Kwon et al. BMC Cancer 2010, 10:392 http://www.biomedcentral.com/1471-2407/10/392 Page 6 of 10 Figure 3 Cinnamon extract treatment down-regulates the levels of NF B and AP1 and their target genes. (A) After treatment of cinnamon extract for indicated time periods, the levels of NF B and AP1 in total cell lysates (left) and nucleus extracts (right) were compared by immunoblotting. The effect of cinnamon extract on the activity NF B (B) and AP1 (C) was measured by reporter assay. B16F10 cells were transfected with AP1- or NF B-dependent reporter construct and then stimulated with PMA and ionomycin (P+I) for 4 hours in the absence or presence of cinnamon extract (CE). Gene expression (D) and protein (E) levels of anti-apoptotic genes were measured by quantitative real-time PCR or immunoblotting, respectively. Data are representative of three independent experiments with similar results. One (*), two (**) or three asterisks (***) indicate p < 0.05, p < 0.005 or p < 0.001, respectively. tumor volume was measured throughout the treatment period (Figure 4B). Oral administration of cinnamon extract significantly reduced tumor size compared with control group (Figure 4A and 4B). Consistent with tumor volume, cinnamon treated group showed signifi- cant decrease of tumor weight compared with control groups (cinnamon group; 6.2 g vs. control group; 12.1 g) (Figure 4C). A reduction in tumor size by administration of cinnamon extract significantly increased a survivor rate compared with control group (Figure 4D). These data suggests that oral administration of cinnamon extract has potent anti-tumor activity in vivo. Anti-tumor effect of cinnamon extract is linked to the reduced levels of NF B and AP1 in vivo melanoma model To get the detail action mechanisms of cinnamon in vivo, we tried to investigate whether anti-tumoral effects of cinnamon in vivo melanoma model is also mediated by the similar mechanisms of in vitro system such as active tumor cell death and inhibition of NF B and AP1 activities (Figure 1 and Figure 2). Apoptosis assay was performed with the cells obtained from the tumor tissues of each treatment group, cinnamon extract (CE) and PBS (cont). First, DNA fragmentation assay was performed [28]. Genomic DNAs purified from each treatment group were separated by gel electrophoresis to compare DNA laddering generated by apoptosis. As shown in Figure 5A, DNA isolated from cinnamon extract treated mice (CE) showed a significant increase in DNA fragmentation levels compared with PBS group (Cont) (Figure 5A). Next, we checked the protein levels of caspase-3. Cinnamon extract treatment did not alter the levels of inactive pro-caspase 3 (Figure 5B). Interestingly, however, cinnamon extract treatment significantly increased the level of active caspases-3 compared with the control group (Figure 5B). In addi- tion, the pro-apoptotic effect of cinnamon extract in tumor tissue was further confirmed by tissue staining with Hoechst dye under microscopic observation. Com- pared with the PBS treated group, tumor tissue sections isolated from cinnamon treated group (CE) showed enhancement of apoptotic population showing con- densed chromatin at the nuclear membrane (crescent formation), dissolution of the nuclear membrane, and apoptotic nucleus surrounded by a rim of cytoplasm and plasma membrane [29] (Figure 5C and Additional file 4, Figure S3). In agreement with in vitro data, these data suggest that in vivo anti-tumoral effect of cinna- mon extract is also linked with significant increment of apoptosis in the melanoma tissues. Next, we examined whether in vivo anti-tumoral effect of cinnamon extract is also directly associated with the down-regulation of