| [Background and Objective]With the rapid development of social economy, primary liver cancer-one of the most common malignant neoplasm in digestive tract worldwide-has ranked as the second most common cause of death from cancer, and the incidence is rising year by year in China, which is a serious threat to human health and life safety. In terms of histology, primary liver cancer can be divided into hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (IHCC) and mixed type liver cancer, of which more than90%is HCC in China.Because the etiologies and pathogenesis of HCC are extremely complex, many of its biological behavior are still unknown. Surgical resection is still the first choice and the most effective treatment for HCC in China. Although great progresses have been made in early diagnosis, clinical treatment and basic research, the overall prognosis for HCC population remains poor. Another dismal problem is that about60%-70%of patients more likely to develop recurrence and metastasis in5years, even after radical resection. In recent years, with the development of molecular biology technology as well as the application of lectin in the detection of saccharide chain structure, the association between glycobiology and oncology appears to be an issue. A variety of biological characteristics, such as tumorigenesis, development, proliferation and metastasis have been associated with the abnormal glycosylation on cells surface. Many glycan chain structures were even regarded as tumor associated carbohydrate antigens (TACA). For example, the oligosaccharides containing (31,6-N-acetylglucosamine (β1-6GlcNAc) branching were found to be dramatically increased in human breast cancer, colon cancer and melanoma; Lewis X antigen-a kind of carbohydrate antigen-is a ligand for E-selectin, which mediates the adhesion of several types of human carcinoma cells to endothelial cells, so that it is considered to play an important role in hematogenous metastasis.Fucose which is regarded as one of the terminal structures of oligosaccharides is an important constituent of many sugar chains. Fucosylated glycans which are the important ingredients of glycoproteins and glycolipids on eukaryotic cells membrane have been found biological functions such as cells recognition, adherence, migration and growth. Fucosylated glycans are often altered in the initiation and progression of cancer cells. It may be not only the products of the tumor cell metabolism, but also a critical factor promoting tumor growth, invasion and metastasis. Fucosylated prostatic specific antigen (PSA) is often increased in the serum and urine of prostate cancer patients with metastasis, compared with non-metastasis patients. If the activities of fucosyltransferasel are increased in patients with lung cancer, it may be a sign of metastasis.Patients who suffer from hepatocellular carcinoma often have high activities of fucosidase and fucosyltransferasel. Fucosidase is a hydrolytic enzyme which plays an important function in the catabolism of fucosylated glycans. Like alpha fetoprotein, we can observe high activities of fucosidase in patients with HCC, which is another useful marker for the detection of HCC. It is the exact opposite of the function of fucosyltransferasel which plays an important function in the synthesis of fucosylated glycans. Fucosylation level is primarily determined by the balance and availabilitv of fucosidases and fucosyltransferases. So what will happen to the fucose-the primary substrate of fucosidase and fucosyltransferasel-on the surfuace of hepatocellular carcinoma cells?In order to study the variation of fucose on cell surfuace in HCC development and the relationship between the variation and metastasis, the amounts of fucose expressed on normal hepatic cells, low metastatic potential hepatocellular carcinoma cells and high metastasis potential hepatocellular carcinoma cells were detected.[MATERIALS AND METHODS]1. Object of studyNormal hepatocytes (L-02), low metastatic potential hepatocellular carcinoma cells (MHCC97-L) and high metastasis potential hepatocellular carcinoma cells (HCCLM3) were obtained from Xiangya central experiment laboratory of Central South University. When5×106hepatocellular carcinoma cells were injected subcutaneously into nude mice, tumorigenicity was100%for MHCC97-L and HCCLM3. After orthotopic orthotopic implantation, MHCC97-L caused abdominal wall invasion in20%, diaphragm invasion in0, intrahepatic metastases in o, and pulmonary metastases in100%. These datas were100%,100%,70%and100%for HCCLM3.2. Expansion of cellsL-02, MHCC97-L and HCCLM3were cultured at37℃in a humidified atmosphere of5%CO2and95%air in high glucose DMEM supplemented with15%fetal bovine serum(FBS). Three cell lines were grown in three culture flasks until they were90%confluent, then digested with trypsin(0.25%) and respectively passaged into7culture dishes(3×7=21) containing high glucose DMEM and15%FBS. When cells were near-confluent in culture dishes, they were collected into different centrifuge tubes, after trypsinization and washing thiple with sterile PBS, and cel concentration was adjusted for1×106/ml.3. Groups of experiment21centrifuge tubes (sample tube) were divided into3groups:normal hepatocytes group, low metastatic potential hepatocellular carcinoma cells group and high metastasis potential hepatocellular carcinoma cells group,7tubes for each group. Every group was filled with L-02cells, MHCC97-L cells and HCCLM3cells respectively. Every sample tube has its control tube filled with same cells.4. Determination of fucoseThe study according to the principle that FITC labeled UEA lectins can bind specifically to the fucose. Cells in sample tube were incubated with FITC-UEA (10p.g/ml) and cells in control tube were incubated with PBS, at4℃in a darkroom for1h. One hour later, cells were washed with PBS for three times, and fixed with1%paraformaldehyde.Then cells were observed under fluorescence microscope and flow cytometric analysis was carried out.5. Outcome measures5.1Location and brightness of fluorescent stainingAfter cell suspension smear prepared, cells were observed immediately under fluorescence microscope. The location of fluorescent staining represents the position of fucose, and the brightness of fluorescent staining was a qualitative measure of the amount of fucose detected with the same exposure time.5.2Mean fluorescence intensity on cells surface determined by flow cytometryAfter cells fixatiom, At least106cells (each tube) were acquired and analyzed for the fluorescent signal by using flow cytometry. The mean fluorescence intensity represents the amounts of fucose on cell surface.6. Statistical AnalysisAll data are presented as mean values±standard error of the mean (x±S). Statistical analysis was performed using the SPSS13.0software package with hypothesis testing level a=0.05. The significance test of mean fluorescence intensity between multiple groups was determined by One-way ANOVA and in two comparisons was analyzed by the least-significant difference(LSD).[Results]1. Results of fluorescence microscopyCell membranes were stained in dark green with fluorescein, while cytoplasm and cell nucleus were stained poorly or lack any staining. The brightness of fluorescence in HCCLM3was brighter than L-02and MHCC97-L. Compare to L-02, the brightness of fluorescence in MHCC97-L was greater. These results indicated that the fucose we detected in the study mainly distributed on the membrane of L-02, MHCC97-L and HCCLM3, and its expression increased gradually.2. Results of lowcytometric analysisThe fluorescence intensity in L-0, MHCC97-L and HCCLM3were (11139.29±496.88),(14161.14±960.11) and (19081.43±665.39). The fucose expressed on HCCLM3wasHigher than L-02and MHCC97-L (P<0.05for both comparisons). Compare to L-02, the fucose expressed on MHCC97-L was greater (P<0.05).[Conclusion]The amounts of fucose on cell surface were found to be closely related to the development and metastasis of hepatocellular carcinoma cells. After liver cells canceration, the fucose content on cell surface will increase, and the higher the metastatic potential of cancerous cells, the higher the content of fucose. |
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