Kwon et al. BMC Cancer 2010, 10:392 http://www.biomedcentral.com/1471-2407/10/392 Page 5 of 10 Figure 2 Cinnamon extract treatment induces active cell death in melanoma cells. At the indicated time periods after treatment of cinnamon extract, cells were stained with Annexin V and 7-ADD. Double positive (Annexin V+ and 7-ADD+) (A) or Annexin V+ positive (B) populations were analyzed by FACS. Gene expression (C) and protein (D) level of pro-apoptotic molecules such as Bad, Bim, Bax and Bak were measured by quantitative real-time PCR or immunoblotting, respectively. Error bars indicated SD. One (*), two (**) or three asterisks (***) indicate p < 0.05, p < 0.005 or p < 0.001, respectively. Data are representative of three independent experiments. cell). Like in B16F10 melanoma cells, treatment of cin- namon extract induced apoptosis in Caco2 as well (Additional file 3, Figure S2). From these experiments, we could infer that cinnamon extract induced apoptosis in cancer cells rather than showing simple cytotoxic effects. Next, we tested whether cinnamon extract induced apoptosis is affected by changes in the expres- sion level of apoptosis related genes [25]. Indeed, treat- ment of cinnamon extract significantly increased mRNA expression (Figure 2C) and protein (Figure 2D) levels of pro-apoptotic genes such as Bad, Bim, Bax and Bak. Cinnamon extract inhibits the melanoma growth by inhibiting NF B and AP1 NF B and AP1 have critical roles in tumor cell survival. They regulate the gene expression program of apoptosis and cell cycle [21,26]. Hence, we checked whether a pro-apoptotic activity of cinnamon extract is linked with changes in the levels and activities of NF B and AP1. Interestingly, cinnamon extract decreased the amount of NF B and AP1 proteins in total cell lysates (left panel in Figure 3A) as well as in nucleus extracts (right panel in Figure 3A). To further test whether cinnamon extract also affected on NF B and AP1 activities, luciferase- based reporter assay was performed. B16F10 cells were stimulated with PMA + ionomycin (P+I) to activate NF B and AP1 in the absence or presence of cinnamon extract. Indeed, treatments of cinnamon extract (CE) significantly down-regulated NF B and AP1 activities in a dose dependent manner (Figure 3B and 3C). Next, we checked whether down-regulation of NF B and AP1 levels and their activities by cinnamon treatment could also affect expression level of their target genes related with apoptosis and cell survival such as Bcl-2, BcL-xL and survivin [21,27]. Indeed, cinnamon extract signifi- cantly down-regulated the expression (Figure 3D) and protein (Figure 3E) levels of Bcl-2, BcL-xL and survivin in a time dependent manner. These results suggest that reduction in the levels and activities of NF B and AP1 by cinnamon extract down-regulated their target mole- cules involved in tumor cell survival. Oral administration of cinnamon extract significantly inhibits melanoma progression in vivo To further confirm the anti-tumor effect of cinnamon extract in vivo, we orally administrated cinnamon extract to a mouse melanoma model. Ten days after subcutaneous transplantation of melanoma cells (B16F10), mice were divided into two groups. Mice in each group were daily treated with cinnamon extract (CE; 400 μg/g mouse weight) or same volume of PBS as a sham control group (Cont) for 30 days by oral-admin- istration with catheter (Figure 4). The dose of cinnamon extract in oral administration was based on our previous work [22] that did show any cytotoxic effect in normal mice. To test anti-tumor effects of cinnamon extract,