genistein down-regulates the constitutive activation of nuclear factor-κb in human multiple myeloma...

Copyright © 2008 John Wiley & Sons, Ltd. Phytother. Res. 23, 868–873 (2009)DOI: 10.1002/ptr

868 H. HE ET AL.

Copyright © 2008 John Wiley & Sons, Ltd.

PHYTOTHERAPY RESEARCHPhytother. Res. 23, 868–873 (2009)Published online 23 December 2008 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/ptr.2715

Genistein Down-regulates The ConstitutiveActivation of Nuclear Factor-κκκκκB in HumanMultiple Myeloma Cells, Leading toSuppression of Proliferation and Inductionof Apoptosis

Hui He1,2, Laurie Chen3, Ming Zhai1* and John Z. S. Chen4

1Department of Hematology, The First Affiliated Hospital of China Medical University, Shenyang 110001, China2Department of Hematology, Benxi Central Hospital of China Medical University, Benxi 117000, China3Arizona Oncology Associates, P.C., Tucson, AZ 85718, USA4Associates in Dermatology, P.C., Tucson, AZ 85718, USA

Because of the central role of transcription factor nuclear factor-κκκκκB (NF-κκκκκB) in cell survival and proliferationin human multiple myeloma, the possibility of using it as a target for myeloma treatment was explored usinggenistein, an agent known to have very little or no toxicity in humans. It was found that NF-κκκκκB was constitu-tively active in two human myeloma cell lines examined and that genistein, a chemopreventive agent, down-regulated NF-κκκκκB in two cell lines as indicated by the electrophoretic mobility gel shift assay and prevented thenuclear retention of p65 as shown by immunocytochemistry. Two myeloma cell lines showed constitutivelyactive Akt phosphorylation. Genistein suppressed the constitutive Akt phosphorylation. Genistein also down-regulated the expression of NF-κκκκκB-regulated gene products, including bcl-2, bcl-xl, cyclin D1 and ICAM-1.This led to the suppression of proliferation and induction of apoptosis. Overall, the results indicate thatgenistein down-regulates NF-κκκκκB and phospho-Akt in human myeloma cells, leading to the suppression ofproliferation and induction of apoptosis, thus providing the molecular basis for the treatment of myelomapatients with this pharmacologically safe agent. Copyright © 2008 John Wiley & Sons, Ltd.

Keywords: genistein; NF-κB; Akt; gene expression; myeloma cells.

Received 16 June 2008Revised 3 August 2008

Accepted 9 September 2008

* Correspondence to: Professor Ming Zhai Department of Hematology,The First Affiliated Hospital of China Medical University, 155 NorthNanjing Street, Heping District, Shenyang 110001, P. R. China.E-mail: [email protected]; [email protected]

INTRODUCTION

Multiple myeloma is a B-cell malignancy characterizedby the latent accumulation in bone marrow of secre-tory plasma cells with a low proliferative index andan extended life span. Myeloma accounts for 1% of allcancers and more than 10% of all hematologic cancers(Mulligan and Badros, 2007). Various agents usedfor the treatment of myeloma include combinationsof vincristine, Bis-2-chloroethylni-trosourea, melphalan,cyclophosphamide, doxorubicin and prednisone ordexamethasone. Despite these treatments, this malig-nancy remains incurable, with a complete remissionrate of 5% and a median survival of 3 years (Rajkumaret al., 2002).

One of the potential mechanisms by which myelomacells could develop resistance to apoptosis is throughthe activation of nuclear transcription factor NF-κB.Under normal conditions, NF-κB is present in the cyto-plasm as an inactive heterotrimer consisting of p50, p65and IκB subunits. Upon activation, IκB undergoesphosphorylation, thus exposing nuclear localization

signals on the p50-p65 heterodimer, leading to nucleartranslocation and binding to a specific consensus sequencein the DNA (5-GGGACTTTC-3). The binding activatesgene expression, which in turn results in gene transcrip-tion. The phosphorylation of IκB occurs through theactivation of IκB kinase (IKK). The phosphorylationof Akt activity is critical for the IKK activity. Someof the NF-κB-regulated genes include cyclin D1, bcl-2,bcl-xL, cyclooxygenase-2 (COX-2), interleukin-6 (IL-6) and the adhesion molecules including intercellularadhesion molecule-1 (ICAM-1), vascular cell adhesionmolecule-1 (VCAM-1) and endothelial leukocyteadhesion molecule-1 (ELAM-1) (Sanda et al., 2005).Recently, it was reported that NF-κB is constitutivelyactive in myeloma cells (Tatetsu et al., 2005), leadingto bcl-2 expression, which rescues these cells fromglucocorticoid-induced apoptosis. As myeloma cellsexpress various adhesion molecules, bcl-xl, and bcl-2,4-6,8,19 which are all regulated by NF-κB and becausetheir suppression can lead to apoptosis (Hideshimaet al., 2002), it is proposed that NF-κB is an importanttarget for myeloma treatment.

To identify a pharmacologically safe and effectiveagent that can block constitutive NF-κB in myeloma,genistein was selected (Fig. 1), a predominant isoflavonefound in soybeans, has been shown to inhibit the growthof various cancer cells in vitro and in vivo withouttoxicity to normal cells (Garg et al., 2005).

GENISTEIN ON APOPTOSIS 869


of culture, the dead cells were removed using cellcentrifugation over Ficoll-Hypaque gradient, and theviable cells were recultured in fresh culture medium.

Preparation of nuclear extracts and electrophoreticmobility shift assay (EMSA) for NF-κκκκκB. Briefly, 2 × 106

cells were washed with cold phosphate-buffered saline(PBS) and suspended in 0.4 mL hypotonic lysis buffercontaining protease inhibitors for 30 min. The cells werethen lysed with 12.5 μL 10% Nonidet P-40. The homo-genate was centrifuged and the supernatant containingthe cytoplasmic extracts was stored frozen at −80 °C.The nuclear pellet was resuspended in 25 μL ice-coldnuclear extraction buffer. After 30 min of intermittentmixing, the extract was centrifuged, and the supernatantcontaining nuclear extracts was secured. The proteincontent was measured by the Bradford method. If thenuclear extracts were not used immediately, they werestored at −80 °C. 8 μg nuclear extracts prepared fromgenistein-treated or untreated cells were incubated with32P end-labeled double-stranded NF-κB oligonucleotide(5-AGT TGA GGG GAC TTT CCC AGG C-3; 3-TCA ACT CCC CTG AAA GGG TCC G-5; underlin-ing indicates NF-κB binding site) for 15 min at 37 °C,and the DNA–protein complex was resolved in a 4%native polyacrylamide gel. The radioactive bands fromthe dried gels were visualized and quantitated by thePhosphorImager (Molecular Dynamics, Sunnyvale,CA) with the use of ImageQuant software (MolecularDynamics).

Immunocytochemistry for NF-κκκκκB p65 localization.Myeloma cells were plated on a glass slide by centrifu-gation, air dried for 1 h at room temperature, and fixedwith cold acetone. After a brief washing in PBS, theslides were blocked with 5% normal goat serum for 1 hand then incubated with rabbit polyclonal antihumanp65 antibody (dilution, 1:300). After overnight incuba-tion, the slides were washed and then incubated withgoat anti-rabbit IgG (IgG/Bio) for 15 min, S-A/HRPfor 15 min, DAB for 5 min and counterstained for nucleiwith hematoxylin for 1 min (Bharti et al., 2003). Pictureswere captured microscopically (BX51, Olympus Tokyo,Japan).

Semiquantitative reverse transcription-PCR. Total RNAwas lysed and extracted with Trizol, and convertedto cDNA by M-MLV reverse transcriptase accordingto the manufacturer’s instructions. The PCR mixturewas amplified in a final volume of 25 μL. PCR wasconducted in the automated DNA Thermal CyclerGeneAmp PCR System 9700. After amplification, theproducts were resolved by electrophoresis on 1.0%agarose gel, stained with ethidium bromide and photo-graphed under UV light. The density of the bandscorresponding to ICAM-1, bcl-2, bcl-xl, cyclin D1mRNA was measured with the Image J software (MediaCybernetics, L, P) and normalized against the densityof the bands for GAPDH.

Western blot analysis. First, 30–50 μg cytoplasmicprotein extracts, prepared as described (Chaturvediet al., 2000) were resolved on 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. After electrophoresis, the proteins wereelectrotransferred to a nitrocellulose membrane, blocked

Figure 1. Structural drawing of genistein.

The following reasons can be cited: (1) Genistein hasbeen shown by others to suppress NF-κB activationinduced by various inflammatory stimuli (Kang et al.,2003). (2) Genistein inhibits the activation of Akt acti-vity needed for IKK activation (Gong et al., 2003).(3) Genistein has been shown to down-regulate theexpression of various NF-κB-regulated genes (Sarkaret al., 2006). (4) Administration of genistein in humanshas been shown to be quite safe (Ullmann et al., 2005).

MATERIALS AND METHODS

Materials. Human myeloma cell lines RPMI 8226 wasobtained from the American Type Culture Collection(ATCC; Rockville, MD, USA). Cell lines XG-1 andhuman recombination IL-6 was kindly provided by DrXue-guang Zhang of Su Zhou University Medical School(Su zhou, China). XG-1 and RPMI8226 cells are plas-macytomas of B-cell origin. An immunocytochemistrykit was purchased from Zhongshan Goldenbridge Bio-technology Co., Ltd (Beijing China). The rabbit polyclonalantibodies to p65 were purchased from ZhongshanGoldenbridge Biotechnology Co., Ltd (Beijing, China).A total RNA extraction reagent and RT-PCR kit werepurchased from Bioer Biotechnology Co., Ltd (HangzhouChina). Genistein (5,7,4′-trihydroxyisoflavone, >99% pure),doxorubicin (≥98% pure) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide were purchasedfrom Sigma-Aldrich Chemicals (Sigma, St Louis, MO,USA). Genistein was prepared as a 4 mM solutionin dimethyl sulfoxide and then further diluted in cellculture medium. Doxorubicin was dissolved in PBSto make a 100 μg/mL stock solution. RPMI1640, fetalbovine serum (FBS) and antibiotic–antimycotic mixturewere obtained from Jingmei Biotechnology Co., Ltd(Beijing China). γ-P32-adenosine triphosphate (γ-P32-ATP)was purchased from Furui Biotechnology Co., Ltd(Beijing China). Antibodies against Akt, phospho-AktSer473, nucleoprotein extraction reagent and EMSAkit was purchased from Nanjing KeyGen biotech Co.,Ltd (Nanjing, China).

Cell culture. The human multiple myeloma cell lineswere cultured in RPMI1640 medium containing 1 ×antibiotic–antimycotic. The cell line XG-1 was culturedin 10% FBS with rIL-6 (1 ng/mL), whereas cell lineRPMI 8226 was grown in 20% FBS. Every 3 days,half-culture medium was replaced with fresh culturemedium supplemented for each culture group with theinitial cytokine combination. After 6, 9, 15 and 18 days


870 H. HE ET AL.

Figure 2. Effect of genistein on constitutive nuclear NF-κB inmultiple myeloma cells. Genistein inhibits constitutive nuclearNF-κB in multiple myeloma cells. Dose responses of NF-κBto genistein treatment in XG-1 and RPMI8226 cells. First, 2 ×106 cells/mL were treated with the indicated concentration ofgenistein for 24 h and tested for nuclear NF-κB by EMSAas described. Nuclear extracts were prepared from XG-1 andRPMI8226 cells (2 × 106/mL), incubated for 30 min with anoligonucleotide probe, and then assayed for NF-κB by EMSA.Control (A); genistein 15 μM (B); genistein 30 μM (C); genistein45 μM (D); genistein 30 μM + doxorubicin 0.001 μg/mL (E);doxorubicin 0.001 μg/mL (F). Mean ± SE (n = 4), * p < 0.05 vscontrol, one-way ANOVA followed by Dunnett’s multicomparisontest.

with 5% nonfat milk, and probed with antibodies againsteither protein kinase B (Akt) or phospho-Akt Ser473(1:500) for 1 h. Thereafter, the blot was washed,exposed to HRP-conjugated secondary antibodies for1 h, and finally detected by chemiluminescence (MediaCybernetics, L, P).

Methyl thiazolyl tetrazolium (MTT) assay. The anti-proliferative effects of genistein and doxorubicin againstdifferent myeloma cell lines were determined by theMTT dye uptake method. Briefly, the cells (5000 perwell) were incubated in triplicate in a 96-well plate in afinal volume of 0.2 mL for 24, 48 and 72 h at 37 °C.Thereafter, 0.025 mL MTT solution (5 mg/mL in PBS)was added to each well. After a 5 h incubation at 37 °C,0.1 mL dimethyl sulfoxide was added, incubation wascontinued for 10 min at 37 °C, and then the opticaldensity (OD) at 590 nm was measured by means of a96-well multiscanner autoreader (Biorad Model 550,Chantilly, VA). The following formula was used: Per-centage cell viability = (OD of the experiment samples/OD of the control) × 100.

Flow cytometric analysis. To determine the effect ofgenistein on the cell apoptosis, RPMI8226 cells weretreated for different times, washed and fixed with 70%ethanol. After incubation overnight at −20 °C, the cellswere washed with PBS prior to staining with propidiumiodide (PI) for 40 min. The cells were analysed byfluorescence-activated cell sorted (FACS) Vantage flowcytometer that uses the CellQuest acquisition and analy-sis programs (Becton Dickinson, San Jose, CA).

Statistical analyses. All data are expressed as the mean± SE. Statistical significance was analysed using the one-way ANOVA followed by Dunnett’s multicomparisontest. A value of p < 0.05 was taken as significant.

RESULTS

Genistein suppresses constitutive NF-κκκκκB expressedby multiple myeloma cells

First the NF-κB status in two different myeloma celllines was investigated by EMSA. The results shownin Fig. 2 indicate that the two cell lines expressed con-stitutively active NF-κB. Both cell lines were treatedwith different concentrations of genistein and 0.001 μg/mL doxorubicin for 24 h and then examined for NF-κBby EMSA. Densitometric analysis of the retardedradiolabeled probe showed a decrease in NF-κB DNA-binding activity. These results showed that 30 μM geni-stein was sufficient to suppress the constitutive NF-κBactivation in XG-1 and RPMI8226 compared with thecontrol and 30 μM genistein was sufficient to partlysuppress the NF-κB activation in doxorubicin-treatedXG-1 cells. Suppression of the NF-κB activation indoxoru-bicin-treated RPMI8226 cells was not signifi-cant, but 30 μM genistein improved the effect (Fig. 2).The specificity of NF-κB DNA binding to the DNAconsensus sequence was confirmed by super-shift andcompetition with excess unlabeled oligonucleotide.Noncompeting oligonucleotide did not replace the spe-cific binding.

To confirm that genistein suppresses nuclear retentionof p65, immunocytochemistry was used. Cells treatedwith 30 μM genistein for 24 h and untreated cells werecytospun on a glass slide, immunostained with antibodyp65 as described in ‘Materials and Methods’. The re-sults in Fig. 3 clearly demonstrate that genistein pre-vented the translocation of the p65 subunit of NF-κBto the nucleus in XG-1 cell lines. These cytologicfindings were consistent with the NF-κB inhibitionobserved by means of EMSA.

Genistein inhibits Akt phosphorylation

To investigate whether the inhibitory effect to NF-κBof genistein is mediated through the alteration ofphosphorylation of Akt, XG-1 cells were treated withgenistein for 24 h, and their protein extracts werechecked for phospho-Akt Ser473 and Akt expression.The results in Fig. 4 show that untreated XG-1 cellsconstitutively expressed phospho-Akt Ser473 and Akt.Upon genistein treatment, the phospho-Akt Ser473content decreased rapidly.

Genistein down-regulates the expression ofNF-κB–regulated gene

Because ICAM-1, bcl-2, bcl-xL and cyclin D1 haveall been shown to be regulated by NF-κB, the effect ofgenistein on the expression of these genes was exam-ined by RT-PCR. As depicted in Fig. 5, all four geneswere expressed in XG-1 cells. The treatment of cellswith genistein down-regulated the expression of ICAM-1,



Figure 3. Effect of genistein on p65. Genistein induces redistri-bution of p65. XG-1 cells were incubated alone or with 30 μM

genistein for 24 h and then analysed for the distribution of p65by immunocytochemistry. Buffy stain indicates the localizationof p65, and blue stain indicates nucleus. Control (A, a); genistein30 μM (B, b). Original magnification, ×400.

Figure 5. Effect of genistein on NF-κB-regulated gene, includingbcl-2, bcl-xL, cyclin D1, and ICAM-1. 2 × 106 XG-1 cells weretreated with genistein for 48 h, mean ± SE (n = 4), * p < 0.05,** p < 0.01 vs control, one-way ANOVA followed by Dunnett’smulticomparison test.

and 0.1 μg/mL of doxorubicin resulted in inhibitionof cell proliferation (Fig. 6). The inhibition of cell growthwas dose- and time-dependent, as observed previously(Gong et al., 2003). Genistein contributed toward enhanc-ing the inhibition of cell proliferation of doxorubicin,leading to greater antitumor activity in vitro.

Genistein induces apoptosis in myeloma cells

The study investigated whether suppression of NF-κBin myeloma cells also leads to apoptosis. The genistein-induced apoptosis was examined by the PI method.RPMI8226 cells were treated for 72 h with differentconcentrations of genistein and then stained with PI.The results in Fig. 7 show a dose-dependent increasein cells positive for PI, indicating the onset of apoptosisin genistein-treated cells.

The results demonstrate that two myeloma cell linesexpressed constitutively active NF-κB, which was sup-pressed by genistein through inhibition of Akt activity.This led to down-regulation of expression of gene prod-ucts regulated by NF-κB, thus suppressing prolifera-tion and inducing apoptosis in myeloma cells.

DISCUSSION

Research over the past decade has revealed that NF-κBis an inducible transcription factor for genes involvedin cancer cell survival, cell adhesion, differentiation andgrowth (Bhardwaj et al., 2007; Mitsiades et al., 2002). Theresults indicate that two cell lines (XG-1, RPMI8226)expressed constitutively active NF-κB. These results areconsistent with recent reports by Lee and colleagues(Lee et al., 2004). Because of the central role of NF-κBin myeloma cell survival and proliferation, this transcrip-tion factor was explored as a target for the treatmentof myeloma using genistein. The suppression of cellproliferation by genistein in myeloma cells is consistentwith previous reports that genistein-induced suppres-sion of NF-κB leads to inhibition of cellular prolifera-tion of lymphoma (Mohammad et al., 2003), breast cancer(Valachovicova et al., 2004), pancreatic cancer (Mohammadet al., 2005) and bladder cancer (Singh et al., 2006).

bcl-2, bcl-xL and cyclin D1 in a dose-dependent man-ner (Fig. 5).

Genistein suppresses the proliferation ofmyeloma cells

Because NF-κB has been implicated in cell survival andproliferation, the effect of genistein on proliferationof myeloma cell lines was examined. RPMI8226 andXG-1 cells were cultured in the presence of differentconcentrations of genistein and doxorubicin, and thenumber of viable cells examined by the MTT method.The treatment of RPMI8226 and XG-1 cells for 1–3 dayswith 0, 15, 30 and 45 μM of genistein and 0.001, 0.01

Figure 4. Effect of genistein on phospho-Akt and Akt. XG-1myeloma cells showed constitutively active Akt and phospho-Akt Ser473. Genistein suppressed Akt phosphorylation but notAkt. Mean ± SE (n = 4), * p < 0.05 vs control (0 μM), one-wayANOVA followed by Dunnett’s multicomparison test.


872 H. HE ET AL.

Figure 6. Effect of genistein and doxorubicin on myeloma cell viability. RPMI8226 and XG-1 cells (5000/0.1 mL) were incubated withdifferent concentrations of Genistein for 24, 48, 72 h, and the cell viability was determined by the MTT method, as described in‘Materials and methods’. RPMI8226 (A), XG-1 (B). Mean ± SE (n = 4), * p < 0.05, ** p < 0.01 vs control, one-way ANOVA followedby Dunnett’s multicomparison test.

Figure 7. Flow cytometric analysis of PI stained cells after treat-ment with different concentrations of genistein. RPMI8266 cellswere incubated with the indicated concentrations of genisteinfor 72 h; thereafter, the cells were stained with PI. 0 μM (A);15 μM (B); 30 μM (C); 45 μM (D); 60 μM (E). Mean ± SE (n = 4),* p < 0.05 vs control (0 μM), one-way ANOVA followed byDunnett’s multicomparison test.

These studies have shown that genistein can regulatethe expression of apoptosis-related genes by regulatingNF-κB in myeloma cells. However, the precise molecularmechanism is not clear. At present, several potentialmechanisms could explain the reason for the NF-κBdown-regulation by genistein. First, it has been reportedthat in the NF-κB signaling pathway, IκB is phosphory-lated by IκB kinase (IKK), while IKK is phosphorylatedand activated by the upstream molecule mitogen acti-vated kinase 1 (MEKK1). Shark found that genisteintreatment inhibited MEKK1 kinase activity when testedby the kinase assay. This result demonstrates that genisteininhibits MEKK1 activity, which may be responsible forthe decreased phosphorylation of IκB, thereby result-ing in the inactivation of NF-κB (Sarkar and Li, 2002).Second, the Notch-1 signaling pathway has been shown

to activate NF-κB. Constitutive levels of Notch activityare essential to maintain NF-κB activity in variouscell types. Activation of NF-κB appears to be mediatedby Notch-1, and the inhibition of NF-κB activity bygenistein treatment may be mediated partly by theinhibition of the Notch-1 pathway (Wang et al., 2006).Lastly, Yiwei Li showed that genistein inhibits theactivities of NF-κB via the Akt pathway (Li and Sarkar,2002), this pathway was further confirmed by subse-quent studies (El-Rayes et al., 2006; Gong et al., 2003).It has been reported that activated NF-κB is mediatedthrough the activation of Akt (Nair et al., 2006). Theactivated Akt has also been found to activate IKK,which regulates the NF-κB transcription factor. Basedon these observations, it would be logical to assumethat the reduction in activity of NF-κB is partly due tothe reduction in activity of Akt. It was further exam-ined whether genistein inhibits the phosphorylation ofAkt protein in myeloma cells. Genistein pretreatmentreduced the amount of phosphorylated Akt. Theseresults were confirmed by using western blot analysis(Fig. 4). These data demonstrate that genistein inhibitsthe phosphorylation of Akt, preventing the translocationof NF-κB to the nucleus and down-regulating theexpression of NF-κB-regulated gene products, includ-ing bcl-2, bcl-xl, cyclin D1 and ICAM-1. This processultimately inhibits cell growth.

In conclusion, it was demonstrated that the inactiva-tion of NF-κB by genistein in myeloma cells was partlymediated via Akt pathway, which could be useful forthe rational design of strategies for the treatment ofmyeloma.

Acknowledgements

We would like to thank Professor Xue-guang Zhang, Su Zhou Univer-sity Medical School, for the generous gift of XG-1 cells and informa-tion on culturing.



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