Figure 6

Effects of adrenomedullin antagonist on MDA-MB-231 cells in bone. All indicated P-values are versus control. (A) Growth. Addition of 10 nM adrenomedullin (AM) peptide (black triangles) increased tumor growth as measured by secreted luciferase marker in conditioned media. Significant vs control (*P < 0.05) at 3 and 6 days. Addition of 1 μM AM antagonist 16311 (black squares) dramatically decreased tumor growth vs control after 3 days (**P < 0.001) and 6 days (***P < 0.0001). RLU, Relative light unit. (B) Osteoblast activity. Bone biosynthetic collagen marker, type I collagen (mCol1A1). Exogenous AM increased mouse collagen mRNA in the presence and absence of tumor, whereas tumor alone (with or without 16311 antagonist) modestly suppressed this marker of osteoblast function. (C) Osteoclast activity. The mouse bone marker tartrate-resistant acid phosphatase (TRAP), encoded by mACP5, was increased by tumor cells, further increased by tumor plus exogenous AM and blocked by the 16311 AM antagonist. AM alone had no effect on TRAP mRNA. (D) Receptor activator of nuclear factor κB ligand (RANKL). Samples were analyzed for both mouse and human RANKL expression because both tumor cells and bone cells can express this factor, which regulates osteoclast formation, survival and activity. Control is bone without tumor or treatment, where the expression of mouse RANKL = 1. Levels of human RANKL (gray bars) are adjusted to the equivalent threshold cycle values so that the absolute amounts of mouse and human RANKL are expressed on the same scale. In (D) only, # indicates significance relative to each of the other relevant columns within the mouse or human groups (^#P < 0.0001) by one-way analysis of variance. No other pairwise comparisons were significantly different, and mouse and human datasets were not compared.