Role of Mitochondria and Caspases in Vitamin D-mediated Apoptosis of MCF-7 Breast Cancer Cells*

Carmen J. Narvaez and JoEllen WelshDagger

From the Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556

Received for publication, July 31, 2000, and in revised form, October 2, 2000


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Vitamin D3 compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha ,25-Dihydroxyvitamin D3 (1alpha ,25(OH)2D3), the active form of vitamin D3, induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha ,25(OH)2D3-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha ,25(OH)2D3 on MCF-7 cells was compared with that of tumor necrosis factor alpha , which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha ,25(OH)2D3 induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha ,25(OH)2D3 treatment of a MCF-7 cell clone selected for resistance to 1alpha ,25(OH)2D3-mediated apoptosis. These mitochondrial effects of 1alpha ,25(OH)2D3 do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha ,25(OH)2D3-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha ,25(OH)2D3-mediated cell death is caspase-independent.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1alpha ,25-Dihydroxyvitamin D3 (1alpha ,25(OH)2D3),1 the active form of vitamin D3, acts through the nuclear vitamin D3 receptor (VDR) and is a potent negative growth regulator of breast cancer cells both in vitro and in vivo (1). A variety of synthetic vitamin D3 analogs that induce mammary tumor regression in animals are now undergoing clinical trials in human patients (2, 3). Our laboratory has shown that 1alpha ,25(OH)2D3 induces morphological and biochemical markers of apoptosis (chromatin and nuclear matrix condensation, and DNA fragmentation) in breast cancer cells (MCF-7) (4, 5); however, the precise mechanism by which 1alpha ,25(OH)2D3 and the VDR mediate apoptosis is poorly understood.

To characterize the mechanisms of 1alpha ,25(OH)2D3-mediated apoptosis in breast cancer cells, we compared specific intracellular events in MCF-7 cells after treatment with 1alpha ,25(OH)2D3 or tumor necrosis factor alpha  (TNFalpha ). TNFalpha was chosen as a positive control since this cytokine induces apoptosis in MCF-7 cells by a well defined pathway triggered by tumor necrosis factor receptor 1 (TNFR1), a cell surface death receptor. Death receptors contain homologous cytoplasmic regions termed "death domains," which transmit apoptotic signals through recruitment of adaptor molecules that activate caspases, a family of cysteine proteases involved in cell disassembly. The best characterized death receptors (Fas, TNFR1) use Fas-associated death domain and TNFR1-associated death domain adaptors to recruit and activate caspase-8 (6). Cleavage of specific substrates by caspases during apoptosis promotes the degradation of key structural proteins, including poly(ADP-ribose) polymerase (PARP), and lead to external display of phosphatidylserine (PS), DNA fragmentation, and cellular condensation (7).

Mitochondria play a central role in commitment of cells to apoptosis via increased permeability of the outer mitochondrial membrane, decreased transmembrane potential, release of cytochrome c and apoptosis-inducing factor, and production of reactive oxygen species (ROS) (8, 9). Anti-apoptotic Bcl-2 family members, such as Bcl-2 and Bcl-XL, can block these mitochondrial events, whereas pro-apoptotic Bcl-2 family members, including Bax, can trigger these changes. For example, apoptotic signals induce conformational changes in Bax, which lead to exposure of the pro-apoptotic BH3 domain, and translocation to the mitochondria (10). The effects of pro-apoptotic Bcl-2 family members are achieved by both caspase-dependent and caspase-independent mechanisms (11, 12).

Although the role of caspases in apoptosis triggered by cell surface death receptors such as TNFR1 has been well established, it is not clear if apoptosis triggered by nuclear receptors such as the VDR is mediated via similar caspase-dependent pathways. To probe the mechanisms whereby vitamin D3 signaling modulates apoptosis in MCF-7 cells, we studied the effects of 1alpha ,25(OH)2D3 on mitochondrial function and caspase activity using a cell-permeable inhibitor of caspase-related proteases (z-Val-Ala-Asp-fluoromethylketone, zVAD.fmk). In addition, the effects of 1alpha ,25(OH)2D3 and TNFalpha on a vitamin D3-resistant variant of MCF-7 cells (MCF-7D3Res cells) were examined to identify events that contribute to vitamin D3 resistance (13, 14). The MCF-7D3Res cells do not undergo cell cycle arrest or apoptosis in response to 1alpha ,25(OH)2D3; however, these cells retain sensitivity to other inducers of apoptosis such as TNFalpha and anti-estrogens (13).

Our results indicate that, although caspase inhibition can block some of the late stages of 1alpha ,25(OH)2D3-mediated apoptosis in MCF-7 cells, the commitment to cell death is caspase-independent. These data implicate Bax distribution and mitochondrial disruption as critical caspase-independent events in 1alpha ,25(OH)2D3-mediated apoptosis of breast cancer cells.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells and Cell Culture-- MCF-7 cells (originally obtained from ATCC) were used to generate the vitamin D3-resistant variant (MCF-7D3Res) that has been described previously (13). Both cell lines were cultured in alpha -minimal essential medium (Life Technologies, Inc.) containing 25 mM HEPES and 5% fetal bovine serum (Life Technologies, Inc.). Cells were routinely plated at 5000 cells/cm2 and passaged every 3-4 days. Stock cultures of MCF-7D3Res cells were routinely grown in medium containing 100 nM 1alpha ,25(OH)2D3 (kindly provided by LEO Pharmaceuticals, Ballerup, Denmark). For experiments, MCF-7 and MCF-7D3Res cells were plated in alpha -minimal essential medium containing 5% fetal bovine serum plus antibiotics, and treated with 1alpha ,25(OH)2D3 or ethanol vehicle 2 days after plating. Parallel cultures were treated with TNFalpha (Sigma) 2-3 days before scheduled harvest of 1alpha ,25(OH)2D3-treated dishes. Caspase inhibitors, zVAD.fmk or zDEVD.fmk (Enzyme Systems Products, Livermore, CA) were added at the same time (1alpha ,25(OH)2D3 or TNFalpha ) or 2 days after (1alpha ,25(OH)2D3) initial treatment. For cell growth assays, cells were seeded at 1000 cells/well in 24-well plates, treated for the indicated times, and analyzed by crystal violet assay. Briefly, cells were fixed with 1% glutaraldehyde for 15 min, incubated with 0.1% crystal violet (Fisher Scientific, Pittsburgh, PA) for 30 min, destained with H2O, and solubilized with 0.2% Triton X-100. Absorbance at 562 nm (minus background at 630 nm) was determined on a microtiter plate reader.

Clonogenicity Assay-- MCF-7 cells were incubated with 1alpha ,25(OH)2D3 for 6 days or TNFalpha for 1 day in the presence or absence of 25 µM zVAD.fmk. For 1alpha ,25(OH)2D3 treatment, medium was replaced every 2 days. After treatments, cells were trypsinized, media and washes were pooled, and cells were pelleted by centrifugation and resuspended in fresh medium. The cells were seeded in 96-well plates at 5, 50, 500, and 2500 cells/well in 24 replicates. After 14 days, the cells were fixed and stained with crystal violet as described above, and clonogenic potential was estimated by counting positive wells (15).

Subcellular Fractionation-- Cells were trypsinized, pooled together with media and washes containing floating cells, and pelleted by centrifugation at 500 × g for 3 min at 4 °C. Pellets were resuspended with 3 volumes of Buffer A (20 mM HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine, 1 mM dithiothreitol, 250 mM sucrose, plus protease and phosphatase inhibitors), lysed with a Dounce homogenizer, and fractionated by differential centrifugation (16). Briefly, homogenates were centrifuged twice at 500 × g for 5 min at 4 °C, and the nuclear pellet was resuspended in Buffer A, sonicated 2 × 10 s, and stored at -80 °C in multiple aliquots. The supernatants were combined and further centrifuged at 10,000 × g for 30 min at 4 °C, and the resultant mitochondrial pellets were resuspended in Buffer A, sonicated, and stored at -80 °C in multiple aliquots. The supernatant from the 10,000 × g spin was further centrifuged at 100,000 × g for 1 h at 4 °C. The resulting supernatant was designated S100 (containing cytosol) and stored at -80 °C in multiple aliquots. Protein concentrations were determined by the Micro BCA protein assay (Pierce).

Immunoblot Analysis-- Subcellular fractions isolated as described above were solubilized in Laemmli sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose. Proteins derived from mitochondria and/or S100 extracts were immunoblotted with Bax rabbit polyclonal (13666E; PharMingen, San Diego, CA) or cytochrome c mouse monoclonal antibodies (7H8.2C12; PharMingen) diluted 1:500 or 1:250, respectively, in PBS plus 5% skim milk. S100 extracts were also probed with cytochrome oxidase subunit II mouse monoclonal antibody (clone 12C4-F12; Molecular Probes, Eugene, OR) to exclude mitochondrial contamination. Nuclear extracts were probed with PARP mouse monoclonal antibody (C2.10, Enzyme Systems Products) diluted 1:1000. Specific antibody binding was detected by horseradish peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech) diluted 1:5000 in PBS, 5% skim milk, 0.1% Tween 20 and autoradiography with enhanced chemiluminescence (Pierce).

Immunocytochemistry-- MCF-7 cells grown on Lab-Tek II chamber slides (Fisher Scientific) were treated with ethanol vehicle, 100 nM 1alpha ,25(OH)2D3, or 2.5 ng/ml TNFalpha for 96 h (ethanol, 1alpha ,25(OH)2D3) or 48 h (TNFalpha ) in the presence or absence of 25 µM zVAD.fmk. The cells were fixed in 4% formaldehyde in PBS for 5 min at room temperature, permeabilized in methanol at -20 °C for 5 min, and blocked overnight with PBS plus 1% BSA containing 0.02% sodium azide. The slides were then incubated with cytochrome c mouse monoclonal antibody (6H2.B4; PharMingen), diluted 1:100 in blocking buffer, for 3 h at 37 °C in a humidified chamber. Slides were washed three times for 5 min each time with PBS, followed by incubation for 1 h at room temperature with anti-mouse secondary antibody conjugated to Alexa-488 (a photostable dye with spectral properties similar to fluorescein; Molecular Probes) diluted 1:50 in blocking buffer. Slides were washed three times for 5 min with PBS, incubated for 15 min at room temperature with 1 µg/ml Hoechst 33258 (Sigma), washed five times for 5 min with PBS, rinsed with distilled H2O, and coverslips were applied with an antifade reagent. Fluorescence was detected using an Olympus AX70 microscope equipped with a Spot RT digital camera.

Flow Cytometry-- For analysis of mitochondrial membrane potential and reactive oxygen species, cells harvested by trypsinization were pooled with media plus washes and pelleted by centrifugation. Cell suspensions (1 × 106 cells) were incubated with 1 µM tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) in PBS containing 130 mM KCl to abolish the plasma membrane potential. After incubation for 10 min at 37 °C, cells were washed once in PBS and then analyzed for TMRE red fluorescence by flow cytometry. Live cells rapidly and reversibly take up TMRE, and accumulation of the dye in mitochondria has been shown to be potential driven (17). For analysis of ROS, cell suspensions (5 × 105 cells) were incubated with 4 µM hydroethidine (HE, Molecular Probes) in PBS for 15 min at 37 °C, and conversion of HE to ethidium by superoxide anion was analyzed by flow cytometry.

For analysis of DNA fragmentation, MCF-7 cells were harvested by trypsinization, collected by centrifugation, fixed in 2% formaldehyde in PBS, and permeabilized in 70% EtOH at -20 °C. DNA strand breaks in cells undergoing apoptosis were indirectly labeled with bromodeoxyuridine by terminal transferase (Roche Molecular Biochemicals) and detected by FITC-conjugated monoclonal antibody to bromodeoxyuridine using the APO-BRDU kit according to manufacturer's protocol (Phoenix Flow Systems, San Diego, CA). Cells were counterstained with 5 µg/ml propidium iodide (PI; Sigma) containing RNase A (Roche Molecular Biochemicals) for detection of total DNA, and two-color analysis of DNA strand breaks and cell cycle was achieved by flow cytometry.

For detection of PS externalization, 1 × 106 cells were incubated in the presence of 10 µg/ml annexin V-FITC (BioWhittaker, Walkersville, MD) and 5 µg/ml PI in binding buffer (10 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 2.5 mM CaCl2) for 15 min at 37 °C. Cells were washed twice in binding buffer, fixed in 2% formaldehyde in PBS for 15 min on ice, and then washed two more times in PBS plus 0.2% BSA. Pellets were resuspended in PBS plus 0.2% BSA and analyzed by flow cytometry. There were less than 1% PI+ cells in the population, and they were therefore excluded from analysis.

All flow cytometric analyses were performed on an Epics XL Flow Cytometer (Coulter Corp., Miami, FL) equipped with an argon laser. TMRE and HE were analyzed on FL3 using a 620-nm band pass filter. For DNA fragmentation analysis, FITC was analyzed on FL1 using a 520-nm band pass filter and PI was analyzed on FL3 with no color compensation. For PS externalization, annexin V-FITC was analyzed on FL1 and PI was analyzed on FL2 (580-nm band pass filter) using software color compensation. Data was modeled with the Multiplus AV software (Phoenix Flow Systems).

Caspase Activity Assay-- Caspase activity was analyzed with the ApoAlert CPP32/caspase-3 assay kit according to manufacturer's protocol (CLONTECH, Palo Alto, CA). Briefly, after harvesting by trypsinization, 2 × 106 cells were pelleted and stored at -20 °C. For analysis, cell pellets were lysed, re-pelleted to remove cell debris, and supernatants were incubated with 50 µM DEVD-AFC for 1 h at 37 °C. The samples were analyzed using a fluorescence spectrophotometer with excitation = 380 nm and emission = 508 nm.

Statistical Evaluation-- Data are expressed as mean ± S.E. One-way analysis of variance was used to assess statistical significance between means. Differences between means were considered significant if p values less than 0.05 were obtained with the Bonferroni method using GraphPad Instat software (GraphPad Software, San Diego, CA).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Disruption of Mitochondrial Function, as Determined by Subcellular Localization of Bax and Cytochrome c, and ROS Generation, by 1alpha ,25(OH)2D3-- To identify specific intracellular events involved in 1alpha ,25(OH)2D3-mediated apoptosis, we examined the signaling pathway downstream of the VDR in MCF-7 cells. Since disruption of mitochondrial function is a primary event in apoptosis that can be triggered by translocation of Bax to mitochondrial outer membrane, we first examined the subcellular distribution of Bax after 1alpha ,25(OH)2D3 treatment of MCF-7 cells. As demonstrated in Fig. 1, Bax redistribution from the cytosolic to the mitochondrial fraction occurred after treatment with 1alpha ,25(OH)2D3 or TNFalpha in MCF-7 cells (Fig. 1, top). Not only was Bax translocated to mitochondria, but both 1alpha ,25(OH)2D3- and TNFalpha -treated cells exhibited cleavage of Bax from the intact 21-kDa protein to an 18-kDa fragment, an observation that is consistent with reports of Bax cleavage during drug-induced apoptosis (18). In both 1alpha ,25(OH)2D3- and TNFalpha -treated cells, the Bax cleavage product was detected in mitochondrial, but not cytosolic, fractions, and others have proposed that Bax cleavage enhances homodimerization and its pro-apoptotic properties (19). These are the first data to implicate translocation and cleavage of Bax during 1alpha ,25(OH)2D3-induced apoptosis. To determine the relationship between Bax translocation and sensitivity to 1alpha ,25(OH)2D3-induced apoptosis, we examined the subcellular distribution of Bax in a vitamin D3-resistant variant of MCF-7 cells, which does not undergo apoptosis after treatment with 1alpha ,25(OH)2D3 but retains sensitivity to other triggers, including TNFalpha .2 In the MCF-7D3Res cells, 1alpha ,25(OH)2D3 did not induce translocation or cleavage of Bax (Fig. 1, bottom). However, in these cells, Bax translocation and cleavage was triggered by TNFalpha , indicating that Bax functions appropriately in MCF-7D3Res cells during apoptosis induced by agents other than 1alpha ,25(OH)2D3. The inability of Bax to redistribute to mitochondria in response to 1alpha ,25(OH)2D3 in the vitamin D3-resistant variant suggests that Bax translocation may be a critical initiating event in 1alpha ,25(OH)2D3-mediated apoptosis of MCF-7 cells.


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Fig. 1.   Subcellular distribution of Bax after treatment with 1alpha ,25(OH)2D3 or TNFalpha in parental MCF-7 or MCF-7D3Res cells. MCF-7 or MCF-7D3Res cells were plated at a density of 2 × 105 cells/150-mm dish. Two days after plating, cells were treated with vehicle control (ethanol) or 100 nM 1alpha ,25(OH)2D3 for 96 h, or with 2.5 ng/ml TNFalpha for 48 h. Mitochondria and S100 isolated as described under "Experimental Procedures" were separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with polyclonal antibody to Bax. The results are representative of at least three independent experiments.

Translocation of Bax to mitochondria has been associated with release of cytochrome c, an event that is considered a commitment point for activation of apoptosis. As expected for viable cultures, no cytochrome c was detected in cytosolic fractions from MCF-7 cells treated with ethanol vehicle for up to 120 h (Fig. 2A). In contrast, redistribution of cytochrome c from mitochondria to cytosol was detected within 48 h of 1alpha ,25(OH)2D3 treatment in MCF-7 cells, before any morphological apoptotic features were detected. The absence of cytochrome oxidase in cytosolic fractions confirmed that extracts were free of mitochondrial contamination (data not shown). In MCF-7D3Res cells, 1alpha ,25(OH)2D3 did not trigger release of cytochrome c; however, cytochrome c was detected in cytosolic fractions after TNFalpha treatment of both MCF-7 and MCF-7D3Res cell lines (Fig. 2B; see also Fig. 7).


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Fig. 2.   Cytosolic localization of cytochrome c after treatment with 1alpha ,25(OH)2D3 or TNFalpha . A, time course of cytosolic cytochrome c after treatment with vehicle control (ethanol) or 100 nM 1alpha ,25(OH)2D3 in MCF-7 cells. Cells were plated at a density of 1 × 105 cells/150-mm dish. Two days after plating, the cells were treated with ethanol or 1alpha ,25(OH)2D3 and re-fed 2 days later. S100 fractions prepared at the indicated time points as described under "Experimental Procedures" were separated on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with cytochrome c (7H8.2C12) antibody. B, cytosolic cytochrome c in MCF-7D3Res cells. Cells were plated and treated with ethanol or 1alpha ,25(OH)2D3 as described above, and 2.5 ng/ml TNFalpha was added 3 days before harvest. All the dishes were harvested on day 5 of treatment, and S100 fractions were prepared and immunoblotted as described above. The results are representative of at least three independent experiments.

Long term exclusion of cytochrome c from the electron transport chain can lead to impairment of proton flow and generation of ROS due to incomplete reduction of molecular oxygen. Hence, mitochondrial generation of ROS in response to 1alpha ,25(OH)2D3 and TNFalpha was examined by flow cytometry. Production of superoxide anion was indirectly assessed as oxidation of hydroethidine to ethidium, which fluoresces red upon DNA intercalation. As presented in Fig. 3, ROS production was enhanced by 1alpha ,25(OH)2D3 in MCF-7, but not MCF-7D3Rescells, whereas TNFalpha increased ROS production comparably in both cell lines. Time-course studies have demonstrated that ROS production is enhanced within 72 h of 1alpha ,25(OH)2D3 treatment in MCF-7 cells (data not shown).


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Fig. 3.   ROS production after treatment with 1alpha ,25(OH)2D3 or TNFalpha in parental MCF-7 or MCF-7D3Res cells. Cells were plated at a density of 1 × 105 cells/150-mm dish. Two days after plating, the cells were treated with vehicle control (ethanol), 100 nM 1alpha ,25(OH)2D3, or media only, re-fed 2 days later, and 2.5 ng/ml TNFalpha was added to dishes containing media only. All dishes were harvested on day 5 when ROS generation was assessed by flow cytometry as described under "Experimental Procedures." Data are expressed as the percentage of cells positive for ROS after negative subtraction of data derived from vehicle-treated MCF-7D3Res cells. The results are representative of at least three independent experiments.

1alpha ,25(OH)2D3 Mediates PARP Cleavage, PS Externalization, and DNA Fragmentation in a Caspase-dependent Manner-- Cytochrome c released into the cytosol is thought to trigger caspase activation downstream of mitochondria through binding to Apaf-1 and autoactivation of procaspase-9. Activated caspase-9 can activate additional effector caspases responsible for cell disassembly and events such as PS externalization, PARP cleavage, and DNA fragmentation. To determine the involvement of caspase-dependent proteolysis in 1alpha ,25(OH)2D3-mediated apoptosis, we examined whether a broad spectrum cell-permeable caspase inhibitor (zVAD.fmk) could abrogate the effects of 1alpha ,25(OH)2D3 in MCF-7 cells.

Proteolytic activity associated with caspase activation was analyzed by three distinct methods: cleavage of an endogenous caspase substrate (PARP), and flow cytometric analysis of PS exposure and DNA fragmentation, which others have shown are provoked by caspases (7). As demonstrated in Fig. 4A, PARP was cleaved after treatment of MCF-7 cells with either 1alpha ,25(OH)2D3 or TNFalpha , and in both cases, cleavage was blocked by zVAD.fmk. Furthermore, both 1alpha ,25OH)2D3 and TNFalpha induced PS externalization, which was also completely blocked by zVAD.fmk (Fig. 4B). Finally, the effects of 1alpha ,25(OH)2D3 and TNFalpha on DNA fragmentation was assessed as terminal transferase-mediated incorporation of bromodeoxyuridine, detected by FITC-conjugated anti-bromodeoxyuridine antibody by flow cytometry (Fig. 5). 1alpha ,25(OH)2D3 treatment of MCF-7 cells induced DNA fragmentation primarily in the G1 phase of the cell cycle, with only a small population of cells (5%) accumulating in sub-G1. TNFalpha treatment induced extensive DNA fragmentation in the G1 phase of the cell cycle, with accumulation of 29% of the population in sub-G1. Despite the differences in the magnitude and profiles of DNA fragmentation between 1alpha ,25(OH)2D3- and TNFalpha -treated cells, zVAD.fmk completely blocked DNA fragmentation induced by both agents.


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Fig. 4.   Effects of caspase inhibitor on PARP cleavage and PS exposure after treatment with 1alpha ,25(OH)2D3 or TNFalpha in MCF-7 cells. A, PARP cleavage. Cells were plated and treated as described in Fig. 3 in the presence or absence of 25 µM zVAD.fmk. All the dishes were harvested on day 5. Nuclear extracts prepared as described under "Experimental Procedures" were separated on SDS-PAGE and immunoblotted with mouse monoclonal antibody to PARP. B, PS externalization. Cells plated and treated as described above were incubated with annexin V-FITC and PI as described under "Experimental Procedures." Data are expressed as the percentage of annexin V-FITC-positive cells after negative subtraction of data generated with vehicle treated cells. The results are representative of at least three independent experiments.


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Fig. 5.   Effect of caspase inhibitor on DNA fragmentation after treatment with 1alpha ,25(OH)2D3 or TNFalpha in MCF-7 cells. Cells were plated and treated as described in Fig. 4, and DNA fragmentation was determined by flow cytometry as described under "Experimental Procedures." The results are representative of at least three independent experiments.

Caspase Activity Induced by 1alpha ,25(OH)2D3 Is Not DEVDase-- DEVDase cleavage activity was measured with the fluorogenic substrate DEVD-AFC, which can detect activity of caspases-3 and -7. Since MCF-7 cells do not express functional caspase-3 (data not shown, Ref. 20), any DEVDase activity detected in these cells would most likely correspond to caspase-7. For these experiments, MCF-7 cells were pretreated with 1alpha ,25(OH)2D3 for 2 days before cytosolic extracts were collected for DEVDase activity assays at the indicated time points. As demonstrated in Fig. 6, TNFalpha , but not 1alpha ,25(OH)2D3, induced DEVDase cleavage activity in MCF-7 cells. Even with extended treatment times in additional studies, no DEVDase cleavage activity could be detected after 1alpha ,25(OH)2D3 treatment of MCF-7 cells (data not shown). This observation indicates that other known or as yet unidentified effector caspases may be activated by 1alpha ,25(OH)2D3, which mediate PARP cleavage, PS exposure, or DNA fragmentation.


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Fig. 6.   Time course of DEVDase cleavage activity after treatment with 1alpha ,25(OH)2D3 or TNFalpha in MCF-7 cells. Cells were plated at a density of 2 × 105 cells/150-mm dish. Two days after plating, the cells were pretreated with ethanol, 100 nM 1alpha ,25(OH)2D3, or media only. Two days later, medium was replaced with ethanol (open circle ), 100 nM 1alpha ,25(OH)2D3 (black-square), or 10 ng/ml TNFalpha (black-down-triangle ). Cytosolic extracts of cells harvested at the indicated time points were incubated with DEVD-AFC for 1 h at 37 °C and analyzed on a fluorescence spectrophotometer. Data represent mean ± S.E. of two independent experiments performed in duplicate.

Caspase Inhibition Does Not Block 1alpha ,25(OH)2D3-mediated Cytochrome c Release, Mitochondrial Dysfunction, or Cell Death-- Since zVAD.fmk blocked 1alpha ,25(OH)2D3-mediated caspase-dependent events downstream of mitochondria, we examined the effects of the caspase inhibitor on cytochrome c release and mitochondrial function. As shown in Fig. 7, zVAD.fmk did not abrogate 1alpha ,25(OH)2D3-mediated cytochrome c release or ROS production under the same conditions where PS exposure, PARP cleavage, and DNA fragmentation were blocked. In contrast, the caspase inhibitor effectively blocked cytochrome c release and ROS generation triggered by TNFalpha (Fig. 7, A and C).


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Fig. 7.   Effects of caspase inhibitor on cytochrome c release, mitochondrial membrane potential, and ROS production after treatment with 1alpha ,25(OH)2D3 or TNFalpha in MCF-7 cells. A, cytochrome c release. S100 extracts prepared from cells treated as described in Fig. 4 were separated on SDS-PAGE and immunoblotted with cytochrome c (7H8.2C12) mouse monoclonal antibody. B, mitochondrial membrane potential. Cells plated and treated as described above were incubated with 1 µM TMRE as described under "Experimental Procedures" and analyzed by flow cytometry. Data are expressed as the percentage of cells with reduced mitochondrial membrane potential. C, ROS production. Cells treated as described above were incubated with 4 µM HE as described under "Experimental Procedures" and analyzed by flow cytometry. The data represent the percentage of cells positive for ROS. Each bar represents the mean ± S.E. of two to four independent experiments. **, p < 0.001; *, p < 0.01; treated versus ethanol control as evaluated by analysis of variance. The results (A and B) are representative of at least three independent experiments.

To further probe mitochondrial function, the membrane potential-sensitive probe TMRE was used to detect mitochondrial membrane potential by flow cytometry. 1alpha ,25(OH)2D3 treatment significantly enhanced the percentage of cells with reduced mitochondrial membrane potential, and zVAD.fmk did not block the decrease in mitochondrial membrane potential induced by 1alpha ,25(OH)2D3. TNFalpha treatment also enhanced the percentage of cells with decreased mitochondrial membrane potential; however, in contrast to 1alpha ,25(OH)2D3, the effect of TNFalpha was completely blocked by zVAD.fmk (Fig. 7B).

Subcellular localization of cytochrome c protein was examined by fluorescence microscopy to confirm the finding that cytochrome c release can proceed independently of caspase activation after 1alpha ,25(OH)2D3 treatment. In Fig. 8, cytochrome c fluorescence (middle panels) is presented alongside phase contrast (top panels) and Hoechst nuclear staining (bottom panels) to compare cytochrome c localization in individual viable and apoptotic cells. In vehicle-treated control cells, apoptotic morphology was not present, and cytochrome c staining was restricted to punctate cytoplasmic regions, consistent with mitochondrial localization (Fig. 8). After treatment with 1alpha ,25(OH)2D3 or TNFalpha , apoptotic cells identified by phase contrast and Hoechst nuclear staining exhibited chromatin condensation, nuclear fragmentation, and cytosolic vacuolization. In these apoptotic cells, diffuse cytoplasmic cytochrome c staining was detected throughout the cell, which obscured the nuclei, consistent with redistribution of cytochrome c from mitochondria to cytoplasm (21). Consistent with the immunoblotting data (Fig. 7A), treatment with zVAD.fmk failed to prevent 1alpha ,25(OH)2D3-mediated cytochrome c release, as demonstrated by persistence of diffuse cytoplasmic cytochrome c staining in 1alpha ,25(OH)2D3- plus zVAD.fmk-treated cells. However, zVAD.fmk did prevent the morphological signs of apoptosis, including chromatin condensation and nuclear fragmentation, consistent with its ability to block PS redistribution, PARP cleavage, and DNA fragmentation induced by 1alpha ,25(OH)2D3.


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Fig. 8.   Effects of 1alpha ,25(OH)2D3 or TNFalpha on morphology and cytochrome c release in MCF-7 cells. Cells grown on Lab-Tek II chamber slides were treated with ethanol, 100 nM 1alpha ,25(OH)2D3 ± 25 µM zVAD.fmk, or 2.5 ng/ml TNFalpha , fixed after 96 h (ethanol, 1alpha ,25(OH)2D3) or 48 h (TNFalpha ), immunostained with cytochrome c (6H2.B4) mouse monoclonal antibody, and visualized with Alexa-488-conjugated secondary antibody. Nuclei were counterstained with Hoechst 33258. The images were taken with an Olympus AX70 fluorescence microscope (original magnification, ×400). Top, phase contrast; middle, cytochrome c (green); bottom, Hoechst (blue). The results are representative of at least two independent experiments.

Since zVAD.fmk did not block cytochrome c release or mitochondrial dysfunction induced by 1alpha ,25(OH)2D3, but did protect MCF-7 cells from morphological signs of apoptosis, including DNA fragmentation, we examined whether zVAD.fmk-treated cells actually remained viable and/or maintained clonogenic potential. As shown in Fig. 9, both zVAD.fmk and zDEVD.fmk caspase inhibitors rescued MCF-7 cells from TNFalpha -mediated cell death, as demonstrated by total cell numbers, with zVAD.fmk offering the greater protection. However, neither zVAD.fmk nor zDEVD.fmk caspase inhibitors could protect MCF-7 cells from 1alpha ,25(OH)2D3-mediated apoptosis, since the reduction in total cell number was not abrogated by either inhibitor (Fig. 9). Finally, the clonogenic potential was determined after treatment of cells with 1alpha ,25(OH)2D3 or TNFalpha in the presence or absence of zVAD.fmk followed by re-plating at limiting dilutions in fresh medium. In vehicle control-treated cultures, at least 1 out of 5 cells had the ability to produce clones. In TNFalpha -treated cultures, clonogenicity was less than 1 out of 500 cells (f < 0.002) but in the presence of zVAD.fmk, the frequency of cells with clonogenic potential was significantly increased (f >=  0.2). In 1alpha ,25(OH)2D3 treated cultures, clonogenicity was less than 1 out of 50 cells (f < 0.02), and this was not enhanced in the presence of zVAD.fmk.


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Fig. 9.   Effect of caspase inhibitors on MCF-7 cell number after treatment with 1alpha ,25(OH)2D3 or TNFalpha . Cells were seeded at a density of 1000 cells/well in 24-well plates. Two days after plating, cells were treated with ethanol, 100 nM 1alpha ,25(OH)2D3, or 10 ng/ml TNFalpha for 5 days ± 25 µM zVAD.fmk (broad spectrum, bottom) or zDEVD.fmk (caspase-3/7-specific, top). Total cell number was determined by crystal violet assay as described under "Experimental Procedures." Data represent mean ± S.E. of four replicate determinations. The results are representative of at least two independent experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Here we report for the first time that 1alpha ,25(OH)2D3 induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is accomplished by translocation of Bax to mitochondria, and increased permeability of the outer mitochondrial membrane. Of particular interest, neither Bax translocation nor the mitochondrial disruption is induced by 1alpha ,25(OH)2D3 in a variant line of MCF-7 cells selected for resistance to 1alpha ,25(OH)2D3-mediated apoptosis (13). Collectively, these data implicate an essential role for mitochondrial signaling in the induction of apoptosis by 1alpha ,25(OH)2D3 and identify the pro-apoptotic protein Bax as an important downstream target of the VDR in MCF-7 cells.

In addition to Bax translocation, we report that treatment of MCF-7 cells with 1alpha ,25(OH)2D3 induces cytochrome c release and ROS generation, events that have been observed in cells induced to undergo apoptosis by overexpression of Bax (22, 23). These data further support the concept that 1alpha ,25(OH)2D3-mediated apoptosis may be driven by Bax translocation. A role for the pro-apoptotic protein Bax in 1alpha ,25(OH)2D3-mediated apoptosis is consistent with previous studies that support a role for Bcl-2, the anti-apoptotic antagonistic partner of Bax, in mediating the effects of 1alpha ,25(OH)2D3 on both breast and prostate cancer cells. Thus, 1alpha ,25(OH)2D3 down-regulates Bcl-2 (24, 25) and overexpression of Bcl-2 blocks 1alpha ,25(OH)2D3-induced apoptosis (26, 27). Since Bcl-2 and Bax act antagonistically in the regulation of apoptosis, these data suggest that down-regulation of Bcl-2 in conjunction with translocation of Bax may be necessary for 1alpha ,25(OH)2D3-mediated apoptosis. Further studies will be necessary to identify the signals generated by 1alpha ,25(OH)2D3 that induce Bax translocation to mitochondria. Since events upstream of Bax translocation to mitochondria in response to 1alpha ,25(OH)2D3 are abrogated in the vitamin D3-resistant MCF-7 variant, comparison of early events in VDR signaling in these cells will be an important subject for future studies.

Examination of events downstream of mitochondria indicated that 1alpha ,25(OH)2D3 induced features of apoptosis associated with caspase activation, such as PARP cleavage, PS exposure, and DNA fragmentation. To determine whether caspase activation was required for 1alpha ,25(OH)2D3-mediated apoptosis, we used the broad spectrum, cell-permeable caspase inhibitor zVAD.fmk. We observed that 1alpha ,25(OH)2D3 signaling on mitochondria does not require caspase activation, since zVAD.fmk was unable to block 1alpha ,25(OH)2D3-induced cytochrome c release, decrease in mitochondrial membrane potential, or ROS production. Again, this is consistent with apoptosis driven by Bax translocation, which promotes cytochrome c release via caspase-independent pathways (28-31). Our data also complement that of Mathiasen et al. (27), who reported that inhibition of caspase activity by overexpression of CrmA, a cowpox-derived caspase inhibitor, or caspase inhibitory peptides (Ac-DEVD-CHO, Ac-IETD-CHO, and zVAD.fmk) did not block vitamin D3-mediated growth arrest or apoptosis.

Although caspase inhibition did not block mitochondrial events induced by 1alpha ,25(OH)2D3, zVAD.fmk did block events downstream of mitochondria such as PARP cleavage, external display of PS, and DNA fragmentation. These findings are similar to reports of Bax-induced apoptosis, where caspase inhibitors had no effect on Bax-induced cytochrome c release or mitochondrial disruption, but prevented cleavage of nuclear and cytosolic substrates and DNA degradation (28-31). However, our data conflict with that of Mathiasen et al. (27), who observed that zVAD.fmk did not block 1alpha ,25(OH)2D3-mediated DNA fragmentation in MCF-7 cells. This discrepancy may reflect differences in doses (1 µM versus 25 µM) or experimental design between the two studies. The lower dose of zVAD.fmk (1 µM) used by Mathiasen et al. may have been insufficient to block mitochondrial-initiated caspases (caspase-9) (32).

The data presented in this paper indicate that 1alpha ,25(OH)2D3 triggers both caspase-independent and caspase-dependent pathways in MCF-7 cells, and suggest that 1alpha ,25(OH)2D3 can activate downstream effector caspases. Since cytochrome c release has been associated with autoactivation of procaspase-9, 1alpha ,25(OH)2D3 may activate caspase-dependent pathways via cytochrome c release. However, no DEVDase activity was detected in 1alpha ,25(OH)2D3-treated cytoplasmic extracts, suggesting that other, possibly unidentified, effector caspases may be activated by 1alpha ,25(OH)2D3, or that caspase-dependent events occur at later stages in the apoptotic program. Although blocking caspase activation prevented some of the morphological aspects of 1alpha ,25(OH)2D3-mediated apoptosis, MCF-7 cells were not rescued from death by zVAD.fmk. This finding is consistent with reports that many cell types eventually die by a slower, non-apoptotic cell death if caspases are inactivated (8). These data support the concept that mitochondrial damage represents a cell death commitment step in the course of apoptosis induced by many stimuli (33), including 1alpha ,25(OH)2D3.

In summary, 1alpha ,25(OH)2D3 mediates apoptosis of MCF-7 cells through mitochondrial signaling, which involves ROS generation, and is regulated by the Bcl-2 family of apoptotic regulators. Caspases act solely as executioners to facilitate 1alpha ,25(OH)2D3-mediated apoptosis, and caspase activation is not required for induction of cell death by 1alpha ,25(OH)2D3. Our data suggest distinct differences in the mechanisms of apoptosis induced by 1alpha ,25(OH)2D3 and TNFalpha , since inhibition of caspases was able to rescue MCF-7 cells from TNFalpha -mediated, but not 1alpha ,25(OH)2D3-mediated, cell death. Although caspase inhibition blocked biochemical changes associated with caspase activation downstream of mitochondrial perturbations and loss of cytochrome c, the commitment of MCF-7 cells to 1alpha ,25(OH)2D3-mediated apoptosis is clearly caspase-independent.

    ACKNOWLEDGEMENT

We thank Thomas Waterfall for technical assistance.

    FOOTNOTES

* This work was supported by Army Breast Cancer Research Program Grant DAMD17-97-1-7183 and NCI National Institutes of Health Grant CA69700.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556. Tel.: 219-631-3371; Fax: 219-631-7413; E-mail: jwelsh3@nd.edu.

Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M006876200

2 C. J. Narvaez and J. Welsh, unpublished data.

    ABBREVIATIONS

The abbreviations used are: 1alpha , 25(OH)2D3, 1alpha ,25-dihydroxyvitamin D3; z, benzyloxycarbonyl; Ac, acetyl; VAD, Val-Ala-Asp; DEVD, Asp-Glu-Val-Asp; IETD, Ile-Glu-Thr-Asp; fmk, fluoromethylketone; CHO, aldehyde; AFC, 7-amino-4-trifluoromethylcoumarin; DEVDase, DEVD-cleavage specific caspase; TNFalpha , tumor necrosis factor alpha ; TNFR1, tumor necrosis factor receptor 1; VDR, vitamin D3 receptor; PAGE, polyacrylamide gel electrophoresis; PARP, poly(ADP-ribose) polymerase; ROS, reactive oxygen species; PS, phosphatidylserine; PI, propidium iodide; PBS, phosphate-buffered saline; TMRE, tetramethylrhodamine ethyl ester; HE, hydroethidine; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin.

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