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J Thorac Cardiovasc Surg 2006;132:1356-1362
© 2006 The American Association for Thoracic Surgery

General Thoracic Surgery

Gossypol, a phytochemical with BH3-mimetic property, sensitizes cultured thoracic cancer cells to Apo2 ligand/tumor necrosis factor–related apoptosis-inducing ligand

Wen-Shuz Yeow, PhD, Aris Baras*, Alex Chua*, Duc M. Nguyen*, Shailen S. Sehgal, BS{dagger}, David S. Schrump, MD, FACS, Dao M. Nguyen, MD, MS, FRCSC, FACS*

Section of Thoracic Oncology, Surgery Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Md.

Read at the Eighty-sixth Annual Meeting of The American Association for Thoracic Surgery, Philadelphia, Pa, April 29-May 3, 2006.

Received for publication May 5, 2006; revisions received May 19, 2006; accepted for publication July 12, 2006.

* Address for reprints: Dao M. Nguyen, MD, Room 4W-4-3940, 10 Center Dr, Bethesda, MD 20892. (Email: dao_nguyen{at}nih.gov).


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
OBJECTIVES: Chemotherapeutic agents sensitize cancer cells to Apo2 ligand/tumor necrosis factor–related apoptosis-inducing ligand (Apo2L/TRAIL) via recruitment of the mitochondria-dependent activation of caspase and induction of apoptosis. This study was designed to evaluate whether gossypol, a phytochemical compound with BH3-mimetic property that functions as an inhibitor of Bcl2/BclXL, would sensitize cultured thoracic cancer cells to this death-inducing ligand.

METHODS: Cancer cell lines from the lung (H460, H322), the esophagus (TE2, TE12), and the pleura (H290, H211) or primary normal cells were treated with gossypol+Apo2L/TRAIL combinations. Cell viability and apoptosis were evaluated by (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assays, respectively. Caspase 9 and 3 specific proteolytic activity in combination-treated cells was determined by fluorometric enzymatic assay.

RESULTS: Gossypol, selectively cytotoxic to cancer cells and not primary normal cells, significantly sensitized thoracic cancer cells to Apo2L/TRAIL as indicated by 1.5- to more than 10-fold reduction of Apo2L/TRAIL 50% inhibitory concentration values in cells treated with gossypol+Apo2L/TRAIL combinations. Whereas less than 20% of cancer cells exposed to either gossypol (5 µmol/L) or Apo2L/TRAIL (20 ng/mL) were dead, more than 90% of cells treated with the drug combinations were apoptotic. Combination-induced cytotoxicity and apoptosis was completely abrogated either by overexpression of Bcl2 or by the selective caspase 9 inhibitor. This combination was not toxic to normal cells.

CONCLUSION: Gossypol profoundly sensitizes thoracic cancer cells to the cytotoxic effect of Apo2L/TRAIL via activation of the mitochondria-dependent death signaling pathway. This study provides evidence for the profound anticancer activity of this drug combination and should be further evaluated as a novel targeted molecular therapeutic for thoracic cancers.

Abbreviations and Acronyms IC50 = inhibitory concentration of 50%; MTT = (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide; TNF = tumor necrosis factor; TRAIL = tumor necrosis factor–related apoptosis-inducing ligand; TUNEL = terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Better understanding of the molecular basis of carcinogenesis and elucidation of signal transduction pathways regulating cell growth and death in normal cells and their roles in the process of malignant transformation offers great opportunities for the development of novel molecularly targeted anticancer therapy. Within this context, therapeutic strategies aiming at direct induction of cell death by activation of the TRAIL receptor-mediated signal transduction pathways using the recombinant protein Apo2L/TRAIL (the Zn++-coordinated homotrimer of the extracellular domain of TRAIL)1Go or the human agonistic anti-DR4 monoclonal antibodies2Go have attracted a great deal of attention for clinical development, as these biologics are selectively cytotoxic to cancer cells.1-4Go In fact, they are currently being tested in phase 1 clinical trials. Binding of Apo2L/TRAIL to its cognate functional receptors DR4 and/or DR5 activates the apical caspases 8 and 10, which either directly (type I pathway) or indirectly, via the mitochondria-dependent death-signaling cascade (type II pathway), activate the downstream executioner caspases 3, 6, and 7 to mediate apoptotic cell death.5Go

Despite expressing adequate levels of functional receptors DR4/DR5 for Apo2L/TRAIL, significant percentages of cancer cells exhibit resistance to the cytotoxic effect of this ligand in vitro. The molecular basis of this phenomenon is complex and yet to be fully elucidated.6Go It is well described that exposure of Apo2L/TRAIL-refractory cancer cells to standard cytotoxic chemotherapeutic agents such as cisplatin and paclitaxel or to experimental targeted anticancer drugs like the histone deacetylase inhibitors (such as depsipeptide or valproic acid) profoundly sensitizes these cells to this death-inducing ligand.7-9Go Our laboratory,7,8Go as well as others,9,10Go has demonstrated that chemotherapeutic drugs sensitize cancer cells to Apo2L/TRAIL via recruitment of the mitochondria-dependent caspase activation cascade. As such, the next logical step in the development of efficient TRAIL-based combination therapy for cancer is to directly target the mitochondria to stimulate its apoptosis-inducing property. The apoptogenicity of the mitochondria is tightly regulated by members of the Bcl2 superfamily, which respond to various intrinsic and extrinsic death-promoting stimuli and ultimately decide the fate of the affected cells.11Go Suppression of antiapoptotic protein expression by antisense or small interfering RNA techniques has been shown to sensitize cancer cells to TRAIL.12Go However, this approach, while valuable in providing proof of concept, has limited clinical application, mainly because of the redundancy of the Bcl2 superfamily antiapoptotic members, the long half-life of Bcl2/BclXL proteins, and the inefficient delivery of ribonucleic acid sequences to every cancer cells in vivo. The antiapoptotic proteins Bcl2 or BclXL sequester the proapoptotic proteins Bad, Bid, Bim, Bax, and Bak by selective interaction between the BH3 domains of these proteins with their own BH3-binding pockets to prevent their translocation to the mitochondria, where they perform their death-inducing function.13Go Intense research and development of small molecule inhibitors of Bcl2/BclXL have identified multiple compounds that can interact with the BH3-binding pockets of these proteins to inhibit their antiapoptotic function.14Go These drugs are commonly referred to as BH3 mimetics.

Gossypol, a phytochemical found in cottonseed oil, was initially developed as an antifertility agent and subsequently recognized to have anticancer property15Go (and refs therein). This compound was tested in many small phase 1 clinical trials.16Go Gossypol, furthermore, was identified to be a BH3 mimetic by computer-assisted molecular modeling, by nuclear magnetic resonance imaging study, and also by fluorescence-polarization assay.17Go We hypothesize that gossypol, via its Bcl2/BclXL inhibitory activity as a BH3 mimetic, synergistically interacts with Apo2L/TRAIL to mediate profound cytotoxicity and apoptosis of cultured thoracic cancer cells (cancer cell lines derived from primary tumors of the lung, the esophagus, or the pleura) in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Lines and Reagents
Cultured non–small cell lung cancer cells H460 and H322, esophageal cancer cells TE2 and TE12, and malignant pleural mesothelioma H211 and H290 were maintained in RPMI 1640 cultured medium supplemented with fetal calf serum (10% v/v), L-glutamine (1 mmol/L), streptomycin (100 µg/mL), and penicillin (100 U/mL). Normal human primary fibroblast and normal human bronchial epithelia were purchased from Cambrex (Walkervile, Md) and grown in their special culture media as per instructions of the vendor. Apo2L/TRAIL was obtained from Genentech Inc (South San Francisco, Calif) via a Cooperative Research and Development Agreement. Bcl2–overexpressing stable transfectants of TE2 (TE2Bcl2), TE12 (TE12Bcl2) cells, as well as their respective vector controls, were created and characterized as previously described.8Go Selective caspase 9 inhibitor z-LEHD-fmk was purchased from Calbiochem (San Diego, Calif). Gossypol was obtained from Sigma-Aldrich (St Louis, Mo), dissolved in dimethylsulfoxide (25 mmol/L stock), divided into aliquots, and stored in –20°C. Fresh drug was used for each experiment to avoid repeated freeze-thaws.

Cytotoxicity and Apoptosis Assays
Cells were seeded in 96-well microtiter plates at predetermined plating densities appropriate for each cell line (1.0 to 2.0 x 104 cells/well) to achieve 80% confluence at the time cells were to be treated with Apo2L/TRAIL. After an overnight incubation, cells were treated with either gossypol (2.5, 5.0, or 10.0 µmol/L) or Apo2L/TRAIL (5 to 100 ng/mL) or 1-hour pretreatment with gossypol followed by addition of Apo2L/TRAIL (as 20 µL of 10X stock) for a total of 200 µL per well. Cell viability after treatment with Apo2L/TRAIL alone or with gossypol+Apo2L/TRAIL combinations was calculated as percentage of untreated controls or of gossypol-treated controls (to normalize for growth-inhibitory effect of gossypol, which ranged from 20% to 50% depending on the duration of exposure to this drug), respectively. The Apo2L/TRAIL 50% inhibitory concentration (IC50) values were estimated from respective dose-response curves. Apoptosis after treatments with Apo2L/TRAIL (20 ng/mL) alone or in combination with gossypol (5.0 µmol/L or 10.0 µmol/L) was determined by the TUNEL* -based ApoBrdU assay (both from BD-Pharmigen, Torrence, Calif).

Caspase Activity Assay
Specific enzymatic activity of caspases 9 and 3 at intervals after the onset of treatment with Apo2L/TRAIL (20 ng/mL) with or without gossypol (5 µmol/L) in TE2, TE12, and H460 cells was measured by enzymatic assays using fluorochrome-labeled substrates (R&D Systems, Inc, Minneapolis, Minn). The specific caspase activity, normalized for total proteins of cell lysates (BCA technique; Pierce Biotechnology, Rockford, Ill), was then expressed as fold of the baseline caspase activity of untreated control cells.

Statisical Analysis
Experiments were performed in quadruplicates unless otherwise indicated. Supra-additive cytotoxicity or apoptosis is defined as the cytotoxicity or apoptosis induced by the drug combinations that is, by statistical analysis, significantly greater than the sum of cytotoxicity or apoptosis induced by individual drug treatment. Results were presented as means ± standard errors of the means. The Student t test was performed when indicated.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antiproliferative Effect of Gossypol on Thoracic Cancer Cells
Continuous exposure of 6 cultured cancer cells to gossypol for 24 hours resulted in a significant dose-dependent reduction of cell proliferation as determined by the MTT{dagger} at 36 hours after the onset of drug treatment (Figure 1). The viability of primary normal cells, on the other hand, was not significantly affected by gossypol. Gossypol induced mild cell cycle arrest at G1/S checkpoint in TE2, TE12, and H322 cells but not in H460, H211, or H290 cells (data not shown). Moreover, gossypol mediated apoptosis of thoracic cancer cells, the magnitude of which was clearly dependent on the drug concentrations and the duration of drug exposure (Figure E1).


Figure 1
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  		Figure 1. The effect of gossypol on the viability of thoracic cancer cells and primary normal cells. Cells were treated with gossypol for 24 hours and cell viability was determined by MTT assay at 36 hours after the onset of drug treatment. Data are presented as means ± SEM of 4 independent experiments. NHBE, Normal human bronchial epithelia; NHEK, normal human epidermal keratinocyte; micM, micromoles per liter.

 

Figure 6
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  		Figure E1. Gossypol-mediated induction of apoptosis. Cells were exposed to gossypol (5.0 or 10.0 µmol/L) for either 24 hours or 48 hours and harvested at 48 hours after the onset of gossypol treatment for quantitation of apoptosis using the TUNEL-based ApoBrdU assay. Data are presented as means ± SEM of 4 independent experiments, #P < .001 versus gossypol 5.0 µmol/L x 48 hours, +P < .01 versus gossypol 10.0 µmol/L x 24 hours, *P < .001 versus gossypol 5.0 µmol/L x 24 hours. G, Gossypol; TUNEL, terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling; micM, micromoles per liter.

 
Supra-additive Enhancement of Cytotoxicity by the Gossypol+Apo2L/TRAIL Combination
The intrinsic sensitivity to Apo2L/TRAIL varies significantly between cultured thoracic cancer cells lines, with H460, H322, and H290 being more susceptible to this death-inducing ligand (Apo2L/TRAIL IC50 values of 50 ± 3, 40 ± 2, and 53 ± 3 ng/mL respectively) while TE2, TE12, and H211 are Apo2L/TRAIL-resistant cells (Apo2L/TRAIL IC50 > 100 ng/mL). All of the cancer cell lines used in this study are type II cells.7,8Go Concurrent treatment of cultured thoracic cancer cells to gossypol (2.5-10.0 µmol/L) and Apo2L/TRAIL (5-100 ng/mL) resulted in significant enhancement of Apo2L/TRAIL-mediated cytotoxicity. Gossypol alone, at the treatment conditions used, mediated a cell line–dependent mild-to-moderate reduction of cell viability (20%-50%). The gossypol-mediated sensitization of cancer cells to Apo2L/TRAIL was most easily appreciated when the growth-inhibitory effect of the gossypol+Apo2L/TRAIL combinations was normalized for the gossypol-mediated cytotoxicity, as presented in the respective dose-response curves shown in Figure 2A for TE2, H460, and H211 cells. With Apo2L/TRAIL IC50 values used as indicators of cellular sensitivity to this ligand, it was apparent that there was 1.5- to greater than 10-fold reduction of Apo2L/TRAIL IC50 values in gossypol-treated cells in a dose-dependent manner (Figure 2B). This effect was observed in all cultured thoracic cancer cells regardless of their intrinsic sensitivity to Apo2L/TRAIL and particularly most pronounced in Apo2L/TRAIL-resistant TE2, TE12, and H211 cell lines. Comparable gossypol-mediated enhancement of Apo2L/TRAIL sensitivity was observed in cells treated with either 12 or 24 hours of gossypol+Apo2L/TRAIL, with no further reduction of Apo2L/TRAIL IC50 after 36 hours of exposure to the drug combination except in H211 cells (Figure E2). Whereas gossypol (5 µmol/L) alone or Apo2L/TRAIL (20 ng/mL) alone induced less than 20% apoptosis in representative H460, TE12, and H211 cancer cells, massive supra-additive induction of apoptosis of more than 90% was observed in combination-treated cells (Figure 3). Most important, neither Apo2L/TRAIL alone nor gossypol+Apo2L/TRAIL combination induced apoptosis of primary human fibroblasts and normal human bronchial epithelia (Figures 2A and 3).


Figure 2
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  		Figure 2A. Gossypol sensitizes cancer cells but not primary normal cells to Apo2L/TRAIL-mediated cytotoxicity. Cells were treated with Apo2L/TRAIL, gossypol, or gossypol+Apo2L/TRAIL for 24 hours, and cell viability was assessed by MTT at 36 hours. Data are presented as means ± SEM of 4 independent experiments. NHBE, Normal human bronchial epithelia; micM, micromoles per liter.

 

Figure 2
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  		Figure 2B. Apo2L /TRAIL IC50 values of cultured cancer cells treated with either Apo2L/TRAIL alone or gossypol+Apo2L/TRAIL combinations. These values were estimated from the respective dose-response curves and used as indicators of cellular sensitivity to Apo2L/TRAIL. Data are presented as means ± SEM of 4 independent experiments; #P < .0001 and +P < .01 versus Apo2L/TRAIL alone by analysis of variance and Bonferroni pairwise analysis. micM, Micromoles per liter.

 

Figure 7
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  		Figure E2. The effect of the duration of gossypol+Apo2L/TRAIL treatment on gossypol-mediated reduction of Apo2L/TRAIL IC50 values in representative H211, H460, and TE12 cells. Cells were treated with gossypol+Apo2L/TRAIL combination for 12 hours, 24 hours, or 36 hours, and cell viability was quantified by MTT at 36 hours after the onset of Apo2L/TRAIL exposure. Data are presented as means ± SEM of 4 independent experiments; #P = .0061 by ANOVA with statistically significant difference (P < .01) only between gossypol+Apo2L/TRAIL 12 hours and 36 hours; **P = .071 and +P = .077 by ANOVA and P > .05 by Bonferroni pairwise analysis. G, Gossypol.

 

Figure 3
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  		Figure 3. Profound and supra-additive induction of apoptosis by the gossypol+Apo2L/TRAIL combinations in representative cancer cell lines H460, H211, and TE12. This combination does not induce apoptosis in primary normal cells. Data are presented as means ± SEM of 4 independent experiments. G, Gossypol; NHBE, Normal human bronchial epithelia; TUNEL, terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling; micM, micromoles per liter.

 
Mitochondria-dependent Death Signaling Cascade Plays Important Role in Enhanced Cytotoxicity by Gossypol+Apo2L/TRAIL
As gossypol is a BH3-mimetic drug, it most likely sensitizes cultured thoracic cancer cells to Apo2L/TRAIL via activation of mitochondria and recruitment of the type II death signaling pathway. Time-course evaluation of caspase activation in TE12 cells treated with gossypol, Apo2L/TRAIL, or gossypol+Apo2L/TRAIL combination indicated significant supra-additive enhancement of the specific proteolytic activity of caspase 9 (apical caspase of the mitochondria-mediated caspase activation cascade) and the downstream executioner caspase 3 (Figure E3). Such caspase activation directly contributed to Apo2L/TRAIL- or gossypol+Apo2L/TRAIL-mediated cytotoxicity and apoptosis, as these were significantly abrogated by either the general caspase inhibitor z-VAD-fmk or the caspase 9 inhibitor z-LEHD-fmk (Figure 4 and Figure E4). To further confirm the involvement of the mitochondria-mediated (type II) pathway in this process, we similarly treated Bcl2-overexpressing stable transfectants TE2Bcl2 and TE12Bcl2 cells with Apo2L/TRAIL or gossypol+Apo2L/TRAIL combinations. Bcl2 overexpression completely protected TE2 or TE12 cells from Apo2L/TRAIL-mediated cytotoxicity (as in TE12 cells) and gossypl+Apo2L/TRAIL-mediated cytotoxicity (in both TE2 and TE12 cells) (Figure 5).


Figure 8
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  		Figure E3. Supra-additive activation of caspases 9 and 3 specific proteolytic activity in H460, TE2, and TE12 cells treated with the gossypol+Apo2L/TRAIL combination. Cells were treated with gossypol (5.0 µmol/L), Apo2L/TRAIL (10 ng/mL for H460 cells or 20 ng/mL for TE2 and TE12 cells), or gossypol+Apo2L/TRAIL combination. Representative data of 3 independent experiments are shown here. G, Gossypol.

 

Figure 4
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  		Figure 4. Significant reduction of gossypol+Apo2L/TRAIL-mediated apoptosis by the general caspase inhibitor Z-VAD-fmk or the caspase 9 inhibitor Z-LEHD-fmk in H211 and TE12 cells. Representative data of 3 independent experiments with similar results are shown.

 

Figure 9
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  		Figure E4. Abrogation of gossypol+Apo2L/TRAIL-mediated cytotoxicity by Z-VAD-fmk. Cells were treated with 24 hours of gossypol, Apo2L/TRAIL, or gossypol+Apo2L/TRAIL with or without prior exposure to Z-VAD-fmk (60 µm). Cell viability was determined by MTT 36 hours after the onset of drug exposure. Data are presented as means ± SEM of 4 independent experiments; #P < .0001, +P = .0006 between gossypol+Apo2L/TRAIL with and without Z-VAD-fmk.

 

Figure 5
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  		Figure 5. Overexpression of Bcl2 protects cancer cells against the profound gossypol+Apo2L/TRAIL-mediated cytotoxicity in TE2 and TE12 Bcl2-overexpressing stable transfectants. Vector control stable transfectants were equally susceptible to the cytotoxic effect of this drug combination (data not shown). Data are presented as means ± SEM of 4 independent experiments; #P < .001 between gossypol+Apo2L/TRAIL in parental cells versus in Bcl2-overexpressing stable transfectants. G, Gossypol; GFP, green fluorescent protein.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The intrinsic sensitivity of cancer cells to TRAIL can be enhanced by treating them with standard cytotoxic chemotherapeutics or with investigational drugs or gamma radiation.18Go The molecular basis of such enhancement effect is complex, cell dependent, and not completely elucidated. Several possibilities have been postulated as the basis of chemotherapy-induced potentiation of TRAIL cytotoxicity: upregulation of receptor expression, elevation of TRAIL-initiated signal transduction through the receptor complex and/or the intracellular pathways, and downregulation of natural inhibitor proteins of the caspase cascade after treatment of cancer cells with sensitizers. Whereas the molecular basis of enhanced TRAIL cytotoxicity by these combinations is diverse, functional and/or phenotypic modulation of the mitochondria-regulated caspase-activation cascade leading to amplification of the Apo2L/TRAIL-induced apoptosis appears to be the common theme. Our current working model of chemotherapy-induced sensitization of cancer cells to Apo2L/TRAIL is as follows: Apo2L/TRAIL engagement of its functional receptors DR4/DR5, via the formation of the death-induced signaling complex, results in an initial minute activation of caspase 8, which activates the mitochondria via cleavage of BID to form tBID. In sensitive type II cells, this would be adequate to engage the mitochondrial arm of the caspase activation cascade and the amplification feedback loop to potentiate caspase 8 activation. In resistant cells, the chemosensitizer "primes" the mitochondria via phenotypic or functional alteration of proapoptotic and antiapoptotic proteins of the Bcl2 superfamily to make the mitochondria more susceptible to tBID and thus effectively engage the mitochondria-dependent caspase activation pathway.7,8Go Proofs of principle of such model have been demonstrated by sensitization of cancer cells to TRAIL by targeting the mitochondria using BclXL small interfering RNA12Go or the mitochondriotropic cytotoxic drug betulinic acid.19Go The gossypol+Apo2L/TRAIL drug combination was thus designed on the basis of the observation that mitochondria are essential for the chemotherapy-induced potentiation of Apo2L/TRAIL cytotoxicity and the hypothesis that functional inhibition of Bcl2/BclXL using BH3-mimetic drugs like gossypol would sensitize cancer cells to this death-inducing ligand. Indeed, gossypol synergistically interacts with Apo2L/TRAIL to induce profound induction of apoptosis in cultured thoracic cancer cells and, most important, not in primary normal cells. Experiments are also in progress to elucidate the exact molecular mechanism(s) by which gossypol interacts with Apo2L/TRAIL to mediate profound cytotoxicity in cancer cells.

The recent discovery of small-molecule chemical inhibitors of Bcl2/BclXL by virtue of their ability to interact with the BH3-binding pocket of these proteins has suggested a new approach for cancer therapy. These compounds exhibit strong anticancer activity in many tumor cells, especially in those expressing high levels of Bcl2/BclXL.15Go They also potentiate the tumoricidal effects of standard cytotoxic chemotherapeutics or radiotherapy20,21Go as well as of Apo2L/TRAIL, as reported herein by our group or by other investigators.22,23Go Gossypol interacts with the BH3-binding pockets of 4 antiapoptotic proteins, Bcl2, BclXL, BclW, and Bfl1, displacing BH3 peptide with an IC50 of about 0.5 µmol/L.14Go Naturally occurring gossypol exists as a racemic mixture of (+) and (–) enantiomers. Interestingly, the (–) enantiomer of gossypol possesses higher affinity for Bcl2 and BclXL, reduced serum binding, and greater anticancer activity in in vitro assays than the (+) enantiomer or racemic gossypol.15Go Gossypol, a polyphenolic containing two aldehyde groups, is a highly reactive compound, which may explain some of toxicities originally seen in phase 1 clinical trials of this drug in addition to producing unfavorable pharmacologic properties.14Go Structural modification of gossypol by removal of these ahdehyde groups creates apogossypol, a semisynthetic BH3-mimetic drug with a better pharmacologic profile. The (–) gossypol and apogossypol are being jointly developed as Bcl2-targeted anticancer drugs by the National Cancer Institute with Ascenta Therapeutics, Inc (San Diego, Calif), and the Burnham Institute (La Jolla, Calif), respectively.14Go Evaluation of the therapeutic efficacy and toxicity profile of the (–)gossypol +Apo2L/TRAIL combination in vitro and in vivo animal model of nude mice bearing human cancer xenografts is the current focus of our laboratory effort to facilitate translation of the gossypol+Apo2L/TRAIL drug combination into clinical application. Moreover, our findings form the basis for further evaluation of this combination strategy using synthetic compounds specifically designed as BH3 mimetics such as BH3I-2' or HA14-1 in cultured thoracic cancer cells.

In summary, we report for the first time profound cytotoxicity and apoptosis mediated by the gossypol+Apo2L/TRAIL combination in cultured thoracic cancer cells via a process that is caspase-mediated and dependent on the mitochondria-regulated death signaling pathway. More important, this drug combination is not toxic to primary normal cells. Our study, in conjunction with other reports cited in this manuscript, provides a strong rationale for further development of Apo2L/TRAIL-based therapy in combination with BH3 mimetics as novel targeted molecular therapeutic for cancers.


   Footnotes
 
This research was supported by the Intramural Research Program of the National Cancer Institute, National Institutes of Health.

* Cancer Research Training Award Recipients, National Cancer Institute. Back

{dagger} Fellow of the Clinical Research Training Program, National Institutes of Health. Back

* TUNEL = terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling. Back

{dagger} MTT = (4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide. Back


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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