Eur J Biochem 270, 3795–3805 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03767.x Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line Hui-Ling Qi, Ying Zhang, Jun Ma, Peng Guo, Xia-Ying Zhang and Hui-Li Chen Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College of Fu-Dan University, Shanghai, China 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 metastasisrelated 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 (SLex), but not antibodies to other Lewis antigens, significantly blocked the cell adhesion to HUVECs, cell migra- tion and invasion, suggesting that SLex played a crucial role in the metastatic potential of H7721 cells The upregulation of cell surface SLex and a-1,3-fucosyltransferase-VII (a-1,3 Fuc T-VII, enzyme for SLex synthesis) was also mediated by PI3K and PKB, since LY294002 and AS-PKB also reduced the expressions of SLex and a-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 SLex 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 SLex and a-1,3 FucT-VII Insulin is well known as an endocrine hormone participating in the regulation of glucose and lipid metabolism It has been considered a member of the growth factor superfamily since the discovery of high structural homology among the receptors of insulin and other growth factors All of these transmembrane receptors contain protein tyrosine kinase activity in their intracellular domain [1] The signalling pathway of insulin is similar to that of some other growth factors [2], and the key signalling molecules of insulin are phosphatidylinositide-3-kinase (PI-3K), phosphotidylinositide dependent kinase-1 (PDK-1) and protein kinase B (PKB, also called Akt) [3,4] When insulin receptor binds to insulin, its C-terminal tyrosine residues become autophosphorylated, which promotes the recruitment of PI-3K via the interaction between the SH2 (Src homology 2) domain of PI-3K and the phosphotyrosine of the receptor, resulting in the activation of PI-3K [5] Alternatively, PI-3K can be activated by its binding to phosphorylated insulin receptor substrate (IRS) via SH2 [6] PKB is a downstream signalling molecule of PI-3K, since the products of PI-3K, phosphatidylinositide-3,4,5-triphosphate and phosphatidylinositide3,4-biphosphate bind to the pleckstrin-homology domain of PKB and recruit PKB to plasma membrane, leading to the activation of PKB via the phosphorylation at Thr308 by PDK-1 [4,6] Full activation of PKB requires another PKB kinase, recently identified as integrin-linked kinase (ILK, which is also activated by insulin via IRS-1) ILK combines with an adaptor protein Nck2 and a five LIM domaincontaining protein named PINCH (a particularly interesting new cysteine histidine protein) to form a ternary complex, which directly phosphorylates PKB at Ser473 [7] This ILK pathway is linked to the PI-3K/PDK-1 pathway [8,9] Recent studies showed that the activated PDK-1 Correspondence to H.-L Chen, Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College, Fu-Dan University, Shanghai, 200032, China Fax: + 86 21 64039987, E-mail: hlchen@shmu.edu.cn Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; ECL, enhanced chemiluminescence; EGF, epidermal growth factor; Fuc, fucose; a-1,3 FucT, a-1,3fucosyltransferase; Gal, galactose; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GlcNAc, N-acetylglucosamine; GnT-V, N-acetylglucosaminyltransferase; HRP, horseradish peroxidase; HUVEC, human umbilical vein endothelial cell; ILK, integrin-linked kinase; Lex, Lewis X [Galb1–4 (Fuc a-1,3) GlcNAc-]; IRS, insulin receptor substrate; PDK-1, phosphotidylinositide dependent kinase-1; PI-3K, phosphstidylinositide-3-kinase; PKB, protein kinase B; PVDF, polyvinylidene difluoride; SA, sialic acid; SLea, sialyl Lewis A [SAa-2,3 Galb1,3 (Fuca1,4) GlcNAc-]; SDLex, sialyl dimeric (difucosyl) Lewis X [SAa-2,3 Galb1,4 (Fuc a-1,3)GlcNAcb1,3Galb1,4 (Fuc a-1,3) GlcNAcb-1,3-]; SH2, Src homology Note: H.-L Qi and Y Zhang contributed equally to this manuscript (Received 10 June 2003, revised 20 July 2003, accepted 25 July 2003) Keywords: insulin; metastasis-related phenotype; protein kinase B; sialyl Lewis X; a-1,3-fucosyltransferase Ó FEBS 2003 3796 H.-L Qi et al (Eur J Biochem 270) phosphorylated PKB at both Thr308 and Ser473 under certain conditions [10] It was also reported that Ser473 of PKB could be auto-phosphorylated [11], and the phosphorylation of tyrosine residue(s) of PKB was also required for its full activation [12] The insulin receptor/IRS/PI-3K/ PDK-1/PKB signalling pathway crosstalks with the typical growth factor signalling pathway, such as receptor protein tyrosine kinase/growth factor receptor binding protein 2-son of sevenless protein/Ras/Raf/mitogen activated protein kinase kinase/mitogen activated protein kinase pathway [13] Another function of the PKB signalling pathway is the promotion of cell survival or inhibition of cell apoptosis [3,14] PKB is a general mediator of survival signals, and several of its mechanisms have been reported by different authors [15,16] PKB may phosphorylate the pro-apoptotic molecule BAD to prevent it from binding to and inhibiting the survival proteins Bcl-XL and Bcl2, or phosphorylates IjB kinase to induce the nuclear translocation of the transcription factor NKjB for activating the survival genes PKB also phosphorylates caspase and blocks its activation by cytochrome c released from mitochondria In addition, PKB has been shown to phosphorylate Fockhead family members, including Fas ligand and block apoptosis through regulation of death genes It is reasonable to assume that insulin has an anti-apoptotic effect, since PKB is an important signal transducer of insulin In our laboratory, Wang et al [17] found that in a human hepatocarcinoma cell line, H7721, both insulin and epidermal growth factor (EGF) stimulated the activity of a metastasis-related enzyme, N-acetylglucosaminyltransferase V (GnT-V) [18,19], which synthesizes a b1,6 N-acetyl-glucosamine (GlcNAc) branch on the asparagine (Asn or N)-linked sugar chains (N-glycans) of glycoproteins Based on the similarities between insulin and EGF on signal transduction, GnT-V stimulation and anti-apoptotic effect [14], we supposed that insulin might also display a metastasis promoting effect In the present investigation, studies were carried out on the effect of insulin on some metastasis-related phenotypes of H7721 cell line, such as cell adhesion, chemotactic migration and invasion, as well as their mechanisms Whether these effects were mediated by PI-3K/PKB signalling pathway was also investigated by using H7721 cells treated with the specific inhibitor of PI-3K or transfected with antisense cDNA of PKB Lewis antigens are a series of fucosylated Galb1–3/b1– 4GlcNAc- (sialylated or not sialylated) oligosaccharides on the cell surface, mainly located at the outer chains of glycolipids and O-linked glycans of glycoproteins It has been well documented that the interaction between the sialyl Lewis antigens expressed on cancer cell surface and the E- or P-selectin on vascular endothelial cells was the initial step of cancer cells adhering to and penetrating the endothelium before haematogeneous metastasis [20,21] Moreover, the expressions of sialyl Lewis antigens are often positively correlated with the metastatic potential of some cancers [22,23] Therefore, sialyl Lewis X (SLex) and the enzyme responsible for its synthesis, a1,3 fucosyltransferase (a-1,3 FucT)-VII [24,25], were selected as metastasis-related molecules to study whether they are regulated by insulin The results indicate that insulin promotes the metastatic potential of H7721 cells ex vivo via the upregulation of SLex/a-1,3 FucT-VII, and its effects are mainly mediated by the PI-3K/PKB signalling pathway Materials and methods The H7721 and human umbilical vein endothelial cell (HUVEC) lines were obtained from the Institute of Cell Biology, Academic Sinica RPMI 1640, Dulbecco’s modified Eagle’s medium (DMEM) and Matrigel were from Gibco/BRL mAbs KM93 (anti-SLex), CA19-9 (anti-SLea) and plasmid pUC19/FucT-VII were kindly provided by H Narimatsu (Soka University, Tokyo, Japan) mAb FH6 (anti-SDLex) was a gift from S I Hakomori (University of Washington, Seattle, USA) CD15 (mAb anti-Lex) and horseradish peroxidase (HRP)-labelled goat anti-(mouse IgG) IgG were from Dako The plasmid containing human PKB-a (pSGS-PKBGAG) was a gift from P Coffer (University Hospital Utrecht, the Netherlands) The polyclonal rabbit anti-(human PKB) Ig was from Biolabs Akt/PKB assay kit was from New England Biolabs Monoclonal anti(human b-actin) Ig was from Santa Cruz Technology Fluorescein isothiocynate-conjugated goat anti-(mouse IgM), HRP-labelled goat anti-(rabbit IgG), insulin, L-poly(L-lysine), laminin, LY294002 and tumour necrosis factor-a (TNF-a) were from Sigma TRIzol, DNA restriction endonucleases and random primer labelling kit were from Promega Hybond-N+ nylon membrane, poly(vinylidene difluoride) membranes, enhanced chemiluminescence plus (ECL+) Western blotting detection system and [a-32P]dATP were from Amersham Corp Insert (transwell) and cell culture plates were from NUNC Other reagents were commercially available in China The construction of plasmids containing sense or antisense cDNA of PKB-a was performed in our laboratory as published previously [26] Briefly, the pSGS-PKBGAG (6.5 kb) was digested with EcoRI and BglII to form a 2.6kb fragment containing GAG-PKB cDNA This fragment was then digested with NcoI to cut off the cDNA of GAG, and the 1.8-kb fragment of PKB cDNA was separated, purified, and made blunt-ended After it was ligated with EcoRI linkers, the fragment was inserted into the EcoRI site of pBluescript-SK (2.96 kb) by T4 DNA ligase The direction of the inserted PKB cDNA was determined by PstI digestion of the recombinant plasmid: there was a PstI site near to the 5¢ end of the PKB cDNA and another PstI site 1.7 kb downstream Hence, a 1.7-kb fragment could be cut out by PstI digestion This pBluescript-SK/PKB plasmid was isolated and digested with HindIII, and the linearized plasmid was then treated with BamHI In the construction of sense PKB-a plasmid, pcDNA3/ S-PKB, the PKB cDNA in the HindIII (5¢)–BamHI (3¢) fragment (1.8 kb) was isolated and ligated with eukaryotic expression plasmid pcDNA3, which was also cut with HindIII and BamHI Alternatively, in the construction of antisense PKB-a plasmid, pcDNA3/AS-PKB, the HindIII linearized pBluescript-SK/PKB plasmid was made blunt ended and digested with BamHI, followed by separation of the PKB cDNA and ligation of it with pcDNA3, which was cut with BamHI and EcoRV Finally, the recombinant plasmids were verified by sequencing Transfection of these constructed plasmids or the vector pcDNA3 into H7721 Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLex (Eur J Biochem 270) 3797 cells was performed by electroporation using Gene Pulser at 250 V/0.4 cm and 1000 lF [26] Cells were selected by G418 and the neomycin-resistant cells were obtained after 2–3 weeks and re-cloned by serial dilution The stable cells transfected with sense and antisense PKB were named S-PKB/H7721 and AS-PKB/H7721 Cell culture and treatment Cells were cultured for 48 h at 37 °C, 5% (v/v) CO2 in RPMI-1640 medium containing 10% (w/v) foetal bovine serum, penicillin and streptomycin as described previously by our laboratory [27,28] Insulin and/or LY294002 (dissolved in dimethylsulfoxide) were added to the culture medium at a final concentration of nM and 15 lM, respectively, determined by the previous dose-dependence assays The same final concentration (0.15%) of dimethylsulfoxide was in the ÔcontrolÕ medium for LY294002 treated cells, but dimethylsulfoxide was omitted from the ÔcontrolÕ medium for insulin treated cells However, there was no difference in the results between the ÔcontrolÕ cells cultured in the presence or absence of dimethylsulfoxide Assay of cell adhesion to HUVECs Cell adhesion to HUVECs was assayed by the method of Takada et al [21] with modification [29,30] Briefly, HUVECs were coated on to a 96-well plate and stimulated with 200 ngỈmL)1 of tumour necrosis factor (TNF)-a for h Then 105 cells in 0.1 mL were added to each well and further incubated for 30 at °C After being washed five times with NaCl/Pi, the cells were fixed with 4% (v/v) formaldehyde, and the number of cells adhered to HUVECs was counted in eight high power fields of view (· 200) Assay of cell adhesion to laminin The cell adhesion experiment was performed with the methods previously published by our laboratory [31] In brief, the wells of culture plate were coated with 0.1 mL of different concentrations of laminin in duplicate In addition, 0.1% poly(L-lysine) or 1% (w/v) BSA was each coated on to two wells as maximal and minimal adhesion controls, respectively After being washed twice, the plate was incubated at 37 °C for h, and blocked by 1% (w/v) BSA at 37 °C for 0.5 h Cells (1 · 105) in 0.1 mL were added to each coated well and incubated for h at 37 °C The cells were then washed twice, fixed with 4% (v/v) formaldehyde and stained with crystal violet After the absorbance at 595 nm (A595) was measured, the relative cell adhesion to the coated wells was calculated using a formula reported in our previous paper [31] Determination of cell migration and invasion The chemotactic cell migration was assayed using 24-well transwell units with polycarbonate filters of 8-lm pore size by the method of Yu et al [32] and described by Liu et al [29,30] Each lower compartment of the transwell contained 600 lL 0.5% (w/v) foetal bovine serum in DMEM as the chemoattractant, or 0.5% (w/v) BSA as the negative control Cells (2 · 104) in 0.1 mL DMEM/0.1% (w/v) BSA were added into the upper compartment of the transwell unit and incubated for h at 37 °C in a humidified atmosphere containing 5% CO2 The cells were then fixed with 4% formaldehyde and stained with crystal violet Then the number of cells that had migrated to the lower side of the polycarbonate filter was counted in eight high power fields of view (· 200) Each sample was assayed in duplicate The assay of chemotactic cell invasion was the same as that for chemotactic cell migration assay except that the upper side of polycarbonate filter was coated with a continuous thin layer of matrigel (20 lg per filter) [29–31] Cells (1 · 105) in 0.1 mL were added, and the incubation time was extended to 36 h Then the cells were fixed, stained and countered as described above, and the number of cells that had migrated to the lower side of the matrigel-coated filter was a measure of the invasive activity of the cells Inhibition of cell adhesion, migration and invasion with monoclonal antibody For inhibition of cell adhesion, migration and invasion, H7721 cells were preincubated with 10 ngỈmL)1 or 20 ngỈmL)1 different mAbs against Lewis antigens for 30 at °C Each antibody was assayed in duplicate Subsequently, the cells were added to the monolayer HUVECs for the adhesion assay, or to the transwells for the migration and invasion assay Detection of Lewis antigen SLex with flow cytometry The cells were detached with mM EDTA, washed and resuspended in NaCl/Pi containing 1% (w/v) BSA Then 106 cells were incubated with : 50 diluted KM93, the mAb for SLex, for 30 at °C After two washes, the cells were incubated for 45 at °C with : 200 diluted fluorescein isothiocyanate-conjugated goat anti-mouse IgM, then the cells were washed again and subjected to flow cytometry (1 · 104 cellsỈsample)1) for fluorescence analysis [29,30] A negative control sample without the addition of the first mAb was set up in each run to determine the background of fluorescence FACS (fluorescence activated cell sorting) spectra were drawn automatically, and the left- or right-shift of the curve or its peak indicated the decrease or increase of the mean fluorescence intensity (MFI), respectively, as indicated by the ÔM1Õ bar in the figures Quantitative data were expressed as the relative MFI (the MFI value of the control or mock-transfected cells was set at 100%) Western blot analysis The cells were homogenized and centrifuged according to the method previously described by us [30] The protein concentration was determined by Lowry’s method [33] Western blotting was performed according to a modified method of Kudo et al [34] Aliquots of 50 lg protein were separated by SDS/PAGE on 10% acrylamide and electroblotted on to a poly(vinylidene difluoride) membrane, which was then blocked with fat-free milk in Tris-buffered saline pH 7.4 containing 0.05% Tween 20 The membrane was treated with : 1000 diluted rabbit polyclonal anti-human PKB or : 800 diluted monoclonal anti-human b-actin in Ó FEBS 2003 3798 H.-L Qi et al (Eur J Biochem 270) 5% fat-free milk/Tris-buffered saline, followed by incubation with HRP-labelled goat anti-(rabbit IgG) (1 : 200) or anti-(mouse IgG) (1 : 500), and stained with ECL reagent Densitometric scanning of the exposed X-ray film was used for quantitative measurement of the protein bands The relative expression of PKB was calculated by the intensity ratio of PKB band and b-actin band Assay of PKB activity PKB assay was performed with a widely used assay kit of Akt kinase according to the instruction manual Briefly, cell lysate (500 lg protein) was mixed with immobilized PKB antibody and incubated at °C for h to immunoprecipitate the PKB The pellet was suspended in 40 lL kinase buffer (25 mM Tris/HCl pH 7.5, M b-glycerol phosphate, mM dithiothreitol, 0.1 mM Na3VO4, 10 mM MgCl2) and used as the enzyme preparation, which was supplemented with the substrates, ATP (200 lM) and lg GSK-3a/b fusion protein (paramyosin fused to GSK-3a/b crosstide corresponding to residues surrounding Ser21/9 of GSK-3a/b, CGPKGPGRRGRRRTSSFAEG) After incubation at 30 °C for 60 min, the phosphorylated GSK3a/b fusion protein was subjected to Western blotting and detected by using phospho-GSK-3a/b (Ser21/9) antibody and ECL reagents Finally, the intensity of the GSK bands on X-ray film was quantified by densitometric scanning Statistical analysis Statistical analysis was performed with SPSS software using student’s t-test or the Cochran–Cox test when the data was uniform or not uniform, respectively Results Time course of insulin action and alteration in cell adhesion after treatment with insulin, LY294002 or transfection with AS-PKB cDNA At first, we determined the time course of insulin action When cell adhesion to HUVECs was selected as an example of metastatic phenotypes, we could not find any significant change 10 after insulin treatment The apparent elevation of cell adhesion to HUVECs was only detected 24 h after the treatment of insulin, and, in fact, the increase after 48 h became very significant (P < 0.01) The 48-h incubation was therefore adopted in all of our following experiments Fig shows that adhesion of insulin (2 nM)treated H7721 cells to HUVECs was increased to 164.9% that of the untreated control cells (UnC) In the cells treated with 15 lM LY294002 (the specific inhibitor of PI-3-K [6]), and transfected with pcDNA3/AS-PKB, the adhesion to HUVECs was deceased to 54.1% of the UnC and 51.2% of Northern blot analysis of a-1,3 FucT-VII mRNA The probe for detection of a-1–3 FucT-VII mRNA was the 1.2-kb full-length a-1–3 FucT-VII cDNA inserted into plasmid pUC19/FucT-VII The cDNA was isolated by digestion with BamH1 and EcoRI, purified by agarose electrophoresis, followed by extraction with Tris-saturated phenol/chloroform and precipitation with ethanol as described previously [30] The probe was labelled with [a-32P]dATP using random primer labelling kit from Promega according to manufacturer’s instructions Glyceraldehyde3-phosphate dehydrogenase (GAPDH) cDNA was labelled by the same method and used as an intrinsic standard Total RNA was extracted from cells using TRIzol according to the manufacturer’s protocol (Promega) Northern blot analysis was carried out according to the method of Sagestrom and Sieve [35] as described by Liu et al [30] In brief, total RNA (30 lg) was separated by formaldehyde denatured electrophoresis, then transferred to Hybond-N+ nylon membrane, and prehybridized for 4–6 h at 65 °C in 0.2 M sodium phosphate buffer (pH 7.4)/1 mM EDTA/1% (w/v) BSA/7% (w/v) SDS/15% (v/v) formamide Hybridization was performed at 65 °C for 16–20 h in the same hybridization solution containing a-32P labelled probe of a-1,3 FucT-VII The hybridized membrane was washed three to five times with 40 mM sodium phosphate buffer pH 7.4/1% (w/v) SDS/1 mM EDTA for 30 at 65 °C, followed by autoradiography The intensities of the a-1,3 FucT-VII bands were quantified by densitometric scanning and compared with the intensities of GAPDH bands on the same membrane, which was re-hybridized by GAPDH probe The magnitude of expression was indicated as the ratio of the intensity of a-1,3 FucT-VII band to the intensity of GAPDH band Fig Alteration in cell adhesion to HUVECs after treatment with insulin, LY294002 or transfection with AS-PKB cDNA HUVEC, human umbilical vein endothelial cells; UnC, untreated control H7721 cells; Ins, H7721 cells treated with nM insulin; LY294002, H7721 cells treated with 15 lM LY294002; LY294002 + Ins, H7721 cells treated with both LY294002 and insulin; Mock, H7721 cells transfected with pcDNA3 vector; AS-PKB, H7721 cells transfected with pcDNA3/ AS-PKB; AS-PKB + Ins, AS-PKB/H7721 cells treated with nM insulin Data are expressed as the mean ± SD of three independent experiments The value of the UnC group (130 ± 24 cells per high power field of view) was set at 100% **P < 0.01 compared with the UnC or Mock group; #P < 0.01 compared with the Ins group, but P > 0.05 compared with the LY294002 group; ##P < 0.01 compared with the Ins group, but P > 0.05 compared with the AS-PKB group The incubation time for all the cell groups was 48 h Experimental procedures were as described in Materials and methods Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLex (Eur J Biochem 270) 3799 Fig Alteration in cell adhesion to laminin after treatment with insulin, LY294002 or transfection with AS-PKB cDNA UnC, Ins, LY294002, LY294002 + Ins, Mock, AS-PKB, AS-PKB + Ins, as in Fig Data are expressed as the mean ± SD of three independent experiments **P < 0.01 compared with the UnC group; *P < 0.05 compared with the UnC or Mock group; #P < 0.01 compared with the Ins group, but P > 0.05 compared with the LY294002 group; ##P < 0.01 compared with the Ins group, but P > 0.05 compared with the AS-PKB group The incubation time for all the cell groups was 48 h Experimental procedures were as described in Materials and methods the ÔmockÕ (cells mock transfected with the vector pcDNA3) value, respectively After insulin stimulation, the adhesion of LY294002 treated cells and AS-PKB/H7721 cells to HUVECs elevated only very slightly Cell adhesion to laminin was increased with the concentrations of the coated laminin, and enhanced after insulin treatment at different concentrations of laminin By contrast, it was reduced after treatment with LY294002 or transfection with pcDNA3/AS-PKB (Fig 2) Insulin did not significantly elevate the cell adhesion of LY294002 treated cells and AS-PKB/H7721 cells Alteration in cell migration and invasion after treatment with insulin, LY294002 or transfection with AS-PKB cDNA In insulin treated H7721cells, the abilities of both chemotactic migration through transwell and chemotactic invasion through matrigel were elevated to 200.0% and 166.1%, respectively, as compared with the UnC cells (Fig 3) In the presence of LY294002, both cell migration and invasion were reduced to about 45% of the UnC level These two parameters also declined to 41.4% and 52.5% in AS-PKB/ H7721 cells when compared with the mock cells, and increased only slightly after insulin treatment in LY294002 treated and AS-PKB/H7721 cells Inhibition of cell adhesion to HUVECs, migration and invasion by different antibodies against Lewis antigens When different mAbs against Lewis antigens were added to block the surface Lewis antigens, it was found that the cell Fig Alteration in cell migration and invasion after treatment with insulin, LY294002 or transfection with AS-PKB cDNA UnC, Ins, LY294002, LY294002 + Ins, Mock, AS-PKB, AS-PKB + Ins, as in Fig Data are expressed as the mean ± SD of three independent experiments The values of the UnC group (31 ± and 60 ± cells per high power field of view for migration and invasion, respectively) were set at 100% **P < 0.01 compared with the UnC or Mock group; #P < 0.01 compared with the Ins group, but P > 0.05 compared with the LY294002 group; ##P < 0.01 compared with the Ins group, but P > 0.05 compared with the AS-PKB group The incubation time for all cell groups was 48 h Experimental procedures were as described in Materials and methods adhesion to HUVECs of both untreated and insulin treated cells was markedly suppressed by KM93 (anti-SLex mAb) only (Fig 4A) KM93 also significantly inhibited the chemostatic cell migration and invasion of insulin treated H7721 cells (Fig 4B) FH6 (anti-SDLex mAb) slightly inhibited these processes but it was not statistically significant In contrast, other antibodies (CD15 and CA19-9) did not show any obvious blocking effects These findings indicate that sialyl Lewis antigen, especially SLex, plays a critical role in the cell adhesion to HUVECs, cell migration and invasion Effect of insulin on the expression of SLex on differently treated and transfected H7721 SLex was selected as the representative of Lewis antigens to study its regulation by insulin because it is the only abundant and metastasis-related Lewis antigen on the H7721 cell surface [29,30] As shown in Fig 5, insulin significantly upregulated the expression of SLex to 430% of the untreated control value, while LY294002 down regulated SLex to 54.9% of the UnC value In the presence of LY294002, insulin treatment was no longer to show any up regulatory effects on SLex When mock cells were treated with insulin, the expression of SLex increased to the same level as the insulin-treated parent control cells Transfection of sense or antisense PKB to H7721 cells increased or decreased SLex expression to 527.5% or 30.9% of the mock value, respectively (Fig 6) After the S-PKB/H7721 cells were treated with insulin, the SLex expression was further increased to 836.2% of the 3800 H.-L Qi et al (Eur J Biochem 270) Ó FEBS 2003 Fig Effect of monoclonal antibodies on the cell adhesion to HUVECs, migration and invasion (A) Inhibition by mAbs of the adhesion to HUVECs of untreated control and insulin treated cells (B) Inhibition by monoclonal antibodies of the migration and invasion of insulin treated H7721 cells Non, Cells not treated with any antibodies; CA-19-9, mAb to SLea; CD15, mAb to Lex; KM93, mAb to SLex; FH6, mAb to SDLex; UnC, untreated control H7721 cells; Ins, H7721 cells treated with nM insulin The data are expressed as the mean ± SD of three independent experiments **P < 0.01 compared with the Non group The cells were incubated for 48 h Experimental procedures for (A) and (B) were as described for Figs and 3, respectively, except that the cells were pretreated with different mAbs against different Lewis antigens (10 ngỈmL)1 for UnC, 20 ngỈmL)1 in insulin treated cells) for 30 at °C Fig Effects of insulin and LY294002 on the expression of SLex on H7721 cells (A) Flow cytometry (–) Control, without the addition of the first antibody (B) Calculation of relative expression of SLex The data are expressed as the mean ± SD of three independent experiments The value of UnC was set at 100% *P < 0.05 compared with UnC; **P < 0.01 compared with UnC; #P < 0.01 compared with the Ins group, but P > 0.05 compared with LY-294002.UnC, Ins, LY294002, LY294002 + Ins, as in Fig The incubation time for all the cell groups was 48 h Experimental procedures were as described in Materials and methods mock level In contrast, the SLex expression on AS-PKB/ H7721 cells was only slightly increased after insulin treatment, by 39.2% of the mock value Effect of insulin on the expression of PKB protein and PKB activity in differently treated and transfected H7721 cells In the previous dose–response assays we found that treatment with nM insulin for 48 h was enough to stimulate the expression of PKB protein and activity in untreated control cells The effect of nM was greater, but the difference between nM and nM was not apparent Therefore, we chose nM of insulin concentration in all of our experiments This concentration was close to the physiological concentration of insulin in serum Moreover, the expression of PKB in the untreated control and sense or antisense PKB transfected cells before and after the insulin treatment was determined to further verify that the upregulation of SLex by insulin was mediated by PKB The results in Fig 7A show that the expressions of PKB protein in differently treated and transfected cells were generally in accordance with SLex expression After densitometric scanning of the Western blot profiles, it was found that the expression of PKB protein was elevated to 201% and 253% of the UnC and mock values, respectively, after insulin treatment The PKB protein was also increased to 233% and decreased to 55% of the mock values in S-PKB/ Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLex (Eur J Biochem 270) 3801 Fig Effects of transfection of sense or antisense PKB cDNA and insulin on the expression of SLex (A) Flow cytometry (–) Control: without the addition of the first monoclonal antibody (B) Calculation of relative expression of SLex Data are expressed as the mean ± SD of three independent experiments The value of UnC was set at 100% *P < 0.01 compared with Mock; **P < 0.01 compared with Mock + Ins or S-PKB; #P < 0.01 compared with Mock + Ins, and P > 0.05 compared with AS-PKB UnC, Mock, Mock + Ins, AS-PKB, AS-PKB + Ins, as in Fig S-PKB, Sense-PKB cDNA transfected H7721 cells; S-PKB + Ins, S-PKB/H7721 cells treated with nM insulin The incubation time for all the cell groups was 48 h Experimental procedures were as described for Fig H7721 and AS-PKB/H7721 cells, respectively The treatment of insulin further increased the PKB protein to the double level in S-PKB transfected cells, but did not significantly elevate PKB in AS-PKB cells Insulin treatment also upregulated PKB activity to 223%, while LY294002 downregulated it to 20% of the UnC value However, insulin could not elevate PKB activity to a level higher than that of UnC in LY294002-treated cells (Fig 7B) In addition, PKB activity was elevated to 202% and reduced to 24% of the mock value in S-PKB and ASPKB transfected cells, respectively After insulin treatment, PKB activity was almost doubled in the mock and S-PKB/ H7721 cells, but it increased not so obviously in AS-PKB/ H7721 cells (Fig 7C) These results concerning the expression and activity of PKB protein were compatible with the SLex expression as shown in Figs and Alteration in a-1,3 FucT-VII expression after treatment with insulin, LY294002 or transfection with AS-PKB cDNA Lewis antigens are synthesized by a set of a-1,3/4 fucosyltransferases (a-1,3/4 FucTs) At least six human a-1,3/4 FucTs (FucT-III, IV, V, VI, VII and IX) have been cloned Among them, FucT-IV and IX prefer nonsialylated neutral acceptors, while FucT-III, V, VI and VII efficiently fucosylate sialylated acceptors FucTIII has two different activities (a-1,3 and a-1,4 fucosyla- tion), leading to the generation of a-1,3 fucosyl-containing SLex and a-1,4 fucosyl containing SLea, respectively, but FucT-VII catalyses only the synthesis of SLex [24,25] In order to clarify the role of FucT-VII in the synthesis of SLex, the expressions of FucT-VII mRNA were determined with Northern blot using the cDNA of FucT-VII as the probes The results showed that the untreated control cells expressed a moderate amount of FucT-VII The mRNA of FucT-VII was about 2.3 kb The expression of FucT-VII mRNA was decreased in LY294002 treated or AS-PKB cDNA transfected cells Insulin treatment obviously upregulated the expression of FucTVII mRNA in parent H7721 cells, but could not significantly elevate the mRNA in LY294002 treated and AS-PKB transfected cells (Fig 8A) Densitometric analysis showed that the level of FucT-VII mRNA expression in insulin treated cells was 188.6% of the untreated control level, while in LY294002 treated and AS-PKB transfected cells, it was only 43.8% of UnC and 25.5% of the mock value, respectively Insulin treatment only slightly increased the FucT-VII mRNA in LY294002 treated and AS-PKB transfected cells (Fig 8B) These results were in accordance with the above findings that the SLex expression was lower in LY294002 or AS-PKB cDNA transfected cells, and insulin could significantly stimulate SLex expression in UnC or mock cells, but not in LY294002 treated and AS-PKB transfected cells 3802 H.-L Qi et al (Eur J Biochem 270) Ó FEBS 2003 Fig Expression of PKB protein and activity in differently treated and transfected cells (A) Expression of PKB protein and b-actin (loading control) in differently treated and transfected cells (Western blot) (B) PKB activity in insulin and LY294002 treated H7721 cells (C) PKB activity in sense or antisense PKB transfected cells before and after insulin treatment UnC, Mock, UnC + Ins or Ins, Mock + Ins, S-PKB, S-PKB + Ins, AS-PKB, AS-PKB + Ins, LY294002, LY294002 + Ins, as in Figs and The incubation time for all the cell groups was 48 h The procedures for Western blot and assay of PKB activity were as described in Materials and methods Three independent experiments of (A), (B) and (C) were performed and the results were reproducible Fig Alteration in a-1,3 FucT-VII expression after treatment with insulin, LY294002 or transfection with AS-PKB cDNA (A) Northern blotting The result shown is representative of three reproducible experiments (B) Densitometric analysis Ratio of the absorbance units of a-1,3 FucT-VII to GAPDH UnC, Ins, LY, LY + Ins, Mock, AS-PKB, AS-PKB + Ins, as in Fig Data were expressed as the mean ± SD of three independent experiments **P < 0.01 compared with the UnC or Mock group; #P < 0.01 compared with the Ins group, but P > 0.05 compared with the LY294002 group; ##P < 0.01 compared with the Ins group, but P > 0.05 compared with AS-PKB group The incubation time for all the cell groups was 48 h Experimental procedures were as described in Materials and methods Discussion In this study, the assay of cell adhesion to TNF-a stimulated HUVECs mimics the interaction between the surface Lewis antigens on malignant cells and the E- and/or P-selectin on vascular endothelium in vivo The cell invasion assay is similar to the cell penetration through the vascular membrane in vivo, since matrigel is an artificial extracellular interstitial membrane Therefore, these parameters can be used to assess the metastatic potential ex vivo The ability of cell adhesion to laminin is often positively correlated to the other metastatic phenotypes, such as cell adhesion to HUVECs as well as cell migration and invasion This correlation was also observed in H7721 cells transfected with the metastasis-promoting gene, H-ras or v-sis [27] or the metastasis-suppressive gene, nm23-H1 [28] The Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLex (Eur J Biochem 270) 3803 upregulation of insulin on metastasis-associated phenotypes described in this paper provides the evidence that insulin also displays a metastasis-promoting function in addition to its metabolism-regulating and anti-apoptotic effects The effect of insulin on metastasis-related phenotypes is a long duration action It is believed that not only the phosphorylation of the signalling molecules but also the transcription of some genes and the synthesis of some proteins will be altered during 48 h treatment with insulin The increase of endogenous PKB protein (Fig 7A) and a-1,3 FucT-VII mRNA (Fig 8) after insulin treatment suggested that these two enzymes were induced by insulin as some enzymes in glycolysis and fatty acid synthesis pathways The induced synthesis of PKB protein was also observed in our lab after H7721 cells were treated with foskolin, an up-regulator of cell cyclic-AMP, for 48 h [36] The expression of PKB protein was parallel to the PKB activity After densitometric analysis, we found that the percentage of the alteration in PKB protein was similar to that in PKB activity (Fig 7), suggesting that the insulininduced PKB protein was almost fully phosphorylated and activated Whether insulin was internalized by the cells remains unknown, but the results that cell adhesion to HUVECs as well as PKB protein and activity was still high in cells treated with nM insulin for 48 h, suggested that the internalization and degradation of PKB, if any, would not significantly influence the results From Figs 1, and 3, it is evident that the effects of insulin on the metastatic potential were mainly mediated by PI3K/PKB signalling pathway Moreover, based on the findings shown in Figs 4, and 6, it can be concluded that the increased cell adhesion to HUVECs, migration and invasion were probably due to the increased expression of SLex The specific inhibitory effect of SLex antibody (KM93) on the metastatic phenotypes was observed not only in untreated and insulin treated H7721 cells, but was also found in metastatic potential upregulated H7721 cells after they were treated with EGF or phorbol myristate acetate [29], or transfected with the c-erbB2/neu oncogene or a-1,3 FucT-VII cDNA [30] as well as in cells treated with forskolin [36] These results indicated that SLex was the key molecule on H7721 cells for adhesion to HUVECs, migration and invasion The structure–function relationship between Lewis antigens and metastatic phenotypes is being investigated in our laboratory Recently, it was found in our laboratory that the a-2,3 sialyl residue of SLex molecule was more important than the a-1,3 fucosyl residue in the promotion of metastatic phenotypes, and this finding was consistent with the observation that the antibody of Lex (CD15) showed no inhibitory effect on metastatic phenotypes We did not determine the effects of antibodies on the cell adhesion to laminin, as it was reported that the laminin receptor is integrin [37], but not Lewis antigens It is suggested that FucT-VII is probably the main enzyme responsible for the synthesis of SLex in the H7721 cell line, as FucT-V is not expressed in many tissues, including liver cells [24], and FucT-III is expressed at low levels in H7721 cells as we reported previously [30] Furthermore, the expression of FucT-VII was positively correlated to the expression of SLex in the insulin or LY294002 treated and AS-PKB transfected H7721 cells as shown in this study, and in the different clones of erbB2/neu transfected H7721 cells [30] In addition, the transfection of FucT-VII cDNA into H7721 cells resulted in a remarkable upregulation of SLex with simultaneous enhancement of the above-mentioned metastasis-related phenotypes [30] However, it is possible that FucT-III and VI, especially FucT-VI also play an important role in the synthesis of SLex in H7721 cells, as it was reported that FucT-VI exhibited the strongest relative activity for SLex synthesis, approximately 6.4-fold higher than that of FucT-III, and 1.5-fold higher than that of FucT-VII [38] The reduction of metastatic phenotypes in AS-PKB/ H7721 cells was not due to the possible apoptosis of the cells, since we had not found obvious signs of apoptosis in the cells even after treatment with insulin for 48 h No subG1 cell peak was observed on the spectra of flow cytometric analysis, and the abnormal nuclei or nuclear fragments were not detected after Hoechest staining Only very few positive cells were found in a TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling) test In addition, our plasmid contained only antisense cDNA of PKB-a, so the expression of PKB-b and c in the cells should not be downregulated by the antisense PKB-a plasmid However, the cell growth rate was decreased and the susceptibility to apoptosis induced by all-trans retinoic acid was increased in AS-PKB/H7721 as compared with the mock-transfected cells Therefore, the decrease of metastatic phenotypes was considered to result from the downregulation of PKB-a in the cells The findings shown in Figs 5, and suggest that the regulation on the expressions of SLex and a-1,3 FucT-VII by insulin was also mediated by PI3K/PKB signalling pathway The changes in the expressions of PKB protein and activity were generally correlated to the alterations in sialyl Lewis X/a-1,3 FucT-VII and the metastatic phenotypes However, the increase rate in SLex was far greater than that of PKB protein and activity in the insulin treated and S-PKB cells, indicating that SLex might be regulated by other insulin-induced factor(s) in addition to PKB signalling pathways It is not clear how the signal goes from PKB to a-1,3 FucT-VII It has been reported that some transcription factors, such as E2F, cAMP responsive element binding protein, and AP-1, b-catenin/Tcf/LEF are the downstream signalling molecules of PKB or GSK-3, and PKB can induce initiation of mRNA translation through phosphorylation of 4E-BP and activation of eIF-4E [39] Another transcription factor, elk-1 can also be induced by insulin [40] Recently, it was reported that the human a-1,3 FucTIV gene is regulated by elk-1 in the U937 cell line [41] Which factor(s) is responsible for the regulation of gene transcription of a-1,3 FucT-VII induced by insulin remains to be studied In summary, our findings reveal that the insulin/PI3K/ PKB signalling pathway enhances the 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Forskolin up-regulates metastasis-related phenotypes and molecules via protein kinase B, but not PI-3K, in H7721 human hepato-carcinoma cell line Mol Cell Biochem (in press) 37 Giancotti, F.G & Mainiero,... value in S-PKB and ASPKB transfected cells, respectively After insulin treatment, PKB activity was almost doubled in the mock and S-PKB/ H7721 cells, but it increased not so obviously in AS-PKB/ H7721. .. treated and transfected cells (Western blot) (B) PKB activity in insulin and LY294002 treated H7721 cells (C) PKB activity in sense or antisense PKB transfected cells before and after insulin treatment