Targeted Killing of CSCs by CDF sphere formation may be associated with the inhibition of CD44 and EpCAM expression. molecular mechanisms by which CDF elicits its anti-tumor activity against PC tumors. CDF in combination with Gemcitabine inhibited Pancreatic Tumor Growth in vivo much more than curcumin combination We have used a subcutaneous xenograft tumor model where the tumor was induced by MIAPaCa-2 cells in CB17-SCID mice. CDF treatment in combination with gemcitabine significantly inhibited tumor growth in MIAPaCa-2 tumors much more than curcumin and gemcitabine combination (Figure 7A) as well as compared to either untreated controls or those treated with a single drug. The mice did not show any weight loss during the treatment period (30 days), suggesting that these treatment had no major adverse effects on animals. CDF with Gemcitabine significantly decreased NF-kB Activation in vivo NF-kB activation was determined in the CDF or curcumin, and/or gemcitabine treated tumor remnants derived from MIAPaCa-2 cells induced tumors as shown above. CDF and curcumin as single agent down-regulated NF-kB activation whereas gemcitabine activated NF-kB level, which was abrogated in combination treatment with CDF. The combination treatment of CDF with gemcitabine showed a significant decrease in NF-kB level compared to curcumin and gemcitabine treatment (Figure 7B), suggesting that the inactivation of NF-kB could be one of the CDF effects on protein expression in vivo The COX-2, PTEN, and b-actin expression was determined by Western blot. A significant down-regulation in the expression of COX-2 was observed in both the combination, but the effect was more pronounced in CDF combination group. The expression of phosphatase and tension homolog (PTEN), a tumor suppressor gene was found to be decreased in MIAPaCa-2 cells; however, the expression of PTEN was up-regulated when treated with CDF (Figure 7C). These results suggest that CDF is much more effective than curcumin. Since PTEN is a known target of miR-21, which has been reported to be up-regulated in PC [13–15], we assessed the expression levels of miR-21 in tumor remnants as shown below. Modulation in the expression of miR-21 and miR-200 family in vivo We determined the expression levels of miR-21, miR-200b and miR-200c in MIAPaCa-2 tumors by real time RT-PCR. Over- expression of miR-21 was observed in MIAPaCa-2 tumors whereas we found a significant reduction in the expression of miR-21 in tumors treated with either CDF alone or in combination with CDF and gemcitabine (Figure 7D). We further determined the expression levels of miRNA-200b and miR-200c in tumor tissues which are known regulators of EMT and found to be significantly low in Figure 6. CDF treatment decreased the expression of CD44 and EpCAM, the known markers of CSCs. Expression in pancreatospheres of AsPC-1 and AsPC-1-GTR cells was assessed by confocal microscopy (Magnification X250). doi:10.1371/journal.pone.0017850.g006 PLoS ONE | www.plosone.org 7 March 2011 | Volume 6 | Issue 3 | e17850