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

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(SLe
x
), but not antibodies to other Lewis antigens, signi-
ficantly blocked the cell adhesion to HUVECs, cell migra-
tion and invasion, suggesting that SLe
x
played a crucial role
in the metastatic potential of H7721 cells. The upregula-
tion of cell surface SLe
x
and a-1,3-fucosyltransferase-VII

(a-1,3 Fuc T-VII, enzyme for SLe
x
synthesis) was also
mediated by PI3K and PKB, since LY294002 and AS-PKB
also reduced the expressions of SLe
x
and 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
SLe
x
expression. Taken together, the insulin/PKB signalling
pathway participated in the enhancement of metastatic
potential of H7721 cells, which was mediated by the upreg-
ulation of the expression of SLe
x
and 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:
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
,LewisX[Galb1–4 (Fuc a-1,3)
GlcNAc-]; IRS, insulin receptor substrate; PDK-1, phosphotidyl-
inositide dependent kinase-1; PI-3K, phosphstidylinositide-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-];
SDLe
x
, 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)
Eur. J. Biochem. 270, 3795–3805 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03767.x
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.
LewisantigensareaseriesoffucosylatedGalb1–3/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 (SLe
x
) 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
SLe
x
/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-SLe
x
), CA19-9 (anti-SLe
a
)
and plasmid pUC19/FucT-VII were kindly provided by
H. Narimatsu (Soka University, Tokyo, Japan). mAb FH6
(anti-SDLe
x
) was a gift from S. I. Hakomori (University of
Washington, Seattle, USA). CD15 (mAb anti-Le
x
)and
horseradish peroxidase (HRP)-labelled goat anti-(mouse
IgG) IgG were from Dako. The plasmid containing human
PKB-a (pSGS-PKB
GAG
) 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-
32
P]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-PKB
GAG
(6.5 kb) was digested with EcoRI and BglII to form a 2.6-
kb fragment containing GAG-PKB cDNA. This fragment
wasthendigestedwithNcoI 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
3796 H L. Qi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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) CO
2
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 n
M

and 15 l
M
,
respectively, determined by the previous dose-dependence
assays. The same final concentration (0.15%) of dimethyl-
sulfoxide 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 ngÆmL
)1
of tumour necrosis factor (TNF)-a for
4h.Then10
5
cellsin0.1mLwereaddedtoeachwelland
further incubated for 30 min at 4 °C. After being washed
five times with NaCl/P
i
, 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 1 h, and blocked by 1% (w/v)
BSA at 37 °C for 0.5 h. Cells (1 · 10
5
)in0.1mLwere
added to each coated well and incubated for 2 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 (A
595
) 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 · 10
4
) 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 37 °C in a humidified

atmosphere containing 5% CO
2
. 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 · 10
5
) 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 4 °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 SLe
x
with flow cytometry
The cells were detached with 2 m
M
EDTA, washed and
resuspended in NaCl/P
i
containing 1% (w/v) BSA. Then
10
6
cells were incubated with 1 : 50 diluted KM93, the mAb
for SLe
x
,for30minat4°C. After two washes, the cells
were incubated for 45 min at 4 °C with 1 : 200 diluted
fluorescein isothiocyanate-conjugated goat anti-mouse
IgM, then the cells were washed again and subjected to
flow cytometry (1 · 10
4
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 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 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
Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLe
x
(Eur. J. Biochem. 270) 3797
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 4 °C for 3 h to immuno-
precipitate the PKB. The pellet was suspended in 40 lL
kinase buffer (25 m
M
Tris/HCl pH 7.5, 1
M
b-glycerol
phosphate, 2 m
M
dithiothreitol, 0.1 m
M
Na
3
VO
4
,10m
M
MgCl
2
) and used as the enzyme preparation, which was
supplemented with the substrates, ATP (200 l
M
)and1lg
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 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 65 °Cin
0.2
M
sodium phosphate buffer (pH 7.4)/1 m
M
EDTA/1%
(w/v) BSA/7% (w/v) SDS/15% (v/v) formamide. Hybrid-
ization was performed at 65 °C for 16–20 h in the same
hybridization solution containing a-

32
P labelled probe of
a-1,3 FucT-VII. The hybridized membrane was washed
threetofivetimeswith40m
M
sodium phosphate buffer
pH 7.4/1% (w/v) SDS/1 m
M
EDTA for 30 min 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.
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 n
M
)-
treated H7721 cells to HUVECs was increased to 164.9%
that of the untreated control cells (UnC). In the cells treated
with 15 l
M
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
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,H7721cellstreatedwith2 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 compared 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.
3798 H L. Qi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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 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 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
adhesion to HUVECs of both untreated and insulin treated
cells was markedly suppressed by KM93 (anti-SLe
x
mAb)
only (Fig. 4A). KM93 also significantly inhibited the
chemostatic cell migration and invasion of insulin treated
H7721 cells (Fig. 4B). FH6 (anti-SDLe
x
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 SLe
x
,playsa
critical role in the cell adhesion to HUVECs, cell migration
and invasion.
Effect of insulin on the expression of SLe
x
on differently treated and transfected H7721
SLe
x
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 SLe
x

to 430% of
the untreated control value, while LY294002 down regula-
ted SLe
x
to 54.9% of the UnC value. In the presence of
LY294002, insulin treatment was no longer to show any up
regulatory effects on SLe
x
.
When mock cells were treated with insulin, the expression
of SLe
x
increased to the same level as the insulin-treated
parent control cells. Transfection of sense or antisense PKB
to H7721 cells increased or decreased SLe
x
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
SLe
x
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)
weresetat100%.**P < 0.01 compared with the UnC or Mock
group; #P < 0.01 compared with the Ins group, but P >0.05com-
pared with the LY294002 group; ##P < 0.01 compared 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.
Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLe
x
(Eur. J. Biochem. 270) 3799
mock level. In contrast, the SLe
x
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 1 n
M
insulin for 48 h was enough to

stimulate the expression of PKB protein and activity in
untreated control cells. The effect of 2 n
M
was greater, but
the difference between 2 n
M
and 4 n
M
was not apparent.
Therefore, we chose 2 n
M
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 SLe
x
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 SLe
x
expression. 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 SLe
a
;CD15,mAbtoLe
x
;KM93,mAbtoSLe
x
;FH6,mAbtoSDLe
x
;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
)1
for
UnC, 20 ngÆmL
)1
in 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.

3800 H L. Qi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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
SLe
x
expression 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
SLe

x
and a-1,4 fucosyl containing SLe
a
, respectively, but
FucT-VII catalyses only the synthesis of SLe
x
[24,25]. In
order to clarify the role of FucT-VII in the synthesis of
SLe
x
, 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
SLe

x
expression was lower in LY294002 or AS-PKB
cDNA transfected cells, and insulin could significantly
stimulate SLe
x
expression 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 SLe
x
. (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.
Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLe
x
(Eur. J. Biochem. 270) 3801
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 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.
3802 H L. Qi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
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
observedinourlabafterH7721cellsweretreatedwith
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 n
M
insulin 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
SLe
x
. The specific inhibitory effect of SLe
x
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 SLe
x
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 SLe
x
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 Le
x
(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 SLe
x
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 SLe
x
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 SLe
x
with 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 SLe
x
in
H7721 cells, as it was reported that FucT-VI exhibited the

strongest relative activity for SLe
x
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 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 SLe
x
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 pheno-
types. However, the increase rate in SLe
x
was far greater
than that of PKB protein and activity in the insulin treated
and S-PKB cells, indicating that SLe
x
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 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, SLe
x
.

Acknowledgement
This work was supported by the grant from National Natural Science
Foundation of China (No.39870169 and 30170219).
Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLe
x
(Eur. J. Biochem. 270) 3803
References
1. Yardan, Y. & Ullrich, A. (1988) Growth factor receptor tyrosine
kinase. Ann. Rev. Biochem. 57, 443–447.
2. Toker, A. & Cantley, L.C. (1997) Signalling through the lipid
products of phospho-inositide-3-OH kinase. Nature 387, 673–676.
3. Flanke, T.F., Kaplan, D.R. & Cantley, L.C. (1997) PI-3K:
Downstrean AKTion blocks apotosis. Cell 88, 435–437.
4. Fillipa, N., Sable, C.L., Hemmings, B.A. & van Obberghen, E.
(2000) Effect of phospho- inositide-dependent kinase 1 on protein
kinase B translocation and its subsequent activation. Mol. Cell.
Biol. 20, 5712–5721.
5. Hemming, B.A. (1997) PtdIdns(3,4,5)
3
gets its message across.
Science 277, 534.
6. Cohen,P.,Dario,R.,Darren,A.&Cross,A.E.(1997)PDK1,one
of the missing links in insulin signal transduction? FEBS Lett. 410,
3–10.
7. Dedhar, S., Benjamin, W. & Gregory, H. (1999) Integrin-linked
kinase (ILK): a regulator of integrin and growth-factor signalling.
Trend Cell Biol. 9, 319–323.
8. Delcommenne,M.,Tan,C.,Gray,V.,Rue,L.,Woodgett,J.&
Dedhar, S. (1998) Phosphoinositide-3OH. kinase-dependent
regulation of glycogen synthetase 3 and protein kinase B/AKT

by the integrin-linked kinase. Proc. Natl Acad. Sci. USA 95,
11211–11216.
9. Krasilnikov, M.A. (2000) Phosphotidylinositol-3-kinase depend-
ent pathway: the role in control of cell growth, survival, and
malignant transformation. Biochemistry (Mosc) 65, 59–67.
10. Balendran, A., Casamayor, A., Deak, M., Paterson, A., Gaffney,
P.A., Downes, C.P. & Alessi, D.R. (1999) PDK-1 aquires PDK-2
activity in the presence of a synthetic peptide derived from the
carboxy terminus of PRK-2. Curr. Biol. 9, 393–404.
11. Toker, A. & Newton, A.C. (2000) Akt/protein kinase B is regu-
lated by autophosphorylation at hypothetical PDK-2 site. J. Biol.
Chem. 275, 8271–8274.
12.Chen,R.,Kim,O.,Yang,J.,Sato,K.,Eisenmann,K.M.,
McCarthy, J., Chen. H. & Qiu, Y. (2001) Regulation of Akt/PKB
activation by tyrosine phosphorylation. J. Biol. Chem. 276, 31858–
31862.
13. Lopez-Ilasaca, M., Crespo, P., Pellici, P.G., Gutkind, J.S. &
Wetzker, R. (1997) Linkage of G protein-coupled receptor to the
MAPK signalling pathway through PI 3-kinase gamma. Science
275, 394–397.
14. Gibson, S., Tu, S., Oyer, R., Anderson, S.M. & Johnson, G.L.
(1999) Epidermal growth factor protects epithelial cells against
Fas-induced apoptosis. Requirement for Akt activation. J. Biol.
Chem. 274, 17612–17618.
15. Datta, S.R., Brunet, A. & Greenberg, M.E. (1999) Cellular sur-
vival: a play in three Akts. Genes Dev. 13, 2905–2927.
16. Kennedy, S.G., Kandel, E.S., Cross, T.K. & Hay, N. (1999)
Akt/protein kinase B inhibits cell death by preventing the release
of cytochrome c from mitochondria. Mol. Cell. Biol. 19, 5800–
5810.

17. Wang, Q.H., Zhou, D.P., Shao, D.M., Shen, Z.H. & Gu, J.X.
(1997) Effects of epidermal growth factor and insulin on the
activity of N-acetylglucosaminyl-transferase V. Biochem. J. 324,
543–545.
18. Dennis, J.W., Lafertes, S., Waghorne, C., Breitman, M.L. &
Kerbel, R.S. (1987) b1,6 branching of Asn-linked oligosaccharides
is directly associated with metastasis. Science 236, 582–588.
19. Yamamoto, H., Swoger, J., Greene, S., Saito, T., Hurh, J.,
Sweeley, C., Leestma, J., Mkirdichian, E., Cerullo, L., Nishi-
kawa, A., Ihara, Y., Taniguchi, N. & Moskal, J.R. (2000) b-1,6-
N-acetylglucosaamine-bearing N-glycans in human gliomas:
implications for a role in regulating invasivity. Cancer Res. 60,
134–142.
20. Hakomori, S.I. (1996) Tumor malignancy defined by aberrant
glycosylation and sphingo (glyco) lipid metabolism. Cancer Res.
56, 5309–5318.
21. Takada, A., Ohmori, K., Yoneda, T., Tsuyuoka, K.,
Hasegawa, A., Kiso, M. & Kannagi, R. (1993) Contribution of
carbohydrate antigens sialyl Lewis A and sialyl Lewis X to
adhesion of human cancer cells to vascular endothelium. Cancer
Res. 53, 354–361.
22. Nakamori, S., Kameyama, M., Imaoka, S., Furukawa, H., Ishi-
kawa, O., Sasaki, T., Kabuto, T., Iwanaga, T., Matsushita, Y. &
Irimura, T. (1993) Increased expression of sialyl Lewis X antigen
correlates with poor survival in patients with colorectal carcinoma:
clinico-pathological and immunohistochemical study. Cancer Res.
53, 3632–3637.
23. Jorgensen, T., Berner, A., Kaalhus, O., Tveter, K.J., Danielsen,
H.E. & Bryne, M. (1995) Up-regulation of the oligo-saccharide
sialyl Lewis X: a new prognosis parameter in metastatic prostate

cancer. Cancer Res. 55, 1817–1819.
24. Narimatsu, H. (1998) Human fucosyltransferases, Tissue distri-
bution of blood group antigens, cancer assotiated antigens and
fucosyltransferase. Protein, Nucl. Acid, Enz. 48, 2394–2403 (in
Japanese).
25. Sasaki, K., Kurata, K., Funayama, K., Nagata, M., Watanabe,
E., Ohta, S., Hanat, N. & Nishi, T. (1994) Expression cloning of a
novel a1,3-fucosyltransferase that is involved in biosynthesis of the
sialyl Lewis X carbohydrate determinants in leukocytes. J. Biol.
Chem. 269, 14730–14737.
26. Guo, H.B., Shen, Z.H., Huang, C.X., Ma, J., Huang, Y. & Chen,
H.L. (2000) Modulation of the basal activity of N-acetyl-
glucosaminyltransferase V by phosphatidylinositol-3-kinase/
protein kinase B signaling pathway in human hepatocarcinoma
cells. Glycoconj J. 17, 315–322.
27. Guo,H.B.,Zhang,Q.S.&Chen,H.L.(2000)EffectsofH-ras and
v-sis over-expression on N-acetylglucosaminyltransferase V and
metastasis-related phenotypes in human hepatocarcinoma cells.
J. Cancer Res. Clin. Oncol. 126, 263–270.
28. Guo, H.B., Liu, F., Zhao, J.H. & Chen, H.L. (2000) Down-
regulation of N-acetylglucosaminyltransferase V by tumorigen-
esis- or metastasis-suppressor gene and its relation to metastatic
potential of human hepatocarcinoma cells. J. Cell Biochem. 79,
370–385.
29. Liu, F., Qi, H.L. & Chen, H.L. (2001) Regulation of differentia-
tion- and proliferation inducers on Lewis antigens, a-fucosyl-
transferase and metastatic potential in hepatocarcinoma cells. Br.
J. Cancer 84, 1556–1563.
30. Liu, F., Qi, H.L., Zhang, Y., Zhang, X.Y. & Chen, H.L. (2001)
Transfection of c-erbB2/neu gene up-regulates the expression of

sialyl Lewis X, a1,3 fucosyl-transferase VII and metastatic
potential in human hepatocarcinoma cell line. Eur. J. Biochem.
268, 3501–3512.
31. Liu, F., Qi, H.L. & Chen, H.L. (2000a) Effects of all-trans retinoic
acid, epidermal growth factor on the expression of nm23–H1 in
human hepatocarcinoma cells. J. Cancer Res. Clin. Oncol. 126,
85–91.
32. YuD., Wang, S.S., Dulki, K.M., Tsai, C.M., Nicolson, G.L. &
Hung, M.C. (1994) C-erbB2/neu overexpression enhances
metastatic potential of human lung cancer cells by induction
of metastasis-associated properties. Cancer Res. 54, 3260–
3266.
33. Lowry, O.H., Rosebrough, N.J., Farr, A.L. & Randall, R.J.
(1951) Protein measurement with the Folin-phenol reagent.
J. Biol. Chem. 193, 265–271.
34. Kudo, T., Ikehara, Y., Togayachi, A., Morozumi, K., Watanabe,
M., Nakamura, M., Nishihara, S. & Narimatsu, H. (1998)
Up-regulation of a set of glycosyltransferase genes in human
colorectal cancer. Lab. Invest. 78, 797–811.
3804 H L. Qi et al. (Eur. J. Biochem. 270) Ó FEBS 2003
35. Sagestrom, C.G. & Sive, H.L. (1996) RNA blot analysis. In: A
Laboratory Guide to RNA. Isolation, Analysis, and Synthesis.
(Krieg, P.A., eds), pp. 83–103. Wiley-Liss Inc. London.
36. Wu, S.L., Ma, J., Qi, H.L., Zhang, Y., Zhang, X.Y. & Chen, H.L.
(2003) 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, F. (1990) Integrin-mediated adhesion
and signaling in tumorigenesis. Biochim. Biophys. Acta. 190,
47–56.

38. Togayachi, A., Kudo, T., Ikehara, Y., Iwasaki, H., Nishihara, S.,
Andoh, T., Higashiyama, M., Kodama, K., Nakamori, S. &
Narimatsu, H. (1999) Up-regulation of Lewis enzyme (FucTIII)
and plasma-type a1,3 fucosyltransferase (Fuc-TVI) expression
determines the argmented expression of sialyl Lewis X antigen in
non-small cell lung cancer. Int. J. Cancer 83, 70–79.
39. Kandel, E.S. & Hay, N. (1999) The regulation and activities of the
multifunctional serine/threonine kinase Akt/PKB. Exp. Cell Res.
253, 210–229.
40. Marais, R., Wynne, J. & Treisman, R. (1993) The SRF accessory
protein Elk-1 contains a growth factor-regulated transcriptional
activation domain. Cell 73, 381–393.
41. White, D.A. & Hakomori, S.I. (2000) Human alpha (1,3)-fuco-
syltransferase IV (FUTIV) gene expression is regulated by elk-1 in
the U937 cell line. J. Biol. Chem. 275, 40588–40593.
Ó FEBS 2003 Insulin upregulates metastasis via PKB and SLe
x
(Eur. J. Biochem. 270) 3805