Antitumor effect of berberine against primaryeffusion lymphoma via inhibition of NF-jB pathwayHiroki Goto,1 Ryusho Kariya,1 Masako Shimamoto,1 Eriko Kudo,1 Manabu Taura,1 Harutaka Katano2 andSeiji Okada1,3

1Division of Hematopoiesis, Center for AIDS Research, Kumamoto University, Kumamoto; 2Department of Pathology, National Institute of InfectiousDiseases, Tokyo, Japan

(Received September 1, 2011 ⁄ Revised December 15, 2011 ⁄ Accepted January 4, 2012 ⁄ Accepted manuscript online February 9, 2012 ⁄ Article first published online February 21, 2012)

Primary effusion lymphoma (PEL) is an infrequent and distinctentity among the aggressive non-Hodgkin B cell lymphomas thatoccurs predominantly in patients with advanced AIDS. It showsserous lymphomatous effusion in body cavities, and is resistantto conventional chemotherapy with a poor prognosis. Thus, theoptimal treatment for PEL is not well defined and there is a needfor novel agents. PEL has been recognized as the tumor causedby Kaposi sarcoma-associated herpes virus/human herpes virus-8(KSHV/HHV-8), and nuclear factor (NF)-jB activation plays a criti-cal role in the survival and growth of PEL cells. In this study, weassessed the antitumor effect of berberine, a naturally occurringisoquinoline alkaloid, on this pathway. The methylthiotetrazoleassay showed that cell proliferation in the PEL cell lines wasinhibited by berberine. Berberine also induced caspase-depen-dent apoptosis and suppressed NF-jB activity by inhibiting IjBkinase (IKK) phosphorylation, IjB phosphorylation and IjB degra-dation, upstream targets of the NF-jB pathway, in PEL cells. In axenograft mouse model that showed ascites and diffuse organinvasion of PEL cells, treatment with berberine inhibited thegrowth and invasion of PEL cells significantly compared withuntreated mice. These results show that the suppression of NF-jBis a molecular target for treating PEL, and berberine is a potentialantitumor agent for PEL. (Cancer Sci 2012; 103: 775–781)

P rimary effusion lymphoma (PEL) is a rare and distinctsubtype of non-Hodgkin lymphoma that was originally

identified in patients with advanced AIDS.(1,2) PEL arisesexclusively in body cavities (pleura, peritoneum and pericar-dium) and is caused by Kaposi sarcoma-associated herpesvirus/human herpes virus-8 (KSHV/HHV-8).(2) It is generallyresistant to chemotherapy, with a median survival of only3 months;(3) therefore, there is a need to develop new thera-pies. PEL displays constitutive activity of many signaling path-ways in survival and growth, including the NF-jB, JAK/STATand PI3K/Akt pathways.(4–6) Inhibition of NF-jB inducesapoptosis in PEL cells and this pathway represents a moleculartarget for this disease.(4,7)

Berberine, an isoquinoline alkaloid from a plant used in tra-ditional Chinese and Ayurvedic medicine, is an active compo-nent of Berberis aquifolium (Oregon grape), Berberis aristata(turmeric tree), Berberis vulgaris (barberry), Coptis chinensis(coptis or golden thread) and Hydrastis canadensis (goldenseal). Berberine has a wide range of biological effects, includ-ing antidiarrheal, antihypertensive, antiarrhythmic, cholesterol-lowering, antimicrobial and anti-inflammatory activities.(8–13)

In addition, berberine possesses antitumor activities againstvarious tumor cells.(14–17) The suppression of NF-jB by ber-berine has been demonstrated in several tumor cell lines;(18–21)

however, the specific target of the NF-jB pathway is not fullyunderstood, and the antitumor ability in vivo of berberine is

limited.(21–23) Further studies in animal models are requiredto identify the potential effects and clinical application ofberberine.In this study, we investigated the effect of berberine on pro-

liferation and apoptosis in PEL cells and clarified the targetmolecules of berberine in the NF-jB pathway against PELcells in vitro. The suppression of upstream molecules of theNF-jB pathway led to the inhibition of NF-jB activity. Wealso assessed the in vivo effect of berberine, showing the ratio-nale for a clinical study. These findings provide insights intothe molecular target of PEL and the antitumor mechanism ofberberine against PEL cells.

Materials and Methods

Cell lines and reagents. Human PEL cell lines, BC-1,(24)

BCBL-1,(25) TY-1(26) and human non-PEL cell line, U937,(27)

were maintained in RPMI1640 supplemented with 10% heat-inactivated FCS, penicillin (100 U/mL) and streptomycin(100 lg/mL) in a humidified incubator at 37°C and 5% CO2.Berberine chloride was obtained from Sigma-Aldrich (St.Louis, MO, USA).

Tetrazolium dye MTT assay. The antiproliferative activities ofberberine against PEL cell lines were measured using theMTT method (Sigma-Aldrich). Briefly, 2 9 104 cells wereincubated in triplicate in a 96-well microculture plate in thepresence of different concentrations of berberine in a final vol-ume of 0.1 mL for 24 h at 37°C. Subsequently, MTT (0.5 mg/mL final concentration) was added to each well. After 3 h ofadditional incubation, 100 lL of a solution containing 10%SDS plus 0.01 N HCl was added to dissolve the crystals.Absorption values at 595 nm were determined with an auto-matic ELISA plate reader (Multiskan; Thermo Electron, Van-taa, Finland). Values are normalized to untreated (control)samples.

Annexin V assay. Apoptosis was quantified using AnnexinV-Alexa fluor 647 (AF647) (BioLegend, San Diego, CA,USA). Briefly, after treatment with berberine for 24 h, cellswere harvested, washed and then incubated with AnnexinV-AF647 for 60 min in the dark, before being analyzed on anLSR II cytometer (BD Bioscience, San Jose, CA, USA). Datawere analyzed using FlowJo software (Tree Star, San Jose,CA, USA).

Analysis of DNA fragmentation by agarose gel electro-phoresis. To detect apoptosis and DNA damage, DNA ladderassays were performed as previously described.(28) Briefly,BCBL-1 cells were cultured in the presence or absence ofberberine at 37°C for 48 h. After incubation, 1 9 106 cellswere lysed in 100 lL of 10 mM Tris–HCl buffer (pH 7.4)

3To whom correspondence should be addressed.E-mail: okadas@kumamoto-u.ac.jp

doi: 10.1111/j.1349-7006.2012.02212.x Cancer Sci | April 2012 | vol. 103 | no. 4 | 775–781© 2012 Japanese Cancer Association

containing 10 mM EDTA and 0.5% Triton X. After centrifuga-tion for 5 min at 20 000g, supernatant samples were treatedwith RNase A (Qiagen, Valencia, CA, USA) and Proteinase K(Wako Pure Chemical, Osaka, Japan). Subsequently, 20 lL of5 M NaCl and 120 lL isopropanol were added to the sampleand kept at �20°C for 6 h. Following centrifugation for15 min at 20 000g, the pellets were dissolved in 20 lL TEbuffer (10 mM Tris–HCl and 1 mM EDTA) as loadingsamples. To assess the DNA fragmentation pattern, sampleswere loaded onto 1.5% agarose gel and electrophoreticallyseparated.

Western blot analysis. For whole cell extraction, BCBL-1cells treated with 100 lM berberine for 0, 1, 3 and 6 h werecollected and washed in cold PBS before the addition of100 lL cold lysis buffer (25 mM HEPES, 10 mMNa4P2O7·10H2O, 100 mM NaF, 5 mM EDTA, 2 mM Na3VO4

and 1% Triton X-100). After rotation for 2 h at 4°C, wholecell extracts were obtained by centrifugation at 20 000g for15 min. For nuclear extraction, BCBL-1 cells treated with100 lM berberine for 0, 1, 3 and 6 h were collectedand washed in cold PBS before the addition of 400 lL coldbuffer A (10 mM HEPES-KOH pH 7.9, 1.5 mM MgCl2,10 mM KCl, 0.1% NP-40, 0.5 mM DTT, 0.5 mM PMSF,2 lg/mL pepstatin A, 2 lg/mL aprotinin and 2 lg/mL leupep-tin). After incubation on ice for 10 min, the samples were vor-texed for 10 sec. Nuclei were pelleted by centrifugation at2000g for 1 min and washed once with buffer A. Then, 50 lLof buffer C (50 mM HEPES-KOH pH 7.9, 10% glycerol, 420mM KCl, 5 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mMPMSF, 2 lg/mL pepstatin A, 2 lg/mL aprotinin and 2 lg/mLleupeptin) was added to the nuclei, before incubating on icefor 30 min. Nuclear extracts were obtained by centrifugationat 20 000g for 15 min. Whole or nuclear extracts (40 lg pro-tein) were separated by 10% SDS-PAGE and blotted onto aPVDF membrane (GE Healthcare, Tokyo, Japan). Detectionwas performed using the Enhanced ChemiluminescenceWestern Blotting Detection System (ECL; GE Healthcare Bio-Science, Buckinghamshire, UK).Primary antibodies were as follows: anti-cleaved caspase-3

(9661), anti-caspase 9 (9502), anti-phospho (Ser180/181)-IKKa/b (2681), anti-phospho (Ser32/36)-IjBa (9246), anti-p65(536) (Cell Signaling Technology, Danvers, MA, USA), anti-IKKa/b (H-470), anti-IjBa (C-21) and anti-c tublin (C-20)(Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Hsc70(SPA-815) (Stressgen Bioreagents, Ann Arbor, MI, USA).

Electrophonic mobility shift assay. An EMSA was performedusing a second generation DIG Gel Shit Kit (Roche Diagnos-tics, Mannheim, Germany). Briefly, double-stranded oligonu-cleotide probes containing the immunoglobulin kappa (Igj)light chain NF-jB site and the Oct-1 binding site were pur-chased from Promega (Madison, WI). The oligonucleotide was3′ end-labeled with a digoxigenin-11-ddUTP. The nuclearextract (10 lg protein) from BCBL-1 cells was incubated with1 lg poly[d(I-C)], 0.1 lg poly-L lysine and DIG-labeled oligo-nucleotide in binding buffer (20 mM HEPES pH 7.6, 1 mMEDTA, 10 mM (NH4)2SO4, 0.2% Tween20 and 30 mM KCl)for 15 min at 25°C. After incubation, 59 loading buffer(0.259 TBE and 60% glycerol) was added, and the sampleswere separated on 6% acrylamide gel in 0.59 TBE buffer.The oligonucleotide was electroblotted onto a positivelycharged nylon membrane (Roche Diagnostics) and immunode-tected using anti-digoxigenin-AP.

Xenograft mouse model. NOD Rag-2-deficient (Rag-2�/�)mice and NOD Jak3-deficient (Jak3�/�) mice were establishedby crossing Rag-2�/� mice or Jak3�/� mice with the NOD strainfor 10 generations, respectively. NOD Rag-2/Jak3 double-defi-cient (Rag-2�/�Jak3�/�) mice (NRJ mice) were established bycrossing NOD Rag-2�/� mice and NOD Jak3�/� mice, and were

housed and monitored in our animal research facility accordingto institutional guidelines. All experimental procedures and pro-tocols were approved by the Institutional Animal Care and UseCommittee at Kumamoto University. Twelve week-old NRJmale mice were inoculated i.p. with 7 9 106 BCBL-1 cells sus-pended in 200 lL PBS. The mice were then treated with i.p.injections of PBS or berberine (10 mg/kg, three times a week).Tumor burden was evaluated by measuring the body weight andvolume of ascites. For assessment of overall survival, Kaplan–Meier analysis was performed and P-values were determined bytwo-tailed analysis using the log-rank test.

Immunohistochemistry. To investigate the expression ofKSHV/HHV-8 ORF73 (LANA) protein, tissue samples werefixed with 10% neutral-buffered formalin, embedded in paraf-fin and cut into 4-lm sections. The sections were deparaffi-nized by sequential immersion in xylene and ethanol, andrehydrated in distilled water. They were then irradiated for15 min in a microwave oven for antigen retrieval. Endogenousperoxidase activity was blocked by immersing the sections inmethanol/0.6% H2O2 for 30 min at room temperature. Affin-ity-purified PA1-73N antibody,(29) diluted 1:3000 in PBS/5%BSA, was then applied, and the sections were incubated

Fig. 1. Chemical structure of berberine chloride.

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Fig. 2. Berberine inhibits the proliferation of primary effusion lym-phoma (PEL) cells. PEL cell lines (BC-1, BCBL-1 and TY-1) and non-PELcell line (U937) were incubated with 0, 3, 10, 30, 10 and 100 lM ber-berine for 24 h. A cell proliferation assay was carried out using MTTas described in the Materials and Methods. One representative resultfrom three independent experiments is shown.

776 doi: 10.1111/j.1349-7006.2012.02212.x© 2012 Japanese Cancer Association

overnight at 4°C. After washing in PBS twice, the second andthird reactions and the amplification procedure were performedusing kits according to the manufacturer’s instructions (Cata-lyzed Signal Amplification System; DAKO, Copenhagen,Denmark). The signal was visualized using 0.2 mg/mL diam-inobenzidine and 0.015% H2O2 in 0.05 M Tris–HCl, pH 7.6.

RT-PCR. Total RNA was extracted from the cells using Trizol(Invitrogen, Carlsbad, CA, USA). First-strand cDNA was syn-thesized from RNA using a PrimeScript RT-PCR kit (TakaraBio, Otsu, Japan) with random primers. The PCR productswere analyzed by 1.5% agarose gel electrophoresis and ethi-diumbromide staining. Primer sequences were as follows:ORFK13(v-FLIP): 5′-ATTGACATTAGGGCATCC-3′ and

5′-AAAGGAGGAGGGCAGGTT-3′,(30) ORF73(LANA): 5′-GAAGTGGATTACCCTGTTGTTAGC-3′ and 5′-TTGGATCT-CGTCTTCCATCC-3′,(30) mouse G3PDH: 5-TGAAGGTCGGT-GTGAACGGATTTGGC-3′ and 5′-CATGTAGGCCATGAGG-TCCACCAC-3′.(31)

Statistical analysis. Data are expressed as the mean ± SD.The statistical significance of the differences observed betweenexperimental groups was determined using Student’s t-test, andP < 0.05 was considered significant.

Results

Berberine inhibits proliferation and induces apoptosis inprimary effusion lymphoma cells. The chemical structure of ber-berine is shown in Figure 1 and has a molecular weight of371.8. We first determined whether treatment with berberineleads to the inhibition of PEL cell proliferation using MTTassay. Three PEL cell lines (BC-1, BCBL-1 and TY-1) werecultured with varying concentrations of berberine (0, 3, 10, 30

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Fig. 3. Berberine induces apoptosis in BCBL-1 cells. (a) Berberine-induced apoptosis was detected by Annexin V staining. A primary effusionlymphoma cell line, BCBL-1, was treated with berberine (30, 100 lM) for 24 h and was subsequently stained with Annexin V-Alexa fluor 647before being analyzed by flow cytometry. (b) Berberine caused DNA fragmentation of nuclei. BCBL-1 cells were treated with berberine (30,100 lM) for 48 h. (c) BCBL-1 cells were treated with 100 lM berberine for 0, 1, 3 and 6 h and total proteins were extracted for western blotting.

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Fig. 4. Inhibitory effects of berberine on the expression of NF-jBpathways. (a) A primary effusion lymphoma cell line, BCBL-1, wastreated with 100 lM berberine for 0, 1, 3 and 6 h and total proteinswere extracted for western blotting. (b) BCBL-1 cells were treatedwith 100 lM berberine for 0, 1, 3 and 6 h and nuclear proteins wereextracted for western blotting to detect NF-jB p65. (c) BCBL-1 cellswere treated with 100 lM berberine for 0, 12, 24 and 48 hand assessed for NF-jB DNA binding activity by EMSA using an NF-jB-specific oligonucleotide probe.

Goto et al. Cancer Sci | April 2012 | vol. 103 | no. 4 | 777© 2012 Japanese Cancer Association

and 100 lM) for 24 h, and proliferation was analyzed byMTT assay. Figure 2 shows that as the dose of berberineincreased from 3 to 100 lM, cell growth inhibition increasedin a dose-dependent fashion in all PEL cell lines. The IC50(50% inhibitory concentration) for BC-1, BCBL-1 and TY-1were 13.56, 29.17 and 32.82 lM. In contrast, the IC50 valueis >100 lM for non-PEL cell line, U937. In subsequent experi-ments, we determined whether the observed suppressive effectsof berberine in MTT assay were due to the induction of apop-tosis. We used Annexin V staining and DNA ladder formationto detect apoptosis. As shown in Figure 3(a), 30 and 100 lMberberine treatment for 24 h caused apoptosis in BCBL-1. Asshown in Figure 3(b), berberine treatment for 48 h causedDNA fragmentation, which is a characteristic of apoptosis celldeath. Next, we analyzed cleaved caspase 3 and cleaved cas-pase 9 to further confirm that berberine induced apoptosis inPEL cells. As shown in Figure 3(c), berberine treatment ofBCBL-1 induced time-dependent cleavage of caspase 3 andcaspase 9, hallmarks of cells undergoing apoptosis, in westernblotting.

Berberine suppresses NF-jB activity in PEL cells. It wasreported previously that NF-jB was required for the survivaland proliferation of PEL cells.(4,32,33) Because NF-jB is con-stitutively active in PEL cells,(34) we examined whether ber-berine inhibited NF-jB activation. BCBL-1 constitutively

expressed both total and phosphorylated IKK and IjB,upstream of NF-jB. When BCBL-1 was treated with 100 lMberberine for 0, 1, 3 and 6 h, berberine treatment reducedphosphorylated IKK and phosphorylated IjB, whereas totalIjB was increased (Fig. 4a), suggesting that inhibition of IKKphosphorylation leads to the accumulation of IjB by blockingthe phosphorylation and degradation of IjB protein. Next, wefractioned nuclear protein and analyzed the expression of p65by western blotting (Fig. 4b) to confirm p65 NF-jB suppres-sion by berberine. When PEL cell lines were treated with100 lM berberine for 6 h, the amount of nuclear p65 NF-jBprotein was reduced, indicating that berberine suppresses NF-jB activity. Thus, berberine mainly suppresses p65 NF-jBnuclear translocation by inhibiting the upstream of NF-jB. Toconfirm that berberine could inhibit NF-jB activity in PELcells, we performed EMSA with DIG-labeled double-strandedNF-jB oligonucleotides. Berberine also suppresses constitutiveNF-jB binding activity for 24 and 48 h (Fig. 4c). Theseresults demonstrate that berberine inhibits the constitutiveNF-jB activity of PEL cells.

In vivo effect of berberine in severe immunodeficient miceinoculated i.p. with BCBL-1. As the in vitro results suggest thatberberine could be an effective treatment against PEL, weassessed the in vivo effects of berberine in the immunodefi-cient mouse model. Severe immunodeficient, NRJ mice were

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Fig. 5. Treatment of NOD/Rag-2/Jak-3-deficient mice with berberine suppresses the development of PEL in vivo. (a) Photograph of berberine-treated and untreated ascites-bearing mice 4 weeks after inoculation with BCBL-1 i.p. (b) The body weight of mice 4 weeks after inoculationwith BCBL-1 cells in berberine-treated or untreated mice is shown as the mean ± SD of 6 mice. (c) The volume of ascites 4 weeks after inocula-tion with BCBL-1 cells in mice is shown as the mean ± SD of 6 mice. (d) Overall survival curve. Treatment with berberine prolongs survivalin vivo.

778 doi: 10.1111/j.1349-7006.2012.02212.x© 2012 Japanese Cancer Association

inoculated i.p. with 7 9 106 BCBL-1 cells. BCBL-1 producedprofuse ascites within 4 weeks of inoculation (Fig. 5a). Aspatients with PEL show lymphomatous effusion in body cavi-ties without a definable tumor mass,(2,35) these mice could beclinically equivalent to the PEL model. A dose of 10 mg/kgberberine or PBS alone was administrated via i.p. injection onday 3 after cell inoculation, and then three times a week. The50% lethal dose of berberine from i.p. injections has alreadybeen reported and is 57.6103 mg/kg.(36) Hence, the dosage ofberberine in vivo in our experiment was expected to be safe.Berberine-treated mice appeared to stay healthy, and the bodyweight did not change, whereas the body weight of untreatedmice significantly increased compared to that of berberine-trea-ted mice on day 28 (36.6 ± 2.7 g vs 30.7 ± 1.7 g, n = 6,P < 0.01; Fig. 5b). Moreover, the volume of ascites wassignificantly lower than in untreated mice on day 28(3.8 ± 0.6 mL vs 0.5 ± 0.8 mL, n = 6, P < 0.01; Fig. 5c). Asshown in Figure 5d, treatment with berberine significantly pro-longed survival of the mice (log-rank test, P < 0.01). Theseresults indicate that treatment with berberine delays or inhibitsthe growth of PEL cells and produces a survival benefit.Organ invasion by PEL cells on day 28 was evaluated by

hematoxylin–eosin staining and LANA immunostaining. Wefound that mice inoculated i.p. with BCBL-1 exhibited inva-sion into the liver, lung and spleen without macroscopic lym-phoma formation (Fig. 6a). The number of LANA-positivecells in berberine-treated mice was significantly reduced(0–1 cells per field magnification, 940) compared to untreatedmice (10–20 cells per field magnification, 940). The mRNAexpression levels of vFLIP and LANA were downregulated inthe spleen of berberine-treated mouse (Fig. 6b). These datademonstrated that berberine significantly inhibits the growth

and infiltration of PEL cells in vivo and could be a potentiallytherapeutic agent in patients with PEL.

Discussion

The clinical course of PEL is very aggressive and generallyrefractory to conventional chemotherapy; hence, novel thera-peutic strategies such us molecular targeting therapy areneeded. In the present study, we investigated the antitumoreffects of a naturally occurring isoquinoline alkaloid, berber-ine, on PEL cells both in vitro and in vivo, and showed thatberberine inhibited the NF-jB pathway with the suppression ofIKK phosphorylation, IjB phosphorylation and IjB degrada-tion. In KSHV/HHV-8-infected cells, vFLIP, a homologue ofcellular FLIP protein, has the ability to activate the NF-jBpathway by binding to the IKK complex.(33,37,38) Moreover,inhibition of NF-jB activity leads to the apoptosis of KSHV-infected PEL cells.(4,32) These results suggest that inhibition ofNF-jB is an effective target for the treatment of PEL. Activa-tion of NF-jB is involved in various kinds of cancer develop-ment and progression,(39,40) indicating that NF-jB is a goodmolecular target for cancer treatment.Berberine has long been used as a stomachic, an anti-

diarrheal agent, an antibiotic and an anti-inflammatory inAsian countries and has been shown to have few sideeffects.(8,9,13) Berberine has been reported to have variouspharmacological effects, including an arresting effect on cellcycle progress, inhibition of tumor cell proliferation and theinduction of apoptosis, and the mechanism of antitumor activ-ity differs among cell lines.(14–17,41,42) Several reports have dem-onstrated that berberine inhibits cancer cell migration bysuppressing COX-2, MMP-2, MMP-9 and urokinase-plasminogen

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Fig. 6. Invasion of primary effusion lymphoma cells into the organs of BCBL-1-inoculated mice on day 28. (a) Hematoxylin–eosin (HE) stainingand immunohistochemical staining using anti-LANA (PA1-73N antibody) was performed to detect BCBL-1 in the liver, lung and spleen. (b) Viralgene expression after treatment with berberine. Viral gene expression in spleen of berberine treated and untreated mouse was examined by RT-PCR. A representative result from two experiments is shown.

Goto et al. Cancer Sci | April 2012 | vol. 103 | no. 4 | 779© 2012 Japanese Cancer Association

activator,(19–21) downstream molecules of NF-jB. We showedhere that berberine induced the apoptosis of PEL by inhibitingIKK phosphorylation, the upstream target of the NF-jB path-way. Consequently, berberine abrogates the phosphorylation ofIjB (Fig. 4a), NF-jB nuclear translocation (Fig. 4b) and DNAbinding activity (Fig. 4c). Previously, we reported biscoclau-rine alkaloid cepharanthine-induced apoptosis of PEL cellsmainly via inhibiting p65 activation. In this study, berberineinhibits IKK activation, the upstream of the NF-jB pathway,and causes efficient apoptosis of PEL cells. Inhibiting IKKactivation is also considered to be a rational pharmacologictarget because vFLIP activates IKK in PEL cells.We also confirmed the therapeutic effect of berberine against

PEL in a xenograft mouse model. We used NRJ mice, whichdisplayed rapid and efficient engraftment of PEL cells, as asmall animal system. NRJ mice display not only complete defi-ciency in mature T/B lymphocytes and complement proteinbut also complete deficiency of NK cells, such as in NOD/Scid/common c-deficient (NOG) mice(43,44) and NOD/Scid/Jak3-deficient (NOJ) mice,(45) and provide the ideal microenvi-ronment for the propagation and increase of PEL cells.Although both scid and Rag mutations prevent the recombina-tion of genes required for functional B and T cell receptors,the Prkdc gene disrupted by the scid mutation is expressedbroadly and is involved in DNA repair, while expression ofrag genes is limited to hematopoietic cells and is involved onlyin the DNA recombination of T and B cell receptor genes.Thus, scid mice are more sensitive to radiation-induced ordrug-induced DNA damage than their Rag mutation counter-parts. In addition, the scid mutation is known to show a leakyphenomenon in which functional T and B cells are producedwith aging and ionized irradiation.(46) Taken together, NRJmice are expected to be more convenient recipients of humancell xeno-transplantation.The formation of malignant ascites without solid lymphoma

formation displayed in PEL xenograft NRJ mice reflects theclinical nature of human PEL and they could be a quite usefulin vivo model for studying PEL and HHV-8 pathogenesis. Ber-berine has been reported to suppress tumor invasion(21) andphorbol-ester-induced tumor promotion,(22) chemical-inducedcarcinogenesis(23) in vivo; however, the direct antitumor effect

and doses of berberine used in animal studies are unclear. Inthis study, we observed that administration of 10 mg/kg ber-berine three times a week showed significant reduction of asci-tes and tumor invasion with no apparent adverse effects onNRJ mice (Figs 5,6). Tumor invasion is related to some targetgenes of NF-jB, such as MMP and vascular endothelialgrowth factor.(39) We confirmed that suppressing NF-jB wasalso effective for invasion of PEL cells in vivo. Further studiesin animals suggest a new direction in the treatment of refrac-tory malignancies such as PEL.The effects of berberine on PEL cells other than the NF-jB

pathway are expected because berberine also affects NF-jB-independent tumors and exerts diverse pharmacologicaleffects.(9,13,15) Elucidating the pharmacological diversity ofberberine could lead to the development of novel effectivetherapies for a variety of malignancies as well as PEL.Berberine has been reported to have antiretroviral activityagainst HIV(47) and to reduce endoplasmic reticulum stress bypreventing an HIV protease inhibitor-induced inflammatoryresponse.(48) In AIDS patients who develop PEL, concomitanttreatment with berberine could contribute to not only antitumorand tumor-preventing activities, but also antiretroviral therapy.In conclusion, our data have shown the ability of berberine

to induce cell death by blocking the NF-jB pathway in PELcells.

Acknowledgments

We thank Ms I. Suzu for technical assistance and Ms K. Tokunaga forsecretarial assistance. This work was supported in part by a Health andLabour Sciences Research Grant from the Ministry of Health, Labour,and Welfare of Japan (H22-AIDS-I-002), and by the Global Center ofExcellence program “Global Education and Research Center Aiming atthe Control of AIDS,” and Grants-in-Aid for Science Research (Nos.21107522 and 21591209) from the Ministry of Education, Science,Sports, and Culture of Japan.

Disclosure Statement

The authors have no conflicts of interest to declare.

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Goto et al. Cancer Sci | April 2012 | vol. 103 | no. 4 | 781© 2012 Japanese Cancer Association