Background
Multiple myeloma (MM) is a B-cell malignancy characterized by the accumulation of malignant plasma cells in the bone marrow. Despite the use of conventional or high-dose chemotherapy or autologous stem-cell transplantation, tumor cells invariably generate a resistance to the various treatments. Chemoresistance of MM cells remains the primary obstacle in developing a satisfactory treatment [
1]. Therefore, to improve outcomes and extend the length of survival, the establishment of more effective treatments that can overcome or circumvent chemoresistance has become a priority.
Casein kinase 2 (CK2) is a ubiquitous cellular serine-threonine kinase with a broad spectrum of substrates. CK2 participates in the regulation of multiple biologic processes and plays an important role in regulating multiple cellular functions, including transcription, translation, signal transduction and metabolism [
2,
3]. The expression and activity of CK2 are frequently elevated in cancer cells, which provides a growth advantage because its activity counteracts apoptosis and sustains the cell cycle [
4,
5]. It has been shown that MM cell lines and highly purified malignant plasma cells in patients with MM expressed higher protein and CK2 activity levels than normal plasma cells and B lymphocytes [
6]. In this regard, using siRNA to inhibit CK2 activity induced apoptosis and enhanced the cytotoxic effect of melphalan on MM cells. It was proposed that CK2 might play a pivotal role in controlling survival and sensitivity to chemotherapeutics of MM cells [
6]. The exact mechanisms governing the pleiotropic activity of CK2 have not been well defined. However, some recent studies have demonstrated that CK2 controls Hsp90 chaperone machinery by phosphorylating a kinase-targeting molecular co-chaperone, Cdc37 [
7,
8].
Among Hsp90 co-chaperones, Cdc37 is unique because it interacts with a subset of client kinase proteins within Hsp90 complexes and plays a specialized role as a primary partner in kinome maintenance [
9]. Cdc37 plays a role in protein kinase quality control not only by protecting nascent polypeptide chains from degradation and by promoting posttranslational maturation [
10]. CK2-mediated phosphorylation of Cdc37 on a conserved Ser13 in the N-terminal region is important for efficient binding to client kinases and for recruiting Hsp90 to the kinase-Cdc37 complex [
7,
8]. Therefore, CK2 activity also depends on Cdc37; there is a positive feedback loop between CK2 and Cdc37 which positively regulates multiple protein kinases [
11]. Hsp90 binds to and protects CK2 from self-aggregation and enhances its kinase activity [
12]. Strikingly, several crucial anticancer targets, including EGFR, PDGFR, Aurora B, Src, Raf-1, AKT, IKK, Cdc2, Cdk2, Cdk4, and Cdk6 are Cdc37 client kinases
http://www.picard.ch/downloads/Cdc37interactors.pdf. Because the function of Hsp90/Cdc37 determines the stability and activity of these kinases, the dependency of the cancer cell kinome on Hsp90/Cdc37 makes the CK2-Cdc37-Hsp90 trinity a promising anti-cancer drug target [
13].
Cdc37 is overexpressed in several types of cancers, including multiple myeloma [
9]. Previous studies have shown that RNA interference-mediated downregulation of Cdc37 enhances the cytotoxic effects of Hsp90 inhibitors in prostate cancer cells and colon cancer cells by reducing client kinase activity and decreasing survival signaling [
14,
15]. Treating cells with 4, 5, 6, 7-Tetrabromobenzotriazole (TBB), which is a specific chemical inhibitor of CK2, induces a decline in phosphorylation of Cdc37 and decreases the intracellular levels of Cdc37-dependent protein kinases [
8]. However, an evaluation of the strategies of killing cancer cells by inhibiting CK2-dependent phosphorylation of Cdc37 has not been reported.
The flavonoid apigenin is abundant in common fruits and vegetables. Apigenin has gained attention because it has notable anti-inflammatory, antioxidant and anti-carcinogenic properties [
16]. Apigenin has been shown to be remarkable in inhibiting growth, arresting cell cycle and inducing apoptosis of human prostate cancer, breast cancer and leukemia [
17‐
21]. Possible mechanisms mediating its anticancer effects include modulation of various kinase activities [
17], inactivation of NF-κB [
18], inhibition of proteasomal activity [
19] and induction of proteasomal degradation of the Her2/neu proteins [
20]. As a selective CK2 kinase inhibitor, apigenin has been reported to induce cell death to a greater extent in CK2α-high AML than in CK2α-low AML or normal BM samples [
21]. However, the detailed mechanism by which targeting CK2 leads to apoptosis and inactivation of survival signals has not been defined. Given that MM cells also exhibit high CK2 activity, it was of interest to determine the ability of apigenin to kill MM cells. In the present study, we have investigated the effects of apigenin on MM cell lines and purified primary MM cells. We found that apigenin inhibited the proliferation of MM cells, and induced apoptosis of MM cells through the suppression of CK2 kinase and the reduction of Cdc37 phosphorylation. These effects disrupted the Hsp90 chaperone function and downregulated multiple client kinase proteins, and as a consequence, induced apoptosis in MM cells.
Methods
Reagents and antibodies
Apigenin, MG132, Geldanamycin and α-tubulin antibody were obtained from Sigma-Aldrich (St Louis, MO, USA), and suberoylanilide hydroxamic acid (SAHA or Vorinostat) was donated by AstraZeneca (Macclesfield, SK, UK). These reagents were dissolved in DMSO (less than 0.1%, v/v). Recombinant human (rh) IL-6 and rhIGF-1 were purchased from PeproTech (Rocky Hill, NJ, USA). Antibodies against phospho-AKT (Ser473), AKT, phospho-ERK (Thr202/Tyr204), ERK, phospho-STAT3 (Tyr705), STAT3, phospho-IκB-α (Ser32), phospho-PDK1, PDK1, phospho-MEK, MEK, phospho-IKK, poly (ADP-ribose) polymerase (PARP), and XIAP were obtained from Cell Signaling Biotechnology (Beverly, MA, USA). Antibodies against Survivin, Mcl-1, IKK and Cdc37 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-β-actin, phosphoserine, CK2α antibodies and tetrabromobenzotriazole (TBB) were obtained from Calbiochem (San Diego, CA, USA). Anti-Raf-1, Bcl-2, Bcl-xL and Cdk4 antibodies were purchased from BD Biosciences (San Diego, CA, USA). The anti-Src antibody was purchased from Upstate Biotechnology (Lake Placid, NY, USA). The anti-Hsp90 antibody was obtained from Stressgen Biotechnologies (Victoria, BC, Canada). The anti-RIP1 antibody was purchased from Abcam (Cambridge, MA, USA).
Cell lines and clinical samples
The human MM cell lines (U266 and RPMI 8226) were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin. The human cervical carcinoma cell line (HeLa) were cultured in DMEM medium with 10% FBS. Bone marrow samples were obtained from patients with MM that underwent treatment at the General Hospital of PLA (Beijing, China), and approval was obtained from the hospital institutional review board for these studies. Informed consent was obtained from all patients in accordance with the Declaration of Helsinki. The CD138+ cells were separated by immunomagnetic bead selection (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). The purity of isolated CD138-positive plasma cells was approximately 95% as assessed by flow cytometry using phycoerythrin (PE)-conjugated monoclonal CD138 antibodies. To generate peripheral blood mononuclear cells (PBMCs), 5 ml of whole blood was collected from five healthy donors. PBMCs were enriched by density centrifugation over Ficoll-Paque density gradient. The mononuclear cell fraction was collected and washed three times in sterile PBS and was immediately used in the cytotoxicity assays.
Cell Viability Assay
The cell viability was determined by the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay according to the manufacturer's instructions (CellTiter96 Aqueous Non Radioactive Cell proliferation Assay kit; Promega, Madison, WI, USA). MM cell lines were cultured in complete medium containing the vehicle DMSO (control, less than 0.1%, v/v) or complete medium supplemented with various concentrations of apigenin or TBB for 24 h and 48 h. At the end of the incubation period, 20 μl of the combined MTS/PMS solution was added into each well of the 96-well plate. Following 4 h of incubation at 37°C and 5% CO2, absorbance was detected at a wavelength of 490 nm. The results are presented as means ± SD from three independent experiments. Inhibition graphs were plotted using mean values obtained from each concentration relative to control values.
Cell cycle analysis
Log-phase U266 and RPMI 8226 cells were seeded in 6-well plates and treated with varying dose of apigenin or vehicle DMSO for 24 h. The cells were harvested, washed with PBS and fixed with 70% ethanol containing 1% FBS at -20°C overnight. After an additional washing step, cells were incubated with RNase A (20 μg/ml) at 37°C for 30 min, stained with propidium iodide (PI, 100 μg/ml) for 10 min, and analyzed by flow cytometry.
Apoptosis assay
Apoptosis was determined with the Annexin-V-FLUOS staining kit (Roche, Indianapolis, IN, USA) according to the manufacturer's instructions. Briefly, the vehicle DMSO control and the apigenin-treated cells were collected by centrifugation and were washed one time with PBS. The cells were subsequently stained with fluorescein and PI for 15 min at room temperature and analyzed by flow cytometry.
CK2 kinase activity assay
CK2 kinase activity in cell lysates was measured by using the Casein Kinase-2 Assay Kit (Upstate Biotechnologies, Temecula, CA, USA) as described before [
21]. Briefly, 20 μg whole-cell lysates were tested in Assay Dilution Buffer I (ADBI) plus with 200 μM substrate peptide (RRRDDDSDDD), 2 μM PKA inhibitor peptide(PKI-[
6‐
22]-NH
2), and 100 μCi [γ-
32P] ATP. The reaction mixtures were incubated with agitation for 10 min at 30°C. Reactions were stopped by addition of 40% trichloroacetic acid (TCA). Samples (25 μl) were then transferred onto phosphocellulose filter paper square P81, and the radiolabeled substrate was allowed to bind to the paper for 30 sec. The paper was immersed in 0.75% phosphoric acid and mixed gently on a rotator; followed by washing six times with 0.75% phosphoric acid and one wash with acetone for 1 min. Radioactivity incorporated into the substrate peptide was determined by scintillation counting.
Immunofluorescence analysis
The vehicle-only control and apigenin-treated cells were fixed for 10 min in PBS containing 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 for 10 min. After washing 3 times with PBS, the cells were immersed in 1% bovine serum albumin (BSA) for 30 min and were incubated with primary anti-CK2α antibody (1:50) overnight at 4°C. After additional washing with PBS, the cells were incubated with secondary antibody conjugated with FITC for 1 h in the dark at room temperature. The cells were examined either by flow cytometry or by fluorescent microscopy at total 1000× magnification under immersion oil using a LSM 510 META ZEISS fluorescent microscope. The fluorescence intensity of CK2α protein was quantified using SoftWoRx Explore 1.2 (Applied Precision, Issaquah, USA).
RNA interference
Small interfering RNA (siRNA) oligonucleotides were synthesized by GeneChem Co., Ltd (Shanghai, China). The sequence for CK2α was 5'-GAUGACUACCAGCUGGUUCdTdT -3' and the control siRNA sequence was 5'- UUCUCCGAACGUGUCACGUTT-3'. The siRNAs were introduced into HeLa and MM cells by RNAiFect Transfection Reagent or electroporation respectively. HeLa cells were transfected with 40 nM siRNA using the RNAiFect Transfection Reagent (Qiagen, Germany) according to the manufacturer's instructions. Log-phase U266 and RPMI 8226 cells were harvested, washed once and resuspended in serum-free RPMI1640 medium at a concentration of 1 × 107/ml. Control siRNA (75 pM) or CK2α-siRNA (75 pM) was added to 200 μl cell suspension. Next, the mix was transferred directly into a 2 mm gap electroporation cuvette and was electroporated with an Electro Square Porator ECM830 (BTX, San Diego, CA) at 250 V and 500 μs. Immediately after the pulse, the cell suspension was incubated on ice for 10 min, and the cells were resuspended in complete medium for 48 h. The cells were harvested and subjected to western blotting with the indicated antibodies.
Immunoprecipitation and western blotting
Immunoprecipitation experiments were performed as previously described [
22]. Briefly, samples (500 μg of total protein) were incubated with 2 μg primary antibody overnight at 4°C, after which 20 μl of protein A/G-Plus-Agarose (Santa Cruz Biotechnology, CA, USA) was added to the mixture and incubated for 2 h at 4°C. The immunoprecipitated protein complexes were washed one time with lysis buffer and twice with ice-cold PBS. After discarding the supernatant, the antibody-protein complexes were resuspended in 20 μl Laemmli Sample Buffer (Bio-Rad Laboratories, CA, USA) and boiled for 5 min. The entire sample was separated by 10% SDS-PAGE and assayed by protein immunoblotting.
For western blotting, vehicle control and apigenin-treated cells were lysed in Laemmli Sample Buffer. After electrophoresis, the proteins were electrotransfered to PVDF membranes, blotting with antibodies indicated and visualized by SuperSignal West Dura Extended Duration Substrate (PIERCE, Rockford, IL, USA).
Statistical analysis
ANOVA was employed for comparisons across multiple groups. The mean of the control was compared with the mean of each individual treatment group by Dunnett's test. All statistical analyses were performed with the Prism 5 software (GraphPad Software Inc., San Diego, CA). Significance was set at p < 0.05.
Discussion
In this study we have shown that a natural dietary flavonoid, apigenin, inhibited the proliferation of MM cell lines and primary MM cells, arrested cell cycle progression, and induced programmed cell death. We demonstrated that apigenin inhibited CK2 activity, thereby leading to inactivation of multiple kinases, including the constitutive and inducible STAT3, AKT, ERK, IκB and their upstream kinase partners PDK, MEK and IKK. Apigenin also downregulated antiapoptotic Bcl-2 family proteins and IAP proteins. We have also shown that the inhibition of CK2-mediated Cdc37 phosphorylation disrupted the Hsp90/Cdc37 chaperone function and led to the degradation of multiple Hsp90/Cdc37 client proteins via the proteasome pathway, which may be the primary mechanism mediating the anticancer activities of apigenin.
Although it is known that apigenin has a selective inhibitory effect on CK2, it has not known if apigenin kills cancer cells through its capacity to interfere with Cdc37 phosphorylation and to disrupt Hsp90 chaperone function. As had been previously reported [
6], we observed that primary MM cells and all MM cell lines express constitutively activated CK2. We found that treatment with apigenin downregulated kinase activity in both MM cell lines and the primary MM cells, confirming the suppression of CK2 (Figure
1, Figure
6). In MM cells, the ability of apigenin to inhibit cell proliferation and to induce cell death correlated with its ability to inhibit CK2 activity. It was previously reported that highly CK2α-positive leukemia cells are more sensitive to apigenin-induced cell death than are CK2α leukemia cells with relatively low levels of CK2α [
21]. However, in this study, we observed that the sensitivity of MM cells to apigenin-induced cell death depended on whether apigenin effectively inhibited CK2 kinase activity, decreased CK2α protein levels, decreased the phosphorylation of Cdc37 and induced the degradation of Hsp90/Cdc37 client kinases. Consistent with these observations, one of the primary MM cell samples in our analysis (No. 9) exhibited high CK2α expression but had low sensitivity to apigenin, whereas the CK2α low U266 cells were more sensitive to apigenin than CK2α high RPMI 8226 cells. We are currently investigating possible explanations for the failure of apigenin to suppress CK2 activity in particular MM cells.
Importantly, apigenin did not inhibit CK2 activity or exhibit any cytotoxic effects in PBMCs (Figure
6). Apigenin-mediated suppression of CK2 activity was accompanied by reduced phosphorylation of Cdc37 in MM cells, leading to the disassociation of Hsp90/Cdc37/client protein complexes and inducing the degradation of client kinase proteins including RIP1, Raf-1, Src, Cdk4, and AKT via the ubiquitin-proteasome pathway (Figure
4,
5,
6). Since some kinases, such as RIP1, Raf-1 and Src, locate at the upstream of various signal pathways, the degradation of these kinase proteins could lead to the abrogation of their downstream pathways. These findings help to explain how apigenin can inhibit many signaling pathways. In addition to apigenin, resveratrol and epigallocatechin-3-gallate (EGCG) have been reported to induce apoptosis by significantly downregulating CK2 activity in both ALVA-41 and PC-3 prostate cancer cells [
27]. Bioactive polyphenolic and flavonoid compounds have demonstrated potential in cancer therapy and cancer chemoprevention, and further studies are needed to determine if CK2 is the common target of these compounds. The possibility that Cdc37 is a secondary target also requires further assessment.
Among the kinases affected by apigenin treatment, receptor interacting protein 1 (RIP1) is of special interest. It has not been determined if RIP1 is a Cdc37 client kinase, but it has been shown that the stability of RIP1 is dependent on Hsp90 chaperone function [
28]. Recent studies have demonstrated that RIP1 kinase is a key protein in the cellular decision of cells to live or die upon exposure to different stress signals [
29]. Depending on the cellular context and stimulation, RIP1 kinase may participate in three different signal complexes, which have various functions with respect to mediating the activation of NF-κB, apoptosis, or necroptosis [
30,
31]. Recent studies have reported that apigenin functions as either a pro-apoptotic or anti-apoptotic mediator via suppression of NF-κB activation in malignant cells, such as in pancreatic cancer cells [
32] and in various models of inflammation including T cell resistance to activation-induced cell death [
33], lipopolysaccharide-stimulated monocytes and macrophages [
34], and pancreatic beta-cells [
35]. Depletion of the RIP1 protein may be an important mechanism by which apigenin inhibits NF-κB activation to mediate various functions.
The resistance of MM cells to apoptosis involves high expression of members of the Bcl-2 family. These antiapoptotic proteins protect against permeabilization of the mitochondrial outer membrane. The combined total level of Bcl-2, Bcl-xL, and Mcl-1 in the outer membrane determines the resistance of cells to apoptosis [
36,
37]. In this work, we have shown that apigenin can downregulate multiple antiapoptotic proteins, including Mcl-1, XIAP, Survivin, Bcl-2 and Bcl-xl (Figure
2). Compared with other antiapoptotic proteins, Mcl-1 plays a more important role in the aberrant survival of MM cells [
37,
38]. As an antiapoptotic protein, Mcl-1 functions either by sequestering Bak on the outer mitochondrial membrane or by heterodimerizing with activated BH3-only proteins including tBid, PUMA, and Bim [
39]. Normally, Mcl-1 is constitutively expressed in many MM cells [
40,
41]. Various extra-cellular stimuli, including interleukins, growth factors, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) and IFN, can upregulate Mcl-1 expression via activation through different signaling pathways [
42,
43]. Previous studies have shown that down-regulation of Mcl-1 by antisense oligonucleotides is sufficient to induce apoptosis in MM cells and to enhance cancer cell sensitivity to TRAIL, suggesting that Mcl-1 might be a potential therapeutic target for the treatment of several human malignancies, including MM [
37,
44]. In MM, tumor cells accumulate within the bone marrow by binding to the extracellular matrix proteins and bone marrow stromal cells (BMSCs). The interaction between MM cells and BMSCs induces secretions of various interleukins and growth factors by both cells to promote MM development. Among these interleukins is IL-6, which then triggers VEGF secretion [
45]. Although IL-6 and VEGF activate multiple signaling pathways, including Jak-STAT3, ERK and PI3K/AKT, the upregulation of Mcl-1 expression is their main mechanism of mediating survival and proliferation in MM cells [
46]. Ideally, the IL-6/VEGF loop ideally supports MM cell growth within the BM microenvironment. A previous study has shown that apigenin can inhibit the expression of VEGF [
47]. In the current study, we have demonstrated that apigenin not only suppresses constitutively activated STAT3, ERK, AKT and NF-κB, but it also blocks exogenous IL-6-induced activation of STAT3, and inhibits IGF-1-induced activation of AKT and ERK. These survival signals are important for initiating transcription of Mcl-1 and other antiapoptotic proteins and for maintaining their stability [
42]. The inhibitory effect of apigenin may be indirect, as many upstream kinases, such as MEK and IKK, were inactivated as well. The ability of apigenin to suppress constitutive and inducible signaling pathways and to downregulate Mcl-1 also contributes to its cytotoxicity in MM cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
MZ carried out the kinase activity assay, Immunoprecipitation and western blotting, participated in analysis and interpretation of data, and helped to draft the manuscript. JM participated in the design of the study and helped to draft the manuscript. HYZ collected the clinical samples and purified the primary MM cells. XHZ participated in the immunofluorescence analysis. ZYD carried out the cytotoxicity assay. YJX carried out the cell cycle and apoptosis assays. XDY designed the study, participated in analysis and interpretation of data, and drafted the manuscript. All of the authors read and approved the final version of this manuscript.