inhibited cell growth, DNA-binding activity of NF-kB, Akt, COX- 2, and the production of PGE2 and VEGF, and caused induction of miR-200 and inactivation of miR-21 in PC cells [12]. Since miR-200 is associated with the acquisition of epithelial to mesenchymal transition (EMT), which is also believed to be associated with CSCs or cancer stem-like cells, here we investigated the effects of CDF on CSC function. Here we report, for the first time, that CDF could inactivate many functions of CSCs including self-renewal capacity as demonstrated by the inhibition of sphere-forming (pancreato- spheres) ability of drug-resistant PC cells, which was consistent with inactivation of CSC biomarkers such as CD44 and EpCAM. We also showed anti-tumor activity of CDF alone and in- combination with gemcitabine, which was consistent with inactivation of miR-21, and consequently increased expression of PTEN, attenuation of the DNA binding activity of NF-kB inhibition in the expression of COX-2, and activation in the expression of miR-200 in tumor remnants of a xenograft mouse model of human PC, all of which provide convincing in vivo activity of CDF which is consistent with in vitro findings. Results AsPc-1 and MIAPaCa-2 cell lines and their clones were chosen for this study because of their relatively resistant nature. The CSC characteristics of these cell lines using stem cell markers’ Lin28B and Nanog by RT-PCR, and EpCAM and CD44 by western blot showed an increase in expression level in the PC-GTR cell lines compared to their parental cell lines (Figure 1). Hence we chose these to test our hypothesis that CDF is more effective than curcumin even in resistant cell lines and also their resistant clones–GTR. Targeted Killing of CSCs by CDF CDF strongly prevents clonogenicity and invasion of PC cells compared to gemcitabine and curcumin We selected the concentration of 20 nmol/L of gemcitabine and 4 mmol/L of curcumin or CDF to conduct clonogenic assay following our previous publication [12]. The results demonstrated that there was a significant reduction in clonogenicity of AsPC-1 and MIAPaCa-2 cells treated with curcumin and CDF, but not with gemcitabine (Figure 2A). However, CDF treatment had a much greater and significant reduction in colony formation compared to curcumin. AsPC-1-GTR and MIAPaCa-2-GTR cells had an 80% reduction of clonogenicity with CDF treatment, whereas, only 20–30% reduction of clonogenicity was observed with gemcitabine or curcumin treatment (Figure 2A). Overall, CDF treatment showed a significant reduction in clonogenicity of human PC cells, suggesting the superiority of CDF. CDF or curcumin treatment decreased PC cell migration and invasion. The results showed that 4 mmol/L of curcumin had minimal inhibition of invasion whereas similar concentration of CDF showed significant inhibition of invasion (Figure 2B). The basal level of ABCG2 expression was found in parental cell lines (de novo drug resistant cells); however, the level of expression of ABCG2 was further increased in drug resistant (acquired drug resistant) cell lines (Figure 2C). CDF inhibited viability of human PC cells more than curcumin and gemcitabine as evaluated by MTT assay Initially MTT assay was conducted to examine the effect of different concentrations of gemcitabine (1 to 50 nmol/L), and curcumin or CDF (2–6 mmol/L) on cell survival after 72h of treatment (data not shown). Subsequently, 4 mmol/L of CDF or Figure 1. Comparative expression of Lin28B (A) and Nanog (B) mRNA by qRT-PCR showed increased expression in resistant cell lines compared to parental cell lines, supporting the CSC characteristics of these cell lines. The characteristics of CSCs were further confirmed by the protein expression of EpCAM and CD44 (C). doi:10.1371/journal.pone.0017850.g001 PLoS ONE | www.plosone.org 2 March 2011 | Volume 6 | Issue 3 | e17850