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.

7ND protein exerts inhibitory effects on both osteoclast differentiation in vitro and lipopolysaccharide‑induced bone erosion in vivo

“…Then, the cells were cultured in 0.5 ml of α-MeM supplemented with 10% FBS and 1% penicillin-streptomycin solution in a 24-well plate at a density of 3x10 5 cells/well and incubated at 37˚C in a humidified atmosphere with 5% CO 2 for 1 day. on the second day, the complete medium was replaced, floating cells were discarded, and the adherent cells were identified as PBMCs (12). PBMCs were cultured and used for subsequent experiments.…”

Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) Drives Activation of Bone Remodelling and Skeletal Metastasis

“…Expression of MCP-1 higher in poorly differentiated NPC cells compared to highly differentiated NPC cells, suggesting that MCP-1 plays a role in the maturation and progression of these cancerous cells [74] Multiple myeloma MCP-1 is over-expressed -MCP-1 expression in bone marrow stromal cells is upregulated by human myeloma cells [75] Oral squamous cell carcinoma Associated with elevated expression of MCP-1 [42••] Inhibition of MCP-1 with its dominant-negative form, 7ND, successfully inhibited osteoclast differentiation and limited the capacity of OSCC to invade surrounding bone [76] Myeloid leukaemia Implicated in cell transmigration and proliferation; however, not in chemotherapy resistance [77] breast cancer progression, MCP-1 targeted monotherapy presents itself as a useful therapeutic. Bonapace et al (2014) found, nonetheless, that cessation of MCP-1 inhibition resulted in a rapid increase in metastasis and accelerated death.…”

Immunomodulatory aspects in the progression and treatment of oral malignancy

“…MCP-1 mediates osteoclastogenesis of mononuclear cells, and promotes osteoclast development in cancers [99]. Furthermore, a deletion mutant of MCP-1 inhibits osteoclast differentiation of stromal monocytes and reduces bone invasion of OSCC cells [14]. Overexpression of leukocyte-associated immunoglobulin-like receptor (LAIR)-1 in OSCC cells is associated with advanced pathological grade and correlates with immune suppressive features of the tumor tissue; in fact, the expression is tightly associated with immune suppressive markers of TAM and MDSC, i.e., CD11b, CD163, CD33, CD68, two immune checkpoints (B7-H3 and VISTA) and indoleamine 2,3-dioxygenase (IDO) [100].…”

“…In animal models, CSF-1 receptor inhibition strongly reduces tumor-associated macrophages and increase the CD8+/CD4+ T cell ratio [13]. The dominant-negative deletion-mutant of MCP-1 inhibits osteoclast differentiation of monocyte and reduces the bone invasion by OSCC cells in vivo [14]. The blockade of MCP-1 could also prevent M2 phenotype polarization of macrophages.…”

“…The most promising approach activating antitumor immunity is the blockade of the PD-1/PD-L1 axis. As novel therapeutic approaches manipulating the polarization of pro-tumorigenic macrophages using specific ligand to TLR3, bisphosphonate, and blockings of specific cytokines have been reported [[11], [12], [13], [14]].…”