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Advances in Brief |
Departments of Gastrointestinal Medicine and Nutrition [X. T., Y. J. S., E. H., F. S.] and Molecular Pathology [M. T. K.], University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT IntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Introduction |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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716 mice, which resulted in a dramatic reduction in intestinal polyp burden in double knockouts (4)
. Forced COX-2 expression has been shown to increase cellular adhesion and to confer resistance to butyrate-induced apoptosis in rat intestinal epithelial cells (5)
. Furthermore, COX-2 overexpression can reduce antineoplastic drug and Fas-mediated apoptosis in human colon and cholangiocarcinoma cells, respectively (6
, 7)
. To date, the mechanisms by which COX-2 can inhibit apoptosis are poorly understood. Apoptosis is controlled via two major pathways, including one that originates at the cell membrane and another that involves the mitochondria (8
, 9)
. The membrane DR pathway involves DRs such as Fas, TNF-R1, DR-3, DR-4, and DR-5, that are activated by their respective ligands and engage the intracellular apoptotic machinery (9
, 10)
. TRAIL (also known as Apo2L) is a newly identified member of the TNF ligand family that can induce a rapid caspase-dependent apoptosis in transformed cells but not in most normal cells (11
, 12)
. Accordingly, TRAIL is being evaluated in human studies as a potential anticancer agent. TRAIL activates DR-4 (DR-4/TRAIL-R1) and DR-5 (DR-5/TRAIL-R2), whereas TNF-
, Fas ligand, and Apo3L are ligands for TNF-R1, Fas, and DR-3, respectively (10)
. Activation of DR-4 and DR-5 results in the recruitment of the intracellular adaptor molecule Fas-associated death domain that engages proximal caspase-8 at the receptor site (13)
. Loss of caspase-8 activation can contribute to resistance to TRAIL-induced apoptosis (14)
. Furthermore, the combination of TRAIL and certain anticancer drugs induced apoptosis to a greater extent than did either drug alone (15)
. Most anticancer drugs induce apoptosis by engaging the cytochrome c-dependent, mitochondrial apoptotic pathway (8) . This pathway is negatively regulated by the antiapoptotic Bcl-2 protein through suppression of cytochrome c release (8 , 16) . We (6) and others (17) have shown that NSAIDs engage the mitochondrial pathway and our data indicate that COX-2 overexpression attenuates apoptosis induction by NSAIDs and 5-fluorouracil, and is associated with Bcl-2 up-regulation. NSAIDs inhibit COX enzymes and can induce apoptosis and inhibit cell proliferation in cultured colorectal cancer cell lines (18 , 19) and in intestinal epithelia from animal models of colon cancer (20) and in humans (21) , suggesting that modulation of these processes contributes to their antitumor effects in vivo. NSAIDs are effective chemopreventive agents against experimental colon cancer and in patients with familial adenomatous polyposis were shown to reduce the number and size of colorectal polyps relative to placebo (reviewed in Ref. 22 ). Furthermore, epidemiological studies have shown a 40–50% reduction in the incidence of colorectal cancer in subjects taking NSAIDs for extended periods of time (reviewed in Ref. 22 ). In this report, we determined the effect of ectopic expression of COX-2 upon TRAIL-mediated apoptosis in HCT-15 colon cancer cells and examined the apoptotic pathway involved in such modulation. Specifically, we determined whether forced COX-2 expression alters DR-4 and DR-5 levels and/or caspase activation by TRAIL. The effect of NSAIDs on DR-4 and DR-5 levels and the effect of the combination of NSAIDs and TRAIL upon cell death were also examined.
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Materials and Methods |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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Transfection of COX-2 cDNA.
Cells (3 x 105 in 2 ml of RPMI 1640) were plated in 6-well Costar tissue culture plates. Twenty-four h later, cells were transfected with vector alone or with 2.5 µg of pSG5-COX-2 plasmid, which contains a full-length COX-2 cDNA in the pSG expression vector (a gift of Dr. Robert Kulmacz, University of Texas Medical School, Houston, TX) and 0.5 mg of pcDNA1, which contains a neomycin-resistant marker, as described previously (23)
. A similar procedure was previously followed for stable transfection of SW480 cells with the Bcl-2 cDNA (PC3-Bcl-2; gift of Dr. Timothy McDonnell, University of Texas M. D. Anderson Cancer Center). Transfection was performed with Lipofectamine reagent (Life Technologies, Inc.) according to the vendor’s instructions. Positive transfectants were selected in RPMI 1640 containing 500 µg/ml geneticin (Life Technologies, Inc.). Cell lines were established from individual colonies using cloning cylinders.
cDNA Preparation and Quantitative Multiplex PCR.
Total RNA was extracted and purified from cultured cells using the RNeasy Mini kit (Qiagen, Valencia, CA), per the manufacturer’s instructions. The RNA was quantified by determining absorbance at 260 nm. One µg of total RNA from each sample was reverse transcribed into cDNA using Superscript II reverse transcriptase (Life Technologies, Inc.) in a volume of 20 µl. The cDNA product was diluted to 100 µl and then amplified by PCR using a Gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA) with ß-actin as an internal control. To produce an optimal PCR amplification, a linear correlation between PCR cycle numbers and products was established. Ten µl of PCR reaction mixture contained 1 µl of diluted cDNA sample, 1 unit of Hotstart polymerase (Qiagen, Valencia, CA), 0.1 mM deoxynucleotide triphosphates, 1 µl of DMSO, 50 ng of primers of DR-4 (forward, 5-CGA TGT GGT CAG AGC TGG TAC AGC-3; reverse, 5-GGA CAC GGC AGA GCC TGT GCC ATC-3'), DR-5 (forward, 5-AAG ACC CTT GTG CTC GTT G-3; reverse, 5-TCA CCT GAA TCA CAC CTG G-3), and ß-actin (forward, 5'-GTT GCT ATC CAG GCT GTG C-3'; reverse, 5'-GCA TCC TGT CGG CAA TGC-3'). The reaction was performed using the following program: 94°C for 15 min to activate the polymerase, 30 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 45 s, and then an additional extension step of 72°C for 7 min. The PCR products were electrophoresed on a 2.5% agarose gel and stained with ethidium bromide, photographed under UV light, and then quantified by scanning densitometry relative to the absorbance of ß-actin.
Western Blotting.
Cells were washed with cold PBS and suspended in a lysis buffer containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10 µg/ml phenylmethylsulfonyl fluoride, 30 µg/ml aprotinin, and 50 mM Tris-HCl (pH 8.0). The samples were placed on ice for 60 min and then centrifuged at 14,000 rpm for 30 min to remove cellular debris. The protein concentration in the samples was measured using a Bradford protein assay kit (Bio-Rad, Richmond, CA). Samples containing 50 µg of protein were added to a SDS-PAGE loading buffer and then loaded in a 12% denaturing polyacrylamide gel. Proteins were then transferred electrophoretically to Hybond-C nitrocellulose membranes (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ). Membranes were blocked using 5% nonfat dry milk in 10 mM PBS for 1 h at room temperature and then incubated for 2 h at room temperature with monoclonal antibodies against DR-4, DR-5 (1:500; ProSci, Inc., Poway, CA), COX-2 (1:1000; Cayman Chemical, Ann Arbor, MI), Bcl-2 (1:100; Dako, Copenhagen, Denmark), or cytochrome c (1:800; PharMingen, San Diego, CA). Anti-ß-actin (1:5000; Sigma, St Louis, MO) was used as an internal control for protein loading. Blots were washed three times with PBS containing 0.1% Tween 20 for 15 min each and incubated with a second antibody supplied with the ECL kit (Amersham-Pharmacia) for 1 h at room temperature. The signal was detected by chemiluminescence using the ECL detection kit. Scanning densitometry was conducted for protein quantification.
Cytochrome c Release from Mitochondria.
Untreated and TRAIL-treated HCT-15 cells transfected with either vector alone or the COX-2 expression vector were gently homogenized in an ice-cold buffer containing 0.1 mM phenylmethylsulfonyl fluoride, 1 mM DTT, 2 mM HEPE-KOH (pH 7.5), 1 mM KCl, 1.5 mM MgCl2, 1 mM sodium-EDTA, 1 mM sodium EGTA, 0.25 M sucrose, proteinase inhibitor (1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin). The homogenate was centrifuged at 900 x g for 10 min at 2°C, and the supernatant was centrifuged at 8000 x g for 30 min at 2°C. The resulting supernatant solution was centrifuged at 100,000 x g for 30 min at 2°C to remove any mitochondrial contamination. The protein concentration in the cytosolic fraction was determined, and the fraction was denatured in Laemmli buffer (250 mM Tris-HCl, 4% SDS, 10% glycerol, 0.006% bromophenol blue, and 2% ß-mercaptoethanol, pH 6.8) at 100°C. Immunoblotting was performed as described above using a 15% polyacrylamide gel.
Northern Blot.
Five µg of total RNA extracted from cultured cells were electrophoresed on 1% formaldehyde-agarose gel and transferred to a nylon membrane (Scheicher & Schuell, Keene, NH). The cDNA for DR-5 was synthesized and 32P-labeled ([-32P]dCTP, 3,000 Ci/mmol; ICN, Costa Mesa, CA) using PCR primers (same as described for RT-PCR). Blots were hybridized in Rapid-hyb buffer (Amersham-Pharmacia) overnight at 65°C. Blots were then autoradiographed, stripped, and reprobed with ß-actin to normalize for differences in loading and transfer.
MTT Assay.
For analysis of cell viability after TRAIL treatment, 4 x 103
cells of each cell line were seeded in triplicate in 96-well plates in 0.1 ml of medium for 24 h. Medium was then replaced with media containing TRAIL (30–300 ng/ml) and incubated for an additional 24 h. Incubation of cells with sulindac sulfide (20 µM) or NS398 (50 µM) for 24 h followed by TRAIL (0–40 ng/ml) for 24 h was also performed. Medium was then removed, and 100 µl of fresh medium containing 10 µl of MTT (Sigma; 5 mg/ml in PBS) were added. Cells were incubated at 37°C for 3 h, medium was aspirated, and the cells were washed with PBS. Then, 0.15 ml of DMSO was added to each well, and absorbance at a wavelength of 540 nm was measured using a Spectra Count microplate reader (Perkin Elmer Life Sciences, Boston, MA). Cell survival was calculated: (%) = 100 x Atreatment/Acontrol.
Caspase Activation Assays.
Cells (2 x 107) in 10-cm dishes were treated with TRAIL (30 ng/ml) for 24 h and then harvested by scraping in lysis buffer (Calbiochem). The cell lysates were placed on ice for 10 min and centrifuged at 10,000 x g for 10 min. The protein concentration in supernatants was determined. Caspase-8 activity was determined with the Caspase-8 Assay kit (Calbiochem) using Ac-IETD-pNA as a substrate with incubation at 37°C for 2 h. For caspase-3, cells were treated as described above, and caspase-3 activity was determined using the Caspase-3 Cellular Activity Assay kit (Calbiochem) using Ac-DEVD-NA as a substrate and a reaction time of 2 h at 37°C. A specific inhibitor of caspase-3, Ac-DQMD-CHO (Biomol Research Labs, Plymouth Meeting, PA), was used. Caspase-9 activity was determined using the caspase-9/Mch6 Colorimetric Protease Assay kit (MBL, Nagoya, Japan) using LEHD-NA as a substrate with incubation at 37°C for 2 h. For all caspase activation assays, 100 µg of extracted protein were mixed with the reaction buffer and substrate (included in kits) in 96-well plates and incubated for 5 h at 37°C. Samples were then read at a wavelength of 405 nm, and absorbances at 3 h of reaction were recorded. Caspase activity was evaluated by the absolute absorbance of TRAIL-treated cells subtracted from the absolute absorbance of untreated control cells.
DNA Fragmentation.
After treatment with 200 ng/ml of TRAIL for 24 h, both floating and attached cells were harvested. Cells were pelleted by centrifugation and resuspended in Tris-EDTA buffer (pH 8.0). Cells were lysed on ice in a mixture of 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, and 0.5% Triton X-100 for 15 min. The lysate was centrifuged at 12,000 x g for 15 min to separate the soluble (fragmented) from the pellet (intact genomic) DNA. Soluble DNA was treated with RNase A (50 µg/ml) at 37°C for 1 h, followed by treatment with proteinase K (100 µg/ml) in 0.5% SDS, at 50°C for 2 h. The residual material was extracted with phenol/chloroform, precipitated in ethanol, electrophoresed on a 1.8% agarose gel, and then stained with ethidium bromide. Gels were photographed under UV light.
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Results and Discussion |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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. COX-2 transfectants had elevated PGE2 levels relative to parental cells (data not shown). The effect of forced COX-2 expression on cell viability after TRAIL treatment was determined. As shown in Fig. 1B
, the ligand TRAIL reduced cell viability in HCT-15 ± COX-2 cells in a dosage-dependent manner. Overexpression of COX-2 was found to significantly attenuate this effect. At 120 ng/ml of TRAIL, COX-2 transfectants showed a 20% reduction in cell viability versus a 40% reduction observed for parental cells (Fig. 1B)
. To determine whether the reduction in cell viability induced by TRAIL was attributable to apoptosis, a DNA fragmentation assay was performed oligonucleosomal. DNA laddering was seen in TRAIL-treated HCT-15 ± COX-2 cells consistent with apoptosis (Fig. 1C)
; COX-2 overexpression appeared to reduce DNA fragmentation by TRAIL. Suppression of COX-2 enzymatic activity by NSAIDs induces apoptosis, whereas ectopic COX-2 expression, as shown here, can inhibit apoptosis. Consistent with our findings are data in cholangiocarcinoma cells where increased COX-2 or PGE2 were shown to confer resistance to Fas-mediated apoptosis, and this effect was reversed by the selective COX-2 inhibitor, NS-398 (7)
. To determine the mechanism of COX-2-mediated resistance to TRAIL-induced apoptosis, we analyzed membrane DR expression levels and caspase activation. DR-4 and DR-5 mRNA and protein expression were determined in two clones (C5 and C9) overexpressing COX-2, using RT-PCR and immunoblotting, respectively, with quantification by scanning densitometry. HCT-15 cells expressed DR-4 and DR-5, and both clones overexpressing COX-2 showed reduced DR-5 and to a lesser extent DR-4, mRNA (Fig. 2A)
. DR-5 protein expression was also reduced (Fig. 2B)
. Furthermore, Northern blotting demonstrated that COX-2 transcriptionally repressed DR-5 expression (Fig. 2C)
. We then determined whether the NSAID sulindac sulfide can modulate DR-4 and DR-5 expression levels. COX-2-overexpressing cells were treated with sulindac sulfide (20 µM for 24 h), and DR-4 and DR-5 mRNA levels were analyzed by RT-PCR. We found that sulindac sulfide increased both DR-4 and DR-5 mRNA levels in these cells (Fig. 3A)
. Similarly, Huang et al. (24)
found that sulindac sulfide treatment increased DR-5 expression in human colon cancer cells. Induction of DR-5 in HCT-15 cells is p53 independent because these cells contain mutant p53 proteins. DR-5 induction has been shown to occur in both p53-positive and p53-negative colon cancer cell lines (24)
. These data suggest that increased DR levels by sulindac sulfide may contribute to the proapoptotic effect of this drug. In this regard, certain anticancer drugs have been shown to induce apoptosis through induction of DRs (25)
. Activation of DR-5 using an agonistic DR-5 antibody (TRA-8) has been shown to increase TRAIL-induced apoptosis in vitro and in vivo, indicating that modulation of the level of DR-5 regulates the apoptotic response to TRAIL (26)
. The finding of increased DR-5 expression by sulindac sulfide suggests that inhibition of COX-2 enzymatic activity by NSAIDs can reverse COX-2-mediated down-regulation of DR-5. To test this hypothesis, we determined the effect of the combination of sulindac sulfide and TRAIL treatment upon cell viability. We found that the combination reduced cell viability to a greater extent than did either drug alone (Fig. 3B)
, indicating that inhibition of COX-2 enhances apoptotic susceptibility to TRAIL.
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. Caspase-9 activation by TRAIL was also found in PC-3 prostate cancer cells, suggesting that multiple caspase pathways are involved in TRAIL-induced cell death (27)
. Forced COX-2 expression significantly attenuated TRAIL-induced caspase-8, caspase-3, and caspase-9 activation relative to parental HCT-15 cells (Fig. 4A)
. An 
30% reduction in caspase activation was found in COX-2 transfectants after drug treatment. Furthermore, the pan-caspase inhibitor, z-Val-Ala-Asp-fluoromethyl ketone, was shown to restore cell viability after TRAIL treatment (Fig. 4B)
. Thus, COX-2 attenuates TRAIL-mediated apoptosis by inhibiting caspase activation and DR expression. Given that caspase-9 is triggered by cytochrome c and Apaf1 binding (28)
, we analyzed cytochrome c release by TRAIL. TRAIL treatment induced cytochrome c expression in COX-2-overexpressing cells and in parental cells to an equivalent extent (Fig. 4C)
. Induction of cytochrome c by TRAIL is likely attributable to caspase-8-related cleavage of BID, which has been reported to translocate to mitochondria and to induce cytochrome c release (29)
. Bcl-2 expression was then analyzed, given its known role as an inhibitor of cytochrome c release and subsequent apoptosis (16)
. COX-2 transfectants showed up-regulation of Bcl-2 mRNA and protein (Fig. 2, A and B
). This finding is consistent with results in rat intestinal epithelial cells where ectopic COX-2 expression was associated with up-regulation of Bcl-2 (30)
. Furthermore, exogenous PGE2 was shown to induce Bcl-2 expression and to inhibit apoptosis induced by a selective COX-2 inhibitor in colon cancer cells (30)
. In our study, COX-2-transfected cells overexpressing Bcl-2 did not show attenuated cytochrome c induction by TRAIL at 4 h (Fig. 4C)
. Similarly, Walczak et al. (14)
and Keogh et al. (31)
found that Bcl-2 failed to block cytochrome c release after exposure to TRAIL in Jurkat cells. To determine whether Bcl-2 can block TRAIL-mediated cell death, we used SW480 colon cancer cells stably transfected with the Bcl-2 cDNA. We found that the Bcl-2-overexpressing cells were protected from TRAIL; however, TRAIL was still able to kill cells overexpressing Bcl-2 (Fig. 5A)
. Partial protection from TRAIL-mediated cell death is consistent with data in PC-3 prostate cancer cells (27)
and in non-small cell lung cancer cells (32)
. In contrast, Bcl-2 failed to protect 8226 and ARP-1 myeloma cells from TRAIL-induced apoptosis (33)
. Interestingly, Bcl-2 was shown to block activation of caspase-8, a component of the DR complex, by the chemopreventive agent curcumin (34)
. To exclude an effect of Bcl-2 on the level of DR-5, we examined DR-5 expression in Bcl-2-overexpressing SW480 cells. DR-5 protein expression was not altered by forced Bcl-2 expression (Fig. 5B)
, indicating that COX-2-mediated down-regulation of DR-5 is independent of Bcl-2.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a Kadoorie Human Cancer Genetics Award and the National Colorectal Cancer Alliance. 
2 To whom requests for reprints should be addressed, at Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Phone: (507) 284-2175; Fax: (507) 284-5486; E-mail: sinicrope.frank{at}mayo.edu
3 The abbreviations used are: COX, cyclooxygenase; DR, death receptor; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2; RT-PCR, reverse transcription-PCR; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Received 6/24/02. Accepted 7/17/02.
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REFERENCES |
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TopABSTRACTIntroductionMaterials and MethodsResults and DiscussionREFERENCES |
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716 knockout mice by inhibition of cyclooxygenase 2 (COX-2). Cell, 87: 803-809, 1996.[Medline]
-glutamylcysteine synthetase gene expression by nonsteroidal anti-inflammatory drugs in human colon cancer cells. Biochem. Biophys. Res. Commun., 290: 1427-1433, 2002.[Medline]
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