Effects of integrin α6ß1 on migration of hepatocellular carcinoma cells.

In this study, we applied specific blocking antibodies for integrin α6 or ß1 subunit, and evaluated the in vitro effects of integrins α6ß1 on the adhesion, chemotaxis and migration of hepatocellular carcinoma (HCC) cell line SMMC-7721 to type IV collagen. The adhesion force and cell migration, as measured by a micropipette aspiration system and Boyden chamber assay respectively, was dramatically reduced when either integrin subunits was blocked. The chemotaxis, as determined using a dual-micropipette system, was only affected by the antibody against ß1 subunit. This study suggests that integrin α6ß1 is an important cell surface receptor that mediates the adhesion of SMMC-7721 to type IV collagen. But the α6 subunit has minimal effect on pseudopod formation in response to type IV collagen. Therefore, the integrin α6ß1-mediated cell migration is, at least in part, through the regulation on the cell adhesion step.

Effects of integrins on laminin chemotaxis by hepatocellular carcinoma cells.

To quantitatively evaluate the effects of integrins alpha1beta1, alpha2beta1, alpha3beta1, alpha4beta1, alpha5beta1, and alpha6beta1 on the chemotaxis of hepatocelluar carcinoma (HCC) cell line SMMC-7721 to laminin (LN). A modified dual-micropipette system was used to dynamically and quantitatively monitor the formation of pseudopod protrusion of HCC cells toward LN in the presence or absence of specific antibodies against integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and beta1. Additionally, the expression levels of different integrin subunits on the surface of the cells were determined via flow cytometry analysis. In response to equal concentrations of LN in both micropipettes, HCC cells form symmetrical pseudopod protrusions on both sides. Addition of antibodies against alpha3, alpha6, or beta1 into one micropipette leads to significant reduction of pseudopod formation on that side, while antibodies against alpha1, alpha2, alpha4, and alpha5 do not affect the symmetrical formation of pseudopods in either micropipette. The percentages of HCC cells positive for expression of integrins alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and beta1 were 95.07, 23.17, 95.55, 2.47, 34, 14.29, and 95.78%, respectively. Integrins alpha3beta1 and alpha6beta1 are important cell surface receptors that mediate the chemotaxis of HCC cells toward LN.

Integrin beta1 mediates hepatocellular carcinoma cells chemotaxis to laminin.

BACKGROUND: Chemotaxis is an important step during the invasion of carcinoma cells. And integrins are most important receptors mediating interaction between cells and extracellular matrix (ECM). This study was designed to study integrin beta1 mediating chemotaxis of hepatocellular carcinoma (HCC) cells to laminin(LN). METHODS: A micropipette technique was adopted to investigate the effect of blockade of integrin beta1 on pseudopod protrusion of HCC cells in response to LN stimulation. Chemotactic pseudopod protrusion of a HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with LN solution were positioned in close contact with the same cell, and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured and plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells were analyzed by flow cytometry. RESULTS: In dual pipette chemotaxis experiment, when the two pipettes were filled with LN(50 microg/ml, 200 microg/ml), pseudopods extended from the HCC cell into each of the pipettes nearly symmetrically, ie, with nearly identical maximum pseudopod length and similar pseudopod growth curves. Upon addition of anti-CD29 (20 microg/ml) to one of the pipettes, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette became more evidently, with a larger maximum length. Expression of integrin beta1 was up to 95.78% to cells chosen in the experiment. CONCLUSION: Integrin beta1 subunit was an important constituent receptor subunit for mediating chemotactic pseudopod protrusion of HCC cell to LN.

[Integrin beta1 mediates hepatocellular carcinoma cells chemotaxis to laminin].

OBJECTIVE: To study the effects of integrin beta1 on the chemotaxis of hepatocellular carcinoma (HCC) cells to laminin (LN). METHODS: A micropipette technique was adopted to investigate the effect of integrin beta1 blockade on pseudopod protrusion of HCC cells in response to LN stimulation. Chemotactic pseudopod protrusion of a HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with LN solution were positional in close contact with the same cell, and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured, then plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells was analyzed by flow cytometry. RESULTS: In dual pipette chemotaxis experiment, when the two pipettes were filled with LN (50microg/ml, 200microg/ml), pseudopods extended from the HCC cells into each of the pipettes nearly symmetrically. Upon addition of anti-CD29 (20microg/ml) to one of the pipettes, the pseudopod protrusion was blocked almost completely, while the pseudopod protrusion into the opposite pipette became more evidently, with larger maximum length. The expression rate of integrin beta1 on the cells was up to 95.78%. CONCLUSION: Integrin beta1 subunit is the important receptor for mediating HCC cells chemotaxis to laminin.

[Integrin alpha3beta1 mediates hepatocellular carcinoma cell adhesion and chemotaxis to type IV collagen].

OBJECTIVE: To investigate the effects of Integrin alpha(3)beta(1) on the adhesion and chemotaxis of hepatocellular carcinoma (HCC) cells to type IV collagen (Col IV). METHODS: (1) HCC cells were culture and suspension of HCC cells was made. Anti-alpha(3) and Anti-beta(1) were added into the HCC cell suspension. Flow cytometry was used to determine the expression of integrin alpha(3)beta(1) on the surface of HCC. (2) 5 micro g/ml Col IV was used to coat a cell with the diameter of 25 mm. Digested HCC cells were added. Anti-alpha(3) and Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml respectively were added into the cell suspension. Before and after the addition of Anti-alpha(3) and Anti-beta(1), micropipette technique was used to measure the adhesion force of HCC on Col IV-coated surface, as function of the square of internal radius of micropipette and the critical negative pressure needed to detach a single HCC cell away from the substrate. (3) Col IV of the concentration of 600 micro g/ml was added into the dual micropipettes. Then the dual micropipettes were led towards the HCC cells. A HCC cell was made to seal the openings of the 2 micropipettes with different parts of the cell contacting Col IV in different micropipettes. The pseudopod protrusion was observed dynamically and recorded with tape recorder. The length of pseudopod was measured and plotted against the chemotactic time so as to obtain a pseudopod growth curve. RESULTS: (1) The expression rates of integrin subunit alpha(3) and beta(1) on the surface of HCC cells were 95.55% and 95.78% respectively. (2) The adhesion force of HCC cells to the 5 micro g/ml Col IV-coated surface was 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of the HCC cells with Anti-alpha(3) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 42% and 49%, to 536 +/- 122 (x 10(-10) N, n = 60) and 476 +/- 63 (x 10(-10) N, n = 60) respectively. Upon treatment of the HCC cells with Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 52% and 76%, to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 60) respectively. (3) The length of pseudopod increased along with the chemotactic time. The pseudopod length and growth curve were almost identical in the dual micropipettes when they were filled with Col IV. When Anti-alpha(3) or Anti-beta(1) was added into one of the dual micropipettes, the HCC cell pseudopod protrusion was almost blocked completely, while the HCC cell pseudopod in the opposite micropipette became more evident. CONCLUSION: Integrin alpha(3)beta(1) is an important constituent receptor in mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.