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Cell and Tumor Biology |
Departments of 1 Cancer Biology, 2 Molecular Pathology, 3 Surgical Oncology, and 4 Gastrointestinal Medical Oncology, University of Texas M.D. Anderson Cancer Center, Houston, Texas and 5 Department of Microbiology and Immunology, Sylvester Cancer Center, University of Miami School of Medicine, Miami, Florida
Requests for reprints: David J. McConkey, Department of Cancer Biology, 173, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-8591; Fax: 713-792-8747; E-mail: dmcconke{at}mdanderson.org.
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(eif2), both of which are key events in translational suppression. These effects resulted in an accumulation of ubiquitylated proteins leading to protein aggregation and proteotoxicity. Peptide inhibitor or small interfering RNA targeting ER-resident caspase-4 blocked DNA fragmentation, establishing a central role for caspase-4 in bortezomib-induced cell death. The translation inhibitor cycloheximide abrogated bortezomib-induced protein aggregation, caspase-4 processing, and all other characteristics of apoptosis. Because malignant cells have higher protein synthesis rates than normal cells, they may be more prone to protein aggregation and proteotoxicity and possess increased sensitivity to bortezomib-induced apoptosis. Taken together, the results show that bortezomib induces a unique type of ER stress compared with other ER stress agents characterized by an absence of eif2 phosphorylation, ubiquitylated protein accumulation, and proteotoxicity. (Cancer Res 2005; 65(24): 11510-9) |
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B degradation leading to inactivation of the prosurvival transcription factor, nuclear factor-B (NF-B). However, recent findings indicate that inhibition of NF-B activity accounts for only a small fraction of the anticancer properties of bortezomib (7, 8), indicating that the mechanisms of action of the drug remain to be fully characterized. Proteasome inhibition may prevent the clearance of misfolded proteins by the endoplasmic reticulum (ER)–associated degradation (ERAD) pathway, resulting in ER stress (9, 10). The ER serves several important functions, including post-translational modification, folding and assembly of newly synthesized proteins, and regulated calcium storage (11). These functions are essential to cell survival, such that their perturbation can result in cellular damage, ER stress, and apoptosis (11). ER stress may be induced by agents, such as thapsigargin, which inhibits the sarcoplasmic/endoplasmic Ca2+-ATPase resulting in ER Ca2+ depletion, or tunicamycin, which blocks N-linked protein glycosylation.
The ER has evolved highly specific signaling pathways to ensure that its protein-folding capacity is not overwhelmed (12). These pathways are collectively termed the unfolded protein response (UPR), and they are required for cell survival in the face of ER stress. Four functionally distinct components of the UPR have been identified (10, 13, 14). The first one involves up-regulation of ER chaperone proteins (BiP/GRP78 and GRP94) to increase protein-folding activity and to prevent protein aggregation. The second consists of translational attenuation to reduce the load of new protein synthesis and to prevent further accumulation of misfolded proteins. Inhibition of translation is accomplished by PKR-like ER kinase (PERK), which phosphorylates and inhibits the subunit of eukaryotic initiation factor 2 (eif2) in response to ER stress. The third component is an increase in the degradation of proteins misfolded in the ER by ERAD. The ERAD pathway signals to increase the expression of numerous genes involved in proteasomal-mediated protein degradation. The fourth element of the UPR involves induction of apoptosis, which occurs when cytoprotective functions of the ER are overwhelmed (15).
Pancreatic epithelial cells have a highly developed ER due to a heavy engagement in insulin and digestive enzyme secretion (15), and it has been suggested that they may be particularly sensitive to ER stress-induced apoptosis (16, 17). In previous studies, we showed that bortezomib potently stimulates apoptosis in pancreatic tumor cells (18, 19). Here, we tested the hypothesis that the cytotoxic effects of bortezomib results from ER stress in human pancreatic cancer cells. We showed that bortezomib induces ubiquitin-conjugated protein accumulation by blocking proteasomal degradation while allowing continued protein synthesis through inhibition of PERK activity. The large accumulation of ubiquitylated proteins leads to protein aggregation, which mediates the cytotoxic activity of bortezomib characterized by caspase-4 cleavage. Consistent with these data, inhibition of translation blocks bortezomib-induced protein accumulation and apoptosis. In addition, the high translation rate of malignant cells (compared with low translation rates of normal cells) may make them selectively sensitive to bortezomib-induced proteotoxicity and apoptosis.
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, phosphorylated eif2, phosphorylated c-Jun, c-Jun, c-Jun NH2-terminal kinase (JNK), caspase-12, and phosphorylated PERK (Cell Signaling, Beverly, MA); anti-CHOP/GADD153, ubiquitin, protein phosphatase 1 (PP1), PERK, and GADD34 (Santa Cruz Biotechnology, Santa Cruz, CA); anti-actin (Sigma Chemical, St. Louis, MO); anti-caspase-4 and calreticulin (StressGen, Victoria, British Columbia, Canada); and anti-phosphorylated JNK (Biosource, Camarillo, CA). Alexa Fluor 488 goat anti-mouse, Texas red goat anti-rabbit, and To-Pro-3 were obtained from Molecular Probes (Eugene, OR). Bortezomib was kindly provided by Millennium Pharmaceuticals (Boston, MA). Propidium iodide (PI), thapsigargin, tunicamycin, staurosporine, and cycloheximide were obtained from Sigma. Z-VAD-fmk and Ac-LEVD-CHO were purchased from A.G. Scientific (San Diego, CA). Recombinant human tumor necrosis factor–related apoptosis-inducing ligand (rhTRAIL) was purchased from R&D Systems (Minneapolis, MN).
Reverse transcription-PCR. Total RNA was extracted from L3.6pl pancreatic cancer cells using the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA using the Omniscript RT kit (Qiagen). Primer sequences used for PCR amplification are as follows: GADD153 sense 5'-GCCTTTCTCTTCGGACACTG-3' and antisense 5'-TCACCATTCGGTCAATCAGA-3', GRP78 sense 5'-TGCAGCAGGACATCAAGTTC-3' and antisense 5'-CGCTGGTCAAAGTCTTCTCC-3', and glyceraldehyde-3-phosphate dehydrogenase sense 5'-CGGAGTCAACGGATTTGGCC-3' and antisense 5'-GTGCAGAGATGGCATGGAC-3'. Reactions were carried out in 50 µL volumes composed of 
50 ng cDNA, 0.2 µmol/L each primer (Sigma-Genosys, The Woodlands, TX), 0.2 mmol/L each of deoxynucleotide triphosphates (Roche, Indianapolis, IN), 1x Gold PCR buffer, 1.5 mmol/L MgCl2, 2.5 units AmpliTaq Gold (Applied Biosystems, Foster City, CA), and nuclease-free H2O (Promega, Madison, WI). PCR amplification of all three genes was done in a Robocycler (Stratagene, La Jolla, CA) using the following cycling conditions: 1 cycle of 3 minutes at 94°C, 3 minutes at 54°C, and 3 minutes at 72°C followed by 35 cycles of 1 minute at each temperature. The final cycle was modified to allow for a 7-minute extension at 72°C. PCR products were electrophoresed at 100 V for 45 minutes on a 1.2% agarose gel and imaged using a gel documentation system (Alpha Innotech, San Leandro, CA). Equal quantities of input cDNA were used for all amplifications.
Immunoblotting. Cells (1 x 105) were incubated with 100 nmol/L bortezomib unless otherwise stated, 50 µmol/L cycloheximide, 1 µmol/L thapsigargin, 100 ng/mL rhTRAIL, 5 µg/mL tunicamycin, or drug combinations. For the combination studies, cells were pretreated with cycloheximide for 2 hours before bortezomib treatment. Cells were collected using a cell scraper at 4°C and then lysed as described previously (22). Total cellular protein (25 µg) from each sample was subjected to SDS-PAGE, proteins were transferred to nitrocellulose membranes, and the membranes were blocked with 5% nonfat milk in a TBS solution containing 0.1% Tween 20 for 1 hour. The blots were then probed overnight with relevant antibodies, washed, and probed with species-specific secondary antibodies coupled to horseradish peroxidase. Immunoreactive material was detected by enhanced chemiluminescence (West Pico, Pierce, Inc., Rockville, IL).
Measurement of intracellular Ca2+ levels. Cells were grown in medium with or without 100 nmol/L bortezomib for 12 hours. Cells were collected, washed in PBS, and incubated with 1 µmol/L calcium green-1 (Molecular Probes) for 30 minutes. Flow cytometric analysis of stained cells was done with a Becton Dickinson FACSCalibur (Becton Dickinson, San Jose, CA).
Quantification of DNA fragmentation. DNA fragmentation was measured by PI staining and fluorescence-activated cell sorting (FACS) analysis as described previously (23). Cells were plated in six-well plates (1 x 104 per well). Following incubation with indicated drugs, cells were harvested, pelleted by centrifugation, and resuspended in PBS containing 50 µg/mL PI, 0.1% Triton X-100, and 0.1% sodium citrate. For the combination studies, cells were pretreated with cycloheximide for 2 hours before bortezomib treatment. Cells were incubated with the PI solution for 1 hour, and flow cytometric analysis of stained cells was done with a Becton Dickinson FACSCalibur (Becton Dickinson, Mountain View, CA).
Caspase-3 assay. Cells (1 x 105) were plated in 10-cm dishes with 10% FBS/MEM and allowed to attach for 24 hours. Cells were treated as described for DNA fragmentation analysis above. Following treatment, cells were washed in PBS and resuspended in appropriate buffers as described in the FITC-conjugated monoclonal active caspase-3 antibody apoptosis kit (PharMingen, San Diego, CA). Cells were fixed, permeabilized, and stained with FITC-conjugated caspase-3 antibody as directed by the kit manufacturer. Flow cytometric analysis of stained cells was done with a Becton Dickinson FACSCalibur (Becton Dickinson, Mountain View, CA).
Confocal microscopy. Pancreatic cancer cells were treated on chamber slides. After drug exposure, cells were fixed with 4% paraformaldehyde, permeabilized using 0.2% Triton X-100, and incubated overnight with indicated primary antibodies. Fluorescent secondary antibodies were used to visualize protein localization. To-Pro-3 was used to counterstain the nucleus. Images were obtained using a Zeiss LSM510 confocal microscope (Zeiss, Oberkochen, Germany).
Preparation and transfection of small interfering RNAs. The annealed double-stranded caspase-4 small interfering RNAs (siRNA) were obtained from Dharmacon (Lafayette, CO): caspase-4 siRNA sense 5'-GGACUAUAGUGUAGAUGUAUU-3' and antisense 5'-UACAUCUACACUAUAGUCCUU-3'. For control, siRNA directed against firefly luciferase was used. Cells were transfected with 100 nmol/L of the above siRNA using Oligofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. Transfected cells were incubated at 37°C for 40 hours without changing the medium. Efficiency of RNA interference (RNAi) was measured by immunoblotting using an antibody to anti-caspase-4.
Transmission electron microscopy. Transmission electron microscopy of cells was done as described previously (24).
Metabolic labeling. To measure global translation, equal number of cells were plated in six-well plates, allowed to attach, washed with PBS, and placed in [35S]cysteine- and [35S]methionine-free medium containing 5% dialyzed FBS. Cells were pretreated with bortezomib, thapsigargin, or cycloheximide for the indicated times. The cells were then labeled with [35S]methionine and [35S]cysteine (Translabel, ICN, Irvine, CA) for the last 30 minutes of treatment. Cells were then harvested and lysed and protein was separated by SDS-PAGE followed by autoradiography to visualize radiolabeled proteins. In parallel, each lysate was subjected to trichloroacetic acid precipitation and scintillation counting was done to measure [35S]methionine and [35S]cysteine incorporation into proteins.
Immunoprecipitation. Cells (1 x 105) were incubated on 100 x 20–mm plates with 100 nmol/L bortezomib for 24 hours. Cells were lysed on the plates at 4°C with a 1% Triton X-100 lysis buffer containing 150 mmol/L NaCl, 25 mmol/L Tris (pH 7.5), 1 mmol/L NaF, 1 mmol/L sodium orthovanadate, 10 mmol/L ß-glycerophosphate, and a Complete Mini protease inhibitor tablet. After centrifugation, the clarified supernatant was incubated with anti-GADD34 antibody for 1 hour followed by incubation with protein A/G-Sepharose beads for 12 hours at 4°C. The immunocomplex was washed thrice with lysis buffer followed by addition of SDS sample buffer. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose membranes. The blots were incubated with the indicated primary antibody followed by secondary antibody coupled to horseradish peroxidase and the signal was revealed by chemiluminescence.
Preparation of cytosol for cytochrome c measurement. Cells were treated with cycloheximide 2 hours before bortezomib treatment for 24 hours. Cells were harvested and washed twice in PBS, resuspended in lysis buffer [20 mmol/L HEPES (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, and a protease inhibitor tablet], and incubated on ice for 15 minutes. Cells were centrifuged at 10,000 x g for 15 minutes at 4°C. Supernatants were collected and subjected to immunoblotting.
Statistical analyses. Statistical significance of differences observed in drug-treated and control samples were determined using the Tukey-Kramer comparison test. Differences were considered significant in all experiments at P < 0.05 (significantly different from untreated controls).
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(Fig. 3A), a key event of the UPR that mediates inhibition of translation (28). Therefore, we investigated whether bortezomib had any effect on protein synthesis. Bortezomib failed to suppress translation in cells pulsed with [35S]cysteine and [35S]methionine, whereas a more conventional inducer of ER stress (thapsigargin) dramatically reduced [35S]cysteine and [35S]methionine incorporation into proteins (Fig. 3B). The ability of bortezomib to inhibit proteasomal degradation while allowing steady protein synthesis resulted in an accumulation of ubiquitin-conjugated proteins. Analysis of bortezomib-treated cells by immunofluorescent anti-ubiquitin staining and confocal microscopy revealed the appearance of perinuclear ubiquitin-containing aggregate structures characterized previously as aggresomes (ref. 29; Fig. 3C). The protein aggregates were organized as electron dense particles in a central core when they were visualized by transmission electron microscopy (Fig. 3C).
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phosphorylation. Bortezomib decreased the low basal levels of phosphorylated eif2 observed in pancreatic cancer cells and induced the expression of GADD34, the regulatory subunit of an eif2-specific holophosphatase complex (refs. 30–32; Fig. 3D). Coimmunoprecipitation analyses showed that GADD34 formed a complex with PP1 in bortezomib-treated cells (Fig. 3D). However, bortezomib also blocked thapsigargin-induced PERK and eif2 phosphorylation at a time point (1 hour) that preceded GADD34 accumulation (Fig. 3D). Consistent with these results, knockdown of GADD34 by RNAi did not attenuate translation or bortezomib-induced apoptosis (data not shown). Therefore, the ability of bortezomib to inhibit PERK activation itself seems to be critically involved.
Cycloheximide inhibits bortezomib-induced protein accumulation and apoptosis. To examine whether loss of translational control was essential for bortezomib-induced cell death, we examined the effects of the translation inhibitor, cycloheximide, on aggresome formation, ER stress, JNK activation, and apoptosis in L3.6pl cells. Cycloheximide blocked [35S]cysteine and [35S]methionine incorporation into proteins in cells exposed to bortezomib (Fig. 4A) and consequently prevented the accumulation of ubiquitylated proteins (Fig. 4B) and aggresome formation (Fig. 4C). Cycloheximide also restored eif2 phosphorylation and blocked the bortezomib-induced increase in GADD34 expression (Fig. 4D). It also inhibited bortezomib-induced JNK phosphorylation and subsequent c-Jun phosphorylation (Fig. 5A). Due to the absence of protein aggregate accumulation, bortezomib-induced caspase-4 processing (Fig. 5B), cytochrome c release (Fig. 5C), caspase-3 activation (Fig. 5D), and DNA fragmentation (Fig. 5D) were inhibited by cycloheximide. These results show that the failure of bortezomib to induce eif2 phosphorylation and inhibit translation play critical roles in its cytotoxic activity in pancreatic cancer cells.
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In a first series of experiments, we characterized the effects of bortezomib on markers of ER stress in the L3.6pl pancreatic cancer cell line. Bortezomib induced BiP and CHOP expression and increased intracellular calcium levels, JNK activation, and caspase-12 processing, all of which are indicative of ER stress (15). A recent study has implicated the involvement of caspase-4 during ER stress-induced apoptosis (39). Therefore, we investigated whether caspase-4 was processed during bortezomib-induced apoptosis. Our data indicate that caspase-4 resides in the ER and is cleaved following bortezomib treatment and that its inhibition abrogates bortezomib-induced apoptosis. Consistent with a role of caspase-4 in bortezomib-induced apoptosis, its processing was only observed in cell lines that were sensitive to bortezomib-induced apoptosis. In addition, peptide- or siRNA-mediated inhibition of caspase-4 blocked bortezomib-induced apoptosis. Taken together, the data show that induction of ER stress-induced apoptosis by caspase-4 mediates bortezomib-induced apoptosis.
Interestingly, bortezomib treatment did not induce phosphorylation of eif2, a key cytoprotective component of the UPR that is required for translational attenuation (40). This prompted us to more directly examine the effects of bortezomib on translation. Consistent with their effects on eif2 phosphorylation, thapsigargin attenuated protein synthesis, whereas bortezomib did not. A previous study reported that thapsigargin-treated NIH 3T3 cells resume translation after several hours (41). Interestingly, we did not observe translational recovery in L3.6pl cells that were exposed to thapsigargin continuously, but protein synthesis was restored after several hours when cells were washed and incubated in thapsigargin-free medium. Our findings suggest that bortezomib is uniquely effective in inducing ER stress by virtue of the fact that it does not inhibit protein synthesis.
Several reports have identified a negative feedback loop that relieves translational inhibition during ER stress via GADD34-mediated PP1 activation (30–32). Our results showed a relationship between GADD34 induction and a decrease in eif2 phosphorylation in cells treated with bortezomib and we found that GADD34 directly interacted with PP1. However, we did not observe an initial decrease in translation followed by protein synthesis recovery, suggesting that bortezomib prevents translational attenuation before GADD34 induction. We showed that bortezomib inhibits eif2 phosphorylation before GADD34 induction by blocking PERK autophosphorylation. Regardless of the mechanism, the ability of bortezomib to inhibit eif2 phosphorylation allows protein synthesis in the face of proteasomal inhibition causing ubiquitin-conjugated protein accumulation and aggregation. This result is in contrast to two recent studies showing that proteasome inhibitors lead to autophosphorylation of PERK in a head and neck squamous cell carcinoma model (42) and eif2 phosphorylation in mouse embryonic fibroblasts (43). Although we agree with the authors in that bortezomib induces ER stress, we argue that eif2 remains dephosphorylated due to PERK inhibition and/or GADD34 stabilization. Our results are consistent with a study in multiple myeloma showing that proteasome inhibitors disrupt the UPR (44). It is possible that the discrepancies between the studies lie in differences between the model systems.
Studies using PERK–/– cells have indicated that it plays an important role in cytoprotection, as these cells were more sensitive to ER stress due to the inability of the cells to regulate protein synthesis (28). Because PERK activation and translational suppression are critical for cell survival in the presence of ER stress, it is likely that bortezomib can synergize with other agents that induce ER stress. Consistent with this idea, the ER stress inducers thapsigargin and tunicamycin enhanced bortezomib-induced apoptosis in our model.6 Therefore, we investigated whether inhibition of translation would relieve the proteotoxicity and subsequent ER stress induced by bortezomib. The translation inhibitor cycloheximide prevented bortezomib-induced ubiquitylated protein accumulation, aggresome formation, dephosphorylation of eif2, and corresponding GADD34 induction. Furthermore, cycloheximide also blocked events associated with bortezomib-induced apoptosis, including JNK activation, cytochrome c release, caspase-4 and caspase-3 activation, and DNA fragmentation. In fact, cycloheximide similarly protected PERK–/– cells under ER stress conditions, indicating the importance of translational regulation to cell survival (28).
We have established that continued translation in the presence of proteasome inhibition leads to the formation of ubiquitin-conjugated protein aggregates and induction of ER stress in pancreatic cancer cells. The majority of pancreatic cancer cells package these protein aggregates into structures termed aggresomes. We determined that the majority of cells possessing aggresomes were void of ER stress and resistant to bortezomib-induced apoptosis.6 These observations are consistent with a recent report showing that aggresomes may be cytoprotective storage areas for large accumulations of undegraded proteins (45). Furthermore, disruption of aggresome formation by inhibiting histone deacetylase 6 greatly enhances bortezomib-induced ER stress and apoptosis (46).6 Another resistance mechanism to bortezomib-induced apoptosis is up-regulation of chaperone proteins, such as heat shock proteins 27 and 70, which both prevent protein aggregation (47, 48). Taken together, the ability of bortezomib to induce protein aggregation seems to be critical for the stimulation of ER stress and contributes to the cytotoxic activity of the drug.
Several reports have found that bortezomib has tumor-selective activity (49–51). However, the molecular explanation advanced to explain this phenomenon is that cycling cells might be generally more sensitive to proteasome inhibition than resting cells, perhaps because so many crucial cell cycle checkpoints are regulated by the proteasome. Our results provide an alternative explanation. We show here that pancreatic cancer cells display a higher baseline rate of translation compared with immortalized pancreatic epithelial cells, which should make them more vulnerable to the protein buildup and subsequent ER stress caused by proteasome inhibition. In parallel studies, we showed that normal murine pancreatic epithelial cells were also more resistant to bortezomib-induced aggresome formation and cell death than were adjacent cancer cells within orthotopic pancreatic tumors exposed to bortezomib in vivo, and we have obtained similar results in comparisons of the effects of bortezomib in EBV-transformed normal B lymphocytes and multiple myeloma cells.7 Therefore, the unique effects of bortezomib on the UPR may explain its selectivity for malignant cells, because elevated translation is probably a general feature of cancer. Clinical trials using bortezomib for the treatment of advanced solid tumors are currently under way. We have submitted a letter of intent to the Cancer Therapeutics Evaluation Program of the National Cancer Institute (NCI) to investigate bortezomib in combination with suberoylanilide hydroxamic acid.
In summary, our results show that bortezomib activates a novel pathway of ER stress-induced apoptosis characterized by unregulated translation leading to the accumulation of ubiquitin-conjugated, aggregated proteins (Fig. 6C). By targeting the ER, bortezomib may be an effective therapy for pancreatic cancer and other cancers derived from secretory cells, which may be hypersensitive to protein aggregation and subsequent ER stress-mediated apoptosis.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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7 S. Nawrocki, manuscript submitted.
Received 7/11/05. Revised 8/26/05. Accepted 9/12/05.
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B as a therapeutic target in multiple myeloma. J Biol Chem 2002;277:16639–47.B mutations or activation of the CD30, CD40, and RANK receptors. Clin Cancer Res 2004;10:3207–15. kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol Cell Biol 2002;22:3864–74.. J Cell Biol 2001;153:1011–22. to the endoplasmic reticulum and promotes dephosphorylation of the subunit of eukaryotic translation initiation factor 2. Mol Cell Biol 2003;23:1292–303.-subunit of the eukaryotic initiation factor-2 (eIF2) reduces protein synthesis and enhances apoptosis in response to proteasome inhibition. J Biol Chem 2005;280:14189–202.-synuclein and synphilin-1 are cytoprotective. J Biol Chem 2004;279:4625–31.This article has been cited by other articles:
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P. Pyrko, A. Kardosh, W. Wang, W. Xiong, A. H. Schonthal, and T. C. Chen HIV-1 Protease Inhibitors Nelfinavir and Atazanavir Induce Malignant Glioma Death by Triggering Endoplasmic Reticulum Stress Cancer Res., November 15, 2007; 67(22): 10920 - 10928. [Abstract] [Full Text] [PDF]
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 M. Rahmani, E. M. Davis, T. R. Crabtree, J. R. Habibi, T. K. Nguyen, P. Dent, and S. Grant The Kinase Inhibitor Sorafenib Induces Cell Death through a Process Involving Induction of Endoplasmic Reticulum Stress Mol. Cell. Biol., August 1, 2007; 27(15): 5499 - 5513. [Abstract] [Full Text] [PDF]
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 W.-X. Ding, H.-M. Ni, W. Gao, T. Yoshimori, D. B. Stolz, D. Ron, and X.-M. Yin Linking of Autophagy to Ubiquitin-Proteasome System Is Important for the Regulation of Endoplasmic Reticulum Stress and Cell Viability Am. J. Pathol., August 1, 2007; 171(2): 513 - 524. [Abstract] [Full Text] [PDF]
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 H.-Q. Wang, Z.-X. Du, H.-Y. Zhang, and D.-X. Gao Different Induction of GRP78 and CHOP as a Predictor of Sensitivity to Proteasome Inhibitors in Thyroid Cancer Cells Endocrinology, July 1, 2007; 148(7): 3258 - 3270. [Abstract] [Full Text] [PDF]
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C. P. Miller, K. Ban, M. E. Dujka, D. J. McConkey, M. Munsell, M. Palladino, and J. Chandra NPI-0052, a novel proteasome inhibitor, induces caspase-8 and ROS-dependent apoptosis alone and in combination with HDAC inhibitors in leukemia cells Blood, July 1, 2007; 110(1): 267 - 277. [Abstract] [Full Text] [PDF]
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 N. A. Kaniuk, M. Kiraly, H. Bates, M. Vranic, A. Volchuk, and J. H. Brumell Ubiquitinated-Protein Aggregates Form in Pancreatic {beta}-Cells During Diabetes-Induced Oxidative Stress and Are Regulated by Autophagy Diabetes, April 1, 2007; 56(4): 930 - 939. [Abstract] [Full Text] [PDF]
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W.-X. Ding, H.-M. Ni, X. Chen, J. Yu, L. Zhang, and X.-M. Yin A coordinated action of Bax, PUMA, and p53 promotes MG132-induced mitochondria activation and apoptosis in colon cancer cells Mol. Cancer Ther., March 1, 2007; 6(3): 1062 - 1069. [Abstract] [Full Text] [PDF]
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S. Meister, U. Schubert, K. Neubert, K. Herrmann, R. Burger, M. Gramatzki, S. Hahn, S. Schreiber, S. Wilhelm, M. Herrmann, et al. Extensive Immunoglobulin Production Sensitizes Myeloma Cells for Proteasome Inhibition Cancer Res., February 15, 2007; 67(4): 1783 - 1792. [Abstract] [Full Text] [PDF]
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 C. Wojcik, M. Rowicka, A. Kudlicki, D. Nowis, E. McConnell, M. Kujawa, and G. N. DeMartino Valosin-containing Protein (p97) Is a Regulator of Endoplasmic Reticulum Stress and of the Degradation of N-End Rule and Ubiquitin-Fusion Degradation Pathway Substrates in Mammalian Cells Mol. Biol. Cell, November 1, 2006; 17(11): 4606 - 4618. [Abstract] [Full Text] [PDF]
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 Y. Ishii, A. Pirkmaier, J. V. Alvarez, D. A. Frank, I. Keselman, D. Logothetis, J. Mandeli, M. J. O'Connell, S. Waxman, and D. Germain Cyclin d1 overexpression and response to bortezomib treatment in a breast cancer model. J Natl Cancer Inst, September 6, 2006; 98(17): 1238 - 1247. [Abstract] [Full Text] [PDF]
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C. Yu, B. B. Friday, J.-P. Lai, L. Yang, J. Sarkaria, N. E. Kay, C. A. Carter, L. R. Roberts, S. H. Kaufmann, and A. A. Adjei Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways. Mol. Cancer Ther., September 1, 2006; 5(9): 2378 - 2387. [Abstract] [Full Text] [PDF]
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X. Dolcet, D. Llobet, M. Encinas, J. Pallares, A. Cabero, J. A. Schoenenberger, J. X. Comella, and X. Matias-Guiu Proteasome Inhibitors Induce Death but Activate NF-{kappa}B on Endometrial Carcinoma Cell Lines and Primary Culture Explants J. Biol. Chem., August 4, 2006; 281(31): 22118 - 22130. [Abstract] [Full Text] [PDF]
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S. E. Canfield, K. Zhu, S. A. Williams, and D. J. McConkey Bortezomib inhibits docetaxel-induced apoptosis via a p21-dependent mechanism in human prostate cancer cells. Mol. Cancer Ther., August 1, 2006; 5(8): 2043 - 2050. [Abstract] [Full Text] [PDF]
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A. Yacoub, M. A. Park, D. Hanna, Y. Hong, C. Mitchell, A. P. Pandya, H. Harada, G. Powis, C.-S. Chen, C. Koumenis, et al. OSU-03012 Promotes Caspase-Independent but PERK-, Cathepsin B-, BID-, and AIF-Dependent Killing of Transformed Cells Mol. Pharmacol., August 1, 2006; 70(2): 589 - 603. [Abstract] [Full Text] [PDF]
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S. Ruiz, Y. Krupnik, M. Keating, J. Chandra, M. Palladino, and D. McConkey The proteasome inhibitor NPI-0052 is a more effective inducer of apoptosis than bortezomib in lymphocytes from patients with chronic lymphocytic leukemia. Mol. Cancer Ther., July 1, 2006; 5(7): 1836 - 1843. [Abstract] [Full Text] [PDF]
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S. T. Nawrocki, J. S. Carew, M. S. Pino, R. A. Highshaw, R. H.I. Andtbacka, K. Dunner Jr., A. Pal, W. G. Bornmann, P. J. Chiao, P. Huang, et al. Aggresome disruption: a novel strategy to enhance bortezomib-induced apoptosis in pancreatic cancer cells. Cancer Res., April 1, 2006; 66(7): 3773 - 3781. [Abstract] [Full Text] [PDF]
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