Functions And Mechanisme Of Galectin-3Involved In Tongue Cancer Invasion And Metastasis

Objectives:Squamous cell carcinoma of tongue (TSCC) is one of the most common malignant neoplasms in oral cavity, accounting for approximately40%of all oral cancers in China. Despite the advances in the therapeutic management of TSCC over the last few decades, the5-year survival rate is still remaining unimproved. The most common reseaons of the failure of the therapy are tumor recurrence and local regional lymph node metastasis. Thus, studies of the mechanisms that involved in tumor cell migration and invasion have become foci of this field. Galectin-3(Gal-3), a member of β-galactoside-binding protein family, has been reportedly implicated in diverse biological functions including tumor cell migration and metastasis. Several studies have linked this protein to tongue cacer progression. However, the effects of Gal-3expression and its mechanisms in tongue carcinoma are still unclear. β-catenin, an important factor in canonical Wnt signaling pathway, makes great contributions on cancer invasion and metastasis. Recently, studies have found an association between Gal-3and β-catenin expression in several cancers. According to these findings, we postulate that Gal-3may play a potential role in tumor cell migration and invasion by modulating Wnt signaling pathway in tongue carcinoma. In the present study, a total of76patients with TSCC were investigated immunohistochemically. χ2test was employed to investigate the correlation between expression of Gal-3and clinicopathological parameters. Gal-3-siRNA was transfected into tongue cancer cell lines, and the effecs of Gal-3on the Wnt/β-catenin signaling pathway as well as the mechanisms involved were determined to explore how Gal-3manipulates tongue cancer progression and metastasis.Methods:1. For immunohistochemical staining. SP method was employed on sections of76patients with TSCC. All these patients had received curative resection at the Department of Oral and Maxillofacial Surgery, Qilu Hospital, between2005and2010.χ2test was employed to investigate the correlation between expression of Gal-3and clinicopathological parameters.2. Tongue cancer cell lines, SCC-4and CAL27, which were stored in liquid nitrogen, were quickly melted in a water-bath at37℃. Tumor cells were resuspended in a fresh medium, and maintained at37℃in a5%CO2atmosphere in RPMI1640and DMEM respectively, containing10%fetal bovine serum with penicillin and streptomycin. Cells in logarithmic growth phase were used in experiments.3. Cells were divided into3groups:blank control, negative control and experimental group. Transient siRNA transfection was performed in negative control and experimental group with control-siRNA and Gal-3-siRNA respectively by cationic liposome (LipofectamineTM RNAiMAX, Invitrogen, USA) according to the manufacturer’s instructions. Cells in blank control group remained untreated.4. Total RNA was extracted from cells of all3groups48h after transfection using a Trizol method. Template cDNA was synthesized from1μg of total RNA with a Quantscript RT Kit. random primers and a ribonuc lease inhibitor.1μl of the cDNA samples was mixed with19μl of mixed solutions. PCR was performed for40cycles with denaturation at95℃for15sec, annealing at60℃for1min with a RealMasterMix Kit (SYBR Green), using a7500Real Time PCR System. β-actin was used as an internal reference. The following primer sets were used:Gal-3,5’-GGCCACTGATTGTGCCTTAT-3’(forward) and5’-TGCAACCTTGAAGTG-GTCAG-3’(reverse); β-actin,5’-CTCCTCCTGAGCGCAAGTACTC-3’(forward) and5’-TCCTGCTTGCTGATCCACATC-3’(reverse).5. Cells were harvested after being treated with control-and Gal-3-siRNA for48h. Total protein was extracted by a RIPA cell lysate. Protein concentrations were measured using a BCA Protein Assay Kit.50μg protein was resolved by12%SDS-PAGE and electroblotted onto polyvinylidene difluoride plus membrane. The membrane was probed with anti-Gal-3antibody. Anti-β-catin was used to monitor equal loading in each lane. Immunoreactivity was detected using an enhanced chemiluminescence detection system. For densitometric analysis of WB, Alpha Image was used.6. Cell proliferation was measured by an MTT assay. Cancer cells (2.0×103cells/well) were seeded in96-well microtiter plates in a total volume of100μl/well. Transfection was performed when cells were30～50%confluent.10μl/well of CCK-8was added and cells were incubated under a humidified5%CO2atmosphere at37℃for4h. The absorbance of each well was determined at450nm using a microtiter plate reader. The results are expressed as mean±SD from triplicate cultures. Determinations were made in triplicate.7. Transfected cells were seeded on a24-well plate with their respective culture media. After the growing cell layers had reached confluence, a straight line in each well was made using a pipette tip. The cells were cultured in RPMI1640/H-DMEM containing1%volume fraction of FBS for24h. and then the media was replaced with RPMI1640/H-DMEM containing10%volume fraction of FBS. The filling of the wounds were evaluated at48h after scratching using a bright-field microscopy. All experiments were performed in triplicate.8. Invasion assays were performed using a BD BioCoat Matrigel Invasion Chamber. Briefly, cells were harvested after being transfected for48h. Cells were resuspended in serum-free DMEM and then added to the upper chamber at a density of2×105cells/well. After incubation for8h at37℃, the cells were fixed with ethanol and stained with hematoxylin and eosin. We subsequently counted the cells that migrated through the pores to the lower surface of each filter under a microscope and evaluated based on the mean values from five fields of view at×200magnifications.9. Cells were harvested48h after transfection. Same methods were applied for β-catenin protein level detecting. Then β-catenin expression was detected at the transcriptional level. Primers for β-catenin used in the experiments were as follows.5’-GCCGGCTATTGTAGAAGCTG-3’(forward) and5’-GAGTCCCAAGGAGAC-CTTCC-3’(reverse). Immunofluorescence method was used to investigate the distribution of β-catenin in SCC-448h after RNAi. 10. Akt, pAkt, GSK-3β and pGSK-3β protein expression were detected at48h after Gal-3silencing. And time-lapse WBs for Gal-3, β-catenin. pAkt and pGSK-3β protein were performed at the indicated times of0h,12h,24h and48h after transfection.11. To confirm the necessity of Akt for Gal-3-mediated Wnt signaling and β-catenin regulation. Akt inhibitor was used in SCC-4cells transfected with or without Gal-3-siRNA. Total Akt, pAkt, and β-catenin protein levels were detected with WB.12. To test if MMP-9might be a mediator of β-catenin-induced migration and invasion in SCC-4cells, RT-PCR and WB were employed48h after Gal-3silencing to investigate the expression of MMP-9.Results:1. Gal-3was positively expressed in tumor cells as well as in cancer-associated stromal cells. Gal-3expression was predominant in cytoplasm. The level of Gal-3expression was found to be positively correlated with lymph node status and clinical stage. No significant relationship was found between Gal-3expression and age, gender, tumor size, and histological grade.2. Fouty-eight hours after transient gene silencing of Gal-3, significant inhibition of Gal-3mRNA as well as protein expression were dectected in experimental groups. Gal-3silencing achieved99.4%and98.6%knockdown in SCC-4and CAL27cell lines respectively. As for tumor cell biological behavior, the results showed that there were no differences in cell proliferation between control and experimental groups for both of the cell lines. However, wound-healing assay demonstrated that cell migration was drastically decreased in experimental groups after Gal-3-siRNA transfection. We found that the time required for wound closure of Gal-3-silenced tongue cacer cells was significantly longer than that of control cells. Furthermore, we also confirmed that Gal-3gene silencing affected cell invasion similarly to migration of tongue carcinoma cell lines. Cell invasion was decreased by approximately two folds in cells treated with Gal-3-siRNA, when compared with the control groups.3. In this study, the results showed that inhibition of Gal-3gene expression can affect the protein level of β-catenin in SCC-4and CAL27cells, causing a significant downregulation of β-catenin expression. To clarify whether β-catenin is manipulated at the transcriptional level, we also evaluated the mRNA level of in tumor cells. The experiments revealed that though Gal-3silencing can down-regulate protein level of β-catenin. its RNA level did not change in both cell lines. The distribution of β-catenin after Gal-3silencing in SCC-4cells was examined by dual-fluorescence confocal microscopy. Reduced production of P-catenin was observed in the cytoplasm and in the nucleus.4. To investigate the Gal-3-mediated regulation of β-catenin expression, we evaluated Akt and GSK-3β protein in total protein and phosphorylation levels in tumor cells of both control and experimental groups. Our study revealed that there were no changes detected of Akt and GSK-3β protein in total protein level, while the phosphorylated forms of both of the two proteins were significantly decreased after Gal-3silencing. To the contrary, in the control groups, we found no significant changes of pAkt and pGSK-3β expression. Additionally, we also assessed the time-dependent alterations these proteins in cells of experimental groups. We found that the decreases of both pAkt and pGSK-3β occurred at12h after transfection. while downregulation of β-catenin expression was detected at48h. Densitometric analysis of time-lapse WB was performed, which revealed that the protein expression levels of Gal-3, pAkt, pGSK-3β and β-catenin significantly reduced in transfected SCC-4cells. Meanwhile, using an Akt inhibitor leads to β-catenin repression, which is similar to observations made using Gal-3-siRNA. Gal-3-siRNA cannot suppress β-catenin levels further after treatment with an Akt inhibitor, indicating that Akt plays an important role in Gal-3/β-caten in function.5. We analyzed MMP-9levels by RT-PCR and Western blotting. MMP-9mRNA and protein levels were significantly lower in Gal-3-siRNA treated cells than in the corresponding controls.Conclusions:1. Gal-3is correlated with tumor invasion and metastasis in TSCC.2. Gal-3RNAi inhibits migration and invasion by human tongue cancer cells (SCC-4and CAL27). 3. Gal-3silencing induces a downregulation of the protein level of β-catenin in both cell lines, but dose not change its mRNA expression. Reduced production of β-catenin was observed in the cytoplasm and in the nucleus.4. Gal-3mediates cell migration and invasion by activating Akt, which regulates GSK-3β phosphorylation and β-catenin degradation.