Interactive effects of fatty acid and butyrate-induced mitochondrial Ca²⁺ loading and apoptosis in colonocytes

Abstract

BACKGROUND

The combination of fish oil-derived docosahexaenoic acid (DHA, 22:6, n-3) and butyrate (4:0), a fiber fermentation product, synergize to enhance colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca^2+-dependent (intrinsic) pathway.

METHODS

In this study, we probed the specificity of n-6 and n-3 polyunsaturated fatty acid induction of Ca²⁺-dependent proapoptotic events in immortalized YAMC colonocytes. We also determined whether combinations of polyunsaturated fatty acid and butyrate trigger endoplasmic stress (ER) stress conditions, thereby promoting mitochondrial Ca²⁺ overload. Cultures were treated with 0–50 μM of DHA (22:6, n-3), EPA (20:5, n-3), AA (20:4, n-6), LA (18:2, n-6) or OA (18:1, n-9) for a total of 72 h ± RU-360, to inhibit the mitochondrial Ca²⁺ uniporter, for 30 min prior to butyrate (0 or 5 mM) co-treatment.

RESULTS

DHA and butyrate combination maximally induced apoptosis and mitochondrial-to-cytosolic Ca²⁺ levels. In comparison, EPA, a precursor to DHA, was minimally effective. Similarly, AA and OA in combination with butyrate had no effect on mitochondrial Ca²⁺ or apoptosis compared to butyrate alone. DHA ± butyrate co-treatment minimally altered ER stress regulated genes, CHOP and eIF2α.

CONCLUSION

These data indicate that butyrate and DHA, but not EPA, work coordinately to trigger an ER-independent, Ca²⁺-dependent intrinsic mitochondrial-mediated apoptotic pathway in colonocytes.

INTRODUCTION

There is substantial experimental, epidemiological and clinical evidence indicating that fish oil-containing diets rich in n-3 polyunsaturated fatty acids (PUFA), e.g. docosahexaenoic acid (DHA, 22:6^{Δ 4,7,10,13,16,19}) and eicosapentaenoic acid (EPA, 20:5^{Δ 5,8,11,14,17}), are protective against colon tumorigenesis (1–9). In a major recent finding, it was demonstrated that EPA reduced rectal polyp number and size in patients with familial adenomatous polyposis (FAP) (10). Most impressive was the fact that fish oil derived EPA suppressed FAP to a degree similar to the selective COX-2 inhibitor celecoxib. Collectively, these data indicate that n-3 PUFA hold promise as a chemoprevention agent for FAP and sporadic colon cancer. In part as a consequence of these studies, fish oil is now being extensively promoted in the unregulated nutraceutical sector. However, some of the fundamental aspects of n-3 PUFA action need to be understood before it can be developed as a clinically viable cancer prevention therapy. These areas pertain to the mechanism of EPA and DHA action, which is still not fully defined in molecular terms. Indeed, it is becoming increasingly clear that these fatty acids have distinct biological properties (11–13).

The balance between proliferation and apoptosis is critical to the maintenance of steady-state number for cell populations in the colon. In general, dysregulation of this mechanism can disrupt homeostasis, resulting in clonal expansion, with the resultant over production of affected cells (14,15). The enhancement of “targeted” apoptosis combined with the reduced formation of DNA adducts during the initiation and late phases of tumorigenesis, in part, accounts for the observed protective effect of n-3 PUFA against experimentally induced colon cancer (16-18). A very exciting outcome of our studies was the demonstration that the pleiotropic bioactive components generated by fermentable fiber (butyrate) and fish oil (DHA) work coordinately to protect against colon tumorigenesis (5,19,20). Although it has been well documented that butyrate is an inhibitor of histone deacetylases and can activate the Fas receptor mediated extrinsic death pathway (21,22), the role of these mechanisms in the induction of colonocyte apoptosis may be a secondary consequence of its ability to promote cellular oxidation (23). With respect to molecular triggers for apoptosis, Ca²⁺ is one of the most versatile and universal signaling mediators in cells and is required for the activation of many cellular processes. Increasing evidence indicates that alterations in the finely tuned intracellular homeostasis and compartmentalization of Ca²⁺ can lead to cell death either through apoptosis or necrosis (24). Eukaryotic cells can increase their cytosolic Ca²⁺ levels via 2 mechanisms: release of Ca²⁺ from intracellular stores or influx via plasma membrane channels. Although the importance of the endoplasmic reticulum (ER) as the major storage organelle is indisputable, growing evidence indicates that functional compartmentalization of Ca²⁺ exists within the various cellular organelles. Recent studies have identified the contributions of the mitochondria in maintaining intracellular Ca²⁺ homeostasis and cellular physiologic function (25). Given the central role of mitochondria in the commitment to apoptosis, we demonstrated that DHA and butyrate interactively promote apoptosis by triggering changes in mitochondrial Ca²⁺ levels that contribute to caspase activation and colonocyte cell death. For example, isogenic p53 wild type and deficient human colon tumor cell lines as well as an immortalized mouse colonocytes were used to show that DHA modulates intracellular Ca²⁺ compartmentalization and store-operated channel (SOC) entry to induce colonocyte apoptosis (26). These results corroborate previous observations (19,27) and demonstrate that DHA and butyrate synergistically enhance both mitochondrial Ca²⁺ accumulation and membrane lipid peroxidation which serve as triggers for apoptosis in a p53-independent manner (28).

In this study, we further probed the specificity of n-6 and n-3 PUFA induction of Ca²⁺-dependent proapoptotic events in immortalized colonocytes. In addition, since ER stress-dependent apoptotic pathways can be activated by fatty acids (29, 30), we also determined whether combinations of PUFA and butyrate trigger ER stress conditions, thereby promoting mitochondrial Ca²⁺ overload.

EXPERIMENTAL PROCEDURES

Materials

Two-well Lab-Tek Chambered Coverglassslides were purchased from Nunc, Inc. (Naperville, IL). RPMI 1640 and Hanks' balanced salt solution (HBSS) were from Mediatech (Herndon, VA). Insulin, transferrin, selenium without linoleic acidwas purchased from Collaborative Biomedical Products (Bedford, MA). Fetal bovine serum (FBS) was from Hyclone(Logan, UT). Recombinant mouse interferon-γ (IFN-γ), Glutamax and Leibowitz media werepurchased from GIBCO BRL (Grand Island, NY). Fatty acids were obtained from NuChek (Elysian, MN). Ca²⁺ sensitive dyes Fluo-4 AM and Rhod-2 AM were from Molecular Probes (Eugene, OR). Soluble recombinant Fas:Fc was from BD Biosciences. Mitochondrial Ca²⁺ inhibitor RU-360 was from Calbiochem (San Deigo, CA). Cell death detection ELISA kit was obtained from Roche Applied Science (Indianapolis, IN). Sodium butyrate, IgG₁ from human serum and all other reagents were obtained from Sigma (St. Louis, MO). Stock solutions of Fluo-4, AM (1 mM) and Rhod-2 (4 mM) were prepared in DMSO and diluted to 3.0 μM and 2.0μM, respectively for cell culture treatment (final concentration of the vehicle DMSO was maintained at 0.1–0.3% in culture). RU-360 (1 mM stock) was prepared in degassed water and diluted in media to a final concentration of 10 μM.

Cell Culture

Conditionally immortalized YAMC cells were originally obtained from R. H. Whitehead, Ludwig Cancer Institute (Melbourne, Australia). These cells are immortalized by virtue of expression of a temperature-sensitive simian virus 40 large (SV40) T antigen (31). YAMC cells were cultured in RPMI 1640 supplemented with 5% FBS, 1% insulin- transferrin-selenium, 2 mM Glutamax and 5,000 units/liter of recombinant IFN-γ. Cells were cultured under permissive (33°C with interferon-γ) or non-permissive conditions (39°C) as previously described (26). For all fluorescence measurements, cells (passages 12–18) were seeded onto borosilicate 2-chambered cover glass slides at a density of 5–7 x 10³ to achieve a 50–70% confluence. For apoptosis assays, cells were seeded onto 6 well plates at a density of 35 x10³. Following 24 h of plating select cultures were treated with 0-50 μM of Bovine serum albumin (BSA) complexed DHA (22:6, n-3), EPA (20:5, n-3), AA (20:4, n-6), LA (18:2, n-6) or OA (18:1, n-9) for a total of 72 h as previously described (26,28). In select experiments, cultures were pre-incubated with 0 or 10 μM RU-360 in RPMI 1640, to inhibit the mitochondrial uniporter, for 30 min prior to butyrate and fatty acid co-treatment. In other experiments cells were co-incubated with Fas:Fc or IgG₁ (3 μg/ml) along with sodium butyrate for the final 24 h of fatty acid pre-treatment following which apoptosis and mitochondrial-to-cytosol Ca²⁺ ratio were measured.

Analysis of Mitochondrial Ca²⁺

Following a 24 h fatty acid and butyrate treatment period, cells were washed with Leibowitz media, free of serum and phenol red. Cells were then coincubated with 3 μM Fluo-4 and 2 μM Rhod-2 for 1 h at 33°C. Cultures were subsequently washed with Leibowitz media without serum and the mitochondrial-to-cytosolic Ca²⁺ ratio was measured. Quantification of Fluo-4 and Rhod-2 fluorescence was performed by excitation at 488 nm and 550 nm, and fluorescence emission was monitored at 530 nm and 580 nm, respectively, as previously described (26,28). The ratio of mitochondrial-to-cytosolic Ca²⁺ level was calculated. Data from five-twelve areas per well and from at least six to eight wells per treatment group were collected in eachexperiment. Mitochondrial Ca²⁺ accumulation observed by Rhod-2 fluorescence was corroborated by inhibiting the mitochondrial uniporter using RU-360 (10 μM) for 30 min prior to butyrate co-treatment. In addition, in previous experiments, MitoTracker, a mitochondrial targeting dye, was used to validate the subcellular localization of Rhod-2 fluorescence (26).

Quantitative Reverse Transcriptase PCR

Total RNA was isolated using the RNAqueous 4PCR isolation kit from Ambion (Austin, TX). The isolated total RNA was subsequently treated with DNase to remove contaminating DNA. RNA was reverse-transcribedto cDNA using SuperScript II (Gibco BRL). Real-time PCR was performed using the ABI 7900 (Applied Biosystems, Foster City, CA). Pre developed Taqman gene expression assay for mouse CHOP (NM-008929) was obtained from Applied Biosystems.

Immunoblotting

Cell lysates from treated YAMC cells were immunoblotted with eIF2α or phospho-eIF2α antibody using the method of Davidson et al. (32) to evaluate the protein expression level. Briefly, samples were treated with SDS sample buffer and subjected to electrophoresisin a 4–20% pre-cast Tris–glycine gel. After electrophoresis, proteins were electroblotted onto PVDF membranes using a HoeferMighty Small Transphor Unit (Pharmacia, Piscataway, NJ) at 400mA for 100 min. After transfer, membranes were incubated with rabbit anti-eIF2α (9722) or rabbit anti-phospho-Ser51-eIF2α (9721, Cell Signaling, Danvers, MD) antibody overnight at 4°C, followed by peroxidase labeled goat anti-rabbit IgG incubation for 1 h at room temperature. Bands were detected using SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL), and the blots were scanned using a Bio-Rad Fluor-S MaxMultiImager System (Hercules, CA).

Cell Death Assay

Apoptosis was determined using nucleosomal fragmentation enzyme linked immuno-sorbent (ELISA) (Roche). Floating cells were harvested, washed, lysed and centrifuged to sediment nuclei. The supernatant containing mono and oligonucleosomes was incubated with substrate and analyzed by ELISA. Absorbance values were measured at 405 nm and subsequently normalized to the number of adherent cells per dish as previously described (22). To determine the association between mitochondrial-to-cytosolic Ca²⁺ ratio and apoptosis, select cultures were pre-incubated with RU-360 (10 μM) for 30 min prior to butyrate exposure. Cells were washed and treated with 5 mM butyrate and apoptosis was measured following a 24 h incubation period. Additionally, the association between the extrinsic pathway and apoptosis was determined by co-incubating select cultures with Fas:Fc or IgG₁ with sodium butyrate for the final 24 h. In complimentary apoptosis experiments, adherent and floating cells were harvested, washed, pelleted by centrifugation and lysed.

Statistical Analysis

The effect of independent variables (treatment effects) was assessed using SuperAnova. Differences among means were determined using t/F type tests of contrast. P-values less than 0.05 were considered to be statistically significant.

Results

The findings in Figure 1 indicate that butyrate alone as well as EPA (n-3 PUFA), AA (n-6 PUFA control) and OA (n-9 monounsaturated control) in combination with butyrate significantly (p<0.05) enhanced apoptosis (~80%) compared to untreated control. In comparison, DHA and butyrate maximally enhanced apoptosis (170%) compared to untreated control. Fatty acid treatment alone had no effect on the apoptotic index (Figure 1 inset). The levels of butyrate and fatty acid are considered physiologically relevant because they lie within the range found in the colonic lumen (33) and blood (34), respectively. With regard to the effect of treatment on intracellular calcium levels, the combination of EPA and butyrate modestly (8%) increased (p<0.05) the mitochondrial-to-cytosolic Ca²⁺ ratio compared to untreated control (Figure 2). In comparison, the combination of DHA and butyrate maximally (47%) induced mitochondrial-to-cytosolic Ca²⁺ levels compared to untreated control. Results in Figure 2 inset indicate that fatty acid treatment alone had no effect on intracellular Ca²⁺ homeostasis.

Figure 1. Induction of apoptosis by fatty acid and butyrate co-treatment.

YAMC cells were treated with (50 μM) DHA, EPA, AA or OA for a total of 72 h and 0 or 5 mM butyrate for the final 24 h. Non-adherent cells were harvested and apoptosis was measured by nucleosomal fragmentation assay. C, control- no fatty acid or butyrate; B, butyrate only; DB, DHA and butyrate; EB, EPA and butyrate; AB, AA and butyrate; OB, OA and butyrate. Apoptotic index data are expressed as a percentage of untreated control. Data are from n = 6-12 wells per treatment, n = 2- 4 independent experiments. Mean values ± S.E. not sharing common letters are significantly different, p<0.05. Inset, Apoptotic index measured in cultures treated with fatty acid (50 μM for 72 h) only. D, DHA only; E, EPA only; A, AA only; O, OA only.

Figure 2. Fatty acid and butyrate co-treatment modulate mitochondrial Ca²⁺ levels.

YAMC cells were exposed to 50 μM fatty acid for 72 h in the absence or presence of 5 mM butyrate for the final 24 h. Cells were co-loaded with Fluo-4 (3 μM) and Rhod-2 AM (2 μM) and the ratio of mitochondrial-to-cytosolic Ca²⁺ was measured as described in the Experimental Procedures Section. Data are means ± S.E., 20–60 cells per treatment, n = 2 independent experiments. Data represent mitochondrial-to-cytosolic Ca²⁺ levels as a percentage of untreated control. Bars not sharing common letters are significantly different, p< 0.05. Inset, Effect of fatty acid treatment alone on mitochondrial Ca²⁺ levels. See Figure 1 for legend details. Data are means ± S.E. from a representative experiment, n=2 independent experiments.

Having established that both DHA and EPA when combined with butyrate perturb intracellular Ca²⁺ homeostasis, we subsequently investigated the ability of an inhibitor of the mitochondrial Ca²⁺ uniporter, RU360 (28), to suppress the induction of apoptosis. In addition, since Fas receptor signaling transduction is involved in butyrate induction of apoptosis in colonocytes (22), we asked how YAMC cells would respond when cultured with antagonistic soluble Fas:Fc. As shown in Figure 3A, RU-360 partially blocked DHA and butyrate induced apoptosis while Fas:Fc had no effect. The combination of RU-360 + Fas:Fc partially reversed the proapoptotic effect of DHA and butyrate combination (DB). In contrast, Fas:Fc + RU-360 treatment combination (DBRF) did not exhibit any significant difference in apoptosis compared to the RU-360 treatment (DBR) group. In cells not exposed to fatty acid (Figure 3A inset), results indicate that butyrate enhanced apoptosis compared to untreated control cells (C). Fas:Fc alone (BF) and the combination of RU-360 + Fas:Fc (BRF) completely blocked the butyrate-induced increase in apoptosis in the absence of fatty acid treatment. In contrast, RU-360 alone had no effect on butyrate-induced apoptosis. Results in Figure 3B demonstrate the DHA and butyrate co-treatment significantly increased mitochondrial-Ca²⁺ accumulation compared to untreated control. Fas:Fc (DBF) had no effect on mitochondrial-to-cytosolic Ca²⁺ levels compared to DHA + butyrate (DB) treated cells. As expected, RU-360 pre-treated cultures (DBR, DBRF) exhibited a significant (p<0.05) reduction in mitochondrial-to-cytosolic Ca²⁺ levels. In cells not exposed to fatty acid (Figure 3B inset), Fas:Fc treatment (BF, BRF) reduced mitochondrial-to-cytosolic Ca²⁺ levels. In contrast, RU-360 alone (BR) had no effect on mitochondrial-Ca²⁺ accumulation, consistent with the lack of an effect of butyrate on intracellular Ca²⁺ homeostasis (26).

Figure 3. Fas-inhibitor and RU-360 block DHA and butyrate-induced changes in apoptosis and mitochondrial Ca²⁺.

(Panel A) YAMC cells were treated with DHA (50 μM) for 48 h. Subsequently, select cultures were pre-incubated with RU-360 (10 μM) for 30 min. RU-360 treated cells were rinsed and subsequently co-incubated with fatty acid + 5 mM butyrate only (DBR treatment) or DHA + butyrate + Fas-inhibitor (3 ug/ml Fas:Fc) for an additional 24 h (DBRF treatment). Select cultures not pre-treated with RU-360 were co-incubated with fatty acid + butyrate + Fas:Fc (DBF group). Non-adherent cells were harvested and nucleosomal fragmentation was used to quantify apoptosis. Data are means ± S.E., n = 6-12 wells per treatment from 2 separate experiments. Inset, Apoptotic index in cells treated with 5 mM butyrate in the absence of fatty acid (B), butyrate + RU-360 (BR), butyrate + Fas:Fc (BRF) or combination of butyrate + RU-360 + Fas:Fc (BRF). (Panel B) To evaluate mitochondrial-Ca²⁺ levels, cultures were co-loaded with Fluo-4 (3 μM) and Rhod-2 AM (2 μM), and the mitochondrial-to-cytosolic Ca²⁺ ratio was measured. Data are means ± S.E., n= 18-90 cells per treatment, n = 3 independent experiments. Inset, Mitochondrial-to-cytosolic Ca²⁺ levels in cells treated with butyrate, RU-360, Fas:Fc or the combination of RU-360 and Fas:Fc in the absence of fatty acid. Refer to Panel A inset for legend details.

We have previously demonstrated that the immortalized YAMC cell line is a good model system to examine the properties of bioactive chemoprotective with respect to apoptosis (19,22,34). In comparative experiments examining EPA, RU-360 partially blocked EPA and butyrate induced apoptosis while Fas:Fc had no effect (Figure 4A). The combination of RU-360 + Fas:Fc partially reversed the proapoptotic effect of EPA and butyrate combination (EB). In contrast, Fas:Fc + RU-360 treatment combination (EBRF) did not exhibit any significant difference in apoptosis compared to RU-360 treatment (EBR) group. In cells not exposed to fatty acid or butyrate (Figure 4A inset), the addition of cell death modulators alone or in combination (RU-360, Fas:Fc or Ru-360+Fas:Fc) had no effect on apoptosis compared to untreated control cells (C). Analysis of mitochondrial-to-cytosolic Ca²⁺ ratios following EPA treatment is shown in Figure 4B. EPA + butyrate combination significantly (p<0.05) enhanced mitochondrial-to-cytosolic Ca²⁺ levels, which was inhibited by RU-360 (EBR) and the combination of Fas:Fc and RU-360 (EBRF). In contrast, Fas:Fc treatment in the absence of RU-360 (EBF) had no effect on mitochondrial-to-cytosolic Ca²⁺ accumulation. As shown in Figure 4B inset, for cells treated with RU-360 (CR), Fas:Fc (CF) or the combination of RU-360 and Fas:Fc CRF) in the absence of fatty acid and butyrate, only RU-360 and Fas:Fc treatment (CRF) reduced mitochondrial-to-cytosolic Ca²⁺ levels as compared to untreated control cells.

Figure 4. Fas-inhibitor and RU-360 block EPA and butyrate-induced changes in apoptosis and mitochondrial Ca²⁺.

(Panel A) YAMC cells were treated with EPA (50 μM) for 48 h after which select cultures were incubated with RU-360 (10 μM) for 30 min to inhibit mitochondrial Ca²⁺ uptake. RU-360 pre-treated cells were rinsed and subsequently co-incubated with EPA and butyrate (5 mM) only (EBR treatment) or EPA + butyrate + Fas:Fc (3 ug/ml) for the final 24 h (EBRF treatment). Select cultures not pre-treated with RU-360 were co-incubated with EPA + butyrate + Fas:Fc (EBF treatment). Data are means ± S.E., n = 6 wells per treatment. Inset, Mitochondrial-to-cytosolic Ca²⁺ levels in untreated cells (no fatty acid or butyrate), RU-360, Fas:Fc or the combination of RU-360 and Fas:Fc. C, untreated control cells-no fatty acid or butyrate added; CR, RU-360 pretreatment; CF, Fas:Fc co-treatment; CRF, RU-360 and Fas;Fc co-treatment. (Panel B) Analysis of mitochondrial-to- cytosolic Ca²⁺ ratios following EPA treatment. Cultures were co-loaded with Fluo-4 (3 μM) and Rhod-2 AM (2 μM), and the mitochondrial-to-cytosolic Ca²⁺ ratio was measured. See panel A for treatment details. Inset, Mitochondrial-to-cytosolic Ca²⁺ ratios in untreated control cells-no fatty acid or butyrate added.

Since Ca²⁺ is stored in the ER, and ER stress increases Ca²⁺ transfer to mitochondria, thereby amplifying the PERK-eIF2α-ATF4-CHOP pathway (36), we further examined the effect of fatty acid and butyrate treatment on ER stress regulated genes. Both butyrate alone and butyrate-fatty acid combinations modestly increased CHOP mRNA expression relative to basal untreated control (Figure 5). Thapsigargin, an inhibitor of sarco/ER Ca²⁺⁻ATPase (SERCA) activity, known to induce ER stress, was used as a positive pharmacological control (26,37). Examination of the ER stress protein phosphorylation (phosphorylated alpha-subunit of eukaryotic initiation factor 2 (P-eIF2α)) revealed no induction of the ER stress response (Figure 6). Collectively, these data suggest that the proapoptotic synergy between DHA and butyrate involving mitochondrial Ca²⁺ loading is not mediated by ER stress.

Figure 5. Butyrate exposure modestly increases CHOP expression.

YAMC cells were treated with butyrate (5 mM), DHA (50 μM), LA (50 μM, n-6 PUFA control) or combinations of butyrate and DHA (But + DHA), butyrate and LA (But + LA), or Thapsigarain (10 μM) (ER stress positive control) for 24 h. Total RNA was isolated and the CHOP mRNA levels were measured using Taqman real-time PCR. Data are means ± SE, expressed as fold of basal CHOP expression, n=3-6 wells. Bars not sharing common letters are significantly different, p<0.05.

Figure 6. Inability of butyrate and fatty acid combination to alter phospho- and total-eIF2α protein levels.

YAMC cells were treated with DHA (50 μM), LA (50 μM), a combination of butyrate (5 mM) and DHA (But + DHA), butyrate and LA (But + LA), or Thapsigargin (10 μM,Th) (ER stress positive control) for 24 h. Cell lysates were prepared and phopho-eIF2α and total eIF2α protein levels were measured by immunoblotting. (A) Representative immunoblots of p-eIF2α and total eIF2α. (B) Quantification of immunoblot data, expressed as percentage of phospho/total eIF2α. Data are means ± SE, n=3-6 wells. Bars not sharing common letters are significantly different, p<0.05.

Discussion

An important outcome of our previous studies was the demonstration that the anti-tumorigenic effect of fish oil (n-3 PUFA) is enhanced when a highly fermentable fiber or its fermentation product-butyrate is added to the diet (4,18–20,38). This chemopreventive effect is mediated in part by the upregulation of targeted apoptosis of DNA adducts during tumor initiation (16,27) and spontaneous apoptosis during promotion (39). With respect to a mechanism of action, pectin is metabolized by bacteria within the lumen of the gut to generate millimolar levels of butyrate and other short chain fatty acids (32). We have recently shown that the combination of DHA and butyrate compared with DHA or butyrate alone further enhances colonocyte apoptosis by inducing a p53-independent, oxidation sensitive, mitochondrial Ca²⁺⁻dependent (intrinsic) pathway (26,28). Our results using YAMC cells are consistent with data generated using the human (HCT116) colon cancer cell line as well as mouse and rat azoxymethane in vivo models (4,19,20,26,28). Therefore, similar results are observed when neoplastic cells are used. Collectively, these data indicate that highly fermentable fiber, which generates butyrate in the colon, only has chemotherapeutic value when n-3 PUFA is the lipid source. This critical observation emphasizes the need to examine both the lipid and fiber composition of diets.

In order to further elucidate specifically which dietary n-3 PUFA in combination with fermentable fiber up-regulate apoptosis effector mechanisms in colonocytes, we contrasted the ability of EPA, AA or OA vs DHA in combination with butyrate to synergistically induce apoptosis. Specifically, n-6 and n-3 PUFA were examined with respect to apoptogenic mitochondrial Ca²⁺ overloading and ER stress. We report that the combination of DHA and butyrate maximally induced apoptosis and mitochondrial-to-cytosolic Ca²⁺ levels. In comparison, EPA, a precursor to DHA, was minimally effective. Similarly, arachidonic acid (20:4n-6) and oleic acid (18:1n-9) in combination with butyrate had no effect on mitochondrial Ca²⁺ or apoptosis compared to butyrate alone. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial-to-cytosolic Ca²⁺ accumulation and also partially blocked apoptosis in DHA and butyrate cotreated cells. Collectively, these data show that only the combination of DHA and butyrate, compared to fatty acid or butyrate alone, synergistically enhances apoptosis by additionally recruiting a Ca^2+-mediated intrinsic mitochondrial pathway. Data from these experiments extend observations in human cell lines and rat colonic crypt cultures (28). Since butyrate is capable of inducing colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway (22,26), we also examined the effect of a CD95 (Fas receptor) antagonist. As expected, Fas:Fc blocked apoptosis in cultures incubated with butyrate alone. However, this antagonist did not block mitochondrial-to-cytosolic Ca²⁺ accumulation and apoptosis in DHA plus butyrate treated cells.

Since select fatty acids can regulate cell death via induction of ER stress in a number of cell types (29,30), we examined whether a PUFA/butyrate combination regimen activates ER stress responses in colonocytes. Intriguingly, we found little evidence that DHA ± butyrate cotreatment increases ER stress regulated genes, CHOP and eIF2α. This is in contrast with a previous report where the cytotoxic effect of DHA was associated with signaling pathways involving ER stress (30). In these studies, DHA was added in ethanol at a higher concentration (70 μM) to the colon cancer cell line SW620. Treatment of these cells leads to the accumulation of numerous large lipid droplets, which is likely cytoxic (30). In order to avoid artifacts related to fatty acid oxidation and toxicity, we routinely complex fatty acids to albumin, a physiologically relevant delivery modality for long chain PUFA use in culture (40).

Acquired resistance to apoptosis is a common defect associated with tumor cells and represents a critical obstacle for therapy (14,41). Therefore, the elucidation of novel apoptotic triggers constitutes an important objective in the effort to develop therapies. It is important to note that chemopreventive interventions using drugs have raised safety concerns. For example, the long-term use of COX-2 selective non-steroidal anti-inflammatory drugs is not feasible due to associated cardiovascular risk (42,43). This has dampened enthusiasm for their extensive use as cancer chemopreventive therapies. Safety problems intrinsic to drugs administered over long periods of time strengthen the interest in diet-related cancer chemoprevention approaches. Therefore, the search for toxicologically innocuous dietary interventions is timely and propitious, and foodstuffs providing both DHA and butyrate-generating fermentable fiber provide an attractive alternative.

In summary, we have demonstrated that the pleiotropic bioactive components generated by fermentable fiber (butyrate) and fish oil (DHA but not EPA) work coordinately to trigger a Ca²⁺-dependent intrinsic mitochondrial-mediated apoptotic pathway in colonocytes. This is significant because n-3 PUFA (1,6,8) and butyrate (20,41,44,45) reduce colon cancer risk in humans, and appear to be ideally suited as a new generation of nontoxic chemotherapeutics to work either alone or in combination with chemoprotective drugs, e.g., NSAIDs, whose long term use is contraindicated (43). Given the critical nature of apoptosis in colon cancer prevention, and the fact that inhibition of apoptosis is an integral component in the genesis of colon cancer, it is imperative to elucidate the precise mechanisms by which n-3 PUFA and butyrate promote apoptosis in the colon.

Abbreviations Used

AA

arachidonic acid

BSA

bovine serum albumin

DHA

docosahexaenoic acid

EPA

eicosapentaenoic acid

ER

endoplasmic reticulum

FAP

familial adenomatous polyposis

FBS

fetal bovine serum

IFNγ

gamma interferon

LA

linoleic acid

OA

oleic acid

SERCA

sarco/ER Ca²⁺ ATPase

YAMC

young adult mouse colonocytes