[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.