Targeting Antiapoptotic Bcl-2 Family Members with Cell-Permeable BH3 Peptides Induces Apoptosis Signaling and Death in Head and Neck Squamous Cell Carcinoma Cells^,

Abstract

Head and neck squamous cell carcinomas (HNSCCs) are frequently characterized by chemotherapy and radiation resistance and by overexpression of Bcl-Xl, an antiapoptotic member of the Bcl-2 protein family. In this report, we examined whether cell-permeable peptides derived from the BH3 domains of proapoptotic Bax, Bad, or Bak could be used to target Bcl-Xl and/or Bcl-2 in HNSCC cells and induce apoptotic death in these cells. To render the peptides cell-permeable, Antennapedia (Ant) or polyarginine (R8) peptide transduction domain was fused to the amino termini. Fluorescence microscopy of peptide-treated HNSCC cells revealed that the BH3 peptides colocalized with mitochondria, the site of Bcl-Xl and Bcl-2 expression. By contrast, a mutant peptide (BaxE BH3) that cannot bind Bcl-Xl or Bcl-2 was diffusely localized throughout the cytoplasm. Treatment of three HNSCC cell lines (1483, UM-22A, and UM-22B) with the wild-type BH3 peptides resulted in loss of viability and induction of apoptosis, as assessed by 3-(4,5-dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays and annexin V staining. In general, Ant-conjugated peptides weremore potent than R8-conjugated peptides and Bad BH3 peptide was typically more potent than Bax BH3 or Bak BH3. Treatment of purified HNSCC mitochondria with BH3 peptides resulted in robust release of cytochrome c. Thus, the relative apoptosis resistance of HNSCC cells is not due to a deficit in this step of the intrinsic, mitochondrialmediated apoptosis pathway. We conclude that cellpermeable BH3 peptides can be used to target Bcl-Xl and/or Bcl-2 in HNSCC and that targeting of these proteins may have therapeutic value in the treatment of this disease.

Introduction

Approximately 40,000 cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed each year in the United States and roughly 500,000 cases are reported worldwide [1]. Standard treatment options for HNSCC include radiation, surgery, and chemotherapy. However, HNSCCs typically exhibit radiation and chemotherapy resistance, and 5-year survival rates have lingered around 50% for several decades [2,3]. Thus, there is a tremendous need to develop new therapeutic strategies for this disease.

The frequent resistance of HNSCC to radiation and chemotherapy is due, in large part, to aberrant inhibition of apoptosis in the tumor cells. A number of signaling proteins and pathways that contribute to cellular survival by blocking apoptosis have been shown to be overexpressed and/or hyperactivated in HNSCC, and include the epidermal growth factor receptor (EGFR) [4,5], Akt kinase [6,7], mammalian target of rapamycin [8], nuclear factor-κB (NF-κB) [9], and signal transducer and activator of transcription-3 (STAT3) [10–12]. Targeting EGFR with cetuximab monoclonal antibody, antisense oligonucleotides, dominant-negative mutants, or the tyrosine kinase inhibitors gefitinib and erlotinib serves to attenuate signaling downstream from the receptor and inhibits the proliferation and survival of HNSCC in vitro and in vivo [5,13–15]. Similarly, targeting of STAT3 or NF-κB with antisense, dominant-negatives, small interfering RNA, or a STAT3 decoy oligonucleotide promotes HNSCC cell apoptosis and enhances the sensitivities of these cells to cisplatin, tumor necrosis factor-α, histone deacetylase inhibitors, and radiation [10,16–22]. In addition, treatment with rapamycin or small molecule inhibitors of Akt has been shown to induce apoptosis and tumor regression in xenograft models of HNSCC [7,8,23]. The success of these molecular targeting strategies in preclinical models of HNSCC has raised hope for their successful application in patients.

HNSCC cell lines and primary specimens are also characterized by frequent overexpression of antiapoptotic members of the Bcl-2 protein family, including Bcl-2 and Bcl-Xl. In particular, Bcl-Xl is overexpressed in a large majority of HNSCC and is associated with chemoresistance in this disease [24]. The bcl-Xl gene is a known downstream target of STAT3 and NF-κB [25,26] and the overexpression of Bcl-Xl in HNSCC may be due to the aberrant activation of these transcription factors. Antisense oligonucleotides directed against Bcl-Xl or Bcl-2 mRNA have been shown to activate apoptosis and sensitize HNSCC cell lines to chemotherapy drugs [27].

The Bcl-Xl and Bcl-2 proteins act to inhibit chemotherapy-and radiation-induced apoptosis by regulating the intrinsic, mitochondrial-mediated pathway of apoptosis. Specifically, Bcl-Xl and Bcl-2 prevent chemotherapy- and radiation-induced release of cytochrome c from mitochondria, thereby preventing activation of the caspase protease cascade and apoptotic destruction of the cell [28]. Bcl-Xl and Bcl-2 exert these effects by binding to the BH3 domains of proapoptotic Bcl-2 family members such as Bax, Bad, and Bak, neutralizing the death-inducing effects of these proteins [28,29]. Recently, the small organic molecule gossypol has been shown to bind to the BH3-binding pocket of Bcl-Xl [30,31]. Treatment of HNSCC cells with (-)-gossypol, the biologically active enantiomer, has been shown to induce apoptosis and promote chemosensitization [32–34]. However, it remains uncertain whether the proapoptotic action of (-)-gossypol in HNSCC is due entirely to the inhibition of Bcl-Xl and/or Bcl-2 [35]. Thus, evaluation of other highly specific agents is needed to clearly validate the utility of Bcl-Xl/Bcl-2 targeting in HNSCC.

In the present study, we evaluated the ability of small peptides derived from the BH3 domains of Bax, Bad, and Bak to promote apoptosis signaling and death in HNSCC cells. Previous studies have shown that short peptides derived from the BH3 domains of proapoptotic proteins can disrupt the heterodimerization of Bcl-Xl and Bcl-2 with proapoptotic binding partners in a highly specific fashion, and induce apoptosis in leukemia and prostate cancer models [36–42]. Our results show that following addition to cells, the cell-permeable BH3 peptides localize to the mitochondria, the subcellular site of Bcl-Xl and Bcl-2 expression, supporting the ability of the peptides to specifically target Bcl-Xl and/or Bcl-2 in intact HNSCC cells. Peptide treatment resulted in disruption of heterodimerization interactions between pro- and antiapoptotic Bcl-2 family members. The cell-permeable peptides also stimulated caspase activation and apoptotic death in HNSCC cells, with Bad BH3 peptide exhibiting the most potent activity. Taken together, our results indicate the potential therapeutic benefit of targeting antiapoptotic members of the Bcl-2 protein family in HNSCC.

Materials and Methods

Cell Lines and Reagents

The UM-22A, UM-22B, and 1483 cell lines were derived from HNSCC and all are of human origin [43–46]. 1483 cells were provided by Dr. Gary Clayman (MD, Anderson Cancer Center, Houston, TX), and UM-22A and UM-22B were provided by Dr. Thomas Carey (University of Michigan, Ann Arbor, MI). Cells were cultured at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Mediatech, Herndon, VA) containing 10% heat-inactivated fetal bovine serum (FBS), 100 µg/ml penicillin-streptomycin, and 0.5 µg/ml amphotericin B. Jurkat T leukemic cells were grown at 37°C and 5% CO₂ in RPMI medium (BioWhitaker, Walkersville, MD) containing 10% FBS, 100 µg/ml penicillin-streptomycin, and 0.5 µg/ml amphotericin B. Peptides were synthesized using a fluoren-9-ylmethoxycarbonyl synthesis protocol on a peptide synthesizer (Pioneer; Applied Biosystems, Foster City, CA) by the University of Pittsburgh Molecular Medicine Institute Peptide Synthesis Facility. Peptides were dissolved in DMSO and stored at -80°C. Hoechst stain solution was purchased from Sigma (St. Louis, MO) and MitoTracker Red fromMolecular Probes (Invitrogen, Carlsbad, CA). Streptavidin-fluorescein isothiocyanate (FITC) was from Molecular Probes as well. 3-(4,5-Dimethythiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reagent was obtained from Promega (Madison, WI). Bio-Rad Protein Assay dye concentrate was purchased from Bio-Rad (Hercules, CA). Anti-Bcl-2 antibody was from DAKO (Carpinteria, CA) and anti-Bcl-Xl antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-poly(ADP)-ribose polymerase (PARP) antibody was from Cell Signaling Technology (Danvers, MA). Anti-β-actin antibody was from Sigma. Annexin V-FITC Apoptosis Detection Kits and anti-cytochrome c antibody were from BD Pharmingen (San Diego, CA).

Fluorescence Microscopy

Fluorescence microscopy was used to examine the uptake and subcellular distribution of BH3 peptides in HNSCC cells. Cells were seeded onto glass coverslips (Fisher Scientific, Pittsburgh, PA) at 1 × 10⁴ cells per well in 24-well plates, grown overnight in 10% FBS/DMEM medium, then treated for varying lengths of time with biotinylated peptides (25 µM) or the peptide diluent DMSO (0.25%). Cells were then rinsed with cold PBS and incubated for 20 minutes at 37°C with 500 nM Mito Tracker Red to stainmitochondria. The cover-slips were then washed three times with 500 µl of cold PBS, and fixed with 3.7% paraformaldehyde for 30 minutes at 37°C. After fixation, cells were washed three times with 500 µl cold PBS, then permeabilized at room temperature with 2% paraformaldehyde containing 0.1%Triton X-100. Following another wash, the cells were incubated with 2 µg/ml streptavidin-FITC for 30 minutes at room temperature. Cells were also stained with 10 ng/ml Hoechst stain solution for 30 minutes at room temperature to identify nuclei. The coverslips were then transferred to slides and cells were visualized by fluorescence microscopy (Nikon Eclipse E800; Quantitative Imaging Corp., Burnaby, British Columbia, Canada).

Cell Viability Assays

For viability assays, cells were seeded at a density of 10,000 cells per well in 96-well plates, incubated overnight in 10% FBS/DMEM, washed with serum-free DMEM medium, then treated for 24 hours with varying doses of peptides in 100 µl of DMEM containing 1% FBS. Following treatment, cells were incubated for 2 hours at 37°C with 20 µl of MTS reagent. Absorbance was measured at 490-nm wavelength using a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Data were analyzed by GraphPad PRISM version 4 software (San Diego, CA) to determine IC₅₀ values.

Immunoblotting

Cells were harvested by centrifugation for 5 minutes at 4°C then washed once with cold PBS. The cell pellets were lysed for 10 minutes on ice in lysis buffer (50mMTris, pH 8.0, 150 mM NaCl, 0.1% SDS, and 1% NP-40) containing 3 µg/ml leupeptin, 20 µg/ml aprotinin, and 1.5 mM PMSF. The lysates were then pelleted at 11,000 × g for 2 minutes at 4°C and supernatants were transferred to new tubes. Protein concentrations were determined using Bio-Rad Protein Assay dye concentrate. For detection of Bcl-2 and Bcl-Xl, proteins (40 µg per lane) were electrophoresed on 12.5% SDS-PAGE gels. For detection of PARP cleavage products, proteins (60 µg per lane) were electrophoresed on 7.5% SDS-PAGE gels. Following electrophoresis, proteins were transferred to nitrocellulose membranes for 3 hours at 45 V. Membranes were blocked in 5% nonfat milk in TBST buffer (10 mM Tris pH 8.0, 150 mM NaCl, and 0.1% Tween 20) at room temperature for 1 hour, briefly washed with TBST buffer, and probed with primary antibody overnight at 4°C. After one 15-minute wash and three 5-minute washes in TBST buffer, the membranes were incubated with secondary antibody for 1 hour at room temperature. The membranes were then washed once with TBST buffer for 15 minutes, followed by three washes for 5 minutes each. The blots were developed using enhanced chemiluminescence reagent, according to the instructions of the manufacturer (PerkinElmer, Boston, MA).

Flow Cytometry Analysis

Externalization of phosphatidylserine was assessed using annexin V-FITC Apoptosis Detection Kits followed by flow cytometry. Cells were seeded at 1.8 × 10⁶ cells per 100-mm dish, grown overnight in 10% FBS/DMEM, washed with serum-free DMEM, then treated for 4 hours with 25 µM of the peptides in 1% FBS/DMEM. Following incubation, cells were washed with cold PBS and removed from the plates by trypsinization. The resuspended cells were washed with cold PBS and stained with FITC-conjugated annexin V antibody and propidium iodide (PI) according to the instructions provided by the manufacturer (Roche Applied Science, Indianapolis, IN). Cells (50,000 per sample) were then subjected to flow cytometric analysis using a flow cytometer (Epics Coulter XL; Beckman-Coulter, Inc., Miami, FL) equipped with a 488-nm argon laser and Expo 32 software (Beckman-Coulter).

Mitochondria Isolation and Cytochrome c Release Assays

Mitochondria were isolated from healthy HNSCC cell lines and Jurkat cells growing in 10% FBS/DMEM. When the cells achieved approximately 80% confluence, 1 × 10⁷ cells were harvested by centrifugation for 8 minutes at 4°C. The cells were washed once with cold PBS, centrifuged again, then resuspended in 100 µl of resuspension buffer (20 mM Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 250 mM sucrose, 1 mM DTT, 1.5 mM PMSF, 3 µg/ml leupeptin, and 20 µg/ml aprotinin). The resuspended cells were then homogenized using a type B tight-clearance dounce homogenizer (Wheaton, Millvale, NJ). The homogenized lysates were centrifuged at 11,000 × g for 30 minutes at 4°C to pellet mitochondria. The pellets were washed in 100 µl of resuspension buffer then centrifuged again at 11,000 × g for 30 minutes at 4°C. The mitochondria-containing pellets were resuspended in 100 µl of resuspension buffer and protein concentrations were determined using Bio-Rad Protein Assay dye concentrate. To perform cytochrome c release assays, resuspended mitochondria (40 µg) were incubated in the absence or presence of 25 µM of the BH3 peptides or DMSO (2%) for 1 hour at 37°C. Following incubation, the treated mitochondria were centrifuged at 11,000 × g for 30 minutes at 4°C and the supernatants were transferred to new tubes. The supernatants, containing proteins released from the mitochondria, were then subjected to a second centrifugation at 11,000 × g for 30 minutes at 4°C to eliminate potential contamination with intact mitochondria. Aliquots of the supernatants (50 µl) were then electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose membranes, and subjected to immunoblotting with anti-cytochrome c antibody.

Results

Cell-Permeable Bax BH3 and Bad BH3 Peptides Localize to Mitochondria in HNSCC Cells

To determine whether peptides corresponding to the BH3 domains of proapoptotic proteins could be used to target Bcl-Xl and/or Bcl-2 in HNSCC cells, fluorescence microscopy was used to examine the subcellular localization of the peptides in treated cells. Bcl-Xl and Bcl-2 are known to be expressed on the outer membrane of mitochondria. Thus, agents that selectively bind and target Bcl-Xl and Bcl-2 would be expected to localize to mitochondria. Figure 1A depicts peptide localization in the HNSCC cell line UM-22B. The peptides were made cell-permeable by conjugating the 16-amino acid Antennapedia (Ant) peptide transduction domain [47] to the amino terminus of the 20-amino acid Bax BH3 peptide and the 21-amino acid Bad BH3 peptide (Table 1) [38,40]. As a control, Ant was also conjugated to the amino terminus of a mutant version of Bax BH3 peptide (termed BaxE), wherein a critical leucine residue in the wild-type peptide was changed to a glutamate residue [38,48]. In full-length Bax protein, mutation of this leucine residue to glutamate has been shown to abolish the ability of Bax to bind to antiapoptotic proteins and promote apoptosis [48]. All three peptides (Ant-Bax, Ant-BaxE, and Ant-Bad) were biotinylated, allowing fluorescent detection by streptavidin-FITC. Mitochondria were stained using MitoTracker Red dye and Hoechst staining was used to show nuclei. The UM-22B cells were treated for 2 or 6 hours with peptides (25 µM) or with 0.25% DMSO, the peptide diluent. As shown (Figure 1A), after 2 hours of treatment, all three peptides were detected in the cytoplasm of the treated cells. In the case of Ant-Bad, and particularly Ant-Bax, the cytoplasmic staining exhibited a punctate pattern similar to that observed for MitoTracker Red staining of mitochondria. Conversely, the mutant control peptide Ant-BaxE demonstrated diffuse cytoplasmic staining. When fluorescence from the peptide (green) and mitochondria (red) staining were merged, substantial yellow fluorescence was observed in cells treated with Ant-Bad and Ant-Bax. This indicated that the Ant-Bad and Ant-Bax peptides were localizing to the mitochondria, the site of Bcl-Xl and Bcl-2 expression. By contrast, no yellow fluorescence was observed in cells treated with Ant-BaxE, which is defective in its ability to bind to Bcl -Xl or Bcl-2. By 6 hours, robust levels of Ant-Bax and Ant-Bad were observed in the cytoplasm and yellow fluorescence indicative of peptide localization at the mitochondria was seen in the merged images. Moreover, cells treated with Ant-Bax or Ant-Bad for 6 hours exhibited altered morphology, with accompanying apoptotic debris, particularly in cells treated with Ant-Bad. Again, no mitochondrial localization (yellow fluorescence) of the control peptide Ant-BaxE was seen after 6 hours, and Ant-BaxE-treated cells did not exhibit any changes in morphology. Taken together, the fact that BH3 peptides capable of binding to Bcl-Xl and Bcl-2 were localized to the mitochondria, whereas a BH3 peptide that fails to bind Bcl-Xl and Bcl-2 did not localize to the mitochondria, indicates that the Ant-Bax and Ant-Bad peptides were targeting Bcl-Xl and Bcl-2 in the HNSCC cells. The disruption of cellular morphology and the appearance of apoptotic debris in the Ant-Bax- and Ant-Bad-treated cells further indicates that targeting of Bcl-Xl and/or Bcl-2 in HNSCC cells results in induction of apoptotic death, a finding explored in greater detail in subsequent experiments.

Figure 1.

Ant-Bax and Ant-Bad colocalize with mitochondria and disrupt Bcl-2/Bax complexes in UM-22B cells. (A) UM-22B cells were seeded onto glass coverslips at 1 × 10⁴ cells per well (24-well plates), grown overnight, and treated with 25 µM biotinylated Ant-Bax, biotinylated Ant-Bad, biotinylated mutant peptide (Ant-BaxE), or with 0.25% DMSO (peptide solvent). After 2 and 6 hours of treatment, cells were incubated with 500 nM MitoTracker Red to stain mitochondria (red). Following fixation and permeabilization, cells were incubated with FITC-conjugated streptavidin to visualize peptide uptake (green). Cells were further incubated with Hoechst stain solution to stain nuclei (blue) and were then examined by fluorescence microscopy. A merged image (yellow) was used to detect colocalization of peptides (green) and mitochondria (red). The results demonstrate colocalization of Ant-Bax and Ant-Bad peptides, but not mutant Ant-BaxE, with mitochondria. Disruption of cellular morphology was also observed in cells treated with Ant-Bax (6 hours) or Ant-Bad (2 and 6 hours). (B)UM-22B cells were seeded at 1 × 10⁶ cells per 100-mm dish, then treated for 2 hours in the absence or presence of 50 µM Ant-Bad, Ant-Bax, or Ant-BaxE mutant peptide, or 0.25% DMSO alone. Whole cell lysates (2 × 10⁶ cells per lane) were prepared, then subjected to immunoprecipitation with anti-Bax polyclonal antibody, and followed by immunoblotting with anti-Bcl-2 monoclonal antibody.

Table 1.

Cell-Permeable BH3 Peptides.

Cell-Permeable Bax BH3 and Bad BH3 Peptides Disrupt Bcl-2/Bax Heterodimerization in Intact HNSCC Cells

To determine whether the cell-permeable BH3 peptides could disrupt physical interactions between pro- and antiapoptotic Bcl-2 family members in intact HNSCC cells, we performed coimmunoprecipitation experiments (Figure 1B). UM-22B cells were treated in the absence or presence of peptides, followed by immunoprecipitation with anti-Bax and immunoblotting with anti-Bcl-2 to detect Bcl-2/Bax heterodimerization complexes. Treatment of the cells with Ant-Bad or Ant-Bax resulted in a substantial decrease in the levels of intracellular Bcl-2/Bax, relative to untreated cells or cells treated with DMSO alone. By contrast, treatment with the mutant control peptide, Ant-BaxE, caused only a modest decline in Bcl-2/Bax heterodimer levels. These findings demonstrate that the wild-type BH3 peptides are capable of displacing proapoptotic proteins bound to antiapoptotic Bcl-2 family members in intact HNSCC cells.

Cell-Permeable BH3 Peptides Promote HNSCC Cell Death

We next examined whether cell-permeable BH3 peptides could induce cell death in HNSCC cell lines (Figure 2). Peptides corresponding to the BH3 domains of Bax, Bad, and Bak, as well as mutant control Bax (BaxE), were made cell-permeable by conjugating the Ant (Figure 2, A, C, and E) or polyarginine (R8; Figure 2, B, D, and F) peptide transduction domains to the amino termini of the peptides (Table 1). The cell-permeable peptides were then used to treat three different HNSCC cell lines, i.e., 1483, UM-22A, and UM-22B [43–45]. In this panel, UM-22A cells represent cells derived from a primary HNSCC tumor, whereas UM-22B cells were derived from a cervical lymph node metastasis in the same patient. 1483 cells were also derived from a primary tumor. Following treatment for 24 hours with varying doses of the peptides, MTS assays were performed, and IC₅₀ values were determined (Table 2). As shown in Figure 2, the mutant control peptides (Ant-BaxE or R8-BaxE) exhibited little, if any, killing activity against the three HNSCC cell lines. By contrast, all three wild-type peptides demonstrated some level of killing activity. In general, peptides conjugated to Ant showed more potent killing activity than the R8-conjugated peptides. In addition, UM-22B cells exhibited greater resistance to the BH3 peptides than 1483 or UM-22A cells, consistent with the fact that UM-22B cells represent a more advanced, metastatic disease stage. In the case of 1483 cells, all three Ant-BH3 peptides promoted cell death, with IC₅₀ values of 18.2 µM, 24.8 µM, and 32.9 µM for Ant-Bax, Ant-Bad, and Ant-Bak, respectively. In UM-22A cells, only Ant-Bad (IC₅₀ = 18.3 µM) and Ant-Bax (IC₅₀ = 45.5 µM) were effective at killing, whereas in UM-22B, only Ant-Bad (IC₅₀ = 40.2 µM) exhibited activity. Similar results were obtained when Trypan Blue exclusion assays were employed (not shown).

Figure 2.

Antennapedia (Ant)- and polyarginine (R8)-conjugated BH3 domains of Bax, Bad, and Bak promote loss of HNSCC cell viability. 1483 (A and B), UM-22A (C and D), and UM-22B (E and F) cells were seeded into 96-well plates, grown overnight, then treated for 24 hours with varying doses of Ant- or R8-conjugated peptides in 1% FBS/DMEM medium. Following treatment, cells were analyzed using MTS assays. Each data point represents the average from triplicate wells and error bars represent standard deviations. Data were plotted as the percent of metabolic activity relative to untreated cells. IC50 values were calculated as described in the Materials and Methods section, and they are shown for Ant-conjugated peptides in Table 2. The experiments were performed three times, with similar results each time.

Table 2.

IC₅₀ Values of Ant-Conjugated Peptides.

To determine whether the differing sensitivities of the three HNSCC cell lines to the BH3 peptides may be due to different levels of expression of Bcl-Xl or Bcl-2, we performed immunoblotting with anti-Bcl-Xl or anti-Bcl-2 (Figure 3). Bcl-Xl has been reported to be frequently overexpressed in primary HNSCC specimens [24]. Consistent with this, all three cell lines were found to express abundant levels of the Bcl-Xl protein. 1483 cells had the lowest Bcl-Xl levels, correlating with the greater sensitivity of this cell line to the BH3 peptides. Bcl-2 levels varied among the three cell lines, with UM-22B cells exhibiting the highest expression. The high levels of Bcl-Xl and Bcl-2 found in UM-22B cells correlated with the greater resistance of these cells to the BH3 peptides. In addition, it is interesting to note that, whereas the Bcl-Xl levels in UM-22A and UM-22B were comparable, Bcl-2 expression was dramatically higher in UM-22B cells. This suggests, in this case, that conversion to a metastatic phenotype was associated with the acquisition of Bcl-2 overexpression status.

Figure 3.

Bcl-2 and Bcl-Xl expression levels in HNSCC cell lines. Whole cell protein extracts (40 µg per lane) from 1483, UM-22A, and UM-22B cells were subjected to immunoblotting with anti-Bcl-2 or anti-Bcl-Xl antibodies. Membranes were stripped and reprobed with anti-β-actin to demonstrate equal loading.

To determine whether the reduced killing activity of R8-conjugated peptides relative to Ant-conjugated peptides was due to reduced cellular uptake of the R8-conjugated peptides, we employed flow cytometry to assess peptide uptake (Figure W1). Cells were left untreated or were treated for 45 minutes with DMSO, biotinylated R8-Bax, or biotinylated Ant-Bax. Streptavidin-FITC and flow cytometry were then used to determine the percentage of cells that had taken up peptide. Surprisingly, whereas 50.4% of cells scored positive for uptake of R8-Bax in this experiment, only 23.0% of cells scored positive for Ant-Bax uptake (see Figure W1). The more efficient uptake, but reduced killing activity, of the R8-conjugated BH3 peptides suggests that these peptides may have reduced affinities for Bcl-Xl and/or Bcl-2 compared with the Ant-conjugated versions.

BH3 Peptides Induce HNSCC Apoptosis

Agents that directly target and inhibit antiapoptotic proteins such as Bcl-Xl and Bcl-2 might be expected to predispose a cell to apoptosis, or to directly activate apoptotic death. To determine whether the cell-permeable BH3 peptides activated an apoptotic form of cell death, HNSCC cell lines were exposed to peptides, then analyzed by flow cytometry for annexin V and PI staining. Annexin V staining serves as a measure of phosphatidylserine externalization, and cells that are annexin V⁺/PI^- represent early apoptotic cells [49,50]. A representative experiment is shown in Figure 4A, in which UM-22A cells were treated with 25 µM peptide for 4 hours. As shown, the appearance of early apoptotic cells (annexin V⁺/PI^-) is seen in cells treated with Ant-Bax, Ant-Bad, or Ant-Bak. By contrast, cells that were either untreated, or were treated with 0.25% DMSO or the control peptide Ant-BaxE (25 µM) showed no appreciable signs of apoptosis induction. After 4 hours of treatment, the Ant-Bad peptide produced the highest levels of apoptosis (31.7%), consistent with the results from MTS assays (Figure 2C). Similar results were seen in cells treated with peptides for 3 hours (data not shown). Longer treatment times were needed to observe high levels of apoptosis in Ant-Bax-treated cells (data not shown). However, Ant-BaxE mutant peptide failed to induce apoptosis even after prolonged incubation.

Figure 4.

Cell-permeable BH3 peptides activate apoptotic signaling and cell death. (A) UM-22A cells were seeded at 1.8 × 10⁶ cells per 100-mm dish, grown overnight, treated for 4 hours with 0.25% DMSO or with 25 µM Ant-Bax, Ant-Bad, Ant-Bak, or Ant-BaxE peptides. Following incubation, cells were stained with annexin V-FITC and PI and subjected to flow cytometric analysis. (B) Cell-permeable BH3 peptides induce caspase protease activation in HNSCC cells. UM-22A cells were seeded at 1.8 × 10⁶ cells per 100-mm dish, grown overnight, and then treated for 90 minutes with 0.5% DMSO or with 50 µM Ant-BaxE, Ant-Bax, Ant-Bad, or Ant-Bak. Following incubation, whole cell lysates were prepared and subjected to immunoblotting with anti-PARP antibody. A representative example of three independent experiments is shown.

The induction of apoptotic death is also characterized by the activation of caspase proteases, and the cleavage of caspase substrate proteins. Caspase-3, a major executioner caspase, is known to cleave PARP, a 116-kDa protein, to fragments of 89 and 24 kDa [51]. To determine whether treatment with the cell-permeable BH3 peptides resulted in caspase protease activation, treated HNSCC cells were analyzed by immunoblotting with anti-PARP antibody. A representative experiment using UM-22A cells treated for 90 minutes is shown in Figure 4B. Appearance of the 89-kDa PARP fragment was clearly visible in cells treated with Ant-Bad or Ant-Bak, although longer treatment times were needed to detect substantial PARP cleavage in Ant-Bax-treated cells. Taken together, our flow cytometric analyses and PARP cleavage analyses demonstrate that the cell-permeable BH3 peptides induce apoptotic cell death in HNSCC cells.

HNSCC Cells Are Not Defective in Mitochondrial-Mediated Apoptosis

We and others have previously reported that BH3 peptides potently kill chemotherapy-sensitive hematopoietic cell lines with IC₅₀ values in the low micromolar range [39,40,52]. As shown in Figure 5A, when Jurkat T leukemic cells were treated for 48 hours with Ant-Bad or Ant-Bax, IC₅₀ values of 3.8 µM and 5.6 µM, respectively, were observed. Much higher concentrations were needed to kill HNSCC cell lines (Figure 2 and Table 2). Because HNSCC cells are typically more resistant to chemotherapy drugs and because chemotherapy-induced apoptosis is known to be mediated by the mitochondrial or intrinsic apoptosis pathway [53], this raised the possibility that HNSCC cells may be defective in mitochondrial-mediated apoptosis signaling. To determine whether the reduced responsiveness of HNSCC cell lines to BH3 peptides was due to defective mitochondrial signaling, we isolated mitochondria from the three HNSCC cell lines and from Jurkat cells. The isolated mitochondria were then treated with peptides and the release of cytochrome c from mitochondria was assessed by immunoblotting of the supernatants (Figure 5B). The release of cytochrome c into cytosol represents a key step during the intrinsic, mitochondrial-mediated pathway of apoptosis [28,54]. As shown in Figure 5B, Ant-Bax, Ant-Bad, and Ant-Bak induced potent release of cytochrome c from the HNSCC mitochondria. By contrast, only negligible release was detected from HNSCC mitochondria treated with DMSO or the control peptide Ant-BaxE. These data were confirmed by immunoblotting of the mitochondrial pellets to show depletion of mitochondrial cytochrome c by the wild-type peptides (Figure 5B). Collectively, these results provide further support for the contention that the wild-type BH3 peptides directly target and inhibit Bcl-Xl and/or Bcl-2 on the surface of HNSCC mitochondria. In the case of isolated Jurkat mitochondria, the background levels of cytochrome c release detected in untreated, DMSO-treated, and Ant-BaxE-treated were significantly elevated. However, when shorter exposures of the gel were used to reduce the intensity of the background bands (compare 20-second exposure for Jurkat mitochondria with 1-minute exposure for HNSCC mitochondria), it can be seen that the induction of cytochrome c release by wild-type BH3 peptides is roughly comparable between Jurkat mitochondria and mitochondria from the HNSCC cell lines. This indicates that HNSCC cells are not defective in this key step in the intrinsic, mitochondrial-mediated apoptosis pathway.

Figure 5.

BH3 peptides induce comparable cytochrome c release from mitochondria isolated from chemosensitive Jurkat cells and chemoresistant HNSCC cells. (A) Cell-permeable BH3 peptides are potently active at killing Jurkat T leukemic cells. Jurkat cells were seeded at 1 × 10⁵ cells/ml in 96-well plates in triplicate, grown overnight, and then treated with varying doses of Ant-Bad, Ant-Bax, or Ant-BaxE mutant peptide, as well as with Ant peptide alone. Following incubation, cells were analyzed using MTS assays as described in Figure 2. (B) Induction of cytochrome c release from mitochondria isolated from Jurkat, UM-22A, 1483, and UM-22B cells. Isolated mitochondria (40 µg per sample) were incubated for 1 hour at 37°C with 25 µM Ant-Bax, Ant-Bad, Ant-Bak, or the mutant control peptide, Ant-BaxE. Following incubation, the mitochondria were pelleted to obtain supernatants containing released cytochrome c. The supernatants and mitochondrial pellets were electrophoresed on 12.5% SDS-PAGE gels, transferred to nitrocellulose, and probed with anti-cytochrome c (Cyt. C). The experiment was performed three times, with similar results each time.

Discussion

HNSCC is a common human cancer with poor 5-year survival rates [1–3]. A major underlying problem in the treatment of HNSCC is the inherent resistance of the cancer cells to chemotherapy and radiation. This resistance is caused, in large part, by the aberrant inhibition of apoptosis signaling. In this report we sought to determine whether direct targeting of Bcl-Xl and/or Bcl-2 would promote apoptosis induction in HNSCC cells. Although previous reports have shown that (-)-gossypol, a small organic molecule that binds Bcl-Xl, promotes apoptosis in HNSCC cells [32–34], it has been difficult to validate Bcl-Xl and/or Bcl-2 as the primary targets of (-)-gossypol in intact HNSCC cells [35]. The BH3 peptides used in our studies offer the advantage that they can be labeled with biotin and be detected with streptavidin-FITC to demonstrate colocalization with the site of Bcl-Xl/Bcl-2 expression. In addition, BH3 peptides derived from proapoptotic proteins are highly specific for binding to antiapoptotic members of the Bcl-2 protein family [38,39,42,53,55–57]. Our results show that wild-type BH3 peptides colocalize with mitochondria in intact HNSCC cells, the site of Bcl-Xl and Bcl-2 expression (Figure 1A). The fact that a mutant BH3 peptide (Ant-BaxE) incapable of binding to Bcl-Xl or Bcl-2 no longer localized to mitochondria strongly supports the contention that the wild-type BH3 peptides directly targeted Bcl-Xl and/or Bcl-2 in intact HNSCC cells. This was supported by the finding that wild-type, and not mutant, peptides caused physical disruption of Bcl-2/Bax complexes in intact cells (Figure 1B). In addition, the wildtype Bax, Bad, and Bak BH3 peptides, and not the BaxE mutant peptide, were able to promote cytochrome c release from isolated HNSCC mitochondria (Figure 5). Together, these experiments indicate direct intracellular targeting of Bcl-Xl and/or Bcl-2 by the cell-permeable BH3 peptides.

Treatment of the three different HNSCC cell lines with the cell-permeable BH3 peptides resulted in activation of apoptosis signaling, as evidenced by phosphatidylserine externalization (annexin V staining) and PARP cleavage (Figure 4). Ultimately, loss of viability and cell death were observed (Figure 2). Thus, utilization of the BH3 peptides provided clear demonstration that direct targeting of Bcl-Xl and/or Bcl-2 in HNSCC cells leads to apoptosis induction. Comparison of the different peptides revealed that Ant-conjugated peptides were generally more efficacious at killing HNSCC cells than the R8-conjugated peptides. By contrast, R8-conjugated peptides appear to be taken up with slightly greater efficiency than the Ant-conjugated peptides. This suggests that conjugation to the Ant or R8 peptide transduction domains may differentially alter the affinities of the BH3 peptides for Bcl-Xl and/or Bcl-2.

We also observed that the cell-permeable Bad BH3 peptide was usually the most potent peptide for inducing apoptosis signaling and cell death. Prior studies have shown that full-length Bad protein and Bad BH3 peptide bind with higher affinity to Bcl-Xl than to Bcl-2 [58,59]. Because Bcl-Xl appears to be more highly overexpressed in the HNSCC cell lines we employed (Figure 3), an agent that exhibits preferential binding for Bcl-Xl might be predicted to exert more potent biological effects, as was observed in the case of Bad BH3. Other studies using in vitro binding assays have shown that Bad BH3 peptide is more efficacious than Bax BH3 peptide at disrupting interactions between Bcl-Xl and Bax proteins [38,40]. Because Bcl-Xl overexpression is more common than Bcl-2 overexpression in HNSCC primary specimens [24,60], it may be useful to focus on the Bad BH3 peptide for future efforts aimed at optimization and generation of derivatives or analogs.

The application of peptidic agents to the treatment of cancer is complicated by issues of size, stability, and uptake of the peptides or peptidomimetic derivatives. Nonetheless, our studies using BH3 peptides demonstrate that specific targeting of antiapoptotic Bcl-2 family members can be used to enhance the apoptotic sensitivities of HNSCC cells. Our findings support investigation in HNSCC of novel small molecule inhibitors of Bcl-Xl and/or Bcl-2, including agents such as ABT-737 and A-385358 which exhibit highly potent activities against solid tumor cell lines [61,62]. The eventual clinical application of highly specific and potent Bcl-Xl/Bcl-2 targeting agents may have significant therapeutic value in the treatment of chemotherapy- and radiation-resistant HNSCC.

Abbreviations

Ant

Antennapedia

DMEM

Dulbecco's modified Eagle's medium

EGFR

epidermal growth factor receptor

FITC

fluorescein isothiocyanate

HNSCC

head and neck squamous cell carcinoma

NF-κB

nuclear factor-kappaB

PARP

poly(ADP)-ribose polymerase

PI

propidium iodide

R8

polyarginine

STAT3

signal transducer and activator of transcription-3