Expre ssion of cyclooxyena se22 protain and it s relationship with HIF21αin HCC

[Abstract ] 　Objective : To investigate the clinical significance of COX22 ( Cyclooxygenase22) expression in HCC ( Primaryhepatocellular carcinoma) and clarify whether COX22 is correlated with hypoxia2inducible factor21α (HIF21α) in the development of HCC.Met hods : Tumor tissues were obtained from 53 patients with HCC. COX22 and HIF21αwere determined by immunohistochemistry. All 53patients were regularly followed up and the data were collected prospectively. Res ults : Immunostaining showed the expression of COX22( n = 33 , 62. 3 %) and HIF21α ( n = 36 , 67. 9 %) in most tumor cells . The level of COX22 was correlated with HIF21αlevels ( r = 0. 440 ,P < 0. 01) . There were significant correlation between clinicopathological features and higher tumor cytosolic COX22 level was in thepresence of multiple tumors ( P = 0. 01) , venous invasion ( P = 0. 03) , advanced tumor stage ( P = 0. 01) , and well2different tumor grade( P = 0. 03) . High tumor cytosolic COX22 level was correlated with patient’s worseprognosis ( P = 0. 0085) . Concl usion : Elevatedtumor COX22 level is correlated with elevated HIF21α levels and invasiveness in HCC , suggesting COX22 plays an important role in theprogression of HCC ,and may be an important therapeutic target in HCC.

 江苏省中医院消化肿瘤外科姜超

[ Key words ] 　cyclooxygenase-2 ; hypoxia-i nduci ble f actor-1α; huma n hep atocell ula r carcinoma

A s we know , cyclooxygenase ( COX) is a rate2limiting enzyme responsible for the oxidation of

arachidonic acid to prostaglandin H2 . The COX at least has two forms : COX-1 and COX-2. COX-1 isconstitutively expressed in many normal tissues , whereasCOX-2 is inducible. Increased COX-2 has been associated with inhibition of apoptosis by antiapoptosis factor , with angiogenesis and with enhance of metastatic ability in human carcinogenesis . However , the role of COX-2 in the HCC remains controversial . Some studiesshowed that the expression of COX22 had nothing to do with invasion of HCC , but just was correlated with tumor differentiation grade [1 ] . While the different voice from Terence C. Tang argued that COX22 had correlated with invasion of HCC , and bad effect on the prognosis of patients after resection of HCC[2 ] . So we employed new separate criteria to study the clinical significance of COX-2 expression in HCC by Immunohistochemistry. In addition , Hypoxia2inducible factor21α ( HIF21α) is a critical transcription factor that regulates the expression of genes encoding factors which influence tumor growth.The vascular endothelial growth factor (VEGF) is thecommon target gene of both COX22 and HIF21α, which could influence the proliferation and metastasis of tumors . To our knowledge , there are only a few researches on the relationship between COX-2 and HIF-1α in tumor[325 ] . We also tried to investigate whether COX-2 was correlated with HIF-1α in the process of HCC development .MATERIALS AND METHODS Materials The tissue samples of 53 primary HCC patients were obtained from Department of Pathology , the First Affiliated Hospital of Nanjing Medical University ,China. Among the patients who underwent HCCresection there were 48 men and 5 women ( aged 40~70years ; mean age 55 years ) . There were 40 patients infected with hepatitis B virus (HBV) , 7 patients infected with hepatitis C virus ( HCV) , and 6 patients never infected with HBV or HCV. All specimens were fixed in 10 % buffered formalin , and embedded in paraffin.Tumor differentiation was classified according to the criteria proposed by the Liver Cancer Study Group of Japan : well2, moderately2, and poorly2differentiated. Tumors were staged using the criteria established by the International Union against Cancer Pathologic Tumor2Node2Metastasis ( PTNM) . All patients were regularly followed up and the data were collected prospectively.Immu nohis t oc hemis t ry Tumor sections were deparaffinized and rehydrated by standard procedures of histology. Slides were put in 10 mmol/ L citrate buffer (pH6. 0) for 10 min for antigen retrieval , and then immersed in 3 % hydrogen peroxide for 15 min at room temperature. After washed in phosphate buffer saline , the specimens were preblocked by biotin blocking reagent for 10 min at room temperature. Then the slides were incubated overnight with mouse antihuman COX22 ( or HIF21α) monoclonal antibody ( dilution , 1 ∶100 ; Beijing Zhongshan Company , China) at 4 ℃. The sections were incubated with rabbit biotinylated antimouse immunoglobulin G for 60 min after washing , and then incubated with streptavidin2biotin complex/ horseradish peroxidase at room temperature for 30 min. The slides were counterstained with hematoxylin after they were washed with phosphate buffer saline. The corresponding tissue slides from the same patient without primary antibody were employed as negative control . When there was undetectable or minimal ( less than 10 % of the tumor cells) staining for COX22 (or HIF21α) , it was defined as negative ( marked as “ - ”) ; Cytoplasm staining was defined as positive when > 10 % of the tumor cells were stained , and moreover 10 %233 % cells stained ranked as“+ ”, and > 33 % stained ranked as “ + + ”. We classified the rank of“ - ”and“ + ”into low intensity of COX-2 team , and the remains into high intensity team.St atis tical a nalysis The SPSS software was employed to analyse the data. χ2. test was used to determine the statistical significance on categorical variables . And the relation of COX-2 and HIF-1αwas determined by the Spearman correlation test( r ) . Survival was computed using the Kaplan2Meier method and compared between groups (the low intensity of COX-2 team and high team) using the log2rank test . A level of P < 0. 05 was considered statistically significant .

RESULTS

The cor relation betwe e n COX-2 levels i n HCC a n d cli nicop at hological p a ramet e rs Immunohistochemical staining of COX-2 in the tumors showed that COX-2 was expressed predominantly in the cytoplasm of tumor cells ( Fig. 1 ) . There were 22 specimens with COX-2 staining intensity of“ + + ”, 31 specimens defined as “ + ” or “ - ”. A significant association was found between high tumor COX-2 levels and the presence of multiple tumors ( P = 0. 01) , venous invasion ( P = 0. 03) , advanced p2TNM stage ( P = 0101) and well differentiated tumor ( P = 0103) . While COX-2 levels did not show a significant correlation with tumor size and varied virus infected ( P > 0. 05 , Table 1) . The relations hip betwe e n COX-2 a n d HI F-1αexp ression i n HCC Of the 53 specimens , positive expression of HIF-1αwas present in 36 (67. 9 %) speciments , and COX22 presentin 33 (62. 3 %) speciments . See table 2. From Table 2 ,we could see that positive correlation between tumor COX22 and HIF21αlevels ( r = 0. 440 , P < 0101) . Relations hip betwe e n t umor COX22 levels a n d p rognosis The 53 patients were stratified into a low intensity (stained as“ - ”“ + ”) tumor cytosol COX22 levels and a high (stained as“ + + ”) tumor cytosol COX22 levels . It was obvious that patients with a high tumor cytosol COX-2 levels had significantly worse long-term survival compared with those with low COX22 levels ( P =010085 , Fig. 2) .

DISCUSSION

Primary hepatocellular carcinoma (HCC) is one of the most common malignant tumors , strongly affected by angiogenesis . Several studies in other cancers revealed that COX-2 had a close relation to angiogenesis and could affect tumor growth , venous invasion , and metastasis [6210] .Our study showed that increased tumor cytosol COX-2 was significantly correlated with presence of multiple tumors ( P = 0101) , portal venous invasion ( P = 0103) , advanced p2TNM stage ( P = 0101) in HCC. Terence C. Tang employed an enzyme immunometric assay ( EIA) , a more quantitative method , and also got the similar result [3 ] . Moreover Prostaglandin E2 , a product of COX-2 , has been found to have the property of promoting migration and adhesion in the human liver cell lines THL E22 , CCL213 and the hepatoma cell line SK2 HEP21 (HTB252) [11 ] . All of the researches demonstrate that high COX-2 can influence the invasion and metastasis of HCC from multiple levels . But some previous studies showed the contrary tendency[2 ,12 ,13 ] . They failed to discover the relationship between COX-2 and tumor features by separating the specimens into negative and positive team by immunohistochemistry. Some important clues may be covered up by the incorrect separate criteria. Our findings imply that only high intensity of COX-2 could influence the HCC invasional ability significantly , while low intensity of COX-2 has no countable effect on the development of HCC.

At the same time , we found increased COX-2 in tumor was closely related with tumor well2differentiated grade ( P = 0. 03) , which was coincident with previous studies that COX22 might be involved in the early stage of hepatocarcinogenesis because of their finding of higher COX-2 expression just in well2differentiated HCC[14 ] .

Unlike COX22 , HIF21αhas not been found correlationwith tumor venous invasion , but previous studies identified HIF21αwas significantly correlated with tumor pro-liferation as well as metastasis and extensively expressed in many tumor such as prostate cancer , breast cancer , HCC and so on[15217 ] . The study conducted by Huang SP had identified the COX-2/ PGE2/ HIF-1α / VEGF pathway possibly contributing to tumor angiogenesis in gastric carcinoma ( GC ) [4 ] . The present study demonstrated a significant correlation between HIF-1α and COX-2 in HCC ( r = 0. 440 , P < 0. 01) . We canhypothesize that COX-2 and HIF-1αhave the cooperative effect on the development of HCC. COX-2 may to induce angiogenesis through up-regulating HIF-1α and finally affect HCC proliferation and metastasis .

A significant correlation between high intensity of tumor COX-2 and worse prognosis of patients ( P = 010085) was found in our experiment . It can be well explained by our findings of significant relationship between high COX-2 and HCC clinicopathological features . Venous invasion , advanced tumor stage and multiple tumors in HCC are all great risk factors of tumor metastasis and recurrence after surgery , which play the negative impact on patient’s long survival . Besides , our finding of close correlation between COX-2 and HIF-1α also suggests that high HIF-1α expression may play an important role on the patient ’s prognosis in the high COX-2 intensity team. However , it is difficult to explain why high intensity of COX-2 specimens which mainly consist of well-differentiated tumors instead of bad-differentiated ones had worse prognosis . The role of COX-2 in HCC is so complex that need further research.

Now medical scientists have found nonsteroidal antiinflammatory drugs especially COX-2 inhibitors have antineoplastic feasibility[18 ,19 ] . Further fundamental researches conducted by Min Yao , had found that

inhibition of Cyclooxygenase-2 by rofecoxib attenuated the growth and metastatic potential of colorectal carcinoma in mice in 2003 [20 ] . And Abiru also found that NS2398 , a selective COX-2 inhibitor , could inhibit hepatocyte growth factor2induced invasiveness of Heg G2 hepatoma cells [21 ] . All these results support that COX-2 can be an important therapeutic target in tumor. Considering the lack of effective chemotherapy for HCC , targeting COX-2 may be an attractive anticancertherapeutic approach worthy of further investigation. REFERENCES

[ 1 ] 　Koga H, Sakisaka S , Ohishi M, et al . Expression of cyclooxygenase-2 in human hepatocellular carcinoma : relevance to tumor dedifferentiation. Hepatology 1999 ;29 :688296.

[ 2 ] 　Tang TC , Poon RT , Lau CP , et al . Tumor cyclooxygenase22 levels correlate with tumor invasiveness in human hepatocellular carcinoma. World J Gastroenterol 2005 ;11 (13) :18962902.

[3 ] 　Huang SP , Wu MS , Shun CT , et al . Cyclooxygenase22 increases hypoxia2inducible factor21 and vascular endothelial growth factor to promote angiogenesis in gastric carcinoma. J Biomed Sci 2005 ; 12 (1) :229241.

[4 ] 　Zhong H, Willard M, Simons J . NS398 reduces hypoxia2inducible factor (HIF)21alpha and HIF21 activity : multiple2level effects involving cyclooxygenase22 dependent and independent mechanisms . Int J Cancer 2004 ;112 (4) :585295.

[5 ] 　Calviello G, Di Nicuolo F , Gragnoli S , et al . n23 PUFAs reduce VEGF expression in human colon cancer cells modulating the COX-2/ PGE2 induced ERK21 and 22 and HIF21alpha induction pathway. Carcinogenesis 2004 ;25 (12) :2303210.

[6 ] 　Li G, Yang T , Yan J . Cyclooxygenase22 increased the angiogenic and metastatic potential of tumor cells . Biochem Biophys Res Commun 2002 ;299 :886290.

[ 7 ] Cianchi F , Cortesini C , Bechi P , et al . Up2regulation of cyclooxygenase 2 gene expression correlates with tumor angiogenesis in human colorectal cancer . Gastroenterology 2001 ;121 :1339247.

[ 8 ] Gallo O , Masini E , Bianchi B , et al . Prognostic significance of cyclooxygenase22 pathway and angiogenesis in head and neck squamous cell carcinoma. Hum Pathol 2002 ;33 :708214.

[ 9 ] 　Konno H, Baba M, Shoji T , et al . Cyclooxygenase22 expression correlates with uPAR levels and is responsible for poor prognosis of colorectal cancer . Clin Exp Metastasis 2002 ;19 :527234.

[ 10 ] Costa C , Soares R , Reis2Filho JS , et al . Cyclo2oxygenase 2 expression is associated with angiogenesis and lymph node metastasis in human breast cancer . J Clin Pathol 2002 ;55 :429234.

[ 11 ] Mayoral R , Fernandez2Martinez A , Bosca L , et al . Prostaglandin E2 promotes migration and adhesion in hepatocellular carcinoma cells . Carcinogenesis 2005 ;26 (4) :753261.

[ 12 ] Kondo M, Yamamoto H, Nagano H, et al . Increased expression of COX-2 in nontumor liver tissue is associated with shorter disease-free survival in patients with hepatocellular carcinoma. Clin Cancer Res 1999 ;5 :4005212.

[ 13 ] Rahman MA , Dhar DK, Yamaguchi E , et al . Coexpression of inducible nitric oxide synthase and COX22 in hepatocellular carcinoma and surrounding liver : possible involvement of COX22 in the angiogenesis of hepatitis C viruspositive cases . Clin Cancer Res 2001 ; 7 :1325232.

[ 14 ] Bae SH, Jung ES , Park YM, et al . Expression of cyclooxygenase22 (COX-2) in hepatocellular carcinoma and growth inhibition of hepatoma cell lines by a COX-2 inhibitor , NS2398. Clin Cancer Res 2001 ;7 :141028.

[ 15 ] Zhong H, Chiles K, Feldser D , et al . Modulation of hypoxia2 inducible factor 1alpha expression by the epidermal growth factor/ phosphatidylinositol 32kinase/ PTEN/ AKT/ FRAP pathway in human prostate cancer cells : implications for tumor angiogenesis and therapeutics . Cancer Res 2000 ;60 (6) :154125.

[ 16 ] Blancher C , Moo re JW , Talk s KL , et al . Relationship of hypoxia2 inducible factor (HIF)21alpha and HIF22 alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines . Cancer Res 2000 ;60 :7106213.

[ 17 ] L Ding , XP Chen , HP Wang. Expression and clinical significance of HIF-1αgene protein in hepatocellular carcinoma and paracancerous tissues . J ournal of Hepatobiliary Surgery 2004 ;12 (1) :3224.

[ 18 ] Xu XC. COX22 inhibitors in cancer treatment and prevention , a recent development . Anticancer Drugs 2002 ;13 :127237.

[ 19 ] Liu XH, Kirschenbaum A , Yao S , et al . Inhibition of cyclooxygenase-2 suppresses angiogenesis and the growth of prostate cancer in vi2 vo. J Urol 2000 ;164 :82025.

[ 20 ] Min Yao , Stacia Kargman , Eugene C. Lam , et al . Inhibition of Cy2 clooxygenase-2 by Rofecoxib Attenuates the Growth and Metastatic Potential of Colorectal Carcinoma in Mice. Cancer Research 2003 ; 63 :586292.

[21 ] Abiru S , Nakao K, Ichikawa T , et al . Aspirin and NS2398 inhibithepatocyte growth factor2induced invasiveness of human hepatoma cells . Hepatology 2002 ;35 (5) :1117224.