Salinomycin Inhibits Wnt Signaling and Selectively Induces Apoptosis in Chronic Lymphocytic Leukemia Cells

Salinomycin inhibits Wnt signaling and selectively induces apoptosis in chronic lymphocytic leukemia cells

Desheng Lu1,2, Michael Y. Choi1, Jian Yu1, Januario E. Castro, Thomas J. Kipps, and Dennis A. Carson2

University of California at San Diego Moores Cancer Center, La Jolla, CA 92093-0820

Contributed by Dennis A. Carson, June 28, 2011 (sent for review May 18, 2011) Salinomycin, an antibiotic potassium ionophore, has been reported on the Wnt/Fzd/LRP complex. The drug blocks Wnt-induced recently to act as a selective breast cancer stem cell inhibitor, but LRP6 phosphorylation, and causes degradation of the LRP6 the biochemical basis for its anticancer effects is not clear. The Wnt/ protein. Treatment of primary CLL cells with salinomycin de- β-catenin signal transduction pathway plays a central role in stem pressed LRP6 levels, down-regulated the expression of some Wnt cell development, and its aberrant activation can cause cancer. In target genes, and impaired cell survival. These results suggest that this study, we identified salinomycin as a potent inhibitor of the the anticancer effects of salinomycin may be at least partially at- Wnt signaling cascade. In Wnt-transfected HEK293 cells, salinomy- tributable to Wnt inhibition. cin blocked the phosphorylation of the Wnt coreceptor lipoprotein receptor related protein 6 (LRP6) and induced its degradation. Results Nigericin, another potassium ionophore with activity against can- Salinomycin Suppresses Wnt1-Induced Signaling Pathway. An initial cer stem cells, exerted similar effects. In otherwise unmanipulated screen of known drugs, performed by using the TOPflash cell re- chronic lymphocytic leukemia cells with constitutive Wnt activation porter system (16, 17), identified the ionophores salinomycin, nanomolar concentrations of salinomycin down-regulated the ex- nigericin, thapsigargin, and ionomycin as inhibitors of the Wnt pression of Wnt target genes such as LEF1, cyclin D1, and fibronec- signaling pathway. To further characterize the Wnt inhibitory ef- tin, depressed LRP6 levels, and limited cell survival. Normal human fect of salinomycin, HEK293 cells were transfected with the peripheral blood lymphocytes resisted salinomycin toxicity. These SuperTOPflash reporter and a Wnt1 expression plasmid. Expres- results indicate that ionic changes induced by salinomycin and re- sion of Wnt1 increased the transcriptional activity of the Super- lated drugs inhibit proximal Wnt signaling by interfering with LPR6 TOPflash reporter more than 400 fold versus basal levels (Fig. 1A). phosphorylation, and thus impair the survival of cells that depend Wnt1 overexpression also caused β-catenin accumulation in on Wnt signaling at the plasma membrane. HEK293 cells (Fig. 1D). Salinomycin strongly suppressed Wnt1- stimulated reporter activity with an IC50 of 163 nM (Fig. 1A), and canonical Wnt antagonist | LRP6 stability | B-cell neoplasm reduced β-catenin levels (Fig. 1D). In control experiments, salinomycin did not inhibit the luciferase activity of an AP1-Luc nt signaling plays a crucial role in embryonic development reporter, and actually enhanced the activity of an NFAT-Luc re- Wand cancer (1–4). The Wnt/β-catenin signaling cascade is porter (Fig. 1 B and C). initiated when a Wnt molecule binds to the frizzled (Fzd) receptor and to the lipoprotein receptor-related protein 5 or 6 (LRP5/6) Effects of Salinomycin on Different Components of Wnt Signaling coreceptors, forming a ternary complex at the cell surface. The Cascade. To determine the mechanism by which salinomycin β fl Fzds are seven-pass transmembrane receptors that have conserved inhibits Wnt/ -catenin signaling, the SuperTOP ash reporter was extracellular cysteine-rich domains, which are responsible for Wnt– activated proximally by Wnt1or Fzd5/LRP6, or more distally by β Fzd interactions. The LRP coreceptors are long single-pass trans- -catenin, in transient transfection assays. Although nanomolar membrane proteins that are also essential for Wnt-induced signal concentrations of salinomycin inhibited Wnt1-induced signaling, – the drug had very little effect on reporter gene activity in cells that transduction (5 7). The formation of a Wnt/Fzd/LRP complex β B C triggers phosphorylation of LRP5 or LRP6 at highly conserved overexpressed Fzd5/LRP6 or -catenin (Fig. 2 and ). Much higher concentrations (4 μM) of salinomycin were needed to block PPPSPxP motifs in the intracellular domain. The phosphorylated D LRP proteins recruit axin to the plasma membrane and undergo the Wnt signaling induced by the downstream activators (Fig. 2 ). endocytosis. The eventual result is the inactivation of the β-catenin Thus, salinomycin can suppress Wnt signaling by at least two dif- destruction complex, leading to β-catenin accumulation, nuclear ferent mechanisms, depending on drug dosage. translocation, and expression of Wnt target genes (8, 9). Salinomycin Blocks Wnt1-Induced LRP6 Phosphorylation. To test Mutations and deletions in various components of the in- whether salinomycin directly targets the Wnt or LRP6 molecules, tracellular β-catenin destruction complex are common causes of Wnt1, Wnt3 or dickkopf-related protein 2 (DKK2) were used to human cancers (1, 10). However, other cancers without Wnt activate Wnt signaling. Although DKK2 can act as an agonist or pathway mutations may depend upon exogenous Wnt signaling for antagonist of Wnt signaling, it has been shown to activate Wnt survival. Included among the latter malignancies are chronic signaling when LRP6 is overexpressed (18). Wnt1, Wnt3, and lymphocytic leukemia (CLL) and other B-cell neoplasms. DKK2 each enhanced the activity of the SuperTOPflash reporter, Increasing evidence also suggests that self-renewing stem-like and these effects were antagonized by 250 nM salinomycin (Fig. cells within a tumor, which are resistant to conventional chemo- therapy and are especially virulent, may also be controlled by Wnt/

β-catenin signaling (11, 12). Thus, drugs targeted to the Wnt/ MEDICAL SCIENCES β Author contributions: D.L. and D.A.C. designed research; D.L., M.Y.C., and J.Y. performed -catenin pathway may be useful as anticancer agents. research; D.L., M.Y.C., J.Y., J.E.C., T.J.K., and D.A.C. analyzed data; and D.L. and D.A.C. Salinomycin is a monocarboxylic polyether antibiotic widely wrote the paper. used as an anticoccidiosis agent in chickens (13, 14). A recent The authors declare no conﬂict of interest. study showed that salinomycin kills breast cancer stem cells at least 1D.L., M.Y.C., and J.Y. contributed equally to this work. 100 times more effectively than paclitaxel in mice (15). However, 2To whom correspondence may be addressed: E-mail: [email protected] or dcarson@ucsd. the mechanism by which salinomycin interferes with cancer stem edu. ﬁ cell survival is still unclear. In the present study, we identi ed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. salinomycin as an inhibitor of Wnt/β-catenin signaling, which acts 1073/pnas.1110431108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1110431108 PNAS | August 9, 2011 | vol. 108 | no. 32 | 13253–13257 Downloaded by guest on September 25, 2021 Fig. 1. Salinomycin specifically inhibits the Wnt/β-catenin signaling pathway initiated by Wnt1. (A) HEK293 cells were transfected with a Super- TOPflash reporter and empty vector or Wnt1 expression plasmid. (B) An AP1- luciferase reporter was transfected with a vector expressing constitutively active Ha-Ras (RasV12) into HEK293 cells. (C) HEK293 cells were transfected Fig. 3. Salinomycin selectively targets the Wnt/Fzd/LRP complex. (A) Sali- with an NFAT-luciferase reporter along with NFATc expression plasmid. The nomycin selectively inhibited Wnt signaling mediated by Wnt1/Fzd5/LRP6 or fl transfected cells were treated with salinomycin at increasing concentrations Wnt3/Fzd5/LRP6 or DKK2/Fzd5/LRP6. The SuperTOP ash reporter assays (from 4 nM to 1,250 nM) for 24 h. The luciferase activities were measured were performed on HEK293 cells transfected with the indicated expression and values were normalized to β-gal activity. Results are expressed as fold vectors. The cells were treated with either vehicle (DMSO) or salinomycin induction compared with empty vector control. (D) HEK293 cells were (250 nM and 500 nM) for a further 24 h. The cells were harvested and lu- transfected with empty vector or Wnt1 expression plasmid. Then cells were ciferase activity was measured as described in Materials and Methods.(B) treated with the indicated amounts of salinomycin for 16 h. The β-catenin The relative inhibition rate (as percentage) was used to compare the in- and β-actin levels were detected by Western blot analysis. hibitory effects of salinomycin on Wnt signaling induced by Fzd5/LRP6 or Wnt1/Fzd5/LRP6 or Wnt3/Fzd5/LRP6 or DKK2/Fzd5/LRP6. (C) HEK293 cells were transfected with empty vector and Wnt1 expression vector for 24 h. 3). In contrast, salinomycin had much less effect on Fzd5/LRP6- Then cells were treated with the indicated amounts of salinomycin or vehicle mediated reporter gene activity in cells overexpressing Fzd/LRP6 control for 16 h. The phospho-LRP6 (Ser1490) and total LRP6 were detected β (Fig. 3 A and B). by Western blot analysis. -Actin was used as a loading control. Previous studies have demonstrated that Wnt pathway acti- γ vation requires LRP6 phosphorylation by casein kinase-1 , gly- as assessed by immunoblotting with a specific antibody (Fig. 3C). β cogen synthase kinase-3 , and various other kinases (8, 19). Furthermore, salinomycin treatment caused degradation of the Indeed, nanomolar concentrations of salinomycin blocked Wnt1- LRP6 protein in a dose-dependent fashion (Fig. 3C). induced LRP6 phosphorylation at serine 1490 in HEK293 cells, Nigericin, Like Salinomycin, Inhibits Wnt Signaling Cascade. Nigericin is a potassium ionophore with structural similarity to salinomycin, which is also toxic to breast cancer stem cells (15). Nigericin, like salinomycin, selectively inhibited Wnt1-mediated signaling in HEK293 cells at nanomolar concentrations, but had little effect on reporter activity induced by β-catenin overexpression (Fig. 4). These results point to a common mechanism underlying the Wnt inhibitory action of the two potassium ionophores.

Effect of Calcium Ionophores. Salinomycin can potentially disrupt Na+/Ca2+ exchange and cause an increase in cytoplasmic calcium levels (20, 21). It has been established that the canonical Wnt/LRP6-mediated signaling cascade is antagonized by ele- vating intracellular calcium levels (16). However, the calcium ionophores thapsigargin (Fig. 4) and ionomycin (Fig. S1) strongly inhibited Wnt pathway activation by Fzd5/LRP6 and, to a lesser degree, β-catenin, at concentrations at which salinomycin was ineffective.

Sensitivity of CLL Cells to Salinomycin. Primary CLL cells produce Wnt proteins, have constitutive Wnt activation, and express high Fig. 2. Salinomycin inhibits Wnt/β-catenin signaling via at least two dif- levels of the Wnt-regulated transcription factor Lymphocyte En- ferent mechanisms. The SuperTOPﬂash reporter was transfected into hancing Factor 1 (LEF1) (22). However, CLL cells do not have HEK293 cells along with expression plasmids encoding Wnt1 (A), Fzd5 and known activating mutations or deletions of the β-catenin tran- β LRP6 (B), and -catenin (C), respectively. After transfection for 24 h, cells scription complex. Hence, CLL cell survival might be sensitive to were treated with indicated amounts of salinomycin for another 24 h. Lu- ciferase activity was measured and presented as fold induction compared compounds that inhibit proximal Wnt signaling by interference with empty vector control. (D) The inhibitory effects of salinomycin on Wnt/ with LRP6 phosphorylation and stability. To test this hypothesis, β-catenin signaling mediated by Wnt1 or Fzd5/LRP6 or β-catenin were primary CLL cells from 13 patients and peripheral blood mono- compared and presented as relative inhibition rate (as percentage). nuclear cells (PBMCs) from seven healthy donors were incubated

13254 | www.pnas.org/cgi/doi/10.1073/pnas.1110431108 Lu et al. Downloaded by guest on September 25, 2021 Fig. 4. Comparison of the Wnt inhibitory effects of salinomycin, nigericin, and thapsigargin. HEK293 cells were cotransfected with the SuperTOPﬂash reporter construct, along with expression plasmids for Wnt1 (A), Fzd5/LRP6 (B), and β-catenin (C), respectively. After transfection for 24 h, the cells were treated with salinomycin (250 nM and 500 nM), thapsigargin (10 nM and 50 nM), and nigericin (125 nM, 250 nM, and 500 nM) as indicated. Luciferase activity is presented as fold induction compared with empty vector control.

with increasing concentrations of salinomycin for 48 h. Induction requires the activity of the vacuolar H+-adenosine triphosphatase of apoptosis was detected by cytofluorometry. Incubation of the (V-ATPase) (30). The prorenin receptor functions as a specific malignant lymphocytes with salinomycin induced apoptosis within adaptor between LRP6 and V-ATPase. The V-ATPase protein 48 h, with a mean IC50 of 230 nM. Under the same conditions, complex localizes in intracellular organelles and at the plasma salinomycin failed to induce apoptosis in PBMC at 100-fold higher membrane, where it pumps protons and acidifies vesicles, thereby concentrations (Fig. 5). This result suggests that salinomycin has promoting endocytosis (31). Inhibition of V-ATPase activity by selective cytotoxicity to CLL cells that are known to depend on bafilomycin blocks Wnt/β-catenin signaling (30). Nigericin has Wnt signaling. been reported to induce intracellular alkalinization (32, 33). It is therefore likely that salinomycin and nigericin may interfere with Salinomycin Reduces LRP6 Protein Levels and Down-Regulates Ex- the proton gradient generated by the V-ATPase, and thus block pression of Wnt Target Genes in CLL Cells. LRP6 expression has the phosphorylation and endocytosis of LRP6 that are required for been detected in primary CLL cells, albeit at low levels (22, 23). initial Wnt signal transduction. As salinomycin could block Wnt-induced LRP6 phosphorylation Previous studies have revealed that salinomycin can also cause and cause the degradation of LRP6 protein in HEK293 cells an increase in intracellular calcium levels by disrupting Na+/Ca2+ (Fig. 3C), we also assayed its effect on the LRP6 protein in CLL exchange (21). The toxicity of salinomycin has been attributed to cells. As predicted, the drug reduced LRP6 levels in the malig- drug-induced accumulation of intracellular calcium (20). As an nant lymphocytes (Fig. 6A). increase in intracellular calcium causes inhibition of the canonical LEF1, cyclin D1,andfibronectin are target genes of the Wnt/ Wnt pathway (16), we tested the effect of other intracellular cal- β-catenin pathway, which are up-regulated in CLL cells compared cium regulators on LRP6-mediated Wnt signaling. Unlike sali- with normal lymphocytes (22, 24). The CLL cells from four nomycin, both thapsigargin and ionomycin blocked Wnt signaling patients were treated with salinomycin for 16 h, and then were downstream of LRP6. Thus, at least at submicromolar concen- analyzed by quantitative PCR for gene expression. At this time trations, salinomycin-induced inhibition of Wnt signaling probably point, the CLL cells were still viable, but all three Wnt-dependent does not depend on its calcium mobilizing activity. transcripts had decreased (Fig. 6B). The LRP5 and LRP6 coreceptors for Wnt signaling have been revealed as potential oncogenic proteins. Aberrant expression of Discussions an LRP5 splicing isoform has been implicated in breast tumor Salinomycin and nigericin are among the first drugs reported to formation (34), and LRP6 is overexpressed in a subset of human act as selective cancer stem cell inhibitors (15). These polyether breast carcinomas (35). Overexpression of LRP6 in the mouse ionophores interfere with transmembrane potassium potential mammary gland induces mammary hyperplasia, whereas down- and promote mitochondrial and cellular potassium efflux (25, 26). regulation of LRP6 inhibits breast cancer tumorigenesis (35, 36). Our experiments demonstrate that salinomycin treatment of cells Furthermore, an LRP antagonist, Mesd (mesoderm development), markedly suppresses breast cancer tumor growth in vivo antagonizes the Wnt signaling cascade. At submicromolar con- fi centrations, the drug inhibits Wnt1-induced LRP6 phosphoryla- without signi cant toxicity (35, 36). These studies strongly suggest tion, and causes the degradation of the LRP6 protein, an essential that LRP6 is a potential therapeutic target in breast cancer. The component of the Wnt receptor complex. At micromolar concentrations, salinomycin also promotes β-catenin degradation. Considering the importance of Wnt/β-catenin signaling in stem cell biology and in various malignancies, the Wnt antagonistic actions of salinomycin may contribute to its toxicity toward breast cancer progenitor cells. The phosphorylation of LRP6 is crucial for activation of Wnt/

β-catenin signaling. Several protein kinases, including glycogen MEDICAL SCIENCES synthase kinase-3 and casein kinase-1γ, have been shown to phos- phorylate this Wnt coreceptor (27). A recent study showed that transmembrane protein 198 can promote LRP6 phosphorylation and Wnt signaling activation by recruitment of casein kinase family proteins (28). Another report showed that salinomycin is Fig. 5. Salinomycin selectively induced apoptosis in CLL cells. Primary CLL able to overcome multidrug resistance mediated by the ATP- cells from 13 patients and the PBMCs from seven normal donors were treated binding cassette transporter p-glycoprotein 170 (29). Cruciat et al. with increasing amounts of salinomycin for 48 h. Apoptosis was measured by

demonstrated that LRP6 phosphorylation and internalization ﬂow cytometry by using DiOC6/PI staining.

Lu et al. PNAS | August 9, 2011 | vol. 108 | no. 32 | 13255 Downloaded by guest on September 25, 2021 at earlier time points. Interestingly, we observed that 250 nM salinomycin was a high enough dose to block LRP6 phosphorylation induced by overexpressing Wnt1 in HEK293 cells. Con- sidering multiple kinsaes responsible for LRP6 phosphorylation (27), our results suggest that salinomycin may speciﬁcally suppress Wnt-induced LRP6 phosphorylation. Furthermore, we showed that salinomycin down-regulates the expression of Wnt target genes and exerts selective toxicity to primary CLL cells compared with normal PBMCs. Thus, drugs that inhibit LRP6 phosphorylation or stability may be therapeutically active in CLL. Materials and Methods Human Samples. Samples were collected by the CLL Research Consortium after informed consent was obtained from patients fulﬁlling diagnostic criteria for CLL. Institutional review board approval was obtained from the University of California, San Diego, for the procurement of patient samples in this study, in accordance with the Declaration of Helsinki. Buffy coat samples from healthy volunteers were obtained from the San Diego Blood Bank.

Reagents and Plasmids. Salinomycin, nigericin, thapsigargin, and ionomycin were purchased from Sigma-Aldrich. A Gen-plus collection of 960 known drugs was obtained from Microsource. The reporter plasmid TOPﬂash and the β-catenin expression plasmid were gifts from H. Clevers (University of Utrecht, Utrecht, The Netherlands). The SuperTOPﬂash reporter construct was from Karl Willert (University of California, San Diego, San Diego, CA). The NFAT-Luc and AP1-Luc reporters were purchased from BD Biosciences. The expression plasmid for human DKK2 was from Origene Technologies. The expression plasmids encoding Wnt1, Wnt3, LRP6, NFATc, H-rasV12, and β-gal have been described previously (22, 42, 43).

Cell Culture and Transfection. HEK293 cells were maintained in DMEM sup- Fig. 6. Salinomycin reduced the LRP6 protein level and down-regulated the plemented with 10% FBS and 100 μg/mL penicillin and 100 μg/mL streptomycin. expression of LEF1, cyclin D1, and fibronectin in CLL cells. (A) Primary CLL HEK293 cells were transfected in 12-well plates by using FuGENE (Roche), and cells from two patients were treated with indicated amounts of salinomycin 0.5 μg of reporter plasmid, 50 to 100 ng of the control plasmid pCMXβgal, 100 for 16 h. The phospho-LRP6 (Ser1490), total LRP6, and β-actin were detected to 200 ng of the various expression plasmids, and carrier DNA pcDNA3 plasmid, by Western blot. (B) Primary CLL cells from four patients were treated with for a total of 1 μg per well. After overnight incubation, the cells were treated increasing concentrations of salinomycin for 16 h. The mRNA levels of LEF1, with the different concentrations of drugs or vehicle. For luciferase assays, cells cyclin D1, and fibronectin were measured by real-time PCR as described in were lysed in isotonic potassium phosphate buffer, pH 7.8, containing 1% Materials and Methods. Triton X-100, and luciferase activities were assayed in the presence of substrate by using a multiple-mode detection reader (Infinite M200; Tecan). The luciferase values were normalized to the activity of the β-gal, and are expressed as fact that salinomycin can block Wnt-induced LRP6 phosphoryla- percentage of control luciferase or fold stimulation of luciferase activity tion and cause degradation of LRP6 protein, indicates that LRP6 compared with the basal level. All of the transfection results are representa- is a “druggable” target for anticancer therapy. Small-molecule tive of a minimum of three independent transfections. drugs that alter LRP6 phosphorylation or stability may have Western Blot Analysis. HEK293 cells and primary CLL cells were treated with therapeutic potential in malignancies with abnormal Wnt activa- the indicated amounts of salinomycin as described in the figure legends. Cells tion at the cell surface. They would be expected to be less active in were washed twice with PBS solution and resuspended in 0.5 mL lysis buffer β cancers with mutational activation of the -catenin destruction (20 mM Tris-HCl, pH 8.0/10% glycerol/5 mM MgCl2/0.15 M KCl/0.1% Nonidet complex, such as colorectal carcinomas. P-40 with protease inhibitors). Equal amount of proteins from each sample CLL is the most frequent form of human leukemia in the was resolved by SDS/PAGE followed by immunoblotting with anti-LRP6 an- Western world. Several activating Wnt proteins are overex- tibody, phospho-LRP6 (Ser1490) antibody (Cell Signaling Technology), pressed in primary CLL cells, whereas physiological inhibitors of anti–β-catenin antibody (Santa Cruz Biotechnology), and β-actin (Chemicon Wnt signaling are partly inactivated in this disease (22, 37). International). Horseradish peroxidase-conjugated anti-IgG was used as the During Wnt activation, β-catenin is up-regulated and cooperates secondary antibody. The membranes were developed by using a chem- with the transcription factors T-cell factor and LEF1 to alter the iluminescence system (ECL detection reagent; Amersham Pharmacia). production of proteins important for cell proliferation and sur- RNA Isolation and Real-Time PCR. Total RNA was isolated from 1 × 106 primary vival, including Myc, cyclin D1, and several antiapoptotic pro- fi LEF1 CLL cells by TRIzol reagent (Invitrogen). The RNA samples were further puri ed teins. In comparison with normal blood B cells, is the most by using a RNeasy Protect kit (Qiagen). The mRNA levels were quantified in highly up-regulated mRNA in CLL cells (38). Knockdown of triplicate by real-time PCR on a 7900HT Fast Real-Time PCR System (Applied LEF1 by siRNA leads to increased apoptosis of CLL cells (39, Biosystems) by using the following primer sets to detect the expression of LEF1, 40). Treatment with two small-molecule inhibitors of Wnt/ cyclin D1, and fibronectin. LEF1 sense, 5′-AGGAACATCCCCACACTGAC-3′;and β-catenin signaling (CGP049090 and PKF115-584) induces apo- LEF1 antisense, 5′-AGGTCTTTTTGGCTCCTGCT-3′. Cyclin D1 sense, 5′-AATGA- ptosis in CLL cells in vitro and in vivo (39). Moreover, our pre- CCCCGCACGATTTC-3′; and cyclin D1 antisense, 5′-TCAGGTTCAGGCCTTGCAC- ′ ′ ′ fi vious results revealed that ethacrynic acid and other Michael 3 . Fibronectin sense, 5 -ACCTACGGATGACTCGTGCTTT-3 ;and bronectin anti- ′ ′ acceptors exhibit selective toxicity to CLL cells, in part by an- sense, 5 -TTCAGACATTCGTTCCCACTCA-3 . PCR was performed by using Power β SYBR Green PCR Master Mix (Applied Biosystems) according to the manu- tagonism of Wnt/ -catenin signaling (17, 41). In the present study, facturer’s instructions. The PCR conditions were 2 min at 50 °C (one cycle), we demonstrated that salinomycin reduces phospho-LRP6 and 10 min at 95 °C (one cycle), and 15 s at 95 °C and 1 min at 60 °C (40 cycles). The total LRP6 protein levels, whereas a higher concentration (more data were analyzed by using the comparative Ct method, where Ct is the cycle than 500 nM) of salinomycin is required for its maximum effect on number at which fluorescence first exceeds the threshold. The ΔCt values LRP6 phosphorylation and stability in unmanipulated CLL cells from each tissue were obtained by subtracting the values for 18S Ct from the

13256 | www.pnas.org/cgi/doi/10.1073/pnas.1110431108 Lu et al. Downloaded by guest on September 25, 2021 sample Ct. One difference of Ct value represents a twofold difference in the lyzed within 30 min by ﬂow cytometry by using a FACSCalibur (Becton

level of mRNA. Dickinson). Fluorescence was recorded at 525 nm (FL-1) for DiOC6 and at 600 nm (FL-3) for PI. The apoptotic cells were determined by calculating the − + Cell Apoptosis Assays. Apoptosis was determined by the analysis of mito- percentages of the DiOC6 /PI CLL populations. chondrial transmembrane potential (ΔΨm) using 3,3′-dihexyloxacarbocy- anine iodine (DiOC6) and by cell membrane permeability to propidium io- ACKNOWLEDGMENTS. We thank Laura Rassenti for technical assistance and 6 dide (PI). Primary CLL cells were cultured at a density of 5 × 10 cells/mL in Nancy Noon for secretarial support. T.J.K. and D.A.C. are members of the RPMI with 10% FBS containing salinomycin at concentrations ranging from Chronic Lymphocytic Leukemia Research Consortium. This work was sup- 0to10μM. After treatment for 48 h, 100 μL of the cell culture was collected ported in part by Leukemia and Lymphoma Society SCOR Grant CA81534-06, and transferred to FACS tubes containing 100 μL FACS buffer with 60 nM National Cancer Institute–Drug Development Group Grant CA113318-01,

DiOC6 and 10 μg/mL PI. Cells were incubated at 37 °C for 30 min and ana- and National Institutes of Health Grant PO1-CA081534 (to D.A.C.).

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