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10.1245/s10434-006-9298-z
Annals of Surgical Oncology 14:1763-1771 (2007)
© 2007 Society of Surgical Oncology
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Original Article

TAT-Bim Induces Extensive Apoptosis in Cancer Cells

Hiroyuki Kashiwagi1, Jonathan E. McDunn1,2, Peter S. Goedegebuure1,5, Mary C. Gaffney1, Katherine Chang2, Kathryn Trinkaus3, David Piwnica-Worms4, Richard S. Hotchkiss2 and William G. Hawkins1,5

1 Department of Surgery, Washington University School of Medicine, St. Louis, MO 63110, USA
2 Department of Anesthesiology, Washington University School of Medicine, St. Louis, MO 63110, USA
3 Division of Biostatistics, Washington University School of Medicine, St. Louis, MO 63110, USA
4 Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
5 Alvin J. Siteman Cancer Center, St. Louis, MO 63110, USA

Correspondence: Address correspondence and reprint requests to: William G. Hawkins, Department of Surgery, WUMS, 660 S. Euclid Avenue, Campus Box 8109, St. Louis, MO 63110, USA; E-mail: hawkinsw{at}wustl.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: Suppression of apoptosis is central to the development of cancer and is associated with resistance to modern adjuvant treatments. Therefore, molecules and pathways of apoptotic processes are critical targets for the development of anti-cancer therapeutics. Since apoptosis is executed by intracellular proteins, molecular approaches must incorporate a method to deliver the treatment into the tumor cells.

Methods: We utilized a peptide that contains two domains, a peptide transduction domain derived from the HIV-1 TAT protein and a biological effector domain, the BH3 domain from the pro-apoptotic Bcl-2 family member Bim. We examined whether this construct (TAT-Bim) induced apoptosis in several cancer cell lines (T-cell lymphoma (EL4), pancreatic cancer (Panc-02), and melanoma (B16)) and whether TAT-Bim treatment synergized with radiation. A mutant TAT-Bim peptide with no biologic activity (TAT-Bim-inactive) was used as a control. C57/BL6 mice were challenged with syngeneic cancer cell lines and the effects of intratumoral TAT-Bim injection on tumor growth and host survival were determined.

Results: TAT-Bim was internalized by all cancer cells within two hours. TAT-Bim resulted in apoptosis in a dose dependent fashion in all cell lines and sublethal irradiation augmented the effects of TAT-Bim induced apoptosis. TAT-Bim significantly slowed tumor growth in murine models of pancreatic cancer and melanoma.

Conclusion: TAT-Bim exemplifies a strategy for cancer therapy that involves inducing apoptosis by antagonizing the endogenous anti-apoptotic machinery. Small peptide therapeutics, in combination with traditional adjuvant therapies such as radiation, may provide a valuable ‘second hit’ and drive tumor cells into programmed cell death.

Key Words: Apoptosis • Tat-fusion protein • Bim • Peptide therapeutic


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Programmed cell death or apoptosis is an essential process in cell homeostasis and eukaryotic development.1,2 Failure to appropriately activate the apoptotic program is associated with inflammatory and autoimmune disorders, while premature acceleration of apoptosis has been linked to neurodegenerative disease.3–7 Recent studies have shown that suppression of apoptosis is both central to the development of cancer and related to resistance toward modern adjuvant treatment such as chemo- or radiation therapies.8–10 As a result, there is considerable interest in targeting the molecular pathways of apoptosis as a component of cancer therapy.8,11,12

Although different cell types undergo apoptosis in response to different stimuli, there are two predominant pathways by which cells initiate apoptosis, the intrinsic (mitochondrial) pathway and the extrinsic (death receptor) pathway. These pathways converge in the activation of the executioner caspases including caspase-3. The prominent role of dysregulated apoptosis in many cancers is underscored by the identification of many oncogenes as regulators of apoptosis such as Bcl-2 and Mcl-1. Bcl-2 was the first-identified member of a family of proteins that regulate the intrinsic pathway in response to cellular stress such as anticancer drugs13 (chemotherapy), DNA-damaging agents14 (UV and ionizing radiation15,16), kinase inhibitors17,18, hypoxia19, and growth factor withdrawal.17

The Bcl-2 family is characterized by the presence of one or more conserved domains termed Bcl-2 homology domains (BH1, BH2, BH3, BH4) corresponding to four {alpha}-helical segments of Bcl-2. The Bcl-2 family is further divided into three groups based on their BH domain composition. The antiapoptotic members (including Bcl-2, Bcl-xL, Mcl-1) contain all four BH domains. There are two groups of proapoptotic members of the Bcl-2 family subdivided into BH3-only proteins (Bim, Bid, Bad, PUMA, etc.) and BH1-BH3 multidomain proteins (Bax, Bak, Bok, etc).20 Bcl-2 family members regulate the intrinsic apoptotic pathway in part by participating in homotypic and heterotypic interactions. The BH3-only proteins antagonize the anti-apoptotic members of the Bcl-2 family by binding to their BH4 domains. Saturating the BH3-sequestering capacity of anti-apoptotic Bcl-2 family members enables the multi-domain pro-apoptotic Bcl-2 family members to induce mitochondrial cytochrome C release and initiate apoptosis.20,21

Bim, a BH3-only protein, is expressed by cells of hematopoietic, neuronal and epithelial lineage, and it is essential for homeostasis.22 Recent studies indicated that Bim is a crucial antagonist of Bcl-2-mediated survival. Over-expression of Bim in cancer cells activated Bax and Bak, which mediate the release of cytochrome C from mitochondria resulting in tumor death.23,24 Bim has also been associated with cancer death induced by radiation therapy or chemotherapy, including paclitaxel, which is among the most effective chemotherapeutic agents for patients with lung, prostate and breast cancer.25,26 These studies indicate that Bim can act as a tumor suppressor and that cancer cells can be driven to undergo apoptosis simply by increasing the intracellular concentration of Bim.

We utilized a novel multidomain peptide that activates apoptosis in cancer cells.23 Because of the well-documented role of Bim in tumor cell apoptosis, we used the BH3 domain of Bim as the biological effector domain. To deliver this peptide intracellularly, we fused it to a peptide based on the polycationic peptide from the HIV-1 TAT (Trans-Activator of Transcription) protein that has been shown to transport large (>10 kDa) cargo across cell membrane.27,28

Here we tested whether TAT-Bim can penetrate tumor cells and induce apoptosis. As chemotherapy and radiation therapy also induce apoptotic stress, we tested whether TAT-Bim-induced apoptosis was augmented by radiation. Finally we attempted to treat established tumors in murine models of cancer with intratumoral injection of our constructs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Peptide Synthesis
TAT-Bim was purchased from the Peptide Synthesis Group of the Tufts University Core Facility (analytical scale, 5–10 mg per lot) and Global Peptide Services (semi-preparative scale, 100 mg per lot, Fort Collins, Colorado, USA). Briefly, the peptide was synthesized using standard HOBt/Fmoc chemistry, purified by reverse phase HPLC to >95% purity and the amino acid composition was verified using amino acid analysis and MALDI TOF mass spectrometry. Trifluoroacetate was replaced with acetate using ion exchange chromatography. The variant TAT sequence is composed of D-amino acids and has a glutamine to ornithine substitution that has been shown to increase uptake 10-fold.29 We designed a negative control peptide containing Bim with two point mutations L152A and D157A that have been shown to abrogate binding to BH4-domain containing proteins.23 The amino acid sequences of TAT-Bim and TAT-Bim-inactive used in this study are:

TAT-Bim: Ac-rkkrr-orn-rrr-EIWIAQELRRIGDEF NAYYAR-OH

TAT-Bim Inactive: Ac-rkkrr-orn-rrr-EIWIAQEARR IGAEFNAYYAR-OH

Cell Lines
Mouse T cell lymphoma (EL4), pancreatic cancer (Panc-02), and melanoma (B16) cell lines were examined. All cell cultures were maintained in supplemented RPMI containing glutamine (2 mmol/L), Pyruvate (1 mmol/L), penicillin and streptomycin (100 IU/mL), and 10% FBS.

Cell Permeability of TAT-Bim
To evaluate permeability of TAT-Bim, the peptide was synthesized with an N terminal fluorescein. Cancer cells were incubated in 1.0 ml culture medium for 2 hours with fluorescein labeled Bim peptide alone or with fluorescein labeled TAT-Bim. The tumor cells were examined for intracellular expression of Bim using a fluorescence microscope and FACS analysis.

Evaluation of Apoptosis
The tumor cells were seeded at a density of 0.5 to 1 x 106 cells per well in 12-well plates in 1.0 ml culture medium. TAT-Bim peptide was dissolved in either PBS or RPMI and added to the cell culture medium. The cells were then incubated for 18 hours at 37°C in humidified 5% CO2. Apoptosis was detected by FACS analysis after staining cells for active caspase-3 (Cell Signaling, Boston, MA).30 Briefly, cells were washed with PBS, treated with trypsin-EDTA to remove adhesion molecules, and washed again. After centrifugation at 1,200 rpm for 10 min, cells were fixed with 1 % paraformaldehyde and washed with PBS. After centrifugation at 1,800 rpm for 10 min, cells were fixed with 90% methanol at –20°C and washed with PBS. Each pellet was resuspended in 1:100 anti-active caspase-3 antibody (1 µg/ml caspase 3, PBS with 5% FBS) and incubated overnight at 4°C. After washing, each pellet was resuspended in 1:100 FITC labeled secondary antibody or 500 µl 7AAD buffer and incubated for 1 hour at room temperature. Cell fluorescence signals were determined using a FACScan flow cytometer (BD Biosciences) and analyzed with CellQuest software (BD Biosciences) to measure the percentage of activated caspase-3.

Double Staining of Bim and Active Caspase-3
To determine whether intracellular abundance of Bim was associated with tumor cell apoptosis, double staining for Bim and active caspase-3, was performed. The staining protocol (vide supra) was modified in the following way: after fixing cells with 90% methanol, FITC- and PE- conjugated antibodies were combined and used to detect Bim and active caspase-3 respectively.

Multimodality Therapy
To determine if TAT-Bim would augment a standard anti-cancer therapy, we tested our construct in combination with sublethal irradiation. Tumor cell lines were irradiated at room temperature in complete media in doses ranging from 0 to 2000 rads. Cells were than divided into groups and left untreated or incubated with TAT-Bim in concentrations ranging from 2 µM to 10 µM. After 18 hours of incubation, cells were washed and tested for apoptosis as described above.

In Vivo Antitumor Effect of TAT-Bim
Female C57BL/6 mice (5–6 weeks old) were purchased from the NCI and housed in the Animal Care Facilities. All mice were acclimated for at least 1 week before tumor implantation. All studies were performed in accordance with an animal protocol approved by the Washington University Institutional Animal Care. Subcutaneous tumor-bearing C57BL/6 mice were used as an animal model for evaluation of TAT-Bim for its anticancer activity. All mice were injected in the right flank with 200 µl of a single cell suspension containing 0.5 x 106 Panc-02 or B16 cells. Treatment of the tumors started 1 week after tumor implantation when their size reached 3–5 mm of mean diameter. Each experimental group contained 9 to 10 mice. The mice were treated with either TAT-Bim (200 µg) or vehicle control twice a day for 7 days by local injection. The tumor diameter was measured three times per week. All mice were euthanized when the tumors reached a size of 20 mm of mean diameter or when the tumors became ulcerated.31 One mouse that rejected tumor implantation was censored from the analysis.

Statistical Analysis
Analysis of survival rate was conducted by a log-rank statistical test, based on the Kaplan-Meier method. Tumor sizes and FACS results were analyzed by a two-tailed Mann-Whitney nonparametric test or a two-tailed Student test where appropriate. Dunnett’s test was used to analyze data from the combined radiation and TAT-Bim treatment groups.32 A P-value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TAT-Bim Is Taken Up by Cancer Cells
Uptake of TAT-Bim was determined using fluorescence microscopy and FACScan. All cell lines were incubated with fluorescently labeled TAT-Bim (F-TAT-Bim) or Bim (F-Bim) for 2 hours. Intracellular F-TAT-Bim was detected by fluorescence microscopy in all cancer cell lines (Fig. 1AGo). FACS analysis revealed that all the cancer cell lines were stained by F-TAT-Bim (Fig. 1BGo). F-Bim treated groups also demonstrated fluorescence compared to untreated cells, though the intensity was at least one log less than that observed in F-TAT-Bim treated cells (Fig. 1BGo). Both EL4 and Panc-02 cell populations exhibited uniform uptake of F-TAT-Bim while B16 cells appear to contain two distinct subpopulations with respect to F-TAT-Bim peptide uptake.


Figure 1
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  		FIG. 1. TAT-Bim penetrates the membrane of cancer cells. Cellular accumulation of fluorescently labeled Bim and TAT-Bim in EL4, B16, and Panc-02 cells examined by fluorescence microscope (A) and FACS (B). (A) Representative fluorescent microscopy image of EL4 cells incubated for 2h with 5 µM F-TAT-Bim. (B) FACS histograms of fluorescein signals from five cell populations: no treatment (0 µM), F-Bim treated (5 µM, 10 µM), and F-TAT-Bim treated (5 µM, 10 µM) for each cell line studied.

 
TAT-Bim Causes Cancer Cell Apoptosis and Augments the Cytotoxicity of Radiation
The cytotoxicity of TAT-Bim was determined by measuring active caspase-3 in TAT-Bim treated cancer cells using FACS analysis (Fig. 2Go). That the BH3 domain was responsible for this effect was shown by using an inactive mutant BH3 domain, TAT-Bim-Inactive. In all TAT-Bim treated cells caspase-3 activity significantly increased in a dose dependent fashion, while TAT-Bim-Inactive had no effect (P < 0.0001).


Figure 2
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  		FIG. 2. TAT-Bim induced cancer cell apoptosis. Tumor cell apoptosis caused by TAT-Bim (diamonds) or TAT-Bim-inactive (squares) for each cell line (A) EL4, (B) B16, (C) Panc-02. Cells were stained for active caspase-3 and apoptosis was quantified by FACS analysis.

 
The cytotoxicity of TAT-Bim was assessed in combination with sublethal radiation therapy. Double staining for Bim and active caspase-3 was performed with or without sublethal radiation (2000 rad). Representative data for the EL4 cell line is shown in Fig. 3Go. Consistent with Fig. 2Go, TAT-Bim induced tumor cell apoptosis in a dose-dependent manner (Fig. 3AGo). Radiation-induced Bim expression in the absence of TAT-Bim treatment (Fig. 3BGo). TAT-Bim augmented radiation-induced apoptosis in EL4 cells in a dose-dependent fashion.


Figure 3
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  		FIG. 3. TAT-Bim induced apoptosis is augmented by adjuvant irradiation. FACS profiles of EL4 cells stained for active caspase-3 (y-axis) and Bim (x-axis) in the presence of increasing concentrations of TAT-Bim (A) or after irradiation (2000 rad) and TAT-Bim (B). The percentage of positive cells in each quadrant is indicated in parentheses.

 
We evaluated the cytotoxic effects of combined treatment using a range of doses of both radiation (0, 1000, 2000 rad) and TAT-Bim (0, 2, 5, 10 µM) against all three cell lines (Table 1Go). Either treatment alone caused significant amounts of apoptosis in a dose-dependent fashion in all cell lines (EL4, B16 and Panc-02; P < 0.0001, P = 0.027 and P < 0.0001, respectively). When combined, radiation and TAT-Bim caused an additive increase in the percentage of cells undergoing apoptosis in all cell lines (EL4, B16 and Panc-02; P < 0.0001, P = 0.001 and 0.0016, respectively). Although a similar maximal apoptotic response could be achieved with extreme doses of TAT-Bim or extreme doses of radiation, the additive effect was most noticeable at the lower and intermediate doses depicted in Table 1Go.


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  		TABLE 1. TAT-Bim induced apoptosis is augmented by radiation
 
TAT-Bim Therapy Slows Tumor Growth and Confers a Survival Advantage In Vivo
TAT-Bim was evaluated in vivo in an established syngeneic tumor model using Panc-02 cells and B16 in C57/BL6 mice. When the tumors were consistently palpable (3–5mm diameter) mice were started on a treatment regimen of intratumoral injection of TAT-Bim, TAT-Bim Inactive, or vehicle twice a day for seven days. Results for both cell lines were similar. Figure 4Go depicts the results from the Panc-02 experiment. Some tumors shrank during and immediately following treatment. Three to five days after treatment was stopped, tumor regrowth occurred in all groups (Fig. 4AGo). Although regrowth occurred in the TAT-Bim treatment group, there was a significant delay when compared to PBS or TAT-Bim Inactive groups (P < 0.0001). Using tumor burden of 20 mm or ulceration as a surrogate endpoint for survival, we found a significant survival benefit in the TAT-Bim treatment group (Fig. 4BGo; P < 0.02). There was no systemic toxicity noted in any animal by behavior, weight loss or on necropsy. Peripheral blood mononuclear cells were not different in quantity or percentage of CD4+, CD8+ T-cells or B-cells as measured by flow cytometry when groups were compared or when experimental animals were compared to age matched controls (data not shown).


Figure 4
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  		FIG. 4. Effects of TAT-Bim treatment in an in vivo cancer model. (A) Tumor growth after TAT-Bim injection (n = 10; dashed line) compared to PBS injection (n = 10; solid line) and TAT-Bim Inactive injection (n = 9; dotted line) (P < 0.0001). The double-headed arrow denotes the treatment period. (B) Survival of the TAT-Bim injected group compared to the control groups (P < 0.02). Tumor diameter > 20 mm or ulceration was used as a surrogate for survival.31

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Dysregulation of apoptosis is a pre requisite for the development of cancer. High Bcl-2 expression has been shown in a variety of human cancers including breast, colon, and lung, and correlates with uncontrolled cell expansion and decreased sensitivity of these cells to adjuvant therapies.33–35 As a result, there is considerable interest in targeting the intracellular apoptotic apparatus for cancer therapy. Although their mechanisms of action are not fully understood, anti-apoptotic Bcl-2 family members are believed to inactivate pro-apoptotic Bcl-2 family members by binding to their BH3 domains and sequestering them from disrupting the mitochondrial membrane. Recent studies have shown that Bim, a BH3-only Bcl-2-like protein, is an antagonist of the anti-apoptotic Bcl-2 family that sensitizes cells to Bak- or Bax-mediated apoptosis.36 Furthermore, Bim deficient cells have a lower sensitivity to paclitaxel, and tumors without Bim have poor prognosis in vivo.10

We utilized a dual domain peptide, TAT-Bim, which induces apoptosis in several cancer cell lines in a dose-dependent fashion. Its apoptotic effect is augmented by radiation therapy, a known inducer of apoptosis. We showed that each domain of our peptide is necessary for the molecule to function. While F-Bim was detectable in cells after 2 hours of coculture, the TAT domain enhanced peptide uptake by at least one order of magnitude. Likewise, a functional BH3 domain is required to induce apoptosis. While EL4 and Panc-02 cells exhibit uniform uptake of F-TAT-Bim, there appear to be two distinct subpopulations of B16 cells (Fig. 1BGo). A similar difference between B16 and the other cell lines (EL4 and Panc-02) was also observed in their sensitivity to TAT-Bim induced apoptosis. Both Panc-02 and EL4 cells showed no resistance toward TAT-Bim. However, there was no appreciable increase in the amount of apoptosis in B16 cells when the TAT-Bim dose was doubled from 5 to 10 µM. It may be the case that the two populations of B16 cells apparent in Figure 1BGo are responsible for this effect. Indeed there are several reports of distinct subpopulations of B16 cells with different metastatic potentials and resistance to chemotherapy.37,38

Radiation treatment induced Bim expression in EL4 lymphoma cells, Panc-02 pancreas cancer cells, and at higher doses, in B16 melanoma cells (data not shown). Apoptosis was similarly increased. When given in combination with TAT-Bim, an augmentative effect on apoptosis was observed in all cell lines, but did not appear to be dose-dependent in B16 cells. These effects appear additive and a high degree of apoptosis is achievable with lower doses of radiation or TAT-Bim than with either treatment alone. B16 cells have previously been characterized as radiation-resistant.39 Therefore TAT-Bim treatment may not sensitize all cancers to radiation to the same degree. These results underscore the need to better understand how cells integrate various pro-apoptotic signals and how different tumors lose sensitivity to these stimuli. These data suggest that TAT-Bim and radiation are two distinct injuries that are integrated in EL4 and PanO2 cells. Although our data do not address whether all B16 cells are refractory to the additive effect of the combined stimuli or whether there are sensitive and refractory subpopulations of B16 cells, the literature suggests that B16 cells have multiple phenotypes. Identification of the mechanism(s) that render certain B16 cells insensitive to apoptosis-inducing treatment may lead to additional targets for cancer therapy.

TAT-Bim treatment of Panc-02 and B16 tumors in C57/BL6 mice led to tumor stability and even regression in some animals. Although all tumors grew shortly after treatment was stopped, TAT-Bim treated tumors grew more slowly than tumors in control treated animals and vehicle-injected control animals. This result suggests tumor-directed therapy with TAT-Bim was successful in reducing tumor mass and/or altering the course of tumor growth after therapy. Because tumor site injection is not always possible in patients and can not address metastatic disease, we plan to incorporate molecular methods that target the delivery of therapeutic peptides specifically to tumor cells. This approach might allow systemic administration but local activation of the therapeutic peptide. Such a strategy has the potential to reduce side effects and improve the therapeutic/toxic index.

Small peptide therapeutics work by incorporating endogenous protein domains and can activate or antagonize cellular processes such as apoptosis. Alternative approaches modulate the expression of critical components of the endogenous apoptotic machinery. Antisense RNA and siRNA have been used to knock down the expression of Bcl-2 in cancer models.40,41 Other groups have used retroviral gene delivery to induce the expression of granzyme B in cancer cell lines.42 The ability to modulate peptide activity by making single amino acid substitutions provides a level of control that is not available with siRNA or antisense RNA. Viral-mediated gene therapy has additional hurdles to surmount including toxicity, stimulation of an inflammatory response, and control of gene expression. Optimizing the intracellular delivery of any of these compounds is an important technical challenge that is under active investigation with liposomes, engineered protein transduction domains and strategies to take advantage of receptor mediated endocytosis being relatively mature.

In our next generation of compounds we will place an emphasis on cancer specific targeting strategies. We believe peptide therapeutics have the potential to selectively deliver cytotoxic small molecules to cancer cells. Our results showing cell type-specific cytotoxicity underscore the need to understand the manner in which apoptotic pathways are dysregulated in various cancers. Armed with this information, the physician will be able to select an optimal therapeutic agent coupled to the appropriate targeting moiety. The realization of this strategy will enable the personalization of cancer therapy and has the potential to reduce side effects and be more effective than contemporary chemotherapeutic and adjuvant therapies.


   ACKNOWLEDGMENTS
 
This work was supported in part by a Barnes Jewish Hospital Foundation Grant (WGH), AACR-PanCAN Career Development Award in Pancreatic Cancer Research, in Memory of Skip Viragh (WGH), GM055194 (RSH) and GM044118 (RSH), P50 CA94056 (DPW) and CA082841 (DPW).


   FOOTNOTES
 
This work was presented in part at the Annual Meeting of the Society for Surgical Oncology Cancer Forum, San Diego 2006

Received for publication July 13, 2006. Accepted for publication November 9, 2006.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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