Cancer Research Translational Cancer Medicine 2008: Cancer Clinical Trials and Personalized Medicine Candidate Pathways, Whole Genome Scans
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[Cancer Research 59, 1584-1591, April 1, 1999]
© 1999 American Association for Cancer Research
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[Cancer Research 59, 1584-1591, April 1, 1999]
© 1999 American Association for Cancer Research

Tumor Biology

Initiation of Growth Arrest and Apoptosis of MCF-7 Mammary Carcinoma Cells by Tributyrin, a Triglyceride Analogue of the Short-Chain Fatty Acid Butyrate,Is Associated with Mitochondrial Activity1

Barbara G. Heerdt2, Michele A. Houston, Gillian M. Anthony and Leonard H. Augenlicht

Albert Einstein Cancer Center, Bronx, New York 10467


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the effects of tributyrin, a triglyceride analogue of the short-chain fatty acid butyrate and an approved food additive, establishing induction of growth arrest and apoptosis of MCF-7 human mammary carcinoma cells. Transient increased mitochondria-associated bax, dissipation of the mitochondrial membrane potential ({Delta}{Psi}m), and caspase-3-independent cleavage of poly(ADP-ribose) polymerase are evident as early as 4 h after treatment of cells with tributyrin. These events are followed by the transient accumulation of mitochondrial cytochrome c in the cytosol and, finally, the generation and accumulation of cells with subdiploid DNA content. During the period in which mitochondria-associated bax levels are elevated, the {Delta}{Psi}m is disrupted, and cytochrome c is detected in the cytosol, we show induction of p21WAF1/Cip1 in the absence of increased p53 and arrest of cells in G2-M. Thus, early mitochondria-associated events may play a key role in initiating and/or coordinating tributyrin-mediated growth arrest and apoptosis of wild-type p53 MCF-7 cells. Because effective chemoprevention has been associated with agents that restore or maintain the balance between proliferation and apoptosis, dietary tributyrin, particularly during the critical period of mammary gland development, may be a promising chemopreventive agent.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Unlike many other organs, the human mammary gland continues to develop after birth, undergoing rapid growth and partial differentiation at puberty (1) . In the nonlactating breast, this partially differentiated state is maintained through sequential expansion and apoptosis of mammary epithelial cells in concert with the menstrual cycle (2, 3, 4) . Thus, for decades during a woman’s life, mammary homeostasis is dependent upon establishing and maintaining a balance between cycles of mammary epithelial cell proliferation and death by apoptosis.

Risk for spontaneous mammary cancer has been attributed to enhanced proliferation of mammary epithelial cells, which leads to the increased likelihood of accumulation of genetic alterations, a paradigm for tumorigenesis in solid tissues. Consequently, chemoprevention of mammary cancer has focused primarily on reducing exposure to substances that stimulate proliferation. However, because mammary gland homeostasis is two-sided, achieved and maintained through the balance between proliferation and apoptosis, it is equally likely that defective apoptotic pathways play a pivotal role in risk for mammary tumorigenesis (5) . Effective chemopreventive agents, therefore, may prove to be those that promote both growth arrest and apoptosis.

Butyric acid is a SCFA3 produced during the fermentation of fiber by endogenous intestinal bacteria and is also present in fruits, vegetables, and milk fat. The sodium salt of butyric acid, sodium butyrate, has potent effects on a variety of cell lines in vitro, including both malignant and normal mammary epithelial cells (6 , 7) . Despite its potency in vitro, butyric acid is rapidly metabolized in vivo, making it difficult to achieve and maintain effective levels in the serum, even when its sodium or arginine salts are administered by continuous i.v. infusion (8 , 9) . Moreover, i.v. infusion carries with it the possibility of complications due to concomitant elevations in serum sodium or arginine (10 , 11) . Therefore, the effectiveness of butyric acid salts as a chemotherapeutic or chemopreventive agents is compromised.

Tributyrin, a triglyceride analogue of butyric acid (butyryl triglyceride), is similar in structure to 95% of the fatty acids found in foods (12) , can be p.o. administered, is well tolerated (13) even by young children (14) , and is an approved food additive in the United States. Moreover, because tributyrin contains three butyric acid moieties esterified to glycerol, when completely hydrolyzed by cellular lipases or esterases, it yields 3-fold more butyric acid than sodium butyrate (15) , producing and maintaining higher serum butyrate levels than p.o.-administered butyric acid salts (16 , 17) .

We have investigated the effects of tributyrin on MCF-7 human mammary carcinoma cells, establishing that both growth arrest and apoptosis pathways are induced. As early as 4 h after treatment, cells exhibit a transient increase in mitochondria-associated bax, dissipation of the mitochondrial membrane potential ({Delta}{Psi}m), and caspase-3-independent cleavage of PARP. These events are followed by a transient increase in cytosolic cytochrome c and, finally, the generation and accumulation of cells with subdiploid DNA content, a terminal event in apoptosis. During the period in which mitochondria-associated bax levels are elevated, the {Delta}{Psi}m is disrupted, and cytochrome c is detected in the cytosol, we demonstrate increased p53-independent p21WAF1/Cip1 induction and G2-M arrest, suggesting that early mitochondria-associated events may play a role in the initiation of, and/or coordination between, tributyrin-mediated growth arrest and apoptosis of wild-type p53 MCF-7 cells.

Therefore, dietary tributyrin, particularly during the critical period of mammary gland development, has the potential of reducing the risk for breast cancer by promoting both growth arrest and apoptosis pathways in mammary epithelial cells.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture.
MCF-7 human mammary carcinoma cells (18) were obtained from the ATCC and maintained in MEM with Earl’s salts supplemented with 10% fetal bovine serum, 0.1 mM MEM nonessential amino acids, 2 mM glutamine, 10 mM HEPES, 1 unit/ml penicillin, and 1 µg/ml of streptomycin (all obtained from Life Technologies, Inc.) by feeding every 3–4 days. To minimize a contribution of growth factor depletion on an effect of tributyrin, confluent cultures were fed 24 h before exposure to 5 mM tributyrin (Sigma Chemical Co.) in fresh media.

Cell Cycle Parameters and Apoptosis.
Cells were stained with PI, and the fluorescence of individual nuclei (10,000 events) was analyzed by flow cytometry (Becton Dickinson FACScan) using LYSIS II Software (BDIS) for instrument control and data acquisition as we have described previously (19 , 20) . DNA fluorescence signal pulse processing (pulse area versus pulse width) was used to exclude doublets and aggregates from analysis. The percentage of cells in the sub-G0/G1 region and the G0/G1, S and G2-M phases of the cell cycle was determined using ModFIT software (Verity Software House, Topsham, ME).

Quantitation of Steady-State mRNA.
Total RNA was extracted from tributyrin-treated and untreated control cells, and replicate RNA dot blots were generated and processed as we have described (21) . Signals produced after hybridization to 32PdCTP-labeled GAPDH, pHCGAP NR (ATCC 57091) or p21WAF1/Cip1, pCMV-Cip1 (ATCC 79928) were analyzed by Phosphor- Imager and quantitated using ImageQuant software (Molecular Dynamics).

Quantitation of p53 Protein by ELISA.
Quantitation of p53/µg of total cellular protein was determined using pantropic p53 ELISA kits as described by the manufacturer (Oncogene Sciences, Cambridge, MA) and the Bradford method of protein determination (22) . Data are expressed relative to untreated control.

Quantitation of p21WAF1/Cip1 Protein by ELISA.
WAF1 ELISA kits (Calbiochem, La Jolla, CA) were used to quantify units of p21WAF1/Cip1. The Bradford method (22) was used to determine protein levels in each lysate, and data are expressed as units of p21WAF1/Cip1/µg of total protein.

Mitochondrial Membrane Potential.
Alterations in the {Delta}{Psi}m were analyzed by flow cytometry using the mitochondrial membrane potential sensitive dye JC-1 (Molecular Probes, Eugene, OR) as we have described (19) . Briefly, after induction, cells were harvested, washed once in PBS, resuspended in MEM without phenol, and incubated with 1 µM JC-1 at 37°C for 10 min. Stained cells were then washed once in PBS and held at 4°C until evaluated by flow cytometry. A minimum of 10,000 cells/sample were analyzed using a Becton Dickinson FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Data were acquired in list mode and evaluated using WinList software (Verity Software House). Forward scatter (FSC) versus side scatter (SSC) was used to gate the viable population of cells. JC-1 monomers emit at 527 nm (FL-1 channel), and J-aggregates emit at 590 nm (FL-2 channel).

Isolation of S-100 Fractions and Mitochondria.
S-100 (cytosolic) fractions and mitochondria were prepared as described (23) with modifications. Briefly, untreated and tributyrin-treated MCF-7 cells were harvested in PBS at 4°C. Cell pellets were resuspended in five volumes of buffer A [20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 10 µg/ml leupeptin, 1 µg/ml pepstatin A, and 250 mM sucrose] and held at 4°C for 10 min. The cells were then disrupted with 30 strokes of a sandpaper homogenizer, and nuclei and cellular debris were removed by two consecutive centrifugations at 750 x g for 10 min at 4°C. Mitochondria were removed from the supernatant by centrifugation at 10,000 x g for 15 min at 4°C, resuspended in buffer A, and held at -80°C. Cytosolic proteins were extracted by centrifugation of the mitochondrial supernatant at 100,000 x g for 60 min at 4°C. These final supernatants, referred to as S-100 fractions, were concentrated using Amicon centriplus concentrator units (Amicon Inc., Beverly, MA), aliquoted, and stored at -80°.

Immunoblot Analyses.
Twenty µg of S-100 fractions proteins or 10–30 µg of mitochondria were size fractionated on 15% acrylamide gels by SDS-PAGE (Bio-Rad, Hercules, CA), blotted onto Hybond ECL nitrocellulose (Amersham, Arlington Heights, IL), and blocked according to the manufacturer’s protocol. Blots were then incubated for 60 min at room temperature with the following antibodies: mouse monoclonal anti-human actin (Boehringer Mannheim, Indianapolis, IN; clone C4) at 500 ng/ml; cytochrome c (PharMingen, San Diego, CA; clone 7H8.2C12) at 1 µg/ml; cytochrome c oxidase, subunit II (Molecular Probes, Eugene, OR; clone 12C4-F12) at 1 µg/ml; cytochrome c oxidase, subunit IV (Molecular Probes, Eugene, OR; clone 20E8-C12) at 1 µg/ml; or CPP32 (Transduction Laboratories, Lexington, KY; clone 19) at 1:1000; or with rabbit polyclonal anti-human PARP (Boehringer Mannheim, Indianapolis, IN) at 1:1000; or anti-human bax (Upstate Biotechnology, Lake Placid, NY) at 1:1,000. Reactions were detected by enhanced chemiluminescence (Amersham, Arlington Heights, IL) according to the manufacturer’s instructions and quantitated by densitometry using a PhosphorImager and ImageQuant software (Molecular Dynamics).

Quantitation of Caspase-3 Protease Activity.
Activation of caspase-3 was quantified using two commercially available methods, ApoAlert (Clontech; Palo Alto, CA) and PhiPhiLux-G6D2 (OncoImmunin; College Park, MD). For the ApoAlert kit, 2 x 106 cells/sample were lysed and processed according to the manufacturer’s instructions. Briefly, cells were incubated for 60 min at 37°C with a labeled substrate, DEVD-pNA (p-nitroanilide), in reaction buffer containing 7 µM DTT. Activated caspase-3 cleaves the substrate, releasing pNA, which results in an increased absorbance of 405 nm. Reaction buffer plus distilled water served as a reference.

For the PhiPhiLux-G6D2 kit, 1 x 106 cells/sample were reacted with 10 µM of substrate solution (supplied by manufacturer). Each molecule of substrate contains two rhodamine moieties that, when cleaved by activated caspase-3 and exposed to light at 530 nm, emit at 560 nm. A minimum of 10,000 cells/sample were analyzed by flow cytometry measuring fluorescent emission at 560 nm in the FL-2 channel.

Statistical Analyses.
Tributyrin-treated and untreated control cells from at least three individual experiments were compared with two-sample Student’s t tests using individual groups and pooled variances (GB-Stat Computer-Aided Statistics, Version 1.0; Dynamic Microsystems; Silver Springs, MD).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tributyrin Initiates Cell Cycle Arrest in MCF-7 Human Mammary Carcinoma Cells.
To maintain genetic integrity and viability, cells with damaged DNA typically arrest at the G1-S or G2-M checkpoints in the cell cycle to repair defective DNA before undergoing mitosis or before entering an apoptotic pathway. To examine its effect on the cell cycle, wild-type p53 MCF-7 cells (24) were exposed to 5 mM tributyrin for 4–48 h, stained with PI, and analyzed for cell cycle parameters by flow cytometry. As shown in Fig. 1Citation , beginning ~16 h after exposure, a significant number of cells are lost from S phase and accumulate in G2-M. With continued treatment, the tributyrin-mediated G2-M arrest is sustained for up to 48 h.



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  		Fig. 1. Tributyrin initiates G2-M arrest. MCF-7 cells were treated with 5 mM tributyrin for 4–48 h, stained with PI, and analyzed by flow cytometry for quantitation of cell cycle parameters. Data are expressed as the means of at least three independent determinations at each time point; bars, SD. *P8, P <= 0.01 compared with untreated cells calculated by Student’s t test.

 
The tumor suppressor protein p53 plays a key role in the control of cell proliferation through its induction of p21WAF1/Cip1 that, by binding to and inhibiting cyclin-dependent kinases and proliferating cell nuclear antigen, causes cells to arrest in G0/G1 and G2-M (25, 26, 27) . To determine whether the tributyrin-mediated G2-M arrest was associated with p53-mediated p21WAF1/Cip1 induction, MCF-7 cells were treated with tributyrin, and p53 and p21WAF1/Cip1 protein levels were quantitated using ELISA. In addition, total RNA was extracted from cells, and relative steady-state mRNA levels of GAPDH and p21WAF1/Cip1 were rigorously quantitated as we have described (21) .

Despite the presence of wild-type p53, exposure of cells to tributyrin for up to 48 h was ineffective in inducing p53 and, rather, produced a modest, but not statistically significant, decrease in p53 levels relative to untreated cells (Table 1)Citation . However, similar to its induction of G2-M arrest, steady-state levels of p21WAF1/Cip1 mRNA and protein (Fig. 2, A and BCitation , respectively) were significantly increased ~16 h after tributyrin treatment and, with continued exposure, maintained for up to 48 h. Finally, as shown in Fig. 2CCitation , tributyrin-mediated induction of p21WAF1/Cip1 protein exhibited excellent correlation with arrest of cells in G2-M over the entire time course.


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  		Table 1 Relative levels of p53 protein

 


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  		Fig. 2. Tributyrin initiates p21WAF1/Cip1induction. MCF-7 cells were treated with 5 mM tributyrin for 4–48 h. A, total RNA was extracted, and steady-state levels of nuclear GAPDH and p21WAF1/Cip1 were rigorously quantitated using RNA dot blots. B, cell lysates were prepared, and units of p21WAF1/Cip1 protein/µg of total cellular protein were quantitated using ELISA. Relative levels of steady-state mRNA and mean units of p21WAF1/Cip1 protein/µg cellular protein were calculated from at least three independent determinations at each time point; bars, SD. *, P <= 0.01 compared with relative steady-state levels of GAPDH mRNA (A) or with untreated cells (B), calculated by Student’s t test. C, regression analysis comparing the percentage of cells in G2-M and units of p21WAF1/Cip1 protein/µg of cellular protein.

 
Tributyrin Initiates and Activates an Apoptotic Cascade in MCF-7 Cells.
Because arrest of cells at G1-S or G2-M transitions may ultimately lead to death by apoptosis, we next asked whether tributyrin initiated an apoptotic cascade in MCF-7 cells. On the basis of evidence demonstrating that escape from, or delayed, apoptosis is associated with increased or stabilized {Delta}{Psi}m (28, 29, 30) whereas commitment to, or activation of, apoptotic cascades may be linked to disruption of the {Delta}{Psi}m (19 , 31, 32, 33, 34, 35) , we first investigated the effect of tributyrin on the {Delta}{Psi}m.

JC-1 is a mitochondrial membrane potential sensitive dye that, in the absence of, or at low {Delta}{Psi}m, exists as a monomer, emitting at 527 nm, within the green range of visible light. At higher {Delta}{Psi}m, JC-1 forms "J-aggregates" that emit at 590 nm, within the orange range of visible light (19 , 36 , 37) . After treatment with tributyrin, MCF-7 cells were stained with JC-1, examined using fluorescence microscopy, and analyzed by flow cytometry.

As shown by the representative photomicrographs (Fig. 3A)Citation and FACS analyses (Fig. 3B)Citation , untreated MCF-7 cells have a relatively high {Delta}{Psi}m, reflected by mitochondria that appear orange and by the intense clustering of cells with fluorescence emission at 590 nm. In contrast, monomeric JC-1 is seen in cells 4 h after treatment with tributyrin, demonstrated by green staining and fluorescence emission at 527 nm. However, 24 h after treatment, cells exhibited orange-staining mitochondria and fluorescence emission similar to that of untreated cells, suggesting that tributyrin initiates a transient dissipation of the {Delta}{Psi}m in MCF-7 cells.



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  		Fig. 3. Tributyrin initiates transient dissipation of the {Delta}{Psi}m. MCF-7 cells were treated with 5 mM tributyrin for 2–48 h, stained with JC-1, and analyzed by flow cytometry. A, representative photomicrographs of JC-1-stained cells 4 and 24 h after tributyrin treatment. B, representative FACS analysis of JC-1-stained cells 4 and 24 h after tributyrin treatment. C, quantitation of the percentage of cells staining positive for J-aggregates (i.e., emitting at 590 nm). Data are expressed as the mean of at least three independent determinations at each time point; bars, SD. *, P <= 0.01 compared with untreated cells, calculated by Student’s t test.

 
Quantitation of the percentage of cells staining positive for J-aggregates (i.e., emitting at 590 nm; Fig. 3C)Citation confirms that tributyrin mediates a transient dissipation of the {Delta}{Psi}m. A significant decrease in J-aggregates is evident as early as 2 h after exposure to tributyrin, but by 20 h, dissipation is lost, and the {Delta}{Psi}m in tributyrin-treated cells is comparable with that of untreated cells.

To investigate the relationship between tributyrin-mediated dissipation of the {Delta}{Psi}m and activation of an apoptotic cascade, S-100 cytosolic proteins and mitochondria were extracted from MCF-7 cells. Cytosolic levels of cytochrome c and proteolytic activation of caspase-3 and cleavage of PARP were quantitated by densitometric scanning of immunoblots. Blots from a representative experiment and a summary of the data obtained from at least two additional experiments are shown in Fig. 4, A and BCitation , respectively.



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  		Fig. 4. Tributyrin initiates transient cytosolic accumulation of cytochrome c and cleavage of PARP in the absence of caspase-3 activation. MCF-7 cells were treated with 5 mM tributyrin, and 20 µg of cytosolic proteins (S-100 fraction) from untreated (-) or tributyrin-treated (+) cells were fractionated by SDS-PAGE, transferred to nitrocellulose, and probed (see "Materials and Methods"). Reactions were quantitated by densitometry. A, representative immunoblots. Caspase-3 positive control: Jurkat cell lysate. B, summary of quantitative analyses. Relative levels of each antigen are expressed as the mean of at least three independent determinations at each time point; bars, SD. * and x,P <= 0.01 and P <= 0.05, respectively, compared with actin, calculated by Student’s t test.

 
As demonstrated by the data presented in Fig. 4BCitation , which shows the mean relative level of each antigen quantitated from at least three independent determinations, 16 h after treatment with tributyrin, cells exhibited a significant (P <= 0.05), albeit modest, accumulation of cytochrome c in the cytosol. However, after 24 h of treatment, levels of cytosolic cytochrome c declined and became comparable with those of untreated cells, suggesting that the accumulation of cytosolic cytochrome c was transient.

Despite the variability in the amount of cytosolic cytochrome c, as indicated by the large SD at the 16-h time point, COII, a protein encoded by the mitochondrial genome, synthesized on mitochondrial ribosomes and localized to the inner mitochondrial membrane, was not detected in the S-100 fractions (Fig. 4A)Citation . Thus, it is unlikely that detection of cytochrome c in the cytosol was due to damage of mitochondria during the fractionation of cells.

Accumulation of cytochrome c in the cytosol has been implicated in the subsequent proteolytic activation of caspase-3 (38) . However, neither native nor activated caspase-3 was detected on immunoblots (Fig. 4A)Citation , and the proteolytic activity of cleaved caspase-3 was not detected by two commercially available assay systems (not shown). These data are consistent with the recent report demonstrating that, due to a functional deletion within exon 3 of the CASP-3 gene, MCF-7 cells lack caspase-3 (39) .

Despite the absence of caspase-3, MCF-7 cells treated with tributyrin exhibited transient cleavage of PARP, a substrate of proteolytically activated caspase-3 (40) . Although intact PARP is involved in DNA repair and maintenance of the nuclear genome, cleavage of the native Mr 116,000 enzyme into Mr 85,000 and Mr 24,000 fragments results in the loss of this function. PARP cleavage was detected as early as 4 h after tributyrin treatment, demonstrated by a significant increase in the Mr 24,000 cleavage product. However, after 24 h of treatment, levels of proteolytically cleaved PARP decreased, returning to those comparable with untreated cells (Fig. 4)Citation .

Disruption of the {Delta}{Psi}m and the accumulation of cytosolic cytochrome c have been linked to caspase activation-dependent redistribution of bax from the cytosol to the mitochondria (41, 42, 43, 44, 45, 46) . To determine whether tributyrin initiated PARP cleavage, dissipation of the {Delta}{Psi}m and detection of cytochrome c in the cytosol were associated with alterations in mitochondria-associated proteins, blots of size-fractionated purified mitochondria were probed with antibodies directed against bax, cytochrome c, COII, or COIV (nuclear encoded cytochrome c oxidase subunit IV), and reactions were quantitated by densitometry. As shown in Table 2Citation , mitochondria-associated bax levels increased almost 3.5-fold in cells treated with tributyrin for 4 h before returning to levels similar to those in untreated cells after 24 h. Mitochondria-associated cytochrome c levels decreased, albeit modestly, in cells treated with tributyrin for 16 h. Therefore, despite the absence of caspase-3 in MCF-7 cells, tributyrin mediated dissipation of the {Delta}{Psi}m (Fig. 3)Citation , PARP cleavage and detection of cytosolic cytochrome c (Fig. 4)Citation coincide with increased mitochondria-associated bax.


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  		Table 2 Relative levels of mitochondria-associated proteins

 
Unlike its transient cytosolic accumulation, levels of mitochondrial cytochrome c continue to decrease with exposure of cells to tributyrin. Levels of mitochondria-associated COII and COIV also progressively decline, suggesting general mitochondrial dysfunction associated with extended tributyrin treatment.

Finally, because apoptotic cascades typically conclude with the generation of nonrandom fragmentation of nuclear DNA, tributyrin-treated and untreated cells were stained with PI and analyzed by flow cytometry to quantitate the number of cells with subdiploid DNA content. We have demonstrated previously that the percentage of cells localizing in this sub-G0/G1 region exhibits excellent correlation with the appearance of nonrandomly fragmented DNA detected on agarose gels and with the percentage of DNA fragmentation quantified by the diphenylamine reaction (47 , 48) . As shown in Fig. 5Citation , 20 h after tributyrin treatment, a significant number of cells have entered the terminal stages of apoptosis, localizing in the subdiploid DNA peak. 



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  		Fig. 5. Tributyrin initiates terminal events in apoptotic cascades. MCF-7 cells were treated with 5 mM tributyrin for 4–48 h, stained with PI, and analyzed by flow cytometry to quantitate cells with subdiploid DNA content, a terminal event in many apoptotic cascades. Data are expressed as the means of at least three independent determinations at each time point; bars, SD. *, P <= 0.01 compared with untreated cells calculated by Student’s t test.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Homeostasis of the resting mammary gland demands that the equilibrium between cyclic mammary epithelial cell proliferation and apoptosis is tightly and consistently regulated over decades of a woman’s life time. In this report, we have demonstrated that tributyrin, a triglyceride analogue of butyrate and an approved food additive, induces cell cycle arrest and apoptosis in MCF-7 human mammary carcinoma cells.

We have shown that within 16 h of exposure, tributyrin mediates accumulation of cells in G2-M of the cell cycle coincident with its induction of p21WAF1/Cip1 mRNA and protein. Moreover, despite the presence of wild-type p53 in MCF-7 cells (24) , p21WAF1/Cip1 was induced in the absence of increased p53 levels. Although p21WAF1/Cip1 induction is typically coupled with its transactivation by p53, we have shown previously that butyrate induces p21WAF1/Cip1 in a p53 mutant colonic carcinoma cell line (19) . Thus, it is likely that SCFAs have the capacity to activate p21WAF1/Cip1 expression through alternative pathways and that such a pathway is triggered by tributyrin in MCF-7 cells.

Although our data clearly demonstrate that tributyrin induced G2-M arrest, other authors have reported that within 48 h, butyrate (49) and a stabilized derivative of butyrate, esterbut-3 (6) , mediate G0/G1 arrest in MCF-7 cells. In the studies reported here, MCF-7 cells were grown to confluence by feeding cultures every 3–4 days with media containing 10% fetal bovine serum. To minimize a contribution of variation in levels of serum-derived growth factors, media was replaced 24 h before its substitution with tributyrin-containing media. Twenty-four h later, the mean percentage of cells in G2-M in tributyrin-treated cultures was ~1.7 times greater than that in untreated cultures, a highly significant increase (P < 0.0001).

The study reporting butyrate-mediated G0/G1 arrest of MCF-7 cells used a cell line that had been adapted to grow in serum-free media, and the percentage of cells in G0-G1 of the cell cycle increased ~25% 24 and 48 h after exposure to the SCFA (49) . In the study using the butyrate derivative, esterbut-3 was added to cultures immediately after seeding. The percentage of cells in G0-G1 increased ~22% and 75% after 24 and 48 h treatment, respectively (6) . Therefore, the G2-M arrest induced by tributyrin, as opposed to the previously reported G0-G1 arrest induced by butyrate or esterbut-3, may reflect variations in the MCF-7 cells used, cell density, levels of serum-derived growth factors present at the time of treatment, or as our preliminary work suggests,4 a unique property of the triglyceride analogue of the SCFA.

In this report, we have established that, despite an elevated {Delta}{Psi}m that characterizes mammary tumors in vivo and MCF-7 human mammary carcinoma cells in culture (50, 51, 52) , and the absence of caspase-3 (39) , tributyrin also induces apoptosis. The apoptotic cascade is characterized by a transient increase in mitochondria-associated bax coincident with cleavage of PARP; dissipation of the {Delta}{Psi}m; accumulation of cytosolic cytochrome c; decreased levels of mitochondria-associated cytochrome c, COII, and COIV; and finally, the generation of cells with subdiploid DNA content.

Apoptosis requires the activation and action of a set of cysteine proteases, or caspases. Synthesized as inactive precursors, caspases must be proteolytically cleaved to become active enzymes (53) . Once activated, they have the capacity to induce dissipation of the {Delta}{Psi}m (54, 55, 56) , which may be linked to the release of cytochrome c from the mitochondrial intermembranous space to the cytosol (57) . In the cytosol, cytochrome c interacts with Apaf-1, promoting the sequential activation of caspases-9 and -3 (38 , 58 , 59) . Activated caspase-3 can then function in a feed-back loop, amplifying {Delta}{Psi}m dissipation, and a feedforward loop, mediating the cleavage of substrates involved in terminal apoptosis such as PARP.

Although bax does not induce apoptosis, it increases the susceptibility of MCF-7 cells to undergo apoptosis induced by exogenous agents (60) . Recent evidence demonstrates that the proapoptotic properties of bax are tightly linked to its caspase activation-dependent redistribution from the cytosol to the mitochondrial membrane. Insertion of bax homodimers into the mitochondrial membrane promotes {Delta}{Psi}m dissipation, liberation of cytochrome c, and the subsequent caspase activation and cleavage of substrates involved in the events of terminal apoptosis (41, 42, 43, 44, 45, 46 , 54 , 57) . Thus, caspase activation and bax redistribution complement one other, amplifying the initiation of, and mediating successful progression through, an apoptotic cascade.

Consistent with acquisition of its proapoptotic properties, we have shown that mitochondria-associated bax levels substantially increase in tributyrin-treated cells coincident with caspase-3-independent PARP cleavage, {Delta}{Psi}m dissipation, and the detection of cytochrome c in the cytosol. Although it is not yet known how the absence of caspase-3 may compromise amplification of the tributyrin-mediated apoptotic cascade, we are addressing this question.

Tributyrin-mediated alterations in the mitochondria are also likely associated with the substantial decrease in mitochondria-associated COII and COIV detected 24 h after treatment. The stability and synthesis of mitochondria-encoded proteins, such as COII, are influenced by the {Delta}{Psi}m (61 , 62) , as is the mitochondrial import of nuclear-encoded polypeptides, such as COIV (63) . Although the {Delta}{Psi}m is not required for transport of cytochrome c, the mitochondrial NADH-dependent attachment of heme groups is required for its translocation from the outer mitochondrial membrane into the intermembranous space (64 , 65) . Consequently, although decreases in mitochondria-associated COII, COIV, and cytochrome c may be linked to tributyrin-mediated {Delta}{Psi}m dissipation, the progressive decrease in these proteins, in spite of the return of {Delta}{Psi}m to basal levels, may reflect mitochondrial dysfunction that typically accompanies progression through apoptotic cascades.

Finally, in contrast to reports using other agents (66) , we have shown that tributyrin induces nonrandom DNA fragmentation in a significant number of MCF-7 cells. However, because the detection of fragmented DNA coincided with the return of mitochondria-associated bax, {Delta}{Psi}m, cytosolic cytochrome c, and PARP cleavage to levels comparable with those of untreated cells, it is likely that completion of an apoptotic cascade occurred in a subpopulation of cells. It is not yet known whether this, as well as the transient effects on MCF-7 cells, reflects termination or deficiencies in amplification of the apoptotic cascade due to the absence of caspase-3 or properties unique to tributyrin-induced apoptosis. Moreover, whereas PARP cleavage, dissipation of the {Delta}{Psi}m, and accumulation of cytochrome in the cytosol may have been achieved through redistribution of bax and activation of caspases other than caspase-3, the function of cytosolic cytochrome c in the absence of caspase-3 is not yet known.

We have established previously that mitochondrial activities are essential for the initiation of butyrate-induced apoptosis, as well as cell cycle arrest, of colonic carcinoma cells (19) . Here we show that in MCF-7 cells, the tributyrin-mediated increase in mitochondria-associated bax precedes, and induction of p21WAF1/Cip1 and G2-M arrest coincides with, dissipation of the {Delta}{Psi}m and accumulation of cytochrome c in the cytosol. Thus, similar to our previous work (19) , these data suggest that the molecular and biochemical mechanisms responsible for early alterations in mitochondrial activities may play a fundamental role in initiating and/or coordinating SCFA-mediated cell cycle arrest and apoptotic cascades.

Although the mechanisms linking cell cycle arrest and apoptotic cascades in MCF-7 cells are yet to be fully understood, we have established that the triglyceride analogue of the SCFA butyrate, tributyrin, a nontoxic, well-tolerated food additive, initiates entry of mammary carcinoma cells into both pathways. Thus, by promoting a balance between cell proliferation and apoptosis, dietary tributyrin, particularly during the critical period of mammary gland development, may have substantial potential as a chemopreventive agent.


   ACKNOWLEDGMENTS
 
We thank Dave Gebhard for assistance with flow cytometry.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Cancer Institute Grants R29 CA59932, RO1 CA75246, and P30 CA13330 and American Institute for Cancer Research Grant 95B025. Back

2 To whom requests for reprints should be addressed, at Albert Einstein Cancer Center, Montefiore Medical Center, Department of Oncology/Hofheimer 511, 111 East 210th Street, Bronx, NY 10467. Phone: (718) 920-2750; Fax: (718) 882-4464. Back

3 The abbreviations used are: SCFA, short-chain fatty acid; PARP, poly(ADP-ribose) polymerase; ATCC, American Type Culture Collection; PI, propidium iodide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JC-1, 5,5',6,6'-tetrachloro-1,1,3,3' -tetraethylbenzimidazolylcarbocyanine iodide; COII and COIV, cytochrome c oxidase subunits II and IV, respectively; FACS, fluorescence-activated cell sorting. Back

4 B. G. Heerdt and M. A. Houston, unpublished data. Back

Received 9/30/98. Accepted 2/ 2/99.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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