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Cyclooxygenase-2 inhibitors suppress angiogenesis and growth of gastric cancer xenografts
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Cyclooxygenase-2 inhibitors suppress angiogenesis and growth of gastric cancer xenografts Y.-L. Wu, S.-L. Fu *, Y.-P. Zhang, M.-M. Qiao, Y. Chen Department of Gastroenterology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
Abstract Many researches have confirmed the tumor-prophylactic effects of cyclooxygenase-2 (COX-2) inhibitors. We previously observed their anti-cancer effects in vivo in nude mice and found that sulindac, a traditional non-steroidal anti-inflammatory drug (NSAID), and celecoxib, a selective COX-2 inhibitor, depressed the growth of SGC7901 xenografts via altering cell kinetics. Then we deeply studied the relationship between the two drugs and angiogenesis in gastric cancer. The results showed both sulindac and celecoxib decreased the micro-vessel density (MVD), which was labeled by either CD34 or VWF staining, within xenografts. Expression of both vascular endothelial growth factor (VEGF) and FGF-1 was suppressed. In addition, a positive correlation between MVD and the volume of SGC7901 xenografts was found. The effect of selective COX-2 inhibitor was stronger than non-special one despite of the insignificant difference. These results demonstrate that COX-2 plays an important role in angiogenesis of cancer. Apart from interfering cell kinetics, decreasing the expression of angiogenic factors and then inhibiting tumor angiogenesis could also be one of the mechanisms that COX-2 inhibitors suppress the growth of gastric cancer. These findings offer another theory basis for the future clinical application of NSAIDs against cancer. © 2005 Elsevier SAS. All rights reserved. Keywords: Cyclooxygenase-2; Gastric cancer; Angiogenesis
1. Introduction Gastric cancer is one of the commonest malignancies of human being. A recent research [14] showed an increasing trend of gastric cancer mortality in China in the past 20 years, especially in rural areas and among aged people. The worldadjusted mortality rate of gastric cancer ranks first in both male and female. There is important significance to find out certain drugs to prevent it or to at least lower its incidence. Non-steroidal anti-inflammatory drugs (NSAIDs) have been put into clinical application for more than 100 years and there have been studies about their anti-cancer effects in rodent animals since two decades ago [10]. Several epidemiologic studies also showed NSAIDs' prophylactic effects on colorectal cancer [6]: Waddell et al. [18] began to administrate sulindac to Gardner's patients to treat colon polyposis and gained curative effect in 1983. Since cyclooxygenase-2, a newly-discovered COX isoenzyme catalyzing the production of prostaglandins, was dis-
* Corresponding author. E-mail address: [email protected] (S.-L. Fu). © 2005 Elsevier SAS. All rights reserved.
covered in early 1990s [21], its correlation with neoplasm has been widely studied. It has been accepted that NSAIDs play an anti-cancer role by inhibiting COX-2 activity. But traditional NSAIDs unselectively inhibit COX-1 at the same time and are prone to cause serious complications such as gastric ulcer, perforation and so on. However, such fatal complications can be avoided by using newly-invented selective COX-2 inhibitors instead of non-selective ones. SGC7901, a human middle-differentiated gastric cancer cell line, has been proved to express COX-2 [19]. We have shown that sulindac can restrain its growth in vitro in our past studies [20]. Now this study goes further so as to observe the anti-cancer effects in vivo of COX-2 inhibitors and clarify their relationship with tumor angiogenesis. 2. Materials and methods
2.1. Animal experiment SGC7901 cells were dissociated, collected and suspended in PBS at a density of 5 x 107 cells per ml after amplified to a certain amount. Thirty male athymic mice (BALB/c nu/nu, 6-week-old, 17-20 g) were purchased from Shanghai Exper-
$290
Y.-L. Wu et al. / Biomedicine & Pharmacotherapy 59 (2005) $289-$292
imental Animal Center of Chinese Academy of Sciences. They were maintained under specific pathogen-free conditions and fed with sterilized food and autoclaved water. Experiments started after 3 d of acclimatization. Each mouse was inoculated with a subcutaneous injection of SGC7901 cells 5 x 106 in 0.1 ml PBS. They were then randomized into control, sulindac, and celecoxib groups, respectively. From the same day, mice were orally administered different agents once a day: gum Arabic (50 mg/kg in body weight), sulindac (8 mg/kg, Sigma inc.), and celecoxib (10 mg/kg) [2]. Mice's diet, activity, stool, urine, and tumor growth were observed daily while shortest and longest diameters of xenografts were measured weekly. The tumor volume was deduced according to the following formula: volume (mm 3) = (the shortest diameter) 2 x (the longest diameter)/2. Both body weight and tumor size of each mouse were measured again just before they were killed by cervical dislocation on the 32nd day. All tumors were dissected and weighed, then divided along the longest diameter into two halves, one half frozen in liquid nitrogen and the other half fixed in 10% phosphate-buffered formalin. 2.2. Micro - vessel density assay
The formalin-fixed tissues were embedded in paraffin and sectioned successively at a thickness of 4 p.m. The sections were deparaffinized and hydrated for the detection of CD34 (BD) and VWF (Dako) expression by immunohistochemistry with EnVision kits (GeneTech Co.) Tests were performed according to the two-step procedure mentioned in the instrument brochure. Sections were incubated with primary antibodies VWF (1/500; Dako) and CD34 (1/100; BD) at 4 °C overnight and further treated with EnVision kits for 30 min at room temperature. Then they were visualized by diaminobenzidin (DAB) and counter-stained by hematoxylin. TBS took the place of primary antibodies and acted as a negative control. Sections were observed under microscope and results of staining were analyzed and evaluated with American Image-Pro Plus software. The average number of vessel with positive CD34 and VWF staining in three hot regions was calculated as MVD.
difluoride membrane. The membrane was incubated with human COX-I, COX-2, FGF-1, VEGF, and [3-actin (as a reference, Sigma) antibodies in blocking buffer at 4 °C overnight. The blots were further detected with Vectastain ABC kit (Vector Laboratorie, Inc.) and visualized with DAB/Ni substrate solution (Vector). The protein bands were exposured and photoed with Gel Documentation System (Bio-Rad), then quantified and analyzed with Quantity One software. Additionally, the mRNA level of VEGF was evaluated by RT-PCT assay. Total RNA was extracted from frozen tissue with TRIzol (Invitrogen) after homogenized and dissolved in DEPC water. 260/280 nm absorbance ratio of the sample was measured and concentration calculated by DU Series 500 spectrophotometer (Beckman). 3 ktg RNA from each sample was taken to be reversely transcribed into cDNA under the catalysis of M-MLV reverse transcriptase (Promega). PCR technique was then applied to amplify target gene with cDNA as template. The primer sequence of target gene ~-actin (as an inter-reference) and VEGF was listed as below. [3-actin (281 bp): 5'-GCC AGC TCA CCA TGG ATG AT-3' 5'-AGA AGG TGT GGT GCC AGA T-3' VEGF (200 bp): 5'-TGC A T r CAC ATI" TGT TGT GC-3' 5'-AGA CCC TGG TGG ACA TCT TC-3' Same volume of PCR products of two genes were synchronously loaded in 2% agarose gel for electrophoresis. The DNA bands were captured of image with Gel Documentation System and the gray scale ratio of VEGF/[3-actin was quantified and analyzed with Quantity One software. 2.4. Statistical analysis
Data were analyzed by software of SAS 6.12 and shown in a default form of mean __.S.D. Comparison among groups was performed using ANOVA test. 3. Results 3.1. Growth o f neoplasm
2.3. Expression o f COX-l, COX-2, FGF-1 and VEGF
The expression of COX-I, COX-2, FGF-1, and VEGF (Santa Cruz Biotech) was detected by immunohistochemical assay (see above) and Western blot. For Western blot analysis, the homogenate was immediately centrifugated and total proteins were extracted into 1 x SDS sample buffer after frozen tumor pieces were homogenized in 1.0 ml icecold PBS. The protein content was determined with BCA protein assay reagent kit (Pierce Chemical). Twenty micrograms of protein from every sample was mixed with 10 x loading buffer and boiled at 100 °C for 5 min The mixture was subjected to electrophoresis in 9% polyacrylamide gel and then electrophoretically transferred to polyvinylidene
During the experiment, the growth of xenografts in treatment groups was strikingly suppressed. Volume of tumors in treatment groups was significantly smaller than that in the controls from the second week to the end of experiment (P < 0.01) [8] while weight of tumors in sulindac (1.02 ___1.23) and celecoxib (0.54 _+0.84) groups was significantly lighter than that in control group (2.30 _+0.97; F = 27.95, P < 0.01) at the end of experiment, but no significant difference was shown between sulindac and celecoxib group in either volume or weight of tumors. Deeply stained tumor ceils with big nuclei were arranged tightly with no cavum structure seen under microscope, which coincided with the characteristics of histological manifestation of tumor (Tables 1 and 2).
E-L. Wu et al. / Biomedicine & Pharmacotherapy 59 (2005) $289-$292 3.4. Expression o f FGF-1 and VEGF
Table 1 Tumor volume of three groups (mean _+S.D.) control
sulindac
celecoxib
Volume of tumor (mm3)
2984.5-+589.3* 1589.7 -+ 1008.8"* 891.2 -+763.6
Vessels of VWF staining
7.7 -- 3.9*
Vessels of CD34 staining
4.5 _+2.8*
2.1 _+2.0**
1.8 _+1.4
MVD
5.6 -+3.0*
2.8 _+2.2**
2.5 _+2.3
4.0 + 2.4**
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Both FGF-1 and V E G F were expressed in cytoplasm of neoplasm. Their expression level were both suppressed by both drugs. V E G F m R N A level was also decreased in two treated groups.
3.5 - 2.8
4. Discussion and conclusion
* P < 0.01, vs. two drug groups; ** P > 0.05, vs. celecoxib group. Table 2 Tumor volume of three groups (mean + S.D.) control FGF-1 by staining
sulindac
34.5 _+19.8" 22.7_+16.6"*
celecoxib 19.4 ___13.5
FGF-1 by Western blot 26.0 _+13.4"
14.9_+7.4**
13.1 _+7.8
VEGF by staining
54.2 _ 25.8*
36.5 + 22.0**
31.8 _+21.6
VEGF by Western blot
176.5 _+78.3* 136.1 _+72.0** 130.8 _+61.4
VEGF mRNA
75.4 _+44.6*
50.5 _+27.3**
43.8 _+20.5
* P < 0.01, vs. two drug groups; ** P > 0.05, vs. celecoxib group.
3.2. Tumor angiogenesis Endothelial cells were stained brown by either CD34 or V W F staining. CD34 staining was mainly used to reveal bigger vessels which exhibit their cavum structures apparently under microscope while V W F staining were used to show tiny vessels and even single endothelial cell. Vessels visioned by V W F staining outnumbered those by CD34 staining of same specimen, but there was a correlation between the results given by the two methods (r = 0.82, P < 0.05). W e took the average value from V W F and CD34 staining as M V D to perform a comparison. The results revealed that M V D of two drug groups was both apparently lower than that of control group (P < 0.01). M V D is lower in celecoxib than in sulindac group with no significant statistical difference. W e also analyzed the correlation between M V D and tumor volume and found a positive correlation (r = 0.88, P < 0.01). 3.3. Expression o f COX-1 and COX-2 Regional distributed cytoplasmic pigmentation of tumor cells was revealed by immunohistochemical staining of COX-1. COX-2 was extensively expressed in nucleus and perinuclear area. Both COX-1 and COX-2 exhibited a sustained expression in Western blot. There was no significant difference of expression level of the two isoenzymes among all groups with both assays.
Although many researches showed prophylactic effect of N S A I D s in preventing carcinogenesis of gastrointestinal tract, one risks the lack of maneuverability in the clinical application in this field. Nevertheless N S A I D s have demonstrated their heartening therapeutic effects against tumor in both clinical trials [7] and animal experiments [2,8,11]. They suppress the growth of various xenografts by inducing apoptosis and disturbing cell kinetics. While involved mechanisms kept unclear, COX-2 is considered to play an important part in it. COX-2 and the prostaglandins catalyzed by it promote proliferation of tumor cell and inhibit their apoptosis [15,17]. In addition, many evidences have demonstrated the involvement of COX-2 in tumor angiogenesis [8,12,16]. Expression of COX-2 exists in gastric cancer and the positive rate could reach 61.4% [3]. Human moderately differentiated gastric cancer cell SGC7901 can be transplanted easily for neoplasia and has been used extensively in domestic studies. In our study SGC7901 xenografts were seen to have a stable expression of COX-2 and their growth was strikingly suppressed by orally administered N S A I D s sulindac and celecoxib. The volume and weight values of tumors were both significantly smaller than those in the controls. But besides influencing cell kinetics via inducing Bcl-2 gene expression [13], with what mechanism was COX-2 concerned in tumorigenesis? The growth o f tumor cells depends on nutrition supply, which largely relies on angiogenesis. Ischemia can induce tumor cell apoptosis, speeding up necrosis and cell extinction. Many researches have verified the relation between COX-2 and angiogenesis [5,9,12,16]. W e evaluated angiogenesis by using the combination of CD34 and V E G F staining to label endothelial cell, having observed that both sulindac and celecoxib decreased the level of M V D of xenografts obviously. Especially, there existed a positive correlation between M V D and tumor volume. It seemed that COX-2 inhibitors realized their anti-cancer effects by reducing angiogenesis in stomach carcinoma, thereby impairing the nutrition supply of the tumor, further inhibiting proliferation and inducing apoptosis of neoplasm cells. FGF-1 and V E G F are two important growth factors and played a pivotal role in tumor angiogenesis [1,4]. Neoplasms overexpressing COX-2 often express V E G F and FGF-1 at the same time [5]. W e observed their expression after N S A I D s treatment in SGC7901 xenografts and found the expression level was remarkably decreased when compared with control group. The m R N A level of V E G F was
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Y.-L. Wu et al. / Biomedicine & Pharmacotherapy 59 (2005) $289-$292
also declined. So we could speculate that NSAIDs inhibit COX-2 activity, thereby reducing expression of angiogenic factors and MVD and resulting in ischemia, which induces apoptosis and depresses the growth of tumor. Celecoxib is the first selective COX-2 inhibitor proved by FDA for clinical application. Its effects were stronger than sulindac in this study with no significant statistical difference shown. Our study offered new evidences for NSAIDs' therapeutic effects against tumor. However, further specially designed experiments are expected to be carfled out to demonstrate the potential advantages of selective COX-2 inhibitors in preventing tumorigenesis when compared with non-selective NSAIDs.
Acknowledgement Supported by a grant from the Natural Science Foundation of Shanghai Science and Technology Committee, No. 02ZB 14042.
[8]
[9]
[10]
[ 11]
[12]
[13]
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Affara NI, Robertson FM. Vascular endothelial growth factor as a survival factor in tumor-associated angiogenesis. In Vivo 2004; 18:525. Fu SL, Wu YL, Zhang YP, Qiao MM, Chen Y. Anti-cancer effects of COX-2 inhibitors and their correlation with angiogenesis and invasion in gastric cancer. World J Gastroenterol 2004; 13:1971. Joo YE, Oh WT, Rew JS, Park CS, Choi SK, Kim SJ. Cyclooxygenase-2 expression is associated with well-differentiated and intestinaltype pathways in gastric carcinogenesis. Digestion 2002;66:222. Kelpke SS, Zinn KR, Rue LW, Thompson JA. Site-specific delivery of acidic fibroblast growth factor stimulates angiogenic and osteogenic responses in vivo. J Biomed Mater Res 2004;71A:316. Koga T, Shibahara K, Kabashima A. Overexpression of cyclooxygenase-2 and tumor angiogenesis in human gastric cancer. Hepatogastroenterology 2004;51:1626. Kune GA, Kune S, Watson LF. Colorectal cancer risk, chronic illnesses, operations, and medications: case contral results from the Melbourne Colorectal Cancer Study. Cancer Res 1988;48:4399. Labayle D, Fischer D, Vielh P, Drouhin F, Pariente A, Bories C, Duhamel O, Trousset M, Attali P. Sulindac causes regression of rectal polyps in familial adenomatous polyposis. Gastroenterology 1991;101:635.
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Liu XH, Kirschenbaum A, Yao S, Lee R, Holland JF, Levine AC. Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vivo. J Uro12000; 164:820. Liu XH, Kirschenbaum A, Yao S, Steams ME, Holland JF, Claffey K, Levine AC. Upregulation of vascular endothelial growth factor by cobalt chloride-simulated hypoxia is mediated by persistent induction of cyclooxygenase-2 in a metastatic human prostate cancer cell line. Clin Exp Metastasis 1999; 17:687. Pollard M, Luckert PH. Effect of indomethacin on intestinal tumors induced in rats by the acetate derivative of dimethylnitrosamine. Science 1981;214:558. Sawaoka H, Kawano S, Tsuji S, Tsujii M, Gunawan ES, Takei Y, Nagano K, Hori M. Cyclooxygenase-2 inhibitors suppress the growth of gastric cancer xenografts via induction of apoptosis in nude mice. Am J Physiol 1998;274:G1061. Sawaoka H, Tsuji S, Tsujii M, Gunawan ES, Sasaki Y, Kawano S, Hori M. Cyclooxygenase inhibitors suppress angiogenesis and reduce tumor growth in vivo. Lab Invest 1999;79:1469. Sheng H, Shao J, Morrow JD, Beauchamp RD, DuBois RN. Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 1998;58:362. Sun XD, Mu R, Zhou YS, Dai XD, Zhang SW, Huangfu XM, Sun J, Li LD, Lu FZ, Qiao YL. [Analysis of mortality rate of stomach cancer and its trend in 20 years in China]. Zhonghua Zhong Liu Za Zhi 2004;1:4 [Chinese]. Tsujii M, DuBois RN. Alterations in cellular adhension and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell 1995;83:493. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell 1998;93:705. Tsuji S, Kawano S, Sawaoka H, Takei Y, Kobayashi I, Nagano K, Fusamoto H, Kamata T. Evidences for involvement of cyclooxygenase2 in proliferation of two gastrointestinal cancer cell lines. Prostaglandins Leukot Essent Fatty Acids 1996;55:179. WaddeU WR, Loughry RW. Sulindac for polyposis of the colon. J Surg Oncol 1983;24:83. Wu H, Wu K, Yao L, Xu M, Fan D. [Antisense inhibition of cyclooxygenase-2 reduces malignant phenotype of gastric cancer cells[. Zhonghua Nei Ke Za Zhi 2002;41:534 ]Chinese]. Wu YL, Sun B, Zhang XJ, Wang SN, He HY, Qiao MM, Zhong J, Xu JY. Growth inhibition and apoptosis induction of sulindac on human gastric cancer cells. World J Gastroenterol 2001;7:796. Xie WL, Chipman JG, Robertson DL, Erikson RL, Simmons DL. Expression of a mitogen-reponsive gene encoding prostaglandin synthase is regulated by mRNA splicing. Proc Natl Acad Sci USA 1991 ;88:2692.
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Biomedicine & Pharmacotherapy 59 (2005) $289-$292 http://france.elsevier.com/direct/BIOPHA/
Cyclooxygenase-2 inhibitors suppress angiogenesis and growth of gastric cancer xenografts Y.-L. Wu, S.-L. Fu *, Y.-P. Zhang, M.-M. Qiao, Y. Chen Department of Gastroenterology, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China
Abstract Many researches have confirmed the tumor-prophylactic effects of cyclooxygenase-2 (COX-2) inhibitors. We previously observed their anti-cancer effects in vivo in nude mice and found that sulindac, a traditional non-steroidal anti-inflammatory drug (NSAID), and celecoxib, a selective COX-2 inhibitor, depressed the growth of SGC7901 xenografts via altering cell kinetics. Then we deeply studied the relationship between the two drugs and angiogenesis in gastric cancer. The results showed both sulindac and celecoxib decreased the micro-vessel density (MVD), which was labeled by either CD34 or VWF staining, within xenografts. Expression of both vascular endothelial growth factor (VEGF) and FGF-1 was suppressed. In addition, a positive correlation between MVD and the volume of SGC7901 xenografts was found. The effect of selective COX-2 inhibitor was stronger than non-special one despite of the insignificant difference. These results demonstrate that COX-2 plays an important role in angiogenesis of cancer. Apart from interfering cell kinetics, decreasing the expression of angiogenic factors and then inhibiting tumor angiogenesis could also be one of the mechanisms that COX-2 inhibitors suppress the growth of gastric cancer. These findings offer another theory basis for the future clinical application of NSAIDs against cancer. © 2005 Elsevier SAS. All rights reserved. Keywords: Cyclooxygenase-2; Gastric cancer; Angiogenesis
1. Introduction Gastric cancer is one of the commonest malignancies of human being. A recent research [14] showed an increasing trend of gastric cancer mortality in China in the past 20 years, especially in rural areas and among aged people. The worldadjusted mortality rate of gastric cancer ranks first in both male and female. There is important significance to find out certain drugs to prevent it or to at least lower its incidence. Non-steroidal anti-inflammatory drugs (NSAIDs) have been put into clinical application for more than 100 years and there have been studies about their anti-cancer effects in rodent animals since two decades ago [10]. Several epidemiologic studies also showed NSAIDs' prophylactic effects on colorectal cancer [6]: Waddell et al. [18] began to administrate sulindac to Gardner's patients to treat colon polyposis and gained curative effect in 1983. Since cyclooxygenase-2, a newly-discovered COX isoenzyme catalyzing the production of prostaglandins, was dis-
* Corresponding author. E-mail address: [email protected] (S.-L. Fu). © 2005 Elsevier SAS. All rights reserved.
covered in early 1990s [21], its correlation with neoplasm has been widely studied. It has been accepted that NSAIDs play an anti-cancer role by inhibiting COX-2 activity. But traditional NSAIDs unselectively inhibit COX-1 at the same time and are prone to cause serious complications such as gastric ulcer, perforation and so on. However, such fatal complications can be avoided by using newly-invented selective COX-2 inhibitors instead of non-selective ones. SGC7901, a human middle-differentiated gastric cancer cell line, has been proved to express COX-2 [19]. We have shown that sulindac can restrain its growth in vitro in our past studies [20]. Now this study goes further so as to observe the anti-cancer effects in vivo of COX-2 inhibitors and clarify their relationship with tumor angiogenesis. 2. Materials and methods
2.1. Animal experiment SGC7901 cells were dissociated, collected and suspended in PBS at a density of 5 x 107 cells per ml after amplified to a certain amount. Thirty male athymic mice (BALB/c nu/nu, 6-week-old, 17-20 g) were purchased from Shanghai Exper-
$290
Y.-L. Wu et al. / Biomedicine & Pharmacotherapy 59 (2005) $289-$292
imental Animal Center of Chinese Academy of Sciences. They were maintained under specific pathogen-free conditions and fed with sterilized food and autoclaved water. Experiments started after 3 d of acclimatization. Each mouse was inoculated with a subcutaneous injection of SGC7901 cells 5 x 106 in 0.1 ml PBS. They were then randomized into control, sulindac, and celecoxib groups, respectively. From the same day, mice were orally administered different agents once a day: gum Arabic (50 mg/kg in body weight), sulindac (8 mg/kg, Sigma inc.), and celecoxib (10 mg/kg) [2]. Mice's diet, activity, stool, urine, and tumor growth were observed daily while shortest and longest diameters of xenografts were measured weekly. The tumor volume was deduced according to the following formula: volume (mm 3) = (the shortest diameter) 2 x (the longest diameter)/2. Both body weight and tumor size of each mouse were measured again just before they were killed by cervical dislocation on the 32nd day. All tumors were dissected and weighed, then divided along the longest diameter into two halves, one half frozen in liquid nitrogen and the other half fixed in 10% phosphate-buffered formalin. 2.2. Micro - vessel density assay
The formalin-fixed tissues were embedded in paraffin and sectioned successively at a thickness of 4 p.m. The sections were deparaffinized and hydrated for the detection of CD34 (BD) and VWF (Dako) expression by immunohistochemistry with EnVision kits (GeneTech Co.) Tests were performed according to the two-step procedure mentioned in the instrument brochure. Sections were incubated with primary antibodies VWF (1/500; Dako) and CD34 (1/100; BD) at 4 °C overnight and further treated with EnVision kits for 30 min at room temperature. Then they were visualized by diaminobenzidin (DAB) and counter-stained by hematoxylin. TBS took the place of primary antibodies and acted as a negative control. Sections were observed under microscope and results of staining were analyzed and evaluated with American Image-Pro Plus software. The average number of vessel with positive CD34 and VWF staining in three hot regions was calculated as MVD.
difluoride membrane. The membrane was incubated with human COX-I, COX-2, FGF-1, VEGF, and [3-actin (as a reference, Sigma) antibodies in blocking buffer at 4 °C overnight. The blots were further detected with Vectastain ABC kit (Vector Laboratorie, Inc.) and visualized with DAB/Ni substrate solution (Vector). The protein bands were exposured and photoed with Gel Documentation System (Bio-Rad), then quantified and analyzed with Quantity One software. Additionally, the mRNA level of VEGF was evaluated by RT-PCT assay. Total RNA was extracted from frozen tissue with TRIzol (Invitrogen) after homogenized and dissolved in DEPC water. 260/280 nm absorbance ratio of the sample was measured and concentration calculated by DU Series 500 spectrophotometer (Beckman). 3 ktg RNA from each sample was taken to be reversely transcribed into cDNA under the catalysis of M-MLV reverse transcriptase (Promega). PCR technique was then applied to amplify target gene with cDNA as template. The primer sequence of target gene ~-actin (as an inter-reference) and VEGF was listed as below. [3-actin (281 bp): 5'-GCC AGC TCA CCA TGG ATG AT-3' 5'-AGA AGG TGT GGT GCC AGA T-3' VEGF (200 bp): 5'-TGC A T r CAC ATI" TGT TGT GC-3' 5'-AGA CCC TGG TGG ACA TCT TC-3' Same volume of PCR products of two genes were synchronously loaded in 2% agarose gel for electrophoresis. The DNA bands were captured of image with Gel Documentation System and the gray scale ratio of VEGF/[3-actin was quantified and analyzed with Quantity One software. 2.4. Statistical analysis
Data were analyzed by software of SAS 6.12 and shown in a default form of mean __.S.D. Comparison among groups was performed using ANOVA test. 3. Results 3.1. Growth o f neoplasm
2.3. Expression o f COX-l, COX-2, FGF-1 and VEGF
The expression of COX-I, COX-2, FGF-1, and VEGF (Santa Cruz Biotech) was detected by immunohistochemical assay (see above) and Western blot. For Western blot analysis, the homogenate was immediately centrifugated and total proteins were extracted into 1 x SDS sample buffer after frozen tumor pieces were homogenized in 1.0 ml icecold PBS. The protein content was determined with BCA protein assay reagent kit (Pierce Chemical). Twenty micrograms of protein from every sample was mixed with 10 x loading buffer and boiled at 100 °C for 5 min The mixture was subjected to electrophoresis in 9% polyacrylamide gel and then electrophoretically transferred to polyvinylidene
During the experiment, the growth of xenografts in treatment groups was strikingly suppressed. Volume of tumors in treatment groups was significantly smaller than that in the controls from the second week to the end of experiment (P < 0.01) [8] while weight of tumors in sulindac (1.02 ___1.23) and celecoxib (0.54 _+0.84) groups was significantly lighter than that in control group (2.30 _+0.97; F = 27.95, P < 0.01) at the end of experiment, but no significant difference was shown between sulindac and celecoxib group in either volume or weight of tumors. Deeply stained tumor ceils with big nuclei were arranged tightly with no cavum structure seen under microscope, which coincided with the characteristics of histological manifestation of tumor (Tables 1 and 2).
E-L. Wu et al. / Biomedicine & Pharmacotherapy 59 (2005) $289-$292 3.4. Expression o f FGF-1 and VEGF
Table 1 Tumor volume of three groups (mean _+S.D.) control
sulindac
celecoxib
Volume of tumor (mm3)
2984.5-+589.3* 1589.7 -+ 1008.8"* 891.2 -+763.6
Vessels of VWF staining
7.7 -- 3.9*
Vessels of CD34 staining
4.5 _+2.8*
2.1 _+2.0**
1.8 _+1.4
MVD
5.6 -+3.0*
2.8 _+2.2**
2.5 _+2.3
4.0 + 2.4**
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Both FGF-1 and V E G F were expressed in cytoplasm of neoplasm. Their expression level were both suppressed by both drugs. V E G F m R N A level was also decreased in two treated groups.
3.5 - 2.8
4. Discussion and conclusion
* P < 0.01, vs. two drug groups; ** P > 0.05, vs. celecoxib group. Table 2 Tumor volume of three groups (mean + S.D.) control FGF-1 by staining
sulindac
34.5 _+19.8" 22.7_+16.6"*
celecoxib 19.4 ___13.5
FGF-1 by Western blot 26.0 _+13.4"
14.9_+7.4**
13.1 _+7.8
VEGF by staining
54.2 _ 25.8*
36.5 + 22.0**
31.8 _+21.6
VEGF by Western blot
176.5 _+78.3* 136.1 _+72.0** 130.8 _+61.4
VEGF mRNA
75.4 _+44.6*
50.5 _+27.3**
43.8 _+20.5
* P < 0.01, vs. two drug groups; ** P > 0.05, vs. celecoxib group.
3.2. Tumor angiogenesis Endothelial cells were stained brown by either CD34 or V W F staining. CD34 staining was mainly used to reveal bigger vessels which exhibit their cavum structures apparently under microscope while V W F staining were used to show tiny vessels and even single endothelial cell. Vessels visioned by V W F staining outnumbered those by CD34 staining of same specimen, but there was a correlation between the results given by the two methods (r = 0.82, P < 0.05). W e took the average value from V W F and CD34 staining as M V D to perform a comparison. The results revealed that M V D of two drug groups was both apparently lower than that of control group (P < 0.01). M V D is lower in celecoxib than in sulindac group with no significant statistical difference. W e also analyzed the correlation between M V D and tumor volume and found a positive correlation (r = 0.88, P < 0.01). 3.3. Expression o f COX-1 and COX-2 Regional distributed cytoplasmic pigmentation of tumor cells was revealed by immunohistochemical staining of COX-1. COX-2 was extensively expressed in nucleus and perinuclear area. Both COX-1 and COX-2 exhibited a sustained expression in Western blot. There was no significant difference of expression level of the two isoenzymes among all groups with both assays.
Although many researches showed prophylactic effect of N S A I D s in preventing carcinogenesis of gastrointestinal tract, one risks the lack of maneuverability in the clinical application in this field. Nevertheless N S A I D s have demonstrated their heartening therapeutic effects against tumor in both clinical trials [7] and animal experiments [2,8,11]. They suppress the growth of various xenografts by inducing apoptosis and disturbing cell kinetics. While involved mechanisms kept unclear, COX-2 is considered to play an important part in it. COX-2 and the prostaglandins catalyzed by it promote proliferation of tumor cell and inhibit their apoptosis [15,17]. In addition, many evidences have demonstrated the involvement of COX-2 in tumor angiogenesis [8,12,16]. Expression of COX-2 exists in gastric cancer and the positive rate could reach 61.4% [3]. Human moderately differentiated gastric cancer cell SGC7901 can be transplanted easily for neoplasia and has been used extensively in domestic studies. In our study SGC7901 xenografts were seen to have a stable expression of COX-2 and their growth was strikingly suppressed by orally administered N S A I D s sulindac and celecoxib. The volume and weight values of tumors were both significantly smaller than those in the controls. But besides influencing cell kinetics via inducing Bcl-2 gene expression [13], with what mechanism was COX-2 concerned in tumorigenesis? The growth o f tumor cells depends on nutrition supply, which largely relies on angiogenesis. Ischemia can induce tumor cell apoptosis, speeding up necrosis and cell extinction. Many researches have verified the relation between COX-2 and angiogenesis [5,9,12,16]. W e evaluated angiogenesis by using the combination of CD34 and V E G F staining to label endothelial cell, having observed that both sulindac and celecoxib decreased the level of M V D of xenografts obviously. Especially, there existed a positive correlation between M V D and tumor volume. It seemed that COX-2 inhibitors realized their anti-cancer effects by reducing angiogenesis in stomach carcinoma, thereby impairing the nutrition supply of the tumor, further inhibiting proliferation and inducing apoptosis of neoplasm cells. FGF-1 and V E G F are two important growth factors and played a pivotal role in tumor angiogenesis [1,4]. Neoplasms overexpressing COX-2 often express V E G F and FGF-1 at the same time [5]. W e observed their expression after N S A I D s treatment in SGC7901 xenografts and found the expression level was remarkably decreased when compared with control group. The m R N A level of V E G F was
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also declined. So we could speculate that NSAIDs inhibit COX-2 activity, thereby reducing expression of angiogenic factors and MVD and resulting in ischemia, which induces apoptosis and depresses the growth of tumor. Celecoxib is the first selective COX-2 inhibitor proved by FDA for clinical application. Its effects were stronger than sulindac in this study with no significant statistical difference shown. Our study offered new evidences for NSAIDs' therapeutic effects against tumor. However, further specially designed experiments are expected to be carfled out to demonstrate the potential advantages of selective COX-2 inhibitors in preventing tumorigenesis when compared with non-selective NSAIDs.
Acknowledgement Supported by a grant from the Natural Science Foundation of Shanghai Science and Technology Committee, No. 02ZB 14042.
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