Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells
2013; Elsevier BV; Volume: 54; Issue: 4 Linguagem: Inglês
10.1194/jlr.m031450
ISSN1539-7262
AutoresWei Wu, Yi Dong, Peng Shi, Mei Yu, Da Fu, Chun Yi Zhang, Qian Cai, Qian Zhao, Ming Peng, Li Hui Wu, Xing Wu,
Tópico(s)Immunotherapy and Immune Responses
ResumoIntegrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation. Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation. Integrins have been implicated as very important adhesion molecules that are involved in multiple physiological processes, such as cell adhesion, proliferation, and survival (1Schwartz M.A. Assoian R.K. Integrins and cell proliferation: regulation of cyclin-dependent kinases via cytoplasmic signaling pathways.J. Cell Sci. 2001; 114: 2553-2560Crossref PubMed Google Scholar, 2Shimaoka M. Takagi J. Springer T.A. Conformational regulation of integrin structure and function.Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 485-516Crossref PubMed Scopus (443) Google Scholar, 3Zahir N. Weaver V.M. Death in the third dimension: apoptosis regulation and tissue architecture.Curr. Opin. Genet. Dev. 2004; 14: 71-80Crossref PubMed Scopus (129) Google Scholar). Each integrin generally consists of a noncovalently linked α- and β-subunit, with each subunit having a large extracellular domain, a single membrane-spanning domain, and a short, noncatalytic cytoplasmic tail. Integrins seem to be the major receptors by which cells attach to components of the extracellular matrix (ECM), such as vitronectin, etc. (4Gao B. Saba T.M. Tsan M.F. Role of alpha(v)beta(3)-integrin in TNF-alpha-induced endothelial cell migration.Am. J. Physiol. Cell Physiol. 2002; 283: C1196-C1205Crossref PubMed Scopus (56) Google Scholar), and are involved in the metastasis signaling of hepatocellular carcinoma (HCC) (5Li H. Ge C. Zhao F. Yan M. Hu C. Jia D. Tian H. Zhu M. Chen T. Jiang G. et al.Hypoxia-inducible factor 1 alpha-activated angiopoietin-like protein 4 contributes to tumor metastasis via vascular cell adhesion molecule-1/integrin beta1 signaling in human hepatocellular carcinoma.Hepatology. 2011; 54: 910-919Crossref PubMed Scopus (132) Google Scholar). The integrin αV subunit associates with one of five integrin β subunits, β1, β3, β5, β6, or β8, to form five distinct αVβ heterodimers (6McCarty J.H. Alpha v integrins lead the way for colorectal metastases.Clin. Cancer Res. 2008; 14: 6351-6353Crossref PubMed Scopus (8) Google Scholar). The integrin αVβ heterodimers on the cell surface interact with cell adhesive proteins, such as collagen, fibrinogen, fibronectin, and vitronectin. These interactions play an important role in cell adhesion or migration, especially in tumor metastasis. Integrins increase in invasive tumors and distant metastases, characterize the metastatic phenotype, and play a key role in tumor metastasis (7Felding-Habermann B. Fransvea E. O'Toole T.E. Manzuk L. Faha B. Hensler M. Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells.Clin. Exp. Metastasis. 2002; 19: 427-436Crossref PubMed Scopus (121) Google Scholar, 8Hood J.D. Cheresh D.A. Role of integrins in cell invasion and migration.Natl. Rev. Cancer. 2002; 2: 91-100Crossref PubMed Scopus (1476) Google Scholar). Many studies have documented marked differences in the surface expression and distribution of integrins between malignant tumors and preneoplastic tissues. For example, the integrin αVβ3 complex is strongly expressed in the invasive front cells of malignant melanoma and angiogenic blood vessels, but it is weakly expressed on preneoplastic melanomas and quiescent blood vessels (9Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar). Also, it has been demonstrated that αVβ3 integrin is specifically required to sustain neovascularization induced in vivo by fibroblast growth factor-2 (10Rusnati M. Tanghetti E. Dell'Era P. Gualandris A. Presta M. alphavbeta3 integrin mediates the cell-adhesive capacity and biological activity of basic fibroblast growth factor (FGF-2) in cultured endothelial cells.Mol. Biol. Cell. 1997; 8: 2449-2461Crossref PubMed Scopus (133) Google Scholar). Integrin αVβ3 physically associates with phosphorylated and activated insulin-like growth factor receptor, and it may be involved in the HCC cell migration and progression (11Nussbaum T. Samarin J. Ehemann V. Bissinger M. Ryschich E. Khamidjanov A. Yu X. Gretz N. Schirmacher P. Breuhahn K. Autocrine insulin-like growth factor-II stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis.Hepatology. 2008; 48: 146-156Crossref PubMed Scopus (101) Google Scholar). Furthermore, inducing the expression of the integrin αV (7Felding-Habermann B. Fransvea E. O'Toole T.E. Manzuk L. Faha B. Hensler M. Involvement of tumor cell integrin alpha v beta 3 in hematogenous metastasis of human melanoma cells.Clin. Exp. Metastasis. 2002; 19: 427-436Crossref PubMed Scopus (121) Google Scholar) or β3 (12Yurchenco P.D. Basement membranes: cell scaffoldings and signaling platforms.Cold Spring Harb. Perspect. Biol. 2011; 3 (pii)Crossref PubMed Scopus (579) Google Scholar) subunit in melanoma cells increases their metastatic potential. A change of the expression and distribution of integrins on the cell surface can modulate the strength of cell adhesion and migration. The regulation of cell adhesion and migration involves coordinated events of tumor metastasis. Many primary and metastatic cancer cells display altered integrin expression levels and/or activation status, leading to abnormal cell adhesion, growth, and survival, which are pathological hallmarks of cancer. Although the influence of integrin αV and β3 on metastasis has been documented in several studies (13Nip J. Brodt P. The role of the integrin vitronectin receptor, alpha v beta 3 in melanoma metastasis.Cancer Metastasis Rev. 1995; 14: 241-252Crossref PubMed Scopus (53) Google Scholar, 14Sato T. Miwa A. Ets-1 and integrin beta3 for lung metastasis from colorectal cancer.APMIS. 2002; 110: 347-353Crossref PubMed Scopus (30) Google Scholar), there is little information about cerebroside sulfation signaling, and the molecular regulation mechanisms underlying integrin expression have not been elucidated. Sulfatide, the product of galactose-3-O-sulfotransferase 1 (Gal3ST-1; also called cerebroside sulfotransferase, CST), is highly expressed in high metastasis potential HCC cells (MHCC97H) compared with low metastasis potential HCC cells (MHCC97L) (15Zhong W.X. Honke K. Long Z.Y. Liang Z.X. Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice.Int. J. Cancer. 2004; 110: 504-510Crossref PubMed Scopus (36) Google Scholar). After retinoic acid treatment and inhibition of cell migration, the cellular sulfatide production is decreased (15Zhong W.X. Honke K. Long Z.Y. Liang Z.X. Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice.Int. J. Cancer. 2004; 110: 504-510Crossref PubMed Scopus (36) Google Scholar), but the precursor cerebroside is not affected, suggesting the inhibition of CST activity. Hep3B cells overexpressing CST that produces sulfatide significantly promotes the metastasis behaviors in nude mice. Apart from CST (Gal3ST-1), Gal3ST-2 is also observed to be involved in tumor metastasis (16Shi B.Z. Hu P. Geng F. He P.J. Wu X.Z. Gal3ST-2 involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.Biochem. Biophys. Res. Commun. 2005; 332: 934-940Crossref PubMed Scopus (14) Google Scholar). Both genes encode galactose-3-O-sulfotransferases, which catalyze and transfer sulfate to the 3′ hydroxyl group of the galactosyl residue in the glycol chain. This sulfation makes the substrate molecule negatively charged, changes its affinity with binding molecules, and involves HCC metastasis or progression (17Zhang C.Y. Hu P. Fu D. Wu W. Jia C.Y. Zhu X.C. Wu X.Z. 3'-Sulfo-Le(x) is important for regulation of integrin subunit alphaV.Biochemistry. 2010; 49: 7811-7820Crossref PubMed Scopus (8) Google Scholar). The HCC Hep3B cells transfected by CST expressed an elevated level of integrin αV and intensively adhered to vitronectin, the ligand of integrin αVβ3 (15Zhong W.X. Honke K. Long Z.Y. Liang Z.X. Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice.Int. J. Cancer. 2004; 110: 504-510Crossref PubMed Scopus (36) Google Scholar, 18Wu X.Z. Li Z. Shi B.Z. Hu P. Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells.World J. Gastroenterol. 2005; 11: 5763-5769Crossref PubMed Scopus (6) Google Scholar). However, the mechanism by which the CST is involved in regulation of integrin αV and cell adhesion is not fully understood. The relationship between the promotion mechanism of cancer cells and elevated expression of sulfatide remains unknown (19Takahashi T. Suzuki T. Role of sulfatide in normal and pathological cells and tissues.J. Lipid Res. 2012; 53: 1437-1450Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). A recent study showed that sulfatide can serve as a laminin-binding glycolipid and can anchor laminin-1 and laminin-2 to the Schwann cell surface, form a laminin-associated complex, and enable basement membrane assembly to initiate c-Src activation (9Li S. Liquari P. McKee K.K. Harrison D. Patel R. Lee S. Yurchenco P.D. Laminin-sulfatide binding initiates basement membrane assembly and enables receptor signaling in Schwann cells and fibroblasts.J. Cell Biol. 2005; 169: 179-189Crossref PubMed Scopus (117) Google Scholar). Sulfatide was also identified as an interacting partner of P-selectin and promoted a P-selectin-mediated metastasis process in colon cancer cells (20Garcia J. Callewaert N. Borsig L. P-selectin mediates metastatic progression through binding to sulfatides on tumor cells.Glycobiology. 2007; 17: 185-196Crossref PubMed Scopus (66) Google Scholar). Sulfatide and P-selectin interactions led to subsequent platelet aggregation (21Merten M. Beythien C. Gutensohn K. Kuhnl P. Meinertz T. Thiagarajan P. Sulfatides activate platelets through P-selectin and enhance platelet and platelet-leukocyte aggregation.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 258-263Crossref PubMed Scopus (51) Google Scholar) and played an important role in the formation of cancer embolus. Our previous study (15Zhong W.X. Honke K. Long Z.Y. Liang Z.X. Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice.Int. J. Cancer. 2004; 110: 504-510Crossref PubMed Scopus (36) Google Scholar, 18Wu X.Z. Li Z. Shi B.Z. Hu P. Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells.World J. Gastroenterol. 2005; 11: 5763-5769Crossref PubMed Scopus (6) Google Scholar) revealed that hepatoma cells expressed sulfatide after CST transfection. The enzyme CST in HCC can only catalyze the production of sulfatide, which acts as the endogenous sulfated cerebroside. We thus hypothesize that the enzyme product sulfatide is responsible for the regulation of the integrin αV subunit and involves the metastasis process. To test this, we investigated, in this study, the regulatory effect of both exogenous and endogenous sulfatide, the product of CST, and demonstrated that sulfatide, but not cerebroside, played an important role in the regulation of the expression of the integrin αV subunit, which can form the molecule αVβ3 in hepatocellular carcinoma cells. Lactocerebroside (Lacto-Cer), galactocerebroside (Gal-Cer), and sulfatide from bovine brain were obtained from Sigma-Aldrich (St. Louis, MO). ManN propanyl perac (ManN-pro) and cyclo-ManN propanyl perac (cyclo-ManN-pro) were kindly provided by Professor Wermer Ruetter (Institut für Biochemie und Molekularbiologie, Campus Benjamin Franklin, Germany) (22Oetke C. Brossmer R. Mantey L.R. Hinderlich S. Isecke R. Reutter W. Keppler O.T. Pawlita M. Versatile biosynthetic engineering of sialic acid in living cells using synthetic sialic acid analogues.J. Biol. Chem. 2002; 277: 6688-6695Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 23Keppler O.T. Horstkorte R. Pawlita M. Schmidt C. Reutter W. Biochemical engineering of the N-acyl side chain of sialic acid: biological implications.Glycobiology. 2001; 11: 11R-18RCrossref PubMed Scopus (248) Google Scholar). Peptides GRGDSP and GRGESP with 99% purity were provided by China Peptides Co. (Shanghai, China). Polyclonal rabbit, monoclonal mouse anti-human integrin αV antibodies, and polyclonal rabbit anti-human Sp1 antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse O4 antibody (anti-galactosyl sufatide and lactosyl sulfatide) was obtained from Chemicon (Temecula, CA). Monoclonal mouse anti-lactosyl sulfatide antibody was obtained from Seikagaku (Tokyo, Japan). FITC-anti-human integrin αV was obtained from BioLegend (San Diego, CA). Antibodies against Erk, p38, Rac, mTOR, JNK, and phospho-Raf (S338) were obtained from Cell Signaling Technology Inc. (Danvers, MA). Phospho-Raf (Y341) was from Beijing Biosynthesis Biotechnology (Bioss, Beijing, China). Akt antibody was from Bioworld (Atlanta, GA). Anti-phosphoserine antibody was from Millipore (Billerica, MA). Fibrinogen was from Sigma-Aldrich, collagen type I was from Roche (Indianapolis, IN), and fibronectin and vitronectin were from Calbiochem (San Diego, CA). Hepatoma cells (SMMC-7721, BEL-7404), human umbilical vein endothelial cells (HUVEC), HeLa cells, and HEK-293T cells were obtained from the Institute of Cell and Biochemistry Research of the Chinese Academy of Science. CST-overexpressing cells (CST-1, CST-8), CST-knockdown cells (Chp2, Chp5), and their corresponding Mock cells were established by our laboratory previously (18Wu X.Z. Li Z. Shi B.Z. Hu P. Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells.World J. Gastroenterol. 2005; 11: 5763-5769Crossref PubMed Scopus (6) Google Scholar). CST overexpressed HCC cells mainly produced lactosyl sulfatide. The cells were maintained in RPMI 1640 medium supplemented with 10% newborn bovine serum (PAA, Austria) at 37°C under a 5% CO2 atmosphere. For the treatment, cells were cultured in RPMI 1640 medium containing 2 μM sulfatide, lactocerebroside, or galactocerebroside added from stock solution in DMSO. An equal amount of DMSO (0.1% v/v) was added to control group. The short hairpin sequences, including 5′-AGGAGUUGGUGGCAAUAAU-3′ and 5′-UAUUAGGCAUCACUCCAGG-3′, which specifically interfered and targeted Sp1 mRNA, were designed according to the protocol from Ambion (24Mukhopadhyay A. Saddoughi S.A. Song P. Sultan I. Ponnusamy S. Senkal C.E. Snook C.F. Arnold H.K. Sears R.C. Hannun Y.A. et al.Direct interaction between the inhibitor 2 and ceramide via sphingolipid-protein binding is involved in the regulation of protein phosphatase 2A activity and signaling.FASEB J. 2009; 23: 751-763Crossref PubMed Scopus (172) Google Scholar). The synthesized 55 bp forward and reverse oligonucleotides containing the siRNA sequence were annealed and ligated to the pSilencer 4.1 vector. The pcDNA3.0-Sp1 expression plasmid was kindly provided by Dr. Jian-Hai Jiang (Fudan University, P.R. China). A human CST cDNA expression plasmid was previously constructed (15Zhong W.X. Honke K. Long Z.Y. Liang Z.X. Taniguchi N. Lactosylsulfatide expression in hepatocellular carcinoma cells enhances cell adhesion to vitronectin and intrahepatic metastasis in nude mice.Int. J. Cancer. 2004; 110: 504-510Crossref PubMed Scopus (36) Google Scholar, 18Wu X.Z. Li Z. Shi B.Z. Hu P. Inhibitory effects of N-(4-hydrophenyl) retinamide on liver cancer and malignant melanoma cells.World J. Gastroenterol. 2005; 11: 5763-5769Crossref PubMed Scopus (6) Google Scholar). The integrin αV promoter fragments from −1295 to +207 bp, −795 to +207 bp, −309 to +207 bp, and −16 to +207 bp were obtained by PCR from the genomic DNA of SMMC-7721 cells. The following primers were used: integrin αV/Kpn I −1295: 5′- CCCGGTACGGTCCACACAATGCACTTAAA-3′, integrin αV/Kpn I −795: 5′- AAAGGTACGCAAGAGGCTATGCTGGC-3′, integrin αV/Kpn I −309: 5′- AAAGGTACGCCTCCTTCCAGGTCTCC-3′, integrin αV/Kpn I −16: 5′- AAAGGTACGTGGGGCGGGGGGAGGT-3′, integrin αV/Xho I +207: 5′-CCCGTCGAGAGAAATCCACGGCGAA-3′. The PCR products were inserted into the Xho I/Kpn I sites of the pGL3-basic vector (Promega, Madison, MI) and designated as pGL3- integrin αV. All of the constructs were verified by sequencing. Total RNA was extracted from cells with the Trizol reagent according to the manufacturer's instructions and was used as the template for cDNA synthesis. Reverse transcription was then carried out by M-MLV. The following primer sets were used for RT-PCR and real-time PCR: integrin αV subunit (sense 5′-GACAGTCCTGCCGAGTA-3′, anti-sense 5′-CTGGGTGGTGTTTGC-3′); Sp1 (sense 5′-TCACAAGCCAGTTCCAGCTCC-3′, anti-sense 5′-GGGTGCACCTGGATTCCTGAA-3′); Sp3v1 (sense 5′-GAAATGGCTGCCTTGGACG-3′, anti-sense 5′-AGCGGTGACGGCTGAGTGT-3′); Sp3v2 (sense 5′-ACCCCCTCCCCCTGTCTCCCTC-3′, anti-sense 5′-CTCCCATCGGTTTGGTGCTCCT-3′); ETS (sense 5′-TCACAAGCCAGTTCCAGCTCC-3′, anti-sense 5′-GGGTGCACCTGGATTCCTGAA3′); AP2 (sense 5′-GCTGGGCACTGTAGGTC-3′, anti-sense 5′-ACTTGGACAGGGACACG-3′); EGR1 (sense 5′-CAGCAGCCTTCGCTAACC-3′, anti-sense 5′-CATCGCTCCTGGCAAACT-3′); EGR2 (sense 5′-CAGCCTCATCCAGCGTCAC-3′, anti-sense 5′-TCGTCAGAGCGGGAGAACC-3′); β-actin (sense 5′-CTCCATCCTGGCCTCGCTGT-3′, anti-sense 5′-GCTGTCACCTTCACCGTTCC-3′); CST (sense 5′-CAAGTTCGCCTTCCCTAA-3′, anti-sense 5′-CACAGCAGGTCCTTCAG-3′). The PCR amplifications were performed, and the products were analyzed by 2% agarose gel electrophoresis. β-actin was used as an internal control. Real-time PCR was performed using a BIO-RAD IQ5 real-time PCR system (Bio-Rad). For transient transfection, SMMC-7721 cells were seeded in 6-well plates at 5 × 105 cells/well. The cells were then transfected with Sp1 expression vectors for 24 h and then treated with Lacto-Cer and sulfatide for 24 h, respectively. The empty pcDNA3.0 vector was used as a Mock control. The pSilencer 4.1-CMV puro vector (Ambion Inc.) encoding Sp1 siRNA (si-Sp1) was transfected to 80% confluent SMMC-7721 cells. The pSilencer 4.1-CMV puro vector containing scramble oligonucleotides was used as the control. The transient transfection of SMMC-7721 cells was performed using the transfection reagent (SunBio, Shanghai, China) according to the manufacturer's instructions. The adhesion assay was performed as previously reported (16Shi B.Z. Hu P. Geng F. He P.J. Wu X.Z. Gal3ST-2 involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.Biochem. Biophys. Res. Commun. 2005; 332: 934-940Crossref PubMed Scopus (14) Google Scholar). Briefly, 96-well plates were coated at 100 μl/well with collagen type I (40 µg/ml), fibrinogen (20 µg/ml), fibronectin (10 µg/ml), and vitronectin (2 μg/ml) at 4°C overnight. The negative control groups were coated with 1% BSA, and the positive groups were coated with poly-lysine. After blocking with 1% BSA for 1 h at room temperature and washing twice with PBS, 200 µl of the cell suspension (4 × 104/ml) was added to each well. After incubation at 37°C for 1 h, all wells were washed twice with PBS, stained with MTT {3- (4, 5)-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide}, and the absorbance value was measured at 570 nm with a reference wavelength of 630 nm. The adhesion rate was calculated by the formula (A570 value in test group − A570 in negative group) / (A570 in positive group − A570 in negative group) × 100%. The method was based on our previous report (25Wu X.Z. Shi P.C. Hu P. Chen Y. Ding S.S. N-all-trans-retinoyl-L-proline inhibits metastatic potential of hepatocellular carcinoma cells.Cell Biol. Int. 2006; 30: 672-680Crossref PubMed Scopus (14) Google Scholar). Briefly, CST-1, CST-8 and Mock cells (1 × 104) were incubated in a 96-well plate. After 24 h of culture, the cells were washed and fixed with 4% paraformaldehyde in PBS. Endogenous alkaline phosphatase was saturated and consumed with the substrate. The plates were blocked with 1% blocking reagent in PBS overnight at 4°C, incubated with O4 antibody (anti-Sulfo-Lacto-Cer and Sulfo-Gal-Cer) and mouse IgG as control for 2 h at room temperature, washed three times, and incubated with alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. The absorbance was measured at 450 nm after incubation with the alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP). The immunofluorescence method was based on the previous report (26Hu P. Shi B. Geng F. Zhang C. Wu W. Wu X.Z. E-cadherin core fucosylation regulates nuclear beta-catenin accumulation in lung cancer cells.Glycoconj. J. 2008; 25: 843-850Crossref PubMed Scopus (32) Google Scholar). The cells of CST-1 and CST-8 were fixed with 2% paraformaldehydrate and not permeabilized, but exogenous sulfatide-treated cells were permeabilized. After blocking and staining with a mouse anti-sulfatide antibody for 2 h and subsequently with FITC-conjugated secondary antibody for 1 h, the coverslips containing the cells were mounted on glass slides with Vectashield, and the cells were viewed under a fluorescence microscope. HUVECs in 150 μl of medium were seeded onto 96-well plates at 1 × 104 cells per well and incubated overnight at 37°C to confluence. Next, the HUVEC monolayer was stimulated with 10 ng/ml TNF-α for 4 h prior to the addition of the variously treated SMMC-7721 (1 × 105/ml) cells in 200 μl RPMI 1640 medium with 1% newborn bovine serum. After incubation at 37°C for 1 h, unattached cells were vigorously washed off with PBS, and the attached cells were fixed with 4% paraformaldehyde for 10 min. Subsequently, adherent cells were counted under a phase contrast microscope. The numbers of adherent cells in all groups were analyzed and compared to show the cell adhesion to HUVEC as described earlier (16Shi B.Z. Hu P. Geng F. He P.J. Wu X.Z. Gal3ST-2 involved in tumor metastasis process by regulation of adhesion ability to selectins and expression of integrins.Biochem. Biophys. Res. Commun. 2005; 332: 934-940Crossref PubMed Scopus (14) Google Scholar). Data were reported as mean ± SD for at least three wells, and the experiment was repeated three times independently. Based on our previous report (25Wu X.Z. Shi P.C. Hu P. Chen Y. Ding S.S. N-all-trans-retinoyl-L-proline inhibits metastatic potential of hepatocellular carcinoma cells.Cell Biol. Int. 2006; 30: 672-680Crossref PubMed Scopus (14) Google Scholar), subconfluent SMMC-7721 cells were detached with 0.02% EDTA, and the single-cell suspensions were washed and maintained in suspension for 1 h in RPMI 1640 medium with 10% newborn bovine serum. Cells, which were not permeabilized, were then incubated with 2 μg of FITC-labeled primary anti-human CD51 antibody per million cells for 2 h at 4°C with gentle shaking. After washing, the cells were resuspended in 1% paraformaldehyde, and cell surface immunofluorescence was analyzed by a flow cytometer (Becton Dickinson, Mountain View, CA). SMMC-7721 cells were seeded into 96-well plates and the next day transiently transfected with 0.2 μg of specific expression vectors. The cells were incubated for 24 h at 37°C, washed once with PBS, and then lysed in 20 μl of lysis buffer (Promega, Madison, WI) to measure the luciferase reporter gene expression by the luciferase reporter assay system (Promega). The intensity of luminescence was measured by a luminometer (Lumat LB 9507, Berthold, Germany). The data of relative luciferase activities were normalized to the control. The results are presented as the mean of the experiments performed in triplicate. Experiments were repeated at least three times. Western blot analysis was carried out essentially as described previously (17Zhang C.Y. Hu P. Fu D. Wu W. Jia C.Y. Zhu X.C. Wu X.Z. 3'-Sulfo-Le(x) is important for regulation of integrin subunit alphaV.Biochemistry. 2010; 49: 7811-7820Crossref PubMed Scopus (8) Google Scholar). Briefly, 30 μg of protein was resolved on 10% SDS-polyacrylamide gels and electrophoretically transferred to a polyvinylidene fluoride membrane. The membrane was then incubated with a series of antibodies against GAPDH, integrin αV, Sp1, and histone 4. After washing, the blots were incubated with horseradish peroxidase-conjugated secondary antibody, and protein bands were visualized using an enhanced chemiluminescence detection kit. Nuclear extracts were prepared with the nuclear extraction kit (Chemicon, Cat. No. 2900). The concentration of protein was determined by the Lowry method. Electrophoretic mobility shift assay (EMSA) was performed with a modified method according to a previous protocol (27Steiner S. Pfannschmidt T. Fluorescence-based electrophoretic mobility shift assay in the analysis of dna-binding proteins. Chapter 18.in: Pfannschmidt T. Plant Signal Transduction: Methods and Protocols. Humana Press, 2009: 273-289Crossref Scopus (10) Google Scholar). The double-stranded probes were labeled with fluorescence by a 5′ oligolabeling kit (RPN 5755, Amersham Pharmacia Biotech) according to the manufacturer's instruction. EMSA reaction mixtures were incubated in 1× binding buffer [1 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, 50 mM NaCl, 10 mM Tris-HCl (pH 7.5)], 4% glycerol, and nuclear extracts at room temperature for 20 min with or without unlabeled competitor, and then with fluorescence-labeled oligonucleotides for 30 min at room temperature. For the super-shift assays, anti-Sp1 antibody was added to the EMSA reaction mixture prior to the addition of the labeled oligonucleotide probes for 30 min at room temperature. For the competitive binding assay, the nonlabeled probe was added to the binding reaction at 100-fold excess over the labeled probe. The samples were electrophoresed on a native 6% polyacrylamide gel in 0.5× Tris-borate-EDTA buffer at 80 V for 1 h at 4°C and then at 120 V for 60 min. Finally, the binding reactions were visualized and analyzed by a multifunction imaging scanner (Typhoon Tyio). Chromatin isolated from SMMC-7721 cells was used for the chromatin immunoprecipitation (ChIP) assays according to the manufacturer's instructions (EZ-ChIP, Millipore, Temecula, CA). Briefly, SMMC-7721 cells, treated as indicated, were washed and chemically cross-linked using 1% formaldehyde in PBS for 10 min at room temperature. After quenching by the addition of 2 ml of 10× glycine solution (2.5 mol/l), cells were washed twice with ice-cold PBS and collected in 2 ml of PBS containing protease inhibitor cocktail II and centrifuged at 700 g at 4°C for 5 min. Next, the cell pellets were resuspended in 1 ml of SDS lysis buffer containing 1× protease inhibitor cocktail II and sonicated on wet ice by a Bioruptor sonicator (Ningbo, China) to disrupt genomic DNA to an average size of 200–750 bp. After preclearing with protein A-agarose for 1 h at 4°C, the chromatin was incubated at 4°C overnight with a specific antibody against Sp1, with anti-RNA polymerase II as the positive control and anti-IgG as the negative control, followed by incubation with protein A-agarose for 1 h. The precipitates were washed, and the chromatin complexes were eluted. After reversal of the cross-linking at
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