Enhancement of migration and invasion of hepatoma cells via a Rho GTPase signaling pathway

Abstract

Aim: Intrahepatic extension is the main cause of liver failure and death in hepatocellular carcinoma patients. The small GTPase Rho and one of its effector molecules ROCK regulate cytoskeleton and actomyosin contractility, and play a crucial role in cell adhesion and motility. We investigated the role of small GTPase Rho in biological behaviors of hepatocellular carcinoma to demonstrate the importance of Rho in cancer invasion and metastasis.

Methods: Using Western blotting, we quantitated Rho protein expression in SMMC-7721 cells induced by Lysophosphatidic acid (LPA). Furthermore, we examined the role of Rho signaling in regulating the motile and invasive properties of tumor cells.

Results: Rho protein expression was stimulated by LPA. Using the Rhotekin binding assay to assess Rho activation, we observed that the level of GTP-bound Rho was elevated transiently after the addition of LPA, and Y-27632 decreased the level of active Rho. LPA enhanced the motility of tumor cells and facilitated their invasion. Rho played an essential role in the migratory process, as evidenced by the inhibition of migration and motility of cancer cells by a specific inhibitor of ROCK, Y-27632.

Conclusion: The finding that invasiveness of hepatocellular carcinoma is facilitated by the Rho/Rho-kinase pathway is likely to be relevant to tumor progression and Y-27632 may be a new potential effective agent for the prevention of intrahepatic extension of human liver cancer.

Figures

Figure 1

Changes in levels of Rho family protein in SMMC-7721cells induced by LPA. The levels of Rho protein were ana-lyzed by Western blot analysis. The data shown are expressed as -x ± s from three different experiments.

Figure 2

Tumor cell random motility determined by gold-col-loid assay. Cells were seeded onto the coverslips and allowed to adhere for 1 h and then incubated in FBS containing LPA with or without Y-27632 for 24 h. The areas of clearing in the gold colloid corresponding to phagokinetic cell tracks were counted and represented as -x ± s from triplicate experiments. aP < 0.01 LPA treated cells vs SMMC-7721 control cells, bP < 0.01 Y-27632 treated cells vs LPA treated cells.

Figure 3

Inhibition of LPA-induced tumor cell invasion by Y-27632. Serum-starved cells were seeded onto porous filters. After incubation of the filters with Y-27632 in the presence of LPA 25 μM for 4.5 h to permit penetration of the cells, nonmigrating cells were removed from the upper chamber and migrating cells adherent to the underside of the filters were counted in a minimum of 10-high power fields. Data were expressed as relative migration (number of cells/field) and represented as -x ± s from triplicate experiments. aP < 0.01 relative to SMMC-7721 control cells.