Tài liệu Báo cáo khoa học: Insulin/protein kinase B signalling pathway upregulates

Insulin/protein kinase B signalling pathway upregulates

metastasis-related phenotypes and molecules in H7721 human

hepatocarcinoma cell line

Hui-Ling Qi, Ying Zhang, Jun Ma, Peng Guo, Xia-Ying Zhang and Hui-Li Chen

Key Laboratory of Glycoconjugate Research, Ministry of Health, Department of Biochemistry, Shanghai Medical College of

Fu-Dan University, Shanghai, China

The effect of insulin on cancer metastatic potential was

studied in a human hepatocarcinoma cell line, H7721. Cell

adhesion to human umbilical vein endothelial cells

(HUVECs) and laminin as well as chemotactic cell migration

and invasion were selected as the indices of metastasis￾related phenotypes for assessment of metastatic potential

ex vivo. The results indicated that insulin enhanced all of

these metastasis-related phenotypes. After the cells were

treated with specific inhibitor of PI3K (LY294002) or

transfected with antisense cDNA of PKB (AS-PKB), all of

the above phenotypes were attenuated, and they could not

be significantly stimulated by insulin, indicating that the

insulin effect on metastatic potential was mediated by PI3K

and PKB. Only the monoclonal antibody to the sialyl Lewis

X (SLex

), but not antibodies to other Lewis antigens, signi￾ficantly blocked the cell adhesion to HUVECs, cell migra￾tion and invasion, suggesting that SLex played 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 SLex and a-1,3 FucT-VII,

and attenuated the response to insulin. Furthermore, the

alterations in the expressions of PKB protein and activity

were correlated to the changes of metastatic phenotypes and

SLex expression. Taken together, the insulin/PKB signalling

pathway participated in the enhancement of metastatic

potential of H7721 cells, which was mediated by the upreg￾ulation of the expression of SLex 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: [email protected]

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; Lex

, Lewis X [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; SLea

, sialyl Lewis A [SAa-2,3 Galb1,3 (Fuca1,4) GlcNAc-];

SDLex

, sialyl dimeric (difucosyl) Lewis X [SAa-2,3 Galb1,4

(Fuc a-1,3)GlcNAcb1,3Galb1,4 (Fuc a-1,3) GlcNAcb-1,3-];

SH2, Src homology 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)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03767.x