Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2016  |  Volume : 12  |  Issue : 8  |  Page : 291--294

Ezrin expression in the primary hepatocellular carcinoma patients and associated with clinical, pathological characteristics


Debiao Pan, Shi Wang, Hailin Ye, Shengqian Xu, Guanxiong Ye 
 Department of General Surgery, Lishui People's Hospital, Lishui, Zhejiang, P.R. China

Correspondence Address:
Shi Wang
Department of General Surgery, Lishui People's Hospital, Lishui, Zhejiang 323000
P.R. China

Abstract

Objective: The aim of this study was to assess Ezrin expression in the primary hepatic carcinoma patients and associated with clinical, pathological characteristics. Materials and Methods: Fifty-one patients with primary hepatocellular carcinoma (PHC) with completed clinical data were retrospectively analyzed in this study. The Ezrin expression in PHC and normal control liver tissue was tested by immunohistochemical assay. The Ezrin expression and relationship with clinical characteristics were evaluated. Results: The Ezrin positive rate were 66.7% and 15.7% with expression score of 3.21 ± 1.46 and 0.60 ± 1.10, respectively, in the cancer tissue and control tissue with statistical difference (P < 0.05). The Ezrin expression was associated with the metastasis status of the patients (P < 0.05) but not associated with the age (P > 0.05), gender (P > 0.05), differentiation (P > 0.05), and tumor diameter (P > 0.05). Conclusion: Ezrin protein is highly expressed in human PHC tissue which can be used for the prediction of metastasis disease.



How to cite this article:
Pan D, Wang S, Ye H, Xu S, Ye G. Ezrin expression in the primary hepatocellular carcinoma patients and associated with clinical, pathological characteristics.J Can Res Ther 2016;12:291-294


How to cite this URL:
Pan D, Wang S, Ye H, Xu S, Ye G. Ezrin expression in the primary hepatocellular carcinoma patients and associated with clinical, pathological characteristics. J Can Res Ther [serial online] 2016 [cited 2021 Apr 18 ];12:291-294
Available from: https://www.cancerjournal.net/text.asp?2016/12/8/291/200761


Full Text

 Introduction



Primary hepatocellular carcinoma (PHC) is a common malignant tumor of the digestive tract that is characterized by high morbidity and death rates.[1] It ranks third among causes of death of malignant tumors following stomach and esophagus malignant tumor, and places second in villages of certain areas after gastric cancer.[2] The pathogenesis of PHC is highly complicated and remains unclear. In recent years, studies have shown that the occurrence, development, invasion, and metastasis of various malignant tumors of Ezrin protein are related to its expression.[3],[4] Ezrin protein is a membrane cytoskeleton crosslinking protein and its expression is specific to tissues and cells. This protein plays a key role in the connection between the cell membrane and actin cytoskeleton of the cytoplasm. In addition, it is also involved in the adjustment of cell polarity, cell migration, and cell growth.[5] This study adopted the cancer tissues and distal hepatic tissues (>5 cm from the cancer tissue) of PHC patients as the study samples to discuss the clinical, pathological significance of Ezrin protein expression in PHC tissues and its correlation with patient prognosis.

 Materials and Methods



Source of clinical data

A total of 51 patients receiving surgical resection for PHC in Lishui People's Hospital of Zhejiang province (no chemotherapy and radiotherapy before the operation) from January 2010 to January 2016 were included as the study objects, including 40 males and 11 females with an mean age of 48.6 ± 15.6 years. Regarding the degree of tumor differentiation, there were 9 well-differentiated cases, 24 moderately differentiated cases, and 18 poorly differentiated cases. Among the 51 patients, 46 had positive hepatitis B and 5 presented with negative hepatitis B. The tumors of all the patients were excised by surgery, and the cancer tissues and areas at >5 cm from the cancer tissue were obtained as specimens. Continuous paraffin-embedded sections were prepared after the specimens were fixed in 10% neutral formalin.

Reagents and apparatus

Mouse anti-human Ezrin gene protein monoclonal antibody was purchased from Santa Cruz Biotechnology, Santa Cruz, CA. General anti-rabbit/mouse immunohistochemistry detection kit (ChemMate EnVisionr M+/HRP/DAB Rb and Mo) was purchased from Gene Tech (Shanghai) Co., Ltd. and packed by Danish Dako. A paraffin slicing machine was purchased from Leica Germany, and the microscope used was from Olympus Optical Co., Ltd. An electric thermostat box was produced by Shanghai Rongke Testing Instrument Co., Ltd. The model of the microwave oven used was WHIRLPOOL VIP202S (850 W).

Detection of Ezrin protein

Sections were heated at 65°C for 3 h and then dewaxed completely. The sections were washed with distilled water for 2 min, and antigens were retrieved. In this study, the specimens were soaked in citrate buffer solution with weak acidity (pH 6.0), heated in a microwave oven with power of 850 W for 11 min, cooled to room temperature, and washed with distilled water for 2 min. The samples were soaked in hydrogen peroxide with mass concentration of 3% and then heated in a microwave oven at 350 W for 2.5 min to completely eliminate peroxidase activity. Subsequently, the samples were washed with distilled water for 2 min and rinsed with phosphate-buffered saline (PBS) three times for 2 min each. A proportion of the diluted primary antibodies was added to the samples, which were incubated at 37°C for 1 h. The samples were then rinsed three times with PBS for 2 min each. Polymer helper was added to the samples, which were incubated at 37°C for 20 min. The samples were again rinsed with PBS three times for 2 min each. Polyperoxidase anti-mouse/rabbit IgG was added, and the samples were incubated at 37°C for 0.5 h. After washing the samples with PBS three times for 2 min each, color development with diaminobenzidine was conducted for 5 min. Each sample was fully washed with tap water before counterstaining. Finally, the sections were dehydrated, hyalinized, and sealed.

Assessment of results

The cells containing yellow to brown particles in the membrane or (and) nucleus were positive cells. Scoring was performed by referring to the method reported by Kong et al.[6] In brief, the positive cells in ten high-power fields were counted (none: 0, weak: 1, moderate: 2, and strong: 3). The sum of the positive rate of expression (<5%: 0, 5%–10%: 1, 10%–20%: 2, 20%–50%: 3, and >50%: 4) was used as the expression score. Cells with scores from 0 to 2 were regarded as negative cases, and those over 3 were considered positive.

Statistical method

The data in this study were first input into Excel software and analyzed statistically by SPSS18.0 software (http://www-01.ibm.com/software/analytics/spss/). The enumeration data were demonstrated by n and tested by χ2 test Fisher's exact test. Statistical significance was set at P < 0.05.

 Results



Expression of Ezrin protein in hepatic cancer tissues

Ezrin demonstrated a linear distribution in the internal side of the hepatic cancer cell membrane and presented brown particles. The positive expression rates of Ezrin protein in cancer and distal tissues of 51 hepatic cancer patients were 66.7% and 15.7%, respectively, with expression scores of 3.21 ± 1.46 and 0.6 ± 1.1, respectively. The positive rate and expression score of Ezrin protein in cancer tissues were higher than those in normal hepatic tissues (P < 0.05) [Table 1].{Table 1}

Relationship between expression of Ezrin protein and clinicopathologic features in hepatic cancer patients

A correlation was found between the score of Ezrin expression and differentiated degree of PHC tissues with metastasis (P < 0.05), whereas no obvious correlation was detected between the score and patients' age, gender, tumor size, and presence or absence of liver cirrhosis (P > 0.05), [Table 2].{Table 2}

 Discussion



PHC is one of the cancers with the highest morbidity all over the world. It is also a common malignant tumor in China, with a high degree of malignancy, rapid development, and relatively high recurrence rate and death rate.[7] Early treatment is difficult because of the concealed onset, and patients are usually in the advanced stage when the disease is detected. Therefore, early diagnosis and treatment of hepatocellular carcinoma have always been the focus of research at worldwide. Around 110 thousand people die of hepatic cancer in China every year, accounting for 45% of hepatic cancer deaths worldwide.[8] The occurrence of PHC is a synergistic process that involves multiple factors, including viral infection, function of carcinogenic agents, activation of the cancer gene, inactivation of the cancer suppressor gene, apoptosis, and abnormal adjustment of hepatic cell proliferation. Metastasis of PHC is a multigene and multistep chain process, involving cytogenes, growth factors and its receptors, adhesion molecules, extracellular matrix, formation of tumor vessels, and immunity.

Ezrin is a membrane cytoskeleton crosslinking protein of the ezrin, radixin, moesin family. Ezrin protein shows specificity to certain tissues and cells and it is commonly expressed on the inner surface of the cell membrane with moesin and radixin and plays an important role in the maintenance of cellular morphology and survival.[4] Activated C-terminal of Ezrin is linked with actin protein cytoskeleton, and the N-terminal is bound to multiple types of transmembrane molecules, such as CD44, intercellular adhesion molecule-1, and E-cadherin.[5] Ezrin is involved in changes in cellular morphology, construction of the cytomembrane, cell movement, adhesion, and related signal transduction.[9] Therefore, the protein plays an important role in the movement, adhesion, and invasion of tumor cells. It mainly adjusts characteristics of tumor cells by mitigating adhesion molecules on the surface of tumor cells, thereby affecting the differentiation, invasion, and metastasis of tumors. The occurrence, development, metastasis, and invasion of tumors comprise a multifactor and multistep process.[10] Given that some functions of Ezrin protein are necessary in tumor events, the protein is likely to participate in this process. During methylcholanthrene-induced transformation from normal cells of mice into malignant tumor cells, the level of Ezrin expression increases significantly, which indicates that Ezrin plays an important role in tumor occurrence. Pang et al.[11] detected Ezrin expression in a group of intraepithelial neoplasia of human prostate glands through immunohistochemistry and found moderate or high Ezrin expression but negative or weak expression in normal prostate tissues. A significant difference in Ezrin expression was found in the same gland containing both atypical cells and relatively normal cells. These results indicated that Ezrin may played an important role in the early stages of cancer.

In this study, the cancer tissues and distal normal hepatic tissues of PHC patients were used as study samples to discuss the clinical, pathological significance of Ezrin protein expression in PHC tissues and its correlation with patient prognosis. The results of the study confirmed that the positive expression rate of Ezrin protein in PHC tissues was obviously higher than that in distal normal hepatic tissues. Moreover, Ezrin expression was correlated with tumor metastasis and the differentiation degree of tumor tissues but not with the patients' age, gender, and tumor size. The positive ratio of Ezrin in the patents with metastasis and low tumor differentiation was higher than that in the patients without metastasis and high tumor differentiation. Therefore, Ezrin protein could be regarded as a marker of distant metastasis of PHC and poor differentiation of tumor tissues. However, the present study was limited by a small number of patients included, low statistical performance, and lack of prognosis-related data. The sample size should be further increased, and patients' survival data should be analyzed to further discuss the clinical, pathological significance of Ezrin protein expression in PHC tissues and its relationship to patients' prognosis. Further studies may provide more sufficient clinical evidence for the potential of Ezrin protein as a related marker of metastasis or prognosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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