MicroRNA-4461 derived from bone marrow mesenchymal stem cell exosomes inhibits tumorigenesis by downregulating COPB2 expression in colorectal cancer.

Colorectal cancer (CRC) is one of the main cause of cancer-related deaths. It's reported that bone marrow mesenchymal stem cells (BMSCs) affects tumor development through secreting exosomes. This study aims to investigate the function of BMSCs-derived exosome miR-4461 in CRC. The results of qRT-PCR showed that miR-4461 expression in DLD1, HCT116 and SW480 CRC cells and CRC tissues was lower than that in FHC cells and normal tissues, respectively. And COPB2 mRNA expression was negatively correlated with miR-4461. Western blot was used to detect COPB2 protein expression. Dual-luciferase reporter assay results revealed that miR-4461 targeted COPB2. Transwell assay and CCK-8 assay demonstrated that COPB2 knockdown inhibited HCT116 and SW480 cells proliferation, migration and invasion abilities. Furthermore, BMSCs-derived exosome miR-4461 downregulated COPB2 expression and inhibited HCT116 and SW480 cells migration and invasion. The findings demonstrated that miR-4461 could be a potential target for the diagnosis and treatment of colorectal cancer.

[Effect of Metformin on Proliferation Capacity, Apoptosis and Glycolysis in K562 Cells].

OBJECTIVE: To investigate the effect of metformin on the proliferation, apoptosis and energy metabolism of acute myeloid leukemia (AML) K562 cells and the possible mechanism. METHODS: Different doses (0, 5, 10, 20 and 30 mmol/L) of metformin was added into the K562 cells, which were cultivated for 24 h, 48 h and 72 h. The inverted optical microscope was used to observe the cell growth, CCK 8 was used to detect the cell vitality. The appropriate metformin doses (0, 10, 20 and 30 mmol/L) and the best time (48 h) were selected for subsequent experiments. The flow cytometer with Annexin V-FITC /PI doulde staining was used to detect apoptosis; the glucose detection kit and lactate detection kit were used to detect glucose consumption and lactate production; fluorescence quantitative PCR was used to detect glycolysis-related gene expression, and Western blot was used to detect protein expression. RESULTS: Metformin inhibited the proliferation of K562 cells in a dose-dependent manner (r=0.92), and the relative survival in the 30 mmol/L group was as low as 19.84% at 72 h. When treated with metformin for 48 h, the apoptosis rates of 0, 10, 20 and 30 mmol/L groups were 5.14%, 12.19%, 26.29% and 35.5%, respectively. Compared with the control group, the glucose consumption and lactate secretion of K562 cells treated with metformin were significantly reduced (P<0.05), and showed a dose-dependent effect(r=0.94,r=0.93,respectively). Metformin inhibited the expression of GLUT1, LDHA, ALDOA, PDK1, and PGK1 genes of K562 cells (P<0.05) showing a dose-dependent manner(r=0.83，r=0.80，r=0.72，r=0.76，r=0.73,respectively). Metformin inhibited the expression of P-Akt, P-S6, GLUT1, LDHA proteins of K562 cells(P<0.05), showing a dose-dependent relationship(r=0.80，r=0.92，r=0.83，r=0.92,respectively). CONCLUSION: Metformin can inhibit the growth and proliferation of K562 cells and promote the apoptosis of K562 cells by inhibiting glycolysis energy metabolism. PI3K/Akt/mTOR signaling pathway may be one of the molecular mechanisms of metformin on k562 cells.

Acoustic Radiation Force Impulse Technology in the Differential Diagnosis of Solid Breast Masses with Different Sizes: Which Features Are Most Efficient?

PURPOSE: To evaluate diagnostic performance of acoustic radiation force impulse (ARFI) technology for solid breast masses with different sizes and determine which features are most efficient. MATERIALS AND METHODS: 271 solid breast masses in 242 women were examined with ARFI, and their shear wave velocities (SWVs), Virtual Touch tissue imaging (VTI) patterns, and area ratios (ARs) were measured and compared with their histopathological outcomes. Receiver operating characteristic curves (ROC) were calculated to assess diagnostic performance of ARFI for small masses (6-14 mm) and big masses (15-30 mm). RESULTS: SWV of mass was shown to be positively associated with mass size (P < 0.001). For small masses, area under ROC (Az) of AR was larger than that of SWV (P < 0.001) and VTI pattern (P < 0.001); no significant difference was found between Az of SWV and that of VTI pattern (P = 0.906). For big masses, Az of VTI pattern was less than that of SWV (P = 0.008) and AR (P = 0.002); no significant difference was identified between Az of SWV and that of AR (P = 0.584). CONCLUSIONS: For big masses, SWV and AR are both efficient measures; nevertheless, for small masses, AR seems to be the best feature.

B3GNT2, a polylactosamine synthase, regulates glycosylation of EGFR in H7721 human hepatocellular carcinoma cells.

The epidermal growth factor receptor (EGFR) is an important surface receptor with N-glycans in its extracellular domain, whose glycosylation is essential for its function, especially in tumor cells. Here, we demonstrated that polylactosamine is markedly increased in H7721 hepatocellular carcinoma cells after treatment with EGF, while it apparently declined after exposure to all-trans retinoic acid (ATRA). In the study of the enzymatic mechanism of this phenomenon, we explored changes in the expression of poly-N-acetyllactosamine (PLN) branching glycosyltransferases using RT-PCR. Among the four glycosyltransferases with altered expression, GnT-V was most elevated by EGF, while GnT-V and B3GNT2 were most declined by ATRA. Next, we conducted co-immunoprecipitation experiments to test whether B3GNT2 and EGFR associate with each other. We observed that EGFR is a B3GNT2-targeting protein in H7721 cells. Taken together, these findings indicated that the altered expression of B3GNT2 will remodel the PLN stucture of EGFR in H7721 cells, which may modify downstream signal transduction.

Expression of ß1,3-N-acetylglucosaminyltransferases during differentiation of human acute myeloid leukemia cells.

The expressions of ß1,3-N-acetylglucosamonyltransferase-2 and -8 (ß3GnT-2, ß3GnT-8),-the two main glycosyltransferases responsible for the synthesis of poly-N-acetyllactosamine (polyLacNAc) in glycans, and ß3GnT-5 participating in the syntheses of sphingoglycolipids were studied in leukemia cell lines during differentiation using RT-PCR method. ß3GnT-2 and ß3GnT-8 distribute widely in six myeloid and monocytoid leukemia cell lines with different abundances, while ß3GnT-4 was only present in NB4 cells. ATRA (all-trans retinoic acid) and dimethylsulfoxide (DMSO), which induce the differentiation of HL-60 and NB4 (two human acute myeloid leukemia cell lines) to myelocytic lineage, up-regulated these two enzymes with various degrees at 2 and 72 h of treatment. In HL-60 cells treated with ATRA, the increase of ß3GnT-8 was more than ß3GnT-2, while in NB4 cells treated with DMSO, the increase of ß3GnT-2 was more than ß3GnT-8. However, when HL-60 and NB4 were differentiated to monocytic lineage induced by phorbol 12-myristate 13-acetate the expressions of ß3GnT-2 and ß3GnT-8 showed no alterations or the increase of expressions was far less than those in myelocytic differentiation. By means of FITC-labeled tomato lectin affinity staining and flow-cytometry, it was found that the product of ß3GnT-2 and -8, polyLacNAc was also increased on the cell surface of HL-60 and NB4 treated with ATRA or DMSO, but unchanged when treated with PMA. These results were in accordance with the up-regulation of the mRNAs of ß3GnT-2 and -8. The expression of ß3GnT-5, however, was not changed both in myelocytic and monocytic differentiations. The difference in the up-regulation of ß3GnT-2 and -8, especially their products may become a useful index to discriminate the myelocytic and monocytic differentiation of leukemia cells.

alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function.

After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.

Alpha1,3 Fucosyltransferase-VII modifies the susceptibility of apoptosis induced by ultraviolet and retinoic acid in human hepatocarcinoma cells.

The role of alpha1,3fucosyltransferase-VII (alpha1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7,721 cells. After the cells were transfected with alpha1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that alpha1,3FucT-VII is a potential anti-apoptotic factor in H7,721 cells. After "alpha1,3FucT-VII" cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of alpha1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7,721 cells responsible to UV stress) when compared with the "Mock" cells. In contrast, "alpha1,3FucT-VII" cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X(L). The up regulation of alpha1,3FucT-VII mRNA and cell surface SLe(x) (alpha1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that alpha1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively.

Alpha 1,3-fucosyltransferase-VII regulates the signaling molecules of the insulin receptor pathway.

Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of alpha1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively. In alpha1,3-FucT-VII-transfected cells, expression of insulin receptor (InR) alpha- and beta subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLe(x), the product of alpha1,3-FucT-VII) content of the EGFR were unchanged. However the level of SLe(x) on the InR alpha subunit (InR-alpha) was increased dramatically. Tyrosine autophosphorylation of InR-beta , but not EGFR, was elevated. Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser/Thr phosphorylation of protein kinase B (PKB; Akt), p42/44 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and the protein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and beta-catenin were also upregulated. The activities of PKB and transcription factor TCF were concomitantly stimulated. Upregulation of InR signaling molecules and their phosphorylation was correlated with the level of SLe(x) on InR-alpha and alpha1,3-FucT-VII expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different levels of alpha1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLe(x). Furthermore, insulin-induced signaling was facilitated in alpha1,3-FucT-VII-transfected cells, particularly FucTVII-H. These findings provide strong evidence that alpha1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling pathway. These effects are likely mediated by its product, SLe(x), on the glycans of the InR. This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling.

Expressions of polypeptide: N-acetylgalactosaminyltransferase in leukemia cell lines during 1,25-dihydroxyvitamin D3 induced differentiation.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)(2)D3] on two leukemia cell lines, K562 and SHI-1, and its relation to the expression of different subtypes of polypeptide: N-acetylgalactosaminyltransferase (pp-GalNAc-T) was studied. With morphological and cell flow-cytometric method, it was found that 1,25(OH)(2)D3 induced the differentiation of both leukemia cell lines toward monocytic lineage, but not affected the cell growth and apoptosis. The expressions of different subtypes of pp-GalNAc-T, the initial glycosyltransferase in O-glycan synthesis, were studied with RT-PCR before and after the treatment of different concentrations of 1,25(OH)(2)D3. Among fourteen subtypes of pp-GalNAc-T (T1 approximately T14), K562 cells obviously expressed pp-GalNAc-T2, T4, T5, T7 (T2 was the highest) and SHI-1 cells apparently expressed pp-GalNAcT1, T2, T3 and T4 (T4 was the highest) only. After K562 cells were treated 1, 25(OH)(2)D3 for 72 h, pp-GalNAc-T2, T4, T5, T7 were increased in a dose dependent manner. In contrast, pp-GalNAc-T1 and T2, especially T1, were up-regulated in SHI-1 cells by 1,25(OH)(2)D3, but T3 was unchanged and T4 was down-regulated. The different alterations of pp-GalNAc-Ts in these two cell lines were probably related to the different structural changes of O-glycans during 1,25(OH)(2)D3 induced differentiation.

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7,721 cells.

N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7,721) was constructed from 7,721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7,721 and parental 7,721 cells. The data indicated that GnT-V-AS/7,721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7,721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2alpha pathway, the activation of ER eIF2alpha kinase PERK was observed. To confirm the results from GnT-V-AS/7,721 cells, the key molecules in the UPR were examined again in 7,721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7,721 cells. Rate of (3)H-Man incorporation corrected with rate of (3)H-Leu incorporation in GnT-V-AS/7,721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7,721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.

Down regulation of N-acetylglucosaminyltransferase V facilitates all-transretinoic acid to induce apoptosis of human hepatocarcinoma cells.

After N-acetylglucosaminyltransferase V (GnT-V) activity was down-regulated by the transfection of its antisense cDNA(GnTV-AS), apoptosis of H7721 cells was appeared and the apoptosis induced by 80 microM all-transretinoic acid (ATRA) was facilitated, while ATRA itself could not induce apparent apoptosis in mock cells transfected with the vector. In the study of the molecular mechanism of this phenomenon, it was found that GnTV-AS reduced the expressions of anti-apoptotic proteins, such as phosphorylated protein kinase B and phosphorylated Bad as well as Bcl-2 and Bcl-X (L), and elevated those of pro-apoptotic proteins, including Bax, full length caspase-3 and its activated fragments as well as anti-oncoprotein p53. In the contrast, ATRA up regulated the expressions of Bax and activated caspase-3 fragments only. After the GnTV-AS transfected cells were treated with ATRA, phosphorylated PKB and Bad were further decreased, while Bax and activated caspase-3 fragment were further increased, leading to the enhanced apoptosis in flow-cytometry analysis when compared with GnTV-AS cells not treated with ATRA. It was speculated that the decreased phospho-Bad resulted from the reduced phospho-PKB and the up regulation of p53 caused the elevated activity of Bax. The increased active caspase-3 was the consequence of the elevated Bax/ Bcl-2(Bcl-X(L)) activity ratio in the cells.

Regulation of metastasis-suppressive gene Nm23-H1 on glycosyl-transferases involved in the synthesis of sialyl Lewis antigens.

By using reverse transcriptase-polymerase chain reaction (RT-PCR), the mRNA expressions of three families of glycosyltransferases involved in the synthesis of sialyl Lewis antigens were determined in H7721 human hepatocarcinoma cell line before and after the transfection of metastasis-suppressive gene nm23-H1. These glycosyltransferases included alpha1,3fucosyltransferase (alpha1,3FucT)-III, -IV, -VI, -VII, and -IX, alpha2,3-sialyltransferase (ST3Gal)-I, -II, -III, and -IV as well as O-glycan core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT)-I and -II. In mock cells transfected with the vector, the expression-order of alpha1,3FucTs was IV>VI>III>VII>IX, that of ST3Gals was IV>I>II>III, and that of C2GnT was I>II. Nm23-H1 downregulated the mRNA expressions of all five subtypes of alpha1,3FucT and -I, -III, -IV subtypes of ST3Gal, but not ST3Gal-II and C2GnT-I, II. On the other hand, the expressions of cell surface sialyl Lewis X (SLe(x)) and alpha2,3 sialyl residues were decreased on nm23-H1 transfected cells as detected with monoclonal antibody of SLe(x) and enzyme-labeled lectins, respectively. Since SLe(x) was reported to be a metastasis-associated glycan structure, the reduced expressions of SLe(x) and some enzymes related to its synthesis may be one of the mechanisms to explain the metastasis-suppressive effect of nm23-H1.

Expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-12 in gastric and colonic cancer cell lines and in human colorectal cancer.

OBJECTIVE: The expression of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase-12 (pp-GalNAc-T12) was studied in 3 normal human tissues (stomach, small intestine and colon), 3 stomach and 6 colon cancer cell lines, as well as in the resected cancer tissues and normal tissues (control) from 19 patients with colorectal cancer. METHODS: Marathon Ready cDNAs were used as the templates of normal tissues. mRNA was extracted from the cell lines and resected tissues, and reverse-transcribed to cDNA. The expression of pp-GalNAc-T12 was determined with a real-time polymerase chain reaction (PCR). RESULTS: It was found that the expression of pp-GalNAc-T12 was strong in 3 normal tissues, weak or negligible in 9 cancer cell lines, and down-regulated in all of the colorectal cancer tissues as compared with normal control samples. Moreover, the expression of pp-GalNAc-T12 tended to inversely correlate with the TNM stage, and statistically was much lower in the samples with metastasis than in those without. However, the expression in the tissues did not correlate with the concentration of serum CA 19-9 routinely applied in the diagnosis and assessment of prognosis in patients with colonic cancers. CONCLUSION: the expression of pp-GalNAc-T12 seems to be a negative marker especially of metastatic gastric and colorectal cancer.

Relations of the type and branch of surface N-glycans to cell adhesion, migration and integrin expressions.

The relations between the structure of cell surface N-glycans to cell behaviors were studied in H7721 human hepatocarcinoma cell line, which predominantly expressed complex-type N-glycans on the surface. 1-Deoxymannojirimycin (DMJ) and swaisonine (SW), the specific inhibitor of Golgi alpha-mannosidase II or I, were selected to block the processing of N-glycans at the steps of high mannose and hybrid type respectively. All-trans retinoic acid (ATRA) and antisense cDNA of N-acetylglucosaminyltransferase-V (GnT-V) were used to suppress the expression of GnT-V and decreased the GlcNAc beta1,6-branching or tri-/tetra-antennary structure of surface N-glycans. The structural alterations of N-glycans were verified by sequential lectin affinity chromatography of [3H] mannose-labeled glycans isolated from the cell surface. The cell adhesions to fibronectin (Fn) and human umbilical vein epithelial cell (HUVEC), as well as cell migration (including chemotaxis and invasion) were selected as the parameters of cell behaviors. It was found that cell adhesion and migration were significantly decreased in SW and DMJ treated cells, suggesting that complex type N-glycan is critical for the above cell behaviors. ATRA and antisense GnTV enhanced cell adhesion to Fn but reduce cell adhesion to HUVEC and cell migration. These results reveal that cell surface complex-type N-glycans with GlcNAc beta1,6 branch are more effective than those without this branch in the cell adhesion to HUVEC and cell migration, but N-glycan without GlcNAc beta1,6-branch is the better one in mediating the cell adhesion to Fn. The integrin alpha5beta1 (receptor of Fn) on cell surface was unchanged by DMJ and SW. In contrast, ATRA up regulated alpha5, but not beta1, and antisense GnT-V decreased both alpha5 and beta1. This findings suggest that both the structure of N-glycan and the expression of integrin on cell surface are two of the important factors in the determination of cell adhesion to Fn, a complex biological process.

Alteration in N -acetylglucosaminyltransferase activities and glycan structure in tissue and bile glycoproteins from extrahepatic bile duct carcinoma.

The activities of three N -acetylglucosaminyltransferases (GnT)-III, IV and V, as well as the structural alterations of N-glycans on the glycoproteins in cancer tissues and bile specimens from 28 cases of extrahepatic bile duct carcinoma (EBDC) were compared with those from 18 cases of benign biliary duct diseases (BBDD). GnT activities were determined with fluorescence-labeled substrate using a HPLC method. It was found that GnT-III and GnT-V activities in EBDC were increased to 3.14 and 15.96 times respectively of the mean BBDD values, but GnT-IV remained unchanged. The activity of GnT-V was correlated with the grade of differentiation and TMN stage of EBDC. The up-regulation of GnT-III resulted in the increased bisecting-GlcNAc on the N-glycans of glycoproteins in cancer tissues and a 201 kDa bile glycoprotein when analyzed with HRP-labeled E(4)-PHA. The increased GnT-V activity led to the elevation of the beta1,6GlcNAc branch (or antennary number) on the N-glycans in cancer tissue glycoproteins and 201, 163, 122 kDa proteins in the bile as probed with HRP-labeled DSA. These findings suggest that the alteration in GnT activities may be involved in the malignant transformation and development of EBDC, resulting in the aberrant glycosylation of some tissue and bile proteins. The latter was expected to be used in the clinical diagnosis and prognosis evaluation in EBDC patients.

Correlation of glycosyltransferases mRNA expression in extrahepatic bile duct carcinoma with clinical pathological characteristics.

BACKGROUND: The incidence of extrahepatic bile duct carcinoma (EBDC) has been increasing, especially in aged people, but the glycobiology of the tumor is not elucidated. In this study we investigated the expressions of three glycosyltransferases in 35 patients with EBDC and 35 patients with benign biliary duct disease (BBDD) as well as their clinicopathological significance. METHOD: The patients were divided into several subgroups by tumor differentiation, TNM stage, and invasion by the standards recommended by UICC. Tumor samples were immediately frozen in liquid nitrogen after resection, followed by mRNA determination of enzymes in the tissue using a mRNA selective reverse trancriptase-polymerase chain reaction kit. The mRNA levels of different groups were semi-quantitatively compared. RESULTS: The mRNA levels of N-acetylglucosaminyltransferase V (GnT-V) and a subtype of alpha 2,3 sialyltransferases for N-glycans, ST3Gal-III were elevated 7.75 and 5.39 times in EBDC as compared with BBDD, respectively, and they were correlated to several clinicopathological factors including tumor advancement, differentiation, metastasis, and invasiveness. The mRNA expression of another sialyltransferase, ST6Gal-I, was also 0.63-fold higher in EBDC than in BBDD, but not involved in the clinicopathological characteristics. CONCLUSION: The elevated expression of these three glycosyltransferases can be considered as an important molecular event in the occurrence and progression of EBDC.

Effect of N-acetylglucosaminyltransferase V on the expressions of other glycosyltransferases.

Transfection of sense cDNA of N-acetylglucosaminyltransferase V (GnTV) into H7721 human hepatocellular carcinoma cells resulted in the decreased expression of surface sialyl Lewis X (SLe(x)), a sialylated fucose-containing antigen. The enzymatic mechanisms were speculated to be the concomitantly decreased expression of alpha1,3-fucosyltransferase (FucT)-III, -VI, -VII and the branching enzyme of O-glycans, core 2-beta1,6-N-acetylglucosaminyltransferase (C2GnT)-I, -II. These two glycosyltransferase families were suggested to be the key enzymes in the synthesis of SLe(x). The expression of alpha2,3-sialyltransferase (ST3)-IV, but not ST3-I, -II and -III was elevated by sense GnTV. However, it did not cause the increase of SLe(x) synthesis. Transfection of antisense GnTV into H7721 cells showed entirely opposite effects on the expression of above-mentioned SLe(x) and glycosyltransferases as the sense GnTV.

Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B, but not PI-3K, in H7721 human hepato-carcinoma cell line.

Forskolin (FSK) is known as an up-regulator of intracellular cAMP and inhibitor of cancer growth and metastasis. The effects of FSK on the metastasis potential and its mechanisms were studied using a human hepatocarcinoma cell line, H7721. It was found that FSK stimulated cell growth, increased cAMP in the cells, and enhanced the metastasis-related phenotypes, including adhesion to laminin (Ln) and human umbilical vein epithelial cells (HUVEC), chemotactic migration and invasion. These effects were supposed to result from the increase of the SLex expression induced by FSK, since only the monoclonal antibody of SLex showed a significant attenuation of the enhanced metastasis-associated phenotypes. Using H7721 cells transfected with the sense or antisense cDNA of protein kinase B (PKB) and some inhibitors of signal transduction, it was discovered that FSK up-regulated the expression of SLex via PKB, but it was independent of phosphotidylinositide-3-kinase (PI-3K). A subtype of atypical protein kinase C (a-PKC) might also participate in the up-regulation of SLex expression by FSK, and cAMP/PKA pathway is a negative regulator of SLex expression on H7721 cells. It can be concluded that FSK shows a metastasis-promoting effect ex vivo.

Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line.

The effect of insulin on cancer metastatic potential was studied in a human hepatocarcinoma cell line, H7721. Cell adhesion to human umbilical vein endothelial cells (HUVECs) and laminin as well as chemotactic cell migration and invasion were selected as the indices of metastasis-related phenotypes for assessment of metastatic potential ex vivo. The results indicated that insulin enhanced all of these metastasis-related phenotypes. After the cells were treated with specific inhibitor of PI3K (LY294002) or transfected with antisense cDNA of PKB (AS-PKB), all of the above phenotypes were attenuated, and they could not be significantly stimulated by insulin, indicating that the insulin effect on metastatic potential was mediated by PI3K and PKB. Only the monoclonal antibody to the sialyl Lewis X (SLe(x)), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migration and invasion, suggesting that SLe(x) played a crucial role in the metastatic potential of H7721 cells. The upregulation of cell surface SLe(x) and alpha-1,3-fucosyltransferase-VII (alpha-1,3 Fuc T-VII, enzyme for SLe(x) synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLe(x) and alpha-1,3 FucT-VII, and attenuated the response to insulin. Furthermore, the alterations in the expressions of PKB protein and activity were correlated to the changes of metastatic phenotypes and SLe(x) expression. Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upregulation of the expression of SLe(x) and alpha-1,3 FucT-VII.