Proteasome Activity In Cells Research Articles

PSMD11 promotes the proliferation of hepatocellular carcinoma by regulating the ubiquitination degradation of CDK4

BackgroundThe 26S proteasome non-ATPase regulatory subunit 11 is a multiprotein complex involved in the ATP-dependent degradation of ubiquitinated proteins, and PSMD11 plays a key role in the regulation of embryonic stem cell proteasome activity. However, the role of PSMD11 in hepatocellular carcinoma has not been studied. In this study, it was found that the expression of PSMD11 in HCC tissues was significantly higher than that in para-cancerous tissues, and was associated with poor prognosis. The results of in vitro experiments showed that PSMD11 knockdown could effectively inhibit the proliferation and apoptosis of hepatoma cell lines, and flow cytometry showed that the G0/G1 phase was significantly prolonged. Through protein spectrometry, immunoprecipitation and in vitro experiments, it was found that PSMD11 can promote the proliferation of hepatocellular carcinoma through regulating the ubiquitination of CDK4 and enhancing its protein stability. This study explores the mechanism of action of PSMD11 in hepatocellular carcinoma and provides new insights for the treatment of hepatocellular carcinoma.

Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

A Novel Indirubin-3-Monoxime Derivative Functions As Regulator of Proteostasis Via Targeting TRIM28 in Multiple Myeloma

Introduction: Multiple myeloma (MM) remains an incurable plasma cell malignancy. Agents disturbing the proteostasis play indispensable roles in MM treatment. Our previous studies have shown that the indirubin-3-monoxime (I3MO) works as a novel proteasome inhibitor that effectively inhibits MM cell growth. In this study, we aim to investigate the improvement of I3MO combined with histone deacetylase 6 (HDAC6) inhibitors, and clarified the molecular mechanisms. Methods: We constructed a compound 8b combined with I3MO and HADC6 inhibitor. The anti-MM effects of compound 8b were investigated in vivo and in vitro studies. Western blots, immunofluorescence, and proteasome activity assay were utilized to investigate the effects of 8b on MM cell proteasome activity, aggresome and autophagosome formation. RNA-seq and Dual-luciferase reporter gene assay were performed to identify the downstream targets of 8b treatment. We constructed MM cell lines with TRIM28 overexpression or shRNA knockdown. We further explored the function of TRIM28, one of the targets of 8b treatment, by ChIP-seq and IP-MS. Results: The novel I3MO derivative 8b more efficiently suppressed myeloma cell growth in vitro and in vivo. Compound 8b not decreased proteasome activity but inhibited the formation of aggresomes and autophagosomes. Interesting, we found that compound 8b efficiently inhibited TRIM28 promoter activity and repressed TRIM28 transcription level further. GEO data analysis demonstrated a positive correlation between TRIM28 and the expression of proteasome subunits as well as autophagy-related genes in MM. Further, we utilized ChIP-seq to confirm that TRIM28 could bind to the promoter region of multiple proteasome subunits-encoding genes, including PSMB1. Our data indicated that TRIM28 knockdown not only decreased the proteasome activity, but inhibited the formation of autophagosomes. Knockdown TRIM28 induced a higher sensitivity to BTZ treatment in MM cells. Furthermore, IP-MS with Co-IP analysis showed that TRIM28 interacts with 14-3-3ζ. TRIM28 acts as an E3 ligase in mediating the ubiquitination and degradation of 14-3-3ζ, a well-known negative regulator of autophagy. 14-3-3ζ degradation induced by TRIM28 activates autophagy in MM cells. These data suggested that TRIM28 suppressed by compound 8b would involve in proteostasis by regulation of proteasome activity and autophagy. Conclusions: Our study identified a novel compound 8b, I3MO combined with HDAC6i, efficiently suppressed MM cells survival. Additionally, we for the first time provided novel important insights for the roles of TRIM28 in MM pathobiology by regulation of proteasome activity and autophagy.

Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity

Proteasomal protein degradation is a promising target for cancer therapy. Here, we developed a positron emission tomography (PET) technique based on the sodium-iodide symporter (NIS) gene fused with the carboxyl-terminal of ornithine decarboxylase (cODC) that noninvasively images cancer cells with inhibited proteasome activity. A retroviral vector was constructed in which the murine cODC degron was fused to the human NIS gene (NIS-cODC). Transiently transduced CT26 and HT29 colon cancer cells and stably expressing CT26/NIS-cODC cells were prepared. In cancer cells transiently transduced with NIS-cODC, NIS expression and transport activity was low at baseline, but NIS protein and 125I uptake was significantly increased by inhibition of proteasome activity with bortezomib. Stable CT26/NIS-cODC cells also showed increased cytosolic and membrane NIS by bortezomib, and four different stable clones displayed bortezomib dose-dependent stimulation of 125I and 99mTc-04− uptake. Importantly, bortezomib dose-dependently suppressed survival of CT26/NIS-cODC clones in a manner that closely correlated to the magnitudes of 125I and 99mTc-04− uptake. CT26/NIS-cODC tumors of bortezomib-treated mice demonstrated greater 124I uptake on PET images and increased NIS expression on tissue staining compared to vehicle-injected animals. NIS-cODC PET imaging may allow noninvasive quantitative monitoring of proteasome activity in cancer cells treated with bortezomib.

Bufalin derivative BF211 inhibits proteasome activity in human lung cancer cells in vitro by inhibiting β1 subunit expression and disrupting proteasome assembly.

Bufalin is one of the active components in the traditional Chinese medicine ChanSu that is used to treat arrhythmia, inflammation and cancer. BF211 is a bufalin derivative with stronger cytotoxic activity in cancer cells. The aim of this study was to identify the putative target proteins of BF211 and the signaling pathways in cancer cells. A549 human lung cancer cells were treated with BF211. A SILAC-based proteomic analysis was used to detect the protein expression profiles of BF211-treated A549 cells. Cellular proteasome activities were examined using fluorogenic peptide substrates, and the binding affinities of BF211 to recombinant proteasome subunit proteins were evaluated using the Biacore assay. The expression levels of proteasome subunits were determined using RT-PCR and Western blotting, and the levels of the integral 26S proteasome were evaluated using native PAGE analysis. The proteomic analysis revealed that 1282 proteins were differentially expressed in BF211-treated A549 cells, and the putative target proteins of BF211 were associated with various cellular functions, including transcription, translation, mRNA splicing, ribosomal protein synthesis and proteasome function. In A549 cells, BF211 (5, 10, and 20 nmol/L) dose-dependently inhibited the enzymatic activities of proteasome. But BF211 displayed a moderate affinity in binding to proteasome β1 subunit and no binding affinity to the β2 and β5 subunits. Moreover, BF211 (0.1, 1, and 10 nmol/L) did not inhibit the proteasome activities in the cell lysates. BF211 (5, 10, and 20 nmol/L) significantly decreased the expression level of proteasome β1 subunit and the levels of integral 26S proteasome in A549 cells. Similarly, knockdown of the β1 subunit with siRNA in A549 cells significantly decreased integral 26S proteasome and proteasome activity. BF211 inhibits proteasome activity in A549 cells by decreasing β1 subunit expression and disrupting proteasome assembly.

Inhibition of metalloproteinase and proteasome activities in colon cancer cells by citrus peel extracts.

Citrus peels are consumed in the form of infusions, candy or wine, based on their well-documented nutritional and medicinal properties. This study sought to investigate the effect of some citrus peels' [grapefruit (Citrus paradisii), orange (Citrus sinensis) and shaddock (Citrus maxima)] extracts on matrix metalloproteinase (MMP) and proteasome activities in primary human colonic tumor (Caco-2) and the metastatic cell lines (LoVo and LoVo/ADR) in a bid to explain the possible mechanism by which the peels could manage/prevent colon cancer. The inhibition of MMP and proteasome activities in the cells by the peel extracts, as well as the identification of phenolic compounds using high-performance liquid chromatography with diode-array detection (HPLC-DAD), was determined. Orange peel extracts had the strongest inhibition of MMP in Caco-2 and LoVo cells, while shaddock had the least. Shaddock peel extracts also had the least MMP inhibition in LoVo/ADR lysates. Grapefruit had the least proteasome inhibition in Caco-2 and LoVo lysates, while there was no significant (p>0.05) difference in the proteasome inhibition of the peel extracts in LoVo/ADR lysates. The extracts inhibited proteasome activity in extract-treated cells, and HPLC fingerprinting of the extracts revealed the presence of some phenolic compounds such as quercetin, caffeic acid, kaempferol, catechin and naringin. The inhibition of MMP and proteasome activities in colon cancer cell lines suggests the potential use of citrus peels as functional food in the management and/or prevention of colon cancer.

Negatively Charged Metal Oxide Nanoparticles Interact with the 20S Proteasome and Differentially Modulate Its Biologic Functional Effects

The multicatalytic ubiquitin-proteasome system (UPS) carries out proteolysis in a highly orchestrated way and regulates a large number of cellular processes. Deregulation of the UPS in many disorders has been documented. In some cases, such as carcinogenesis, elevated proteasome activity has been implicated in disease development, while the etiology of other diseases, such as neurodegeneration, includes decreased UPS activity. Therefore, agents that alter proteasome activity could suppress as well as enhance a multitude of diseases. Metal oxide nanoparticles, often developed as diagnostic tools, have not previously been tested as modulators of proteasome activity. Here, several types of metal oxide nanoparticles were found to adsorb to the proteasome and show variable preferential binding for particular proteasome subunits with several peptide binding "hotspots" possible. These interactions depend on the size, charge, and concentration of the nanoparticles and affect proteasome activity in a time-dependent manner. Should metal oxide nanoparticles increase proteasome activity in cells, as they do in vitro, unintended effects related to changes in proteasome function can be expected.

Abstract 746: Acquired bortezomib resistance in non-small cell lung cancer is associated with overexpression of the mutated β5 proteasome subunit

Abstract The proteasome inhibitor bortezomib (Velcade®) is registered for the treatment of multiple myeloma, but has limited activity in solid tumors such as non-small cell lung cancer (NSCLC). Bortezomib predominantly inhibits proteasome subunit β5 and mutations in this subunit were recently associated with bortezomib resistance in leukemic cell line models. Herein we studied the molecular mechanisms underlying intrinsic and acquired bortezomib resistance in NSCLC cells. H460, A549 and SW1573 NSCLC cells demonstrated differential intrinsic sensitivities towards bortezomib with IC50 values of 12.6, 9.0 and 2.1 nM, respectively. Basal proteasome activities in these cell lines measured by an intact cell proteasome activity assay revealed that high basal levels of chymotrypsin- and caspase-like proteasome activities strongly correlate with intrinsic bortezomib resistance (R2=0.99, P<0.05 and R2=0.99, P<0.06, respectively).We established a model for acquired bortezomib resistance in H460, A549 and SW1573 cells by exposing cells to gradually increasing concentrations of bortezomib starting from 5nM to 80 or 200 nM for H460, 40 or 100 nM for A549 as well as 30 or 150 nM for SW1573. These bortezomib-resistant cell lines, named H460BTZR80, H460BTZR200, A549BTZR40, A549BTZR100, SW1573BTZR30 and SW1573BTZR150 displayed 14 to 57-fold bortezomib resistance. Consistantly, bortezomib-resistant cells required higher bortezomib concentrations to induce a G2/M cell cycle arrest and activation of apoptosis. Overall activation of apoptosis was reduced even at higher bortezomib concentrations when compared to parental cells treated with equitoxic drug doses. Furthermore, bortezomib-resistant cells exhibited increased levels of both constitutive β-subunits and immuno-β-subunits. Moreover, sequence analyses of the bortezomib binding pocket encoded by exon 2 of the PSMB5 gene revealed a previously described Ala49Thr mutation and two newly identified Met45Val and Cys52Phe mutations. Nevertheless, alterations in the proteasome subunits did not affect basal catalytic proteasome activities, although higher bortezomib concentrations were required in bortezomib-resistant cells to achieve comparable inhibition levels of proteasome activity. Cross-resistance was also observed to other proteasome inhibitors that specifically target the β-subunits of the proteasome. Interestingly, no cross-resistance was found to 5AHQ, a novel non-competitive proteasome inhibitor that targets the α7-subunit. Taken together, these findings establish a correlation between bortezomib-sensitivity and low basal levels of proteasome activity. In addition, acquired resistance to bortezomib in NSCLC was associated with expression of mutant β5-subunits that may compromise bortezomib binding. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 746. doi:10.1158/1538-7445.AM2011-746

The Over‐expression of the β2 Catalytic Subunit of the Proteasome Decreases Homologous Recombination and Impairs DNA Double‐Strand Break Repair in Human Cells

By a human cDNA library screening, we have previously identified two sequences coding two different catalytic subunits of the proteasome which increase homologous recombination (HR) when overexpressed in the yeast Saccharomyces cerevisiae. Here, we investigated the effect of proteasome on spontaneous HR and DNA repair in human cells. To determine if the proteasome has a role in the occurrence of spontaneous HR in human cells, we overexpressed the β2 subunit of the proteasome in HeLa cells and determined the effect on intrachromosomal HR. Results showed that the overexpression of β2 subunit decreased HR in human cells without altering the cell proteasome activity and the Rad51p level. Moreover, exposure to MG132 that inhibits the proteasome activity reduced HR in human cells. We also found that the expression of the β2 subunit increases the sensitivity to the camptothecin that induces DNA double-strand break (DSB). This suggests that the β2 subunit has an active role in HR and DSB repair but does not alter the intracellular level of the Rad51p.

Abstract 1063: The activity of the 26S proteasome is modulated during the different phases of the cell cycle

Abstract The 26S proteasome is a multi-catalytic protein complex responsible for the degradation of almost all short-lived and most long-lived proteins. Aberrations of the ubiquitin/proteasome system contribute to many human diseases, including cancer. Many aspects of its structural organization and regulation are still unknown. Among many other functions, the 26S proteasome as the major proteolytic machinery in the cell plays a central role in all phases of the cell cycle. At present, the activity of the 26S proteasome is considered static throughout the cell cycle with protein degradation only depending on their ubiquitination status. We hypothesized that regulation of the activity of the proteasome complex itself could be an additional layer of regulation of cell cycle progression. To test our hypothesis we synchronized two human cell lines, PC-3 prostate cancer cells and MDA-MB-231 breast cancer cells, by treatment with Mimosine and measured 26S proteasome activity in these cells in the presence of the drug and 3, 6, 12 and 24h after restarting the cell cycle. Cells cycle distribution was assessed by Propidium Iodide staining and analyzed by flow cytometry. In parallel, using a proteomics approach we investigated changes of the expression levels and posttranslational modifications of proteasome subunits. Therefore, intact 26S proteasomes from PC3 prostate cancer cell stably expressing a 19S subunit, Rpn11, tagged with 6xHis-TEV-biotin-6xHis, were pulled down from synchronized and untreated (normally cycling) cells. Proteasome subunits and proteasome interacting proteins were separated by 2-dimensional gel electrophoresis (2DE). Differences between synchronized and un-treated cells were analyzed by specific staining for total protein and posttranslational modifications. Differentially expressed or post-translationally modified spots were identified by mass spectrometry. Using this approach, we observed that all three activities of the proteasome were regulated in a similar fashion with down regulation of 26S proteasome activity in cells arrested in the G1-phase of the cell cycle. Treatment of isolated proteasome with Mimosine did not affect its function, excluding that the drug could serve as proteasome inhibitor. After restarting the cell cycle, all three activities sharply increased with maximum activities in cells in the G2/M phase of the cell cycle, which exceeded the 26S proteasome activity of non-synchronized cells about 1.5fold. 2DE and mass spectrometry revealed that proteasome subunit expression was not affected. However, cell cycle synchronization caused specific changes in the phosphorylation pattern of 20S alpha proteasome subunits. In conclusion, we demonstrated that protein degradation and accumulation throughout the cell cycle is not only controlled at the level of proteins involved in cell cycle progression but also by fine-tuning of the activity of the 26S proteasome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1063.

4′-Chloro-3,5-dihydroxystilbene, a resveratrol derivative, induces lung cancer cell death

To examine the antitumor effect of 4'-chloro-3,5-dihydroxystilbene, a resveratrol derivative, on lung adenocarcinoma A549 cells. The cytotoxic IC(50) was determined by direct cell counting. Flow cytometry, monodansylcadaverine (MDC) staining, transfection, Western blot and a proteasome activity assay were used to study the cellular mechanism of 4'-chloro-3,5-dihydroxystilbene. A xenograft nude mouse model was used to analyze the antitumor effect in vivo. 4'-Chloro-3,5-dihydroxystilbene induced a rapid and persistent increase in the intracellular reactive oxygen species in the cells, but the cell death could not be inhibited by two antioxidant agents. The derivative caused sub-G(1) formation, a decrease in the mitochondria membrane potential and poly (ADP-ribose) polymerase degradation, and the caspase inhibitor Z-VAD-FMK could partially prevent cell death. It also induced a significant increase in intracellular acidic vacuoles, LC3-II formation and intracellular GFP-LC3 aggregation. An autophagic inhibitor partially reversed cell death. Additionally, 4'-chloro-3,5-dihydroxystilbene induced the accumulation of ubiquitinated conjugates and inhibited proteasome activity in cells. In an in vivo study, 4'-chloro-3,5-dihydroxystilbene retarded tumor growth in nude mice. These data suggest that the resveratrol derivative 4'-chloro-3,5-dihydroxystilbene could be developed as an anti-tumor compound.

Cytomegalovirus Promoter Up-regulation Is the Major Cause of Increased Protein Levels of Unstable Reporter Proteins after Treatment of Living Cells with Proteasome Inhibitors

Fluorescent unstable proteins obtained by the fusion of a fluorescent protein coding sequence with specific amino acid sequences that promote its fast degradation have become popular to gauge the activity of the ubiquitin/proteasome system in living cells. The steady-state levels of expression of these unstable proteins is low in agreement with their short half-lives, and they accumulate in the cell upon treatment with proteasome inhibitors. We show here that this accumulation is mainly due to transcriptional up-regulation of the cytomegalovirus promoter by proteasome inhibitors and mediated, at least in part, by AP1 transactivation. These simple facts put under quarantine conclusions reached about the activity of the ubiquitin/proteasome pathway in animal cells in culture or in transgenic mice, where popular cytomegalovirus-driven constructs are used, as transcriptional regulation of the expression of the reporter protein construct and not degradation of the unstable protein by the ubiquitin/proteasome system may contribute significantly to the interpretation of the results observed.

Aggregate formation of mutant protein kinase C gamma found in spinocerebellar ataxia type 14 impairs ubiquitin‐proteasome system and induces endoplasmic reticulum stress

Several causal missense mutations in protein kinase C gamma (gamma PKC) gene have been found in spinocerebellar ataxia type 14 (SCA14), an autosomal dominant neurodegenerative disease. We previously demonstrated that mutant gamma PKC found in SCA14 is susceptible to two types of aggregation, cytoplasmic dot-like and perinuclear massive aggregation, and causes cell death in Chinese hamster ovary cells. Long-term time-lapse imaging revealed that firstly accumulated dot-like aggregation of mutant gamma PKC-green fluorescent protein (GFP) gradually formed perinuclear massive aggregations, followed by cell death. However, it remains unclear how aggregate formation of mutant gamma PKC causes cell death. In the present study, we examined whether these mutant aggregations affect the ubiquitin-proteasome system (UPS) and endoplasmic reticular (ER) stress. Two mutant gamma PKC-GFPs (S119P and G128D) were strongly ubiquitinated, and dot-like aggregations of these mutants were ubiquitin-positive and colocalized with proteasome 20S. Furthermore, proteasome activity in cells with aggregates, especially massive ones, was significantly decreased. Aggregate formation of mutant gamma PKC-GFP induced phosphorylation of PERK (PKR-like ER kinase) and nuclear expression of CHOP (C/EBP homologous protein), hallmarks of ER stress and subsequently activated caspase-3. These results indicate that aggregate formation of mutant gamma PKC found in SCA14 impairs UPS and induces ER stress, leading to apoptotic cell death.

Clioquinol Promotes Cancer Cell Toxicity through Tumor Necrosis Factor α Release from Macrophages

Copper has an important role in cancer growth, angiogenesis, and metastasis. Previous studies have shown that cell-permeable metal ligands, including clioquinol (CQ) and pyrrolidine dithiocarbamate, inhibit cancer cell growth in cell culture and in vivo. The mechanism of action has not been fully determined but may involve metal-mediated inhibition of cancer cell proteasome activity. However, these studies do not fully account for the ability of cell-permeable metal ligands to inhibit cancer cell growth without affecting normal cells. In this study, we examined the effect of CQ on macrophage-mediated inhibition of HeLa cancer cell growth in vitro. When CQ was added to RAW 264.7 macrophage-HeLa cell cocultures, a substantial increase in HeLa cell toxicity was observed compared with CQ treatment of HeLa cells cultured alone. Transfer of conditioned medium from CQ-treated macrophages to HeLa cells also induced HeLa cell toxicity, demonstrating the role of secreted factors in the macrophage-mediated effect. Further investigation revealed that CQ induced copper-dependent activation of macrophages and release of tumor necrosis factor (TNF) alpha. In studies with recombinant TNFalpha, we showed that the level of TNFalpha released by CQ-treated macrophages was sufficient to induce HeLa cell toxicity. Moreover, the toxic effect of conditioned medium from CQ-treated macrophages could be prevented by addition of neutralizing antibodies to TNFalpha. These studies demonstrate that CQ can induce cancer cell toxicity through metal-dependent release of TNFalpha from macrophages. Our results may help to explain the targeted inhibition of tumor growth in vivo by CQ.

Secondary Bortezomib-Resistance Is Characterized by Upregulation of Proteasomal Subunits and Their Insufficient Inhibition by Bortezomib, Which Can Be Overcome by Alternative Proteasome Inhibitors Combined with Induction of the ER Stress Response.

Resistance towards proteasome inhibition by Bortezomib (Velcade®) represents a challenge for myeloma therapy. Its biology has not yet been characterized in detail. We have demonstrated that Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. We here present the first analysis of proteasome biology and activity, alternative proteolytic pathways, ubiquitin-specific proteases (USP) and the ER stress response (unfolded protein response, UPR) upstream of the proteasome, as well as in vitro cytotoxicity of conventional cytotoxic drugs, alternative proteasome inhibitors and agents that target the UPR in Bortezomib-resistant (BR) cells, compared to wild type (WT) controls. BR cells had higher activities of all subunits of the constitutive and the immunoproteasome, as deferred from turnover of fluorogenic substrates as well as affinity-labelling of active proteasome subunits in intact cells. This was mirrored by increased levels of proteasomal β1 and β2, but especially β5 polypeptides, implicating a homeostatic system that senses and corrects low proteasome activity in cells chronically exposed to Bortezomib. While the vinylsulfone-type proteasome inhibitor NLVS abrogated detectable proteasome activity in both BR and WT cells, Bortezomib at therapeutic concentrations eliminated proteasomal β1 and β5-type activity only in WT cells, while BR cells retained residual activity. These changes in proteasome biology appear to be the molecular hallmark of required Bortezomib resistance, since no changes were observed between WT and BR cells in alternative cytosolic or lysosomal proteolytic pathways, UPR activity as well as the gross activity pattern of USP. As expected, this translated into sensitivity against cytotoxic drugs in vitro: BR cells were less sensitive towards alternative proteasome inhibitors. However, while the IC50 for pan-proteasome inhibitors was only roughly doubled in BR cells, it was nearly tenfold elevated for the β5-preferring vinylsulfone inhibitor NLVS. By contrast, sensitivity towards anthracyclines or cytotoxicity induced by ER stressors as well as the synergy between proteasome inhibitors and UPR-activators remained unaffected in BR cells. Based on our data, proteasome inhibitors with activity profiles different from that of Bortezomib, alone or in combination with induction of the UPR, may represent an appropriate concept to overcome secondary Bortezomib resistance.

Biochemical and Cellular Characterization of the Novel Proteasome Inhibitor PR-171.

Recent clinical studies have identified the proteasome as an important therapeutic target for hematologic malignances. The proteasome inhibitor, bortezomib, has been approved for the treatment of relapsed or refractory multiple myeloma and ongoing clinical trials suggests a potential benefit for the treatment of Non-Hodgkin's lymphoma. PR-171 is a novel epoxomicin derivative that is a potent and irreversible inhibitor of the human proteasome. It inhibits the chymotrypsin-like activity of purified human 20S proteasome with a kinact/Ki of 34,000 M−1s−1 and is >300-fold selective over the other proteasome catalytic activities. In addition, PR-171 has been found to exhibit minimal activity in a broad diversity panel of biochemical assays that includes 67 receptor/ligand and 37 enzyme assays. PR-171 retains its potency for inhibition of the proteasome chymotrypsin-like activity in mammalian cells displaying IC50 values <10 nM in multiple tumor cell lines. Although PR-171 is a covalent irreversible inhibitor, proteasome activity in cells recovers with a t1/2 of approximately 24 hr after removal of the compound. This recovery is likely due to induction of de novo synthesis since other proteasome inhibitors have been shown to promote transcription of multiple proteasome subunit genes. The cellular consequences of proteasome inhibition by PR-171 include accumulation of polyubiquitinated proteins, cell cycle arrest, and induction of apoptosis. Potent cytotoxic activity of PR-171 is observed across a broad panel of human tumor cell lines (IC50 range: 2–40 nM). Pulsatile exposure studies designed to mimic the anticipated drug exposure in vivo have demonstrated that cytotoxicity is dependent upon the magnitude and duration of proteasome inhibition. These studies have also shown that hematological tumor lines are most sensitive to brief PR-171 exposure, with solid tumor lines exhibiting intermediate sensitivity and non-transformed cells being the least sensitive to such treatment. Finally, PR-171 has been found to retain its cytotoxic potential on cells made resistant to bortezomib in vitro. These studies motivate the clinical investigation of PR-171 in hematological malignances.

Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo.

The proteasome is an important multicatalytic enzyme complex that degrades misfolded and oxidized proteins, signal transduction factors, and antigenic peptides for presentation. We investigated the in vitro effects of peroxynitrite (PN) on the peptidase activity of both crude 20S and 26S and purified 20S proteasome preparations from rat liver as well as proteasome activity in Hep G2 cells and in mouse liver. Crude and purified proteasome preparations were exposed to PN or to the PN donor, 3-morpholinosydnonimine hydrochloride (SIN-1), and then assayed for chymotrypsin-like activity. For in vivo experiments, mice were treated with molsidomine, which is metabolized to SIN-1 in liver. PN and SIN-1 dose-dependently modulated the chymotrypsin-like activity of the 20S proteasome: lower concentrations enhanced proteasome activity, and higher concentrations caused its decline. The NO donor S-nitroso-N-acetylpenicillamine (SNAP), at all concentrations, suppressed 20S proteasome activity. We observed similar results when liver soluble fractions (S-100) were treated with PN, SIN-1, or SNAP, except that enzyme activity in S-100 fractions was less sensitive than the purified enzymes to these agents. Treatment of Hep G2 cells with 0.01 or 0.1 mmol/L SIN-1 stimulated in situ proteasome activity in these cells, while 1 mmol/L SIN-1 suppressed it. SNAP treatment did not affect proteasome activity in Hep G2 cells. Mice treated with molsidomine had enhanced liver proteasome activity 6 hours after treatment, but after 24 hours enzyme activity declined below control levels. In conclusion, PN dose-dependently modulated proteasome activity, regulating protein degradation by the proteasome in liver cells.

Oxidative Stress, Induced by 6-Hydroxydopamine, Reduces Proteasome Activities in PC12 Cells: Implications for the Pathogenesis of Parkinson's Disease

Mutations in familial Parkinson's disease (PD) have been associated with the failure of protein degradation through the ubiquitin-proteasome system (UPS). Impairment of proteasome function has also been suggested to play a role in the pathogenesis of sporadic PD. We examined the proteasome activity in PC12 cells treated with 6-hydroxydopamine (6-OHDA), the dopamine synthetic derivate used in models of PD. We found that 6-OHDA treatment increased protein oxidation, as indicated by carbonyl group accumulation, and increased caspase-3 activity. In addition, there was an increase in trypsin-, chymotrypsin-, and postacidic-like proteasome activities in cells treated with 10-100 microM 6-OHDA, whereas higher doses caused a marked decline. 6-OHDA exposure also increased mRNA expression of the 19S regulatory subunit in a dose-dependent manner, whereas the expression of 20S- and 11S-subunit mRNAs did not change. Administration of the antioxidant N-acetylcysteine to 6-OHDA-treated cells prevented the alteration in proteasome functions. Moreover, reduction in cell viability owing to administration of proteasome inhibitor MG132 or lactacystin was partially prevented by the endogenous antioxidant-reduced glutathione. In conclusion, our data indicate that mild oxidative stress elevates proteasome activity in response to increase in protein damage. Severe oxidative insult might cause UPS failure, which leads to protein aggregation and cell death. Moreover, in the case of UPS inhibition or failure, the blockade of physiological reactive oxygen species production during normal aerobic metabolism is enough to ameliorate cell viability. Control of protein clearance by potent, brain-penetrating antioxidants might act to slow down the progression of PD.

A natural musaceas plant extract inhibits proteasome activity and induces apoptosis selectively in human tumor and transformed, but not normal and non-transformed, cells

Animal studies have demonstrated that a dietary polyphenol known as tannic acid (TA) exhibits anticarcinogenic activity in chemically induced cancers. Most recently, we have reported that TA and ester-bond containing green tea polyphenols are potent proteasome inhibitors in vitro and in vivo. We hypothesize that CellQuest, a patented formula which contains high level of TA obtained from a musaceas (plantain) plant extract, will inhibit the tumor cell proteasome activity. Here, we report that a partially purified CellQuest fraction, S3, potently inhibits the proteasomal chymotrypsin-like activity of Jurkat T cell extracts in a concentration-dependent manner. Inhibition of the proteasome by S3 in leukemia Jurkat T, simian virus 40-transformed and prostate cancer LNCaP cells results in accumulation of ubiquitinated proteins and the natural proteasome substrate p27Kip1, followed by induction of apoptosis. In contrast, non-transformed, immortalized human natural killer cells and normal human fibroblasts are resistant to S3-mediated proteasome inhibition and apoptosis induction. Our present study suggests that CellQuest targets and inhibits the proteasome selectively in tumor cells, which may contribute to the claimed anticancer activity.

Synthetic Analogs of Green Tea Polyphenols as Proteasome Inhibitors

Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.