Expression of Ezrin, HGF, C-met in pancreatic cancer and non-cancerous pancreatic tissues of rats

Xing-Guo Tan and Zhu-Lin Yang

Changsha, China

Expression of Ezrin, HGF, C-met in pancreatic cancer and non-cancerous pancreatic tissues of rats

Xing-Guo Tan and Zhu-Lin Yang

Changsha, China

BACKGROUND:Recent studies have confirmed that the expression of Ezrin, hepatocyte growth factor (HGF) and its receptor (C-met) is related to the genesis, progress, invasion and metastasis of some malignant tumors. Researches have also found that the biological function of Ezrin is closely related to HGF/C-met in malignant tumors. However, there is no report on the expression levels of Ezrin, HGF and C-met in rat pancreatic cancer induced by dimethylbenzanthracene (DMBA). This study aimed to detect the expression of Ezrin, HGF and C-met in rat pancreatic cancer and non-cancerous pancreatic tissues, and assess its effect in cancer induction by DMBA.

METHODS:Ninety Sprague-Dawley rats were divided into 3 groups randomly: 40 in a pancreatic cancer model group (group A), 40 in a trichostatin A (TSA) intervention group (group B), and 10 in a control group (group C). DMBA was directly implanted into the parenchyma of rat pancreas in group A+group B. The rats of group B were treated with 1 ml of TSA saline solution (1 μg/ml) via intraperitoneal injection weekly. The carcinogenesis of rats executed within 3-5 months in groups A and B was observed by macrograph and microscopy. Meanwhile, the rats in group C were executed within 5 months. The EnVisionTMimmunohistochemistry for detecting the expression levels of Ezrin, HGF and C-met was used in paraffinembedded sections of the pancreatic specimens.

RESULTS:The incidence of pancreatic cancer in group A was 48.6% and in group B 33.3%. The maximal diameter of tumor mass was significantly larger in group A than that in group B (P<0.05). No pathological changes were observedin the pancreas of group C and other main organs of groups A and B. The positive rates of Ezrin, HGF and C-met were significantly higher in ductal adenocarcinoma than in noncancerous pancreatic tissues of groups A and B (P<0.01). The positive rates of Ezrin, HGF and C-met were significantly higher in ductal adenocarcinoma of group A than those in noncancerous pancreatic tissues of group A (P<0.05), but there was no significant difference in group B (P>0.05). The positive rates of Ezrin, HGF and C-met in non-cancerous pancreatic tissues proved mild to severe atypical hyperplasia of the ductal epithelia. The pancreas of group C and 2 cases of fibrosarcoma showed the negative expression of Ezrin, HGF and C-met. There was a trend of consistency in the expression of Ezrin, HGF and C-met in ductal adenocarcinoma (P<0.05 orP<0.01).

CONCLUSIONS:DMBA directly implanted into the parenchyma of the pancreas can produce a model of pancreatic cancer with a high incidence in a short time. TSA might inhibit the carcinogenesis and growth of pancreatic cancer, and its effects may be related to the inhibition of the expression of Ezrin, HGF and C-met during the process. Ezrin, HGF and C-met may have positive effects on the carcinogenesis of rat pancreas.

(Hepatobiliary Pancreat Dis Int 2010; 9: 639-644)

pancreatic neoplasms; animal model; Ezrin; hepatocyte growth factor; C-met

Introduction

Pancreatic cancer is one of the solid malignancies because of its rapid growth and propensity to invade adjacent organs and metastasize. Around the world, pancreatic cancer causes exproximately 213 000 deaths each year. The 1-year survival rate is around 20%, and the 5-year survival rate is less than 5% in spite of aggressive therapies.[1]But investigation of molecular pathogenesis has been useful in the diagnosis and treatment of pancreatic cancer. In the last two decades research has shown that pancreatic cancer is fundamentally a geneticdisease caused by inherited germline and acquired somatic mutations in cancer-associated genes. To build useful models studying the pathological molecular mechanisms of pancreatic cancer, Rivera et al[2]directly implanted dimethylbenzanthracene (DMBA) into the parenchyma of the pancreas of rats and found a pancreatic cancer incidence of 39% within 10 months. Bockman et al[3]reported similar studies.

Trichostatin A (TSA) is one of a variety of histone deacetylase inhibitors that have a broad spectrum of epigenetic activities. It can up-regulate several gene expressions and restrain other gene expression, thus intervening the genesis and development of tumor.In vivoorin vitroexperiments have confirmed that TSA could restrain the genesis of some tumors and control the progress of the tumor by restraining the angiogenesis and changing tumor microenvironment.[4]Many studies showed that TSA acted as a tumor suppressor in human pancreatic cancer cell lines.[5,6]

Ezrin plays a positive role in maintaining cell shape and polarity and participates in cell migration, signaling, growth regulation, and differentiation. It is also actively involved in regulating the growth and metastatic capacity of cancer.[7-14]Hepatocyte growth factor (HGF) is a kind of multifunctional cytokine and it has multiple biological function after integrating with its receptor (C-met). Previous studies[15,16]showed that HGF/C-met plays important roles in angiogenesis and tumor growth. Inhibitors of this signaling pathway have been shown to inhibit angiogenesis inin vitroandin vivomodels. The HGF/C-met signaling pathway is now recognized as a promising target in cancer by inhibiting angiogenesis, tumor growth, invasion, and metastasis.[17-19]Recent studies have found that the biological function of Ezrin is closely related to HGF/C-met in malignant tumor.[7,20,21]

Since no studies have examined the levels of Ezrin, HGF and C-met inpancreatic cancer induced by DMBA and non-cancerous pancreatic tissues in rats, little is known about the effects of Ezrin, HGF and C-met on rat pancreatic cancer induced by DMBA. In this study, DMBA was directly implanted into the parenchyma of the pancreas of rats to establish a pancreatic cancer model, and TSA injection was given to establish the intervention group. After that we detected the expression levels of Ezrin, HGF and C-met in pancreatic cancer and non-cancerous pancreatic tissues, and their effect in the process of inducing cancer by DMBA.

Methods

Animal modelNinety Sprague-Dawley rats (no sex limit, weighing 150-200 g) were divided randomly into 3 groups: 40 in pancreatic cancer model group (group A), 40 in TSA group (group B), and 10 in control group (group C). The rats were preoperatively fasted for 24 hours (except water), and 2% amyl-barbital was injected into the abdomen for anesthesia. Then the abdomen of the rats was dissected by a 2-cm incision, and the pancreas was found. Subsequently the parenchyma of the pancreas was dissected (1 mm). DMBA (9 mg) was directly implanted into the parenchyma of the pancreas in groups A and B, and then the pancreas was sutured. The rats were raised in common conditions after operation, and in group B the rats were injected 1 ml of TSA (1 μg/ ml) weekly via the abdomen. Except natural death, the rats were executed randomly in the third month after operation (7 rats in group A and 6 rats in group B), in the fourth month after operation (10 rats in each group), and in the fifth month after operation (20 rats each group). Rats in group C were treated with no DMBA implanting, and were executed in the fifth month after operation.

Macrography and pathological observation

The liver, gallbladder, stomach, intestine and lung of the rats in groups A and B were observed by macrography. And pancreatic tissues and tissues of the liver, gallbladder, stomach, intestine and lung of rats were put into 4% formaldehyde for 16-18 hours. Then the tissues were paraffin-embedded and sectioned. The sections were stained with HE and observed under a microscope.

Immunohistochemical staining

Rat anti-human Ezrin monoclonal antibody and rabbit anti-human HGF and C-met polyclonal antibody were purchased from Santa Cruz, USA. EnVisionTMkit was from Dako Company in Sweden. Ezrin, HGF and C-met was stained by the EnVisionTMtwo step staining method. The main procedures for staining were as follows: 1) The sections were deparaffined in distilled water; 2) 3% H2O2methanol liquid was used for 10 minutes and the sections were washed with lotic water for 5 minutes; 3) 0.05% pancreatin was used for digestion for 20 minutes, then the sections were washed with lotic water for 5 minutes and with distilled water for 5 minutes, washed with 0.01 mol/L PBS liquid (pH7.4) for 3 minutes (two times); 4) Antibody liquid was dropped to the sections for incubation for 60 minutes (37 ℃), and then washed with 0.01 mol/L PBS liquid for 3 minutes (three times); 5) A liquidwas dropped to the sections for incubation at 37 ℃for 30 minutes. The sections were then washed with 0.01 mol/L PBS liquid for 3 minutes (three times); 6) The secions were colored with colorant for 15 minutes (preparation of the colorant: add 20 μl C liquid to 1 ml B liquid in EnVisionTMkit and use it instantly after blending uniformly), then washed with lotic water; 7) The sections were stained with hematoxylin for one minute, and washed with tap water for 15 minutes; 8) The sections were dehydrated clarified and mounted with neutral balsam. Cytoplasm and/or cell membranes containing brown-yellow granules were defined as positive cells. The rate of cells in the sections was considered ≥25% for positive cases, while <25% for negative cases. The positive controls were the positive pancreatic cancer sections that had been confirmed by sub-staining for several times, while the negative controls were the 0.01 mol/L PBS liquid (pH7.4) substituting the first antibody.

Statistical analysis

All statistical analyses were performed using SPSS13.0 for Windows. The Chi-square test and Fisher's exact test were used to analyze the expressions of Ezrin, HGF and C-met in pancreatic duct adenocarcinoma and non-cancerous pancreatic tissues. The rank-sum test was used to compare the mean of maximal diameter of tumors. APvalue of less than 0.05 was considered statistically significant.

Results

Macrography

In group A, 2 rats died after operation within one month and one rat died within 2 months. In group B, 3 rats died after operation within one month and one rat died within 2 months. These rats were excluded for statistical analysis, and 37 rats in group A and 36 in group B were analyzed at last. During 3 to 5 months after operation, 18 rats developed pancreatic tumors in group A (18/37, 48.6%). Among these rats, 2 showed tumors in 3 months (2/7, 28.6%), 4 in 4 months (4/10, 40.0%), 12 in 5 months (12/20, 60.0%) and one metastasis to the liver. Twelve rats developed tumors in the pancreas in group B (12/36, 33.3%). Among these rats, 1 developed tumors in 3 months (1/6, 16.7%), 3 in 4 months (3/10, 30.0%), 8 in 5 months (8/20, 40.0%) and one epiploon metastasis. The incidence of pancreatic cancer in group A was higher than that in group B (P<0.05). The maximal diameter of tumor mass in group A varied: 0.5-1.0 cm (7 rats), 1.0-2.0 cm (10), and >2.0 cm (1); and the maximal diameter of tumor mass in group B: 0.5-1.0 cm (9 rats), 1.0-2.0 cm (2), and >2.0 cm (1). The mean of maximal diameter of tumors in group A was higher than that in group B as was shown by the rank-sum test (P<0.05). No pathological changes were found by macrography in the pancreas of group C and main organs (except the pancreas) of groups A, B and C.

Pathological observation

The pancreatic specimens taken from group C after HE staining showed normal tissues. Among 18 rats with pancreatic tumor in group A, 17 had ductal adenocarcinoma (6 well-differentiated, 7 moderatelydifferentiated, 4 poorly-differentiated) and 1 had fibrosarcoma (Fig. 1). Among 12 rats with pancreatic tumor in group B, 11 had ductal adenocarcinoma (6 well-differentiated, 4 moderately-differentiated, 1 poorlydifferentiated) and 1 had fibrosarcoma (Fig. 2). In groups A and B, one rat had fibrosarcoma with metastasis to the liver or epiploon. Non-tumor and peri-tumor pancreatic tissues in groups A and B showed hyperplasiaand atypical hyperplasia in the ductual epithelia and interlobular ductual endepidermis. The non-tumor tissues in group A showed mild atypical hyperplasia (5/19, 26.3%) and moderate to severe atypical hyperplasia (10/19, 52.6%; Fig. 1). Whereas the non-tumor tissues in group B which showed mild atypical hyperplasia (10/24, 41.6%) and moderate to severe atypical hyperplasia (8/24, 33.3%; Fig. 2). No statistical differences were found between the two groups (P>0.05). No pathological changes were observed under a microscope in the pancreas in group C and in major organs (but pancreas) of groups A and B.

Fig. 1. Expression of pancreatic tumor in group A (HE, original magnification ×200). A: poorly-differentiated pancreatic duct adenocarcinoma; B: fibroma sarcomatosum; C: severe atypical hyperplasia in ductual epithelia.

Expressions of Ezrin, HGF and C-met in pancreatic duct adenocarcinoma and non-tumor tissues

Fig. 2. Expression of pancreatic tumor in group B (HE, original magnification ×200). A: well-differentiated ductal adenocarcinoma; B: moderate atypical hyperplasia in ductual epithelia.

The immunohistochemical reaction of Ezrin, HGF or C-met occurred in cell membrane and/or cytoplasma (Fig. 3). The positive rate of Ezrin, HGF or C-met was significantly higher in pancreatic duct adenocarcinoma than in non-tumor tissues in groups A and B (P<0.01). The ductal epithelia of non-cancerous pancreases with positive expressions of Ezrin, HGF or C-met in groups A and B showed moderate to severe atypical hyperplasia. The pancreatic tissues in group C and fibrosarcoma showed negative expressions of Ezrin, HGF or C-met. The positive rate of Ezrin, HGF or C-met was significantly higher in pancreatic duct adenocarcinoma than in nontumor tissues in group A (P<0.05). The positive rates of Ezrin, HGF and C-met were higher in pancreatic duct adenocarcinoma than in non-tumor tissues in group B, (P>0.05)(Table). The expressions of Ezrin, HGF nad C-met were not correlated with the size and differentiation degree of the tumor (P>0.05). Of 18 rats with positive expression of Ezrin, 14 showed positive expression of HGF and 13 showed positive expression of C-met. The expression of Ezrin was consistent with the expressions of HGF and C-met (PHGF=0.032,PC-met=0.044). Among 17 rats with positive expression of HGF, 12 showed positive expression of C-met (P=0.029).

Table. Expressions of Ezrin, HGF and C-met in pancreatic duct adenocarcinoma and non-cancerous pancreatic tissues

Fig. 3. A: positive expression of Ezrin, group A moderate-differentiated ductal adenocarcinoma (EnVisionTM immunohistochemical method, original magnification ×200); B: positive expression of HGF, group B well-differentiated ductal adenocarcinoma (EnVisionTMimmunohistochemical method, original magnification ×200); C: positive expression of C-met, group A poorly-differentiated ductal adenocarcinoma (EnVisionTMimmunohistochemical method, original magnification ×200).

Discussion

Ezrin is a connectin of the theca cytoskeleton which is encoded by Villin 2 and it is a member of the ERM (Ezrin/Radxin/Moesin) family. Accordingly, a series of cellular functions generate such as cellular shaping, cellular movement, immigration, caryocinesia, etc. Ezrin is normally located in cellular membrane and at the top of microvilli. With the cell surface of actin, it has an adhering function of normal cell. Ezrin is located in cytoplasma when it is activated or cells are changed to be malignant. Recent studies[9,11,12,14]showed high expression in malignant tumors and low expression in benign lesions such as cancer of the breast, stomach, pancreas, liver, prostate, etc. Furthermore, recent studies have confirmed that malignant tumors with high expression of Ezrin are always invasiveness with poor-differentiation, rapid progress, easy metastasis and poor prognosis.[7-10,17,22,23]The results of the present study indicated that Ezrin could contribute to the carcinogenesis induced by DMBA, but its underlying mechanism needs further investigation. In addition, the inhibitory effects of TSA might be related to the inhibition of Ezrin expression.

HGF belongs to the cytokine family of dissolubility. Its receptor C-met exists in endothelial cell and it is a trans-membrane PTK receptor. After the combination of HGF with C-met, the receptor is activated and autophosphorylation accordingly induces phosphorylation of many substrate proteins and a series of biological, functional changes. The system of HGF/C-met significantly affects the interreaction of the epithelia and mesenchyma as well as regulation of cell multiplication, differentiation and movement.Recent studies[7,17,20,24,25]have found that the system of HGF/C-met has an important effect on genesis, progress, invasion and metastasis of tumors. The expression of HGF and C-met is obviously higher in malignant tumors than in benign lesions and normal tissues. The results of the present study indicated that HGF and C-met might affect the carcinogenesis induced by DMBA and that TSA might suppress the carcinogenesis of the pancreas by inhibiting the expression of HGF and C-met. But its mechanism needs further research.

In conclusion, the biological function of Ezrin is probably related to HGF/C-met, apart from the effect of E-cadherin and cell surface receptor CD44v. Ezrin can up-regulate the expression of HGF/C-met probably by the interaction with CD44.[14,20,21]The expression of Ezrin, HGF and C-met contributes to the induction of pancreatic duct carcinoma induced by DMBA, but Ezrin can up-regulate the expression of HGF/C-met. These speculations need further study.

Funding:None.

Ethical approval:Not needed.

Contributors:YZL proposed the study. TXG and YZL wrote the first draft. TXG did most of the experiments and analyzed the data. Both authors contributed to the design and interpretation of the study and to further drafts. YZL is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

1 Jemal A, Siegel R, Ward E, Murray T, Xu J, Smigal C, et al. Cancer statistics, 2006. CA Cancer J Clin 2006;56:106-130.

在吳鈞陶《愛麗絲奇境歷險記》漢譯本中，他在翻譯地名的時候采取了音譯加注釋的方法，既可以保留源語言的文化信息又可以在一定程度上解決文化沖突。

2 Rivera JA, Graeme-Cook F, Werner J, Z'graggen K, Rustgi AK, Rattner DW, et al. A rat model of pancreatic ductal adenocarcinoma: targeting chemical carcinogens. Surgery 1997;122:82-90.

3 Bockman DE, Guo J, Büchler P, Müller MW, Bergmann F, Friess H. Origin and development of the precursor lesions in experimental pancreatic cancer in rats. Lab Invest 2003;83: 853-859.

4 Monneret C. Histone deacetylase inhibitors. Eur J Med Chem 2005;40:1-13.

5 García-Morales P, Gómez-Martínez A, Carrato A, Martínez-Lacaci I, Barberá VM, Soto JL, et al. Histone deacetylase inhibitors induced caspase-independent apoptosis in human pancreatic adenocarcinoma cell lines. Mol Cancer Ther 2005;4:1222-1230.

6 Zhang S, Cai X, Huang F, Zhong W, Yu Z. Effect of trichostatin a on viability and microRNA expression in human pancreatic cancer cell line BxPC-3. Exp Oncol 2008; 30:265-268.

7 Mallikarjuna K, Pushparaj V, Biswas J, Krishnakumar S. Expression of epidermal growth factor receptor, ezrin, hepatocyte growth factor, and c-Met in uveal melanoma: an immunohistochemical study. Curr Eye Res 2007;32:281-290.

8 Torer N, Kayaselcuk F, Nursal TZ, Yildirim S, Tarim A, Nòyan T, et al. Adhesion molecules as prognostic markers in pancreatic adenocarcinoma. J Surg Oncol 2007;96:419-423.

9 Musial J, Sporny S, Nowicki A. Prognostic significance of E-cadherin and ezrin immunohistochemical expression in prostate cancer. Pol J Pathol 2007;58:235-243.

10 K?bel M, Gradhand E, Zeng K, Schmitt WD, Kriese K, Lantzsch T, et al. Ezrin promotes ovarian carcinoma cell invasion and its retained expression predicts poor prognosis in ovarian carcinoma. Int J Gynecol Pathol 2006;25:121-130.

11 Bruce B, Khanna G, Ren L, Landberg G, Jirstr?m K, Powell C, et al. Expression of the cytoskeleton linker protein ezrin in human cancers. Clin Exp Metastasis 2007;24:69-78.

12 Yeh TS, Tseng JH, Liu NJ, Chen TC, Jan YY, Chen MF. Significance of cellular distribution of ezrin in pancreatic cystic neoplasms and ductal adenocarcinoma. Arch Surg 2005;140:1184-1190.

13 Ogino W, Takeshima Y, Mori T, Yanai T, Hayakawa A, Akisue T, et al. High level of ezrin mRNA expression in an osteosarcoma biopsy sample with lung metastasis. J Pediatr Hematol Oncol 2007;29:435-439.

14 Hunter KW. Ezrin, a key component in tumor metastasis. Trends Mol Med 2004;10:201-204.

15 Maulik G, Shrikhande A, Kijima T, Ma PC, Morrison PT, Salgia R. Role of the hepatocyte growth factor receptor, c-Met, in oncogenesis and potential for therapeutic inhibition. Cytokine Growth Factor Rev 2002;13:41-59.

16 Comoglio PM, Giordano S, Trusolino L. Drug development of MET inhibitors: targeting oncogene addiction and expedience. Nat Rev Drug Discov 2008;7:504-516.

17 Hara S, Nakashiro K, Goda H, Hamakawa H. Role of Akt isoforms in HGF-induced invasive growth of human salivary gland cancer cells. Biochem Biophys Res Commun 2008;370: 123-128.

18 Trovato M, Vitarelli E, Grosso M, Alesci S, Benvenga S, Trimarchi F, et al. Immunohistochemical expression of HGF, c-MET and transcription factor STAT3 in colorectal tumors. Eur J Histochem 2004;48:291-297.

19 Murai M, Shen X, Huang L, Carpenter WM, Lin CS, Silverman S, et al. Overexpression of c-met in oral SCC promotes hepatocyte growth factor-induced disruption of cadherin junctions and invasion. Int J Oncol 2004;25:831-840.

20 Orian-Rousseau V, Morrison H, Matzke A, Kastilan T, Pace G, Herrlich P, et al. Hepatocyte growth factor-induced Ras activation requires ERM proteins linked to both CD44v6 and F-actin. Mol Biol Cell 2007;18:76-83.

21 Crepaldi T, Gautreau A, Comoglio PM, Louvard D, Arpin M. Ezrin is an effector of hepatocyte growth factor-mediated migration and morphogenesis in epithelial cells. J Cell Biol 1997;138:423-434.

22 Sarrió D, Rodríguez-Pinilla SM, Dotor A, Calero F, Hardisson D, Palacios J. Abnormal ezrin localization is associated with clinicopathological features in invasive breast carcinomas. Breast Cancer Res Treat 2006;98:71-79.

23 Martin TA, Harrison G, Mansel RE, Jiang WG. The role of the CD44/ezrin complex in cancer metastasis. Crit Rev Oncol Hematol 2003;46:165-186.

24 Klosek SK, Nakashiro K, Hara S, Li C, Shintani S, Hamakawa H. Constitutive activation of Stat3 correlates with increased expression of the c-Met/HGF receptor in oral squamous cell carcinoma. Oncol Rep 2004;12:293-296.

25 Nakashiro K, Hayashi Y, Oyasu R. Immunohistochemical expression of hepatocyte growth factor and c-Met/HGF receptor in benign and malignant human prostate tissue. Oncol Rep 2003;10:1149-1153.

February 11, 2010

Accepted after revision September 2, 2010

Author Affiliations: Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tan XG and Yang ZL)

Zhu-Lin Yang, MD, Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha 410011, China (Tel: 86-731-85292169; Fax: 86-731-85533525; Email: yangzhulin8@sina.com)

? 2010, Hepatobiliary Pancreat Dis Int. All rights reserved.