ABSTRACT
1. Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf-536, (+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf-536 in inhibition of tumour metastasis, we analysed the effect of Wf-536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems. 2. In a coculture system of human fibrosarcoma HT1080 cells plated on a monolayer of human ECV304 cells, Wf-536 (0.3-3 micromol/L) inhibited the paracellular infiltration of tumour cells. 3. Wf-536 (3-30 micromol/L) inhibited the invasion of tumour cells through the reconstituted basement membrane (Matrigel) layer. 4. Wf-536 (10-30 micromol/L) inhibited the migration of tumour cells. At 0.3-3 micromol/L, Wf-536 also restrained hepatocyte growth factor/scatter factor (HGF)-induced increases in paracellular permeability of the ECV304 cell layer. 5. These results suggest that Wf-536 suppresses tumour metastasis by both enhancing the barrier function of host cell layers and inhibiting tumour cell motility at the stage of host tissue penetration by metastatic tumour cells.
Subject(s)
Benzamides/pharmacology , Cell Movement/drug effects , Neoplasm Metastasis/prevention & control , Pyridines/pharmacology , Tight Junctions/drug effects , Basement Membrane/drug effects , Basement Membrane/physiology , Benzamides/therapeutic use , Cell Movement/physiology , Coculture Techniques/methods , Dose-Response Relationship, Drug , Humans , Intracellular Signaling Peptides and Proteins , Neoplasm Metastasis/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/metabolism , Pyridines/therapeutic use , Tight Junctions/physiology , Tumor Cells, Cultured , rho-Associated KinasesABSTRACT
PURPOSE: Rho-associated coiled-coil-forming protein kinase (ROCK) is pivotally involved in invasion by tumor cells and their evolution to metastasis. We have developed a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride]. In the present study, we investigated its effect on in vitro invasion and in vivo pulmonary metastasis of B16 melanoma. METHODS: The following were evaluated: the anti-invasive effect of Wf-536 against the motility of mouse B16BL6 melanoma cells through a culture insert layered with reconstituted basement membrane (Matrigel); the cytotoxic effect of Wf-536 in the same cell line; the antimetastatic effect of Wf-536, administered by osmotic pump, on spontaneous pulmonary metastasis following subcutaneous injection of B16BL6 melanoma in mice; and the inhibitory effect of orally administered Wf-536, alone or in combination with the antineoplastic drug paclitaxel, on pulmonary metastasis of intravenously injected B16F10 melanoma in mice. RESULTS: Wf-536 inhibited in vitro invasion by B16BL6 cells significantly and in a concentration-dependent manner and displayed an anti-invasive effect under conditions of both chemotaxis and chemokinesis. No cytotoxic effect was observed at any of the concentrations used. In vivo, Wf-536 administration suppressed tumor colony formation on the lung surface in a dose-dependent manner (0.3-3 mg/kg per day), with a metastasis inhibition rate of 95% at 3 mg/kg per day. In experimental metastasis of B16F10 melanoma, oral administration of Wf-536 significantly decreased tumor colony formation in the lung, with an inhibition rate of 41% at 3 mg/kg per day. The inhibition rate of paclitaxel (5 mg/kg per day) was 27%. The combination of Wf-536 and paclitaxel produced a synergistic effect on B16F10 metastasis and a 68% inhibition rate. Wf-536 administration at the doses used did not alter body weight, blood pressure or the health of treated animals as compared to vehicle-treated controls. CONCLUSION: The results suggest that Wf-536 is a potentially valuable drug for preventing tumor metastasis both in monotherapy and in combination with an antineoplastic drug.
Subject(s)
Antineoplastic Agents/pharmacology , Benzamides/pharmacology , Enzyme Inhibitors/pharmacology , Lung Neoplasms/drug therapy , Lung Neoplasms/secondary , Melanoma, Experimental/drug therapy , Melanoma, Experimental/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Animals , Antineoplastic Agents, Phytogenic/therapeutic use , Cell Line, Tumor , Cell Movement/drug effects , Cell Survival/drug effects , Intracellular Signaling Peptides and Proteins , Mice , Neoplasm Invasiveness/prevention & control , Paclitaxel/therapeutic use , rho-Associated KinasesABSTRACT
The signaling pathway of Rho and Rho-associated coiled-coil forming protein kinase (ROCK) is involved in tumor metastasis. In the present study, we investigated the suppressive effect of a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride], on spontaneous tumor metastasis in vivo and analyzed its action on tumor cell motility and angiogenesis to clarify its action mechanism. Wf-536 (0.3-3 mg/kg/day) was found to inhibit Lewis lung carcinoma (LLC) metastasis and LLC-induced angiogenesis in orally treated mice; in vitro, it inhibited both invasion and migration by LLC cells and invasion, migration, and formation of capillary-like tubes on Matrigel by endothelial cells, without cytotoxicity or anti-proliferative action in either cell type. We conclude that Wf-536 has tumor anti-metastatic activity which may depend on inhibition of tumor motility and angiogenesis. The findings support its further clinical development as an anti-metastatic agent.
