Fig 1.
Representative images of radiation-induced mucositis used for scoring are shown. See details in the Methods.
Fig 2.
Inhibitory effects of AMP peptide and AMP-18 on HNC cells.
SCC-25 (A) or SCC-61 (B) cells were left untreated, exposed to AMP-18 (2 μg/ml), cisplatin (2 μM for SCC-25 cells, 10 μM for SCC-61 cells) only, cisplatin in the presence of 2 μg/ml AMP peptide, or rhAMP-18 for 48 h. Cell viability was assayed by CellTiter-Blue reagent. Experiments were performed in quadruplicate. Values are mean ± SE. Adding either AMP peptide or rhAMP-18 amplified the cytotoxicity of cisplatin by inhibiting growth of SCC-25 cells by >30% (AMP peptide, P = 0.013; rhAMP-18, P = 0.03), and SCC-61 cells by 65% (P<0.05) and 75% (P<0.02), respectively.
Fig 3.
Effect of AMP peptide and radiation treatment in an orthotopic model of HNC cancer in the tongue assessed using IVIS.
An orthotopic model of HNC cancer was established by inoculating bioluminescent SCC-25-LUC cells into the anterior tongue of nude mice. Tumor size in different groups was assessed and compared on Days 5 and 8 as well as Days 10 and 14 using IVIS after injection of D-luciferin. Mice in Group 1 and Group 3 were not irradiated, whereas animals in Groups 2 and 4 received 30 Gy on Day 0 of the study. Treatment with PBS (Group 2) or AMP peptide (Group 4) was administered by injection s. c. once daily. In the irradiated mice, treatment with AMP peptide was associated with much lower IVIS radiance compared to treatment with PBS, indicating that AMP peptide reduced tumor growth. Representative IVIS images of tongue tumors from 32 mice are shown.
Fig 4.
Effect of AMP peptide and radiation treatment of HNC tumors in the anterior tongue of nude mice with time assessed by measuring tumor IVIS radiance.
Irradiated animals were treated with AMP peptide or PBS, and IVIS radiance was measured on Days 5 and 8, and then on Days 10 and 14. Means ± SE were compared. In mice given PBS, IVIS radiance appeared to increase between Days 5 and 8 and 10 and 14, whereas radiance in animals treated with AMP peptide was unchanged between Days 5 to 14. On Days 10 and 14, treatment with AMP peptide significantly reduced tumor size compared to treatment with PBS (* P = 0.03). In non-irradiated mice, the orthotopic tumors in the tongue grew progressively; administration of AMP peptide did not change tumor size significantly (not shown).
Fig 5.
Mean daily mucositis scores in irradiated mice.
Irradiation of the tongue and oral mucosa was followed by daily treatment with AMP peptide or vehicle (PBS). Ulcerative oral mucositis that developed was scored as described in Methods. A mucositis score of 3.0 (broken line) indicates mucosal ulceration that correlates with significant human oral mucositis. Animals treated with the vehicle reached a score of 3 on Day 12, whereas mice treated with AMP peptide did not. Mucositis score was significantly lower on Days 11 (* P = 0.003) and 12 (** P = 0.03), but not Day 13 (P = 0.1) in animals treated with AMP peptide compared to those given the vehicle. Values are means ± SE.
Fig 6.
Differential effects of AMP-18 on cleavage of caspase 3 in HaCaT and SCC-25 cells.
Nontransformed human keratinocyte (HaCaT cells) and human HNC (SCC-25) cell cultures were each exposed to TNF-α for 6 h to induce apoptosis displayed as increased cleavage of caspase 3; 8.1-fold in HaCaT, and 2.8-fold in SCC-25 cells. Treatment with rhAMP-18 appeared to block TNF-α –induced cleavage of caspase 3 in HaCaT (50–75%, * P = 0.02) but not in HNC cells. Each immunoblot represents one of three experiments.
Table 1.
Hierarchically arranged lists of pathways differentially targeted by AMP-18 in SCC-61 and HaCaT cells.
SCC-61 and HaCaT cells were treated with AMP-18 for 2 h, total RNA was purified and subjected to RNA microarray analysis. Differentially expressed pathways were identified as stated in the Methods with no AMP-18 treatment as a control.