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Cyclooxygenase-2-selective antagonists do not inhibit growth of colorectal carcinoma cell lines
Cancer Letters 122 (1998) 25–30
Cyclooxygenase-2-selective antagonists do not inhibit growth of colorectal carcinoma cell lines Vincent J. Murphy a, Zhiyu Yang b, Kristy A. Rorison b, Graham S. Baldwin a,b,* a
Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia University Department of Surgery, Austin Campus, Melbourne, Victoria, Australia
b
Received 1 July 1997; accepted 1 July 1997
Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colorectal carcinoma. We now report that the potent cyclooxygenase-1 inhibitor indomethacin had no effect on the growth of human colorectal carcinoma cell lines in vitro at concentrations up to 30 mM. The selective cyclooxygenase-2 inhibitors L-745337 and NS-398 reduced cyclooxygenase activity, but had no effect on cell growth at concentrations as high as 100 mM. Our results provide direct evidence that inhibition of cyclooxygenase activity does not necessarily inhibit the growth of colorectal carcinoma cell lines and imply that the growth-inhibitory effects of NSAIDs in vitro are not mediated by inhibition of cyclooxygenases. 1998 Elsevier Science Ireland Ltd. Keywords: Colorectal carcinoma; Cyclooxygenase; NSAIDs
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal tumour growth in several experimental models. Indomethacin and other NSAIDs inhibited the development of chemically-induced colon cancers in rats and mice [1,2]. Sulindac reduced the size and number of colorectal polyps in patients with familial adenomatous polyposis (FAP) [3] and inhibited tumour formation in a murine model of
* Corresponding author. Department of Surgery, A & RMC, Austin Campus, Locked Bag 25, Heidelberg, Victoria 3084, Australia. Tel.: +61 3 94965592; fax: +61 3 94581650; e-mail: [email protected]
FAP [4]. In addition, several epidemiological studies have revealed that NSAIDs and in particular aspirin reduced the risk of colorectal carcinoma by approximately 50% [5,6]. Finally, several NSAIDs have been shown to reversibly inhibit the growth of colorectal carcinoma cell lines in vitro [7,8]. The effect of NSAIDs on colon cancer has generally been accepted to be the result of inhibition of cyclooxygenases [9]. The two forms of cyclooxygenase, which are key enzymes in prostaglandin biosynthesis [9], differ in inducibility, tissue distribution and susceptibility to NSAIDs [10]. Levels of mRNA for the inducible cyclooxygenase-2 [11] and of cyclooxygenase-1 and -2 immunoreactivity [12] were significantly higher in human colorectal tumour samples than in the accompanying normal mucosa. In
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0361-3
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V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
addition, treatment of rats with a selective cyclooxygenase-2 inhibitor significantly reduced the induction of aberrant crypt foci by the carcinogen azoxymethane [13]. Several indirect lines of evidence have recently suggested that cyclooxygenases may not be the only target of the anti-proliferative effects of NSAIDs. Firstly, treatment of colorectal carcinoma cell lines with prostaglandins did not reverse the inhibitory effects of NSAIDs on cell growth [14]. Secondly, sulindac sulphoxide, which does not inhibit prostaglandin synthesis, has been shown to inhibit the formation of aberrant crypt foci in carcinogen-treated rats [15] and the growth of human colorectal carcinoma cell lines in vitro [7]. Thirdly, sulindac sulphoxide and several other NSAIDs reduced the amounts and activities of the cyclin-dependent kinases, with a concomitant increase in the proportion of cells in the G0/G1 phase [8,16]. Fourthly, NSAIDs have been shown to induce apoptosis in the rectal epithelium of patients with FAP [17] and in the human colon carcinoma cell line HT 29 [8,16,18]. To investigate the role of cyclooxygenases in the anti-proliferative effects of NSAIDs on colon cancer, we studied the expression of cyclooxygenases and the concentration dependence of growth inhibition by NSAIDs and cyclooxygenase-2-selective antagonists in two colorectal cancer cell lines. Our results suggest that cyclooxygenases are not the target for the inhibitory effects of NSAIDs on colorectal cancer cell growth in vitro.
2. Materials and methods 2.1. Materials The human colorectal carcinoma cell lines LIM 1215 [19] and LIM 1899 [20] were obtained from Dr R.H. Whitehead, Ludwig Institute for Cancer Research, Melbourne. NSAIDs were from Sigma (St. Louis, MO). The cyclooxygenase-2-selective inhibitors NS-398 [21] and L-745 337 [22] were generously provided by Taisho (Tokyo, Japan) and Merck, Frosst (Pointe-Claire-Dorval, Canada), respectively, and sulindac sulphide and sulphone were from Merck, Sharp and Dohme (West Point, PA).
2.2. Cyclooxygenase assay Total cyclooxygenase activity was measured by determining arachidonic acid-stimulated production of prostaglandin E2 with an enzyme-linked immunoassay kit (Cayman Chemical, Ann Arbor, MI) as described by Chan et al. [22]. 2.3. Cell growth assay A colorimetric assay [23] was used to measure cell growth. Briefly 104 cells were seeded in a 96-well plate in RPMI 1640 medium containing 10 mM thioglycerol, 25 units/ml insulin, 1 mg/ml hydrocortisone and 10% foetal calf serum. Fresh medium containing the above additives, 10% foetal calf serum and the substance under investigation was added 24 h later. After incubation at 37°C for 20 h in a humidified atmosphere of 10% CO2, 10 ml of 5 mg/ml MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) was added per well and the plate was incubated for a further 4 h before the medium was discarded. Then 200 ml 0.04 M HCl in isopropanol was added to lyse the cells and the absorbance at 560 nm was read on a Titertek Multiscan MCC 1340 (Labsystems, Helsinki, Finland). 2.4. PCR of cyclooxygenase 1 and 2 Randomly primed cDNA was prepared from 10 mg of total RNA from human colorectal carcinoma cell lines and normal human fibroblasts as described previously [24] and used as PCR template with final reagent concentrations of 2.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 250 nM of each of the appropriate pair of primers and 2.5 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Scoresby, Australia) in a total volume of 20 ml. The sequences of specific primers were taken from the indicated references as follows: human cyclooxygenase-1 [25], 5′-TGCCCAGCTCCTGGCCCGCCGCTT-3′ (sense) and 5′-GTGCATCAACACAGGCGCCTCTTC-3′ (anti-sense); human cyclooxygenase-2 [26], 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′ (sense) and 5′-AGATCATCTCTGCCTGAGTATCTT-3′ (anti-sense). The PCR reaction was started with a 9 min heating step at 95°C followed by either 30 or 42 repetitive cycles (94°C for 30 s, 55°C
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V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
for 30 s and 72°C for 30 s) in a DNA thermal cycler (Corbett Research, Australia). PCR products were separated on a 7.5% acrylamide gel and visualized by staining with ethidium bromide.
3. Results The colorectal carcinoma cell lines LIM 1215 and LIM 1899 were screened for the expression of
Table 1 IC50 values for inhibition of colorectal carcinoma cell growth by NSAIDs NSAID
IC50 (mM) LIM 1215 cell growth
Acetaminophen 21000 ± 14000 Aspirin 1770 ± 640 Diclofenac 120 ± 110 Ibuprofen 290 ± 160 Indomethacin 200 ± 130 Naproxen 660 ± 460 Salicylate 1750 ± 400 NS-398 >100 L-745 337 >100 Sulindac sulphide 190 ± 180 Sulindac sulphoxide 660 ± 520 Sulindac sulphone 580 ± 140
Cyclooxygenase-1a
Cyclooxygenase-2a
17.9b 1.7 1.6 4.8 0.03 9.6 219 >100a >10a
132b 278 1.1 73 1.7 5.7 625 3.8a 0.023a
Growth of LIM 1215 cells in the presence of increasing concentrations of NSAIDs was measured with the MTT assay as described in Section 2. IC50 values (mean ± SEM of at least three separate experiments) were obtained with the program LIGAND. a IC50 values for inhibition of cyclooxygenases-1 and -2 by NSAIDs and by the selective cyclooxygenase-2 antagonists NS-398 and L745337 are taken from Refs. [10,21,22], respectively. b IC30 values are shown because 50% inhibition of cyclooxygenase2 was not achieved by concentrations as high as 1 mg/ml [10].
Fig. 1. Colorectal carcinoma cell lines express cyclooxygenase mRNA. Expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in LIM 1215 cells (COX, tracks 2 and 7; GAPDH, track 3), LIM 1899 cells (COX, tracks 3 and 8; GAPDH, track 4) and human fibroblasts (COX, tracks 4 and 9; GAPDH, track 5) was measured by RT-PCR for either 30 or 42 cycles as described in Section 2. Reaction products were separated on a 7.5% acrylamide gel and visualized by staining with ethidium bromide. The expected sizes of the cyclooxygenase-1, cyclooxygenase-2 and glyceraldehyde-3-phosphate dehydrogenase PCR products were 304, 305 and 153 bp, respectively. The control sample (COX, tracks 1 and 6; GAPDH, track 2) had no cDNA in the PCR reaction. The sizes of molecular weight markers (COX, track 5; GAPDH, track 1) were 501, 489, 404, 331 and 242 bp.
cyclooxygenase-1 and -2 mRNA by RT-PCR. Levels of cyclooxygenase-2 mRNA in LIM 1215 cells were similar to levels in human fibroblasts (Fig. 1). Levels of cyclooxygenase-2 mRNA in LIM 1899 cells and of cyclooxygenase-1 mRNA in both LIM 1215 and LIM 1899 cells were considerably lower than levels in human fibroblasts (Fig. 1). Levels of cyclooxygenase activity in either LIM 1215 or LIM 1899 cells measured by enzyme-linked immunoassay of arachidonic acid-stimulated prostaglandin E2 production were approximately 200-fold lower than in human fibroblasts. All of the NSAIDs tested inhibited growth of LIM 1215 cells (Table 1). IC50 values ranged from 120 mM for diclofenac and sulindac sulphide to 21 mM for acetaminophen (Table 1). Comparison with the IC50 values previously reported for inhibition of cyclooxygenases-1 and -2 in intact bovine aortic endothelial cells and murine macrophages, respectively, revealed no correlation in either case (Table 1). The potent and selective cyclooxygenase-2 inhibitors NS-398 [21] and L-745337 [22] did not inhibit
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V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
4. Discussion
Fig. 2. Cyclooxygenase-2 selective antagonists do not inhibit growth of colorectal carcinoma cell lines. Growth (open bars) of LIM 1215 cells in the presence of increasing concentrations of the cyclooxygenase-2-selective antagonists L-745337 and NS-398 was measured with the MTT assay as described in Section 2, except that cells were synchronized by treatment with media lacking foetal calf serum for 24 h before addition of the antagonists, which were in fresh medium containing 1% foetal calf serum. Total cyclooxygenase activity (hatched bars) in LIM 1215 cells following treatment with the same antagonists was measured by determining arachidonic acid-stimulated production of prostaglandin E2 with an enzyme-linked immunoassay kit [22].
growth of LIM 1215 (Fig. 2) or LIM 1899 cells (data not shown) at concentrations as high as 100 mM. Both NS-398 and L-745337 inhibited cyclooxygenase activity (Fig. 2). Indomethacin is a potent and selective inhibitor of cyclooxygenase-1 (Table 1). Although indomethacin inhibited growth of LIM 1215 (Table 1) and LIM 1899 cells (data not shown), the IC50 values were more than 6000-fold greater than the IC50 values for inhibition of cyclooxygenase-1 (Table 1).
Herein we present evidence that inhibition of cyclooxygenases does not necessarily inhibit the growth of the colorectal carcinoma cell lines LIM 1215 and LIM 1899. Firstly, the potent cyclooxygenase-1 inhibitor indomethacin (IC50 30 nM) was only a moderately effective inhibitor of colorectal carcinoma cell growth (IC50 300 mM). Secondly, the potent and selective cyclooxygenase-2 inhibitors NS-398 and L745337 (IC50 values of 3.8 mM and 23 nM for cyclooxygenase-2, respectively) had no effect on colorectal carcinoma cell growth at concentrations as high as 100 mM (Fig. 2). The lack of inhibition was not due to the absence of cyclooxygenase-1 and -2 from these cells, since cyclooxygenase activity and mRNA for cyclooxygenase-1 and -2 was detected in both cell lines (Fig. 1), although the levels present were lower than in human fibroblasts. The observation that both NS-398 and L-745337 inhibited cyclooxygenase activity by at least 80% in both cell lines suggested that levels of cyclooxygenase-2 were at least four times higher than cyclooxygenase1 and that cyclooxygenase-2 was fully accessible to the inhibitors. The above results imply that inhibition of the growth of colorectal carcinoma cell lines by other NSAIDs may not be mediated by cyclooxygenases. This conclusion is supported by the observation that there is no correlation between the IC50 values for inhibition of LIM 1215 cell growth by NSAIDs and the IC50 values for inhibition of cyclooxygenase-1 or 2 (Table 1). The values for both cyclooxygenases were determined in intact cell preparations in the presence of 10% foetal calf serum [10] and are therefore directly comparable with the values for LIM 1215 cell growth, if it is assumed that there are no major differences in NSAID affinities between species. Interestingly, acetaminophen, which has a similar potency to aspirin as an inhibitor of the inducible cyclooxygenase-2 with an IC30 value of 130 mM [10], is relatively ineffective as an inhibitor of LIM 1215 cell growth with an IC50 of 21 mM. Epidemiological studies have revealed that acetaminophen use, in contrast to aspirin use, was not associated with a decreased risk of any gastrointestinal tract cancer [6]. Furthermore, both sulindac sulphoxide and the biologically irreversibly oxidized product sulindac sulphone inhibited cell
V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
growth with IC50 values only three-fold lower than the IC50 value for sulindac sulphide. Of the three oxidation states the sulphone is inactive in anti-inflammatory assays in vivo and neither the sulphoxide nor the sulphone is able to inhibit cyclooxygenase activity in vitro [18]. Similar IC50 values have been reported previously for the inhibition of growth of three other colorectal carcinoma cell lines by the three oxidation states of sulindac [7]. Although the studies reported herein demonstrate directly that cyclooxygenase activity is not required for the in vitro growth of colorectal carcinoma cell lines, at least two possible roles for cyclooxygenase1 or -2 in colorectal carcinoma growth in vivo can be envisaged. Firstly, prostaglandins produced by inflammatory cells within a tumour might stimulate the growth of cancer cells in a paracrine manner. Secondly, cyclooxygenase-1 or -2 might be upregulated in the cancer cells themselves by inflammatory mediators released from other cells within a tumour. The prostaglandins produced might then stimulate the growth of the cancer cells in an autocrine manner. Indeed, cyclooxygenase-2 expression has been observed previously in carcinoma cells within a colorectal tumour by immunohistochemistry [12]. In either case blockade of prostaglandin synthesis could then contribute to the observed inhibition of tumour growth. Although clarification of the relative importance of cyclooxygenases and other proteins as targets for the inhibitory effects of NSAIDs on colorectal tumours in vivo is an urgent priority, it appears that NSAIDs inhibit the growth of colorectal cancer cell lines in vitro by a mechanism independent of cyclooxygenase-1 or -2.
Acknowledgements We thank Dr R.H. Whitehead for the colorectal carcinoma cell lines and for helpful discussions. This work was supported in part by grants no. 920527 and 960182 from the Australian NHMRC.
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Cyclooxygenase-2-selective antagonists do not inhibit growth of colorectal carcinoma cell lines Vincent J. Murphy a, Zhiyu Yang b, Kristy A. Rorison b, Graham S. Baldwin a,b,* a
Ludwig Institute for Cancer Research, Melbourne, Victoria, Australia University Department of Surgery, Austin Campus, Melbourne, Victoria, Australia
b
Received 1 July 1997; accepted 1 July 1997
Abstract Non-steroidal anti-inflammatory drugs (NSAIDs) reduce the incidence of colorectal carcinoma. We now report that the potent cyclooxygenase-1 inhibitor indomethacin had no effect on the growth of human colorectal carcinoma cell lines in vitro at concentrations up to 30 mM. The selective cyclooxygenase-2 inhibitors L-745337 and NS-398 reduced cyclooxygenase activity, but had no effect on cell growth at concentrations as high as 100 mM. Our results provide direct evidence that inhibition of cyclooxygenase activity does not necessarily inhibit the growth of colorectal carcinoma cell lines and imply that the growth-inhibitory effects of NSAIDs in vitro are not mediated by inhibition of cyclooxygenases. 1998 Elsevier Science Ireland Ltd. Keywords: Colorectal carcinoma; Cyclooxygenase; NSAIDs
1. Introduction Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit colorectal tumour growth in several experimental models. Indomethacin and other NSAIDs inhibited the development of chemically-induced colon cancers in rats and mice [1,2]. Sulindac reduced the size and number of colorectal polyps in patients with familial adenomatous polyposis (FAP) [3] and inhibited tumour formation in a murine model of
* Corresponding author. Department of Surgery, A & RMC, Austin Campus, Locked Bag 25, Heidelberg, Victoria 3084, Australia. Tel.: +61 3 94965592; fax: +61 3 94581650; e-mail: [email protected]
FAP [4]. In addition, several epidemiological studies have revealed that NSAIDs and in particular aspirin reduced the risk of colorectal carcinoma by approximately 50% [5,6]. Finally, several NSAIDs have been shown to reversibly inhibit the growth of colorectal carcinoma cell lines in vitro [7,8]. The effect of NSAIDs on colon cancer has generally been accepted to be the result of inhibition of cyclooxygenases [9]. The two forms of cyclooxygenase, which are key enzymes in prostaglandin biosynthesis [9], differ in inducibility, tissue distribution and susceptibility to NSAIDs [10]. Levels of mRNA for the inducible cyclooxygenase-2 [11] and of cyclooxygenase-1 and -2 immunoreactivity [12] were significantly higher in human colorectal tumour samples than in the accompanying normal mucosa. In
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0361-3
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addition, treatment of rats with a selective cyclooxygenase-2 inhibitor significantly reduced the induction of aberrant crypt foci by the carcinogen azoxymethane [13]. Several indirect lines of evidence have recently suggested that cyclooxygenases may not be the only target of the anti-proliferative effects of NSAIDs. Firstly, treatment of colorectal carcinoma cell lines with prostaglandins did not reverse the inhibitory effects of NSAIDs on cell growth [14]. Secondly, sulindac sulphoxide, which does not inhibit prostaglandin synthesis, has been shown to inhibit the formation of aberrant crypt foci in carcinogen-treated rats [15] and the growth of human colorectal carcinoma cell lines in vitro [7]. Thirdly, sulindac sulphoxide and several other NSAIDs reduced the amounts and activities of the cyclin-dependent kinases, with a concomitant increase in the proportion of cells in the G0/G1 phase [8,16]. Fourthly, NSAIDs have been shown to induce apoptosis in the rectal epithelium of patients with FAP [17] and in the human colon carcinoma cell line HT 29 [8,16,18]. To investigate the role of cyclooxygenases in the anti-proliferative effects of NSAIDs on colon cancer, we studied the expression of cyclooxygenases and the concentration dependence of growth inhibition by NSAIDs and cyclooxygenase-2-selective antagonists in two colorectal cancer cell lines. Our results suggest that cyclooxygenases are not the target for the inhibitory effects of NSAIDs on colorectal cancer cell growth in vitro.
2. Materials and methods 2.1. Materials The human colorectal carcinoma cell lines LIM 1215 [19] and LIM 1899 [20] were obtained from Dr R.H. Whitehead, Ludwig Institute for Cancer Research, Melbourne. NSAIDs were from Sigma (St. Louis, MO). The cyclooxygenase-2-selective inhibitors NS-398 [21] and L-745 337 [22] were generously provided by Taisho (Tokyo, Japan) and Merck, Frosst (Pointe-Claire-Dorval, Canada), respectively, and sulindac sulphide and sulphone were from Merck, Sharp and Dohme (West Point, PA).
2.2. Cyclooxygenase assay Total cyclooxygenase activity was measured by determining arachidonic acid-stimulated production of prostaglandin E2 with an enzyme-linked immunoassay kit (Cayman Chemical, Ann Arbor, MI) as described by Chan et al. [22]. 2.3. Cell growth assay A colorimetric assay [23] was used to measure cell growth. Briefly 104 cells were seeded in a 96-well plate in RPMI 1640 medium containing 10 mM thioglycerol, 25 units/ml insulin, 1 mg/ml hydrocortisone and 10% foetal calf serum. Fresh medium containing the above additives, 10% foetal calf serum and the substance under investigation was added 24 h later. After incubation at 37°C for 20 h in a humidified atmosphere of 10% CO2, 10 ml of 5 mg/ml MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, St. Louis, MO) was added per well and the plate was incubated for a further 4 h before the medium was discarded. Then 200 ml 0.04 M HCl in isopropanol was added to lyse the cells and the absorbance at 560 nm was read on a Titertek Multiscan MCC 1340 (Labsystems, Helsinki, Finland). 2.4. PCR of cyclooxygenase 1 and 2 Randomly primed cDNA was prepared from 10 mg of total RNA from human colorectal carcinoma cell lines and normal human fibroblasts as described previously [24] and used as PCR template with final reagent concentrations of 2.5 mM MgCl2, 0.2 mM each of dATP, dCTP, dGTP and dTTP, 250 nM of each of the appropriate pair of primers and 2.5 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer, Scoresby, Australia) in a total volume of 20 ml. The sequences of specific primers were taken from the indicated references as follows: human cyclooxygenase-1 [25], 5′-TGCCCAGCTCCTGGCCCGCCGCTT-3′ (sense) and 5′-GTGCATCAACACAGGCGCCTCTTC-3′ (anti-sense); human cyclooxygenase-2 [26], 5′-TTCAAATGAGATTGTGGGAAAATTGCT-3′ (sense) and 5′-AGATCATCTCTGCCTGAGTATCTT-3′ (anti-sense). The PCR reaction was started with a 9 min heating step at 95°C followed by either 30 or 42 repetitive cycles (94°C for 30 s, 55°C
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V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
for 30 s and 72°C for 30 s) in a DNA thermal cycler (Corbett Research, Australia). PCR products were separated on a 7.5% acrylamide gel and visualized by staining with ethidium bromide.
3. Results The colorectal carcinoma cell lines LIM 1215 and LIM 1899 were screened for the expression of
Table 1 IC50 values for inhibition of colorectal carcinoma cell growth by NSAIDs NSAID
IC50 (mM) LIM 1215 cell growth
Acetaminophen 21000 ± 14000 Aspirin 1770 ± 640 Diclofenac 120 ± 110 Ibuprofen 290 ± 160 Indomethacin 200 ± 130 Naproxen 660 ± 460 Salicylate 1750 ± 400 NS-398 >100 L-745 337 >100 Sulindac sulphide 190 ± 180 Sulindac sulphoxide 660 ± 520 Sulindac sulphone 580 ± 140
Cyclooxygenase-1a
Cyclooxygenase-2a
17.9b 1.7 1.6 4.8 0.03 9.6 219 >100a >10a
132b 278 1.1 73 1.7 5.7 625 3.8a 0.023a
Growth of LIM 1215 cells in the presence of increasing concentrations of NSAIDs was measured with the MTT assay as described in Section 2. IC50 values (mean ± SEM of at least three separate experiments) were obtained with the program LIGAND. a IC50 values for inhibition of cyclooxygenases-1 and -2 by NSAIDs and by the selective cyclooxygenase-2 antagonists NS-398 and L745337 are taken from Refs. [10,21,22], respectively. b IC30 values are shown because 50% inhibition of cyclooxygenase2 was not achieved by concentrations as high as 1 mg/ml [10].
Fig. 1. Colorectal carcinoma cell lines express cyclooxygenase mRNA. Expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in LIM 1215 cells (COX, tracks 2 and 7; GAPDH, track 3), LIM 1899 cells (COX, tracks 3 and 8; GAPDH, track 4) and human fibroblasts (COX, tracks 4 and 9; GAPDH, track 5) was measured by RT-PCR for either 30 or 42 cycles as described in Section 2. Reaction products were separated on a 7.5% acrylamide gel and visualized by staining with ethidium bromide. The expected sizes of the cyclooxygenase-1, cyclooxygenase-2 and glyceraldehyde-3-phosphate dehydrogenase PCR products were 304, 305 and 153 bp, respectively. The control sample (COX, tracks 1 and 6; GAPDH, track 2) had no cDNA in the PCR reaction. The sizes of molecular weight markers (COX, track 5; GAPDH, track 1) were 501, 489, 404, 331 and 242 bp.
cyclooxygenase-1 and -2 mRNA by RT-PCR. Levels of cyclooxygenase-2 mRNA in LIM 1215 cells were similar to levels in human fibroblasts (Fig. 1). Levels of cyclooxygenase-2 mRNA in LIM 1899 cells and of cyclooxygenase-1 mRNA in both LIM 1215 and LIM 1899 cells were considerably lower than levels in human fibroblasts (Fig. 1). Levels of cyclooxygenase activity in either LIM 1215 or LIM 1899 cells measured by enzyme-linked immunoassay of arachidonic acid-stimulated prostaglandin E2 production were approximately 200-fold lower than in human fibroblasts. All of the NSAIDs tested inhibited growth of LIM 1215 cells (Table 1). IC50 values ranged from 120 mM for diclofenac and sulindac sulphide to 21 mM for acetaminophen (Table 1). Comparison with the IC50 values previously reported for inhibition of cyclooxygenases-1 and -2 in intact bovine aortic endothelial cells and murine macrophages, respectively, revealed no correlation in either case (Table 1). The potent and selective cyclooxygenase-2 inhibitors NS-398 [21] and L-745337 [22] did not inhibit
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4. Discussion
Fig. 2. Cyclooxygenase-2 selective antagonists do not inhibit growth of colorectal carcinoma cell lines. Growth (open bars) of LIM 1215 cells in the presence of increasing concentrations of the cyclooxygenase-2-selective antagonists L-745337 and NS-398 was measured with the MTT assay as described in Section 2, except that cells were synchronized by treatment with media lacking foetal calf serum for 24 h before addition of the antagonists, which were in fresh medium containing 1% foetal calf serum. Total cyclooxygenase activity (hatched bars) in LIM 1215 cells following treatment with the same antagonists was measured by determining arachidonic acid-stimulated production of prostaglandin E2 with an enzyme-linked immunoassay kit [22].
growth of LIM 1215 (Fig. 2) or LIM 1899 cells (data not shown) at concentrations as high as 100 mM. Both NS-398 and L-745337 inhibited cyclooxygenase activity (Fig. 2). Indomethacin is a potent and selective inhibitor of cyclooxygenase-1 (Table 1). Although indomethacin inhibited growth of LIM 1215 (Table 1) and LIM 1899 cells (data not shown), the IC50 values were more than 6000-fold greater than the IC50 values for inhibition of cyclooxygenase-1 (Table 1).
Herein we present evidence that inhibition of cyclooxygenases does not necessarily inhibit the growth of the colorectal carcinoma cell lines LIM 1215 and LIM 1899. Firstly, the potent cyclooxygenase-1 inhibitor indomethacin (IC50 30 nM) was only a moderately effective inhibitor of colorectal carcinoma cell growth (IC50 300 mM). Secondly, the potent and selective cyclooxygenase-2 inhibitors NS-398 and L745337 (IC50 values of 3.8 mM and 23 nM for cyclooxygenase-2, respectively) had no effect on colorectal carcinoma cell growth at concentrations as high as 100 mM (Fig. 2). The lack of inhibition was not due to the absence of cyclooxygenase-1 and -2 from these cells, since cyclooxygenase activity and mRNA for cyclooxygenase-1 and -2 was detected in both cell lines (Fig. 1), although the levels present were lower than in human fibroblasts. The observation that both NS-398 and L-745337 inhibited cyclooxygenase activity by at least 80% in both cell lines suggested that levels of cyclooxygenase-2 were at least four times higher than cyclooxygenase1 and that cyclooxygenase-2 was fully accessible to the inhibitors. The above results imply that inhibition of the growth of colorectal carcinoma cell lines by other NSAIDs may not be mediated by cyclooxygenases. This conclusion is supported by the observation that there is no correlation between the IC50 values for inhibition of LIM 1215 cell growth by NSAIDs and the IC50 values for inhibition of cyclooxygenase-1 or 2 (Table 1). The values for both cyclooxygenases were determined in intact cell preparations in the presence of 10% foetal calf serum [10] and are therefore directly comparable with the values for LIM 1215 cell growth, if it is assumed that there are no major differences in NSAID affinities between species. Interestingly, acetaminophen, which has a similar potency to aspirin as an inhibitor of the inducible cyclooxygenase-2 with an IC30 value of 130 mM [10], is relatively ineffective as an inhibitor of LIM 1215 cell growth with an IC50 of 21 mM. Epidemiological studies have revealed that acetaminophen use, in contrast to aspirin use, was not associated with a decreased risk of any gastrointestinal tract cancer [6]. Furthermore, both sulindac sulphoxide and the biologically irreversibly oxidized product sulindac sulphone inhibited cell
V.J. Murphy et al. / Cancer Letters 122 (1998) 25–30
growth with IC50 values only three-fold lower than the IC50 value for sulindac sulphide. Of the three oxidation states the sulphone is inactive in anti-inflammatory assays in vivo and neither the sulphoxide nor the sulphone is able to inhibit cyclooxygenase activity in vitro [18]. Similar IC50 values have been reported previously for the inhibition of growth of three other colorectal carcinoma cell lines by the three oxidation states of sulindac [7]. Although the studies reported herein demonstrate directly that cyclooxygenase activity is not required for the in vitro growth of colorectal carcinoma cell lines, at least two possible roles for cyclooxygenase1 or -2 in colorectal carcinoma growth in vivo can be envisaged. Firstly, prostaglandins produced by inflammatory cells within a tumour might stimulate the growth of cancer cells in a paracrine manner. Secondly, cyclooxygenase-1 or -2 might be upregulated in the cancer cells themselves by inflammatory mediators released from other cells within a tumour. The prostaglandins produced might then stimulate the growth of the cancer cells in an autocrine manner. Indeed, cyclooxygenase-2 expression has been observed previously in carcinoma cells within a colorectal tumour by immunohistochemistry [12]. In either case blockade of prostaglandin synthesis could then contribute to the observed inhibition of tumour growth. Although clarification of the relative importance of cyclooxygenases and other proteins as targets for the inhibitory effects of NSAIDs on colorectal tumours in vivo is an urgent priority, it appears that NSAIDs inhibit the growth of colorectal cancer cell lines in vitro by a mechanism independent of cyclooxygenase-1 or -2.
Acknowledgements We thank Dr R.H. Whitehead for the colorectal carcinoma cell lines and for helpful discussions. This work was supported in part by grants no. 920527 and 960182 from the Australian NHMRC.
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