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Immunohistochemical Analysis of Cyclooxygenase-2 Expression in Pancreatic Tumors

Takatomo Koshiba,* Ryo Hosotani, Yoshiharu Miyamoto, Michihiko Wada, Jeon.Uk Lee, Koji Fujimoto, Shoichiro Tsuji,

Sanae Nakajima, Ryuichiro Dot, and Masayuki Imamura I)epartment o1 Surgery and Surt~ical Basic Science, Kvoto I/niver.~ity. Kvoto 606-4897, Japan

Summary

Background. A considerable amount of evidence collected from several experimental systems and clin- ical slttdies with nonstcroidal Anti-inllammatory drugs (NSAIDs) indicates that Cox-2 may play a major role in colorcctal tumorigenesis, but little information about Cox-2 expression in pancreatic tumors is avail- able. In this study, we inve',tigatcd Cox-2 expression by means of both immunohistochernical analysis and i|nn|unoblot analysis in pancreatic tumors.

Methods. Fifty invasive ductal adenocarcinomas and 26 intraductal papillary-mutinous tumors (IPMTs) were used for immunohistochcmical analysis, and five pancreatic cancer tissues and five pancrcatic cancer cell lines for immunoblot analysis.

Results. Cox-2 was expressed in 72r)f of the invw, ive ductal adcnocarcinomas, 31% of intraductal pap- illary-mutinous adenocarcinomas, and none of intradt|ctal papillary-|nucinous adenomas. The expression rate of Cox-2 in intraductal papillary-mutinous adenocarcinomas was significantly higher than that in intra- duccal papillary-mutinous adenomas, and that in inYasive ductal adcnocarcinomas was significantly higher than that in intraductal papillary-mutinous carcinomas. However, there was no significant tonclarion between Cox-2 expression and the prognosis and clinicopathological factors. Immunoblot analysis identified Cox- 2 in all of pancreatic cancer tissues and 60% of cell lines.

Conclusion, The biological role of cyciooxygenase-2 (Cox-2) in carcinoma cells should be investi- gated with reference to the cancer progression of the pancreas.

Key Words: Immunohistoche|nistry: inmmnohlol analysis: Cox-2; pancreatic cancer: intraductal papil- l a r y - n l u c h l o u s t tmlor .

Introduction

Epidemiological studies have shown a 40-50r reduction in mortali ty from colorectal carcinoma in individuals who are continuous users of aspirin or

R e c e i v e d S c p l e m h e r 29, 1998: R e v i s e d Apri l I. 1 9 9 9 Accepted June 16, 1999.

*Aulhor h~ whom all cnrrespondence and reprint requesls ~hould be addressed: I)eparlmenl olSur@er~, and Surgical Baxic Sciel~cc. K volo LJniversi~3,. 54 -Shogo in Kawara-cho, Sakyo, Kyoto 606-4~97, Japan. E-mail: uriri @ kuhpkyo to -u . ac . j p

69

other nonsteroidal anti- inflamatory drugs (NSAIDs) (1-3). Other studies have shown that NSA1Ds caused a remarkable reduction in size and number of tumors in patients with familial polyposis (4-6). It is well known that cyc looxygenase (Cox) is a key enzyme in the conversion of arachidonic acid to prostanoids. Recent ly , two i soforms of Cox have been iden- tiffed as Cox-I and Cox-2. Cox-1 is consti tutively expressed in a large number of cell types and tis- sues, whereas Cox-2 is induced in various cells by in f l ammatory media tors , such as cy tok ines and

70 Koshiba et aL

growth factors (7,8). Recent studies have demon- strated that overexpression of Cox-2 mRNA or pro- tein level increased in carcinomas and adenoma compared to paired normal mucosa in the gastro- in tes t ina l t ract (9-11). Other s tudies us ing azoxymethane-treated rat models provided evidence that Cox-2 mRNA and protein expression increased in colonic tumors and that NSAIDs reduced tumor occurrence and growth (12,13). Furthermore, one report demonstrated that Cox-2 gene knockouts and Cox-2 inhibitor reduced the number and size of the intestinal polyps in adenomatous polyposis colt (APC) gene knockout mice (14). This clinical and experimental evidence suggests that Cox-2 plays an important role in colon carcinogenesis.

A recent gene transfection study demonstrated that constitutive expression of Cox-2 in human colon car- cinoma cells increased the potential for metastasis through matrix metalloproteinase-2 (MMP-2) acti- vation and this potential and MMP-2 activation were reduced by sulindac sulfide (15). Since MMP-2 degrades the type 1V collagen, which is a main com- ponent of the basement membrane, it is thought to be one of the key molecules for cancer invasion and metastasis. Another study showed that a biologically aggressive and invasive breast cancer cell line, in comparison with another one, possessed a high con- stitutive level of Cox-2 expression (16). These find- ings suggest Cox-2 expression may be related to cancer invasion by colorectal carcinomas as well.

Prognosis for patients with pancreatic cancer, par- ticularly ductal cell adenocarcinoma, is the least favorable among solid tumors, probably reflecting its invasive characteristic of frequent metastasis, which it is still small. There have been no reports, however, on the expression of Cox-2 proteins in con- nection with the pancreatic cancer. In this study, we used immunohistochemical method and immunoblot analysis to investigate the expression of Cox-2 in pancreatic ductal cell adenocarcinomas. In addition to examining the expression of Cox-2 proteins in intraductal papillary-mucinous tumors (IPMTs), we also compared benign and malignant IPMTs.

Materials and Methods

Materials

Paraffin-Embedded Tissue Sections

Samples of tumor tissue containing 50 invasive ductal adenocarcinomas and 26 intraductal papil-

lary mucinous tumors were obtained from patients with primary pancreatic tumors who underwent resection surgery at the Department of Surgery and Surgical Basic Science, Kyoto University, between 1991 and 1996. Samples were fixed in 4% para- formaldehyde or 10% formalin and embedded in paraffin, and 4-~tm sections were cut and placed on silane-coated slides for immunohistochemical stud- ies. Histological identification of the pancreatic tumors yielded 50 invasive ductal adenocarcinomas, as well as 26 IPMTs, consisting of 10 adenomas and 16 adenocarcinomas. The clinicopathologic charac- teristics of the 50 patients with ductal adenocarci- nomas of the pancreas who were the subjects of this study are summarized in Table 1. There were 26 males and 24 females, with an age range from 41 to 79 yr (median age: 63.1). Approximately 3-mm sec- tions from each tumorous tissue and dissected lymph node were examined histologically to confirm the diagnosis and pTNM classification of International Union Against Cancer (UICC), and the cancer was staged accordingly. The histopathological examina- tion revealed a well-differentiated adenocarcinoma in 5 patients, moderately differentiated adenocarci- noma in 37, poorly differentiated adenocarcinoma in 4, and others in 3. Six patients were at UICC stage I, 9 at stage II, 18 at stage III, and 17 at stage IV.Seven patients underwent pylorus-preserving pancreato- duodenectomies, 24 patients pancreatoduodenec- tomies, 18 patients distal pancre-atectomies, and one patient total pancreatectomies.

Frozen Tissues Five pancreatic cancer tissues, diagnosed as inva-

sive ductal adenocarcinomas, were obtained at the time of surgery, and immediately frozen with liquid nitrogen and stored at -80~

Cell Lines Five human pancreatic cancer cell lines (CFPAC-

1, BxPC-3, HPAC, AsPC- 1, and MIAPaCa-2) were purchased from American Type Culture Collection. The monolayer culture was maintained in an appro- priate medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 ~tg/mL strep- t o m y c i n at 37~ in a humid a t m o s p h e r e of 5%/CO2/95% air.

Antibody and Positive Control Monoclonal mouse antihuman Cox-2 antibody

(Cayman Chemical, MI; clone No. CX229) was used for immunohistochemical analysis and immunoblot

International Journal of Pancreatology Volume 26, 1999

Expression of Cox-2 in Pancreatic Tumor

Table 1 Comparison Between Expression of Cox-2 Proteins and Clinicopathological Features

in Pancreatic Ductal Adenocarcinomas (n = 50)"

71

Cox-2 expression

Variables No. of patients Negative, n = 14 Positive, n = 36

Grade (UICC) h G1 5 1 4 G2 37 12 25 G3 4 0 4 G4 4 1 3

pT (UICC) T1 3 0 3 T2 13 4 9 T3 34 10 24

pN (UICC) NO 19 6 13 N1 31 8 23

pM (UICC] M0 35 11 24 MI 15 3 12

Stage (UICC) I 6 2 4 II 9 4 5 III 18 4 14 IV 17 4 13

MST (months)" 10.8 10.6

0.367

0.358

0.660

0.400

0.643

NS

a �9 �9 �9

p Values were obtained from the chl-square test (two-sided). b UICC TNM Classification of Malignant Tumors, 4th ed. 1987, Springer-Verlag, Berlin. ~'MST = median survival time: NS = no significant difference by Kaplan Meier curves together with a log-rank test.

analysis. This antibody has no cross reactivity with Cox- 1 (17). The prostagrandin H synthase 2 (ovine) electropboresis standard (Cayman Chemical, MI) was used as a positive control for immunoblot analysis.

Immunohistochemical Analysis

We performed immunohistochemical staining as described previously (18). Briefly, paraffin sections were dewaxed and pretreated in 0.01 M sodium cit- rate buffer (pH 6.0) for 20 min at 95~ to unmask tissue antigen. These sections were incubated with 1% hydrogen peroxide in methanol for 15 rain at room temperature to block endogenous peroxidase and then with phosphate-buffered saline (PBS) containing 5% normal goat serum for 30 rain at room temperature to block nonspecific reaction. Immunostaining was per- formed with antiCox-2 antibody (diluted 1:200) at

4~ overnight. The sections were incubated with goat ant imouse IgG biot inyla ted second antibodies (Dakopatts) diluted 1:300 for 1 h at room tem- perature and thereafter incubated in streptavidin- peroxidase complex for 30 min. The sections were developed with diaminobenzidine-tetra-hydrochlo- ride (0.03%) as the substrate for 5 min, counterstained with Mayer's hematoxylin, and mounted.

The intensity of positive cancer cells was graded on a scale of four grades: 0, no staining of cancer cells; 1, weak staining; 2, moderate staining; 3, strong staining. The percentage of staining cancer cells was also graded on a scale with four grades: 0, none; 1, 1-33%; 2, 34-66%; 3, 67-100%. The intensity rating was multiplied by the percent stain rating to obtain an overall score. The specimens with a score of more than 4 were regarded as positive, and those with a score of <3 as negative.

International Journal of Pancreatology Volume 26, 1999

72 Koshiba et al.

lmmunoblot Analysis

The extracted protein from pancreatic carcinoma tissue or cancer cell lines was subjected to immunoblot analysis, details of which have been described previously (19). Briefly, 100 ~tg aliquots taken from the total quantity of protein were size- fractionated to a single dimension by sodium dode- cyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (10% gels) and transblotted to 0.45 ~m polyvinylidene difluoride membrane (Bio-Rad, Richmond, CA) in a semidry electroblot apparatus (Bio-Rad). Blots were preblocked overnight at 4~ in a blocking solution (TBST buffer [Tris-buffered saline {TBS} {10 mAC Tris-HC1, 150 mM NaC1}, 0.05% Tween 20] containing 5% [w/v] dried nonfat milk, 0.05% sodium azide, 2% bovine serum albu- min, 1% normal goat serum, and 5% BlockAce [Yuki- jirushi Ltd., Sapporo, Japan]). After blocking, the blots were incubated for 1 h with anti-Cox-2 anti- body (diluted 1:400) at room temperature for 1 h, and then incubated for 1 h at room temperature with 0.2 ~tg/mL of alkaline phosphatase conjugated goat antimouse IgG (Pierce, Rockford, IL). Thereafter, color development was achieved by incubation for about 5 min with AP solution (l 00 mM Tris-C1 [pH 9.5], 100 mM NaC1, 5 mM MgC12, 0.03% nitroblue tetrazolium, and 0.017% 5-bromo-3-indolylphos- phate p-toluidine salt).

Statistical Analysis

The distribution of categorical data in pancreatic tumors and clinicopathologic characteristics was assessed by chi-square test. The Kaplan-Meier method was used to calculate survival curves, and the log-rank and generalized Wilcoxon test were per- formed to compare differences in survival rates among the patient groups. A value of p <0.05 was deemed significant.

Results

Immunohistochemical Analysis Figure 1 shows representative immunostainings

of pancreatic tumors. In the invasive ductal adeno- carcinoma and intraductal papillary-mucinous car- cinoma, the staining of Cox-2 protein was identified in the cytoplasm and and/or nuclear membrane of cancer cells (Fig. IA,B,D,E). To confirm the speci- ficity of the results, we exposed peptide-absorbed

Cox-2 antisera as the primary antibody to several tissue specimens of invasive ductal adenocarcinoma and intraductal papillary-mucinous carcinoma, but they showed no immunoreaction. The staining of Cox-2 protein was faint or not identifiable in intra-

International Journal of Puncreatology Volume 26, 1999

Expression o f Cox-2 in Pancreatic Tumor 73

Fig. 1. (starting on opposite page) Representative photomicrographs of immunostaining results for Cox-2 in pan- creatic tumors. (A) Invasive ductal adenocarcinoma, (B) high-power magnification of (A), (C) serial section of (A) stained with peptide-absorbed Cox-2 antisera, (D) papillary-mucinous carcinoma, (E) high-power magnification of (D), (F) serial section of (D) stained with peptide-absorbed Cox-2 antisera, (G) intraductal papillary-mucinous ade- noma. Immunostaining was performed with ABC technique. Counterstained with hematoxylin. Original magnification is x l00. High-power magnification is x400.

ductal papillary-mucinous adenoma (Fig. IF). The pattern of staining in the cancer tissues showed great variation between specimens. Some sections showed labeling of the vast majority of cancer cells, whereas others showed only small areas of the tumor. We also observed nontumorous lesions containing chronic pancreatitis and normal pancreas in pancreatic tumor tissues. Negative or weak staining for Cox-2 protein was observed in chronic pancreatitis lesions and neg- ative staining in normal pancreatic lesions (data not shown).

The results of the immunohistochemical analysis of Cox-2 in the 50 tissues of invasive ductal adeno-

carcinomas, 16 tissues of intraductal papillary-muci- nous adenocarcinomas, and 10 tissues of intraduc- tal papillary-mucinous adenomas are summarized in Table 2. For specimens with a positive staining score of more than 4, the immunopositive rate for invasive ductal adenocarcinomas was 72% (36/50), for intraductal papillary-mucinous carcinomas 31% (5/16), and for intraductal papillary-mucinous car- cinomas 0% (0/10). The immunopositive rate for invasive ductal adenocarcinomas was significantly higher than that for intraductal papillary-mucinous carcinomas (chi-square test; p = 0.037) and that for intraductal papillary-mucinous carcinomas was sig-

bzternational Journal of Pancreatology Volume 26, 1999

74 Koshiba et al.

Table 2 Cox-2 Expression in Invasive Ductal Adenocarcinomas and IPMT"

Cox-2 expression

Negative Positive P

Invasive ductal adenocarcinomas

IPMT (adenocarcinoma)

IPMT (adenoma)

14 36 (72%) - - 1

l l 5(18%) _

10 0 (0%) __A

0.04

0.018

"p Values were obtained from the chi-square test.

nificantly higher than that for intraductal papillary- mucinous adenomas (chi-square test; p = 0.018).

Table I summarizes the relationship between Cox- 2 expression and clinicopathological features of 50 invasive ductal adenocarcinoma. There was no sig- nificant correlation between the expression of Cox- 2 protein and the cl inicopathological variables examined, that is, grade, pT, pN, pM, and UICC stage. The survival rates for the 28 patients with Cox- 2 protein-positive tumors and the 22 with Cox-2 pro- tein-negative tumors were not significantly different.

Immunoblot Analysis

Immunoblot analysis of tissue lysates identified the spec i ficity of the antibodies for detection of Cox - 2 protein (Fig. 2). The Cox-2 protein was detected in all tissue specimens (5/5) as a single band corre- sponding to a molecular size of 72 kDa, which is compatible with the molecular size of Cox-2 (Fig. 2A). Cell lysates were used for immunoblot analy- sis to confirm the existence of the Cox-2 proteins in pancreatic cancer cells (Fig. 2B). Cox-2 protein was detected at 72 kDa in 60% (3/5) of the examined cell line specimens.

Discussion

In this study, we used immunohistochemical meth- ods to examine Cox-2 expression in pancreatic tumors. Immunoreactive Cox-2 was found at the cytoplasm and/or perinucleus of invasive ductal adenocarcinoma cells and intraductal papillary- mucinous adenocarcinoma cells. Although Cox-2 staining in pancreatic carcinoma tissue was hetero- geneous and showed great variation between spec- imens, it was found mainly in carcinoma cells. These

A M 2 3 4 5 6

103kDa ~ ~ : : i : ~i!:::::' ~ i ~

B M 1 2 3 4 5 6

103kDa :, : �9 " ~: ":: ~ :~

Fig. 2. Immunoblot analysis of Cox-2 proteins in five invasive ductal adenocarcinomas and in five pancreatic cancer cell lines (100 gg total protein/lane). (A) M: mol- wt marker, Lane 1 : positive control, Lane 2-Lane 6: inva- sive ductal adenocarcinoma. (B) M: mol-wt marker, Lane 1: positive control, Lane 2: BxPC-3, lane 3: CFPAC-1, lane 4: HPAC, lane 5: AsPC-I, lane 6: MIAPaCa-2. The prostagrandin H synthase 2 (ovine) electrophoresis stan- dard was used as positive control

results were in agreement with those reported by Ristm~iski et al., who demonstrated that Cox-2 stain- ing was cytoplasmic in gastric carcinoma cells, but not in the surrounding stroma (11). Immunoblot analysis of cancer tissues and cancer cell lines was

International Journal of Pancreatology Volume 26. 1999

Expression of Cox-2 in Pancreatic Tumor 75

performed in order to verify the specificity of the antibodies for detection of Cox-2 proteins. Major bands, except for the band that is compatible with the molecular size of Cox-2, were undetectable in specimens for pancreatic cancer tissues. The results confirmed that the protein recognized by the anti- body used for immunostaining was Cox-2, and this antibody has little crossreactivity for other proteins. Immunoblotting of the purified pancreatic cancer cell lines indicated that the degree of intensity of the band varied among cell lines. The expression rate of Cox-2 in invasive ductal adenocarcinoma was con- siderably high at 72%. IPMT of the pancreas is a rel- atively new and increasingly reported entity. In 1982, Ohhashi et al. first described "mucous secreting pan- creatic cancer," defined as diffuse dilatation of the main pancreatic duct with filling defects identified on endoscopic retrograde cholangiopancreatography (ERCP) and extrusion of mucin through a bulging papilla of Vater (20). On the basis of the new World Health Organization (WHO) classification of pan- creatic neoplasm, we divided IPMT into benign and malignant in this study; borderline tumors with dys- plasia were classified as adenoma. It is also known that intraductal papillary-mucinous carcinomas rep- resent relatively poor invasiveness to surrounding tis- sues, whereas invasive ductal adenocarcinomas often represent strong invasiveness. The current immuno- histochemical study demonstrated that the expres- sion rate of Cox-2 in intraductal papillary-mucinous adenocarcinomas (31%) was significantly higher than that in intraductal papillary-mucinous adenomas (0%) (Table 1 ). Takahashi et al. reported that indomethacin and other NSAIDs significantly reduced the devel- opment of buffalo orphan prototype (BOP)-induced pancreatic cancer when administered during the pos- tinitiation phase in golden hamsters (21). In the IPMT of the pancreas, the expression of Cox-2 may be asso- ciated with the process of adenoma-carcinoma sequence. Furthermore, we demonstrated that the expression rate of Cox-2 in invasive ductal adeno- carcinomas (72%) was significantly higher than that in intraductal papillary-mucinous carcinomas (31%). These findings suggest that Cox-2 expression may be associated with the degree of histological malig- nancy. However, the role of Cox-2 in the pathogen- esis of adenomas and their progression to adenocarcinomas needs to be further clarified.

Tsujii et al. reported that constitutive expression of Cox-2 in human colon carcinoma cells activated

pro-MMP-2 and promoted cancer cell invasion, whereas sulindac sulfide, one of the NSAIDs, inhib- ited MMP-2 activation and subsequent cancer cell invasion (15). Their results suggests that MMP-2 activation might be modulated by an intracellular Cox-2-mediated pathway. We have previously reported that MMP-2 activation in pancreatic carci- nomas was deeply involved in tumor invasion and metastasis; therefore, we hypothesized that Cox-2 expression might also be involved in invasion and metastasis by pancreatic carcinomas (19,21). How- ever, there was no significant correlation between Cox-2 expression and the prognosis of the patients and clinicopathological factors, such as pT (tumor extension to extrapancreatic tissues), pN (regional lymph node metastasis), pM (distant metastasis), and tumor staging (Table 1). Furthermore, there was no correlation between MMP-2 activation and Cox- 2 expression in the same pancreatic carcinoma tissue specimens (data not shown). Recent experimental studies on carcinoma cells demonstrated that over- expression and persistent expression of Cox-2 were involved in aggressive and metastatic phenotypes (15,16). Cox-2 expression may not be involved in the invasiveness and metastatic potential of pancre- atic ductal adenocarcinomas.

We have presented evidence of Cox-2 expression in human pancreatic adenocarcinomas. The biolog- ical role of this enzyme in carcinoma cells, particu- larly downstream signal transduction, should be investigated with reference to progression of cancer of the pancreas. Chemoprevention by means of NSAIDs on pancreatic carcinoma as well as col- orectal carcinoma also needs to be further clarified.

Acknowledgments

This study was supported in part by a grant from the Ministry of Education of Japan. Part of this study was presentated in the 8th meeting of the Interna- tional Association of Pancreatology.

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