• BMB Reports
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• v.49 no.5
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• pp.270-275
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• 2016

The Ubiquitin proteasome system (UPS) plays roles in protein degradation, cell cycle control, and growth and inflammatory cell signaling. Dysfunction of UPS in cardiac diseases has been seen in many studies. Cholesterol acts as an inducer of cardiac hypertrophy. In this study, the effect of proteasome inhibitors on the cholesterol-induced hypertrophic growth in H9c2 cells is examined in order to observe whether UPS is involved in cardiac hypertrophy. The treatment of proteasome inhibitors MG132 and Bortezomib markedly reduced cellular surface area and mRNA expression of β-MHC in cholesterol-induced cardiac hypertrophy. In addition, activated AKT and ERK were significantly attenuated by MG132 and Bortezomib in cholesterol-induced cardiac hypertrophy. We demonstrated that cholesterol-induced cardiac hypertrophy was suppressed by proteasome inhibitors. Thus, regulatory mechanism of cholesterol-induced cardiac hypertrophy by proteasome inhibitors may provide a new therapeutic strategy to prevent the progression of heart failure.

• Tuberculosis and Respiratory Diseases
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• v.54 no.4
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• pp.403-414
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• 2003

Background : Proteasome inhibitors can promote either cell survival or programmed cell death, depending on both the specific type and proliferative status of the cell. However, it is not well known whether inhibition of proteasome activity is related to apoptosis in lung cancer cells. In addition, the exact mechanisms responsible for apoptosis induced by proteasome inhibition are not well understood. In the present study, we have examined the effect of proteasome inhibition on lung cancer cells and tried to test the mechanisms that may be associated with the apoptosis of these cells. Methods : We examined the effect of proteasome inhibition with MG132 or PS-341 on cell survival in A549 and NCI-H157 lung cancer cells using MTT assay, and analyzed the cleavage of PARP by Western blot analysis to find evidence of apoptosis. Next, we evaluated the activation of caspase 3 by Western blot analysis and the activity of JNK by immunocomplex kinase assay. We also examined the changes in anti-apoptotic pathways like ERK and cIAP1 by Western blot analysis after inhibition of proteasome function. Results : We demonstrated that MG132 reduced cell survival by inducing apoptosis in A549 and NCI-H157 cells. Proteasome inhibition with MG132 or PS-341 was associated with activation of caspase 3 and JNK, reduced expression of activated ERK, and downregulation of cIAP1. Conclusion : Apoptosis induced by proteasome inhibition may be associated with the activation of pro-apoptotic pathways like caspase 3 and JNK and the inactivation of anti-apoptotic pathways in lung cancer cells.

• International Journal of Oral Biology
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• v.33 no.1
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• pp.25-31
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• 2008

Inhibition of proteasome activity may reduce many types of cancer, so it's pathway is effective in cancer as well as in clinical fields. Here the author has carried out experiment targeting on the elevation of apoptosis in oral cancer cells by combination of proteasome inhibitor, lactacystin, and DNA replication inhibitor, etoposide. The growth of KB cells was measured by MTT methods and apoptosis was analyzed by DNA fragmentation and Hochest nucleus staining. The proteasome activity was analyzed by fluorescent tagged peptide and cellular protein expression was detected by Western hybridization. Though lactacystin and etoposide inhibited KB cell growth alone, but low combined doses inhibited cell growth more strongly and induced apoptosis. The proteasome activity was also seriously inhibited by the combination of both chemicals. Tumor suppressor proteins and apoptosis inducing proteins were highly increased under the combination of both chemicals. From above studies we can conclude that proteasome inhibitors may be used for the treatment of oral cancer and proteasome inhibitors with DNA replication inhibitors may be effective in clinical trials of oral cancer.

• Bulletin of the Korean Chemical Society
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• v.30 no.6
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• pp.1385-1387
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• 2009

• BMB Reports
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• v.44 no.3
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• pp.182-186
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• 2011

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) and phosphatidylinositol-3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the $Ub^{K63}$ chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.

• Biomedical Science Letters
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• v.10 no.3
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• pp.179-186
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• 2004

We examined a role of proteasome, the non-lysosomal multicatalytic protease complex,on the differentiation of chick embryonic myoblasts in culture. The peptide hydrolyzing activities of proteasome were found to change; the hydrolyzing activity against N-succinyl-Leu-Leu- Val- Tyr-7 -amido-4-methy1coumarin (SLLVY-AMC) was prominent and increased with myogenic differentiation. Proteasome inhibitors, N-carbobenzoxy-Leu-Leu-norvalinal (MG115) and N-carbobenzoxy-Ile-Glu (O-t-butyl)-Ala-Leucinal (PSI), blocked membrane fusion of myoblasts as well as the SLLVY-AMC hydrolyzing activity. Those inhibitory activities of the agents occurred in parallel, but were reversible and both cell fusion and the peptidase activity were restored when the agents were withdrawn from the culture medium. On the other hand, the agents caused accumulation of the ubiquitinylated proteins in the cytoskeletal proteins. These results suggest that each of the peptide hydrolyzing activities of proteasome is independently regulated during the myogenic differentiation and the chymotrypsin-like activity may play an important role in that process.

• Bulletin of the Korean Chemical Society
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• v.35 no.12
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• pp.3571-3575
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• 2014

Proteasome, a multicatalytic protease complex, has been validated as a promising therapeutic target in oncology. Carfilzomib (Kyprolis$^{(R)}$), a tetrapeptide epoxyketone, irreversibly inhibits the chymotrypsin-like (CT-L) activity of the proteasome and has been recently approved for multiple myeloma treatment by FDA. A chemistry effort was initiated to discover the compounds that are reversibly inhibit the proteasome by replacing the epoxyketone moiety of carfilzomib with a variety of ketones as reversible and covalent warheads at the C-terminus. The newly synthesized compounds exhibited significant inhibitory activity against CT-L activity of the human 20S proteasome. When the compounds were tested for cancer cell viability, 14-8 was found to be most potent in inhibiting Molt-4 acute lymphoblastic leukemia cell line with a $GI_{50}$ of $4.4{\mu}M$. Cytotoxic effects of 14-8 were further evaluated by cell cycle analysis and Western blotting, demonstrating activation of apoptotic pathways.

• Molecules and Cells
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• v.19 no.1
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• pp.23-30
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• 2005

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder caused by expansion of the polyglutamine tract in the SCA1 gene product, ataxin-1. Using d2EGFP, a short-lived enhanced green fluorescent protein, we investigated whether polyglutamine-expanded ataxin-1 affects the function of the proteasome, a cellular multicatalytic protease that degrades most misfolded proteins and regulatory proteins. In Western blot analysis and immunofluorescence experiments, d2EGFP was less degraded in HEK 293T cells transfected with ataxin-1(82Q) than in cells transfected with lacZ or empty vector controls. To test whether the stability of the d2EGFP protein was due to aggregation of ataxin-1, we constructed a plasmid carrying $ataxin-1-{\Delta}114$, lacking the self-association region (SAR), and examined degradation of the d2EGFP. Both the level of $ataxin-1-{\Delta}114$ aggregates and the amount of d2EGFP were drastically reduced in cells containing $ataxin-1-{\Delta}114$. Furthermore, d2EGFP localization experiments showed that polyglutamine-expanded ataxin-1 inhibited the general function of the proteasome activity. Taken together, these results demonstrate that polyglutamine-expanded ataxin-1 decreases the activity of the proteasome, implying that a disturbance in the ubiquitin-proteasome pathway is directly involved in the development of spinocerebellar ataxia type1.

• BMB Reports
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• v.55 no.4
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• pp.161-165
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• 2022

The mechanistic target of rapamycin (mTOR) regulates numerous extracellular and intracellular signals involved in the maintenance of cellular homeostasis and cell growth. mTOR also functions as an endogenous inhibitor of autophagy. Under nutrient-rich conditions, mTOR complex 1 (mTORC1) phosphorylates the ULK1 complex, preventing its activation and subsequent autophagosome formation, while inhibition of mTORC1 using either rapamycin or nutrient deprivation induces autophagy. Autophagy and proteasomal proteolysis provide amino acids necessary for protein translation. Although the connection between mTORC1 and autophagy is well characterized, the association of mTORC1 inhibition with proteasome biogenesis and activity has not been fully elucidated yet. Proteasomes are long-lived cellular organelles. Their spatiotemporal rather than homeostatic regulation could be another adaptive cellular mechanism to respond to starvation. Here, we reviewed several published reports and the latest research from our group to examine the connection between mTORC1 and proteasome. We have also investigated and described the effect of mTORC1 inhibition on proteasome activity using purified proteasomes. Since mTORC1 inhibitors are currently evaluated as treatments for several human diseases, a better understanding of the link between mTORC1 activity and proteasome function is of utmost importance.

• Journal of Oral Medicine and Pain
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• v.34 no.3
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• pp.261-276
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• 2009

Lactacystin, a microbial natural product synthesized by Streptomyces, has been commonly used as a selective proteasome inhibitor in many studies. Proteasome inhibitors is known to be preventing the proliferation of cancer cells in vivo as well as in vitro. Furthermore, proteasome inhibitors, as single or combined with other anticancer agents, are suggested as a new class of potential anticancer agents. This study was undertaken to examine in vitro effects of cytotoxicity and growth inhibition, and the molecular mechanism underlying induction of apoptosis in SCC25 human tongue sqaumous cell carcinoma cell line treated with lactacystin. The viability of SCC25 cells, human normal keratinocytes (HaCaT cells) and human gingiva fibroblasts (HGF-1 cells), and the growth inhibition of SCC25 cells were assessed by MTT assay and clonogenic assay respectively. The hoechst staining, hemacolor staining and TUNEL staining were conducted to observe SCC25 cells undergoing apoptosis. SCC25 cells were treated with lactacystin, and Western blotting, immunocytochemistry, confocal microscopy, FAScan flow cytometry, MMP activity, and proteasome activity were performed. Lactacystin treatment of SCC25 cells resulted in a time- and does-dependent decrease of cell viability and a does-dependent inhibition of cell growth, and induced apoptotic cell death. Interestingly, lactacytin remarkably revealed cytotoxicity in SCC25 cells but not normal cells. And tested SCC25 cells showed several lines of apoptotic manifestation such as nuclear condensation, DNA fragmentation, the reduction of MMP and proteasome activity, the decrease of DNA contents, the release of cytochrome c into cytosol, the translocation of AIF and DFF40 (CAD) onto nuclei, the up-regulation of Bax, and the activation of caspase-7, caspase-3, PARP, lamin A/C and DFF45 (ICAD). Flow cytometric analysis revealed that lactacystin resulted in G1 arrest in cell cycle progression which was associated with up-regulation in the protein expression of CDK inhibitors, $p21^{WAF1/CIP1}$ and $p27^{KIP1}$. We presented data indicating that lactacystin induces G1 cell cycle arrest and apoptois via proteasome, mitochondria and caspase pathway in SCC25 cells. Therefore our data provide the possibility that lactacystin could be as a novel therapeutic strategy for human tongue squamous cell carcinoma.