C11, PCP and DMA (Fig. 1a) were initially assayed for their click here ability to inhibit the kinase activity of CK2. As shown in Fig. 1b and summarized in Table 1, C11 inhibited both the CK2α subunit (IC50 4.96 μM) and CK2 holoenzyme (IC50 4.64 μM) in the low micromolar range. In the case of PCP, the IC50 was 3.73 μM and 1.99 μM for CK2α and CK2 holoenzyme, respectively. DMA did not exert

any inhibitory effect on both enzymes indicating that PCP is the active component in the C11 mixture. Further, a kinetic analysis was performed with two different PCP concentrations (i.e. 1 μM and 10 μM, respectively) in order to address the mechanism by which CK2 is inhibited by the aforementioned compounds. As shown in Fig. 1c and Table 1, Km values increased concomitantly to increasing concentrations of Protease Inhibitor Library order PCP, moreover, the double reciprocal plots indicated that the inhibition is consistent with an ATP-competitive binding

of the enzyme. We performed a WST-1 viability assay with two human pancreatic cancer cell lines, i.e. Panc-1 and MIA PaCa-2, for studying the effects of treatment with C11 and its individual components. As shown in Fig. 2a, incubation of cells with increasing concentrations of C11 or PCP for 48 h resulted in progressive cytotoxicity in both cell lines. Similar effects, albeit less pronounced, were obtained when PCP and DMA where combined in a 1:1 ratio. Incubation of cells with DMA alone did not result in reduced metabolic activity indicating that PCP is the component within C11 responsible for the reduced metabolic activity of the cells. A subpopulation of cells within the Panc-1 cell line exhibits features of cancer stem cells (CSCs) such as extensive self-renewal, proliferation, tumorigenesis and high chemoresistance

[19] and [20]. Thus, we asked the question whether PCP induced cytotoxic effects also in this fraction of cells by performing a WST-1 assay. Panc-1 cells were enriched in CSCs expressing the cell surface markers CD44 and CD24. The percentage of CD44+/CD24+ cells was determined by flow cytometry showing an increase from 4.35% to 23.33% (Fig. 2b). A batch of Panc-1 Olopatadine cells as well as a population of depleted and enriched CSCs were left untreated or exposed to C11 and PCP, respectively, for 48 h. Data reported in Figure 2c show that PCP is toxic to all cell populations in a dose dependent manner and independently of the stem-like properties of the cells. Next, flow cytometry analysis was performed to measure the percentage of cells in sub-G1 indicative of cell death, in response to C11 and PCP treatment, respectively, (Fig. 3a). Cells were left untreated or incubated with 100 μM C11, PCP and DMA for the indicated time, respectively. In Panc-1 cells, 72 h incubation with C11 and PCP resulted in 23% and 29% of hypodiploid cells (fraction of cells in sub-G1), respectively.