pathways are known to contributes to drug-resistance of PC cells to chemotherapeutic agents [16–18]. CSCs comprises only a very small proportion of cells in a tumor mass and posses the ability to self-renew and give rise to differentiated tumor cells [3–5,19]. The CSC theory has fundamental clinical implications especially because CSC has been identified in many malignant tumor tissues including pancreatic cancers and considered to be highly resistant to chemo-radiation therapy than differentiated daughter cells [3– 5,20]; however, CSCs isolated from human tumors are usually insufficient for further mechanistic studies. The existence of CSCs provides an explanation for the clinical observation that tumor regression alone may not correlate with patient survival [21] because of tumor recurrence, which is in part due to the presence of CSCs. Therefore, targeting self-renewal pathways and the killing of CSCs might provide more specific approach for eliminating cells that are the root cause of tumor recurrence. A potential challenge in this regard is the development of therapies that selectively affect CSCs while sparing normal stem cells that may rely on similar mechanisms for self-renewal. In this study, we have demonstrated that CDF not only inhibit cell growth of PC cells, but also inhibit CSC self-renewal capacity as assessed by sphere formation (pancreatospheres) assays. Therefore, CDF could have a greater potential to inhibit cancer growth as documented by our xenograft mouse model of gemcitabine resistant PC cells, which appears to be mediated via inhibition of CSC self-renewal capacity. Emerging evidence suggests the role of microRNA (miRNA) in many biological processes [22–25]. Among many miRNAs, miR- 21, commonly considered as an oncogene, is over-expressed in many solid tumors including PC and has been reported to be associated with tumor progression, poor survival and drug resistance [13,14,26,27]. In our previous report, we have demonstrated that the expression of miR-21 is up-regulated in gemcitabine-resistant PC cells and that its expression can be significantly down-regulated by CDF treatment in vitro [12]. The increased expression of miR-21 is known to be associated with inactivation of PTEN, a know tumor suppressor gene, resulting in activation of PI3K/Akt/mTOR signaling pathway, leading to aggressive tumor growth [15,28]. In this study, we confirmed that CDF treatment could results in the down-regulation of miR-21, resulting in the up-regulation of PTEN in vivo, suggesting that the anti-tumor activity of CDF is associated with up-regulation of PTEN resulting from the inactivation of miR-21 expression. In contrast to miR-21, miR-200 family is known as tumor suppressor and they are usually down-regulated in some tumors including PC and the loss of expression of miR-200 family contribute to the acquisition of EMT phenotype and drug resistance. Down-regulation of miR-200 by siRNA technique has been shown to be associated with EMT phenotype while re- expression of miR-200 can result in the reversal of EMT phenotype [29,30]. In our previous publication [12], we demonstrated that CDF treatment could re-express miR-200 in PC cells. Here we showed, for the first time, that CDF can up- regulate miR-200b and miR-200c in tumor remnants in vivo, consistent with significantly greater inhibition of tumor growth in the xenograft mouse model when CDF was used in combination with gemcitabine. These results suggest that the anti-tumor activity of CDF is mediated via re-expression of miR-200 which may potentially results in the reversal of EMT phenotype and could also lead to overcome drug resistance in PC. In conclusion, CDF showed much more pronounced growth inhibitory effect, inhibited CSC self-renewal consistent with inactivation of CSC biomarkers (CD44 and EpCAM) in PC cells Targeted Killing of CSCs by CDF especially in gemcitabine-resistant PC cells compared to curcumin. In xenograft mouse model of human PC tumors induced by MIAPaCa-2 cells, CDF exhibits anti-tumor activity by regulating COX-2, PTEN, miR-21, miR-200, and NF-kB in vivo. These results strongly suggest that CDF could be a novel agent for the treatment of PC in general but gemcitabine-resistant PC in particular by attenuating the behavior of CSCs. Materials and Methods Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Any animal found unhealthy or sick were promptly euthanized. The protocol was approved by the Committee on the Ethics of Animal Experiments of Wayne State University institutional Users Animal Care Committee (Permit Number: A-10-03-08). Cell Culture, Drugs and Reagents Human pancreatic cancer cell lines AsPC-1, and MIAPaCa-2 were purchased from ATCC (Manassas, VA). These two cell lines AsPC-1 and MIAPaCa-2 were exposed to 200 nmol/L of gemcitabine and 5 mmol/L of tarceva (erlotinib) every other week for about 6 months to create gemcitabine and tarceva resistant (GTR) cell lines, named as AsPC-1-GTR and MIAPaCa-2-GTR, respectively. As a result, AsPC-1, AsPC-1-GTR, MIAPaCa-2, and MIAPaCa-2-GTR were chosen for this study based on their differential sensitivities to gemcitabine. All the cell lines have been authenticated (Applied Genomics Technology Center at Wayne State University) on March 13, 2009 and these authenticated cells were frozen for subsequent use. The method used for testing was short tandem repeat profiling using the PowerPlex 16 System from Promega. Gemcitabine and curcumin were purchased from Eli Lilly (Indianapolis, IN) and Sigma-Aldrich (St. Louis, MO), respectively. CDF was synthesized as described in our earlier publication [10,11]. Gemcitabine was dissolved in water, whereas CDF and curcumin were dissolved in DMSO with a final concentration of 0.1% DMSO in medium. Antibodies Antibodies against ABCG2, and PTEN were purchased from Santa Cruz (Santa Cruz, CA). Antibody to COX-2 and b-actin was acquired from Cayman Chemicals (Ann Arbor, MI), and Sigma Chemicals (St. Louis, MO). Clonogenic assay Clonogenic assay was conducted to examine the effect of drugs on cell growth in PC cells, as described previously [12]. 56104 cells were plated in a six-well plate. After 72h of exposure to 20 nmol/L of gemcitabine, 4 mmol/L of CDF or curcumin, the cells were trypsinized, and 1,000 single viable cells were plated in 100- mm Petri dishes. The cells were then incubated for 10 to 12 days at 37uC in a 5% CO2/5% O2/90% N2 incubator. Colonies were stained with 2% crystal violet and counted. Invasion assay The invasive activity of cells was tested by using BD BioCoat Tumor Invasion Assay System (BD Biosciences, Bedford, MA) according to the manufacturer’s protocol as described previously [31]. Briefly 56104 cells were seeded with serum free medium supplemented with curcumin or CDF into the upper chamber and bottom wells were filled with complete medium in the system. The, PLoS ONE | www.plosone.org 10 March 2011 | Volume 6 | Issue 3 | e17850