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Tumor Biology |
Departments of Gastrointestinal Medicine and Nutrition [Y. S., X. M. T., E. H., F. A. S.], and Molecular Pathology [M. T. K.], The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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716mice dramatically reduced intestinal polyp number (11)
. Furthermore, overexpression of the human COX-2 gene in mammary glands of transgenic mice resulted in mammary tumors, indicating that COX-2 was sufficient to induce tumorigenesis (12)
. Epidemiological data indicate that regular and prolonged intake of NSAIDs are associated with a 40–50% reduction in CRC incidence (13, 14, 15, 16) . Treatment of familial adenomatous polyposis patients with the NSAID sulindac or the selective COX-2 inhibitor celecoxib have been shown to effectively regress existing colorectal adenomas relative to placebo (17 , 18) . Selective COX-2 inhibitors were developed given that inhibition of COX-1 is largely responsible for the adverse effects associated with NSAID treatment (19 , 20) . NSAIDs are also potent chemopreventive agents in animal models of colon cancer (11 , 21, 22, 23) . In intestinal tissues from animals and humans treated with NSAIDs, modulation of apoptotic rates was found in association with tumor inhibition/regression, suggesting a mechanism for their chemopreventive effects (24, 25, 26, 27, 28) . Studies by ourselves (6) and others (29, 30, 31) have shown that sulindac sulfide, a nonselective COX inhibitor, and NS398, a selective COX-2 inhibitor, can inhibit growth and induce apoptosis in cultured colon and pancreatic cancer cell lines. However, the molecular and biochemical pathways responsible for the proapoptotic effects of NSAIDs remain poorly understood.
Inhibition of COX and PG synthesis by NSAIDs may not explain many of the experimental results obtained. Evidence for COX-independent effects include the observation that NSAIDs can inhibit the growth of colon and pancreatic cancer cells devoid of COX-2 expression as they do for those producing COX-2 (6 , 31 , 32) . Furthermore, NSAID metabolites lacking the ability to inhibit COX enzymes, i.e., sulindac sulfone, can induce apoptosis in vitro (30) and inhibit experimental colon cancer (33) . In some (34) , but not other (35) reports, the addition of PGE2 to NSAID-treated colon cancer cells reversed their growth inhibitory and proapoptotic effects. Moreover, murine embryonic fibroblast cells with homologous knockout of COX-1 and COX-2 alleles remain sensitive to the antiproliferative and proapoptotic effects of NSAIDs (36) .
Anticancer drugs eliminate tumor cells by inducing their apoptosis (37)
. Therefore, modulation of apoptotic susceptibility is a critical determinant of therapeutic efficacy. Apoptosis is controlled by two major pathways including a mitochondrial (38)
and a membrane DR pathway (39)
. Mitochondrial release of cytochrome c into the cytosol has been shown in cell-free systems to be rate limiting for the activation of caspases and endonucleases (40
, 41)
. Cytosolic cytochrome c activates procaspase-9 by binding to Apaf1 in the presence of dATP, leading to caspase-9 activation and subsequent activation of downstream effector caspases, including caspase-3, with triggering of apoptosis (42)
. Activated caspases perform proteolytic cleavage events involved in apoptosis (38)
. The antiapoptotic Bcl-2 or Bcl-XL proteins act on mitochondria to inhibit cytochrome c release and to prevent opening of the megachannel (38
, 43
, 44)
. Accordingly, Bcl-2 proteins can prevent caspase activation, Bax redistribution to the mitochondria, and apoptosis after exposure to anticancer drugs (41
, 43, 44, 45)
. Caspase-8 is a proximal caspase that plays a critical role in the DR-mediated apoptotic pathway, used by Fas, tumor necrosis factor 
, and tumor necrosis factor-related apoptosis-inducing ligand, that is independent of cytochrome c release (39)
. Membrane DRs are activated by their respective ligands and engage adaptor molecules and caspases, including proximal caspase-8 (39
, 46)
.
In this report, we determined whether forced COX-2 expression can modulate susceptibility to apoptosis induced by NSAIDs and 5-FU. After 4 decades, 5-FU, an inhibitor of DNA synthesis, remains the most active and widely used single agent in the treatment of CRC. We examined whether these drugs use the mitochondrial apoptotic pathway by determining the effects of forced COX-2 expression on cytochrome c release, caspase-9, -3, and -8 activation, and Bcl-2 expression.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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COX-2 cDNA Transfection.
Cells (3 x 105 in 2 ml of RPMI 1640) were plated in six-well Costar tissue culture plates. Twenty-four h later, cells were transfected with 2.5 µg pSG5-COX2 plasmid, which contains a full-length COX-2 cDNA in the pSG expression vector and 0.5 µg pcDNA1, which contains neomycin-resistant marker, as described previously (47)
. Transfection was performed with LipofectAMINE reagent (Life Technologies, Inc.) according to vendor’s instruction. Positive transfectants were selected in RPMI 1640 containing 500 µg/ml geneticin (Gibco Life Technologies, Inc., Carlsbad, CA). Cell lines were established from individual colonies using cloning cylinders.
PGE2 Synthesis.
Medium from 2-day serum-depleted 35-mm cultures were pooled and [14C] arachidonic acid (0.10 µCi/ml media) was added. The dishes were refed with the radioactive medium and incubated for 16 h, as described previously (48)
. PGE2 analysis was performed by Dr. Susan Fischer (M. D. Anderson Cancer Center).
TUNEL Assay.
DNA strand breaks, consistent with apoptosis, were detected by the TUNEL assay, which was performed using the In Situ Cell Death Detection kit (Roche Diagnostics, Mannheim, Germany). Cultured cells were treated for 48 h with NS398 (40–160 µM), sulindac sulfide (40–160 µM), or 5-FU (0.04–0.16 mM). Cells were then harvested and fixed using 4% paraformaldehyde in PBS (pH 7.4) for 1 h at room temperature. Cells were resuspended in 100 µl of 0.1% Triton X-100 in 0.1% sodium citrate for 2 min on ice for permeabilization and then labeled with fluorescein dUTP and stained with propidium iodide, according to the manufacturer’s instructions. Thereafter, the cells were analyzed for apoptosis using a FACScan flow cytometer (Epics Profile; Coulter Corp., Hialeah, FL).
MTT Assay.
Cells were seeded in 96-well plates at a density of 2 x 104 and allowed to attach overnight. The cells were then treated with NS398 (200 µm) for 48 h. Medium was then replaced with 100 µl of MTT solution (Sigma Chemical Co., St. Louis, MO) at a final concentration of 1 mg/ml/well. After incubation at 37°C for 4 h, the supernatant was aspirated, and 100 µl of 10% SDS was added to each well and incubated for 8 h at 37°C. Absorbance was measured at a wavelength of 75 nm using a microplate reader.
Immunoblotting.
Cells were washed with cold PBS and suspended in a lysis buffer [20 mM HEPES (pH 7.5), 100 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM EGTA (pH 7.5), 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride] on ice for 15 min. After removal of cell debris by centrifugation, the protein concentration in cell lysates was determined using a Bradford protein assay (Bio-Rad, Richmond, CA). Samples containing 50 µg of protein were added to SDS-PAGE loading buffer with 5% ß-mercaptoethanol, and loaded in a 10% (COX-2), 12% (Bcl-2), or 15% (cytochrome c) polyacrylamide gels. Proteins were then transferred to nitrocellulose membranes. After blocking the blot with 5% milk in PBS containing 0.1% Tween 20, the membrane was incubated for 1 h at room temperature with an anti-COX-2 (1:1000; Cayman Chemicals), anti-ß-actin (1:3000; Sigma Chemical Co.), anti-Bcl-2 (1:80; Dako Corporation, Denmark), or anticytochrome c (1:1000; PharMingen, San Diego, CA) monoclonal antibodies. Blots were then incubated with secondary antibodies conjugated with peroxidase (Bio-Rad) for 1 h at room temperature. The signal was detected by chemiluminescence using the ECL detection kit (Amersham, Arlington Heights, IL).
RT-PCR.
Total cellular RNA was extracted from HCT15 ± COX-2 cells using the RNeasy Mini kit (Qiagen, Valencia, CA), per the manufacturer’s instructions. The RNA was quantified by determining absorbance at 260 nM. After reverse transcription of 10 µg RNA, the cDNA product was amplified by PCR using a gene Amp PCR System 2700 (Applied Biosystems, Foster City, CA) in a 20 µl final reaction volume for 38 cycles using One Step RT-PCR kit (Qiagen, Hilden, Germany). Amplification of the Bcl-2 and reference gene ß-actin were performed in one reaction. The following primer pairs were used: Bcl-2: sense, 5'-CTTTGGAAGGATGGCGCACGCTGG-3'; antisense, 5'-AGGCACTTCACTTGTGGCTCAGAGG-3'; size: 723 bp; ß-actin sense, 5'-GTTGCTATCCAGGCTGTGC-3'; antisense, 5'-CGTAACGGCTGTCCTACG-3'; size: 541 bp. The cycling conditions were as follows: 30 min at 50°C, 15 min at 95°C, followed by 38 cycles at 94°C for 30 s; 61°C for 1 min, and 72°C for 1 min. The amplified products were separated on 1.5% agarose gels stained with 0.1 µg/ml of ethidium bromide and photographed under UV light.
Analysis of Cytochrome c Release.
Floating and attached cells were collected after incubation with sulindac sulfide (160 µM), NS398 (200 µM), or 5-FU (0.16 mM) for 48 h. Cells were washed once in PBS (pH 7.5) and then added to Buffer A [0.25 M sucrose, 1.0 mM DTT, 2.0 mM HEPES (pH 7.5), 1.0 mM KCl, 1.5 mM MgCl2, 1.0 mM sodium-EDTA, 1.0 mM sodium-EGTA, 0.1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1 µg/ml aprotinin]. Cells were collected, and then homogenized and centrifuged at 1,000 x g for 15 min at 4°C. The supernatants were centrifuged again at 20,000 x g for 45 min at 4°C. The supernatant (cytosolic fraction) was collected, and protein concentration was determined by the Bradford protein assay (Bio-Rad). To determinate cytochrome c release into the cytosol, immunoblotting was performed using anticytochrome c monoclonal antibody (PharMingen).
Caspase Activation Assays.
Cells were treated with NS398 (200 µM), sulindac sulfide (160 µM), or 5-FU (0.16 mM) for 48 h, then harvested and lysed. The protein concentration in the cell lysates was determined, and 200 µg of protein was added to 50 µl of lysis buffer for each assay. 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. Samples were then read at a wavelengt2h of 405 nm. For caspase-3, cells were treated as described above, and 50 µg protein from cell lysates was analyzed for caspase-3 activity using the Caspase-3 Cellular Activity Assay kit (Calbiochem, San Diego, CA) using Ac-DEVD-NA as a substrate and a reaction time of 2 h at 37°C. Samples were read as described above. A specific inhibitor of caspase-3 Ac-DQMD-CHO (Biomol Research Labs, Plymouth Meeting, PA) was used in separate experiments. 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.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. COX-2 transfectants had elevated PGE2 levels relative to parental cells. The effect of forced COX-2 expression on the apoptotic susceptibility of these cells to sulindac sulfide, NS398, and 5-FU was determined. As shown in Fig. 2, A–C
, all three of the drugs induced apoptosis in a dosage-dependent manner. NS398 was less potent as an inducer of apoptosis on an equimolar basis relative to sulindac sulfide (6)
. Overexpression of COX-2 in both clones (2A and 2B) significantly attenuated apoptosis induction by all three of the drugs relative to parental HCT-15 cells. Analysis of PGE2 levels after drug treatment revealed suppression by NSAIDs and no change for 5-FU (data not shown).
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. Forced COX-2 expression inhibited cytochrome c release compared with parental HCT-15 cells (Fig. 3)
. Given that Bcl-2 can inhibit mitochondrial cytochrome c release (41
, 43, 44, 45)
, we hypothesized that COX-2 transfectants may overexpress Bcl-2 with resultant inhibition of cytochrome c release and attenuation of drug-induced apoptosis. To address this issue, we analyzed Bcl-2 expression in COX-2 transfected cells. Interestingly, we found that both Bcl-2 mRNA and protein levels were increased in untreated COX-2 overexpressing cells relative to parental cells (Fig. 4, A and B)
. The increase in Bcl-2 in COX-2 transfectants relative to parental cells was maintained during drug treatment. These data indicate that COX-2 overexpression can up-regulate Bcl-2.
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. In COX-2 overexpressing cells, a significant reduction in drug-induced caspase-9 and -3 activation was observed relative to parental HCT-15 cells. The reduction in caspase-9 activity by COX-2 varied from 1.5- to 2.5-fold after treatment with these three drugs. A 1.8–4.2-fold reduction in caspase-3 was detected in COX-2 transfectants after drug treatment. To verify the role of caspase-3 as a downstream effector of apoptosis in HCT-15 cells, we used the specific caspase-3 inhibitor Ac-DQMD-CHO (2 nM). After treatment with NS398 (200 µM) for 48 h this inhibitor was shown to restore cell survival, using the MTT assay, in COX-2 transfected and parental cells (data not shown). We also analyzed caspase-8 activity given that it plays a critical role in DR-mediated apoptosis that is independent of cytochrome c (39)
. Caspase-8 activity was increased by drug treatment but to a lesser extent than were caspase-9 and -3 (Fig. 5, A–C)
. Forced COX-2 expression attenuated caspase-8 activation induced by sulindac sulfide but not by NS398 or 5-FU.
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. This result is consistent with experiments shown in Fig. 2, A–C
, wherein apoptosis resistance was observed using doses of NSAIDs that have been shown to inhibit COX-2 enzymatic activity and PG production (2)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sulindac sulfide, NS398, and 5-FU engaged the mitochondrial apoptotic pathway involving cytosolic cytochrome c release and subsequent activation of upstream caspase-9 and downstream caspase-3. Importantly, COX-2 overexpression attenuated these effects resulting in an inhibition of apoptosis. Induction of the mitochondrial apoptotic pathway by NSAIDs has also been shown in SW620 and other colon cancer cell lines (52 , 53) . We found that NSAIDs and 5-FU also induced caspase-8 activation but to a much lesser extent than for caspase-9 and -3. Recently, sulindac sulfide was shown to increase expression of membrane DR5 in colon and prostate cancer cells, and to activate caspase-8 (54) . Furthermore, we found that COX-2 overexpression inhibited sulindac sulfide-induced caspase-8 activation. In a separate study, we found that DR5, but not DR4, was transcriptionally repressed in COX-2 overexpressing HCT-15 cells, indicating that COX-2 can modulate the level of DR5, and thereby regulate the membrane DR pathway (55) . This finding provides a potential mechanism for the observed reduction in caspase-8 activation by sulindac sulfide in COX-2 transfected cells. Taken together, we provide evidence for predominant involvement of the mitochondrial pathway but also the caspase-8-dependent apoptotic pathway in NSAID and 5-FU-induced apoptosis in human colon cancer cells.
Evidence indicates that Bcl-2 acts to stabilize mitochondrial membrane integrity by preventing cytochrome c release, and subsequent caspase activation and apoptosis (40 , 41 , 43 , 44) . These effects contribute to the ability of Bcl-2 to inhibit apoptosis-induction by a wide variety of cytotoxic drugs (37) . To determine whether attenuated cytochrome c release in drug-treated COX-2 transfectants was related to alterations in Bcl-2, we analyzed Bcl-2 expression. COX-2 transfectants, in the absence or presence of drug treatment, showed up-regulation of Bcl-2 mRNA and protein levels relative to parental cells. Therefore, increased Bcl-2 may contribute to attenuated cytochrome c release, reduced caspase-9 and -3 activation, and resultant inhibition of apoptosis in COX-2 overexpressing colon cancer cells. Our results are consistent with the finding that forced COX-2 expression in nontransformed rat intestinal epithelial cells was associated with Bcl-2 induction and resistance to butyrate-induced apoptosis (50) . In transgenic mice, forced COX-2 expression in mammary glands was associated with the development of mammary tumors and induction of Bcl-2 expression (12) . Addition of PGE2 to HCA-7 colon cancer cells that constitutively express COX-2 was shown to inhibit apoptosis induced by celecoxib (SC-58125) and to induce Bcl-2 expression (56) . Maintenance of elevated Bcl-2 levels in cells treated with NSAIDs, as shown in this report, suggests that up-regulation of Bcl-2 can occur independently of PGE2. Whereas reduced apoptotic susceptibility mediated by COX-2 is attributable, in part, to up-regulation of Bcl-2 and its inhibition of cytochrome c release, the molecular mechanism underlying COX-2-mediated up-regulation of Bcl-2 is unknown at this time. The Bcl-2 promoter is transcriptionally regulated by wild-type p53 via a p53-dependent response element (57) ; however, HCT-15 cells express mutant p53 proteins (58) . In contrast to Bcl-2, Bax and Bid are believed to promote apoptosis by increasing cytochrome c release (38) . Zhang et al. (53) found that disruption of the Bax gene in human colon cancer cells abrogated cytochrome c-dependent apoptosis induction by NSAIDs. These data indicate that NSAIDs engage the mitochondrial apoptotic pathway in colon cancer cells that is regulated by Bcl-2 and Bax (53) .
In conclusion, our data demonstrate that COX-2 can confer resistance to apoptosis induction by diverse antineoplastic agents in transformed cells and suggest that COX-2 overexpression may represent a novel mechanism of intrinsic drug resistance in human CRCs. NSAIDs and 5-FU engaged the cytochrome c-dependent apoptotic pathway that was inhibited by forced COX-2 expression. Up-regulation of Bcl-2 by COX-2 provides a potential mechanism for reduced apoptotic susceptibility. Given that COX-2 is overexpressed in the majority of human CRCs, our findings have important implications for the prevention and treatment of this disease.
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FOOTNOTES |
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1 Supported by a Kadoorie Human Cancer Genetics Award, an award from the National Colorectal Cancer Alliance (both to F. A. S.), and NIH Grant CA16672 (Institutional Core Facility). 
2 To whom requests for reprints should be addressed, at Mayo Clinic, 200 First Street, SW, Rochester, MN 55905. Phone: (507) 266-0132; Fax: (507) 284-5486; E-mail: sinicrope.frank{at}mayo.edu
3 The abbreviations used are: CRC, colorectal cancer; PG, prostaglandin; COX, cyclooxygenase; NSAID, non-steroidal anti-inflammatory drug; DR, death receptor; 5-FU, 5-fluorouracil; TUNEL, terminal deoxynucleotidyl transferase-mediated nick end labeling; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; RT-PCR, reverse transcription-PCR.
Received 5/ 7/02. Accepted 9/ 5/02.
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—) and COX-2 transfected HCT-15/COX-2-A (—
—), and HCT-15/COX-2-B (—
—) cells were incubated for 48 h with sulindac sulfide (A), NS398 (B), or 5-FU (C). Cells were then harvested, and apoptosis was determined using the TUNEL assay. Induction of apoptosis by all three of the drugs was dosage-dependent. Forced COX-2 expression significantly attenuated drug induced apoptosis compared with parental HCT-15 cells. Data points represent the mean triplicate of value determinations; bars, ±SD.
) showed significantly reduced caspase-9 and -3 activation by these drugs relative to parental HCT-15 cells (