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

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,

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

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

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

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

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

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

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

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

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

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/

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
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

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

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

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
.

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(Eur. J. Biochem. 270) 3805