On these bases, CK2 is presently considered a promising therapeutic target [7,10], also exploiting the fact that, due to the peculiar structure of the CK2 catalytic site [11,12], several very specific inhibitors are available (reviewed in [13]). Many of them have already proved to be able to kill cancer cells and in some cases also employed for successful animal treatment (e.g. [14?8]). The two compounds CX-4945 and CX-5011 are among the most selective and effective CK2 inhibitors developed so far. They are tricyclic ATP-competitive compounds, displaying a Ki in vitro ,1 nM [17,19], and an unprecedented selectivity for CK2, proved by profiling them against a panel of 235 protein kinases [19].Figure 1. Pgp expression. 10 mg of proteins from total lysates of the indicated cells were loaded onto an SDS-PAGE, blotted and analyzed by WB with anti-Pgp or anti-actin. number of cancer cell lines and are effective in reducing tumor size in animal models of cancer [17,20]; CX-4945 is orally bioavailable, and is presently in clinical trial for treatment of different kinds of cancer [17]. However, CX-4945 and CX-5011 have never been tested in cells that are resistant to drug-induced apoptosis.
Apoptosis resistance is a major reason of cancer therapy failure; its mechanisms can be different and multifaceted, and is only partially understood. In many cases it is due to the (over)expression of extrusion pumps of the ABC-transporter family, such as Pgp, which drive drugs outside the cell and reduce their effective concentration [21]. Cells expressing these pumps are selected for their survival in response to treatment with a certain drug, but usually a cross-resistance occurs towards other compounds, even not structurally related; in these cases, cells are indicated as multidrug-resistant (MDR). Many other mechanisms have been reported to be involved in apoptosis resistance, including alteration in genetic features, DNA repair, drug target molecules, metabolic and growth pathways [22,23]. In some cases, specific resistance is observed, such as that towards Imatinib and its derivatives targeting Bcr-Abl tyrosine kinase, frequently due to kinase mutations, but also to epigenetic changes, alternative splicing or induction of compensatory signaling pathways [24]. CK2 has been already associated to the phenomenon of drug resistance: it phosphorylates Pgp [25] and another extrusion pump, MRP1 [26] and its inhibition allows a higher accumulation of drugs in Pgp [27] or MRP1 [26] expressing cells, suggesting that
Figure 2. CK2 activity in CX-4945-treated cells. (A) CK2 activity was measured towards a synthetic specific peptide. 1? mg of proteins from total lysates of the indicated cells were incubated with the peptide and a radioactive phosphorylation mixture (see Materials and Methods). Activity is reported as percentage of that found in vehicle-treated control cells. Mean 6 SE values of four independent experiments are shown. (B) 10 mg of total proteins were analyzed with the indicated antibodies; actin was used to normalize the loading. Representative WB of three independent experiments are shown. carcinoma 2008 cells (normal sensitive, S-2008, and their cisplatinresistant variant, R-2008 [30]); chronic myeloid leukemia (CML) LAMA84 [31], KCL22 [32], and K562 [32] cell lines, sensitive or resistant to Imatinib, kindly supplied by Dr. C. GambacortiPasserini.
Inhibitors
CX-4945 (5-(3-chlorophenylamino)benzo[c][2,6]naphthyridine-8-carboxyliuc acid) and CX-5011 (5-(3-ethynylphenylamino)pyrimido[4,5-c]quinoline-8-carboxylic acid) were synthetized by Cylene Pharmaceutical. Solutions were made in dimethylsulfoxide (DMSO).
Cell culture and treatment
Cells were cultured in an atmosphere containing 5% CO2; CEM, LAMA84, K562 and KCL22 cell lines were maintained in RPMI 1640 medium (Sigma), U2OS and 2008 lines were maintained in D-MEM medium (Sigma); both media were supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM Lglutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell treatments with inhibitors were performed in the culture medium, but with 1% FCS, where not differently indicated. Control cells were treated with equal amounts of the inhibitor solvent. At the end of the incubations, cells were harvested by centrifugation, washed, and lysed as indicated below.
Figure 3. CK2 activity in CX-5011-treated cells. S-CEM and R-CEM cells were treated for 24 h with different concentrations of CX-5011. Cell lysates were analyzed for the activity of CK2 towards a synthetic specific peptide (upper graph), or by WB with the indicated antibodies (lower panel, separate development for S- and R-CEM cells). See details in the legend of Figure 2.
Cell viability
Cell viability was detected by means of MTT (3-(4,5dimethylthiazol-2-yl)-3,5-diphenyltriazolium bromide) reagent: cells (105 cells/100 ml) were incubated for variable times in a 96well plate under the indicated conditions. 1 h before the end of the incubation, 10 ml of MTT solution (5 mg/ml in PBS) were added to each well. Incubations were stopped by addition of 20 ml of lysis solution at pH 4.7, as described elsewhere [33]. Plates were read for OD at l590 nm, in a Titertek Multiskan Plus plate reader (Flow Laboratories). DC50 (concentrations inducing 50% of cell death) values were calculated with Prism 4.0c software (GraphPad Software).
CK2 can up-regulate the Pgp function. Moreover, we have previously found that the CK2 catalytic subunit is overexpressed in a MDR cell line compared to the non-MDR counterpart, and that its overexpression contributes to the maintenance of the resistant phenotype [27]. Here we evaluate the efficacy of the CK2 inhibitors CX-4945 and CX-5011 in a number of different cell lines, available as pairs, each pair containing a variant selected for resistance to druginduced apoptosis, and we demonstrate that these compounds can overcome the problem of drug resistance.
