Effects of thymoquinone on isolated and cellular proteasomes Valentina Cecarini, Luana Quassinti, Alessia Di Blasio, Laura Bonfili, Massimo Bramucci, Giulio Lupidi, Massimiliano Cuccioloni, Matteo Mozzicafreddo, Mauro Angeletti and Anna Maria Eleuteri School of Biosciences and Biotechnology, University of Camerino, Italy Keywords apoptosis; glioblastoma; p53; thymoquinone; ubiquitin proteasome system Correspondence V Cecarini, School of Biosciences and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy Fax: +39 0737 403290 Tel: +39 0737 403247 E-mail: valentina.cecarini@unicam.it (Received 18 November 2009, revised 24 February 2010, accepted 26 February 2010) doi:10.1111/j.1742-4658.2010.07629.x Thymoquinone, a naturally derived agent, has been shown to possess antioxidant, antiproliferative and proapoptotic activities In the present study, we explored thymoquinone effects on the proteasomal complex, the major system involved in the removal of damaged, oxidized and misfolded proteins In purified 20S complexes, subunit-dependent and composition-dependent inhibition was observed, and the chymotrypsin-like and trypsin-like activities were the most susceptible to thymoquinone treatment U87 MG and T98G malignant glioma cells were treated with thymoquinone, and 20S and 26S proteasome activity was measured Inhibition of the complex was evident in both cell lines, but predominantly in U87 MG cells, and was accompanied by accumulation of ubiquitin conjugates Accumulation of p53 and Bax, two proteasome substrates with proapoptotic activity, was observed in both cell lines Our results demonstrate that thymoquinone induces selective and time-dependent proteasome inhibition, both in isolated enzymes and in glioblastoma cells, and suggest that this mechanism could be implicated in the induction of apoptosis in cancer cells Introduction Black cumin seed (Nigella sativa) oil extracts have been used for many centuries in the treatment of several human diseases, and thymoquinone (TQ), its active component, has recently been tested for its efficacy against several diseases, including cancer [1–3] In this regard, TQ was found to inhibit proliferation in a concentration-dependent manner in numerous cell lines [4,5] It has shown significant antineoplastic activity against multidrug-resistant human pancreatic adenocarcinoma, uterine sarcoma and leukemic cell lines, with minimal toxicity for normal cells [6] In a mouse model, the injection of the essential oil into the tumor site significantly inhibited solid tumor development as well as the incidence of liver metastasis, thus improving mouse survival [5] These results indicate that the antitumor activity or cell growth inhibition could in part be due to the effect of TQ on the cell cycle [5] Furthermore, it has been demonstrated that the growth of prostate cancer cells is highly sensitive to the inhibitory effect of TQ, and that this inhibitory action is extremely selective, showing very little effect on the growth of noncancerous prostate epithelial cells in culture, and preventing the growth of human prostate tumors in nude mice [7] Despite awareness of these potential antineoplastic effects, the molecular pathways involved are not Abbreviations AMC, 7-amino-4-methyl-coumarin; BrAAP, branched chain amino acid-preferring; ChT-L, chymotrypsin-like; ECL, enhanced chemiluminescence; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; pAB, 4-aminobenzoate; PGPH, peptidyl-glutamyl peptide-hydrolyzing; PVDF, poly(vinylidene difluoride); Suc, succinyl; T-L, trypsin-like; TQ, thymoquinone; Ub, ubiquitin 2128 FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al completely clear Recent findings suggest that TQ has a strong chemopreventive potential for the inhibition of carcinogenesis by modulating lipid peroxidation and the cellular antioxidant milieu [8,9] In fact, TQ is reported to possess strong antioxidant properties, inhibiting free radical generation [10] Interestingly, according to Gali-Muhtasib et al., TQ is able to trigger apoptosis in several cell lines in a p53-independent or a p53-dependent manner [11,12], and, as recently shown, its proapoptotic effects are linked to its pro-oxidant activity [13] Among the different mechanisms involved in the induction of apoptotic pathways, the tumor suppressor protein p53 plays a pivotal role [14] Under physiological conditions, p53 is maintained at low steady-state levels by the MDM2 protein, an E3 ubiquitin (Ub) ligase, which ubiquitinates and targets p53 for proteasome-mediated degradation [15] Specific stress agents make p53 and MDM2 undergo different post-translational modifications, including phosphorylation, thus disrupting the interaction and leading to activation of p53 [16] At this point, p53 induces a series of downstream events that regulate the transcription of a subset of genes involved in apoptosis, such as that encoding Bax, a member of the Bcl-2 family [17] The Ub–proteasome pathway is a nonlysosomal protein degradation system responsible for degrading both damaged/unfolded proteins dangerous for normal cell growth and metabolism [18], and critical regulatory proteins involved in apoptosis [19], cell cycle regulation [20], gene expression [21], carcinogenesis and DNA repair [22–24] Because of this, studies on the discovery of molecules that are able to modulate proteasome activity have recently been gaining great attention The central core of this system is the 20S proteasome This is a cylindrical structure with an internal cavity, composed of four rings, each containing seven different a subunits and b subunits, resulting in the following arrangement: a1–7b1–7b1–7a1–7 [19] Only three of the seven b subunits, b1, b2, and b5, located inside the main chamber, show proteolytic activity Specifically, b1 is associated with the peptidyl-glutamyl peptidehydrolyzing (PGPH) activity and possesses limited branched chain amino acid-preferring (BrAAP) activity, b2 is associated with the trypsin-like (T-L) activity, and b5 is associated with the chymotrypsin-like (ChTL) activity However, mutational analyses have shown that b5 also has a tendency to cleave after small neutral and branched side chains; therefore, two other activities, BrAAP and small neutral amino acid-preferring (SNAAP), can be assigned to this subunit [25] In certain conditions, such as in the presence of c-interferon, these three b subunits can be replaced by Thymoquinone inhibits proteasome functionality homologous subunits, b1i, b2i, and b5i, resulting in a de novo synthesized proteasomal form, the immunoproteasome, which produces mainly immunogenic peptides in association with major histocompatibility complex class I [19] Malignant gliomas are the most common and lethal tumors of the central nervous system [26] Treatment outcomes, even with an aggressive approach including surgery, radiation therapy, and chemotherapy, are dismal The median survival of treated patients with glioblastoma multiforme is less than year, with fewer than 20% surviving for years [27] There is therefore an urgent need to devise alternative therapeutic strategies with which to fight gliomas In the present work, the effects of TQ on proteasome functionality were investigated both in isolated and in cellular complexes For this purpose, constitutive and immune-isolated proteasomes and two human glioblastoma cell lines, U87 MG and T98G, differing in their p53 gene status, were used Specifically, U87 MG cells present the wild-type form of p53, whereas T98G cells harbor a single p53 mutation [28] Results Nucleophilic susceptibility analysis TQ was examined for sites of electrophilic and nucleophilic susceptibility Computational analysis revealed that TQ possessed two carbons (C1 and C4) with similar nucleophilic susceptibility (Fig 1) that are likely to be the target of a nucleophilic attack [29] TQ effects on isolated 20S proteasomes To test TQ effects on isolated 20S constitutive and immunoproteasome functionality, we incubated purified enzymes with different concentrations of TQ (0.0–100 lm) In particular, the ChT-L, T-L, PGPH and BrAAP activities of the isolated complexes were tested using specific substrates, as described in Experimental procedures As shown in Fig 2, it is possible to highlight that TQ is able to modulate proteasome functionality inducing a subunit and composition-dependent inhibition Of the two complexes, the immunoproteasome was the most susceptible to the presence of TQ, and the ChTL and T-L activities were the components with the highest degree of inhibition The PGPH component was not particularly altered in the presence of TQ; only 16% inhibition was evident at 30 lm Finally, the BrAAP activity was not significantly influenced by the presence of TQ (data not shown) FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2129 Thymoquinone inhibits proteasome functionality A V Cecarini et al B C Lowest susceptibility Highest susceptibility Fig Chemical structure and nucleophilic susceptibility of TQ Chemical structure (A), nucleophilic susceptibility (B) and electrophilic susceptibility (C) of TQ Isosurfaces were calculated with WEBMO C1 and C4 carbonyl were found to be nucleophilically attacked by the OH group of Thr1 Interestingly, the inhibition showed concentrationdependent behavior only up to 20 lm, when the maximum detectable rates of inhibition were 30% and 40% for the ChT-L and T-L components, respectively, of the immunoproteasome Thereafter, an increase in TQ concentrations did not lead to enhanced inhibition Supported by the literature [30], we propose that this U-shaped inhibition depends on the presence of an additional binding site on the proteasomal complex to which TQ binds with a lower affinity than it does to the active site Our model assumes that TQ preferentially binds to the active site at low concentrations, resulting in the observed inhibition, whereas at higher concentrations the binding to the additional site becomes significant, allosterically restoring the activity The fraction of TQ bound to the active site is now released, allowing the substrate to enter it and be successfully degraded, resulting in the activity recovery observed at TQ concentrations higher than 20 lm To verify this hypothesis, we performed an experiment using the peptide aldehyde Z-LLF-CHO, a selective and reversible proteasome inhibitor, with the aim of blocking part of the proteasome active sites [31] After h of incubation of the 20S immunoproteasome with Z-LLF-CHO, TQ at different concentrations was added and the T-L activity was measured (Fig 3) In agreement with the mechanism described above, we observed a recovery in the proteasome activity The Nitro Blue tetrazolium assay, which monitors the formation of quinone adducts, shows the existence of additional TQ-binding sites on the proteasome Figure indicates that the formation of b-subunit–TQ adducts increases at TQ concentrations of and 20 lm, whereas it decreases at a concentration of 100 lm (corresponding to the recovery of proteasome activity) At the same time, increases in TQ concentration resulted in clear enhancement in the levels of a-subunit–TQ adducts, confirming our model of the presence of two different TQ-binding sites on the proteasome complex TQ inhibits cell proliferation Fig Effects of increasing TQ concentrations (0–100 lM) on isolated 20S complexes The ChT-L, T-L and PGPH activities were assayed r, constitutive proteasome; , immunoproteasome 2130 Two cell lines, U87 MG and T98G, derived from human glioblastomas were used as a model They FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al Fig TQ binding to a secondary site of the proteasome complex After Z-LLF-CHO and 20S immunoproteasome preincubation, in order to partially inhibit the enzyme, the effects of increasing concentrations of TQ on the T-L activity were tested Data are reported as percentages relative to proteasome activity in the presence of ZLLF-CHO (mean values ± standard deviations of five independent determinations) A Thymoquinone inhibits proteasome functionality A set of dose–response experiments was performed to compare the effects of TQ on cell viability in U87 MG and T98G cells Cells were incubated in the presence of TQ at concentrations ranging from 0.0 lm to 200 lm Analysis by light microscopy showed that treatment of glioblastoma cells with increasing amounts of TQ resulted in significant alterations in cell morphology and impaired the ability of the cells to become confluent (Fig 5A) Data obtained with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that cell viability was significantly reduced in a dose-dependent and exposure time-dependent manner in both cell lines (Fig 5B) In both cell lines, almost complete loss of viability was seen after exposure to 200 lm TQ At lower concentrations, TQ exerted a stronger inhibitory effect on U87 MG cells than on T98G cells A comparison of IC50 values, reported in Table 1, showed that, after 48 h of treatment with TQ, IC50 values were 38.82 lm for U87 MG cells and 62.48 lm for T98G cells TQ effects on the proteasome functionality of glioblastoma cells B Fig Detection of quinone adducts 20S isolated immunoproteasomes were treated with different concentrations of TQ and lactacystin (see Experimental procedures), resolved by SDS ⁄ PAGE, and electroblotted onto PVDF membranes Adducts were visualized after 45 of incubation with Nitro Blue tetrazolium Lane C represents 20S proteasome loaded without pretreatment with TQ and lactacystin (A) Densitometry related to three different experiments (B) A representative membrane after the Nitro Blue tetrazolium staining carry, respectively, the wild-type and a mutant p53 gene This mutation consists of a single G fi A transition in codon 237, resulting in a missense mutation of methionine to isoleucine [32,33] Interestingly, a study conducted by Van Meir et al on different glioblastoma lines and their p53 status revealed that this mutation in the T98G line results in a transcriptionally inactive form of p53 [34] Considering the major role of the proteasome in mediating numerous cellular pathways, including apoptosis, we wanted to determine whether TQ was able to modulate its functionality in the two glioblastoma cell lines Cells were treated with TQ at 20 lm, the concentration with the greatest effects on isolated proteasomes, for 12, 24, 48 and 72 h Control cells were cultured in parallel in the presence of dimethylsulfoxide Both cell lines had a high level of responsiveness to TQ treatment, showing compromised activities as compared with controls (Figs and 7) Parallel assays run in the presence of specific proteasome inhibitors, Z-GPFLCHO and lactacystin, demonstrated that the contribution to the proteolysis was effectively due to the 20S proteasome (data not shown) Figures and illustrate the presence of timedependent proteasome inhibition, which assumes particular significance after 48 and 72 h of treatment Interestingly, U87 MG cells showed a higher extent of proteasome inhibition, with relevant differences also at 24 h, as evident for the T-L and BrAAP activities Generally, in this cell line, TQ induced a global and stronger decrease in proteasome functionality than that observed in T98G cells We also measured the ChT-L component of the 26S proteasome, whose proteolytic activity is ATP-dependent, and obtained, at 72 h, similar percentages of inhibition in the two lines However, at 48 h, a significant FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2131 Thymoquinone inhibits proteasome functionality V Cecarini et al A Fig TQ effects on U87 MG and T98G cells (A) Morphology of U87 MG and T98G cells grown under standard conditions and treated with 50 lM or 100 lM TQ dissolved in dimethylsulfoxide Dimethylsulfoxide concentrations in treated and control cells did not exceed 0.25% per well Cells were observed by using an inverted microscope 24 h post-treatment (B) Dose–response curve for the effect of TQ on cell viability after 24, 48 and 72 h of exposure Cell viability was determined by the MTT assay, and is reported as the percentage of viable cells Each value is the mean ± standard deviation of three separate experiments performed in triplicate B Table Thymoquinone IC50 values for glioma cell lines after incubation periods of 24, 48 and 72 h CI, confidence interval Incubation period (h) 24 48 72 IC50 (lM) (95% CI) T98G U87 MG 77.73 (71.93–83.99) 62.48 (57.81–67.53) 61.46 (58.58–64.48) 47.08 (41.84–52.97) 38.82 (35.37–41.26) 35.83 (31.64–38.35) difference after TQ exposure was evident for U87 MG cells To verify the above-mentioned proteasome inhibition, we conducted western blot analyses, using antibodies against Ub In fact, the abnormal presence of Ub conjugates is a clear marker of impaired proteasome activity Our findings demonstrate time-dependent accumulation of Ub–protein aggregates, confirming the data on proteasome inhibition (Fig 8) Furthermore, western blot assays performed with an antibody against 20S suggested that the observed inhibition was really due to compromised complex functionality and not to downregulation of its synthesis (Fig 9) These results support the findings regarding the ability of TQ to act directly on the proteasome activity, and remove the possibility of decreased synthesis of the enzyme 2132 TQ effects on p53 and Bax levels In order to strengthen the data on proteasome inhibition, we measured the levels of two proteasome substrates, p53 and Bax, that play an important role in the onset of apoptotic events In both cell lines, time-dependent accumulation of p53 was observed In T98G cells, this increase was significant even after 24 h of treatment, but was particularly evident at 48 h and 72 h (levels that are 2.3-fold and a 2.8-fold higher, respectively, than in controls) In U87 MG cells, instead, the enhancement in protein levels was delayed, and became consistent only after 48 h of TQ exposure (Fig 10) Bax accumulation was more evident in T98G cells than in U87 MG cells Specifically, the former responded in a shorter time, with significant increases at 48 h and 72 h (1.21-fold and 1.42-fold, respectively, that seen in controls), whereas the latter presented significant enhancement only at 72 h, with a 1.44-fold increase as compared with the respective control (Fig 11) Discussion The debate on the use of naturally derived drugs as coadjuvants in the treatment of cancer is of growing interest In fact, owing to concerns about the possible FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al Thymoquinone inhibits proteasome functionality Fig 20S and 26S proteasome functionality in U87 MG cells treated with 20 lM TQ Activities were assayed as reported in Experimental procedures Data are expressed as percentage of activity relative to control cells in each set (mean values ± standard deviations of five independent determinations) Fluorescence due to nonproteasomal degradation was subtracted The asterisks indicate data points that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01) toxic side effects of conventional medicine, the use of natural products as alternatives to such treatments has been increasing TQ is the most abundant constituent of N sativa, and has pivotal roles in several biological processes Numerous studies have demonstrated the antioxidant, antiproliferative and proapoptotic activities of TQ Most notably, TQ is able to induce selective apoptosis, discriminating between tumor and normal cells, in a p53-dependent or p53-independent way For example, previous published data established that osteosarcoma cells [4] and neoplastic keratinocytes [35] are susceptible to TQ treatment, whereas normal cells and mouse primary keratinocytes not exhibit morphological and ⁄ or proliferative alterations [4,35] The observation that proteasome inhibitors are able to induce apoptosis in tumor cells opened the possibility of their use as potential drugs, and numerous studies have been conducted with the aim of finding natural, nontoxic and inexpensive compounds [36–38] FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2133 Thymoquinone inhibits proteasome functionality V Cecarini et al Fig 20S and 26S proteasome functionality in T98G cells treated with 20 lM TQ Activities were assayed as reported in Experimental procedures Data are expressed as percentage of activity remaining relative to control cells in each set (mean values ± standard deviations of five independent determinations) Fluorescence due to nonproteasomal degradation was subtracted The asterisks indicate data points that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01) In this scenario, we decided to investigate the possible interaction between TQ and proteasomes in order to determine whether TQ could modulate the enzyme functionality Considering the data obtained from computational analysis, it is reasonable to think that TQ could behave as a nucleophilic target, resulting in inhibition of proteasome activity To confirm this hypothesis, we tested proteasome functionality after TQ treatment of both isolated and cellular complexes Interestingly, we 2134 observed subunit-dependent and composition-dependent inhibition of both the purified enzymes, with the immunoproteasome being the most sensitive and the ChT-L and T-L components being the most influenced activities We also demonstrated that TQ induces a U-shaped inhibition in proteasome complexes through the binding of two distinct sites with different degrees of affinity Exposure of two human glioblastoma cell lines, U87 MG and T98G, to TQ was able to significantly FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al Thymoquinone inhibits proteasome functionality A B C Fig Detection of Ub–protein conjugates in U87 MG and T98G cells The densitometric analysis from five separate blots, shown as mean values ± standard deviations, and a representative western blot are shown (A, B) Membranes were reprobed with GAPDH antibody to ensure equal protein loading (C) Detection was performed with an ECL western blotting analysis system The asterisks indicate data points that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01) compromise proteasome activity The two cell lines are different with respect to a single mutation in the p53 gene; this characterizes the T98G line, whereas the U87 MG line maintains the wild-type form of the protein As previously shown by other authors, this mutation results in a transcriptionally inactive p53 gene [34] Assaying TQ effects on cell viability, we found that both cell lines showed clear changes in cell morphology, although with different degrees of sensitivity In fact, U87 MG cells were more susceptible to the treatment, as shown by the different IC50 values obtained after the treatments Cells were then treated with TQ at 20 lm, the concentration with the highest effect according to the in vitro data, and both 20S and 26S proteasomes showed changes in their functionality In particular, our assays showed significant, time-dependent but differential sensitivities of U87 MG and T98G cells to TQ treatment T-L, BrAAP and PGPH activities were significantly more affected in U87 MG cells than in T98G cells, with the former showing altered proteasome functionality at 24 h This inhibition was also confirmed by accumulation of Ub–protein conjugates Furthermore, when we tested the 20S expression levels with specific antibodies, we could not detect any differences between control and treated cells, demonstrating the ability of TQ to directly alter proteasome activity without affecting its synthesis Considering our data, it is clear that TQ is able to modulate proteasome activity, inducing global inhibition in the studied models, although to different extents These results are in line with previously published data from our laboratory and others reporting on the ability of small, naturally derived ligands, e.g flavonoids, to inhibit proteasome functionality and selectively modulate its activity, depending on the subunit composition [37,39,40] It has been widely reported that the proteasome, being responsible for the removal of proapoptotic FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2135 Thymoquinone inhibits proteasome functionality V Cecarini et al A B C Fig Detection of the 20S ‘core’ in U87 MG and T98G cells The densitometric analysis from five separate blots, shown as mean values ± standard deviations, and a representative western blot are shown (A, B) Membranes were reprobed with GAPDH antibody to ensure equal protein loading (C) Detection was performed with an ECL western blotting analysis system A B C Fig 10 Detection of p53 in U87 MG and T98G cells The densitometric analysis from five separate blots, shown as mean values ± standard deviations, and a representative western blot are shown (A, B) Membranes were reprobed with GAPDH antibody to ensure equal protein loading (C) Detection was performed with an ECL western blotting analysis system The asterisks indicate data points that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01) proteins, is involved in the induction of programmed cell death [19] Its inhibition, in fact, triggers the accumulation of proteins such as p53 and Bax [41–43] For 2136 this reason, numerous compounds with the ability to modulate proteasome activity have been used in the treatment of malignancies FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al Thymoquinone inhibits proteasome functionality A B C Fig 11 Detection of Bax in U87 MG and T98G cells The densitometric analysis from five separate blots, shown as mean values ± standard deviations, and a representative western blot are shown (A, B) Membranes were reprobed with GAPDH antibody to ensure equal protein loading (C) Detection was performed with an ECL western blotting analysis system The asterisks indicate data points that are statistically significant as compared with the respective untreated control cells (*P < 0.05, **P < 0.01) Our results on the accumulation of both p53 and Bax are in line with the data describing the ability of TQ to inhibit proteasome activity These two proapoptotic proteins are proteasome substrates, and their intracellular levels increase together with proteasome malfunctions It is therefore likely that one of the mechanisms through which TQ triggers apoptosis in cancer cells is the induction of proteasome inhibition In summary, our data demonstrate that TQ is able to modulate proteasome functionality, inducing composition-dependent inhibition both in isolated complexes and in glioblastoma cells This inhibition leads to intracellular increases in the levels of apoptotic proteins such as p53 and Bax, and may be linked to the onset of apoptotic events Such findings represent evidence that this compound, characterized by very low toxicity, deserves further clinical analysis and investigation, mostly for its potential application as an adjuvant in the treatment of cancer and other diseases Experimental procedures Reagents and chemicals Thymoquinone, substrates for assaying the ChT-L, T-L and PGPH activities [succinyl (Suc)-Leu-Leu-Val-Tyr-7amino-4-methyl-coumarin (AMC), Z-Leu-Ser-Thr-Arg- AMC, and Z-Leu-Leu-Glu-AMC], proteasome inhibitors (Z-Gly-Pro-Phe-Leu-CHO and lactacystin), Nitro Blue Tetrazolium and MTT were purchased from Sigma-Aldrich S.r.L (Milan, Italy) The substrate Z-Gly-Pro-Ala-PheGly-4-aminobenzoate (pAB), for testing BrAAP activity, and the proteasome inhibitor Z-LLF-CHO (Cbz-LeuLeu-Phe-CHO) were kind gifts from M Orlowski (Department of Pharmacology, Mount Sinai School of Medicine, New York, NY, USA) Aminopeptidase N (EC 3.4.11.2) for the coupled assay utilized to detect BrAAP activity [44] was purified from pig kidney as reported elsewhere [45,46] TQ was dissolved in dimethylsulfoxide (Sigma Aldrich S.r.l.) U87 MG and T98G human glioblastoma cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) All of the reagents for cell cultures were obtained from Euroclone (Milan, Italy) Rabbit anti-(human 20S proteasome) serum, rabbit anti-(human 20S proteasome b5 subunit) serum and mouse anti-[human 20S a(1, 2, 3, 5, 6, and 7) subunits] serum were purchased from BIOMOL International, L.P The mouse monoclonal antibodies against Ub, p53 and Bax were obtained from Santa Cruz Biotechnology, Inc (Heidelberg, Germany) Membranes for western blot analyses were purchased from Millipore (Milan, Italy) Proteins immobilized on films were detected with the enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech, Milan, Italy) All chemicals and solvents were of the highest analytical grade available FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS 2137 Thymoquinone inhibits proteasome functionality V Cecarini et al Nucleophilic susceptibility analysis The frontier electron density isosurfaces of TQ were created using webmo [47], by performing a Gaussian ab initio and semiempirical calculation of nuclear susceptibility analysis using the pm3 wavefunction Electrophilic (HOMO) and nucleophilic (LUMO) frontier density surfaces were computed from the magnitudes of molecular orbitals available for attack by an electrophile or a nucleophile The results are represented as a ‘bull’s eye’ pattern, with blue representing the highest probability of an attack Measurements of isolated 20S proteasome activity To evaluate the effects of TQ on the 20S constitutive and immunoproteasome peptidase activities, in vitro assays were performed with fluorogenic peptides Suc-Leu-Leu-Val-TyrAMC was used for ChT-L activity, Z-Leu-Ser-Thr-ArgAMC for T-L activity, Z-Leu-Leu-Glu-AMC for PGPH activity, and Z-Gly-Pro-Ala-Phe-Gly-pAB for BrAAP activity [48–50] Isolation and purification of the 20S proteasome from bovine brain and thymus were performed as previously reported [50,51] The incubation mixture contained TQ at concentrations ranging from 0.0 to 100.0 lm, lg of the isolated 20S proteasomes, the appropriate substrate, and 50 mm Tris ⁄ HCl (pH 8.0), up to a final volume of 100 lL Incubation was performed at 37 °C, and after 60 the fluorescence of the hydrolyzed 7-amino4-methyl-coumarin (AMC) and 4-aminobenzoic acid (pAB) was detected (AMC, kexc = 365 nm, kem = 449 nm; pAB, kexc = 304 nm, kem = 664 nm) on a SpectraMax Gemini XPS microplate reader To test the presence of a TQ secondary binding site on the proteasome complex, lg of isolated 20S immunoproteasome was preincubated with lm Z-LLF-CHO for h at 37 °C Then, TQ at different concentrations (0.0– 200 lm) and the appropriate substrate for testing the T-L activity were added After 60 min, the hydrolyzed AMC was detected on a SpectraMax Gemini XPS microplate reader Detection of proteasome–quinone adducts Detection of the TQ-mediated formation of quinone adducts in isolated 20S immunoproteasomes was performed as described by Gallop et al [52] Twenty micrograms of purified complex was preincubated for h at 37 °C with different concentrations of lactacystin (0, 2.5, and 10 lm) Then, TQ (5, 20 and 100 lm) was added to the mixtures and incubated for h at 37 °C Samples were then resolved by 12% SDS ⁄ PAGE and electroblotted onto poly(vinylidene difluoride) (PVDF) membranes The detection was performed by staining the membrane with Nitro Blue 2138 tetrazolium (0.24 mm in m potassium glycinate, pH 10) for 45 in the dark As internal control, 20 lg of isolated immunoproteasome was loaded and stained without treatment with either TQ or lactacystin Proteasome a subunits and b subunits were identified by staining the same membrane with a primary antibody specific for the 20S a1, a2, a3, a5, a6 and a7 subunits, and with a primary antibody specific for the b5 subunit, respectively Cell culture T98G and U87 MG cells were maintained in EMEM with mm l-glutamine, 0.1 mm nonessential amino acids, mm sodium pyruvate, 100 ImL)1 penicillin G, and 100 lgỈmL)1 streptomycin, supplemented with 10% heat-inactivated fetal bovine serum Cells were maintained in a 5% CO2 atmosphere at 37 °C Cell viability assay Cell viability was determined by the standard MTT assay [53] Cells were seeded at an initial density of · 104 cellsỈmL)1 in 96-well microtiter plates (Iwaki, Tokyo, Japan) in 100 lL of growth medium After incubation for 24 h at 37 °C, cells were exposed to different concentrations of TQ (0.0–200 lm) containing 0.25% dimethylsulfoxide, which was applied as a control, for 24, 48 and 72 h in a humidified atmosphere at 37 °C in the presence of 5% CO2 Cell viability was then quantified by the ability of living cells to reduce the yellow dye MTT to a purple formazan product Cells were incubated with MTT for h, the medium was replaced with 100 lL of dimethylsulfoxide, and the attenuance was measured with a Titertek Multiscan microElisa microplate spectrophotometer reader (Labsystems, Helsinki, Finland) at 540 nm The IC50 values were determined using graphpad prism (GraphPad Software, San Diego, CA, USA) TQ treatment Cells were grown in 100 mm tissue culture dishes at an initial concentration of · 104 cells per dish, and were then exposed to 20 lm TQ for 12, 24, 48 and 72 h Control treatments were performed in the presence of dimethylsulfoxide for each time point After removal of the medium and washing with cold NaCl ⁄ Pi, cells were harvested in mL of NaCl ⁄ Pi and centrifuged at 1600 g for The pellet was resuspended in lysis buffer (20 mm Tris, pH 7.4, 250 mm sucrose, mm EDTA, and mm b-mercaptoethanol), and passed through a 25-gauge needle at least 10 times Lysates were centrifuged at 12 000 g for 15 min, and the supernatants were stored at )80 °C The protein concentration in cell lysates was determined by the method of Bradford [54], using BSA as standard FEBS Journal 277 (2010) 2128–2141 ª 2010 The Authors Journal compilation ª 2010 FEBS V Cecarini et al Measurements of proteasome activities in cell lysates Proteasome peptidase activities in cell lysates (1 lg in the mixture) were determined with fluorogenic peptides, as previously described The 26S proteasome ChT-L activity was tested using Suc-Leu-Leu-Val-Tyr-AMC as substrate, and a 50 mm Tris ⁄ HCl (pH 8.0) buffer containing 10 mm MgCl2, mm dithiothreitol, and mm ATP In order to evaluate the effective 20S proteasome contribution to the short peptide cleavage, control experiments were performed using specific proteasome inhibitors, Z-Gly-Pro-Phe-Leu-CHO and lactacystin (5 lm in the reaction mixture) Fluorescence values obtained by analyzing the lysates were then subtracted from the values of control assays in the presence of the two inhibitors to find the effective proteasome contribution BrAAP activity was determined in a coupled test in the presence of aminopeptidase N [49] Incubation was performed at 37 °C for 60 The fluorescence of hydrolyzed AMC and pAB was then measured (AMC, kexc = 365 nm, kem = 449 nm; pAB, kexc = 304 nm, kem = 664 nm) on a SpectraMax Gemini XPS microplate reader Western blot analysis Cell lysates were resolved by 12% SDS ⁄ PAGE and electroblotted onto PVDF membranes Membranes with transferred proteins were incubated with the mouse monoclonal antibodies against p53 and Bax, and with the polyclonal rabbit anti-(human 20S proteasome) serum Cell lysates resolved by 10% SDS ⁄ PAGE were electroblotted and then incubated with mouse monoclonal antibody against Ub The immunoblot detection was performed with an ECL western blotting analysis system, using peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) Each gel was loaded with molecular mass markers, including proteins with molecular masses from 6.5 kDa to 205 kDa (SigmaMarker – Wide Molecular Weight Range; Sigma-Aldrich S.r.l.) Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was utilized as a control for equal protein loading: membranes were stripped and reprobed for GAPDH using a monoclonal antibody diluted : 500 (Santa Cruz Biotechnology Inc.) 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proteasomes To test TQ effects on isolated 20S constitutive and immunoproteasome functionality, we incubated purified... standard deviations of five independent determinations) A Thymoquinone inhibits proteasome functionality A set of dose–response experiments was performed to compare the effects of TQ on cell viability