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Original Studies |
Departments of Medical Biochemistry (K.J.S., A.A.K.), Obstetrics and Gynecology (M.D., R.P.S.), and Pathology (M.D.H.), University of Cape Town Medical School, Observatory, Cape Town 7925, South Africa; and Medical Research Council Human Reproductive Sciences Unit, Center for Reproductive Biology (K.J.S., R.P.M., H.N.J.), Edinburgh, United Kingdom EH3 9ET
Address all correspondence and requests for reprints to: Dr. H. N. Jabbour, Medical Research Council Human Reproductive Sciences Unit, 37 Chalmers Street, Edinburgh, United Kingdom EH3 9ET. E-mail: h.jabbour{at}hrsu.mrc.ac.uk
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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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Cyclooxygenase (COX) enzymes, also called PG endoperoxide synthase, catalyze the rate-limiting step in the conversion of arachidonic acid to PGH2 and other eicosanoids, including PGE (9). There are at least two isoforms of the COX enzyme, COX-1 and COX-2 (10, 11). COX-1 is constitutively expressed in many tissues and cell types and generates PGs for normal physiological function (11). By contrast, the expression of COX-2 is rapidly induced after the stimulation of quiescent cells by growth factors, oncogenes, carcinogens, and tumor-promoting phorbol esters (10, 11, 12). PGE2 elicits its autocrine/paracrine effects on target cells through interaction with seven transmembrane G protein-coupled receptors, which belong to the rhodopsin family of serpentine receptors (13). Four main subtypes of PGE2 receptors have been identified (EP1, EP2, EP3, and EP4); these use alternate and, in some cases, opposing intracellular pathways (14). To date, the roles of the different PGE2 receptors, their divergent intracellular signaling pathways, as well as their target genes involved in mediating the effects of PGE2 on normal or neoplastically transformed cervical epithelium remain to be elucidated.
Recently, a relationship between COX-2, its synthesized product PGE2, and neoplastic transformation of epithelial cells has been established (15, 16). Transcription of COX-2 is up-regulated in numerous cancers, including colon, pancreas, esophagus, lung, prostate, and bladder (17, 18, 19, 20, 21, 22). It has been proposed that COX-2 overexpression and PGE2 synthesis mediate neoplastic transformation of epithelial cells by increasing their proliferation rate, resistance to apoptosis, and invasiveness. These effects are mediated by suppressing the transcription of target genes that may be involved in cellular growth/transformation (e.g. p53) and adhesion (e.g. E-cadherin) (16, 23). Moreover, COX-2 and PGE2 promote cancer development and invasiveness by mediating the transcription of angiogenic factors that promote both the migration of endothelial cells and their arrangements into tubular structures (24, 25).
The present study was designed to investigate whether COX-2 expression and PGE2 synthesis are up-regulated in human squamous cell carcinomas and adenocarcinomas of the cervix. In addition, a possible autocrine/paracrine role for PGE2 in cervical carcinogenesis was assessed by investigating 1) the expression of EP2/EP4 receptors in cervical carcinoma tissue and 2) the effect of exogenous treatment of carcinoma tissue with PGE2 on cAMP turnover.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cervical specimens were obtained at the time of surgery/biopsy
from patients who were attending the Gynecologic Oncology Clinic at
Groote Schuur Hospital (Cape Town, South Africa) and who had previously
been diagnosed with invasive carcinoma of the cervix. Punch biopsies
were taken from the lesion by an experienced gynecologist with a
special interest in oncology. A portion of the biopsy was excised and
fixed in formalin, followed by paraffin wax embedding for
histopathological typing. The remaining portion was snap-frozen in
either dry ice or liquid nitrogen and stored at -70 C for RT-PCR and
Western blot analysis or was transported at 4 C for in vitro
culture and PGE2 stimulation. Histologically
normal cervical samples (N1–N25) were obtained from patients
undergoing Wertheim’s hysterectomy for nonmalignant conditions.
Pathological typing was defined according to the International
Federation of Obstetricians and Gynecologists (26) staging
upon physical examination. The extent of invasiveness and racial
distribution of carcinoma biopsies (C1–C50) are presented in Table 1
. The ages of the patients ranged from
29–81 yr, with a median age of 50.5 yr. The study was approved by the
University of Cape Town research ethics committee, and informed consent
was obtained from all patients before tissue collection. The data in
this study were analyzed by ANOVA using StatView 5.0 (Abacus Concepts,
Berkeley, CA).
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Real-time quantitative RT-PCR was performed to assess COX-2, EP2, and EP4 expression. Ribonucleic acid (RNA) samples were extracted from cervical tissue (squamous cell carcinomas, C1–C8 and C31–C37; adenocarcinomas, C9, C10, and C38; normal cervix, N1–N5 and N11–N15) using Tri-Reagent (Sigma, Dorset, UK) according to the manufacturer’s protocol. RNA samples were reverse transcribed using MgCl2 (5.5 mmol/L), deoxy (d)-NTPs (0.5 mmol/L each), random hexamers (1.25 µmol/L), oligo(deoxythymidine) (1.25 µmol/L), ribonuclease inhibitor (0.4 U/µL), and multiscribe reverse transcriptase (1.25 U/µL; all from PE Applied Biosystems, Warrington, UK). The mix was aliquoted into individual tubes (16 µL/tube) and template RNA was added (4 µL/tube of 100 ng/µL RNA). Samples were incubated for 60 min at 25 C, 45 min at 48 C and then at 95 C for 5 min. A reaction mix was made containing Taqman buffer (5.5 mM MgCl2, 200 µM dATP, 200 µM dCTP, 200 µM dGTP, 400 µM dUTP), ribosomal 18S forward and reverse primers and probe (all at 50 nM), forward and reverse primers for COX-2, EP2 or EP4 receptor (300 nM), COX-2, EP2 or EP4 receptor probe (200 nM), AmpErase UNG (0.01 U/µL) and AmpliTaq Gold DNA Polymerase (0.025 U/µL; all from PE Biosystems). A volume of 48 µL of reaction mix was aliquoted into separate tubes for each complementary DNA sample and 2 µL/replicate of complementary DNA was added. After mixing 23 µL of sample were added to the wells on a PCR plate. Each sample was added in duplicate. A no template control (containing water) was included in triplicate. Wells were sealed with optical caps, and the PCR reaction was run on an ABI Prism 7700 using standard conditions. COX-2 and EP receptor primers and probe for quantitative PCR were designed using the PRIMER express program (PE Applied Biosystems). The sequences of the COX-2 primers and probe were as follows: forward, 5'-CCT TCC TCC TGT GCC TGA TG-3'; reverse, 5'-ACA ATC TCA TTT GAA TCA GGA AGC T-3'; and probe (FAM labeled), 5'-TGC CCG ACT CCC TTG GGT GTC A-3'. The sequences of the EP2 receptor primers and probe were as follows: forward, 5'-GAC CGC TTA CCT GCA GCT GTA C-3'; reverse, 5'-TGA AGT TGC AGG CGA GCA-3'; and probe (FAM labeled), 5'-CCA CCC TGC TGC TGC TTC TCA TTG TCT-3'. The sequences of the EP4 receptor primers and probe were as follows: forward: 5'-ACG CCG CCT ACT CCT ACA TG-3'; reverse, 5'-AGA GGA CGG TGG CGA GAA T-3'; and probe (FAM labeled), 5'-ACG CGG GCT TCA GCT CCT TCC T-3'. The ribosomal 18S primers and probe sequences were as follows: forward, 5'-CGG CTA CCA CAT CCA AGG AA-3'; reverse, 5'-GCT GGA ATT ACC GCG GCT-3'; and probe (VIC labeled), 5'-TGC TGG CAC CAG ACT TGC CCT C-3'. Expression of COX-2, EP2, and EP4 was normalized to RNA loading for each sample using the 18S ribosomal RNA as an internal standard. Relative gene expression in carcinoma tissue compared with normal cervix was calculated by dividing the expression in carcinoma tissue by the expression in normal cervix. The data are presented as the mean ± SEM.
Western blotting
COX-2 protein expression was assessed by Western blotting. Proteins were extracted from cervical tissue (squamous cell carcinomas, C1–C8; adenocarcinomas, C9 and C10; normal cervix, N1–N5) using Tri-Reagent (Sigma, St. Louis, MO) following the manufacturer’s instructions. A total of 100 µg protein were resuspended in 38 µL sample buffer [125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 5% 2-mercaptoethanol, 20% glycerol, and 0.05% bromophenol blue], boiled for 5 min at 95 C, and run on a 10% SDS-polyacrylamide gel. Proteins were transferred onto polyvinylidene difluoride membrane (Millipore Corp., Watford, UK) and subjected to immunoblot analysis. Membranes were blocked for 1 h at 25 C in 5% skimmed milk powder diluted in washing buffer [50 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.05% (vol/vol) Tween-20]. Thereafter, membranes were incubated with goat anti-COX-2 primary IgG antibody (sc-1745, Autogenbioclear, Wiltshire, UK) at a dilution of 1:500 at 4 C for 18 h. Control samples were incubated with goat anti-COX-2 antibody preadsorbed to blocking peptide (sc-1745p, Autogenbioclear) according to the manufacturer’s protocol. Membranes were subsequently incubated for 1 h, respectively, with rabbit antigoat secondary IgG antibody conjugated to biotin (DAKO Corp., High Wycombe, UK; 1:500) and streptavidin-biotin-horseradish peroxidase complex (Amersham Pharmacia Biotech, Aylesbury, UK). Proteins were revealed by chemiluminescence (ECL Plus kit, Amersham Pharmacia Biotech) following the manufacturer’s instructions. The molecular mass of the COX-2 protein was approximately 72 kDa, as determined from the relative mobility on SDS-PAGE compared with the molecular mass standard.
Immunohistochemistry
The site of COX-2 expression and PGE2 synthesis was localized in cervical tissues by immunohistochemistry using archival cervical blocks (squamous cell carcinomas, C11–C20; adenocarcinomas, C21–C30; normal cervix, N5–N10) obtained from the Department of Anatomical Pathology, University of Cape Town (Cape Town, South Africa). Five-micron paraffin wax-embedded tissue sections were cut and mounted onto coated slides (TESPA, Sigma). Sections were dewaxed in xylene, rehydrated in graded ethanol, and washed in water followed by TBS (50 mM Tris-HCl and 150 mM NaCl, pH 7.4) and blocked for endogenous endoperoxidase (1% H2O2 in methanol). Antigen retrieval was performed by pressure cooking for 2 min in 0.01 mol/L sodium citrate, pH 6 (for COX-2 and PGE2). No antigen retrieval was performed for CD34 immunohistochemistry. Sections were blocked using 5% normal rabbit serum (for COX-2), 5% swine serum (for PGE2), or 5% normal goat serum (for CD34) diluted in TBS. Subsequently, the tissue sections were incubated with polyclonal goat anti-COX-2 antibody (sc-1745, Autogenbioclear) at a dilution of 1:400, rabbit anti-PGE2 antibody (supplied by Prof. R. W. Kelly, Medical Research Council Human Reproductive Sciences Unit, Edinburgh, UK) at a dilution of 1:100, or monoclonal mouse antihuman CD34 primary antibody (mca-547, Serotec, Oxford, UK) at a dilution of 1:25 at 4 C for 18 h. The rabbit antiserum that was raised against PGE2-complexed keyhole limpet hemocyanin has been previously characterized (27). Control tissue was incubated with 5% antisera (for CD34) or goat anti-COX-2 antibody preadsorbed to blocking peptide (sc-1745p, Autogenbioclear) according to the manufacturer’s protocol. Control tissue for PGE2 was incubated with rabbit anti-PGE2 antibody preadsorbed to excess exogenous PGE2. Briefly, the PGE2 antibody was incubated together with a 10-fold excess of exogenous PGE2 (Sigma) at 37 C for 2 h. Thereafter, the antibody-ligand mixture was diluted, and immunohistochemistry was performed as described above. After thorough washing with TBS, the tissue sections probed with the goat antihuman COX-2 and rabbit anti-PGE2 primary antibodies were incubated with biotinylated rabbit antigoat secondary IgG antibody (for COX-2; DAKO Corp.) or swine antirabbit secondary IgG antibody (for PGE2; DAKO Corp.) at a dilution of 1:500 at 25 C for 40 min. Thereafter, the tissue sections were incubated with streptavidin-biotin peroxidase complex (DAKO Corp.) at 25 C for 20 min. Tissue sections probed with the mouse antihuman CD34 antibody were developed using a mouse EnVision Kit (DAKO Corp.) according to the manufacturer’s instructions. Color reaction was developed by incubation with 3,3'-diaminobenzidine (DAKO Corp.). The tissue sections were counterstained in aqueous hematoxylin, followed by sequential dehydration using graded ethanol and xylene, before mounting and coverslipping.
PGE2 stimulation and cAMP measurement
Determination of basal cAMP levels in cervical tissues.
Initially, basal cAMP levels were measured in cervical tissue (squamous
cell carcinomas, C39–C44; normal cervix, N16–N20; Fig. 5A). Carcinoma
and normal cervical tissues were obtained on the day of surgery/biopsy,
sectioned finely, and divided equally into three aliquots. The tissue
was transported at 4 C and then incubated in 35-mm tissue culture
dishes containing 2 mL DMEM (Sigma), 10% FCS, 0.3 mg/mL
L-glutamine, 100 IU penicillin, and 100 µg streptomycin
for 1.5 h. One aliquot of tissue was snap-frozen to determine the
basal cAMP concentration in the tissue at the time of collection. The
other two aliquots were incubated overnight at 37 C in humidified 5%
CO2 in the presence or absence of 3 µg/mL
indomethacin (a dual COX enzyme inhibitor). Subsequently, tissue
sections were harvested by centrifugation at 2000 x g.
The supernatant was discarded, and the tissue was homogenized in 0.1
mol/L HCl. The cAMP concentration was quantified by ELISA using a cAMP
kit (Biomol, Affiniti, Exeter, UK) according to the manufacturer’s
protocol and normalized to the protein concentration of the homogenate.
Protein concentrations were determined using protein assay kits
(Bio-Rad Laboratories, Inc., Hemel Hempstead, UK).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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)
and Western blot analysis (Fig. 1B). Expression of COX-2 was
significantly up-regulated in all cases of squamous cell carcinoma and
adenocarcinoma investigated. COX-2 expression as assessed by
quantitative RT-PCR was 150.8 ± 43.18-fold greater in
cervical carcinoma tissues than in normal cervical tissue
(P < 0.05). Western blot analysis on these cervical
carcinomas revealed immunoreactive bands of approximately 72 kDa.
Minimal levels of COX-2 transcript was detected in normal cervical
tissue by quantitative RT-PCR, and no COX-2 protein was detected in any
of the normal cervical samples. Preadsorbing the primary antibody with
the blocking peptide abolished the COX-2 signal in the carcinoma
samples, thus confirming the specificity of detection of the 72-kDa
COX-2 protein in the carcinoma samples.
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, A and
B, respectively), and to neoplastically transformed columnar
epithelium lining the endocervical canal and the glandular epithelium
of the endocervical glands in adenocarcinomas (Fig. 2, C and D,
respectively). In addition, COX-2 and PGE2
immunostaining was observed in endothelial cells lining the vasculature
in all squamous cell carcinoma and adenocarcinoma sections investigated
(Fig. 3, A and B). To confirm that COX-2
expression and PGE2 synthesis were localized to
the endothelial cells of blood vessels, immunohistochemistry was
performed on tissue sections using antibodies raised against the CD34
endothelial cell marker. The pattern of expression with CD34 was
identical to that observed with COX-2 and PGE2,
thus confirming that COX-2 expression and PGE2
synthesis are localized to the endothelial cell layer of blood vessels
in human cervical carcinoma (Fig. 3C). Negligible staining was observed
in the stromal compartment of all carcinoma tissue investigated.
Moreover, little or no staining for COX-2 and
PGE2 was observed in the normal cervical tissues
(Fig. 2, E and F, respectively). Preadsorbing the antibody with the
blocking peptide (COX-2-negative control) or incubating sections with
PGE2 preincubated with exogenous
PGE2 ligand (PGE2-negative
control) abolished the COX-2 and PGE2 signals in
all carcinoma samples (insets in figures show negative
controls for COX-2 and PGE2 performed on serial
sections). No CD34 staining was observed in sections incubated with
nonimmune serum in place of primary antibody (Fig. 3D).
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). Expression of both receptors was
significantly up-regulated in all carcinoma tissues compared with that
in normal cervix (P < 0.01). The relative expressions
of EP2 and EP4 receptor in carcinoma tissue were 14.5 ± 3.2- and
106 ± 25.8-fold (respectively) greater than that in normal
cervix. To assess the activity of the EP2/EP4 receptors in the cervical
tissue, basal levels of cAMP were determined at the time of tissue
collection and after overnight incubation in the absence or presence of
3 µg/mL indomethacin (Fig. 5A
). The
cAMP concentration immediately after tissue excision was significantly
higher in carcinoma compared with normal cervix (77.9 ± 30.9
vs. 32.5 ± 8.7 pmol cAMP/mg protein; P
< 0.05). cAMP concentrations in carcinoma tissue after overnight
incubation in the absence of indomethacin was similar to that detected
in the tissue at the time of excision (64.2 ± 5.1 pmol cAMP/mg
protein), but was significantly reduced when the tissue was cultured in
the presence of indomethacin (2.59 ± 0.64 pmol cAMP/mg protein).
In normal cervical tissue, levels of cAMP were significantly reduced
after overnight incubation in the absence or presence of indomethacin
(11.96 ± 1.35 and 4.0 ± 0.7 pmol cAMP/mg protein,
respectively; P < 0.05). Subsequently, we determined
the effect of exogenous PGE2 and forskolin
treatment on cAMP production in carcinoma and normal cervical tissues
(Fig. 5B). Stimulation of cervical carcinoma tissue with 300 nmol/L
PGE2 or 50 µmol/L forskolin (positive control)
yielded a greater cAMP response than in normal cervical tissue treated
in the same manner. Overall, the inductions of cAMP generation after
PGE2 and forskolin stimulation were 51.1 ±
12.3- and 55.3 ± 15.84-fold, respectively, in cancer tissue and
5.8 ± 1.68- and 9.18 ± 1.59-fold, respectively, in normal
cervix (P < 0.01).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PGE2 acts on target cells through interaction
with G protein-coupled receptors. To date, several of these receptors
have been cloned (termed EP1–EP4) that use alternate intracellular
signaling pathways (14). In this study we investigated a
possible autocrine/paracrine role for synthesized
PGE2 in neoplastic cervical carcinoma tissue. For
this we assessed the expression and functionality of two subtypes of
PGE2 receptors, namely EP2 and EP4, which mediate
their effects on target cells via the protein kinase A pathway by
activating adenylate cyclase and increasing intracellular cAMP levels
via Gs
(13). In vitro
studies have suggested that cAMP is the primary secondary messenger in
regulating COX activity, as cAMP activity accompanies a concomitant
increase in COX activity (9). The data presented in this
study confirm up-regulation of expression of EP2 and EP4 receptors
compared with normal cervical tissue. This is associated with elevated
basal cAMP concentrations in carcinoma tissue compared with normal
cervix. Treatment of cervical tissue with the COX enzyme inhibitor
indomethacin significantly reduced the cAMP concentration. This
suggests that the elevated basal cAMP concentration in the carcinoma
tissue is mediated by COX enzyme products. Moreover, treatment of
cervical carcinoma tissue with exogenous PGE2 or
forskolin after overnight incubation with the COX enzyme inhibitor
indomethacin resulted in a rapid cAMP response that was greater in
carcinoma tissue than in normal cervical tissue. Taken together, these
data confirm that PGE2 synthesized in cervical
carcinoma tissue mediates an autocrine/paracrine effect via interaction
with EP2/EP4 receptors. It is possible that other receptor subtypes may
also be associated with PGE2 function in the
cervical carcinoma tissue. Due to limitations in the sizes of the
biopsies obtained at surgery, it was not possible to investigate other
intracellular signaling pathways that may be associated with
PGE2 function in cervical cancers
(14).
COX-2 inhibitors exhibit dramatic antineoplastic activity in a number of tumor model systems investigated to date, including colon cancer cells implanted into nude mice, tumor production in APC mutant mice, and carcinogen-induced tumors in rats (30, 31, 32). This is mediated partially by reducing PGE2 synthesis in the COX-2-overexpressing cells, which, in turn, down-regulates the survival, metastatic, and angiogenic potentials of the cancerous tissue (23, 24, 29). This has prompted the suggestion that the inhibition of PGE2 secretion by the application of COX-2 inhibitors may have an effect on growth and invasiveness of various carcinomas (24, 25, 29, 30). Such treatments may also be of benefit in regulating the growth of cervical carcinoma. Treatment of cervical carcinoma with NSAIDs will suppress endogenous expression of COX-2 and synthesis of PGE2, which may act in an autocrine/paracrine manner via the EP2/EP4 receptors. However, it is important to emphasize that in sexually active women the use of selective COX-2 inhibitors may be of partial therapeutic benefit. In these women, the growth and invasiveness of neoplastic cells may be under the direct influence of PGE2 present in seminal plasma. The PG concentration in seminal plasma is 10,000 times higher than that at the site of inflammation, and PGE is the predominant type of PG detected (33). Future studies to elucidate the relative contributions of endogenous and seminal plasma PGs on the phenotypic behavior of neoplastically transformed cervical epithelial and endothelial cells may assist in implementing improved therapy for women with cervical carcinomas.
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Acknowledgments |
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Received August 29, 2000.
Revised December 28, 2000.
Accepted January 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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716 knockout mice by
inhibition of cyclooxygenase 2 (COX-2). Cell. 87:803–809.[CrossRef][Medline]
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 T. Schmitz, M.J. Leroy, E. Dallot, M. Breuiller-Fouche, F. Ferre, and D. Cabrol Interleukin-1{beta} induces glycosaminoglycan synthesis via the prostaglandin E2 pathway in cultured human cervical fibroblasts Mol. Hum. Reprod., January 1, 2003; 9(1): 1 - 8. [Abstract] [Full Text] [PDF]
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K. J. Sales, A. A. Katz, R. P. Millar, and H. N. Jabbour Seminal plasma activates cyclooxygenase-2 and prostaglandin E2 receptor expression and signalling in cervical adenocarcinoma cells Mol. Hum. Reprod., December 1, 2002; 8(12): 1065 - 1070. [Abstract] [Full Text] [PDF]
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 R. L. Konger, G. A. Scott, Y. Landt, J. H. Ladenson, and A. P. Pentland Loss of the EP2 Prostaglandin E2 Receptor in Immortalized Human Keratinocytes Results in Increased Invasiveness and Decreased Paxillin Expression Am. J. Pathol., December 1, 2002; 161(6): 2065 - 2078. [Abstract] [Full Text] [PDF]
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 J. M. Wallace Nutritional and Botanical Modulation of the Inflammatory Cascade--Eicosanoids, Cyclooxygenases, and Lipoxygenases-- As an Adjunct in Cancer Therapy Integr Cancer Ther, March 1, 2002; 1(1): 7 - 37. [Abstract] [PDF]
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K. J. Sales, A. A. Katz, B. Howard, R. P. Soeters, R. P. Millar, and H. N. Jabbour Cyclooxygenase-1 Is Up-Regulated in Cervical Carcinomas: Autocrine/Paracrine Regulation of Cyclooxygenase-2, Prostaglandin E Receptors, and Angiogenic Factors by Cyclooxygenase-1 Cancer Res., January 1, 2002; 62(2): 424 - 432. [Abstract] [Full Text] [PDF]
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 K. Yoshimatsu, D. Golijanin, P. B. Paty, R. A. Soslow, P.-J. Jakobsson, R. A. DeLellis, K. Subbaramaiah, and A. J. Dannenberg Inducible Microsomal Prostaglandin E Synthase Is Overexpressed in Colorectal Adenomas and Cancer Clin. Cancer Res., December 1, 2001; 7(12): 3971 - 3976. [Abstract] [Full Text] [PDF]
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)
or 50 µmol/L forskolin (
; positive control) or left unstimulated
(
).
