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Abstract

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Cyclooxygenase (COX)-2 is one of the rate-limiting enzymes in the conversion of arachidonic acid to prostaglandins and other eicosanoids. Recent studies have shown enhanced expression of COX-2 in cancer cells of several tissues. We investigated the expression of COX-2 and prostaglandin (PG) Einline image production in two human skin epidermal cancer cell lines: cutaneous squamous cell carcinoma, HSC-5, and eccrine carcinoma, EcCa. Both COX-2 expression and PGEinline image production were significantly enhanced in cancer cell lines compared with the non-tumorigenic human keratinocyte cell line, HaCaT. In order to determine the role of COX-2 in the proliferation of HSC-5 and EcCa, the growth of untreated cells and cells transfected with COX-2 antisense oligonucleotide was compared using the MTT assay. Transfection with the antisense oligonucleotide suppressed COX-2 protein expression and significantly inhibited cell growth. The effect of a selective inhibitor of COX-2, NS398, was compared with the effect of the antisense oligonucleotide in order to see whether COX-2 expression and prostaglandins have selective effects on cell growth. COX-2 expression was unchanged by NS398 treatment, whereas NS398 inhibited cell growth to a certain extent. The degree of growth inhibition was greater with the antisense oligonucleotide than with NS398. Our findings indicate that COX-2 protein expression is enhanced in skin epidermal cancer cells and that COX-2 plays a pivotal role in regulating cell growth. Furthermore, inhibition of COX-2 expression had a more significant effect on growth suppression than inhibition of COX-2 catalytic activity, suggesting the existence of two different signal pathways via COX-2 in regulating cell growth. Int. J. Cancer 86:667–671, 2000. © 2000 Wiley-Liss, Inc.

The conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2 ), the committed step in prostanoid biosynthesis, is catalyzed by the COX isoenzymes, COX-1 and COX-2, each of which is encoded by a unique gene, located on different chromosomes (Smith et al., 1996). COX-1 is expressed constitutively in most tissues and is thought to perform primarily “housekeeping” functions, such as gastric cytoprotection, regulation of renal blood flow, and platelet aggregation. In contrast, COX-2 message and protein are normally undetectable in most tissues, but its expression can be induced rapidly as part of the inflammatory reaction in response to a wide variety of extracellular stimuli (Herschman, 1996; Smith et al., 1996). In addition to its association with inflammation, COX-2 expression clearly plays a role in regulating cellular proliferation, differentiation and tumorigenesis (Bornfeldt et al., 1997; DuBois et al., 1996). There are indications that intestinal epithelial cells overexpressing the COX-2 gene develop altered adhesion properties and become resistant to undergoing apoptosis (Tsujii and DuBois, 1995). Direct genetic evidence that COX-2 plays a key role in tumorigenesis was provided with a murine model system, which showed that knocking out the COX-2 gene caused a marked reduction in the number and size of intestinal polyps in ApcΔ716 knockout mice, a murine model of familial adenomatous polyposis (Oshima et al., 1996). In humans, increased COX-2 expression is found in carcinomas of various organs including colon, breast, prostate, lung, esophagus, pancreas and mucous membranes of head and neck (Subbaramaiah et al., 1996; Zimmermann et al., 1999). Treatment with highly selective COX-2 inhibitors results in a reduction in the size and number of colonic lesions in several animal models (Kawamori et al., 1998) and suppression of carcinogenesis of human colon, prostate and esophageal cancers (Zimmermann et al., 1999). The mechanism by which COX-2 inhibitors suppress carcinogenesis is attributed to its modulation of prostanoid production which affects cell proliferation, tumor growth and immune responsiveness. PGE2, a major product of COX-2, induces bcl-2 expression and inhibits apoptosis and, conversely, that COX-2 inhibitors induce apoptosis (Sheng et al., 1998a; Zimmermann et al., 1999). While the involvement of prostanoids in carcinogenesis is clear, another study showed that selective inhibition of COX-2 activity suppresses the production of PGE2 but does not alter the progress of the morphological transformation of rat fibroblasts (Sheng et al., 1998b), suggesting the existence of distinct signal pathways, PGE2-dependent and PGE2-independent, in the regulation of carcinogenesis.

Since we and others reported that COX-2 is associated with differentiation and transformation of keratinocytes (Kanekura et al., 1998; Leong et al., 1996), we examined the expression of COX-2 and its role in regulating cell growth using two different skin epidermal cancer cell lines and examined whether prostaglandins and COX-2 expression have selective effects on cell growth using either a COX-2 inhibitor which inhibits its catalytic activity or a COX-2 antisense oligonucleotide that inhibits its expression.

MATERIAL AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Materials

Cell culture reagents were purchased from Life Technologies, Inc. (Grand Island, NY). Anti-PGE2 antibody was purchased from Upstate Biotechnology (Lake Placid, NY). [3H]PGE2 was obtained from DuPont-NEN (Boston, MA). Polyclonal antibodies to human COX-1, COX-2 and cytosolic phospholipase A2 (cPLA2) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). NS398 was purchased from Cayman Chemicals (Ann Arbor, MI). The COX-2 antisense oligonucleotide (GGAAACATCGACAGT) was synthesized by SAWADY (Tokyo, Japan). The sequence of the antisense oligonucleotide corresponded to nucleotides 2263 to 2277 of the sense sequence (Hla and Neilson, 1992), which was determined according to the previous report (Tsujii et al., 1998).

Cell culture

HaCaT cells were kindly provided by Dr. L. Matrisian (Vanderbilt University, Nashville, TN) with the permission of Dr. N. E. Fusenig (German Cancer Research Center, Heidelberg, Germany) and the HSC-5 cell line was a gift from Dr. Y. Hozumi (Yamagata University, Yamagata, Japan). HaCaT, HSC-5 and EcCa were grown as described previously (Boukamp et al., 1988) with some modifications. Cells were cultured at 37°C in a humidified atmosphere containing 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing high glucose and 100,000 units/l Penicillin G, 100 mg/l Streptomycin, 1 mg/l Fungizone, 0.1 mM nonessential amino acids, 292 mg/l glutamine, 50 mg/l ascorbic acid and 10% FCS.

Prostaglandin E2 measurement

Cells were seeded at 1×105 cells/ml in 24-well tissue culture plates (0.5 ml/well) and incubated for 72 hr until confluent. The media was then replaced with fresh DMEM containing 1% FCS. The culture media were collected after 24 hr and analyzed for PGE2 production. PGE2 produced by transfected cells was measured with a little modification because of the suppressed cell growth and the results were normalized by cell numbers of the dishes. PGE2 was measured by the radioimmunoassay described previously (Kanekura et al., 1998). The method is based on the competition of PGE2 in the test sample with [3H]-labeled PGE2 for binding to anti-PGE2 antibody. A 10 to 100 μl aliquot of culture medium was added to RIA assay buffer (0.1 mM phosphate buffer, pH 7.4, containing 0.9% sodium chloride, 0.1% sodium azide, and 0.1% gelatin), mixed with the appropriate amounts of labeled PGE2 and reconstituted antiserum, and incubated overnight at 4°C. The assay tubes were then placed on ice and 1 ml of cold charcoal-dextran suspension was added. After 15 min, the tubes were centrifuged at 2,200g for 10 min at 4°C. The supernatants were decanted into scintillation vials and radioactivity was determined by scintillation spectrometry. Percent binding was compared to a standard curve and the amounts of PGE2 in the samples were calculated.

Western blotting

Cells grown in 10-cm plastic dishes were pelleted in PBS containing 1 μg/ml leupeptin, 0.03% Aprotinin, 2 mM PMSF and 2 mM EDTA, pH 7.2. Proteins were obtained by suspending the pellet in extraction buffer (62.5 mM Tris, 2% SDS, 0.5 mM DTT, 1 mM fresh PMSF, pH 6.8) and denaturing the proteins by incubating the tubes at 95°C for 5 min. Identical amounts of protein were electrophoresed in a 7.5% SDS/polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was blocked with 1% BSA and 4% non-fat dry milk in PBS containing 0.1% Tween 20 and then incubated with either anti-COX-1, anti-COX-2 or anti-cPLA2 antibodies for 1 hr at room temperature. After incubation with anti-goat (COX) or anti-rabbit (cPLA2) alkaline phosphatase linked immunoglobulin for 30 min at room temperature, the immunoreactive bands were detected using an ECL kit (Amersham, Arlington Heights, IL).

Transfection

Transfection was carried out using the Lipofectin reagent (Gibco BRL, Gaithersburg, MD) according to the manufacturer's instructions. HSC-5 and EcCa were seeded at 2× 105 cells/plate into 6-cm dishes and grown in DMEM containing 10% FCS until the cells were 50–70% confluent. Two micrograms of COX-2 antisense oligonucleotide and 20 μl Lipofectin were combined and incubated for 15 min at room temperature. Cells were incubated with the DNA lipid complexes for 12 to 18 hr and fed with DMEM containing 10% fetal calf serum.

Cell growth assays

Cell growth was monitored by a colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay with minor modifications. Cells were seeded at 5 × 103 into 96 well plates. At the indicated times (culture days), 10 μl of MTT solution (2.5 mg/ml in PBS) was added to each well and the plates were incubated at 37°C for 4 hr. Acid-SDS (100 μl of 0.01 N HCl in 10% SDS) was added to all the wells and mixed thoroughly to dissolve the dark blue crystals. The plates were kept at room temperature over night in the dark to ensure that all crystals were dissolved. They were read with a Microplate Reader, using a test wavelength of 570 nm and a reference wavelength of 630 nm.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Increased levels of COX-2 protein expression in skin epidermal cancer cells

The levels of COX-2 expression were studied in 2 different skin epidermal cancer cells, HSC-5 and EcCa. As shown in Figure 1, the skin epidermal cancer cells expressed elevated levels of COX-2 protein compared to the nontumorigenic human keratinocyte cell line, HaCaT, as detected by Western immunoblotting. COX-2 expression was higher in EcCa than in HSC-5 (Fig. 1).

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Figure 1. COX-2 protein expression is enhanced in human skin epidermal cancer cells. Cells were seeded at 1×10inline image cells/ml in 10-cm tissue culture plates (10 ml/plate) and incubated for 72 hr until confluent. The media was then replaced with fresh DMEM containing 1% FCS. Proteins were extracted after 24 hr and 10 μg of each sample was analyzed by Western blotting as described in Material and Methods using anti COX-2 antibody. The lower graph is the quantitaton of each signal in the upper immunoblot. Values are signal intensity relative to the intensity measured in untreated HSC-5.

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Increased PGE2 production proportional to COX-2 expression levels in HSC-5 and EcCa

Because PGE2 is a major COX-2 product, the levels of PGE2 produced by these cells were measured. As shown in Figure 2, HSC-5 and EcCa synthesized 17-fold and 90-fold more PGE2 than HaCaT, respectively. The amounts of PGE2 produced correlated well with the levels of expression of COX-2 shown in Figure 1.

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Figure 2. PGE2 production is increased in human skin epidermal cancer cells. Cells were seeded at 1×10inline image cells/ml in 24-well tissue culture plates (0.5 ml/well) and incubated for 72 hr until confluent. The media was then replaced with fresh DMEM containing 1% FCS. The culture media were collected after 24 hr and analyzed for PGE2 production as described in Material and Methods. The data presented are means ± SE of triplicate determinations and representative of 6 separate experiments. *: significant difference at p < 0.01 vs. HaCaT.

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Decrease in expression of COX-2 protein by transfection with COX-2 antisense oligonucleotide

In order to elucidate the functional role of COX-2 in regulating cell growth, we examined the effect of suppressing its expression on cell growth. We employed a COX-2 antisense oligonucleotide which specifically inhibits COX-2 expression but has no biological function. As shown in Figure 3, Western blotting of extracts confirmed that COX-2 expression was suppressed in HSC-5 and EcCa cells treated with the antisense oligonucleotide.

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Figure 3. Decrease in COX-2 protein expression by treatment with COX-2 antisense oligonucleotide. HSC-5 and EcCa cells were seeded at 2×10inline image cells/ml in 6 cm tissue culture plates and cultured for 48 hr in DMEM containing 10% FCS. At 50–70% confluence, cells were treated with COX-2 antisense oligonucleotide and Lipofectin complexes or Lipofectin alone as a control as described in Material and Methods. Transfected cells were incubated for 24 hr until confluent and the proteins were extracted and 10 μg of each sample was analyzed by Western blotting using anti COX-2 antibody. The lower graph is the quantitaton of each signal in the upper immunoblot. Values are signal intensity relative to the intensity measured in untreated HSC-5.

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Suppression of growth by inhibition of COX-2 expression

When COX-2 expression was inhibited by the antisense oligonucleotide, HSC-5 and EcCa cell growth was suppressed with statistical significance (Fig. 4).

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Figure 4. Suppression of cell growth by COX-2 antisense oligonucleotide. After HSC-5 and EcCa cells were treated with COX-2 antisense oligonucleotide and Lipofectin complexes (open circles) or Lipofectin alone (solid circles), they were seeded at 5 × 10inline image and incubated in 96 well plates. Growth of HSC-5 and EcCa was evaluated by the MTT assay as described in Material and Methods. Data shown are means ± SE of 10 determinations and representative of 3 separate experiments. *: significant difference at p < 0.001 vs. control.

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Suppression of cell growth by inhibition of COX-2 catalytic activity

The effect of a highly selective COX-2 inhibitor, NS398, on PGE2 production and cell growth was examined. COX-2 expression was not reduced by 20 μM NS398 in both HSC-5 and EcCa (Fig. 5). In contrast, as shown in Figure 6, cell growth was suppressed by NS398, which at 20 μM decreased PGE2 production more significantly than the antisense oligonucleotide (Fig. 7).

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Figure 5. Lack of effect of a specific COX-2 inhibitor on COX-2 protein expression. Cells were seeded at 1×10inline image cells/ml in 10 cm tissue culture plates (10 ml/plate) and incubated for 72 hr until confluent. The media was then replaced with fresh DMEM containing 1% FCS. Confluent HSC-5 and EcCa cells were incubated in the absence or presence of 20 μM NS398. Aftr 24 hr, proteins were extracted and 10 μg of each sample was analyzed by Western blotting using anti COX-2 antibody. The lower graph is the quantitaton of each signal in the upper immunoblot. Values are signal intensity relative to the intensity measured in untreated HSC-5.

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Figure 6. Suppression of cell growth by a selective COX-2 inhibitor. HSC-5 and EcCa were seeded at 5 × 10inline image and incubated in 96-well plates in the absence (solid circles) or presence of 20 μM NS398 (open circles). Cell growth was evaluated by the MTT assay as described in Material and Methods. Data shown are means ±SE of 6 determinations and representative of 3 separate experiments. *: significant difference at p < 0.001 vs. control.

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Figure 7. NS398 inhibits PGE2 production more significantly than the antisense oligonucleotide. The amounts of PGE2 produced by the cells transfected with antisense oligonucleotide or cells treated with 20 μM NS398 were measured. For measurement of PGE2 produced by NS398 treated cells, cells were seeded at 1×10inline image cells/ml in 24-well tissue culture plates (0.5 ml/well) and incubated for 72 hr until confluent. The media was then replaced with fresh DMEM containing 1% fetal calf surum. Confluent cells were incubated in the absence or presence of 20 μM NS398. The culture media were collected after 24 hr and analyzed for PGE2 production. For measurement of PGE2 produced by transfected cells, cells were seeded at 2×10inline image cells/ml in 6 cm tissue culture plates and cultured for 48 hr in DMEM containing 10% FCS. At 50– 70 % confluence, cells were treated with COX-2 antisense oligonucleotide and Lipofectin complexes or Lipofectin alone as a control as described in Material and Methods. After 24 hr, transfected cells were seeded at 2×10inline image cells/ml in 24-well tissue culture plates and incubated for 48 hr, at which culture media were replaced with fresh DMEM containing 1% fetal calf serum. After 24 hr, culture media were collected and analyzed for PGE2 production. The data shown are means ± SE of 6 determinations. * and **: significant difference at p <0.01 and p< 0.001 vs. control, respectively.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Elevated COX-2 expression has previously been observed in various carcinomas including skin cancers (Buckman et al., 1998; Muller-Decker et al., 1999; Subbaramaiah et al., 1996; Zimmermann et al., 1999). While COX-2 expression in normal skin was usually very low and restricted to regions of differentiated epidermis (Buckman et al., 1998; Muller-Decker et al., 1999), studies on mouse and human skin carcinogenesis revealed that overexpression of COX-2 contributes to the development of skin cancers and COX-2 expression is constitutively enhanced in skin cancers (Buckman et al., 1998; Muller Decker et al., 1998, 1999). In the present report, we showed that the skin epidermal cancer cells, HSC-5 and EcCa expressed elevated levels of COX-2 protein. PGE2, a major COX-2 product, was synthesized much more by HSC-5 and EcCa than HaCaT. PGE2 is believed to play a role in cell proliferation, differentiation and development of tumors. For example, PGE2 treatment of human colon cancer cells induces bcl-2 expression and inhibits apoptosis (Sheng et al., 1998a). In human skin keratinocytes, PGE2 modulates calcium-induced differentiation (Evans et al., 1993). Our findings support these studies showing that COX-2 and PGs are involved in carcinogenesis.

Conversion of arachidonic acid to PGs is catalyzed by 2 isoenzymes, COX-1 and COX-2. COX-1 is expressed constitutively in most tissues to generate PGs for physiological functions, whereas COX-2 expression is induced by various extracellular stimuli, including growth factors and tumor promoters (Herschman, 1996). In addition to COX, a primary enzyme in PG production is cytosolic phospholipase A2 (cPLA2), which releases arachidonic acid from membrane phospholipids into the cytoplasm. Previous studies revealed that cPLA2 also mediates cell proliferation through mitogen-activated protein kinase and/or protein kinase C pathways (Bornfeldt et al., 1997). Therefore, we also examined COX-1 and cPLA2 expression in HSC-5 and EcCa, but no increase was found compared with their expression in HaCaT cells (data not shown).

When HSC-5 and EcCa were treated with the COX-2 antisense oligonucleotide, COX-2 expression and cell growth were suppressed. This clearly indicates that COX-2 plays an important role in tumor cell growth. One possible mechanism by which COX-2 is thought to function in carcinogenesis is via increased PG biosynthesis resulting from enhanced COX-2 expression, since PGE2 induces cell proliferation (Sheng et al., 1998b), promotes angiogenesis (Tsujii et al., 1998) and inhibits immune surveillance in human cancers.

While it is clear from a number of previous reports that PGs are involved in carcinogenesis, a study by Sheng et al. (1998b) indicates the existence of a pathway of cell transformation which is independent of PGE2. Ras proteins are key molecules in cell proliferation and differentiation, and COX-2 is thought to be an important target gene of Ras (Sheng et al., 1998b; Subbaramaiah et al., 1996). Sheng et al. (1998b) showed that COX-2 was induced by Ras and produced PGE2 in Ras-mediated transformation of rat fibroblasts. In that study, a selective COX-2 inhibitor suppressed PGE2 production but neither delayed the progress nor reduced the extent of morphological transformation, indicating that increased PGE2 production was not required for Ras-mediated morphological transformation. This finding suggests that 2 distinct signal pathways, PGE2-dependent and PGE2-independent, may regulate carcinogenesis. In order to examine whether PGE2 and COX-2 expression have a specific effect on cell growth, the effect of a highly selective COX-2 inhibitor, NS398 on PGE2 production and cell growth was compared with the effect of the antisense oligonucleotide. NS398 inhibits COX-2 catalytic activity but does not suppress its expression (Chinery et al., 1998). COX-2 expression was not reduced by 20 μM NS398 in both HSC-5 and EcCa whereas cell growth was suppressed by NS398, and the extent of growth suppression was more pronounced with the antisense oligonucleotide than with NS398 (see Figs. 4 and 6), despite the fact that 20 μM NS398 inhibited PGE2 production more than the antisense oligonucleotide. These results show that inhibiting COX-2 expression reduces cell growth more effectively than inhibiting its catalytic activity suggesting that there is a PGE2-independent pathway modulating cell growth, which is downstream of COX-2. This proposal may be supported by a previous study that demonstrated that combined treatment of colon cancer cells with an antioxidant and a selective COX-2 inhibitor, each of which reduces tumor growth independently, had an additive or synergistic effect on tumor regression (Chinery et al., 1998). COX is a bifunctional enzyme possessing cyclooxygenase activity, which converts arachidonic acid to PGGinline image, and peroxidase activity, which generates PGH2 , a direct precursor of PGE2, from PGGinline image. In addition to its role in prostaglandin synthesis, the peroxidase activity of COX contributes to superoxide production and subsequent alterations in intracellular redox status, which are associated with perturbations of cell growth and transformation (Chinery et al., 1998). Inhibitors such as NS398 inhibit the cyclooxygenase activity but not the peroxidase activity of COX (Subbaramaiah et al., 1996), whereas the suppression of COX-2 expression might result in the inhibition of both activities. These facts can explain why the antisense oligonucleotide reduced cell growth more effectively than NS398.

Our results show that COX-2 expression is enhanced in skin cancer cells and that cell growth is suppressed by inhibition of COX-2 expression.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIAL AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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