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Expression and clinical significance of galectin-3 in osteosarcoma
GENE-39640; No. of pages: 5; 4C: Gene xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Expression and clinical significance of galectin-3 in osteosarcoma
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Xuesong Zhou, Jie Jing, Wen Mao, Yizhou Zheng, Du Wang, Xin Wang, Zhiming Liu, Xiangming Zhang ⁎
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Department of Orthopaedics, The Third Hospital of Wuhan, 241# Pengliuyang Rd, Wuchang District, Wuhan, Hubei Province 430060, PR China
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Article history: Received 27 December 2013 Received in revised form 23 April 2014 Accepted 28 April 2014 Available online xxxx
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Keywords: Expression Galectin-3 Immunohistochemistry Osteosarcoma Serum
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Galectin-3 is a multifunctional β-galactoside-binding protein which has been shown to play a role in carcinogenesis. However, the involvement of galectin-3 in osteosarcoma remains unclear. In this study, we aimed to examine the serum level of galectin-3 in osteosarcoma patients and healthy controls, and the protein expression of galectin-3 in osteosarcoma tissues and their adjacent non-malignant tissues. We further aimed to investigate the clinical significance of galectin-3 serum and protein expression levels. Galectin-3 serum level was evaluated using ELISA in 132 osteosarcoma patients and 184 healthy controls, while the protein expression of galectin-3 was determined using immunohistochemistry in the malignant and the surrounding non-malignant tissues of the same 132 osteosarcoma patients. Our results showed that the mean galectin-3 serum level was significantly higher in patients than in controls (2.35 ± 0.91 ng/ml vs. 0.86 ± 0.20 ng/ml) (p b 0.0001). Among patients, a higher galectin-3 serum level was significantly associated with the Enneking stage of cancer (p b 0.0001). In addition, we found a significant overrepresentation of high galectin-3 expression in osteosarcoma tissues than in non-malignant tissues (p b 0.0001). Galectin-3 expression in osteosarcoma tissues was also found to be correlated with the Enneking stage of cancer (p b 0.0001) and the occurrence of metastasis (p b 0.0001). In conclusion, galectin-3 could serve as a useful prognostic marker in osteosarcoma. © 2014 Published by Elsevier B.V.
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1. Introduction
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Osteosarcoma is the most common type of cancer involving the human skeletal system. In China, the rate of osteosarcoma is 0.5 incidences per 100,000 individuals (Zhou and Zhang, 2010). The cancer accounts for about 51% of the incidence of childhood cancer, with the majority (50–70%) occur to adolescents of 10–20 years old (Zhou and Zhang, 2010). Generally, the symptoms of osteosarcoma are mild at early stages, but the disease is highly aggressive. As such, nearly 75% of the patients are diagnosed at a more advanced stage of cancer, i.e. Enneking stage IIB or later (Zhou and Zhang, 2010). With modern surgical technology, the average five-year survival rate of osteosarcoma is relatively high at around 66% (Aljubran et al., 2009). However, the survival rate drops to merely 25% with the occurrence of metastasis, primarily lung metastasis, which remains the most significant burden in osteosarcoma-related deaths (Gorlick et al., 2003; Ta et al., 2009). Therefore, identification of biomarkers of the cancer is crucial for the diagnosis, treatment and management of osteosarcoma patients. Galectin-3 is a member of the lectin family, which comprised of carbohydrate-binding proteins with multiple biological functions. The protein is encoded by the LGALS3 gene on chromosome 14q21–q22 (Raimond et al., 1997). Galectin-3 has been thought to play a role in
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Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; HSD, honest significant difference; IHC, immunohistochemistry. ⁎ Corresponding author. E-mail address: [email protected] (X. Zhang).
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carcinogenesis, as it is involved in a variety of cancer-related physiological and pathological processes, including cell growth, apoptosis, cell adhesion, and angiogenesis. Previous studies have shown that inhibition of galectin-3 expression can suppress the growth of human breast cancer (Honjo et al., 2001), and forced expression of galectin-3 can help diffuse large B-cell lymphoma cells to resist apoptosis (Hoyer et al., 2004). Several groups have also found a correlation between galectin-3 expression and clinicopathological characteristics of various cancers (Castronovo et al., 1996; Cheng et al., 2004; Endo et al., 2005; Inohara et al., 1999; Lee et al., 2006; Takenaka et al., 2003; van den Brûle et al., 1995; Zhou et al., 2011). However, there are disagreements between the results reported in different cancers, and little is known about the relationship between galectin-3 expression and its clinical value in osteosarcoma. Therefore, this study intended to determine the clinical significance of galectin-3 serum and tissue expression levels in osteosarcoma.
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2. Materials and methods
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2.1. Serum and tissue samples
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The study was approved by the institutional review board of Wuhan University. Sample collection was performed with informed consent. One hundred and thirty two (132) histopathologically confirmed osteosarcoma patients from Department of Orthopaedics, The Third Hospital of Wuhan who had received no therapy before sample collection were enrolled into the study. The mean age of the patients was 18.6 years
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http://dx.doi.org/10.1016/j.gene.2014.04.066 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
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2.3. Immunohistochemistry
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Immunohistochemistry was performed using UltraSensitive™ SP IHC Kit (Maxim Biotech, Fuzhou, China). Tissue slides were hydrated by conventional dewaxing. The paraffin-embedded tissue sections were then incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. The slides were incubated with normal goat serum for 20 min at room temperature. The slides were then incubated overnight at 4 °C with rabbit anti-human galectin-3 primary antibody (Mshar Bioscience & Technology, Shanghai, China). Specimen with PBS instead of the primary antibody was used as the negative control. The tissues were equilibrated to 37 °C for 45 min, and washed with PBS. The sample was then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody for 4 min at room temperature and allowed for color development. The sections were then hematoxylin-stained, dehydrated, transparentizing in xylene, and observed under an optical microscope. Expression of galectin-3 was evaluated using H score.
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2.4. Statistical analysis
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The difference between galectin-3 serum levels in osteosarcoma patients and healthy controls was determined by using t-test. On the other hand, the difference of galectin-3 protein expression in cancerous and normal tissues was compared by using a chi-square test. The relationships of galectin-3 serum level and protein expressions in patients with clinicopathological characteristics were determined by using one-way ANOVA, followed by Tukey's HSD test. p b 0.05 was considered significant.
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3. Results
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3.1. Galectin-3 serum levels in osteosarcoma patients and healthy controls
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The level of galectin-3 in the serum of the healthy controls varied between 0.0 and 2.14 ng/ml (median, 0.73 ng/ml), while the galectin-3 serum level in osteosarcoma patients varied between 0.43 and 3.70 ng/ml (median, 2.56 ng/ml). The mean galectin-3 serum level in the cancer patients (2.35 ± 0.91 ng/ml) was significantly higher than in the healthy controls (0.86 ± 0.20 ng/ml) (p b 0.0001) (Fig. 1). The maximum galectin-3 serum level in healthy controls was 2.14 ng/ml, and only 47 patients were below this reading. Therefore, with a threshold value of 2.14 ng/ml, the serum level allowed one to discriminate a group of osteosarcoma patients from healthy individuals with a 100% level of specificity and a 64.39% level of sensitivity.
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The serum samples were diluted 1:4 in sample diluent, and the galectin-3 concentration was measured by using Human Galectin-3 ELISA Kit (EIAab Science, Wuhan, China) according to the manufacturer's instructions. Three replicates were done for each sample. A standard curve ranging from 0 to 4 ng/ml of galectin-3 was generated for the ELISA. Galectin-3 sample concentration was determined based on the standard curve generated, by measuring the optical density of each well at 450 nm, and multiplied by the dilution factor.
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Fig. 1. Galectin-3 serum level in osteosarcoma patients and healthy controls. The mean galectin-3 serum level in the cancer patients was 2.35 ± 0.91 ng/ml, which was significantly higher than that in the healthy controls (0.86 ± 0.20 ng/ml) (p b 0.0001).
3.2. Clinical significance of serum galectin-3 level
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The relationship between galectin-3 serum level in osteosarcoma patients and clinicopathological characteristics is shown in Table 1. No significant difference was found between the mean galectin-3 serum levels of different age groups (≤20 years old and N 20 years old) (p = 0.14), genders (males and females) (p = 0.88), sites of cancer (femur, tibia, humerus, fibula, and others) (p = 0.33), histopathology of cancer (osteoblastic, fibroblastic, chondroblastic, telangiectatic, and others) (p = 0.97) and the incidence of metastasis (no metastasis, lung metastasis and metastasis to other organs) (p = 0.26). However, significant difference was observed for the mean galectin-3 serum levels of different Enneking stages (p b 0.0001). Tukey's test showed a significant correlation between more advanced Enneking stages with higher mean serum levels of galectin-3 (p b 0.0001).
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(ranging from 7 to 41 years), comprising of 76 (57.58%) males and 56 (42.42%) females. Cancerous osteosarcoma tissues and the surrounding non-malignant tissues were collected from the patients for immunohistochemical examination of galectin-3 protein expression. Blood samples were also collected from the patients, in addition to 184 healthy controls. The mean age of the controls was 19.1 years (ranging from 12 to 43), comprising 101 (54.89%) males and 83 (45.11%) females. Serum was isolated from the blood samples collected and used for ELISA quantification of galectin-3.
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Age group ≤20 years N20 years Gender Male Female Enneking stage IIA IIB III Site Femur Tibia Humerus Fibula Others Histopathology Osteoblastic Fibroblastic Chondroblastic Telangiectatic Others Metastasis No Lung Others
Galectin-3 serum level
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0.88 2.34 ± 0.90 2.36 ± 0.91 b0.0001 1.53 ± 0.93 2.24 ± 0.62 3.08 ± 0.71 0.33 ± ± ± ± ±
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± ± ± ± ±
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Table 1 Clinical significance of serum galectin-3 level in osteosarcoma patients. Characteristic
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0.26 2.29 ± 0.90 2.47 ± 0.97 2.97 ± 0.06
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
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3.3. Immunohistochemical analysis of galectin-3
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Immunohistochemistry was performed for osteosarcoma tissues and the surrounding non-malignant osteoblastic tissues. Our results showed that the median H score for osteosarcoma tissues was 95, while that for normal tissue was 39. The mean scores for osteosarcoma and normal tissues were 100.90 and 40.64 respectively, and this difference was statistically significant (p b 0.0001). When H scores of ≤100 were categorized as low expression and H N 100 as high expression (Fig. 2), 124 normal tissues fell into the low expression group and only 8 belonged to the high expression group. However, for cancerous tissues, 72 fell into the low expression category and 60 into the high expression category. The difference was statistically significant (p b 0.0001).
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3.4. Clinical significance of galectin-3 expression
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The clinical significance of galectin-3 expression in osteosarcoma tissues is shown in Table 2. A significant association was observed between high galectin-3 expression and Enneking stage (p b 0.0001) as well as metastasis (p b 0.0001). No significant difference was found between the high and weak expression categories in terms of age group (p = 0.63), gender (p = 0.72), and site and histopathology of osteosarcoma (p = 0.22 and p = 0.94 respectively).
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4. Discussion
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Galectin-3 is a β-galactoside-binding protein which plays a role in variety of biological processes, including cell growth and differentiation, cell adhesion, and apoptosis. The role of galectin-3 in these biological processes implicates the protein to the process of cellular malignant transformation. Thus, several studies have investigated and established
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0.63 44 (60.27%) 29 (39.73%)
38 (64.41%) 21 (35.59%)
41 (56.16%) 32 (43.84%)
35 (59.32%) 24 (40.68%)
19 (26.03%) 42 (57.53%) 12 (16.44%)
8 (13.56%) 23 (38.98%) 28 (47.46%)
28 (38.36%) 11 (15.07%) 11 (15.07%) 10 (13.70%) 13 (17.81%)
15 (25.42%) 9 (15.25%) 13 (22.03%) 15 (25.42%) 7 (11.86%)
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23 (31.51%) 23 (31.51%) 13 (17.81%) 6 (8.22%) 8 (10.96%)
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69 (94.52%) 3 (4.11%) 1 (1.37%)
0.94 16 (27.12%) 21 (35.59%) 11 (18.64%) 6 (10.17%) 5 (8.47%) b0.001 34 (57.63%) 22 (37.29%) 3 (5.08%)
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the prognostic importance of galectin-3 expression in different cancers, including cervical cancer (Lee et al., 2006; Zhou et al., 2011), thyroid cancer (Inohara et al., 1999; Takenaka et al., 2003), colorectal cancer (Endo et al., 2005), melanoma (van den Brûle et al., 1995), breast cancer (Castronovo et al., 1996) and gastric cancer (Cheng et al., 2004). However, there have been contradicting findings in this aspect, with some
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High expression, N = 59
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Low expression, N = 73
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Age group ≤20 years N20 years Gender Male Female Enneking stage IIA IIB III Site Femur Tibia Humerus Fibula Others Histopathology Osteoblastic Fibroblastic Chondroblastic Telangiectatic Others Metastasis No Lung Others
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Characteristic
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Table 2 Clinical significance of galectin-3 expression in osteosarcoma tissues.
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Fig. 2. Galectin-3 protein expression in osteosarcoma tissues. The tissues were categorized into low or high expression groups based on their H scores (≤100 and H N 100 respectively). (A) Low expression. (B) High expression.
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29
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The authors declare that they have no conflict of interest.
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Acknowledgments
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The study was supported by a grant from the Wuhan City Health Bureau. The funding agency has no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. The authors thank the doctors who helped in sample collection and the subjects who participated in this study.
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Our results showed that the serum expression level of galectin-3 were significantly higher in osteosarcoma patients than in healthy controls, and the tissue expression levels of galectin-3 were significantly higher in the osteosarcoma tissues than in the adjacent non-malignant tissues. We also demonstrated a correlation between galectin-3 serum and protein expression levels with poorer prognosis in osteosarcoma. Therefore, galectin-3 could serve as a useful biomarker for the evaluation of osteosarcoma progression.
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studies suggested that an increased expression of galectin-3 leads to a poorer prognosis, while some others showed otherwise. The inconsistent finding suggests that the implication of galectin-3 expression in carcinogenesis is cancer-specific. An investigation on the expression and clinical significance of galectin-3 in osteosarcoma is therefore important for the management of the disease. In this study, we present the first report on the serum and protein expression levels of galectin3 in osteosarcoma and its correlation with clinicopathological characteristics of the cancer. We demonstrated that the serum level of galectin-3 was significantly higher in osteosarcoma patients than in healthy individuals. Additionally, we found an increasingly higher galectin-3 serum level as the disease progressed into more advanced stages. These findings indicate the possibility that measurement of serum galectin-3 level could serve as a clinically useful marker in osteosarcoma. The galectin-3 protein expression in osteosarcoma tissues was also found to be significantly higher than in the surrounding non-malignant tissues, and the expression was positively correlated with the Enneking stage of cancer and the incidence of metastasis. This suggests the role of galectin-3 as an important protein that promotes cancer progression and metastasis. It has been shown previously that a possible mechanism for the enhanced expression of galectin-3 is through its interaction with common gammachain signaling molecules (Joo et al., 2001). One class of such gammachain signaling molecules is the various inflammatory cytokines involved in carcinogenesis (Joo et al., 2001). Therefore, we propose that the complex interactions between galectin-3 and inflammatory cytokines led to its overexpression in malignant tissues, which in turn contribute to disease progression by various mechanisms such as through the mediation of cell growth and apoptosis. Apart from interacting with inflammatory cytokines, galectin-3 itself plays a vital role in leukocyte trafficking and activation, which in turn facilitate the release of more inflammatory cytokines to the cancer microenvironment (Gao et al., 2013). In fact, previous studies have shown that galectin-3 could boost the growth, and enhance the anti-apoptosis potential, of cancerous cells (Yang et al., 1996). This is because galectin-3 shares sequence similarity with Bcl-2, and interacts with the latter, which allows the former to effectively suppress cell death and regulate cell growth (Yang et al., 1996). In addition, galectin-3 has also been shown to interact with various components of K-ras signaling pathway, which further implicates a role for the protein in cancer development (Dumic et al., 2006; Shimura et al., 2004). Besides, the observation that galectin-3 expression was significantly correlated to the incidence of metastasis can be explained by the fact that galectin-3 plays an important role in cell–cell adhesion. The elevated levels of galectin-3 expression in osteosarcoma therefore allow the metastatic cells to have a greater adhesion potential to their target organ, hence facilitating metastasis (Pacis et al., 2000). Our findings were in agreement with several previous reports on other cancers. For example, Camby et al. (2001) also found a correlation between levels of galectin-3 expression and the progression of astrocytic glioma. In addition, galectin-3 was found to be lower in the noninvasive parts of xenografted glioblastomas than in the invasive parts, and was shown to induce cell migration in vitro (Camby et al., 2001). On the other hand, Miyazaki et al. (2002) observed a significantly higher galectin-3 expression in gastric cancer compared to the adjacent gastric tissues, and a correlation of galectin-3 expression level with lymph node metastasis. Endo et al. (2005) also reported a prognostic value of galectin3 expression in colorectal cancer. The involvement of galectin-3 in cancer progression and metastasis could be due to its role in regulating angiogenesis and the anti-death activity of cancer cells (Nangia-Makker et al., 2000, 2008). Angiogenesis may reduce the adhesion of cancer cells, which allows local invasiness and metastatic spread of the cells (Tromp et al., 2000). On the other hand, anti-death activity mediated by galectin-3 is critical for the survival of metastatic cells in an anchorage-independent condition in the circulation (Portt et al., 2011). In conclusion, this is the first study demonstrating the clinical significance of galectin-3 serum level and tissue expression in osteosarcoma.
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Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Expression and clinical significance of galectin-3 in osteosarcoma
2Q1
Xuesong Zhou, Jie Jing, Wen Mao, Yizhou Zheng, Du Wang, Xin Wang, Zhiming Liu, Xiangming Zhang ⁎
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Department of Orthopaedics, The Third Hospital of Wuhan, 241# Pengliuyang Rd, Wuchang District, Wuhan, Hubei Province 430060, PR China
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Article history: Received 27 December 2013 Received in revised form 23 April 2014 Accepted 28 April 2014 Available online xxxx
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Keywords: Expression Galectin-3 Immunohistochemistry Osteosarcoma Serum
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Galectin-3 is a multifunctional β-galactoside-binding protein which has been shown to play a role in carcinogenesis. However, the involvement of galectin-3 in osteosarcoma remains unclear. In this study, we aimed to examine the serum level of galectin-3 in osteosarcoma patients and healthy controls, and the protein expression of galectin-3 in osteosarcoma tissues and their adjacent non-malignant tissues. We further aimed to investigate the clinical significance of galectin-3 serum and protein expression levels. Galectin-3 serum level was evaluated using ELISA in 132 osteosarcoma patients and 184 healthy controls, while the protein expression of galectin-3 was determined using immunohistochemistry in the malignant and the surrounding non-malignant tissues of the same 132 osteosarcoma patients. Our results showed that the mean galectin-3 serum level was significantly higher in patients than in controls (2.35 ± 0.91 ng/ml vs. 0.86 ± 0.20 ng/ml) (p b 0.0001). Among patients, a higher galectin-3 serum level was significantly associated with the Enneking stage of cancer (p b 0.0001). In addition, we found a significant overrepresentation of high galectin-3 expression in osteosarcoma tissues than in non-malignant tissues (p b 0.0001). Galectin-3 expression in osteosarcoma tissues was also found to be correlated with the Enneking stage of cancer (p b 0.0001) and the occurrence of metastasis (p b 0.0001). In conclusion, galectin-3 could serve as a useful prognostic marker in osteosarcoma. © 2014 Published by Elsevier B.V.
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1. Introduction
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Osteosarcoma is the most common type of cancer involving the human skeletal system. In China, the rate of osteosarcoma is 0.5 incidences per 100,000 individuals (Zhou and Zhang, 2010). The cancer accounts for about 51% of the incidence of childhood cancer, with the majority (50–70%) occur to adolescents of 10–20 years old (Zhou and Zhang, 2010). Generally, the symptoms of osteosarcoma are mild at early stages, but the disease is highly aggressive. As such, nearly 75% of the patients are diagnosed at a more advanced stage of cancer, i.e. Enneking stage IIB or later (Zhou and Zhang, 2010). With modern surgical technology, the average five-year survival rate of osteosarcoma is relatively high at around 66% (Aljubran et al., 2009). However, the survival rate drops to merely 25% with the occurrence of metastasis, primarily lung metastasis, which remains the most significant burden in osteosarcoma-related deaths (Gorlick et al., 2003; Ta et al., 2009). Therefore, identification of biomarkers of the cancer is crucial for the diagnosis, treatment and management of osteosarcoma patients. Galectin-3 is a member of the lectin family, which comprised of carbohydrate-binding proteins with multiple biological functions. The protein is encoded by the LGALS3 gene on chromosome 14q21–q22 (Raimond et al., 1997). Galectin-3 has been thought to play a role in
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Abbreviations: ANOVA, analysis of variance; ELISA, enzyme-linked immunosorbent assay; HSD, honest significant difference; IHC, immunohistochemistry. ⁎ Corresponding author. E-mail address: [email protected] (X. Zhang).
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carcinogenesis, as it is involved in a variety of cancer-related physiological and pathological processes, including cell growth, apoptosis, cell adhesion, and angiogenesis. Previous studies have shown that inhibition of galectin-3 expression can suppress the growth of human breast cancer (Honjo et al., 2001), and forced expression of galectin-3 can help diffuse large B-cell lymphoma cells to resist apoptosis (Hoyer et al., 2004). Several groups have also found a correlation between galectin-3 expression and clinicopathological characteristics of various cancers (Castronovo et al., 1996; Cheng et al., 2004; Endo et al., 2005; Inohara et al., 1999; Lee et al., 2006; Takenaka et al., 2003; van den Brûle et al., 1995; Zhou et al., 2011). However, there are disagreements between the results reported in different cancers, and little is known about the relationship between galectin-3 expression and its clinical value in osteosarcoma. Therefore, this study intended to determine the clinical significance of galectin-3 serum and tissue expression levels in osteosarcoma.
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2. Materials and methods
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2.1. Serum and tissue samples
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The study was approved by the institutional review board of Wuhan University. Sample collection was performed with informed consent. One hundred and thirty two (132) histopathologically confirmed osteosarcoma patients from Department of Orthopaedics, The Third Hospital of Wuhan who had received no therapy before sample collection were enrolled into the study. The mean age of the patients was 18.6 years
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http://dx.doi.org/10.1016/j.gene.2014.04.066 0378-1119/© 2014 Published by Elsevier B.V.
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
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2.3. Immunohistochemistry
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Immunohistochemistry was performed using UltraSensitive™ SP IHC Kit (Maxim Biotech, Fuzhou, China). Tissue slides were hydrated by conventional dewaxing. The paraffin-embedded tissue sections were then incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. The slides were incubated with normal goat serum for 20 min at room temperature. The slides were then incubated overnight at 4 °C with rabbit anti-human galectin-3 primary antibody (Mshar Bioscience & Technology, Shanghai, China). Specimen with PBS instead of the primary antibody was used as the negative control. The tissues were equilibrated to 37 °C for 45 min, and washed with PBS. The sample was then incubated with horseradish peroxidase (HRP)-labeled goat anti-rabbit secondary antibody for 4 min at room temperature and allowed for color development. The sections were then hematoxylin-stained, dehydrated, transparentizing in xylene, and observed under an optical microscope. Expression of galectin-3 was evaluated using H score.
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2.4. Statistical analysis
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The difference between galectin-3 serum levels in osteosarcoma patients and healthy controls was determined by using t-test. On the other hand, the difference of galectin-3 protein expression in cancerous and normal tissues was compared by using a chi-square test. The relationships of galectin-3 serum level and protein expressions in patients with clinicopathological characteristics were determined by using one-way ANOVA, followed by Tukey's HSD test. p b 0.05 was considered significant.
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3. Results
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3.1. Galectin-3 serum levels in osteosarcoma patients and healthy controls
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The level of galectin-3 in the serum of the healthy controls varied between 0.0 and 2.14 ng/ml (median, 0.73 ng/ml), while the galectin-3 serum level in osteosarcoma patients varied between 0.43 and 3.70 ng/ml (median, 2.56 ng/ml). The mean galectin-3 serum level in the cancer patients (2.35 ± 0.91 ng/ml) was significantly higher than in the healthy controls (0.86 ± 0.20 ng/ml) (p b 0.0001) (Fig. 1). The maximum galectin-3 serum level in healthy controls was 2.14 ng/ml, and only 47 patients were below this reading. Therefore, with a threshold value of 2.14 ng/ml, the serum level allowed one to discriminate a group of osteosarcoma patients from healthy individuals with a 100% level of specificity and a 64.39% level of sensitivity.
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The serum samples were diluted 1:4 in sample diluent, and the galectin-3 concentration was measured by using Human Galectin-3 ELISA Kit (EIAab Science, Wuhan, China) according to the manufacturer's instructions. Three replicates were done for each sample. A standard curve ranging from 0 to 4 ng/ml of galectin-3 was generated for the ELISA. Galectin-3 sample concentration was determined based on the standard curve generated, by measuring the optical density of each well at 450 nm, and multiplied by the dilution factor.
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Fig. 1. Galectin-3 serum level in osteosarcoma patients and healthy controls. The mean galectin-3 serum level in the cancer patients was 2.35 ± 0.91 ng/ml, which was significantly higher than that in the healthy controls (0.86 ± 0.20 ng/ml) (p b 0.0001).
3.2. Clinical significance of serum galectin-3 level
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The relationship between galectin-3 serum level in osteosarcoma patients and clinicopathological characteristics is shown in Table 1. No significant difference was found between the mean galectin-3 serum levels of different age groups (≤20 years old and N 20 years old) (p = 0.14), genders (males and females) (p = 0.88), sites of cancer (femur, tibia, humerus, fibula, and others) (p = 0.33), histopathology of cancer (osteoblastic, fibroblastic, chondroblastic, telangiectatic, and others) (p = 0.97) and the incidence of metastasis (no metastasis, lung metastasis and metastasis to other organs) (p = 0.26). However, significant difference was observed for the mean galectin-3 serum levels of different Enneking stages (p b 0.0001). Tukey's test showed a significant correlation between more advanced Enneking stages with higher mean serum levels of galectin-3 (p b 0.0001).
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(ranging from 7 to 41 years), comprising of 76 (57.58%) males and 56 (42.42%) females. Cancerous osteosarcoma tissues and the surrounding non-malignant tissues were collected from the patients for immunohistochemical examination of galectin-3 protein expression. Blood samples were also collected from the patients, in addition to 184 healthy controls. The mean age of the controls was 19.1 years (ranging from 12 to 43), comprising 101 (54.89%) males and 83 (45.11%) females. Serum was isolated from the blood samples collected and used for ELISA quantification of galectin-3.
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Age group ≤20 years N20 years Gender Male Female Enneking stage IIA IIB III Site Femur Tibia Humerus Fibula Others Histopathology Osteoblastic Fibroblastic Chondroblastic Telangiectatic Others Metastasis No Lung Others
Galectin-3 serum level
0.14
0.88 2.34 ± 0.90 2.36 ± 0.91 b0.0001 1.53 ± 0.93 2.24 ± 0.62 3.08 ± 0.71 0.33 ± ± ± ± ±
0.95 0.82 1.04 0.84 0.77
2.30 2.30 2.42 2.45 2.41
± ± ± ± ±
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t1:3
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Table 1 Clinical significance of serum galectin-3 level in osteosarcoma patients. Characteristic
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0.26 2.29 ± 0.90 2.47 ± 0.97 2.97 ± 0.06
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14 t1:15 t1:16 t1:17 t1:18 t1:19 t1:20 t1:21 t1:22 t1:23 t1:24 t1:25 t1:26 t1:27 t1:28 t1:29
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3.3. Immunohistochemical analysis of galectin-3
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Immunohistochemistry was performed for osteosarcoma tissues and the surrounding non-malignant osteoblastic tissues. Our results showed that the median H score for osteosarcoma tissues was 95, while that for normal tissue was 39. The mean scores for osteosarcoma and normal tissues were 100.90 and 40.64 respectively, and this difference was statistically significant (p b 0.0001). When H scores of ≤100 were categorized as low expression and H N 100 as high expression (Fig. 2), 124 normal tissues fell into the low expression group and only 8 belonged to the high expression group. However, for cancerous tissues, 72 fell into the low expression category and 60 into the high expression category. The difference was statistically significant (p b 0.0001).
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3.4. Clinical significance of galectin-3 expression
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The clinical significance of galectin-3 expression in osteosarcoma tissues is shown in Table 2. A significant association was observed between high galectin-3 expression and Enneking stage (p b 0.0001) as well as metastasis (p b 0.0001). No significant difference was found between the high and weak expression categories in terms of age group (p = 0.63), gender (p = 0.72), and site and histopathology of osteosarcoma (p = 0.22 and p = 0.94 respectively).
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4. Discussion
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Galectin-3 is a β-galactoside-binding protein which plays a role in variety of biological processes, including cell growth and differentiation, cell adhesion, and apoptosis. The role of galectin-3 in these biological processes implicates the protein to the process of cellular malignant transformation. Thus, several studies have investigated and established
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0.63 44 (60.27%) 29 (39.73%)
38 (64.41%) 21 (35.59%)
41 (56.16%) 32 (43.84%)
35 (59.32%) 24 (40.68%)
19 (26.03%) 42 (57.53%) 12 (16.44%)
8 (13.56%) 23 (38.98%) 28 (47.46%)
28 (38.36%) 11 (15.07%) 11 (15.07%) 10 (13.70%) 13 (17.81%)
15 (25.42%) 9 (15.25%) 13 (22.03%) 15 (25.42%) 7 (11.86%)
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b0.0001
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23 (31.51%) 23 (31.51%) 13 (17.81%) 6 (8.22%) 8 (10.96%)
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69 (94.52%) 3 (4.11%) 1 (1.37%)
0.94 16 (27.12%) 21 (35.59%) 11 (18.64%) 6 (10.17%) 5 (8.47%) b0.001 34 (57.63%) 22 (37.29%) 3 (5.08%)
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the prognostic importance of galectin-3 expression in different cancers, including cervical cancer (Lee et al., 2006; Zhou et al., 2011), thyroid cancer (Inohara et al., 1999; Takenaka et al., 2003), colorectal cancer (Endo et al., 2005), melanoma (van den Brûle et al., 1995), breast cancer (Castronovo et al., 1996) and gastric cancer (Cheng et al., 2004). However, there have been contradicting findings in this aspect, with some
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High expression, N = 59
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Low expression, N = 73
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Age group ≤20 years N20 years Gender Male Female Enneking stage IIA IIB III Site Femur Tibia Humerus Fibula Others Histopathology Osteoblastic Fibroblastic Chondroblastic Telangiectatic Others Metastasis No Lung Others
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Characteristic
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Table 2 Clinical significance of galectin-3 expression in osteosarcoma tissues.
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Fig. 2. Galectin-3 protein expression in osteosarcoma tissues. The tissues were categorized into low or high expression groups based on their H scores (≤100 and H N 100 respectively). (A) Low expression. (B) High expression.
Please cite this article as: Zhou, X., et al., Expression and clinical significance of galectin-3 in osteosarcoma, Gene (2014), http://dx.doi.org/ 10.1016/j.gene.2014.04.066
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29
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The authors declare that they have no conflict of interest.
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Acknowledgments
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The study was supported by a grant from the Wuhan City Health Bureau. The funding agency has no role in study design, in the collection, analysis and interpretation of data, in the writing of the report, and in the decision to submit the article for publication. The authors thank the doctors who helped in sample collection and the subjects who participated in this study.
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References
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Aljubran, A.H., Griffin, A., Pintilie, M., Blackstein, M., 2009. Osteosarcoma in adolescents and adults: survival analysis with and without lung metastases. Annals of Oncology 20 (6), 1136–1141. Camby, I., Belot, N., Rorive, S., Lefranc, F., Maurage, C.A., Lahm, H., Kaltner, H., Hadari, Y., Ruchoux, M.M., Brotchi, J., Zick, Y., Salmon, I., Gabius, H.J., Kiss, R., 2001. Galectins are differentially expressed in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas and glioblastomas, and significantly modulate tumor astrocyte migration. Brain Pathology 11 (1), 12–26. Castronovo, V., van den Brûle, F.A., Jackers, P., Clausse, N., Liu, F.T., Gillet, C., Sobel, M.E., 1996. Decreased expression of galectin-3 is associated with progression of human breast cancer. The Journal of Pathology 179, 43–48. Cheng, C.S., Dong, W.G., Luo, H.S., Yu, B.P., 2004. Expression of galectin-3 in gastric carcinoma and its significance. Chinese Journal of Cancer Research 16 (3), 176–181. Dumic, J., Dabelic, S., Flogel, M., 2006. Galectin-3: an open-ended story. Biochimica et Biophysica Acta 1760, 616–635. Endo, K., Kohnoe, S., Tsujita, E., Watanabe, A., Nakashima, H., Baba, H., Maehara, Y., 2005. Galectin-3 expression is a potent prognostic marker in colorectal cancer. Anticancer Research 25, 3117–3122. Gao, P., Simpson, J.L., Zhang, J., Gibson, P.G., 2013. Galectin-3: its role in asthma and potential as an anti-inflammatory target. Respiratory Research 14, 136. Gorlick, R., Anderson, P., Andrulis, I., 2003. Biology of childhood osteogenic sarcoma and potential targets for therapeutic development: meeting summary. Clinical Cancer Research 9, 5442–5453. Honjo, Y., Nangia-Makker, P., Inohara, H., Raz, A., 2001. Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells. Clinical Cancer Research 7 (3), 661–668. Hoyer, K.K., Pang, M., Gui, D., Shintaku, I.P., Kuwabara, I., Liu, F.T., Said, J.W., Baum, L.G., Teitell, M.A., 2004. An anti-apoptotic role for galectin-3 in diffuse large B-cell lymphomas. The American Journal of Pathology 164 (3), 893–902. Inohara, H., Honjo, Y., Yoshii, T., Akahani, S., Yoshida, J., Hattori, K., Okamoto, S., Sawada, T., Raz, A., Kubo, T., 1999. Expression of galectin-3 in fine-needle aspirates as a diagnostic marker differentiating benign from malignant thyroid neoplasms. Cancer 85, 2475–2484. Joo, H.G., Goedegebuure, P.S., Sadanaga, N., Nagoshi, M., von Bernstorff, W., Eberlein, T.J., 2001. Expression and function of galectin-3, a β-galactoside-binding protein in activated T lymphocytes. Journal of Leukocyte Biology 69, 555–564. Lee, J.W., Song, S.Y., Choi, J.J., Choi, C.H., Kim, J., Lee, J.H., Kim, B.G., Bae, D.S., 2006. Decreased galectin-3 expression during the progression of cervical neoplasia. Journal of Cancer Research and Clinical Oncology 132, 241–247. Miyazaki, J., Hokari, R., Kato, S., Tsuzuki, Y., Kawaguchi, A., Nagao, S., Itoh, K., Miura, S., 2002. Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes. Oncology Reports 9 (6), 1307–1312. Nangia-Makker, P., Honjo, Y., Sarvis, R., Akahani, S., Hogan, V., Pienta, K.J., Raz, A., 2000. Galectin-3 induces endothelial cell morphogenesis and angiogenesis. The American Journal of Pathology 156 (3), 899–909. Nangia-Makker, P., Balan, V., Raz, A., 2008. Regulation of tumor progression by extracellular galectin-3. Cancer Microenvironment 1 (1), 43–51. Pacis, R.A., Pilat, M.J., Pienta, K.J., Wojno, K., Raz, A., Hogan, V., Cooper, C.R., 2000. Decreased galectin-3 expression in prostate cancer. The Prostate 44, 118–123. Portt, L., Norman, G., Clapp, C., Greenwood, M., Greenwood, M.T., 2011. Anti-apoptosis and cell survival: a review. Biochimica et Biophysica Acta 1813 (1), 238–259. Raimond, J., Zimonjic, D.B., Mignon, C., Mattei, M., Popescu, N.C., Monsigny, M., Legrand, A., 1997. Mapping of the galectin-3 gene (LGALS3) to human chromosome 14 at region 14q21–22. Mammalian Genome 8 (9), 706–707.
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Our results showed that the serum expression level of galectin-3 were significantly higher in osteosarcoma patients than in healthy controls, and the tissue expression levels of galectin-3 were significantly higher in the osteosarcoma tissues than in the adjacent non-malignant tissues. We also demonstrated a correlation between galectin-3 serum and protein expression levels with poorer prognosis in osteosarcoma. Therefore, galectin-3 could serve as a useful biomarker for the evaluation of osteosarcoma progression.
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studies suggested that an increased expression of galectin-3 leads to a poorer prognosis, while some others showed otherwise. The inconsistent finding suggests that the implication of galectin-3 expression in carcinogenesis is cancer-specific. An investigation on the expression and clinical significance of galectin-3 in osteosarcoma is therefore important for the management of the disease. In this study, we present the first report on the serum and protein expression levels of galectin3 in osteosarcoma and its correlation with clinicopathological characteristics of the cancer. We demonstrated that the serum level of galectin-3 was significantly higher in osteosarcoma patients than in healthy individuals. Additionally, we found an increasingly higher galectin-3 serum level as the disease progressed into more advanced stages. These findings indicate the possibility that measurement of serum galectin-3 level could serve as a clinically useful marker in osteosarcoma. The galectin-3 protein expression in osteosarcoma tissues was also found to be significantly higher than in the surrounding non-malignant tissues, and the expression was positively correlated with the Enneking stage of cancer and the incidence of metastasis. This suggests the role of galectin-3 as an important protein that promotes cancer progression and metastasis. It has been shown previously that a possible mechanism for the enhanced expression of galectin-3 is through its interaction with common gammachain signaling molecules (Joo et al., 2001). One class of such gammachain signaling molecules is the various inflammatory cytokines involved in carcinogenesis (Joo et al., 2001). Therefore, we propose that the complex interactions between galectin-3 and inflammatory cytokines led to its overexpression in malignant tissues, which in turn contribute to disease progression by various mechanisms such as through the mediation of cell growth and apoptosis. Apart from interacting with inflammatory cytokines, galectin-3 itself plays a vital role in leukocyte trafficking and activation, which in turn facilitate the release of more inflammatory cytokines to the cancer microenvironment (Gao et al., 2013). In fact, previous studies have shown that galectin-3 could boost the growth, and enhance the anti-apoptosis potential, of cancerous cells (Yang et al., 1996). This is because galectin-3 shares sequence similarity with Bcl-2, and interacts with the latter, which allows the former to effectively suppress cell death and regulate cell growth (Yang et al., 1996). In addition, galectin-3 has also been shown to interact with various components of K-ras signaling pathway, which further implicates a role for the protein in cancer development (Dumic et al., 2006; Shimura et al., 2004). Besides, the observation that galectin-3 expression was significantly correlated to the incidence of metastasis can be explained by the fact that galectin-3 plays an important role in cell–cell adhesion. The elevated levels of galectin-3 expression in osteosarcoma therefore allow the metastatic cells to have a greater adhesion potential to their target organ, hence facilitating metastasis (Pacis et al., 2000). Our findings were in agreement with several previous reports on other cancers. For example, Camby et al. (2001) also found a correlation between levels of galectin-3 expression and the progression of astrocytic glioma. In addition, galectin-3 was found to be lower in the noninvasive parts of xenografted glioblastomas than in the invasive parts, and was shown to induce cell migration in vitro (Camby et al., 2001). On the other hand, Miyazaki et al. (2002) observed a significantly higher galectin-3 expression in gastric cancer compared to the adjacent gastric tissues, and a correlation of galectin-3 expression level with lymph node metastasis. Endo et al. (2005) also reported a prognostic value of galectin3 expression in colorectal cancer. The involvement of galectin-3 in cancer progression and metastasis could be due to its role in regulating angiogenesis and the anti-death activity of cancer cells (Nangia-Makker et al., 2000, 2008). Angiogenesis may reduce the adhesion of cancer cells, which allows local invasiness and metastatic spread of the cells (Tromp et al., 2000). On the other hand, anti-death activity mediated by galectin-3 is critical for the survival of metastatic cells in an anchorage-independent condition in the circulation (Portt et al., 2011). In conclusion, this is the first study demonstrating the clinical significance of galectin-3 serum level and tissue expression in osteosarcoma.
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van den Brûle, F.A., Buicu, C., Sobel, M.E., Liu, F.T., Castronovo, V., 1995. Galectin-3, a laminin binding protein, fails to modulate adhesion of human melanoma cells to laminin. Neoplasma 42, 215–219. Yang, R.Y., Hsu, D.K., Liu, F.T., 1996. Expression of galectin-3 modulates T-cell growth and apoptosis. Proceedings of the National Academy of Sciences of the United States of America 93, 6737–6742. Zhou, X.S., Zhang, R.L., 2010. Fascin gene expression in osteosarcoma and statistical analysis. Journal of Mathematical Medicine 23 (5), 558–560 (in Chinese). Zhou, Y.Q., Zhou, X.K., Kong, Y.Y., Song, C.Y., Wu, X.H., 2011. Expression of galectin-3 in cervical cancer and its clinical significance. Tumor 31 (2), 148–153 (in Chinese).
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