Proteasome inhibition improves diaphragm function in an animal model for COPD

2011 ◽  
Vol 301 (1) ◽  
pp. L110-L116 ◽  
Author(s):  
Hieronymus van Hees ◽  
Coen Ottenheijm ◽  
Leo Ennen ◽  
Marianne Linkels ◽  
Richard Dekhuijzen ◽  
...  
Keyword(s):  
Animal Model ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Contractile Protein ◽  
Specific Force ◽  
Bortezomib Treatment ◽  
Diaphragm Function ◽  
Generating Capacity ◽  
Diaphragm Weakness

Diaphragm muscle weakness in patients with chronic obstructive pulmonary disease (COPD) is associated with increased morbidity and mortality. Recent studies indicate that increased contractile protein degradation by the proteasome contributes to diaphragm weakness in patients with COPD. The aim of the present study was to investigate the effect of proteasome inhibition on diaphragm function and contractile protein concentration in an animal model for COPD. Elastase-induced emphysema in hamsters was used as an animal model for COPD; normal hamsters served as controls. Animals were either treated with the proteasome inhibitor Bortezomib (iv) or its vehicle saline. Nine months after induction of emphysema, specific force-generating capacity of diaphragm bundles was measured. Proteolytic activity of the proteasome was assayed spectrofluorometrically. Protein concentrations of proteasome, myosin, and actin were measured by means of Western blotting. Proteasome activity and concentration were significantly higher in the diaphragm of emphysematous hamsters than in normal hamsters. Bortezomib treatment reduced proteasome activity in the diaphragm of emphysematous and normal hamsters. Specific force-generating capacity and myosin concentration of the diaphragm were reduced by ∼25% in emphysematous hamsters compared with normal hamsters. Bortezomib treatment of emphysematous hamsters significantly increased diaphragm-specific force-generating capacity and completely restored myosin concentration. Actin concentration was not affected by emphysema, nor by bortezomib treatment. We conclude that treatment with a proteasome inhibitor improves contractile function of the diaphragm in emphysematous hamsters through restoration of myosin concentration. These findings implicate that the proteasome is a potential target of pharmacological intervention on diaphragm weakness in COPD.

scholarly journals Proteasome inhibition improves diaphragm function in congestive heart failure rats

2008 ◽  
Vol 294 (6) ◽  
pp. L1260-L1268 ◽  
Author(s):  
Hieronymus W. H. van Hees ◽  
Yi-Ping Li ◽  
Coen A. C. Ottenheijm ◽  
Bingwen Jin ◽  
Cindy J. C. Pigmans ◽  
...  
Keyword(s):  
Heart Failure ◽  
Congestive Heart Failure ◽  
Proteasome Inhibitor ◽  
Caspase 3 ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Cross Bridge ◽  
Bortezomib Treatment ◽  
Diaphragm Function ◽  
Generating Capacity

In congestive heart failure (CHF), diaphragm weakness is known to occur and is associated with myosin loss and activation of the ubiquitin-proteasome pathway. The effect of modulating proteasome activity on myosin loss and diaphragm function is unknown. The present study investigated the effect of in vivo proteasome inhibition on myosin loss and diaphragm function in CHF rats. Coronary artery ligation was used as an animal model for CHF. Sham-operated rats served as controls. Animals were treated with the proteasome inhibitor bortezomib (intravenously) or received saline (0.9%) injections. Force generating capacity, cross-bridge cycling kinetics, and myosin content were measured in diaphragm single fibers. Proteasome activity, caspase-3 activity, and MuRF-1 and MAFbx mRNA levels were determined in diaphragm homogenates. Proteasome activities in the diaphragm were significantly reduced by bortezomib. Bortezomib treatment significantly improved diaphragm single fiber force generating capacity (∼30–40%) and cross-bridge cycling kinetics (∼20%) in CHF. Myosin content was ∼30% higher in diaphragm fibers from bortezomib-treated CHF rats than saline. Caspase-3 activity was decreased in diaphragm homogenates from bortezomib-treated rats. CHF increased MuRF-1 and MAFbx mRNA expression in the diaphragm, and bortezomib treatment diminished this rise. The present study demonstrates that treatment with a clinically used proteasome inhibitor improves diaphragm function by restoring myosin content in CHF.

scholarly journals Mechanistic Insights into the Enhancement of Adeno-Associated Virus Transduction by Proteasome Inhibitors

2013 ◽  
Vol 87 (23) ◽  
pp. 13035-13041 ◽  
Author(s):  
Angela M. Mitchell ◽  
R. Jude Samulski
Keyword(s):  
Cysteine Protease ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Protease Inhibition ◽  
Adeno Associated Virus

Proteasome inhibitors (e.g., bortezomib, MG132) are known to enhance adeno-associated virus (AAV) transduction; however, whether this results from pleotropic proteasome inhibition or off-target serine and/or cysteine protease inhibition remains unresolved. Here, we examined recombinant AAV (rAAV) effects of a new proteasome inhibitor, carfilzomib, which specifically inhibits chymotrypsin-like proteasome activity and no other proteases. We determined that proteasome inhibitors act on rAAV through proteasome inhibition and not serine or cysteine protease inhibition, likely through positive changes late in transduction.

Selective Inhibition of the proteasome's β2 Catalytic Subunit Alone Does Not Induce Cytotoxicity, but Resensitizes Bortezomib-Refractory Myeloma Cells for Bortezomib Treatment

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2915-2915
Author(s):  
Marianne Kraus ◽  
Bobby Florea ◽  
Jürgen Bader ◽  
Nan Li ◽  
Paul Geurink ◽  
...  
Keyword(s):  
Er Stress ◽  
Cell Lines ◽  
Active Site ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Selective Inhibition ◽  
Catalytic Site ◽  
Myeloma Cells ◽  
Bortezomib Treatment

Abstract Abstract 2915 Bortezomib is a reversible first-generation proteasome inhibitor that inhibits the β5 and to a lesser extent the β1 catalytic site of the proteasome. However, bortezomib does not inhibit the β2 catalytic proteasomal site at clinically relevant concentrations, and bortezomib-resistance is accompanied by upregulation of the β2 subunit, suggesting that increased β2 activity may compensate for the loss of β1/ β5 activity during bortezomib-treatment. The second generation proteasome inhibitor carfilzomib, due to the chemistry of its epoxyketone warhead, has a higher substrate specificity and functions as an irreversible proteasome inhibitor, but is still a β1/ β5 inhibitor that does not affect the β2 active site. We investigated the effect of β2-specific proteasome inhibition on myeloma and acute myeloid leukemia (AML) cells and tested the hypothesis that β2-selective proteasome inhibition may overcome bortezomib-resistance. To this end we have developed a set of epoxyketone- and vinylsulfone-based, cell permeable proteasome inhibitors of which we selected the compounds PR523A and PR671A for further testing in cell-based assays. PR671A is a peptide-vinylsulfone that selectively inhibits the proteasome's β2/ β2i subunit in an irreversible fashion in human cell lines and primary cells at low micromolar concentrations without inhibition of other protease species. PR523A is a β5-selective peptide-epoxyketone with otherwise similar properties. Treatment of myeloma and AML cell lines (AMO-1, U-266, HL-60, THP-1) with PR523A induced ER-stress mediated apoptosis, very similar to bortezomib. The combination of bortezomib with PR523A led to additive, but not synergistic induction of apoptosis, as expected. Selective β2 inhibition by PR671A resulted in the induction of ER stress and the accumulation of poly-ubiquitinated protein, however, this was not effectively translated into apoptotic cell death. This indicates that selective inhibition of the β2 proteasome subunit alone has only a poor cytotoxic effect on myeloma and AML cell lines, suggesting that the function of β2 is largely redundant and can be compensated when the remaining proteasome catalytic subunits (β1 and β5) remain active. However, when the β2 inhibitor PR671A was combined with agents that target the proteasome's β5 active site (PR523A) or the β5 and the β1 site (bortezomib), the combination of either inhibitor with the β2 inhibitor PR671A was highly synergistic for both activation of ER stress and the induction of apoptotic death. Importantly, the bortezomib-resistance in bortezomib-adapted myeloma and AML cell lines could be overcome by combining PR671A with either bortezomib or PR523A, while β2 inhibition by PR671A alone had no effect on the viability of bortezomib-adapted cells. We conclude that PR671A is a β2 selective proteasome inhibitor. Selective Inhibition of the proteasome's β2 subunit has little effect on viability or ER stress both in normal and bortezomib-resistant myeloma and leukemia cells, suggesting that the function of the β2 catalytic site is largely redundant. However, when β1/ β5 proteasome activity is inhibited by drugs like bortezomib or carfilzomib, proper function of the β2 proteasome active site is crucial for cell survival, also in bortezomib-resistant myeloma cells. The use of specific β2 inhibitors like PR671A in combination with β1/ β5 inhibitors like bortezomib or carfilzomib is therefore a promising strategy to overcome resistance against β1/ β5-selective proteasome inhibitors. Disclosures: No relevant conflicts of interest to declare.

The Proteasome Inhibitor BSc2118 Causes Growth Inhibition, Cell Cycle Arrest and Cyclin D1 Degradation in Mantle Cell Lymphoma Cell Lines.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1401-1401
Author(s):  
Christian Jakob ◽  
Jan Sterz ◽  
Ivana von Metzler ◽  
Ulrike Kuckelkorn ◽  
Hannes A. Braun ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cyclin D1 ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Nf Kappa B ◽  
Cycle Arrest ◽  
Dose Dependent

Abstract The ubiquitin-proteasome complex has recently been identified as a novel therapeutic target, particularly in hematological malignancies. An increased proteasome activity has been described in certain types of malignant cells including mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has shown in vitro activity in MCL cell lines and also clinical efficacy in patients with refractory or relapsed MCL. We previously described a novel tripeptide compound, BSc2118 which inhibits all three proteolytic activities of the 20S proteasome and has anti-tumor activity in melanoma and multiple myeloma (Cancer Res2006; 66: 7598–7605). We investigated the anti-tumor effects of BSc2118 in the MCL cell lines HBL-2, JeKo-1 and Granta-519 and studied its effects on cell cycle progression and the expression of the cell cycle regulatory proteins p21, p27 and cyclin D1. Furthermore the inhibition of intracellular proteasome and NF-kappa B activity was analyzed in MCL cells. In MCL cell lines HBL-2, JeKo-1 and Granta-519, BSc2118 caused a time- and dose-dependent growth inhibition, an induction of apoptosis and a dose-dependent inhibition of intracellular proteasome activity. After 24 and 48 hours of incubation with 40 – 260 nM BSc2118 we found a time- and dose-dependent cell cycle arrest in G2/M phase in all three MCL cell lines. Furthermore we could demonstrate a dose-dependent stabilization of p21 and a degradation of cyclin D1 in western blot analysis. No significant changes could be seen in p27 expression under proteasome inhibition with BSc2118. After TNF-alpha stimulation we found a low NF-kappa B activity also in untreated MCL cells, which could be partially inhibited by preincubation with BSc2118. In this study we show the effects of the novel proteasome inhibitor BSc2118 on cell cycle and growth inhibition in MCL cells lines and furthermore demonstrate that inhibition of proteasome activity, cyclin D1 degradation and p21 stabilization are crucial mechanisms of action for this compound in MCL. Since recent trials have shown a clinical efficacy of proteasome inhibition in MCL, our preclinical data suggest to consider BSc2118 as a novel agent in drug development against MCL.

Pharmacodynamic and Efficacy Studies of a Novel Proteasome Inhibitor NPI-0052 in Human Plasmacytoma Xenograft Mouse Model

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3665-3665
Author(s):  
Ajita V. Singh ◽  
G. Kenneth Lloyd ◽  
Michael A. Palladino ◽  
Dharminder Chauhan ◽  
Kenneth C. Anderson
Keyword(s):  
Mouse Model ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Vehicle Control ◽  
Two Dimensions ◽  
Proteasome Activity ◽  
Vascular Tumors ◽  
Xenograft Mouse Model ◽  
Model Animal ◽  
Efficacy Studies

Abstract Background: We recently characterized a novel proteasome inhibitor NPI-0052, a small molecule derived from the fermentation of a marine gram-positive actinomycete Salinispora tropica. NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Importantly, NPI-0052 is distinct from bortezomib in its chemical structure, proteasome inhibition profiles, and mechanisms of action. In the present study, we utilized a human plasmacytoma xenograft mouse model to examine the effect of NPI-0052 on proteasome activity profiles in selected organs and tumors. Our results demonstrate that NPI-0052 rapidly leaves the vascular compartment in an active form after intravenous (IV) administration and inhibits the proteasome in extra-vascular tumors and other organs, excluding brain. NPI-0052 triggers a more sustained proteasome inhibition in tumors than in other organs examined. Importantly, we also confirmed the anti-tumor efficacy of NPI-0052. Methods and Model: Animal studies were approved by the DFCI Institutional Animal Care and Use Committee. Sixty CB-17 SCID-male mice were inoculated with 5.0 × 106 MM.1S cells in 100ul of serum free RPMI-1640 medium. The mice were divided into three different groups: Groups 1 and 2 (25 mice EA group) for pharmacodynamic studies (time course) and Group 3 (10 mice) for drug efficacy study. Tumor size was measured every third day in two dimensions using calipers, and tumor volume was calculated using the formula V = 0.5 a × b2, where a and b are the long and short diameter of the tumor respectively. When tumors were ~250 mm3 (~three-four weeks after injection), mice were treated with 0.15 mg/kg of NPI-0052 (IV) or vehicle control. Proteasome inhibition was assessed after either single NPI-0052 treatment (given at Day1) or three treatments (given at Day1, Day4 and Day8). Mice were euthanized at 10 mins, 1h, 4h, and 24h; and packed whole blood (PWB), liver, spleen, lung, kidney, brain and tumors were analyzed for chymotrypsin-like (CT-L), Caspase-like (C-L), and Trypsin-like (T-L) proteasome activities. For efficacy studies mice were treated with NPI-0052 twice a week for three weeks. Mice were sacrificed when their tumors reached ~1.5 cm3. NPI-0052 was dissolved in 100% DMSO to generate a 10 mg/ml stock solution, aliquoted, and stored frozen at − 80°C. The stock solution was serially diluted with 100% DMSO and for injection with 5% Solutol (Solutol HS, polyethylene glycol 660, 12 hydroxystearate; BASF, Shreveport, LA) yielding a final concentration of 2% DMSO and 98% (5% Solutol). The vehicle control was 2% DMSO and 98% (5% Solutol). The pH of the dosing solutions is between 6–7. Results: Inhibition of all three proteasome activities after a single treatment of NPI-0052 was detectable as early as 10 mins in the liver, lung, spleen, kidney and PWB; Within 24h after either a single or three IV treatments of NPI-0052, proteasome activity recovered in liver, lung, spleen and kidney, but not in tumor or PWB; No significant proteasome inhibition was noted in brain up to 24h after either a single or three IV treatments with NPI-0052; CT-L activity was inhibited within 1h post first dose, and 24h exposure triggered marked inhibition of CT-L, C-L and T-L activities in vivo in the xenografted MM.1S tumors. For example, in 1h CT-L activity was inhibited 34%, T-L activity 6% and C-L activity 16%. After 24h hours, CT-L activity was inhibited 60%, T-L activity 24% and C-L activity 49%; and finally, 6) Inhibition of CT-L, C-L and T-L activities increased in the tumor after the third NPI-0052 treatment compared to the first treatment. For example, at 1h post third dose all three activities were inhibited approximately 70–80%. Additionally, the anti-MM activity of NPI-0052 was associated with significant proteasome inhibition in tumors (P < 0.005). Conclusions: Our findings show that NPI-0052 induces a prolonged inhibition (>24h) of all three-20S proteasome activities in established MM.1S tumor xenografts which correlated with marked anti-tumor activity. In contrast, proteasome inhibition in normal tissues including liver, spleen, kidney and lung markedly recovered within 24h of administration after a single or three treatments with NPI-0052. In addition, no significant inhibition of proteasome activities was detectable in the brain after either treatment schedule.

Proteasome inhibition attenuates hepatic injury in the bile duct-ligated mouse

2006 ◽  
Vol 291 (4) ◽  
pp. G709-G716 ◽  
Author(s):  
Akira Anan ◽  
Edwina S. Baskin-Bey ◽  
Hajime Isomoto ◽  
Justin L. Mott ◽  
Steven F. Bronk ◽  
...  
Keyword(s):  
Bile Duct ◽  
Liver Injury ◽  
Proteasome Inhibitor ◽  
Stellate Cell ◽  
Proteasome Inhibition ◽  
Hepatocyte Proliferation ◽  
Hepatocyte Apoptosis ◽  
Inhibitory Protein ◽  
Bortezomib Treatment ◽  
Duct Ligation

Proteasome inhibition has recently been demonstrated to inhibit hepatic fibrogenesis in the bile duct-ligated (BDL) mouse by blocking stellate cell NF-κB activation. The effect of proteasome inhibition on liver injury, however, is unclear. Our aims were to assess the effect of the proteasome inhibitor bortezomib on liver injury in the BDL mouse. Liver injury was assessed in 7-day BDL mice treated with a single dose of bortezomib on day 4 after bile duct ligation. Despite NF-κB inhibition by bortezomib, liver injury and hepatocyte apoptosis were reduced in treated BDL mice. The antiapoptotic effect of bortezomib was likely mediated by an increase in hepatic cellular FLICE inhibitory protein (c-FLIP) levels, a potent antiapoptotic protein. Unexpectedly, numerous mitotic hepatocytes were observed in the bortezomib-treated BDL mice liver specimens. Consistent with this observation, PCNA immunoreactivity and cyclin A protein expression were also increased with bortezomib treatment. Bortezomib therapy was also associated with a decrease in numbers and activation of Kupffer cells/macrophages. In conclusion, these data suggest that the proteasome inhibitor bortezomib reduces hepatocyte injury in the BDL mouse by mechanisms associated with a reduction in hepatocyte apoptosis, a decrease in Kupffer cell/macrophage number and activation, and increased hepatocyte proliferation.

scholarly journals Heat-shock protein 70 antisense oligomers enhance proteasome inhibitor-induced apoptosis

1999 ◽  
Vol 344 (2) ◽  
pp. 477-485 ◽  
Author(s):  
John D. ROBERTSON ◽  
Kaushik DATTA ◽  
Shyam S. BISWAL ◽  
James P. KEHRER
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Cell Lines ◽  
Heat Shock Protein 70 ◽  
Proteasome Inhibitor ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Caspase Activity ◽  
Protein Levels ◽  
Induced Apoptosis

Recent evidence supports a role for heat-shock protein 70 (hsp70) and the 26 S proteasome in regulating apoptosis, although the precise nature of their involvement is not known. In the present study, control and Bcl-xL-overexpressing, interleukin-3-dependent FL5.12 cell lines were treated with the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Basal proteasome activity appeared to be ≈ 30% lower in bcl-xL cells compared with control cells using a substrate for the chymotrypsin-like activity. However, no difference in proteasome activity was detected using substrates for the trypsin-like or peptidylglutamyl peptide-hydrolysing activities. In addition, protein levels of the 20 S proteasome β-subunit, as determined by Western blot analyses, were similar in control and bcl-xL cells, leading to the conclusion that proteasome activities were the same in these two cell lines. At 24 h after treatment with 500 nM MG132, apoptosis in bcl-xL cells (22%) was less than that observed in control cells (34%). Concomitantly, caspase activity in control cells, as assessed by N-acetyl-L-aspartyl-L-glutamyl-L-valyl-L-aspartyl-7-amino-4-methylcoumarin (Ac-DEVD-AMC), was twice that observed in bcl-xL cells. By 48 h after MG132 treatment, apoptosis and caspase activity in bcl-xLcells were similar to those observed in control cells at 24 h. Proteasome inhibition stimulated increases in hsp70 protein levels in control and bcl-xL cells by 12 h, although the maximal increases found in bcl-xL cells were less. Blocking this induction with hsp70 antisense oligonucleotides potentiated apoptosis after treatment with MG132. Inhibiting caspase activity with a broad-spectrum caspase inhibitor, t-butoxycarbonyl-Asp(OMe)-fluoromethyl ketone, prevented MG132-induced apoptosis. The more specific caspase-3 inhibitor, Ac-DEVD-aldehyde, afforded less protection, although both inhibitors completely inhibited Ac-DEVD-AMC cleavage. These data indicate that both hsp70 and Bcl-xL provide some protection against proteasome inhibitor-induced apoptosis.

In Vitro and In Vivo Proteasome Activity Profiles of Bortezomib and a Novel Proteasome Inhibitor NPI-0052.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3363-3363 ◽  
Author(s):  
Dharminder Chauhan ◽  
Ta-Hsiang Chao ◽  
Laurence Catley ◽  
Benjamin Nicholson ◽  
Mugdha Velanker ◽  
...  
Keyword(s):  
Proteasome Inhibitor ◽  
Proteasome Inhibitors ◽  
Proteasome Inhibition ◽  
Proteasome Activity ◽  
Catalytic Activities ◽  
Proteasome Subunits ◽  
20 Nm ◽  
Whole Blood Cells

Abstract Proteasome inhibition is an effective anti-cancer therapy. Proteasome function is mediated by three catalytic activities: chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L). Kinetics of inhibition of catalytic activities may define the pharmacologic utility of proteasome inhibitors. Here we utilized two structurally distinct proteasome inhibitors Bortezomib, a dipeptide boronic acid; and a non-peptide proteasome inhibitor NPI-0052 to determine their effect on proteasome activities in vitro and in animal model. Examination of the proteasome activity using human erythrocyte 20S proteasomes and fluorogenic substrates shows that NPI-0052 and Bortezomib inhibit all three proteasome activities, albeit at different concentrations: NPI-0052 inhibits CT-L and T-L activities at lower concentrations than Bortezomib (NPI-0052: EC50 = 3.5 ± 0.3 nM versus Bortezomib: 7.9 ± 0.5 nM for CT-L activity; and NPI-0052: EC50 = 28 ± 2 nM versus Bortezomib: EC50 = 590 ± 67 nM for T-L activity); in contrast, higher concentrations of NPI-0052 than Bortezomib are required to inhibit C-L activity (NPI-0052 EC50 = 430 ± 34 nM versus Bortezomib: EC50 = 53 ± 10 nM for C-L activity). We next compared the effects of NPI-0052 and Bortezomib on all three proteasome activities in vivo. Mice were treated with a single MTD dose of NPI-0052 (0.15 mg/kg i.v) or Bortezomib (1 mg/kg i.v); blood samples were collected at 90 mins, 24h, 48h, 72h, or 168h; and whole blood cells were then analyzed for proteasome activity. NPI-0052 completely inhibited CT-L activity by 90 mins, which was recoverable by 168h; whereas Bortezomib-inhibited CT-L activity is recoverable at 24h. T-L activity is significantly inhibited by NPI-0052 at 90 mins, 24h, 48h, and 72h; and is recoverable by 168h; in contrast, Bortezomib enhances T-L activity. Finally, NPI-0052 inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and this activity recovered at 168h, whereas Bortezomib significantly inhibits C-L activity at 90 mins, 24h, 48h, and 72h; and is similarly recoverable at 168h. We next utilized a novel methodology to measure proteasome activity by immunoblotting using dansylAhx3L3VS as a probe (Berkers et al., Nature Methods, 2005), which also allow for determining subunit specificity of a proteasome inhibitor. Multiple myeloma (MM) cells were cultured in the presence or absence of various concentrations of either NPI-0052 (2 nM; 7 nM: IC50; or 20 nM) or Bortezomib (2 nM; 5 nM: IC50; or 20 nM). Competition experiments between either NPI-0052 or Bortezomib and dansylAhx3L3VS revealed that NPI-0052 (7 nM) markedly inhibits the CT-L activity represented by beta-5 subunit of the proteasome and decreased the dansylAhx3L3VS-labeling of the beta-1 (C-L activity) and -2 (T-L activity) subunits. Slightly higher concentrations of Bortezomib are necessary to markedly inhibit beta-5 and -1 subunits, whereas beta-2 subunits are not inhibited. Importantly, both agents trigger apoptosis in MM cells; however, NPI-0052 is remarkably less toxic to normal lymphocytes than Bortezomib. Our data show that NPI-0052, like Bortezomib, targets the proteasome, but triggers a proteasome activity profile distinct from Bortezomib. The mechanistic insights gained from these studies will allow for improved drug design based on targeting specific proteasome subunits.

Bortezomib Resistant Constitutive NF-κB Activation in Mantle Cell Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3465-3465
Author(s):  
David T. Yang ◽  
Ken H. Young ◽  
Brad S. Kahl ◽  
Shigeki Miyamoto
Keyword(s):  
Dna Binding ◽  
Mantle Cell Lymphoma ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Cytotoxic Effect ◽  
Cell Lymphoma ◽  
Proteasome Inhibition ◽  
Bortezomib Treatment ◽  
Mantle Cell ◽  
Synergistic Cytotoxic Effect

Abstract Bortezomib is a proteasome inhibitor whose antineoplastic effects include inhibition of NF-κB, a transcription factor whose deregulation may play a central role in mantle cell lymphoma (MCL) pathogenesis. Bortezomib has shown clinical efficacy in relapsed and refractory cases of MCL with response rates of 40%. NF-κB can be activated through several pathways, including a proteasome inhibitor resistant pathway. It remains unknown whether MCL harbors bortezomib resistant constitutive NF-κB activity, but characterization of this may have important implications in elucidating bortezomib resistance and also in establishing rational therapeutic combinations. We investigated the effect of bortezomib on constitutive NF-κB activity in 3 EBV-negative MCL cell lines (Jeko, Rec-1 and Z138) and 20 MCL patient samples. Electrophoretic mobility supershift assay demonstrated that each of the cell lines had distinct NF-κB complexes with the Jeko and Rec-1 containing mainly p50/p65 and p50/cRel heterodimers, and Z138 comprised almost entirely of p52/RelB heterodimers. At a physiologically achievable dose of bortezomib (20nM), a live cell-based proteasome inhibition assay demonstrated greater than 80% proteasome inhibition in all three cell lines. Treatment of Jeko cells with such a dose resulted in a 50% decrease of NF-κB DNA binding, in contrast to a 10 to 30% increase of DNA binding in Rec-1 and Z138 cells by electrophoretic mobililty shift assay. Of 10 MCL patient samples from which results could be obtained, only 2 demonstrated a greater than 50% decrease in NF-κB DNA binding after treatment with 20nM and 100nM of bortezomib, whereas the remainder showed either no inhibition or even increased binding. Thus, bortezomib resistant constitutive NF-κB activity appears to be present in Rec-1, Z138, and a majority of MCL cases. Cytotoxicity assessed by flow cytometry following staining with propidium iodide showed Rec-1 and Z138 cells had greater resistance to bortezomib induced apoptosis (82 ± 5% and 69 ± 5% viability) than Jeko cells (47 ± 6% viability) after 20nM bortezomib treatment for 24 hours. Combining bortezomib with perillyl alcohol, a known suppressor of proteasome inhibitor resistant NF-κB activation, resulted in a synergistic cytotoxic effect in all 3 cell lines as assessed by the combination index (CI) method with CIs of 0.31, 0.32, and 0.60 for Jeko, Rec-1, and Z138 cells respectively, where CI of 0.1–0.3 is strong synergism, 0.3–0.7 is synergism, 0.7–0.85 is moderate synergism and 0.85–0.9 is slight synergism. In conclusion, our findings suggest that bortezomib resistant NF-κB activity is present in a significant subset of MCL cases, and the combination of bortezomib with a suppressor of proteasome inhibitor resistant NF-κB activity may elicit a synergistic cytotoxic effect in MCL.

Cell Non-Autonomous Modulation Of Tumor Cell Responses To Pharmacological Inhibition Of The Proteasome

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4454-4454
Author(s):  
Eugen Dhimolea ◽  
Richard W.J. Groen ◽  
Catriona A. Hayes ◽  
Jana Jakubikova ◽  
Bariteau Megan ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Tumor Cells ◽  
Cell Lines ◽  
Induced Resistance ◽  
Proteasome Inhibitor ◽  
Bioluminescence Imaging ◽  
Proteasome Inhibition ◽  
Bortezomib Treatment

Extensive preclinical studies of several groups using tumor cells co-cultured with bone marrow stromal cells (BMSCs) has documented that the potent anti-MM activity of the proteasome inhibitor bortezomib is not suppressed by BMSCs (e.g. primary and immortalized BMSCs). Using our compartment-specific bioluminescence imaging (CS-BLI) assays, we extended these observations to larger panels of MM cell lines. We observed, however, a recurrent pattern that primary CD138+ MM tumor cells from bortezomib-refractory patients recurrently exhibited substantial in vitro response to clinically-achievable concentrations and durations of bortezomib treatment. To simulate this clinicopathological observation, MM.1R-Luc+ cells were injected i.v. in SCID-beige mice and treated with bortezomib (0.5 mg/kg s.c. twice weekly for 5 weeks): diffuse MM tumors initially responded to bortezomib, but eventually became refractory. These in vivo-resistant MM cells were isolated from the mice and were treated in vitro with bortezomib, exhibiteing similar responsinveness to this agent as their isogenic bortezomib-naive MM cells, To further address the possibility that this represents a previously underexplored form of a microenvironment-induced alteration in bortezomib responsiveness, we studied how MM cells respond to pharmacological proteasome inhibition after variable times of co-culture with BMSCs prior to bortezomib exposure. We observed that prolonged tumor-stromal co-culture (48-96hrs) prior to initiation of bortezomib treatment did not affect drug sensitivity for many of the MM cell lines (OPM2, H929, UM9, KMS11, KMS18 and RPMI-8226) tested. Notably, prolonged co-cultures with BMSCs prior to bortezomib treatment enhanced the activity of this agent for other MM cell lines (e.g. OPM1, Dox40, OCI-My5, KMS12BM or KMS18). However, MM.1S and MM.1R cells, when exposed to extended co-cultures with BMSCs prior to initiation of drug exposure, exhibited significant attenuation (2-3 fold increase of IC50 values) of their response to bortezomib in several independent replicate experiments. In support of these in vitro results, heterotypic s.c. xenografts of Luc+ MM.1S cells co-implanted with Luc-negative BMSCs did not show significant reduction in MM tumor growth with bortezomib treatment (0.5 mg/kg s.c. twice weekly for 5 weeks) compared to vehicle-treated controls (p=0.13), as quantified by bioluminescence imaging. In co-cultures with BMSCs, MM.1S and MM.1R cells also exhibited suppression of their response to carfilzomib (the degree of this stroma-induced resistance was more pronounced that in the case of bortezomib for these 2 cell lines). Consistent with these observations, in vivo administration of carfilzomib in the orthotopic model of diffuse bone lesions of MM.1R-Luc+ cells was associated with less pronounced reduction in tumor growth, compared to bortezomib treatment (p<0.03). These results suggest that the stroma-induced attenuation of activity against a subset of MM cells represents a class-effect for this group of therapeutics, despite quantitative differences between different proteasome inhibitors. Mechanistically, we determined a distinct transcriptional signature of stroma-induced transcripts which are overexpressed in refractory myeloma patients with significantly shorter overall survival (p<0.03, log-rank tests) after bortezomib treatment. Our results in vitro and in vivo support the notion that the responses of MM cells to proteasome inhibition can exhibit substantial plasticity depending on the specific microenvironmental context with which these MM cells interact. We also identify prognostically-relevant candidate molecular mediators of stroma-induced resistance to proteasome-inhibitor based therapy in MM. Disclosures: No relevant conflicts of interest to declare.

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