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Tài liệu Báo cáo khoa học: Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line pptx

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Tiêu đề Insulin/protein kinase B signalling pathway upregulates metastasis-related phenotypes and molecules in H7721 human hepatocarcinoma cell line
Tác giả Hui-Ling Qi, Ying Zhang, Jun Ma, Peng Guo, Xia-Ying Zhang, Hui-Li Chen
Trường học Shanghai Medical College of Fu-Dan University
Chuyên ngành Biochemistry
Thể loại báo cáo khoa học
Năm xuất bản 2003
Thành phố Shanghai
Định dạng
Số trang 11
Dung lượng 588,41 KB

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Nội dung

Only the monoclonal antibody to the sialyl Lewis X SLex, but not antibodies to other Lewis antigens, signi-ficantly blocked the cell adhesion to HUVECs, cell migra-tion and invasion, sugg

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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

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 (SLex), but not antibodies to other Lewis antigens,

signi-ficantly blocked the cell adhesion to HUVECs, cell

migra-tion and invasion, suggesting that SLexplayed a crucial role

in the metastatic potential of H7721 cells The upregula-tion 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 SLexand 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 SLexexpression Taken together, the insulin/PKB signalling pathway participated in the enhancement of metastatic potential of H7721 cells, which was mediated by the upreg-ulation of the expression of SLexand a-1,3 FucT-VII Keywords: insulin; metastasis-related phenotype; protein kinase B; sialyl Lewis X; a-1,3-fucosyltransferase

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), phosphotidylino-sitide 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 autophos-phorylated, 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, phospha-tidylinositide-3,4,5-triphosphate and phosphatidylinositide-3,4-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 domain-containing 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-acetyl-glucosamine; GnT-V, N-acetylglucosaminyltransferase; HRP,

horseradish peroxidase; HUVEC, human umbilical vein endothelial

cell; ILK, integrin-linked kinase; Le x , Lewis X [Galb1–4 (Fuc a-1,3)

GlcNAc-]; IRS, insulin receptor substrate; PDK-1,

phosphotidyl-inositide dependent kinase-1; PI-3K, phosphstidylphosphotidyl-inositide-3-kinase;

PKB, protein kinase B; PVDF, polyvinylidene difluoride; SA, sialic

acid; SLe a , 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 2.

Note: H.-L Qi and Y Zhang contributed equally to this manuscript.

(Received 10 June 2003, revised 20 July 2003,

accepted 25 July 2003)

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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

phos-phorylation 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 9 and blocks its

activa-tion 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-acetylglucos-aminyltransferase V (GnT-V) [18,19], which synthesizes a

b1,6 N-acetyl-glucosamine (GlcNAc) branch on the

aspa-ragine (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 modi-fied 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 (Uni-versity 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 restric-tion endonucleases and random primer labelling kit were from Promega Hybond-N+ nylon membrane, poly(viny-lidene 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 anti-sense 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

2.6-kb 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 plas-mid 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

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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 37C, 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 2 nM and 15 lM,

respectively, determined by the previous dose-dependence

assays The same final concentration (0.15%) of

dimethyl-sulfoxide was in the control medium for LY294002 treated

cells, but dimethylsulfoxide was omitted from the control

medium for insulin treated cells However, there was no

difference in the results between the control 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 ngÆmL)1of tumour necrosis factor (TNF)-a for

4 h Then 105cells in 0.1 mL were added to each well and

further incubated for 30 min at 4C 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 37C for 1 h, and blocked by 1% (w/v)

BSA at 37C for 0.5 h Cells (1 · 105) in 0.1 mL were

added to each coated well and incubated for 2 h at 37C

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 6 h at 37C 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 ngÆmL)1 or

20 ngÆmL)1 different mAbs against Lewis antigens for

30 min at 4C 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 SLexwith flow cytometry The cells were detached with 2 mM EDTA, washed and resuspended in NaCl/Pi containing 1% (w/v) BSA Then

106cells were incubated with 1 : 50 diluted KM93, the mAb for SLex, for 30 min at 4C After two washes, the cells were incubated for 45 min at 4C with 1 : 200 diluted fluorescein isothiocyanate-conjugated goat anti-mouse IgM, then the cells were washed again and subjected to flow cytometry (1· 104 cellsÆsample)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 (fluores-cence activated cell sorting) spectra were drawn automati-cally, 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 M1 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/PAGEon 10% acrylamide and electro-blotted 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 1 : 1000 diluted rabbit polyclonal anti-human PKB or 1 : 800 diluted monoclonal anti-human b-actin in

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5% fat-free milk/Tris-buffered saline, followed by

incuba-tion 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 4C for 3 h to

immuno-precipitate the PKB The pellet was suspended in 40 lL

kinase buffer (25 mM Tris/HCl pH 7.5, 1M b-glycerol

phosphate, 2 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 1 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 30C for 60 min, the phosphorylated

GSK-3a/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

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

des-cribed previously [30] The probe was labelled with [a-32

P]-dATP using random primer labelling kit from Promega

according to manufacturer’s instructions

Glyceraldehyde-3-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)

Nor-thern 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 65C in

0.2Msodium phosphate buffer (pH 7.4)/1 mMEDTA/1%

(w/v) BSA/7% (w/v) SDS/15% (v/v) formamide

Hybrid-ization was performed at 65C 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 mMEDTA for 30 min at 65C,

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

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 min 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 1 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 lMLY294002 (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

Fig 1 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 2 n M insulin; LY294002, H7721 cells treated with 15 l M 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 2 n M

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 com-pared with the Ins group, but P > 0.05 comcom-pared with the AS-PKB group The incubation time for all the cell groups was 48 h Experi-mental procedures were as described in Materials and methods.

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the mock (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

concen-trations of the coated laminin, and enhanced after insulin

treatment at different concentrations of laminin By

con-trast, 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

chemo-tactic migration through transwell and chemochemo-tactic 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

adhesion to HUVECs of both untreated and insulin treated cells was markedly suppressed by KM93 (anti-SLexmAb) 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 signifi-cant 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 SLexwas 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 SLexto 430% of the untreated control value, while LY294002 down regula-ted 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

Fig 2 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 1 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.

Fig 3 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 1 Data are expressed as the mean ± SD of three independent experiments The values of the UnC group (31 ± 5 and 60 ± 7 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 com-pared with the LY294002 group; ##P < 0.01 comcom-pared with the Ins group, but P > 0.05 compared with the AS-PKB group The incu-bation time for all cell groups was 48 h Experimental procedures were

as described in Materials and methods.

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mock level In contrast, the SLexexpression 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 1 nM insulin for 48 h was enough to

stimulate the expression of PKB protein and activity in

untreated control cells The effect of 2 nMwas greater, but

the difference between 2 nMand 4 nMwas not apparent

Therefore, we chose 2 n 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 SLexby 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 SLexexpression After densi-tometric 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/

Fig 4 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 2 n M 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 1 and 3, respectively, except that the cells were pretreated with different mAbs against different Lewis antigens (10 ngÆmL)1for UnC, 20 ngÆmL)1in insulin treated cells) for 30 min at 4 C.

Fig 5 Effects of insulin and LY294002 on the expression of SLe x on H7721 cells (A) Flow cytometry (–) Control, without the addition of the first antibody (B) Calculation of relative expression of SLe x 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 1 The incubation time for all the cell groups was 48 h Experimental procedures were as described in Materials and methods.

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H7721 and AS-PKB/H7721 cells, respectively The

treat-ment 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

AS-PKB 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

expres-sion and activity of PKB protein were compatible with the

SLexexpression as shown in Figs 5 and 6

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

FucT-III 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 deter-mined 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 expres-sion of FucT-VII mRNA was decreased in LY294002 treated or AS-PKB cDNA transfected cells Insulin treatment obviously upregulated the expression of FucT-VII 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 SLexexpression in UnC or mock cells, but not

in LY294002 treated and AS-PKB transfected cells

Fig 6 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 SLe x 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 1 S-PKB, Sense-PKB cDNA transfected H7721 cells; S-PKB + Ins, S-PKB/H7721 cells treated with 2 n M insulin The incubation time for all the cell groups was 48 h Experimental procedures were as described for Fig 5.

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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

mem-brane 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 trans-fected with the metastasis-promoting gene, H-ras or v-sis [27] or the metastasis-suppressive gene, nm23-H1 [28] The

Fig 8 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 1 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.

Fig 7 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 anti-sense 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 1 and 6 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 per-formed and the results were reproducible.

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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

path-ways 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

insulin-induced 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 2 nMinsulin for 48 h, suggested that the

internalization and degradation of PKB, if any, would not

significantly influence the results

From Figs 1, 2 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, 5 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 SLexwas 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 SLexmolecule 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

pheno-types 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 SLexin 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 SLexwith simultaneous enhancement of the above-mentioned metastasis-related phenotypes [30] How-ever, 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 SLexsynthesis, 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 sub-G1 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 downregula-tion of PKB-a in the cells

The findings shown in Figs 5, 6 and 8 suggest that the regulation on the expressions of SLexand 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 pheno-types 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 SLexmight 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 transcrip-tion 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 phosphory-lation 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

FucT-IV gene is regulated by elk-1 in the U937 cell line [41] Which factor(s) is responsible for the regulation of gene transcrip-tion 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 metastatic potential

of human hepatocarcinoma cells, which is at least partially mediated by the increased expressions of metastasis-related molecules, a-1,3 FucT-VII and its product, SLex

Acknowledgement

This work was supported by the grant from National Natural Science Foundation of China (No.39870169 and 30170219).

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