Mutant monocyte chemoattractant protein-1 protein (7ND) inhibits osteoclast differentiation and reduces oral squamous carcinoma cell bone invasion.

Abstract

The seven-amino acid truncated (7ND) protein is an N-terminal deletion mutant of monocyte chemoattractant protein-1 (MCP-1) and it functions as a dominant-negative inhibitor. 7ND and wild-type MCP-1 form a heterodimer, which binds to MCP-1 receptors and inhibits monocyte chemotaxis. In the present study, the 7ND protein was cloned, expressed and purified. An MTT assay revealed that the proliferation of oral squamous cell carcinoma (OSCC) SCC25 cells was not affected following 3 days of treatment with synthetic 7ND protein. Serial dilutions of the 7ND protein were tested for monocyte migration and osteoclast differentiation, and tartrate-resistant acid phosphatase staining demonstrated that significantly fewer osteoclasts were differentiated from cluster of differentiation 14+ (CD14+) monocytes using magnetic activated cell sorting. Immunofluorescence confirmed these results and significantly less F-actin staining was observed in 7ND-treated osteoclasts. Furthermore, bone invasion was examined by subcutaneously injecting SCC25 cells into the area overlaying the calvariae of nude mice. The results demonstrated that the average tumor volume of SCC25 cells with 7ND protein was similar to the average volume of tumors formed by untreated SCC25 cells. Flow cytometric analysis suggested that the CD14+ subpopulation in the bone marrow of 7ND-treated mice was reduced compared with that of untreated mice. Micro-computed tomography imaging revealed significantly less bone resorption in the calvariae injected with SCC25 cells plus the 7ND protein. Taken together, the results of the present study demonstrated the potential therapeutic value of the 7ND protein. The 7ND MCP-1 variant not only functions in vitro to inhibit osteoclast differentiation, but also reduces the progression of bone invasion by OSCC cells in vivo.