Fig. 4
From: Smac mimetics LCL161 and GDC-0152 inhibit osteosarcoma growth and metastasis in mice
Implanted and spontaneous osteosarcomas contain high concentrations of TNFα. a Seven weeks after subcutaneous 1029H-Luc implantation, mice were administered a single dose of saline, GDC-0152 (50 mg/kg) or LCL161 (50 mg/kg). Six hours later, the mice were culled and their blood and tumors were harvested. Serum and tumor lysates were prepared and TNFα levels were measured and used to calculate TNFα abundance per milliliter of serum or per gram of tumor. One way ANOVAs and Sidak’s post-tests were used to determine if the treatments significantly influenced TNFα levels in blood or tumors (P > 0.05 for all comparisons; n = 5, +/− SEM). b TNFα was quantitated in the serum of mice 6 h following the final administration (after 4 weeks of treatment) of the listed agents to tumor-bearing mice, or tumor-free untreated mice. One-way ANOVA analyses with Sidak’s post-tests were used to estimate the probability that random chance accounted for the differences observed between saline-treated mice and those treated with drugs or tumor-free animals (colored asterisks), and whether doxorubicin significantly altered the TNFα responses to Smac mimetics (black asterisks and “ns” labels) (*** P < 0.001; ns P > 0.05; n = 3–11, +/− SEM). c Lysates from tumors resected from treated and untreated mice were immunoblotted using an antibody that detects both cIAP1 (70 kDa) and cIAP2 (67 kDa). Loading was visualized by immunoblotting for beta actin (42 kDa). d TNFα was quantitated in the serum and tumors of four tumor-bearing Osx-Cre p53fl/fl pRbfl/fl mice, and in the serum of three tumor-free p53fl/fl pRbfl/fl mice. A one-way ANOVA analysis with Sidak’s post-tests was used to estimate the probability that random chance accounted for the differences in TNFα concentrations between the blood of the tumor-bearing mice versus either their tumors or the blood of tumor-free animals (* P < 0.05; ns P > 0.05; n = 3–4, +/− SEM). e 1029H-Luc tumors were resected from six untreated mice. The cells were disaggregated, then cultured alongside in vitro-cultured 1029H-luc cells for 48 h in media containing no drugs, 1 μM or 3 μM doxorubicin, 100 pg/ml murine TNFα and/or 1 μM or 10 μM of GDC-0152. Residual ATP was quantitated using CellTitreGlo (n = 6 +/− SEM for resected tumors)