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Molecular Cancer Therapeutics 6, 1046-1053, March 1, 2007. doi: 10.1158/1535-7163.MCT-06-0577
© 2007 American Association for Cancer Research
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Research Articles: Therapeutics, Targets, and Development

Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis accompanied by up-regulation of noxa in the non–small cell lung cancer cell line NCI-H460

Jens Voortman, Agnieszka Checinska, Giuseppe Giaccone, Jose A. Rodriguez and Frank A.E. Kruyt

Department of Medical Oncology, VU University Medical Center, Amsterdam, the Netherlands

Requests for reprints: Frank A.E. Kruyt, Department of Medical Oncology, CCA 2.36, VU University Medical Center, 1081 HV Amsterdam, the Netherlands. Phone: 31-20-444-33-74; Fax: 31-20-444-38-44. E-mail: kruyt{at}vumc.nl

Abstract

Defects in the apoptotic machinery may contribute to chemoresistance of non–small cell lung cancer (NSCLC) cells. We have previously showed a deficiency in mitochondria-dependent caspase-9 activation in NSCLC H460 cells after exposure to cisplatin, a drug widely used to treat NSCLC. Here we show that, unlike cisplatin, the novel anticancer agent bortezomib efficiently induces caspase-9 activation and apoptosis in H460 cells. A comparative analysis of molecular events underlying cell death in bortezomib-treated versus cisplatin-treated H460 cells revealed that bortezomib, but not cisplatin, caused a rapid and abundant release of cytochrome c and Smac/DIABLO from mitochondria. This was associated with a marked increase in levels of the BH3-only proapoptotic protein Noxa and the antiapoptotic protein Mcl-1. Taken together, our data show that bortezomib, by promoting a proapoptotic shift in the levels of proteins involved in mitochondrial outer-membrane permeabilization, is a potent activator of the mitochondrial pathway of apoptosis in NSCLC cells. Our preclinical results support further investigation of bortezomib-based therapies as a possible new treatment modality for NSCLC. [Mol Cancer Ther 2007;6(3):1046–53]

Introduction

Apoptosis is an evolutionarily conserved and genetically regulated cell death mechanism, playing an essential role in the development and maintenance of tissue homeostasis (1). Many anticancer agents are thought to induce tumor cell death via apoptosis (2). Defects in the process of apoptosis due to overexpression of antiapoptotic proteins and/or loss of proapoptotic proteins are frequent in cancer cells, and apoptosis resistance is thought to contribute to their malignant and chemoresistant phenotype (3–5).

Proapoptotic and antiapoptotic members of the Bcl-2 protein family are key regulators of the mitochondrial apoptotic pathway. Antiapoptotic members, such as Bcl-2, Bcl-xL, and Mcl-1, contain multiple Bcl-2 homology domains. Proapoptotic Bcl-2 family members, on the other hand, can be subdivided into two classes according to the number of Bcl-2 homology domains they bear: multidomain proteins, such as BAK and BAX, or BH3-only proteins, such as Noxa, PUMA, BID, BIM, BAD, BIK, and Hrk (6, 7). BH3-only proteins can be further subdivided in activator (BID, BIM) and sensitizer/derepressor (Noxa, BIK, BAD, Bmf, Hrk) members. Sensitizer/derepressor BH3-only proteins can interact via their BH3 domain with antiapoptotic members, ultimately leading to the activation of BAK and BAX. Activator BH3-only proteins can directly induce oligomerization and activation of BAX or BAK (8). Activated BAX/BAK, in turn, leads to mitochondrial outer membrane permeabilization (MOMP), causing the release of cytochrome c and Smac/DIABLO, which triggers apoptosis by inducing apoptosome-dependent caspase-9 activation and neutralization of inhibitor of apoptosis proteins, respectively (9, 10). Thus, the balanced expression and the interactions between proapoptotic and antiapoptotic Bcl-2 proteins are critical determinants for a cell to undergo apoptosis or not.

Non–small cell lung cancer (NSCLC) constitutes ~85% of all lung cancer cases (11). Platinum-based palliative chemotherapy was long regarded as the standard treatment for advanced NSCLC (12, 13). However, only a minority of patients benefit from this toxic therapy. Therefore, more effective therapies are required (14). In this context, a novel approach to cancer treatment is the use of targeted agents modulating the ubiquitin-proteasome proteolytic pathway, such as bortezomib.

Bortezomib (VELCADE, Millennium Pharmaceuticals, Inc., Cambridge, MA and Johnson & Johnson Pharmaceutical Research and Development, LLC, Raritan, NJ) is a dipeptide boronic acid compound that reversibly inhibits the chymotryptic-like proteolytic activity of the 20 S proteasome (15). Bortezomib has been approved for the treatment of relapsed multiple myeloma and has shown promising antitumor activity, alone or in combination with other cytotoxic drugs, in patients with NSCLC (16–19).

Although bortezomib-induced nuclear factor-{kappa}B inhibition was shown to be pivotal for the observed anticancer activity in multiple myeloma, other mechanisms might be more important for the observed cytotoxicity induced in solid tumor types, such as NSCLC (20). In this regard, recent reports indicate that bortezomib treatment induces a proapoptotic shift of the balance between levels of Bcl-2 family member proteins. Proteasome inhibition by bortezomib was shown to induce cleavage of Bcl-2 and accumulation or up-regulation of proapoptotic members of the Bcl-2 family including the BH3-only proteins BIK, BIM, and Noxa (21–25).

We have previously shown that cisplatin-induced activation of caspase-9 is defective in NSCLC H460 cells, and more recent work suggested the presence of a deficiency upstream of the process of apoptosome formation (26, 27). Here we show that bortezomib, in contrast to cisplatin, efficiently induces activation of caspase-9 and apoptosis in H460 cells, which coincides with a differential effect of both drugs on the levels of Bcl-2 family proteins involved in MOMP. These findings show that bortezomib can overcome the observed resistance to mitochondria-dependent apoptosis in H460 cells seen after exposure to cisplatin. Consequently, bortezomib may be of therapeutic benefit in treating patients with NSCLC.

Materials and Methods

Cell Culture and Drugs
Human NSCLC NCI-H322 (H322) and NCI-H460 (H460) cells were grown in RPMI medium (Cambrex Bioscience, Verviers, Belgium) supplemented with 10% FCS, 100 units/mL penicillin, and 100 µg/mL streptomycin (Invitrogen, Breda, the Netherlands). We have previously generated stably transfected derivatives of H460 cells overexpressing Bcl-2 (26). H460 cells overexpressing Bcl-2 cells were grown in RPMI medium supplemented with 10% FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 1 µg/mL puromycin.

Bortezomib (VELCADE, formerly PS-341) was kindly provided as a pure substance by Millennium Pharmaceuticals and dissolved in DMSO. The stock solution of cisplatin (Bristol-Meyers Squibb, Woerden, the Netherlands) was prepared in PBS.

Growth Inhibition Assay
A 100-µL suspension of 5 x 103 cells was added to each well of 96-well, flat-bottomed plates (Costar, Corning, NY). After 24 h, various concentrations of bortezomib were added to the cells. After 72 h of incubation, growth inhibition was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma Chemical Co., St. Louis, MO) as described previously (26). Absorbance values, measured at 540 nm, were expressed as a percentage of untreated controls. The concentrations resulting in cell-growth inhibition of 50% (IC50) or 80% (IC80) were calculated.

Proteasome Inhibition Assay
To determine the ability of bortezomib to inhibit intracellular proteasome activity of H460 cells, the chymotryptic activity of the proteasome was estimated as described previously (15, 28). H460 cells (3 x 105) were incubated for 24 h in the presence or absence of 10 nmol/L or 100 nmol/L bortezomib. Cells were collected by trypsinization and washed twice in PBS. Cell pellets were incubated with 100 µL of lysis buffer {50 mmol/L HEPES, 5 mmol/L 3-[(3-Cholamidopropyl) dimethylammonio]-1-propane-sulfonate, 0.5 mmol/L EDTA, 0.035% SDS (pH 7.5)} for 1 h on ice. Samples were then centrifuged at 14,000 rpm for 10 min, and supernatants were isolated. Protein (40 µg) was added to 90 µL of lysis buffer, plates were warmed for 10 min at 37°C, and 10 µL of the succinyl-Leu-Leu-Val-Tyr-AMC substrate (BACHEM, King of Prussia, PA) was added to a final concentration of 150 µmol/L. The resultant fluorescence of the released 7-amido-4-methylcoumarin dye was measured on a Spectra Fluor multiwell plate reader (Tecan, Salzburg, Austria) set at an excitation wavelength of 380 nm and emission wavelength of 460 nm.

Western Blot Analysis
Western blot analysis was done as described before (26). Rabbit polyclonal primary antibodies used were anti–caspase-9, anti–caspase-3, anti–Mcl-1, anti-BAX, anti-BAK, anti-BIK (Cell Signaling Technology, Beverly, MA), anti–Bcl-xL/S (Santa Cruz Biotechnology, Santa Cruz, CA), anti-BIM (Axxora, Life Science, Inc., San Diego, CA) and anti-Smac (Imgenex, San Diego, CA). Mouse monoclonal primary antibodies used were anti–Bcl-2 (Dako Norden, Glostrup, Denmark), anti–Mcl-1 (clone 22, BD PharMingen, San Diego, CA), anti–cytochrome c (BD PharMingen), anti-Noxa (Calbiochem, San Diego, CA), and anti–ß-actin (Sigma-Aldrich). As secondary reagents, horseradish peroxidase–conjugated goat antimouse or antirabbit antibodies (Amersham, Braunschweig, Germany) were used.

Protein Interaction Assay
For immunoprecipitation experiments, 1 x 108 cells were washed in ice-cold PBS and rinsed with ice-cold lysis buffer [20 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT] supplemented with 1x protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany), 250 µmol/L phenylmethylsulfonyl fluoride, and 1 mmol/L Na3VO4. Cells were scraped, pelleted, resuspended in lysis buffer, and incubated for 30 min on ice before homogenization with 40 strokes in a 2-mL Dounce homogenizer using pestle B. After centrifugation at 13,200 rpm for 30 min at 4°C, the supernatants were taken as total cell extracts. Protein concentrations were determined with the Protein Assay Dye Reagent Solution (Bio-Rad, Veenendaal, the Netherlands). Extracts were incubated on ice for 15 min and centrifuged for 5 min at 13,200 rpm to remove insoluble particles. Next, extracts were supplemented with 0.1% 3-[(3-Cholamidopropyl) dimethylammonio]-1-propane-sulfonate and 50 mmol/L NaCl, before immunoprecipitation with anti-Noxa monoclonal antibody or anti–Mcl-1 monoclonal antibody and purification with protein A/G sepharose beads (Santa Cruz) O/N at 4°C. Subsequently, beads were washed five times in ice-cold lysis buffer and resuspended in loading buffer, and the immunoprecipitated proteins were subjected to Western blot analysis.

Subcellular Fractionation
Cytosolic and mitochondrial fractions were generated using a previously described digitonin-based subcellular fractionation technique (29). In brief, cells were harvested, washed in ice-cold PBS (pH 7.2), and resuspended at a density of 3 x 107/mL in cytosolic extraction buffer (250 mmol/L sucrose, 70 mmol/L KCl, 137 mmol/L NaCl, 4.3 mmol/L Na2HPO4), supplemented with 1x protease inhibitor cocktail, 250 µmol/L phenylmethylsulfonyl fluoride, and 200 µg/mL digitonin. After incubation for 5 min on ice, cell membrane permeabilization of at least 95% of the cells was confirmed by staining with 0.2% trypan blue solution. After centrifugation at 3,000 rpm for 5 min at 4°C, the cytosolic fraction was recovered. Pellet was solubilized in an equal volume of mitochondrial lysis buffer [50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 2 mmol/L EDTA, 2 mmol/L EGTA, 0.2% Triton X-100, 0.3% NP40], supplemented with 1x protease inhibitor cocktail and 250 µmol/L phenylmethylsulfonyl fluoride. Mitochondrial extracts were recovered by centrifugation at 3,000 rpm for 10 min at 4°C. For the detection of cytochrome c and Smac/DIABLO, 20 µg of cytosolic and mitochondrial fractions were subjected to SDS-PAGE and Western blot analysis.

Flow Cytometric Analysis of Cytochrome c Release
Cells (1 x 105) were harvested and treated with 100 µL digitonin (50 µg/mL in PBS containing 100 mmol/L KCl and 1 mmol/L EDTA) for 5 min on ice until >95% were permeabilized as assessed by trypan blue exclusion. Cells were fixed in 3.7% formaldehyde in PBS for 20 min at room temperature, washed thrice in PBS, and incubated in blocking buffer (3% bovine serum albumin, 0.05% saponin in PBS) for 1 h. The cells were incubated overnight at 4°C with anti–cytochrome c monoclonal antibody (BD PharMingen) diluted 1:200 in blocking buffer, washed thrice, and incubated for 1 h at room temperature with FITC-conjugated goat antimouse (Santa Cruz) diluted 1:200 in blocking buffer. The cells were then analyzed by flow cytometry as described (30).

Cell Death Measurement
Propidium iodide staining and flow cytometry analysis were done as described previously (26). The fraction of cells, with hypodiploid (sub-G1) DNA content, was taken as the apoptotic cell population. The percentage of apoptosis indicated was corrected for background sub-G1 levels found in the corresponding untreated controls.

Caspase Activity Assay
Caspase-9 and caspase-3 activity was determined by measuring cleavage of the fluorogenic substrates LEHD-AFC or DEVD-AFC, respectively, using commercially available kits (MBL, Co., Nagoya, Japan, BD Biosciences Clontech, Mountain View, CA), according to manufacturer's protocols. Fluorescence was detected using a fluorometer equipped with a 400-nm excitation filter and a 505-nm emission filter (Spectra Fluor, Tecan). Relative caspase activity was determined by comparing the level of substrate cleavage in the treated samples versus untreated controls.

Statistical Analysis
When applicable the data were analyzed by the Student's t test. All P values were considered significant when P ≤ 0.05. Statistical analysis was done using the SPSS software program 9.0 (SPSS, Chicago, IL).

Results

Bortezomib Induces Apoptosis in H460 and H322 NSCLC Cells More Efficiently than Cisplatin
H460 and H322 cells were exposed to various concentrations of bortezomib. Figure 1A shows the growth inhibition curves for the two cell lines. The IC50 values of bortezomib were 49 and 69 nmol/L for H322 and H460 cells, respectively. The IC80 values were 1 µmol/L and 100 nmol/L for H322 and H460 cells, respectively. The IC50 and IC80 concentrations of cisplatin are 2 and 7 µmol/L in H460 cells and 4 and 10 µmol/L in H322 cells, as reported previously (26, 31).


Figure 1
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  		Figure 1. Growth inhibition, induction of apoptosis, and proteasome inhibition by bortezomib and cisplatin in NSCLC cells. A, growth curves [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays] of H460 and H322 cells treated with different concentration of bortezomib. B-E, the sub-G1 cell fraction was estimated by propidium iodide staining–based fluorescence-activated cell sorting analysis 48 h after treatment with a concentration range of bortezomib and cisplatin. Columns, mean of at least three independent experiments; bars, SD. F, Intracellular proteasome activity in H460 cells in absence or presence of bortezomib (10 nmol/L or 100 nmol/L).

 
To compare the cytotoxic effect of bortezomib and cisplatin, H460 and H322 cells were treated with a concentration range of bortezomib or cisplatin including the IC80 concentrations of each drug. The percentage of apoptotic cells with sub-G1 DNA content was evaluated at 48 h by propidium iodide staining–based fluorescence-activated cell sorting analysis. As shown in Fig. 1B-E, bortezomib was more effective in inducing apoptotic cell death in H460 and H322 cells than cisplatin. Additionally, a potential synergistic induction of apoptosis by combining bortezomib and cisplatin was tested. Cells were simultaneously or sequentially treated with the IC80 concentration of cisplatin and bortezomib. However, this treatment resulted in a sub-G1 population comparable with treatment with the IC80 concentration of bortezomib alone (data not shown).

Finally, we analyzed the ability of different concentrations of bortezomib to inhibit intracellular proteasome activity in H460 cells. As shown in Fig. 1F, bortezomib at a concentration of 100 nmol/L, but not at 10 nmol/L, was able to reduce proteasome activity to ~25% of its basal level after 24 h of exposure. Because 100 nmol/L is the IC80 concentration and effectively inhibits proteasome activity in H460 cells, subsequent experiments were carried out using this concentration of bortezomib.

Bortezomib Induces Strong Activation of Caspase-9 and Caspase-3 Activity in H460 Cells
The observed superior cytotoxicity of bortezomib in NSCLC cells prompted us to compare the mechanism underlying bortezomib-induced and cisplatin-induced cell death in H460 cells. The processing and activation of caspase-9 and caspase-3 were analyzed in total extracts from H460 cells exposed to IC80 concentrations of either drug. As shown in Fig. 2A , processing of pro–caspase-9 into p35-cleaved and p37-cleaved products was already detectable after 24 h of exposure to bortezomib, further increasing at 48 h. In contrast, in cells exposed to cisplatin, processing of pro–caspase-9 was only detected after 48 h. The predominant cleavage product detected upon cisplatin treatment was a p37 fragment, previously shown to be an inactive form, generated by caspase-3–mediated cleavage of pro–caspase-9 (27, 32).


Figure 2
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  		Figure 2. Bortezomib is an efficient inducer of the processing and activation of caspases in H460 cells when compared with cisplatin. A, processing of caspase-9 and caspase-3 was determined by Western blot analysis in cytosolic extracts of cells treated with bortezomib (100 nmol/L) and cisplatin (7 µmol/L) for 24 and 48 h. B and C, caspase-9 and caspase-3 protease activity upon 24-h and 48-h exposure to bortezomib or cisplatin, as measured by LEHD-AFC or DEVD-AFC cleavage, respectively. Changes in activity relative to activity in control cells. Columns, mean of at least three independent experiments; bars, SD.

 
Furthermore, an almost complete processing of pro–caspase-3 was observed in extracts derived from bortezomib-treated, but not cisplatin-treated, H460 cells after 48 h.

Next, LEHD-AFC and DEVD-AFC cleavage assays were used to monitor the activity of caspase-9 and caspase-3 after 24 and 48 h of treatment with either drug. In line with the processing of pro–caspase-9 observed by Western blot analysis, treatment with bortezomib for 48 h induced a 3-fold increase in caspase-9 activity compared with the untreated control (P < 0.01; Fig. 2B). At the same time point, cisplatin induced an increase in caspase-9 activation that is not statistically significant(P = 0.06). Furthermore, bortezomib treatment also induced a more pronounced and rapid increase in caspase-3 activity than cisplatin. As shown in Fig. 2C, the level of bortezomib-induced caspase-3 activity was ~40% higher than the level of cisplatin-induced caspase-3 activity after 48 h of treatment.

These results show that bortezomib, in contrast to cisplatin, is a potent inducer of the caspase-9–mediated apoptotic route in H460 NSCLC cells.

Bortezomib Treatment Induces Cytosolic Release of Cytochrome c and Smac/DIABLO
We have previously found that the addition of exogenous cytochrome c and dATP to H460 cell extracts induces caspase-9 activation, suggesting that the inability of cisplatin to trigger caspase-9 activation is related to its inability to induce MOMP (27). Therefore, we hypothesized that the increased cytotoxicity and caspase-9 activation induced by bortezomib could be associated with a more pronounced cytosolic release of cytochrome c. To test this hypothesis, subcellular fractions were generated from H460 cells that were treated with a concentration range of either bortezomib or cisplatin (Fig. 3A ) or for different periods of time with IC80 concentrations (Fig. 3B). Western blot analyses showed that cytochrome c release occurred more efficiently after bortezomib treatment than after cisplatin treatment. The amount of cytochrome c released into the cytosol of the cisplatin-exposed cells was only slightly higher than in untreated cells. We quantitated the release of cytochrome c after bortezomib treatment, using flow cytometry (ref. 30; Fig. 3C). Already after 16 h of exposure to bortezomib treatment, >80% of the cells showed reduced fluorescence intensities resulting from diffuse cytoplasmic staining, whereas cisplatin exposure had hardly any detectable effect. Furthermore, we observed that bortezomib, but not cisplatin, promoted the cytosolic release of Smac/DIABLO, which was clearly detectable after 24 h of exposure to bortezomib (Fig. 3B). These results show that bortezomib, in contrast to cisplatin, potently induces the cytosolic release of mitochondrial proapoptotic factors in H460 cells.


Figure 3
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  		Figure 3. Bortezomib more potently than cisplatin promotes the release of mitochondrial proapoptotic proteins. A, cytochrome c and Smac/DIABLO release from mitochondria was determined by Western blot analysis of cytosolic and mitochondrial extracts from a concentration range and a time course treatment of H460 cells with bortezomib or cisplatin. B, abundant cytosolic cytochrome c release upon bortezomib treatment, but not cisplatin treatment, was confirmed by flow cytometry. C, H460 cells were treated with an IC80 concentration of either drug for 6, 16, or 24 h. Loss of cytochrome c from mitochondria resulted in a decrease in fluorescence intensity as determined by flow cytometry analysis. Percentages, proportion of cells with decreased cytochrome c fluorescence intensity as defined by the gate (bar).

 
Differential Effects of Bortezomib and Cisplatin on Bcl-2 Proteins
Furthermore, to determine whether the different efficiencies of bortezomib and cisplatin to induce cytosolic release of cytochrome c and Smac is related to differences in their ability to modulate the levels of proapoptotic and antiapoptotic Bcl-2 family members, we compared the effect of both drugs on the expression of Bcl-2 proteins using Western blot analysis. As shown in Fig. 4A , we observed an increase in the level of Mcl-1 upon treatment with bortezomib. Upon treatment with cisplatin, a slow, time-dependent decrease in the level of Mcl-1 was observed. The levels of Bcl-xL and Bcl-2 were not significantly altered by any of the drugs. Nevertheless the cleavage of Bcl-2 at 48 h posttreatment was clearly more evident upon bortezomib treatment. The levels of the proapoptotic members BAX and BAK were not affected by treatment with either drug. The level of proapoptotic BH3-only proteins, BIK and BIM, were slightly increased upon bortezomib treatment–only. Most notably, the BH3-only protein Noxa was dramatically up-regulated in cells treated with bortezomib but not with cisplatin. This was already evident after 6 h of treatment.


Figure 4
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  		Figure 4. Involvement of the Bcl-2 family in mediating bortezomib-induced apoptosis: Increased levels of Noxa and Mcl-1 proteins after bortezomib treatment. A, time course Western blot analysis of total cell extracts of H460 cells showing the effects of treatment with IC80 concentration of cisplatin (7 µmol/L) and bortezomib (100 nmol/L) on the levels of antiapoptotic and proapoptotic Bcl-2 family member proteins. B, bortezomib-induced binding and interaction of Noxa and Mcl-1 in H460 cells. Noxa or Mcl-1 were pulled down from total cell extracts, untreated or treated with bortezomib (100 nmol/L) for 24 h. Levels of coprecipitating Mcl-1 or Noxa were analyzed using Western blot analysis. Total lysate was included as a control. C, bortezomib (100 nmol/L), but not cisplatin (7 µmol/L), induces apoptosis in H460-derived cells stably overexpressing Bcl-2.

 
These results indicate that bortezomib and cisplatin differentially affect the levels of Bcl-2 family member proteins.

Bortezomib–up-regulated Noxa Displays High Affinity for Mcl-1
The differential effects of bortezomib and cisplatin on Bcl-2 family protein expression may provide an explanation for the potent induction of MOMP by bortezomib in contrast to cisplatin. As shown above, bortezomib increases the expression of both proapoptotic Bcl-2 proteins, BIK, BIM, and especially Noxa, and the antiapoptotic Bcl-2 protein Mcl-1, which is sufficient for the triggering of mitochondria-dependent apoptosis as shown by the increased release of mitochondrial proapoptotic factors and the activation of caspases. Although the level of Mcl-1 is gradually reduced in cisplatin-treated cells, Noxa remains undetectable even upon prolonged cisplatin exposure.

We used a coimmunoprecipitation approach to examine the interaction between Noxa and Mcl-1 in H460 cells treated with bortezomib. Western blot analysis of total cellular extracts confirmed the accumulation of Mcl-1 upon bortezomib treatment. Subsequent pull-down of Noxa shows the binding of Noxa to Mcl-1 (Fig. 4B). Additionally, reverse coimmunoprecipitation of Mcl-1 confirmed the interaction between Noxa and Mcl-1 in bortezomib-treated H460 cells.

This finding suggests that the observed Noxa/Mcl-1 binding in bortezomib-treated cells is able to release proapoptotic Bcl-2 family proteins bound to Mcl-1, such as activator BH3-only proteins or BAK, thereby facilitating MOMP and apoptosis (22, 33).

Bortezomib-Induced Apoptosis Is Partially Inhibited by Ectopic Overexpression of Bcl-2
Finally, to further study the role of Bcl-2 family members in mediating bortezomib-induced apoptosis, H460-derived cells stably overexpressing exogenous Bcl-2 were used. As shown in Fig. 4C, 100 nmol/L of bortezomib induced significant levels of apoptosis in these cells (~20%) when compared with cisplatin that hardly triggered apoptosis. However, the apoptotic potential of bortezomib was reduced by ~50% when compared with the levels obtained in parental H460 cells (~40% of apoptotic cells; Fig. 1B). The strong reduction of cisplatin-induced apoptosis in H460 cells by Bcl-2 (Figs. 1C and 4C) also suggests the involvement of this pathway in mediating cell death by this drug as we reported before, although the mechanism remains elusive, considering the failure of cisplatin to induce MOMP in NSCLC cells (26).

Discussion

Bortezomib is a potent and selective proteasome inhibitor. It is a new, promising antineoplastic therapeutic agent that currently has been approved for the treatment of relapsed multiple myeloma. Bortezomib, as a single agent, achieved a response rate of 10% in unselected advanced NSCLC patients who had already received one prior chemotherapy regimen (34).

Intrinsic apoptosis resistance of NSCLC cells is regarded as one of the prime mechanisms for the observed chemoresistance in this cancer type (35). We and others have provided experimental evidence that suppression of mitochondria-dependent caspase-9 activation contributes to the drug-resistant phenotype, in particular toward DNA damaging agents (26, 27, 36–38), although the exact molecular mechanism underlying this inhibition remains to be elucidated. New therapeutic strategies are needed, which can effectively bypass or restore apoptotic defects and possibly improve the poor survival rates of NSCLC patients. Here, we compared the apoptosis-inducing activity of bortezomib to that of cisplatin in H460 and H322 NSCLC cell lines. We then compared the molecular mechanism of apoptosis induction by bortezomib and cisplatin in one of these cell lines, such as H460. Bortezomib was able to effectively inhibit proteasome activation in these cells. Interestingly, bortezomib, in contrast to cisplatin, induced rapid activation and processing of caspase-9 and caspase-3 activation, which was followed by a strong apoptotic response. Moreover, bortezomib stimulated a pronounced release of the mitochondrial proapoptotic factors cytochrome c and Smac/DIABLO, whereas cisplatin did not. These findings are consistent with a previous report implicating the formation of radical oxygen species and loss of mitochondrial membrane potential in bortezomib-induced cytotoxicity in NSCLC cells (39). Additionally, we have recently reported that the addition of exogenous cytochrome c and dATP induces efficient apoptosome formation and caspase-9 activation in H460 cell extracts, indicating no intrinsic defects in this pathway, which is in agreement with the observed potent activation of this route by bortezomib (27). Taking into account all these observations, we propose that the amount of cytochrome c release upon cisplatin treatment is below the threshold needed to initiate the activation of the intrinsic pathway of apoptosis, whereas the abundant cytochrome c release induced by bortezomib is sufficient to induce caspase-9 activation. Interestingly, bortezomib, but not cisplatin, induced the release of the inhibitor of apoptosis proteins–antagonizing protein Smac/DIABLO, which additionally may contribute to the strong apoptotic response induced by bortezomib treatment (40).

MOMP, which determines the release of cytochrome c into the cytosol, is critically regulated by the balanced expression and interactions between proapoptotic and antiapoptotic members of the Bcl-2 family of proteins (41–43). The treatment with bortezomib or cisplatin revealed distinct effects on the expression level of various Bcl-2 family proteins. Bortezomib treatment altered the balance between Bcl-2 proteins toward apoptosis by inducing a dramatic increase in the levels of BH3-only "sensitizer" proteins Noxa and, to a lesser extent, BIK and by increasing the level of BH3-only "activator" protein BIM. Concomitantly, an increase in the level of antiapoptotic protein Mcl-1 was detected.

Interestingly, here we showed that bortezomib-induced apoptosis in NSCLC cells involves the up-regulation of three BH3-only proteins, Noxa, BIM, and BIK, in combination with accumulation of Mcl-1. Similar observations have been made in other cancer types, for example, bortezomib was reported to increase the expression of NOXA and Mcl-1 in melanoma or mantle-cell lymphoma cells and of BIK and BIM in different cancer cell lines (21–24). In our study, we showed that Noxa and Mcl-1 strongly interact in bortezomib-treated H460 cells. It seems that up-regulated Noxa is able to antagonize the antiapoptotic effect of Mcl-1. This observation is in agreement with a recently published report, showing that the increase of Noxa and Mcl-1 in bortezomib-treated mantle-cell lymphoma cells leads to the release of Bak from its inhibitory binding to Mcl-1, thus triggering apoptosis (22). According to the model that was proposed by Letai et al. (8, 33), up-regulated expression of the sensitizer BH3-only proteins Noxa and BIK can force the replacement of activator BH3-only proteins, such as up-regulated BIM, from binding to antiapoptotic proteins, such as Bcl-2, Bcl-xL, and Mcl-1. Released BIM can subsequently promote the activation and oligomerization of BAK/BAX and induce MOMP, followed by cytochrome c release and induction of cell death.

In line with the above, we showed that Bcl-2 overexpression in H460 cells partially suppresses bortezomib-induced apoptosis, further indicating that the Bcl-2 family plays a substantial role in mediating apoptosis by this agent. On the other hand, because Bcl-2 overexpression did not completely inhibit apoptosis, other not-yet-defined mechanisms are also involved in facilitating bortezomib-induced apoptosis in these cells.

Table 1 summarizes the differences that were detected between bortezomib-induced and cisplatin-induced mitochondrial-dependent apoptosis in NSCLC cells. The detailed molecular events underlying the deficiency of cisplatin to efficiently induce cytosolic cytochrome c release are currently under investigation.


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  		Table 1. Molecular events underlying the differential activation of the mitochondrial apoptotic pathway by cisplatin and bortezomib in H460 cells

 
This study shows the greater potential of bortezomib compared with cisplatin to efficiently activate the mitochondrial pathway of apoptosis in NSCLC H460 cells by modulation of the balance between Bcl-2 family proteins toward apoptosis. When extrapolated to the clinic, these in vitro findings might provide a rationale for the use of bortezomib-based anticancer therapy as a potential treatment strategy for NSCLC.

Footnotes

Grant support: Dutch Cancer Society KWF-NK grant 2001-2509 (A. Checinska) and the Netherlands Organization of Scientific Research ZonMW/NWO-AGIKO grant 920-03-290 (J. Voortman).

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.

Note: J. Voortman and A. Checinska contributed equally to this work.

Received 9/18/06; revised 12/19/06; accepted 1/31/07.

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