We have previously shown that butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. Interestingly, fermentable fiber when combined with fish oil containing docosahexaenoic acid (DHA, 22:6n-3) exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis. To determine the molecular mechanism of action, the effect of DHA and butyrate cotreatment on intracellular Ca2+ homeostasis was examined. Mouse colonocytes were treated with 50 μmol/L DHA or linoleic acid (LA) for 72 h ± butyrate (0–10 mmol/L) for the final 24 h. Cytosolic and mitochondrial Ca2+ levels were measured using Fluo-4 and Rhod-2. DHA did not alter basal Ca2+ or the intracellular inositol trisphosphate (IP3) pool after 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA- and butyrate-treated cultures exhibited a 25% and 38% decrease in cytosolic Ca2+ compared with LA and butyrate. Chelation of extracellular Ca2+ abolished the effect of thapsigargin on the IP3-releasable Ca2+ pool. DHA and butyrate cotreatment compared with untreated cells increased the mitochondrial-to-cytosolic Ca2+ ratio at 6, 12, and 24 h by 73%, 18%, and 37%, respectively. The accumulation of mitochondrial Ca2+ preceded the onset of apoptosis. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial Ca2+ accumulation and also partially blocked apoptosis in DHA and butyrate cotreated cells. Collectively, these data show that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+-mediated intrinsic mitochondrial pathway. [Cancer Res 2007;67(11):5561–8]

There is substantial experimental, epidemiologic, and clinical evidence indicating that fish oil–containing diets rich in n-3 polyunsaturated fatty acids (PUFA), for example, docosahexaenoic acid (DHA, 22:6Δ4,7,10,13,16,19) and eicosapentaenoic acid (EPA, 20:5Δ5,8,11,14,17), are protective against colon tumorigenesis (13). In addition, there is evidence from epidemiologic and observational studies indicating that the consumption of fiber, which increases butyrate levels in the lumen of the colon, is chemoprotective against colorectal cancers (4, 5). In contrast, several systematic reviews have challenged the premise that dietary fiber and fish oil reduce colon cancer risk, fueling a debate regarding the role of dietary fat and fermentable fiber as chemoprotective nutrients (69). To address this apparent conundrum, our laboratory has focused on the question as to why a diet containing highly fermentable fiber is only protective when fish oil is the lipid source. We have shown that the bioactive components generated by fermentable fiber (butyrate) and fish oil (DHA) work coordinately to protect against colon tumorigenesis, primarily by increasing apoptosis rather than decreasing cell proliferation (3, 10, 11). With regard to a molecular mechanism of action, we have shown that DHA alters colonocyte mitochondrial membrane composition and function, thereby creating a permissive environment for apoptosis induced by luminal metabolites, such as butyrate (12, 13). More recently, we have shown that mitochondrial lipid oxidation products, membrane phospholipid-derived hydroperoxides (LOOH), play an important role in DHA and butyrate-induced apoptosis (14). Despite the evidence indicating that the combination of dietary fish oil and fermentable fiber enhance apoptosis and suppress colon cancer, we still lack information regarding the precise molecular mechanisms by which the DHA and butyrate combination protect against colon tumorigenesis.

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. The Ca2+ concentration inside the cell is regulated by the simultaneous interplay of various counteracting mechanisms, which can turn cellular signals “on” and/or “off” (15). Eukaryotic cells can increase their cytosolic Ca2+ levels via two mechanisms: release of Ca2+ from intracellular stores or influx via plasma membrane channels. Channels located in the plasma membrane, for example, store-operated Ca2+ channels (SOC), receptor-operated channels, and voltage-operated channels, regulate the influx of Ca2+ into the cell. Currently, there is a good understanding of the organelles that function as Ca2+ stores and how Ca2+ can be released from stores into the cytosol. 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. More recent studies have identified the contributions of the nuclear envelope, Golgi apparatus, lysosomes, and mitochondria in maintaining intracellular Ca2+ homeostasis and cellular physiologic function (16, 17). In fact, it is now recognized that mitochondria play a key role in both apoptosis and necrosis by regulating energy metabolism, intracellular Ca2+ homeostasis, activation of caspases, and the release of reactive oxygen species (18, 19).

Mitochondria are localized in close proximity to inositol 1,4,5-trisphosphate (IP3)–gated channels situated on the ER. Ca2+ uniporters, which are low-affinity and high-capacity active pumps located in the mitochondrial outer membrane, rapidly take up Ca2+ that is released from the ER stores and recirculate it back into the cytosol via exchangers such as the Na2+/Ca2+ exchanger (20, 21). Therefore, mitochondria can be regarded as critical checkpoints in Ca2+ signaling, acting as membrane-bound Ca2+ buffers. Under normal conditions, mitochondrial Ca2+ uptake serves as a signal to increase cellular metabolism (20). However, in certain situations, mitochondrial Ca2+ accumulation is a trigger for cytochrome c release and the induction of apoptosis (22).

Given the central role of mitochondria in the commitment to apoptosis, we hypothesized that n-3 PUFA and butyrate can promote apoptosis by triggering changes in mitochondrial Ca2+ levels that contribute to caspase activation and colonocyte cell death. We used an immortalized mouse colonocyte (YAMC) cell line to determine whether chemoprotective nutrients modulate intracellular calcium compartmentalization and SOC entry to induce colonocyte apoptosis. The results confirm and extend our previous observations and show that DHA and butyrate combination synergistically alter intracellular Ca2+ compartmentalization by enhancing mitochondrial Ca2+ accumulation through an SOC-mediated mechanism. These outcomes provide clear evidence that an increase in mitochondrial Ca2+ stores contributes to the induction of apoptosis by DHA and butyrate cotreatment.

Materials. RPMI 1640 and HBSS were purchased from Mediatech. Fetal bovine serum was from Hyclone. Insulin, transferrin, and selenium without linoleic acid (LA), were purchased from Collaborative Biomedical Products. Glutamax, recombinant mouse IFN-γ, and Leibovitz medium were from Life Technologies. Fatty acids were obtained from NuChek. Fluo-4 AM, Rhod-2 AM, and Calcium Calibration buffer kit 2 were purchased from Molecular Probes. RU-360 and SKF-96365 were purchased from Calbiochem. Cell death detection ELISA kit was obtained from Roche Applied Science. Two-well Lab-Tek Chambered Coverglass slides were purchased from Nunc, Inc. Thapsigargin, staurosporine, EGTA, sodium butyrate, and all other reagents were obtained from Sigma. Stock solutions of 1.0 mmol/L Fluo-4, AM, and 4.0 mmol/L Rhod-2 were prepared in DMSO and diluted with medium to 3.0 and 2.0 μmol/L, respectively (final concentration of the vehicle DMSO was maintained at 0.1–0.3% in culture). Thapsigargin stock (5.0 mmol/L) was prepared in DMSO and used at a concentration of 5.0 μmol/L (0.1% DMSO). RU-360 (1 mmol/L) stock was prepared in degassed water and diluted to a final concentration of 10 μmol/L for cell treatment. A (1 mmol/L) stock solution of staurosporine was prepared in DMSO and diluted to a final concentration of 1 μmol/L for cell treatment.

Cell culture. Conditionally immortalized YAMC cells were originally obtained from R.H. Whitehead (Ludwig Cancer Institute, Melbourne, Australia). Cells were cultured under permissive (33°C with IFN-γ) or nonpermissive conditions (39°C) as previously described (14, 23). For all fluorescence measurements, cells (passages 12–18) were seeded onto borosilicate two-chambered cover glass slides at a density of 5 × 103 to 7 × 103 to achieve a 50% to 70% confluence. For apoptosis assays, cells were seeded onto 35-mm cell culture dishes or six-well plates at a density of 35 × 103. Bovine serum albumin (BSA)–complexed fatty acids were added to cultures 24 h after cell plating as previously described (23). Select cultures were treated with BSA-complexed DHA (22:6, n-3) or LA (18:2, n-6; 0–200 μmol/L) for 72 h. Cells were coincubated with sodium butyrate (0–10 mmol/L) in RPMI 1640 for the final 6, 12, 24, or 48 h of fatty acid pretreatment.

Quantification of cytosolic Ca2+ in mouse colonocytes. Following fatty acid and butyrate treatment, cells were washed with Leibovitz medium, free of serum and phenol red. Cells were loaded with 3 μmol/L Fluo-4, AM, at 33°C to determine the levels of cytosolic Ca2+. Fluo-4 is a visible wavelength nonratiometric cytosolic Ca2+ indicator that exhibits a 40-fold increase in fluorescence intensity with Ca2+ binding (24, 25). Following a 1-h incubation with the probe, cells were washed with Leibovitz medium and imaged. Images were acquired from groups of 10 to 20 cells, 10 to 12 areas per well, and 4 to 8 wells per treatment using a Stallion Digital Imaging workstation equipped with 300 W xenon fluorescent light source. Fluorescent light was rapidly (<2 ms) switched between excitation wavelengths. Images were captured using a ×20 objective 0.75 numerical aperture and a ROPER CoolSnap HQ camera. For image acquisition, imaging variables were adjusted for maximum detection of fluorescence with minimal cellular photobleaching. Cells were excited at 488 nm, and fluorescence emission from individual cells was collected at 530 nm. Image acquisition frequency was set to one image every 10 s. Fluorescence intensities were recorded for 300 s. A minimum of two experiments done on different days were analyzed. Basal intracellular Ca2+ was measured before the addition of thapsigargin. Thapsigargin is an irreversible sarcoplasmic/ER Ca2+ ATPase (SERCA) pump inhibitor that was used to empty the intracellular Ca2+ store from the ER (25). Fluorescence intensities after thapsigargin stimulation were normalized to basal Ca2+ levels.

To determine the contribution of IP3 Ca2+ pools, extracellular Ca2+ was chelated using 2 mmol/L EGTA followed by cell stimulation with thapsigargin. Chelation of extracellular Ca2+ facilitates quantification of IP3 Ca2+ released from the ER after thapsigargin addition (26, 27). Ca2+ was then added back into the medium and the contribution of the SOC pool was evaluated (25). To further evaluate the association between PUFA and butyrate cotreatment and SOC, cultures were preincubated with 10 μmol/L SKF-96365, a pharmacologic inhibitor of SOC, for 5 min before image acquisition. Calibration of intracellular Ca2+ was done using the Calcium Calibration Buffer kit 2 from Molecular Probes as previously described (28).

Analysis of mitochondrial Ca2+. Cells treated with fatty acid and butyrate were washed with Leibovitz medium and coloaded with 3 μmol/L Fluo-4 and 2 μmol/L Rhod-2 for 1 h at 33°C. Cells were then washed twice with Leibovitz medium, and the mitochondrial-to-cytosolic Ca2+ ratio was measured. For quantification of Fluo-4 and Rhod-2 fluorescence, excitation light was provided at 488 and 550 nm and fluorescence emissions were collected at 530 and 580 nm, respectively. The ratio of the cytosolic-to-mitochondrial Ca2+ level was subsequently calculated. Although accumulation of Rhod-2 dye in the nucleoli was noticed, MitoTracker was used to confirm that dye loading was predominantly localized within the mitochondria as previously described (29). In other experiments, cells were incubated with RU-360 (10 μmol/L), an inhibitor of the mitochondrial uniporter, for 30 min before butyrate cotreatment (30). Cells were then washed and coloaded with fatty acid and butyrate for the final 6, 12, or 24 h after which the mitochondrial-to-cytosolic Ca2+ ratio was determined. Nucleoli were not included in the analysis of mitochondrial Ca2+.

Apoptosis assays. Apoptosis was measured using cellular fragmentation ELISA (Roche) and caspase-3 activity assay (Molecular Probes) as previously described (23). To determine the association between mitochondrial Ca2+ and apoptosis, select cultures were preincubated with RU-360 (10 μmol/L) for 30 min before butyrate exposure. Cells were washed and treated with 5 mmol/L butyrate and apoptosis was measured after a 6-, 12-, or 24-h incubation period.

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 <0.05 were considered to be statistically significant.

DHA and butyrate combination decreases cytosolic Ca2+. Cytosolic Ca2+ levels were initially examined after stimulation with thapsigargin, an irreversible SERCA pump inhibitor used to empty ER Ca2+ stores. Figure 1A shows representative traces of thapsigargin-induced Ca2+ response measured over 300 s. The arrow indicates the time point of thapsigargin addition. Butyrate treatment (5 mmol/L) over a period of 6 to 24 h increased (P < 0.05) cytosolic Ca2+ in cells preincubated with LA (50 μmol/L, a control n-6 fatty acid) by up to 45% compared with untreated control (Fig. 1B–D). In contrast, cells preincubated with DHA consistently exhibited a lower cytosolic Ca2+ level compared with LA and butyrate cotreatment. Control cultures containing either DHA or LA alone, in the absence of butyrate, exhibited a modest increase in cytosolic Ca2+ levels, 8% to 12%, compared with untreated control (Fig. 1C  and D, inset). In comparison, cultures treated with LA and butyrate exhibited a 45% increase compared with untreated control at both time points. Changes in Ca2+ levels were detected as early as 6 h after butyrate and fatty acid cotreatment.

Figure 1.

Effects of fatty acid and butyrate on thapsigargin-induced Ca2+ response. YAMC cells were treated with fatty acid alone (50 μmol/L) for 72 h with butyrate (5 mmol/L) for the final 6 h (B), 12 h (C), or 24 h (D). Cells were subsequently incubated with Fluo-4 (3 μmol/L), a fluorescent cytosolic Ca2+ indicator for 1 h at 33°C, and basal Ca2+ was measured. Representative traces (15–20 cells/trace) are shown in (A). Arrow, point of thapsigargin (5 μmol/L) addition. Normalized intensity is the average intensity at any point in time, t divided by intensity at time 0. Cytosolic Ca2+ was quantified at the single-cell level by choosing at random three to four fields with 10 to 12 cells per field. Representative data are from a single experiment, average of 80 to 120 cells per treatment, n = 4 to 6 independent experiments. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. C, control—no fatty acid or butyrate; B, butyrate only; L, LA only; D, DHA only; LB, LA and butyrate; DB, DHA and butyrate. DHA compared with LA-pretreated cells showed a significant decrease in cytosolic Ca2+ levels with 12 and 24 h of butyrate cotreatment. Inset, cytosolic Ca2+ levels measured in untreated, butyrate only, fatty acid only, and LA and butyrate combination cells at 12 and 24 h (inset in C and D, respectively). Representative data are from a single experiment, average of 80 to 120 cells per treatment, n = 2 to 4 independent experiments. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. DHA-treated cells exhibited no significant difference in cytosolic Ca2+ levels when compared with LA-treated cells.

Figure 1.

Effects of fatty acid and butyrate on thapsigargin-induced Ca2+ response. YAMC cells were treated with fatty acid alone (50 μmol/L) for 72 h with butyrate (5 mmol/L) for the final 6 h (B), 12 h (C), or 24 h (D). Cells were subsequently incubated with Fluo-4 (3 μmol/L), a fluorescent cytosolic Ca2+ indicator for 1 h at 33°C, and basal Ca2+ was measured. Representative traces (15–20 cells/trace) are shown in (A). Arrow, point of thapsigargin (5 μmol/L) addition. Normalized intensity is the average intensity at any point in time, t divided by intensity at time 0. Cytosolic Ca2+ was quantified at the single-cell level by choosing at random three to four fields with 10 to 12 cells per field. Representative data are from a single experiment, average of 80 to 120 cells per treatment, n = 4 to 6 independent experiments. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. C, control—no fatty acid or butyrate; B, butyrate only; L, LA only; D, DHA only; LB, LA and butyrate; DB, DHA and butyrate. DHA compared with LA-pretreated cells showed a significant decrease in cytosolic Ca2+ levels with 12 and 24 h of butyrate cotreatment. Inset, cytosolic Ca2+ levels measured in untreated, butyrate only, fatty acid only, and LA and butyrate combination cells at 12 and 24 h (inset in C and D, respectively). Representative data are from a single experiment, average of 80 to 120 cells per treatment, n = 2 to 4 independent experiments. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. DHA-treated cells exhibited no significant difference in cytosolic Ca2+ levels when compared with LA-treated cells.

Close modal

SOC involvement in maintaining intracellular Ca2+ homeostasis. To examine the role of SOC entry, the increase in plasma membrane influx associated with ER Ca2+ emptying was characterized using a Ca2+ add-back protocol. This standard procedure uses extracellular Ca2+ chelation with EGTA followed by utilization of thapsigargin to induce an initial emptying of the ER Ca2+ pool, followed by repletion of the bathing solution with Ca2+ (26, 27). Consistent with previous experiments (Fig. 1) after incubation with 5 μmol/L thapsigargin, butyrate (24 h)–treated cells preincubated with DHA in the presence of extracellular Ca2+ exhibited a 40% decrease in cytosolic Ca2+ compared with LA-primed cells (P < 0.05; Fig. 2). Subsequently, extracellular Ca2+ was chelated by addition of 2 mmol/L EGTA, which allows for indirect quantification of the ER Ca2+ pool. In these experiments, butyrate-treated cells primed with DHA or LA showed no difference in cytosolic Ca2+ levels (P > 0.05; Fig. 2). Following replacement of the medium with CaCl2 (10 mmol/L), the phenotype was reestablished, implicating the involvement of plasma membrane channels in the propagation of fatty acid–induced Ca2+ signaling.

Figure 2.

Effect of fatty acid on thapsigargin-induced Ca2+ response in the presence or absence of EGTA. YAMC cells were treated with fatty acid (50 μmol/L) for 72 h with or without butyrate (5 mmol/L) for the final 24 h. A, cells were incubated with Fluo-4 (3 μmol/L) for 1 h at 33°C and basal Ca2+ was measured. B, extracellular Ca2+ was chelated using 2 mmol/L EGTA and cells were stimulated with thapsigargin (5 μmol/L). C, CaCl2 (10 mmol/L) was added back to the extracellular medium and cytosolic Ca2+ was quantified. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. Representative data are from a single experiment, n = 2 independent experiments. Refer to Fig. 1 for legend details. In colonocytes with the combination of DHA and butyrate, chelation of extracellular Ca2+ abolished the difference in cytosolic Ca2+ levels and replenishing extracellular medium with Ca2+ reestablished the difference. Taken together, these data show that plasma membrane channel entry contributed to the difference seen in cytosolic Ca2+ level.

Figure 2.

Effect of fatty acid on thapsigargin-induced Ca2+ response in the presence or absence of EGTA. YAMC cells were treated with fatty acid (50 μmol/L) for 72 h with or without butyrate (5 mmol/L) for the final 24 h. A, cells were incubated with Fluo-4 (3 μmol/L) for 1 h at 33°C and basal Ca2+ was measured. B, extracellular Ca2+ was chelated using 2 mmol/L EGTA and cells were stimulated with thapsigargin (5 μmol/L). C, CaCl2 (10 mmol/L) was added back to the extracellular medium and cytosolic Ca2+ was quantified. Columns, mean; bars, SE. Data not sharing common letters are significantly different, P < 0.05. Representative data are from a single experiment, n = 2 independent experiments. Refer to Fig. 1 for legend details. In colonocytes with the combination of DHA and butyrate, chelation of extracellular Ca2+ abolished the difference in cytosolic Ca2+ levels and replenishing extracellular medium with Ca2+ reestablished the difference. Taken together, these data show that plasma membrane channel entry contributed to the difference seen in cytosolic Ca2+ level.

Close modal

To identify the plasma membrane channels involved in eliciting a rise in calcium influx after ER Ca2+ depletion in colonocytes, cultures were incubated with SKF-96365, an inhibitor of nonvoltage-gated, SOC channels (31, 32). Typically, butyrate-treated cells primed with DHA or LA were preincubated with 10 μmol/L SKF for 5 min before basal recording. SKF abrogated the fatty acid–induced effect on cytosolic Ca2+ levels after 12 h butyrate cotreatment (Supplementary Fig. S1A). Similar effects were observed after prolonged butyrate treatment (24 h; Supplementary Fig. S1B). Thus, the activation of SOCs, leading to rapid entry of Ca2+ through the plasma membrane, is likely to contribute to the observed fatty acid and butyrate-induced perturbation in colonocyte Ca2+ homeostasis.

Effect of DHA and butyrate cotreatment on mitochondrial Ca2+ levels. Because the efflux of Ca2+ from the ER can lead to coupled increases in mitochondrial Ca2+ levels, we assessed the effects of fatty acid and butyrate cotreatment on mitochondrial Ca2+ uptake. Butyrate cotreatment for 6 h increased (P < 0.0001) the mitochondrial-to-cytosolic Ca2+ ratio in cells treated with DHA by 73% in comparison with untreated cells (Fig. 3A). In addition, at 12 and 24 h, DHA and butyrate selectively increased (P < 0.0001) mitochondrial Ca2+ by 18% and 37%, respectively (Fig. 3B and C). In contrast to DHA-treated cultures, cells incubated in the presence of LA showed a small response or no change in the mitochondrial-to-cytosolic Ca2+ ratio compared with untreated cells. With respect to the fatty acid–only controls, LA treatment had no effect on the mitochondrial-to-cytosolic Ca2+ ratio. DHA treatment tended to increase the mitochondrial-to-cytosolic Ca2+ ratio at all time points (Fig. 3B, and C, inset). However, in all cases, cultures pretreated with DHA and coincubated with butyrate showed the largest increase in mitochondrial Ca2+ levels (Fig. 3). Representative photomicrographs of untreated cells and cultures coincubated with butyrate (5 mmol/L for 24 h) and DHA or LA (50 μmol/L) are shown in Fig. 3 (D–F).

Figure 3.

Effect of fatty acid and butyrate cotreatment on mitochondrial Ca2+ levels. YAMC cells were exposed to 50 μmol/L fatty acid for 72 h in the absence or presence of 5 mmol/L butyrate for the final 6 (A), 12 (B), or 24 h (C). Cells were coloaded with Fluo-4 (3 μmol/L) and Rhod-2 AM (2 μmol/L), and the ratio of mitochondrial-to-cytosolic Ca2+ was evaluated as described in Materials and Methods. Columns, mean from a representative experiment, n = 3 independent experiments; bars, SE. D to F, representative images from no-treatment (control), LA and butyrate, and DHA and butyrate 24-h cultures. A significant (P < 0.0001) difference between the combination of LA with butyrate and DHA with butyrate existed starting from 6 h. Refer to Fig. 1 for legend details. Inset, effects of fatty acid treatment with or without butyrate on mitochondrial Ca2+ levels at 12 h (B) and 24 h (C), respectively. Columns, means from a representative experiment, n = 2 to 3 independent experiments; bars, SE. Data not sharing common letters are significantly different, P < 0.05.

Figure 3.

Effect of fatty acid and butyrate cotreatment on mitochondrial Ca2+ levels. YAMC cells were exposed to 50 μmol/L fatty acid for 72 h in the absence or presence of 5 mmol/L butyrate for the final 6 (A), 12 (B), or 24 h (C). Cells were coloaded with Fluo-4 (3 μmol/L) and Rhod-2 AM (2 μmol/L), and the ratio of mitochondrial-to-cytosolic Ca2+ was evaluated as described in Materials and Methods. Columns, mean from a representative experiment, n = 3 independent experiments; bars, SE. D to F, representative images from no-treatment (control), LA and butyrate, and DHA and butyrate 24-h cultures. A significant (P < 0.0001) difference between the combination of LA with butyrate and DHA with butyrate existed starting from 6 h. Refer to Fig. 1 for legend details. Inset, effects of fatty acid treatment with or without butyrate on mitochondrial Ca2+ levels at 12 h (B) and 24 h (C), respectively. Columns, means from a representative experiment, n = 2 to 3 independent experiments; bars, SE. Data not sharing common letters are significantly different, P < 0.05.

Close modal

Effects of a mitochondrial uniporter inhibitor on mitochondrial Ca2+ uptake after DHA and butyrate cotreatment. To investigate the role of the mitochondrial uniporter in Ca2+ uptake, cells were treated with RU-360, a mitochondrial Ca2+ uniporter inhibitor (33). RU-360 (10 μmol/L) partially inhibited (P < 0.001) the butyrate-induced increase in mitochondrial Ca2+ in DHA-primed cells upon addition 30 min before butyrate cotreatment at 6 or 12 h (Fig. 4). In comparison, RU-360 had no effect on mitochondrial Ca2+ levels in LA-treated cells.

Figure 4.

Effects of RU-360 on mitochondrial Ca2+ uptake after fatty acid and butyrate cotreatment. Cells were pretreated with fatty acid for a total of 72 h and RU-360 (10 μmol/L) for 30 min before butyrate cotreatment for the last 6 h (A) and 12 h (B), respectively. Cells were coincubated with Fluo-4 (3 μmol/L) and Rhod-2 (2 μmol/L) for 1 h and the mitochondrial-to-cytosolic Ca2+ ratio was measured. Columns, mean from a representative experiment, n = 2 independent experiments; bars, SE. Refer to Fig. 1 for legend details. Data obtained show that in DHA- and butyrate-treated cells, RU-360 partially blocked mitochondrial Ca2+ accumulation.

Figure 4.

Effects of RU-360 on mitochondrial Ca2+ uptake after fatty acid and butyrate cotreatment. Cells were pretreated with fatty acid for a total of 72 h and RU-360 (10 μmol/L) for 30 min before butyrate cotreatment for the last 6 h (A) and 12 h (B), respectively. Cells were coincubated with Fluo-4 (3 μmol/L) and Rhod-2 (2 μmol/L) for 1 h and the mitochondrial-to-cytosolic Ca2+ ratio was measured. Columns, mean from a representative experiment, n = 2 independent experiments; bars, SE. Refer to Fig. 1 for legend details. Data obtained show that in DHA- and butyrate-treated cells, RU-360 partially blocked mitochondrial Ca2+ accumulation.

Close modal

Induction of colonocyte apoptosis by fatty acid and butyrate combination. Using the nucleosomal fragmentation assay, DHA significantly (P < 0.0001) enhanced butyrate-induced apoptosis compared with cells treated with LA plus butyrate or butyrate alone (control) at 12 and 24 h (Supplementary Fig. S2). In contrast, fatty acid treatment alone had no effect on apoptosis (Supplementary Figs. S2C, inset, S3A and B). To further corroborate these observations, complimentary methodology (caspase-3 activity) was also used. Similar results were obtained (Supplementary Fig. S2D). For comparative purposes, staurosporine, a broad-spectrum protein kinase inhibitor, which induces apoptosis in normal and malignant cells, was used as a positive control (34). As expected, cultures preincubated with 1 μmol/L staurosporine for 4 h exhibited an 8-fold increase in apoptotic cells compared with untreated or fatty acid–treated cells (P < 0.0001; Supplementary Fig. S2C, inset).

The mitochondrial Ca2+ uniporter inhibitor, RU-360, suppresses induction of apoptosis after DHA and butyrate cotreatment. To investigate the relationship between mitochondrial Ca2+ uptake and cellular apoptosis, we compared the levels of apoptosis observed in DHA plus butyrate–treated cells in the absence or presence of the mitochondrial Ca2+ uniporter inhibitor RU-360. Results obtained in experiments with butyrate at 12 and 24 h after DHA or LA cotreatment are shown in Fig. 5. RU-360 significantly (P < 0.05) reduced apoptosis by ∼45% after 12-h butyrate cotreatment. Similar results were observed in DHA primed cells after 24-h butyrate cotreatment. In contrast, inhibition of the uniporter had no effect on cells treated with LA plus butyrate or butyrate alone at 12 and 24 h. Collectively, these results show that mitochondrial Ca2+ uptake is required for the enhanced apoptosis associated with DHA and butyrate cotreatment.

Figure 5.

Effect of RU-360 on apoptotic index. YAMC cells were treated with DHA or LA (50 μmol/L) for 48 h and subsequently preincubated with 10 μmol/L RU-360 for 30 min. Treated cells were then coincubated with fatty acid with or without 5 mmol/L butyrate for the final 12 h (A) and 24 h (B). Nonadherent cells were harvested, and nucleosomal fragmentation was used to quantify apoptosis. Columns, mean from two separate experiments, n = 6 wells per treatment; bars, SE. Refer to Fig. 1 for legend details. Data show that RU-360 significantly blocked apoptosis with cells pretreated with the combination of DHA and butyrate.

Figure 5.

Effect of RU-360 on apoptotic index. YAMC cells were treated with DHA or LA (50 μmol/L) for 48 h and subsequently preincubated with 10 μmol/L RU-360 for 30 min. Treated cells were then coincubated with fatty acid with or without 5 mmol/L butyrate for the final 12 h (A) and 24 h (B). Nonadherent cells were harvested, and nucleosomal fragmentation was used to quantify apoptosis. Columns, mean from two separate experiments, n = 6 wells per treatment; bars, SE. Refer to Fig. 1 for legend details. Data show that RU-360 significantly blocked apoptosis with cells pretreated with the combination of DHA and butyrate.

Close modal

Kinetics of DHA and butyrate induction of mitochondrial Ca2+ and apoptosis. To probe the functional proximity of mitochondrial Ca2+ uptake in relation to the induction of apoptosis, we analyzed the effects of treatment over time (6, 12, and 24 h). As shown in Supplementary Fig. S4, cells treated with butyrate and DHA exhibited a significantly (P < 0.0001) higher level of mitochondrial Ca2+ compared with all other treatments as early as 6 h. In contrast, the induction of apoptosis was not observed until 12 h. These results indicate that the increase in mitochondrial Ca2+ preceded the onset of apoptosis and therefore plays an important role in triggering programmed cell death in colonic mucosal cells.

Dietary DHA (22:6n-3) has been extensively studied over the last decade in relation to its role as a bioactive chemopreventive agent. Past results from our laboratory have shown that DHA, when combined with either a fermentable fiber source or butyrate, enhances reactive oxygen species and LOOH production and causes a change in mitochondrial permeability transition in colonocytes (1214, 35). Here, we report that DHA and butyrate cotreatment also synergistically enhance apoptosis by up to 43% in colonocyte cultures compared with butyrate alone (Supplementary Fig. S2). In contrast, colonocytes treated with either DHA or LA alone showed no significant increase in the level of apoptosis compared with untreated cells (Supplementary Fig. S3). From a biological relevance perspective, these data are consonant with animal carcinogen studies showing that the bioactive components of fermentable fiber (butyrate) and fish oil (DHA) coordinately protect against colon tumorigenesis, primarily by increasing apoptosis (3, 10, 11, 36). Further, our studies reveal that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+-mediated intrinsic mitochondrial pathway. Based on our findings, we propose a pathway for the induction of apoptosis in colonic epithelium that involves the synergistic action of DHA and butyrate on enhanced mitochondrial Ca2+ accumulation (Fig. 6).

Figure 6.

Proposed molecular model of DHA and butyrate-induced apoptosis. Butyrate induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. DHA and butyrate, in combination, synergistically perturb intracellular Ca2+, stimulating mitochondrial Ca2+ uptake. This directly or indirectly decreases cytosolic Ca2+ and promotes SOC-mediated entry via plasma membrane channels. Mitochondrial Ca2+ accumulation subsequently triggers the opening of the permeability transition pore (PTP) and release of proapoptotic molecules like cytochrome c and other factors such as apoptosis-inducing factor (AIF), and second mitochondrial activator of caspases (smac/DIABLO). Together, these effects culminate in the induction of procaspases and downstream caspases that execute cellular apoptosis.

Figure 6.

Proposed molecular model of DHA and butyrate-induced apoptosis. Butyrate induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. DHA and butyrate, in combination, synergistically perturb intracellular Ca2+, stimulating mitochondrial Ca2+ uptake. This directly or indirectly decreases cytosolic Ca2+ and promotes SOC-mediated entry via plasma membrane channels. Mitochondrial Ca2+ accumulation subsequently triggers the opening of the permeability transition pore (PTP) and release of proapoptotic molecules like cytochrome c and other factors such as apoptosis-inducing factor (AIF), and second mitochondrial activator of caspases (smac/DIABLO). Together, these effects culminate in the induction of procaspases and downstream caspases that execute cellular apoptosis.

Close modal

Over the last decade, extensive progress has been made in establishing the effects of long-chain PUFA on various ions and ion channels, including Ca2+ (3739). With respect to a molecular mechanism of action, agents that increase mitochondrial reactive oxygen species/LOOH generation have been linked to a proapoptotic cycle involving Ca2+ release from intracellular stores and mitochondrial loading (40). These data suggest that a change in mitochondrial Ca2+ homeostasis may mediate the proapoptotic effect of butyrate and DHA in colonocytes. The current experiments support this hypothesis because the combination of DHA and butyrate produced the highest mitochondrial Ca2+ accumulation (Fig. 3) while simultaneously decreasing free cytosolic Ca2+ levels (Fig. 1C and D). These data are consistent with our previous finding that DHA primes the cell for butyrate-induced lipid oxidation (14).

The role of mitochondria in the regulation of intracellular Ca2+ homeostasis and apoptotic signaling is well established (1618). Recent work has shown that mitochondria are closely juxtaposed with the ER to effectively take up Ca2+ that is released from the ER. There is also evidence of clustering of IP3 receptors on the ER membrane facing the mitochondria (41, 42). Besides close physical association of the ER and mitochondria, IP3 receptor–dependent Ca2+ signals are associated with an increase in mitochondrial Ca2+, which is capable of triggering apoptosis (22, 43). Consistent with these observations, DHA-treated cultures exhibited a significant accumulation of mitochondrial Ca2+ within 6 h of butyrate cotreatment (Fig. 3A), preceding the onset of apoptosis that was not increased until 12 h after butyrate cotreatment (Supplementary Figs. S2B and 4).

To explore the connection between the accumulation of mitochondrial Ca2+ and apoptosis, we examined the effect of a mitochondrial Ca2+ uniporter inhibitor (RU-360). RU-360 significantly blocked mitochondrial Ca2+ uptake after DHA and butyrate cotreatment (Fig. 4) and partially blocked the induction of apoptosis (Fig. 5). These outcomes provide evidence that an increase in mitochondrial Ca2+ levels contribute directly to the induction of apoptosis by DHA and butyrate cotreatment and the change in intracellular Ca2+ homeostasis is not an epiphenomenon.

Despite the difficulties inherent to measuring mitochondrial Ca2+ levels, both imaging and uniporter inhibitor data indicate that Rhod-2 loads primarily into the mitochondria. With respect to how mitochondria translate/interpret Ca2+ signals that ultimately trigger apoptosis, it has been shown that multifactorial cross-talk among Ca2+, ATP, and oxidative stress enhance cytochrome c dislocation from the inner mitochondrial membrane and activate Ca2+-dependent endonucleases, which are responsible for the induction of the DNA fragmentation and apoptosis (44). There is also evidence that mitochondrial Ca2+ sequestration (via the mitochondrial uniporter) results in the opening of the mitochondrial permeability transition pore, which can induce the release of proapoptotic molecules (i.e., Bax, Bak, cytochrome c), resulting in mitochondrial-mediated (intrinsic) apoptosis (42, 45). This scenario correlates well with our finding of a decrease in colonic mitochondrial membrane potential in fish oil– but not corn oil–fed rats (12, 13).

To elucidate the relative contribution of ER Ca2+ store emptying versus SOC Ca2+ entry in maintaining intracellular Ca2+ homeostasis, thapsigargin-evoked Ca2+ responses were measured in a Ca2+-free environment using EGTA. The removal of extracellular Ca2+ effectively abolished the DHA and butyrate-induced decrease in cytosolic Ca2+ levels (Fig. 2). Following replenishment of extracellular Ca2+, the difference in cytosolic Ca2+ between DHA and butyrate versus control (LA and butyrate) treatment groups was reestablished. These data suggest that Ca2+ influx through SOC partly mediates the DHA and butyrate perturbation of intracellular Ca2+. To corroborate the involvement of plasma membrane non–voltage-gated Ca2+ channels, SOC-dependent influx was also antagonized using SKF-96365. SKF pretreatment eliminated the difference in thapsigargin-evoked cytosolic Ca2+ levels between LA plus butyrate and DHA plus butyrate treatment groups. These data are consistent with previous observations in which DHA was found to regulate intracellular signaling by modulating plasma membrane Ca2+ entry (46), suggesting that the combination of DHA and butyrate alters colonocyte-free cytosolic Ca2+ levels in part by modulating SOC entry. Along these lines, growing evidence suggests that the mitochondrial permeability transition pore is activated by both oxidative stress and Ca2+, whereas mitochondrial reactive oxygen species inhibit SERCA pumps (47) and activate SOC (48). In accordance with these findings, we have recently shown in HCT-116 cells that a mitochondrion-specific antioxidant blocks the DHA- and butyrate-induced induction of apoptosis.4

4

In preparation.

Further studies are needed to determine if these effects extend to other members of the n-3 PUFA family, because a number of studies have shown that EPA and DHA have similar effects on biological membranes (1, 12, 13).

We have previously shown that butyrate induces colonocyte apoptosis via a Fas receptor–mediated extrinsic pathway (23). In an extension of our findings, we show for the first time that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca2+-dependent mitochondrial-intrinsic pathway. Notably, DHA-enriched mitochondria were sensitized to rapidly sequester Ca2+, which served to trigger apoptosis in the presence of butyrate. This could, in part, explain why the combination of dietary fish oil (containing DHA) and pectin (which generates butyrate in the lumen of the colon) seems to reduce tumor formation in the colon by promoting apoptosis (3, 10, 13, and 35). Although previous studies have examined the effects of PUFA to mobilize intracellular Ca2+ (46, 49), it is apparent that DHA and butyrate work coordinately in the colon to initiate a proapoptotic cycle involving the activation of SOC, leading to rapid entry of Ca2+ through the plasma membrane and mitochondrial Ca2+ loading (Fig. 6). With regard to putative upstream mediators, we have shown that a DHA-butyrate combination dissipates mitochondrial membrane potential, an effect that was reversed by coincubation with permeability transition pore inhibitors (14). This is consistent with the fact that mitochondrial Ca2+ interacts with cyclophilin D, which can trigger opening of the permeability pore (50). Although the precise chronology of these events have not been elucidated, it is likely that permeability transition pore opening, mitochondrial lipid oxidation, mitochondrial Ca2+ accumulation, and the induction of apoptosis are all intimately linked.

In summary, recent data indicate that chemotherapeutic agents that restore normal apoptotic pathways have the potential for effectively treating cancers that depend on aberrations of the apoptotic pathway to develop. Hence, it is of interest that DHA and butyrate work coordinately in the colon to trigger a previously unrecognized proapoptotic cycle involving mitochondrial Ca2+ loading. This may explain why a diet containing highly fermentable fiber is only protective when fish oil is the lipid source.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: NIH CA59034 and P30ES09106.

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.

We thank Evelyn Callaway for her excellent technical assistance and Dr. Robert Burghardt for his thoughtful advice.

1
Chapkin RS, McMurray DN, Lupton JR. Colon cancer, fatty acids and anti-inflammatory compounds.
Curr Opin Gastroenterol
2007
;
23
:
48
–54.
2
Courtney ED, Matthews S, Finlayson C, et al. Eicosapentaenoic acid (EPA) reduces crypt cell proliferation and increases apoptosis in normal colonic mucosa in subjects with a history of colorectal adenomas. Int J Colorectal Dis 2007. Epub ahead of print.
3
Chang WCL, Chapkin RS, Lupton JR. Fish oil blocks azoxymethane-induced rat colon tumorigenesis by increasing cell differentiation and apoptosis rather than decreasing cell proliferation.
J Nutr
1998
;
128
:
491
–49.
4
Lupton JR. Microbial degradation products influence colon cancer risk; the butyrate controversy.
J Nutr
2000
;
134
:
479
–82.
5
Bingham SA, Day NE, Luben R, et al. Dietary fibre in food and protection against colorectal cancer in the European prospective investigation into cancer and nutrition (EPIC): an observational study.
Lancet
2003
;
361
:
1496
–501.
6
Alberts DS, Martinez ME, Roe DJ, et al. Lack of effect of a high-fiber cereal supplement on the recurrence of colorectal adenomas.
N Engl J Med
2000
;
342
:
1156
–62.
7
Peters U, Sinha R, Chatterjee N, et al. Dietary fibre and colorectal adenoma in a colorectal cancer early detection programme.
Lancet
2003
;
361
:
1491
–5.
8
Park Y, Hunter DJ, Spiegelman D, et al. Dietary fiber intake and risk of colorectal cancer: a pooled analysis of prospective cohort studies.
JAMA
2005
;
294
:
2849
–57.
9
MacLean CH, Newberry SJ, Mojica WA, et al. Effects of ω-3 fatty acids on cancer risk: a systematic review.
JAMA
2006
;
295
:
403
–15.
10
Davidson LA, Brown RE, Chang WC, et al. Morphodensitometric analysis of protein kinase CβII expression in rat colon: modulation by diet and relation to in-situ cell proliferation and apoptosis. Morphodensitometric analysis of protein kinase CβII expression in rat colon: modulation by diet and relation to in-situ cell proliferation and apoptosis.
Carcinogenesis
2000
;
21
:
1512
–9.
11
Hong MY, Lupton JR, Morris JS, et al. Dietary fish oil reduces O6 methylguanine DNA adduct levels in rat colon in part by increasing apoptosis during tumor initiation.
Cancer Epidemiol Biomarkers Prev
2000
;
9
:
819
–26.
12
Chapkin RS, Hong MY, Fan YY, et al. Dietary n-3 PUFA alter colonocyte mitochondrial membrane composition and function.
Lipids
2002
;
37
:
193
–9.
13
Hong MY, Chapkin RS, Barhoumi R, et al. Fish oil increases mitochondrial lipid unsaturation, upregulating reactive oxygen species and apoptosis in rat colonocytes.
Carcinogenesis
2002
;
23
:
1919
–25.
14
Ng Y, Barhoumi R, Tjalkens RB, et al. The role of docosahexaenoic acid in mediating mitochondrial membrane lipid oxidation and apoptosis in colonocytes.
Carcinogenesis
2005
;
26
:
1914
–21.
15
Bootman MD, Collins TJ, Peppiatt CM, et al. Calcium signaling—an overview.
Cell Dev Biol
2001
;
12
:
3
–10.
16
Orrenius S. Mitochondrial regulation of apoptotic cell death.
Toxicol Lett
2004
;
149
:
19
–23.
17
Parekh AB, Putney JW, Jr. Store-operated calcium channels.
Physiol Rev
2005
;
85
:
757
–810.
18
Rizzuto R, Pozzan T. Microdomain of intracellular Ca2+: molecular determinants and functional consequences.
Physiol Rev
2006
;
86
:
369
–408.
19
Nutt LK, Chandra J, Pataer A, et al. Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.
J Biol Chem
2002
;
277
:
20301
–8.
20
Babcock DF, Herrington J, Goodwin PC, Park YB, Hille B. Mitochondrial participation in the intracellular Ca2+ network.
J Cell Biol
1997
;
136
:
833
–44.
21
Hajnoczky G, Csordas G, Yi M. Old players in a new role: mitochondria-associated membranes, VDAC, and ryanodine receptors as contributors to calcium signal propagation from endoplasmic reticulum to the mitochondria.
Cell Calcium
2002
;
32
:
363
–77.
22
Szalai G, Krishnamurthy R, Hajnoczky G. Apoptosis driven by IP(3)-linked mitochondrial calcium signals.
EMBO J
1999
;
18
:
6349
–61.
23
Fan YY, Zhang J, Barhoumi R, et al. Antagonism of CD95 signaling blocks butyrate induction of apoptosis in young adult mouse colonic cells.
Am J Physiol
1999
;
277
:
C310
–9.
24
Gee KR, Brown KA, Chen WN, Bishop-Stewart J, Gray D, Johnson I. Chemical and physiological characterization of fluo-4 Ca(2+)-indicator dyes.
Cell Calcium
2000
;
27
:
97
–106.
25
Barhoumi R, Awooda I, Mouneimne Y, Safe S, Burghardt RC. Effects of benzo-a-pyrene on oxytocin-induced Ca(2+) oscillations in myometrial cells.
Toxicol Lett
2006
;
165
:
133
–41.
26
Denys A, Aires V, Hichami A, Khan NA. Thapsigargin-stimulated MAP kinase phosphorylation via CRAC channels and PLD activation: inhibitory action of docosahexaenoic acid.
FEBS Lett
2004
;
23
:
177
–82.
27
Chan C, Harland ML, Webb SE, Chen J, Miller AL, Barritt GJ. Evaluation, using targeted aequorins, of the roles of the endoplasmic reticulum and its (Ca2+ Mg2+)ATP-ases in the activation of store-operated Ca2+ channels in liver cells.
Cell Calcium
2004
;
35
:
317
–31.
28
Tsien R, Pozzan T. Measurement of cytosolic free Ca2+ with quin2.
Methods Enzymol
1989
;
172
:
230
–62.
29
Boitier E, Rea R, Duchen MR. Mitochondria exert a negative feedback on the propagation of intracellular Ca2+ waves in rat cortical astrocytes.
J Cell Biol
1999
;
145
:
795
–808.
30
An J, Chen Y, Huang ZJ. Critical upstream signals of cytochrome c release induced by a novel Bcl-2 inhibitor.
J Biol Chem
2004
;
279
:
19133
–40.
31
Waldron RT, Short AD, Gill DL. Store-operated Ca2+ entry and coupling to Ca2+ pool depletion in thapsigargin-resistant cells.
J Biol Chem
1997
;
272
:
6440
–7.
32
Gamberucci A, Giurisato E, Pizzo P, et al. Diacylglycerol activates the influx of extracellular cations in T-lymphocytes independently of intracellular calcium-store depletion and possibly involving endogenous TRP6 gene products.
Biochem J
2002
;
364
:
245
–54.
33
Nutt LK, Pataer A, Pahler J, et al. Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2+ stores.
J Biol Chem
2002
;
277
:
9219
–25.
34
Gescher A. Staurosporine analogues—pharmacological toys or useful antitumour agents?
Crit Rev Oncol Hematol
2000
;
34
:
127
–35.
35
Sanders LM, Henderson CE, Hong MY, et al. An increase in reactive oxygen species by dietary fish oil coupled with the attenuation of antioxidant defenses by dietary pectin enhances rat colonocyte apoptosis.
J Nutr
2004
;
134
:
3233
–8.
36
Hong MY, Bancroft LK, Turner ND, et al. Fish oil decreases oxidative DNA damage by enhancing apoptosis in rat colon.
Nutrition Cancer
2005
;
52
:
166
–175.
37
Rao CV, Reddy BS. Inhibitory effect of aspirin on azoxymethane-induced colon carcinogenesis in F344 rats.
Carcinogenesis
1993
;
14
:
1327
–33.
38
Calviello G, Palozza P, Di Nicuolo F, Maggiano N, Bartoli GM. n-3 PUFA dietary supplementation inhibits proliferation and store-operated calcium influx in thymoma cells growing in Balb/c mice.
J Lipid Res
2000
;
41
:
182
–9.
39
Triboulot C, Hichami A, Denys A, Khan NA. Dietary (n-3) polyunsaturated fatty acids exert antihypertensive effects by modulating calcium signaling in T cells of rats.
J Nutr
2001
;
131
:
2364
–9.
40
Jacobson J, Duchen MR. Mitochondrial oxidative stress and cell death in astrocytes-requirement for stored Ca2+ and sustained opening of the permeability transition pore.
J Cell Sci
2002
;
1115
:
1175
–88.
41
Satoh T, Ross CA, Villa A, et al. The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment.
J Cell Biol
1990
;
111
:
615
–24.
42
Yang J, Li X, Bhalla K, et al. Prevention of apoptosis by Bcl2: release of cytochrome-c from the mitochondria blocked.
Science
1997
;
275
:
1132
–6.
43
Hanson J, Bootman MD, Roderick LH. Cell signaling: IP-3 receptors channel calcium to cell death.
Curr Biol
2004
;
14
:
R933
–5.
44
Chinopoulos C, Adam-Vizi V. Calcium, mitochondria and oxidative stress in neuronal pathology. Novel aspects of an enduring theme.
FEBS J
2006
;
272
:
433
–50.
45
Szabadkai G, Rizzuto R. Participation of endoplasmic reticulum and mitochondrial calcium handling in apoptosis: more than just neighborhood?
FEBS Lett
2004
;
567
:
111
–5.
46
Bonin A, Khan NA. Regulation of calcium signalling by docosahexaenoic acid in human T-cells. Implication of CRAC channels.
J Lipid Res
2000
;
41
:
277
–84.
47
Touyz RM. Reactive oxygen species as mediators of calcium signaling by angiotensin II: implications in vascular physiology and pathophysiology.
Antioxid Redox Signal
2005
;
7
:
1302
–14.
48
O'Rourke B, Cortassa S, Aon MA. Mitochondrial ion channels: gatekeepers of life and death.
Physiology
2005
;
20
:
303
–15.
49
Sergeeva M, Strokin M, Wang H, Ubl JJ, Resier G. Arachidonic acid in astrocytes blocks Ca(2+) oscillations by inhibiting store-operated Ca(2+) entry, and causes delayed Ca(2+) influx.
Cell Calcium
2003
;
33
:
283
–92.
50
Basso E, Fante L, Fowlkes L, Petronilli V, Forte MA, Bernardi P. Properties of the permeability transition pore in mitochondria devoid of Cyclophilin D.
J Biol Chem
2005
;
280
:
18558
–61.

Supplementary data