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Mol Cancer Ther. 2004;3:727-736
© 2004 American Association for Cancer Research

Maximal apoptosis of renal cell carcinoma by the proteasome inhibitor bortezomib is nuclear factor-{kappa}B dependent

Jiabin An1, Yiping Sun1, Myrna Fisher1 and Matthew B. Rettig1,2

1 VA Greater Los Angeles Healthcare System and 2 School of Medicine, University of California at Los Angeles, Los Angeles, California

Requests for reprints: Matthew B. Rettig, VA Greater Los Angeles Healthcare System, 11301 Wilshire Boulevard, Building 304, Room E1-113, Los Angeles, CA 90073. Phone: 310-478-3711, ext. 44761; Fax: 310-268-4508. E-mail: matthew.rettig{at}med.va.gov


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Advanced renal cell carcinoma (RCC) is resistant to cytotoxic chemotherapy, and immunotherapy has modest activity. Proteasome inhibitors represent a novel class of anticancer agents that have activity across a wide spectrum of tumor types. We investigated the efficacy of the proteasome inhibitor bortezomib (VELCADE, formerly known as PS-341) in RCC and found that bortezomib potently induces apoptosis of RCC cell lines. Blockade of the nuclear factor-{kappa}B (NF-{kappa}B) pathway is considered a crucial effect in bortezomib-induced apoptosis, but the dependence on NF-{kappa}B inhibition for bortezomib-mediated death has not been formally demonstrated. Thus, we also studied the contribution of NF-{kappa}B inhibition as a mechanism of bortezomib-induced apoptosis in RCC cells, which display constitutive NF-{kappa}B activation. Ectopic expression of the NF-{kappa}B family members, p65 (Rel A) and p50 (NF-{kappa}B1), markedly reduced bortezomib-induced apoptosis. However, when we used selective genetic and chemical inhibitors of NF-{kappa}B, we found that NF-{kappa}B blockade was not sufficient to induce apoptosis of RCC cells. Thus, we conclude that maximal bortezomib-induced apoptosis is dependent on its NF-{kappa}B inhibitory effect, but NF-{kappa}B-independent effects also play a critical role in the induction of apoptosis by bortezomib. This represents the first report to formally demonstrate that bortezomib-induced NF-{kappa}B blockade is required to achieve the maximum degree of apoptosis by this drug.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Renal cell carcinoma (RCC) persists as a major cause of morbidity and mortality in the United States. An estimated 31,900 new cases of and 11,900 deaths from RCC are anticipated for the year 2003 (1). Metastatic disease is present in ~30% of patients at the time of diagnosis and develops in one third of the remainder of patients who have clinically localized disease at presentation. Metastatic RCC is quite resistant to chemotherapy, in large part due to high expression of the multidrug resistance gene (2-4). In addition, immunotherapeutic agents, including interleukin-2 and IFN-{alpha}, are only modestly effective, with response rates in the 10% to 20% range (2-4). Median survival of metastatic RCC remains at only 8 months. Clearly, novel and more effective treatments are required to alter the natural history of advanced RCC.

The 26S proteasome has received much attention as a potential therapeutic target. The proteasome is composed of a large complex of proteins that functions as the principal executioner of cytosolic protein degradation. Proteins degraded by the proteasome are targeted by the process of ubiquitination. The normal degradation of proteins is crucial for maintenance of cellular homeostasis. Thus, the proteasome plays a critical role in modulating intracellular levels of a wide variety of proteins such as tumor suppressor genes (e.g., p53), oncogenes, including c-jun and c-myc, and proteins that are involved in cell cycle regulation, including cyclins and cyclin-dependent kinase inhibitors (5-9).

Proteasome inhibitors have been actively studied for their antitumor effects. Bortezomib is a potent, specific, and reversible boronic acid, dipeptide proteasome inhibitor that has been shown to induce cytotoxicity in many tumor models, including prostate, colon, and pancreatic cancer as well as multiple myeloma (10-13). For reasons that remain to be fully elucidated, neoplastic cells are more sensitive to the cytotoxic effects of proteasome inhibition than their normal cellular counterparts (14). In addition, the activity of proteasome inhibitors does not seem to be influenced by the low growth fractions of tumors, which is in contradistinction to cytotoxic chemotherapy that is more often cell cycle dependent (15, 16). Proteasome inhibitors appear to induce cytotoxicity independent of the expression of the antiapoptotic protein Bcl-2, which is frequently overexpressed in RCC (17, 18), and can overcome the multidrug resistance phenotype, which is also often manifested in RCC (12).

The proteasome also regulates the activity of the nuclear factor-{kappa}B (NF-{kappa}B) pathway, which is thought to represent an essential target of proteasome inhibitors (10, 11, 13, 14, 19). I{kappa}B, the NF-{kappa}B inhibitory protein that binds to and sequesters NF-{kappa}B family members in the cytoplasm, is degraded by the ubiquitin-proteasome pathway (20). When the NF-{kappa}B pathway is activated, I{kappa}B is phosphorylated by I{kappa}B kinase, which phosphorylates I{kappa}B at Ser32 and Ser36 (21). Phosphorylated I{kappa}B is subjected to ubiquitination and proteasome-mediated degradation, which results in the translocation of NF-{kappa}B to the nucleus, where it activates transcription of genes involved in a variety of cellular functions, such as regulation of apoptosis, proliferation, and angiogenesis. NF-{kappa}B activation results in inhibition of apoptosis in most cell systems via induced expression of antiapoptotic proteins, such as A1/Bfl-1 and Bcl-XL (21, 22). NF-{kappa}B activation has also been associated with proliferative responses mediated by induction of expression of cyclin D1, which drives the transition from the G1 to the S phase of the cell cycle (23). NF-{kappa}B also up-regulates expression of proangiogenic factors such as interleukin-8 and adhesion molecules and metalloproteinases that are involved in metastasis development (21, 24). Consequently, the NF-{kappa}B pathway is considered a critical target for bortezomib.

Constitutive activation of NF-{kappa}B has been observed in many solid tumors and hematologic malignancies (25-28). Substantial constitutive NF-{kappa}B activation has been observed in several RCC cell lines (29, 30). Importantly, the frequency of constitutive NF-{kappa}B activation is significantly greater in locally advanced and metastatic compared with localized cases of RCC (31), suggesting that activation of NF-{kappa}B is involved in the progression of RCC. In addition, the degree of constitutive NF-{kappa}B activation in RCC cells may determine sensitivity to apoptotic stimuli, such as tumor necrosis factor (TNF)-related apoptosis-inducing ligand and chemotherapy (29, 30). In the current study, we sought to determine the efficacy of bortezomib as a proapoptotic agent in RCC cell lines. Although inhibition of constitutive NF-{kappa}B activation has been purported to represent a crucial mechanism of bortezomib-induced apoptosis (10, 11, 13, 14, 19), no direct evidence for the requirement for NF-{kappa}B blockade in bortezomib-mediated death has been presented. Thus, we also studied the contribution of NF-{kappa}B inhibition as a mechanism of bortezomib-induced apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and RCC Cell Lines
R11 and 444 RCC cell lines (Dr. A. Belldegrun, University of California at Los Angeles, Los Angeles, CA) were maintained in RPMI 1640 supplemented with 10% fetal bovine serum and penicillin (100 µg/mL) and streptomycin (100 µg/mL) in a humidified 5% CO2 atmosphere at 37°C. All culture media were purchased from Omega Scientific (Thousand Oaks, CA).

Reagents and Vectors
The proteasome inhibitor bortezomib and PS-1145, a specific inhibitor of I{kappa}B kinase, were provided by Millennium, Inc. (Cambridge, MA) and dissolved in DMSO. For all experiments, the final DMSO concentration was maintained at 0.1%. TNF-{alpha} was obtained from BD Biosciences (Bedford, MA) and dissolved in PBS.

The firefly luciferase-based reporter constructs, p{kappa}B-luc and pCRE-luc reporter plasmids, which contain tandem copies of the {kappa}B and cyclic AMP response elements, respectively, were from Clontech Laboratories, Inc. (Palo Alto, CA). The pRL-SV40 plasmid, in which Renilla luciferase is constitutively expressed under the regulation of the SV40 promoter/enhancer, was purchased from Promega (Madison, WI) and was used for normalization of firefly luciferase activity.

The pEGFP-p65 plasmid, which expresses wild-type Rel A (p65) as a fusion protein with the enhanced green fluorescent protein (EGFP) tag at the NH2 terminus of p65, was a gift of Drs. David Baltimore and Jeff Wiezorek (California Institute of Technology, Pasadena, CA). The pCMV4-p50 plasmid, which expresses wild-type p50 (NF-{kappa}B1) under the regulation of the cytomegalovirus promoter, was a kind gift of Dr. Warner Greene (Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA).

An adenoviral vector that contains the I{kappa}B superrepressor (Ad-I{kappa}B-SR) and the control vector (Ad-CMV) were amplified, purified, and titered in 293 cells and were provided by Dr. Raj Batra (University of California at Los Angeles). The I{kappa}B-SR contains mutations at the phosphorylation sites (Ser32 to Ala and Ser36 to Ala), which prevent its phosphorylation, dissociation from NF-{kappa}B, and subsequent degradation by the ubiquitin-proteasome pathway and thereby blocks NF-{kappa}B activity (32). Transgene expression in the Ad-I{kappa}B-SR vector is driven by the cytomegalovirus early/intermediate promoter/enhancer. The control vector, Ad-CMV, is identical to Ad-I{kappa}B-SR but lacks a transgene insert. To estimate transduction efficiency, pilot experiments were performed with the same adenoviral vector containing the EGFP transgene. As estimated by UV light microscopy, these experiments demonstrated that >90% of both 444 and R11 cells expressed EGFP at multiplicities of infection (MOI) ≥ 5.

Western Blots
Protein was resolved by SDS-PAGE and transferred to a nitrocellulose membrane with the Trans-Blot System (Bio-Rad, Hercules, CA). The membrane was blocked with TBS with 1% bovine serum albumin and 1% nonfat powdered milk. Membranes were immunoblotted with relevant primary antibodies. The I{kappa}B-{alpha} antibody (Cell Signaling Technology, Beverly, MA) and the actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) were both used at a 1:500 dilution. Horseradish peroxidase–conjugated secondary antibodies (1:2,000 dilution, Santa Cruz Biotechnology) were used for detection of bands by enhanced chemiluminescence Western blotting detection reagents (Amersham Biosciences, Piscataway, NJ).

Electrophoretic Mobility Shift Assay
To extract nuclear protein, cells were washed with cold PBS, and Buffer A [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, 1 mmol/L DTT, 200 µmol/L phenylmethylsulfonyl fluoride, 1 µmol/L leupeptin, 1 µmol/L aprotinin, and 100 µmol/L EDTA] was added prior to pulverization with a tissue grinder. Subsequently, nuclei were pelleted, lysed with Buffer C [20 mmol/L HEPES (pH 7.9), 0.42 mol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mol/L EDTA, 25% glycerol, 1 mmol/L DTT, and 200 µmol/L phenylmethylsulfonyl fluoride], and passed several times through a 25-gauge needle. Debris was removed by centrifugation.

Wild-type and mutant {kappa}B and activator protein-1 oligonucleotide probes were purchased from Santa Cruz Biotechnology. Nuclear protein (15 µg) was combined with end-labeled, double-stranded oligonucleotide probe, 1 µg poly (deoxyinosinic-deoxycytidylic acid) (Amersham Pharmacia Biotech, Piscataway, NJ), 1 µg bovine serum albumin, and 5 mmol/L spermidine in a final reaction volume of 20 µL for 20 minutes at room temperature. The DNA protein complex was run on a 4% nondenaturing polyacrylamide gel with 0.4x Tris-borate EDTA running buffer prior to subsequent autoradiography. Cold competition experiments were performed with a 100-fold molar excess of double-stranded, cold wild-type or cold mutant {kappa}B oligonucleotide probes.

Transient Transfections and Reporter Gene Assays
To assess the effects of bortezomib on {kappa}B-regulated reporter gene expression, cells were plated at 105 cells per well in 24-well plates the day prior to transfection. Plasmids (p{kappa}B-luc and pCRE-luc) were transfected with Lipofectamine Plus (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The pRL-SV40 plasmid was cotransfected to normalize for transfection efficiency. Three hours after transfection, fresh complete medium was added. Protein was extracted 48 hours after transfection, and firefly and Renilla luciferase were measured on a TD20/20 tube luminometer (Turner Designs, Sunnyvale, CA) using the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions.

Cell Growth Assay
444 and R11 cells were seeded in 96-well plates at 2 x 104 and 1 x 104 cells per well, respectively, in 100 µL of culture medium. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MTT (25 µL, 5 mg/mL) was added to each well for 3 hours at 37°C. Subsequently, 10% SDS-0.01 N HCl (100 µL) was added overnight at 37°C. Absorbance was measured at 570 nm on a microplate reader. All experiments were performed in quadruplicate.

Assessment of Apoptosis and Cell Cycle Analysis
After treatment, cells were harvested and apoptosis was assessed by the terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) method using the FragEL DNA Fragmentation Kit (Calbiochem, La Jolla, CA) according to the manufacturer's instructions. Cells were analyzed on a fluorescence-activated cell sorting caliber flow cytometer (Becton Dickinson, Mountain View, CA) with CellQuest software (BD Biosciences). Alternatively, apoptosis was also measured by Annexin V-FITC staining (ApoAlert Annexin V-FITC Apoptosis Kit, Clontech) and flow cytometry.

For experiments to determine the effects of ectopic expression of p65/p50 on bortezomib-induced apoptosis, cells were transiently transfected with pEGFP-p65 and pCMV4-p50 or the parental control vectors. Twenty-four hours after transfection, bortezomib was added for an additional 24 hours. Apoptosis was measured on EGFP-positive cells using a TUNEL assay kit (In situ Cell Death Detection Kit, tetramethylrhodamine red, Roche Molecular Biomedicals, Indianapolis, IN) by flow cytometry.

Cell cycle analysis was performed by hypotonic propidium iodide (PI) staining. The PI staining buffer was freshly prepared in 250 mL of distilled water containing PI (0.025 g), sodium citrate (0.25 g), Triton X-100 (0.75 mL), and RNase A (5 mg). PI staining buffer (1 mL) was added to 1 x 106 cells and placed on ice protected from light. Cells were analyzed on a fluorescence-activated cell sorting caliber flow cytometer with CellQuest software, and apoptosis was determined as the sub-G1 peak.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bortezomib Induces Apoptosis of RCC Cells in a Dose-Dependent Fashion
We first investigated the ability of bortezomib to induce apoptosis in RCC cell lines. We used R11 and 444 cells, which both display high constitutive NF-{kappa}B activity (see below and Figs. 2B and 4A). As measured by the TUNEL assay, bortezomib induced apoptosis in a dose-dependent fashion in R11 (Fig. 1A) and 444 (Fig. 3E) cells. Apoptosis was minimal at 0.1 µmol/L, highly significant at 1 µmol/L, and massive at 10 µmol/L. As a complementary assay, Annexin V-FITC staining of RCC cells also demonstrated a potent proapoptotic effect induced by bortezomib (Fig. 1B), as did cell cycle analysis with assessment of the sub-G1 peak (see below and Fig. 5D). We also tested the time dependence of bortezomib-mediated apoptosis. Apoptosis was observed at as early as 24 hours and reached a maximum at 48 hours, with little additional apoptosis at 72 hours (data not shown).



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  		Figure 2. Bortezomib inhibits NF-{kappa}B activation in a dose-dependent fashion. A, bortezomib inhibits the degradation of I{kappa}B-{alpha}. R11 cells were exposed to bortezomib (10 µmol/L) for 30 minutes prior to treatment with TNF-{alpha} (20 ng/mL) for the indicated times. Total protein was harvested for Western blotting with I{kappa}B-{alpha} or actin (for loading control) antibodies. B, bortezomib inhibits basal and TNF-{alpha}-stimulated DNA binding of NF-{kappa}B family members in R11 cells by EMSA. Top panel, cells were exposed to bortezomib for 30 minutes and with or without TNF-{alpha} (20 ng/mL) for an additional 20 minutes. Nuclear protein was extracted for EMSA with a hot {kappa}B probe. Cold competition experiments (rightmost two lanes) with excess cold wild-type (Wt) and mutant (Mut) {kappa}B oligonucleotide probes demonstrate the specificity of the bands obtained on EMSA. Bottom panels, NF-{kappa}B and activator protein-1 (AP1) EMSAs, respectively; R11 cells were treated with bortezomib for 24 hours prior to extraction of nuclear protein. C, bortezomib inhibits transcriptional activity of NF-{kappa}B. R11 and 444 cells were transiently transfected with the p{kappa}B-luc (1 µg) and pRL-SV40 (1 ng) plasmids (see Materials and Methods), and a dual luciferase assay was performed at 48 hours. Columns, means of three experiments expressed as relative luminescence units (RLU); bars, SD. Note that relative luminescence units are plotted on a logarithmic scale.

 


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  		Figure 4. Genetic inhibition of NF-{kappa}B with an I{kappa}B-SR is not sufficient to induce apoptosis of RCC cells. A, Ad-I{kappa}B-SR completely inhibits NF-{kappa}B. R11 and 444 cells were transduced with various MOIs of the Ad-I{kappa}B-SR or the control virus (Ad-CMV) for 48 hours. Nuclear protein was extracted for EMSA. B, TUNEL assay of adenovirally transduced R11 cells. Forty-eight hours after adenovirus transduction, cells were analyzed for apoptosis by the TUNEL assay on a flow cytometer. Horizontal axis, apoptosis. C, cell cycle analysis on bortezomib-treated R11 cells. Hypotonic PI staining followed by flow cytometric analysis was performed as described in Materials and Methods. Experiments in A–C were performed in a 10-cm dish format. Results of experiments in B and C show a MOI of 5, because this was the lowest MOI tested that completely abrogated basal NF-{kappa}B activity, as shown in A. Similar results as shown in A–C were obtained for 444 cells. As a positive control for apoptosis for both TUNEL assay and assessment of the sub-G1 peak, cells were treated with bortezomib for 48 hours (see Fig. 5C and D, respectively).

 


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  		Figure 1. Bortezomib induces apoptosis of RCC cells. A, TUNEL assay of bortezomib-treated R11 cells. R11 cells (7 x 106 cells) were seeded in a 10-cm dish and exposed to bortezomib or vehicle control for 48 hours. Apoptosis was measured by the TUNEL assay on a flow cytometer as described in Materials and Methods. As a positive control for the TUNEL assay, cells were treated with DNase I to digest DNA prior to running the TUNEL assay. B, Annexin V-FITC staining of R11 cells exposed to bortezomib. R11 cells were treated as in A and assayed for apoptosis by Annexin V-FITC staining (see Materials and Methods). C, MTT assay of bortezomib-treated R11 and 444 cells. Columns, means of four experiments; bars, SD.

 


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  		Figure 3. Bortezomib requires inhibition of NF-{kappa}B for maximal induction of apoptosis. A and B, determination of transfection conditions/bortezomib concentrations that result in NF-{kappa}B transcriptional activity equivalent to that of untreated/untransfected control cells. R11 (A) and 444 (B) cells were transfected in 24-well plates with the indicated amounts of the pEGFP-p65 and pCMV4-p50 plasmids. Total DNA was held constant (1 µg for R11 cells and 0.5 µg for 444 cells) with empty vector control plasmids. pRL-SV40 (1 ng) was included to normalize for transfection efficiency. Twenty-four hours after transfection, bortezomib was added for an additional 24 hours, and a dual luciferase assay was performed. Columns, means of three experiments expressed as relative luminescence units (RLU); bars, SD. C–E, ectopic expression of p65/p50 inhibits bortezomib-induced apoptosis. R11 (C and D) and 444 (E) cells were transiently transfected in 10-cm dishes with pEGFP-p65 and pCMV4-p50 or respective control vectors (concentrations scaled up proportionally from results of experiments in A and B, which were performed in 24-well plates). Bortezomib was added 24 hours after transfection for an additional 24 hours, and cells were harvested for assessment of apoptosis by the TUNEL assay on a flow cytometer. This TUNEL assay employs tetramethylrhodamine red as the fluorochrome, which allows for simultaneous identification of EGFP-positive and apoptotic (red) cells. C, representative dot plot results demonstrating the percentage of apoptotic (red) R11 cells that express the EGFP transgene. D and E, histograms of TUNEL assay results on R11 and 444 cells, respectively.

 


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  		Figure 5. Chemical inhibition of NF-{kappa}B with PS-1145 does not result in apoptosis of RCC cells. A, PS-1145 specifically inhibits NF-{kappa}B transcriptional activity in a dose-dependent fashion. R11 and 444 cells were plated as in Fig. 4. Cells were transiently transfected the next day with p{kappa}B-luc or pCRE-luc plasmid (1 µg) and pRL-SV40 control vector (1 ng). Three hours later, fresh medium was added with PS-1145 or vehicle control for an additional 48 hours, at which time the dual luciferase assay was performed. Columns, means of three experiments expressed as relative luminescence units (RLU); bars, SD. B, PS-1145 inhibits the basal and TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha}. R11 cells were treated with PS-1145 (20 µmol/L) or bortezomib (10 µmol/L) for 30 minutes prior to exposure to TNF-{alpha} for the indicated time points. Protein was extracted for Western blotting for I{kappa}B-{alpha} or actin (as a loading control). C and D, PS-1145 does not induce apoptosis of R11 cells. R11 cells were treated with the indicated concentrations of PS-1145 or bortezomib for 48 hours and subjected to the TUNEL assay (C) or hypotonic PI staining (D) followed by flow cytometric analysis. Bortezomib treatment served as a positive control for the apoptosis assays. D, a prominent sub-G1 peak indicative of apoptosis is observed in bortezomib-treated cells. All experiments were performed on 444 cells and yielded similar results.

 
We next measured the effect of bortezomib on overall growth of R11 and 444 cells by the MTT assay. Both cell lines displayed sensitivity to bortezomib by the MTT assay, although 444 cells were considerably more sensitive (Fig. 1C). The IC50 values for R11 and 444 cells were 0.67 and 5.5 x 10–3 µmol/L, respectively. Thus, bortezomib represents a potent antitumor agent against RCC cells.

Bortezomib Inhibits Constitutive NF-{kappa}B Activity in RCC Cells in a Dose-Dependent Fashion
Inhibition of NF-{kappa}B has been proposed as a critical mechanism of action of bortezomib-induced apoptosis. Thus, we determined the NF-{kappa}B inhibitory effect of bortezomib in RCC cells. Because bortezomib blocks NF-{kappa}B activity by inhibiting the proteasomal degradation of I{kappa}B, we first assayed the I{kappa}B-{alpha} levels in RCC cells in the presence or absence of bortezomib. Treatment with bortezomib for 30 minutes potently inhibited degradation of I{kappa}B-{alpha} in both basal and TNF-{alpha}-stimulated R11 cells (Fig. 2A).

I{kappa}B binds to and sequesters NF-{kappa}B family members in the cytoplasm and prevents their nuclear localization (21). Thus, by increasing intracellular I{kappa}B levels, bortezomib is expected to decrease the nuclear localization and DNA binding of NF-{kappa}B. Indeed, bortezomib decreased the basal and TNF-{alpha}-stimulated nuclear localization and DNA binding of NF-{kappa}B in R11 cells in a dose-dependent fashion as assayed by electrophoretic mobility shift assay (EMSA; Fig. 2B, left and middle panels). After 30 minutes, there was a modest diminution of basal and TNF-{alpha}-stimulated gel shifted bands at 0.1 µmol/L of bortezomib, with progressive reduction in the intensity of the shifted bands at increasing concentrations of bortezomib up to 10 µmol/L (Fig. 2B, left panel). At 24 hours, bortezomib induced a more marked inhibition of NF-{kappa}B, especially at 1 and 10 µmol/L (Fig. 2B, middle panel). Based on antibody supershift experiments, the bands on the EMSA were represented by a p65/p50 heterodimer and a p50/p50 homodimer (data not shown). An EMSA for activator protein-1 was performed as a protein loading control (Fig. 2B, right panel).

Cellular I{kappa}B levels and nuclear localization of NF-{kappa}B are surrogate markers for the activation state of the NF-{kappa}B pathway. To directly measure the effects of bortezomib on NF-{kappa}B transcriptional activity, we assayed reporter gene expression driven by tandem copies of the {kappa}B response element. After 48 hours, bortezomib induced a potent and dose-dependent inhibition of NF-{kappa}B transcriptional activity (Fig. 2C; note logarithmic scale of vertical axis). The NF-{kappa}B IC50 values for bortezomib, which were normalized to that of the pRL-SV40 internal control, were 45 and 8.7 nmol/L for R11 and 444 cells, respectively.

Induction of Apoptosis by Bortezomib Is Dependent on Inhibition of NF-{kappa}B
Although the NF-{kappa}B inhibitory effect of bortezomib has received much attention as a mechanism of action of this drug (10, 11, 13, 14, 19), it has not been established whether bortezomib-induced apoptosis requires NF-{kappa}B blockade. In support of the notion that NF-{kappa}B inhibition is crucial for bortezomib-induced apoptosis, both the inhibitory concentrations for NF-{kappa}B and growth were higher in R11 cells than in 444 cells; that is, R11 cells, which manifest a greater level of basal NF-{kappa}B activation, also have a higher IC50 for growth.

To directly test whether NF-{kappa}B blockade was required for the proapoptotic effect of bortezomib, we used a strategy to maintain NF-{kappa}B activation in the presence of bortezomib. Thus, if NF-{kappa}B inhibition is required for bortezomib-induced apoptosis, then maintaining NF-{kappa}B activation in the presence of bortezomib ought to abrogate the proapoptotic effect of the drug. To activate NF-{kappa}B, we transiently transfected R11 and 444 cells with expression vectors containing the cDNA for the p65 and p50 NF-{kappa}B family members (pEGFP-p65 and pCMV4-p50) or the respective control vectors in equal amounts. We initially identified combinations of quantities of pEGFP-p65/pCMV4-p50 DNA transfected and bortezomib concentrations that would yield {kappa}B-driven reporter gene expression equivalent to that of control vector transfected, DMSO-treated cells (Fig. 3A and B). Thus, in a 24-well format, 0.5 µg of both pEGFP-p65 and pCMV4-p50 with bortezomib concentrations of 1 and 10 µmol/L yielded reporter gene expression for R11 cells in the range of the vector control transfected, DMSO-treated group (Fig. 3A). Regardless of the quantity of pEGFP-p65 and pCMV4-p50 DNA transfected, a bortezomib concentration of 0.1 µmol/L resulted in NF-{kappa}B-driven reporter gene expression in R11 cells that was significantly greater than the basal level of reporter gene expression (Fig. 3A); therefore, we did not use this concentration for apoptosis studies (see below). For 444 cells, 0.25 µg of both pEGFP-p65 and pCMV4-p50 with bortezomib at 10 µmol/L recapitulated the basal level of NF-{kappa}B transcriptional activity (Fig. 3B).

Having identified the conditions of transfection/bortezomib concentrations that would maintain the approximate degree of NF-{kappa}B activation as control cells, we next tested R11 and 444 cells transfected with pEGFP-p65 and pCMV4-p50 or control vectors for bortezomib-induced apoptosis. After transfection, cells were exposed to bortezomib for 24 hours. Apoptosis of EGFP-positive cells was assayed by flow cytometry with a TUNEL assay that employs tetramethylrhodamine red as the fluorochrome. Compared with control groups, cells with ectopic expression of EGFP-p65/p50 demonstrated ~50% less apoptosis when exposed to bortezomib at a concentration of 1 or 10 µmol/L (Fig. 3C-E). For example, at a bortezomib concentration of 10 µmol/L, 42% of the control vector transfected R11 cells underwent apoptosis compared with only 19% of the EGFP-p65/p50 expressing cells (Fig. 3C and D). In 444 cells exposed to 10 µmol/L bortezomib, control cells manifested 66% apoptosis compared with 33% in the cells with ectopic expression of NF-{kappa}B (Fig. 3E). The dot plot of EGFP (horizontal axis) versus red (TUNEL, vertical axis), shown in Fig. 3C for R11 cells, demonstrates a clear decrease in the proportion of EGFP-positive, apoptotic cells (cells in the upper right quadrant) in the group expressing EGFP-p65/p50 (right panel) compared with the control transfected cells (middle panel). There was no significant difference in the percentage of apoptosis between pEGFP-p65/pCMV4-p50 and control vector transfected cells in the absence of bortezomib (Fig. 3D and E). These results indicate that inhibition of NF-{kappa}B is required for the maximal apoptotic effect of bortezomib. However, the fact that ectopic expression of NF-{kappa}B did not completely abrogate bortezomib-induced apoptosis suggests that NF-{kappa}B-independent effects are operative in the proapoptotic effect of bortezomib.

Inhibition of NF-{kappa}B Is Not Sufficient to Induce Apoptosis of RCC Cells
The above data confirmed that bortezomib-dependent NF-{kappa}B inhibition participates in apoptosis. To determine whether NF-{kappa}B blockade was sufficient to induce apoptosis in RCC cells, we targeted the NF-{kappa}B pathway with the use of an I{kappa}B-SR. The I{kappa}B-SR contains mutations at Ser32 and Ser36 that prevent phosphorylation and degradation of the I{kappa}B-SR and functions as a dominant active inhibitor of NF-{kappa}B activation (32). An adenoviral vector containing the I{kappa}B-SR transgene (Ad-I{kappa}B-SR) or control vector (Ad-CMV) was transduced into R11 and 444 cells. Preliminary studies with an adenoviral vector containing the EGFP (Ad-EGFP) demonstrated that >90% of cells express the EGFP transgene at MOIs as low as 5 (data not shown). We first identified the MOI at which basal NF-{kappa}B was completely abrogated. As measured by EMSA, the Ad-I{kappa}B-SR completely blocked NF-{kappa}B activation in both R11 and 444 cells at a MOI of 5 at 48 hours (Fig. 4A, left and middle panels).

Next, we determined whether inhibition of NF-{kappa}B resulted in apoptosis. We exposed R11 cells to the Ad-I{kappa}B-SR or Ad-CMV (MOI = 5 to 50) and measured apoptosis flow cytometrically by the TUNEL assay. Transduction of the Ad-I{kappa}B-SR did not induce apoptosis as compared with the control adenovirus (Fig. 4B, left panels). Treatment with bortezomib (10 µmol/L) for 48 hours served as a positive control for apoptosis (see below and Fig. 5C and D). As a complementary measurement of apoptosis, we performed cell cycle analysis with hypotonic PI staining on adenovirally transduced R11 cells. Again, no apoptosis was observed as determined by sub-G1 peak analysis in the Ad-I{kappa}B-SR versus the Ad-CMV transduced cells, and there was no change in the S-phase fraction (Fig. 4C).

To confirm that selective inhibition of NF-{kappa}B does not induce apoptosis of RCC cells at time points later than 48 hours, we treated R11 cells with the Ad-I{kappa}B-SR or the Ad-CMV control for 5 days. Similar to the data at 48 hours, the Ad-I{kappa}B-SR completely abrogated NF-{kappa}B activity at 5 days at a MOI of ≥5 (Fig. 4A, right panel) yet did not induce apoptosis (Fig. 4B, right panels).

To confirm the results of the adenovirus experiments, we employed PS-1145, a specific, chemical inhibitor of I{kappa}B kinase. The specificity of PS-1145 has been previously demonstrated; PS-1145 potently inhibits I{kappa}B kinase but has no significant effect on 14 other kinases tested (33). We validated this specificity in reporter gene experiments. PS-1145 inhibited reporter gene expression from the p{kappa}B-luc plasmid (IC50 = 8.0 µmol/L) but had no effect on the pCRE-luc construct (Fig. 5A). Moreover, PS-1145 prevented the basal and TNF-{alpha}-stimulated degradation of I{kappa}B-{alpha} in R11 cells, although PS-1145 was considerably less potent than bortezomib in this regard (Fig. 5B). Thus, PS-1145 specifically inhibits the NF-{kappa}B pathway in RCC cells.

When we tested the proapoptotic effects of PS-1145 (1 to 20 µmol/L), we did not observe apoptosis in R11 or 444 cells as measured by the TUNEL assay (Fig. 5C). Similarly, PS-1145 had no effect on the sub-G1 peak determined by cell cycle analysis (Fig. 5D). Hence, our data with PS-1145 corroborated the results of the adenoviral studies and indicate that specific blockade of NF-{kappa}B activity is not sufficient to induce apoptosis in RCC cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteasome inhibition has received increasing attention as an anticancer therapy. In vitro and animal studies have documented the efficacy of proteasome inhibitors, such as bortezomib, in many tumor models, including multiple myeloma and prostate, ovarian, pancreatic, and colon cancer (10-13). A recent study established the activity of bortezomib in heavily pretreated multiple myeloma patients that led to the approval of this drug by the U.S. Food and Drug Administration (34). Despite the broad activity of proteasome inhibitors, the particular proteins and biochemical pathways that are modulated by proteasome inhibitors that contribute to the proapoptotic effect of this class of drugs have not been clearly defined. In many of the reports of the anticancer activity of proteasome inhibitors, the role of inhibition of NF-{kappa}B activity has been emphasized (10, 11, 13, 14, 19). Given that NF-{kappa}B family members induce the transcription of genes involved in a diversity of functions that determine cell fate, such as apoptosis, proliferation, angiogenesis, and cell adhesion (21-24), there is a clear rationale for considering NF-{kappa}B as a vital target of proteasome inhibitors.

Here, we have shown that the proteasome inhibitor bortezomib potently induces apoptosis of RCC cells and inhibits NF-{kappa}B in a dose-dependent fashion. Importantly, we have demonstrated for the first time that inhibition of constitutive NF-{kappa}B activation is required for the maximal apoptotic effect of bortezomib. Our studies of specific inhibition of constitutive NF-{kappa}B demonstrated that NF-{kappa}B blockade is not sufficient to induce apoptosis of RCC cells, which is in line with a prior report, although this report identified some RCC cell lines that did undergo apoptosis in response to NF-{kappa}B inhibition (29). We used both genetic (i.e., I{kappa}B-SR) and chemical (i.e., PS-1145) means to specifically inhibit NF-{kappa}B, and both of these complementary strategies failed to induce apoptosis. The absence of apoptosis of RCC cells in response to the Ad-I{kappa}B-SR is consistent with responses in most other solid tumor models that also fail to undergo apoptosis upon NF-{kappa}B blockade (35). Our findings that bortezomib induced apoptosis at 48 hours, yet selective inhibition of NF-{kappa}B did not result in apoptosis, clearly indicate that NF-{kappa}B-independent effects play a critical role in the induction of apoptosis by bortezomib. In support of this notion, activation of the c-Jun NH2-terminal kinase by bortezomib is required for bortezomib-induced apoptosis of multiple myeloma cells (36).

Interestingly, some tumor cells with absent basal NF-{kappa}B activity are sensitive to proteasome inhibition. For example, LNCaP prostate cancer cells and colon cancer cell lines, which lack significant basal NF-{kappa}B activation, are sensitive to bortezomib (11, 37). Thus, our finding that bortezomib-induced apoptosis of RCC cells requires NF-{kappa}B blockade does not necessarily signify that a tumor cell must display constitutive NF-{kappa}B activity as a prerequisite for sensitivity to proteasome inhibition. Rather, we take these results to indicate that the absence of NF-{kappa}B activation, whether it is achieved in response to proteasome inhibition or is an intrinsic property of a tumor cell, may represent a requirement for the optimal proapoptotic effect of proteasome inhibitors. Alternatively, non-NF-{kappa}B pathways may function completely independently of NF-{kappa}B to induce apoptosis. In support of this concept, ectopic expression of p65/p50 did not completely abrogate bortezomib-induced apoptosis in RCC cells. Thus, NF-{kappa}B-independent pathways may be particularly relevant in cell types that do not manifest constitutive NF-{kappa}B activation.

Most cases of sporadic and hereditary RCC are associated with loss of function mutations of the von Hippel-Lindau tumor suppressor gene (VHL; refs. 38, 39), the protein product of which normally ubiquitinates hypoxia-inducible factor (HIF) and thereby targets HIF for proteasomal degradation (40). HIF functions as a transcription factor that induces expression of genes that promote angiogenesis and enhance glycolysis, which are classic hallmarks of the malignant phenotype (40), and the aberrant activation of HIFs are a driving force in the oncogenesis of kidney cancer. Interestingly, NF-{kappa}B stabilizes the level of HIF-{alpha} protein (41-43), and in this regard, bortezomib-mediated inhibition of NF-{kappa}B may be particular germane to the treatment of RCC; that is, by inhibiting NF-{kappa}B activity, bortezomib may reduce HIF protein levels. Because heightened HIF expression due to VHL gene mutations is a pervasive phenomenon in hereditary and sporadic RCC, bortezomib may serve as an ideal agent to target this primary, pathophysiologic alteration in RCC.

RCC is notoriously resistant to cytotoxic chemotherapy. Chemotherapy resistance in RCC is in large part attributed to expression of the multidrug resistance gene as well as genes that augment intracellular glutathione levels (2). Interestingly, these genes are NF-{kappa}B inducible (44), which suggests that drugs that inhibit NF-{kappa}B, such as bortezomib, may sensitize RCC cells to chemotherapy. Thus, although bortezomib has recently been shown to have modest activity in RCC patients (45), proteasome inhibition represents a potential strategy in combination with cytotoxic chemotherapy to treat advanced RCC, a disease against which no current therapy has demonstrated consistent activity.


   Footnotes
 
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.

Received 11/ 6/03; revised 4/ 6/04; accepted 4/13/04.


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 Introduction
 Materials and Methods
 Results
 Discussion
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