[Abstract] Objective To observe Smac and Ki67 expression in thyroid cancer, and to explore the relationship and its clinical significance of Smac expression of Ki67. Methods Immunohistochemical method detected 87 cases of thyroid cancer tissue Smac and Ki67 expression, analysis of the of Smac expression and clinicopathological factors and Ki67 relationship. Results Smac positive expression in thyroid carcinomas was 71.3% (62/87), all adenomas next organization were seen in the expression (20/20); of Ki67 proliferation index was 42.74 ± 14.52. Smac in men, age ≥ 45 years, medullary carcinoma, and undifferentiated carcinoma, the primary lesion beyond the capsule, Ⅱ, Ⅲ, IV thyroid cancer patients was significantly lower (P <0.05). Of Smac in patients with positive expression of Ki67 proliferation index is low compared Smac negative expression (40.70 ± 14.68) vs (47.78 ± 13.06), P <0.05]. Conclusion Smac and Ki67 altered expression in thyroid cancer correlation, suggesting that it may play an important role in the development of thyroid cancer and both potentially synergistic.

Key words thyroid cancer; of Smac; Ki67 proliferation index; apoptosis; proliferation

Expression of Smac and Ki67 in Thyroid Carcinoma and Its Clinical Significance

    DENG Wan  kai, CHEN Jian, ZHENG Hong  mei

    Department of Head and Neck Surgery, Hubei Cancer Hospital, Wuhan 430079, China

    Corresponding Author: CHEN Jian, E  mail: chenjian2003@yahoo.com.cnAbstract: Objective To investigate the expression of Smac and Ki67 in thyroid carcinoma and the correlation of Smac and Ki67, the clinical significance as well.Methods Immunohistochemical staining of Smac and Ki67 expression was performed by the standard streptavidin  peroxidase (SP) technique for the paraffin sections of 87 thyroid carcinoma.The relationship between the Smac expression and the clincopathological parameters or Ki67 proliferation index was analyzed.Results The positive rate of Smac in thyroid carcinoma was 71.3% (62/87), all of the normal thyroid tissues expressed Smac (20/20). Ki67 proliferation index in thyroid carcinoma was 42.74 ± 14.52.Smac expression was significantly lower in patients of male, over 45 years old, medullary and anaplastic thyroid carcinoma, stage Ⅱ, Ⅲ and Ⅳ, and in those with invasions over capsular (P <0.05). The Ki67 proliferation index in negative expression of Smac was significantly higher than that in positive expression of Smac [(40.70 ± 14.68) vs (47.78 ± 13.06), P <0.05]. Conclusion Smac and Ki67 expression in thyroid carcinoma were significantly different from those in normal tissues, and they had significant correlation, which suggest that they may play an important role or even have synergic action in carcinogenesis and development of thyroid carcinoma.

    Key words: Thyroid carcinoma; Smac; Ki67 proliferation index; Apoptosis; Cell proliferation

Thyroid cancer is a common malignant tumors of the head and neck, but less than 1% of systemic cancer occurs in women and adolescent patients, statistics show that its upward trend in the incidence in the United States [1]. The Smac Wang Xiaodong, 2000 to a protein found in the mitochondria can be released into the cytoplasm to promote apoptosis [2]. Ki67, a proliferating cell nuclear antigen, and tumor proliferation, invasion, metastatic potential and prognosis [3]. In this paper, immunohistochemistry SP method detection Smac and Ki67 in normal tissue adjacent thyroid cancer and adenoma expression to explore its role in the development of thyroid cancer and their correlation.

    1 Data and methods

    1.1 Data

    Randomly collected in our hospital from 2004 to 2006, the Pathology archive thyroid carcinoma 87 cases, patients had a detailed clinical and pathological data, not all cases preoperative radiotherapy, chemotherapy and immunotherapy. There were 23 males and 64 females, aged 11 to 73 years old, with an average age of 49.4 years. Divided into pathological types: 71 cases of papillary thyroid carcinoma, follicular carcinoma, medullary carcinoma of the seven cases, undifferentiated carcinoma in 6; divided into lymph nodes: lymph node metastasis were 48 cases, 39 cases without lymph node metastasis ; according to the the 2002 UICC sixth edition of TNM classification is divided into: Ⅰ 51 cases, 12 cases of stage Ⅱ, Ⅲ of 13 cases, Ⅳ of 11 cases. Another randomly selected from the same period in thyroid adenoma gland resection peritumoral least 0.5cm thyroid tissue specimens 20 were used as the control.

    1.2 Reagents

    Mouse anti-human of Smac / Diablo monoclonal antibody (Cell Signaling Technology, USA), rabbit anti-human Ki67 monoclonal antibody (Lab Vision Corporation, USA), ready-to-use SP kit (Beijing Zhongshan biotechnology company), DAB staining kit ( Beijing Biosynthesis Biotechnology Company).

    1.3 Methods

    87 patients with surgical resection specimens archived paraffin tissue blocks 4μm serial sections, hematoxylin  eosin staining. SP detected thyroid carcinomas and adenomas beside thyroid tissue of Smac expression of Ki67. The methods are summarized as follows: Paraffin sections xylene dewaxing, graded ethanol hydration; citrate buffer solution (pH 6.0) 95 ° C Fix 10min; 3% H2O2 incubated 10min, phosphate buffered saline (PBS) after washing was added dropwise non-immune animal serum incubated at room temperature for 15min, plus an anti-(anti-Smac antibody 1:200, anti-Ki67 antibody 1:100) at 4 ° C overnight incubation; biotinylated secondary antibody at 37 ℃ for 10min after washing in PBS, horseradish peroxidase-labeled anti-biotin antibody at 37 ℃ for 15min, PBS washing after DAB color, rinse, the hematoxylin, Fengpian, microscope and photographed. Known positive sections as a positive control, PBS instead of primary antibody as a negative control.

    1.4 Results decision

    (1) Smac staining criteria [4]: ​​cytoplasm stained pale yellow to brown defined as positive cells, 5 to 10 randomly selected high-power field (× 400) count of 500 cells per field, the positive cell count of more than 30% of the tissue sections as positive samples. (2) Ki67 proliferation index [3]: nuclei stained light yellow to brown defined as positive cells, 5 to 10 randomly selected high magnification (× 400) view count, counting 500 cells per field, calculated The percentage of Ki67 positive cells to total cell number, namely, the Ki67 proliferative index.

    1.5 statistical methods

    SPSS13.0 statistical software for statistical analysis the measurement column t test, the count column χ2 test, P <0.05 for the difference was statistically significant.

    2 Results

    2.1 Smac and Ki67 expression in thyroid carcinomas

    Smac expression in the cell cytoplasm, pale yellow to brownish yellow distribution, as shown in Figure 1a. 62 cases of positive expression in 87 cases of thyroid cancer tissues, the positive rate of 71.3%, 20 cases of tumor adjacent normal tissues were positive expression. The difference was statistically significant (P <0.05), Smac expression in thyroid cancer than normal tissue.

    Ki67 expression in the nucleus, pale yellow to brownish yellow distribution, as shown in Figure 1b. Ki67 proliferation index for the 87 cases of thyroid cancer tissues (42.74 ± 14.52), 20 patients with peritumoral normal tissues rare Ki67 expression.

    2.2 Smac expression relationship with clinicopathological parameters can be seen from Table 1, the Smc expression and gender, age, pathological type, primary is obviously related to the lesions and clinical stage (P <0.05), Smac in men and ≥ 45 years of age, papillary carcinoma and follicular carcinoma, the primary lesion beyond the envelope, and II, III, IV thyroid cancer expression significantly reduced. Table 1 Smac expression with clinicopathological indicators relationship

    2.3 Smac and Ki67 relationship

    Smac positive expression of Ki67 proliferation index (40.70 ± 14.68) of Smac negative expression group (47.78 ± 13.06), the two groups by the t-test the difference was statistically significant (P <0.05), Ki67 expression in the low the Smac-positive expression group .

    3 Discussion

    The balance of apoptosis and proliferation is the basis for normal growth and development of individual organisms, excessive cell proliferation and (or) blocked apoptosis is an important mechanism in the process of tumor formation. Smac is the recent discovery of a pro-apoptotic protein, along with cytochrome C (cyt c) release from mitochondria in apoptotic signal stimulation apaf  1 and the combined lift apoptosis inhibitory protein (IAPs by binding to and activation play a role) Inhibition of caspase  3,7,9 [2,4]. There are currently study pro-apoptotic activity of Smac build carrier overexpression of Smac sensitizing immune agents or chemotherapy drugs induced a variety of cancer cell apoptosis [4  5].

    Smac in carcinoma of the relevant literature has been reported, but uncommon its expression in thyroid cancer tissues and its expression with various clinical and pathological features article. Yoo et al [6] Smac different expression in different cancer or sarcoma study found decreased expression in a variety of cancer and sarcoma not even express. Bao et al [7] RT  PCR and SP method to detect liver cancer in of Smac mRNA and Smac protein expression was significantly lower (about 40%). Mizutani et al [5] confirmed Smac decreased expression in renal cell carcinoma, and staging, no expression of Smac prognosis is worse; Smac cDNA transfection to renal cell carcinoma cells enhances TRAIL and cisplatin-mediated cytotoxicity. Arellano  Llamas et al [8] study found that Smac expression in cervical adenocarcinoma compared with cervical squamous cell carcinoma, further analysis showed higher expression of Smac in local recurrence of cervical squamous cell carcinoma patients, can be used as prognostic indicators. This study demonstrated that the Smac positive expression in 71.3% (62/87) thyroid carcinoma, further analysis showed that the expression of Smac primary lesion and clinical stage significantly correlated with gender, age, pathological type, suggesting that Smac expression in thyroid cancer may play an important role in the development occurs. Tirrò et al [9] found performing thyroid cancer chemotherapy drug resistance mechanisms, Taxol still alive after 48h thyroid cancer decreased expression of Smac, and only a very small amount of Smac was released from mitochondria. while the over-expression of Smac promotes the cytotoxic effects of Taxol, the study confirmed Smac pro-apoptotic role, but also provides a new approach to the treatment of certain patients with thyroid cancer.

    The Ki67 Is the current study a broader indicator of cell proliferation involved in tumor development, can be expressed in the G0 phase of cell cycle phase. Ki67 expression relationship with thyroid cancer tissue has been reported, but its significance is not yet sure. Cheng Jimin et al [10] found that Ki67 expression was elevated in thyroid cancer, associated with the pathological type. Tisell [3] study found that Ki67 expression elevated in medullary thyroid carcinoma, and metastasis in patients with relapse, age and prognosis. Mar [11] that Ki67 expression in thyroid follicular carcinoma and follicular adenoma did not differ insufficient to help distinguish mild capsule or vascular invasion follicular carcinoma. The study found that increased Ki67 proliferation index in thyroid cancer, and Smac expression was negatively correlated, suggesting that the two may be in the development of thyroid cancer may have a synergistic effect.

    Summary of Smac expression in thyroid carcinomas decreased apoptosis of thyroid cancer cells may be reduced by inhibition of the activity of IAPs with increased Ki67 proliferation index may have a synergistic effect to promote tumor development; Smac decreased expression primary lesions and clinical stage significantly associated with gender, age, histological type, detect its expression to help determine the clinical and pathological features.