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
Trang 1Insulin/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)
Trang 2phosphorylated 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
Trang 3cells 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
Trang 45% 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.
Trang 5the 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.
Trang 6mock 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.
Trang 7H7721 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.
Trang 8In 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.
Trang 9upregulation 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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