosis* ; Blotting, Western ; Caspase 3 ; Cell Line ; Colonic Neoplasms ; HT29 Cells* ; Humans ; Proteasome Endopeptidase Complex ; Proteasome Inhibitors ; Sulindac

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 system (UPS) plays an essential role in eukaryotic protein cycle,the dysregulation of which can lead to tumorigenesis.Increased activities of UPS have been observed in the patients with cancers including leukemia.UPS inhibitors can kill cancer cells by affecting ubiquitin-ligating enzyme E3,deubiquitinase,and protein degradation active sites of UPS.Therefore,UPS inhibitors have emerged as an important therapy for treating hematological malignancies,while they are rarely applied in the treatment of acute myeloid leukemia.This paper summarizes the research progress in the inhibitors affecting the protein ubiquitination at different stages of acute myeloid leukemia,aiming to provide new clues for the clinical treatment of acute myeloid leukemia.
Humans ; Proteasome Inhibitors/therapeutic use* ; Ubiquitin/metabolism* ; Proteasome Endopeptidase Complex/metabolism* ; Ubiquitination ; Leukemia, Myeloid, Acute/drug therapy*

Although much information has been accumulated about the synergistic interaction of proteasome inhibitors and HDAC inhibitors to induce apoptosis in a certain type of cells, much less is known currently about the underlying mechanism. This study was undertaken to explore the combination effect of a histone deacetylase inhibitor, TSA, and a proteasome inhibitor, lactacystin, on the induction of apoptosis. Pretreatment of TSA and subsequent treatment of lactacystin showed the strong antitumor activity and nuclear condensation. Western blot assay showed that combination treatment of TSA and lactacystin increased Bax/Bcl-2 ratio and decreased level of XIAP. Activation of caspase-7 and cleavage of PARP were demonstrated after the combination treatment. In combination treatment group, cell cycle arrest was induced at G2/M phase and abolished increase in proteasome activity. This study is elucidating the mechanims whereby targeting apoptotic machineries may help in directing therapeutic strategies.
Apoptosis ; Blotting, Western ; Caspase 7 ; Cell Cycle Checkpoints ; Histone Deacetylase Inhibitors* ; Histone Deacetylases* ; Histones* ; MCF-7 Cells* ; Proteasome Endopeptidase Complex* ; Proteasome Inhibitors*

The proteasome inhibitor, bortezomib, is ineffective against many solid tumors. Nutlin-3 is a potent antagonist of human homolog of murine double minute 2/p53 interaction exhibiting promising therapeutic anti-cancer activity. In this study, we show that treatment of various p53-defective bortezomib-resistant solid tumor cells with bortezomib plus nutlin-3 induces paraptosis, which is a cell death mode accompanied by dilation of the endoplasmic reticulum (ER) and mitochondria. Bortezomib alone did not markedly alter cellular morphology, and nutlin-3 alone induced only a transient mitochondrial dilation. However, bortezomib/nutlin-3 co-treatment triggered the progressive fusion of swollen ER and the formation of megamitochondria, leading to cell death. Mechanistically, proteasomal-impairment-induced ER stress, CHOP upregulation and disruption of Ca²⁺ homeostasis were found to be critically involved in the bortezomib/nutlin-3-induced dilation of the ER. Our results further suggest that mitochondrial unfolded protein stress may play an important role in the mitochondrial dilation observed during bortezomib/nutlin-3-induced cell death. Collectively, these findings suggest that bortezomib/nutlin-3 perturbs proteostasis, triggering ER/mitochondria stress and irrecoverable impairments in their structure and function, ultimately leading to paraptotic cell death.
Bortezomib* ; Cell Death ; Endoplasmic Reticulum ; Homeostasis ; Humans ; Mitochondria ; Proteasome Inhibitors ; Up-Regulation

Bortezomib (Velcade(R)) is proteasome inhibitor that is used as a first-line therapy for multiple myeloma. It can cause gastrointestinal, hematologic, and neuromuscular side effects, and a cutaneous reaction is one of its common adverse reactions. To date, several bortezomib-induced cutaneous adverse reactions have been reported, including folliculitis-like rash, pruriginous rash, purpuric rash, mouth swelling, stomatitis-mucositis, edema in the lower limbs, telogen effluvium, and vasculitis. In the Korean literature, only one case of vasculitis has been reported earlier. Two patients have presented with multiple plaques on the trunk at our clinic. The lesions developed several days after bortezomib chemotherapy, and disappeared spontaneously in about 1 week. Herein, we report bortezomib-induced drug eruption presenting as multiple plaques on the trunk with a review of the relevant literature.
Drug Eruptions* ; Drug Therapy ; Edema ; Exanthema ; Humans ; Lower Extremity ; Mouth ; Multiple Myeloma ; Proteasome Inhibitors ; Vasculitis ; Bortezomib

Multiple myeloma is a hematologic malignancy characterized by the proliferation of monoclonal plasma cells that produce monoclonal immunoglobulins. Cutaneous involvement is very uncommon in patients with multiple myeloma. It usually appears late in the course of the disease. Bortezomib is a potent proteasome inhibitor, recently introduced in the treatment of multiple myeloma. It has proven to be safe and effective in the treatment of refractory or relapsed multiple myeloma. In this study, we show the high efficacy of a concurrent therapeutic approach with bortezomib and radiation in a patient with a cutaneous plasmacytoma.
Boronic Acids ; Hematologic Neoplasms ; Humans ; Immunoglobulins ; Multiple Myeloma ; Plasma Cells ; Plasmacytoma ; Proteasome Inhibitors ; Pyrazines ; Bortezomib