TheIsoflavone Genistein Inhibits Proliferationand Increases HistamineContent inHuman Leukemic MastCells

Michael G.Alexandrakis, M.D.,* Despina S.Kyriakou, M.D.,* Duraisamy Kempuraj, Ph.D.,# Man Huang, M.D., Ph.D.,# WilliamBoucher, B.Pharm.,# Dimitris Seretakis, M.D.,* and Theoharis C.Theoharides, Ph.D.,M.D.#

ABSTRACT Mastcells exposed to allergens or otherstimuli 2 can release Mastcells are involved in allergic inflammation and some rare manypotent and rapidly acting mediators of inflammation, disorderssuch as systemic mastocytosis and mast cell leukemia. includinghistamine, proteases, prostaglandins, leukotrienes, 1,3–5 Certainnaturally occurring flavonoids have been shown to inhibit andcytokines. Themediator most readily associated 6 mastcell activation and promotematuration of secretory granules. withthe mast cell is histamine. Histamineplays an impor- Here,we report that the isoflavone genistein inhibited the growth tantrole in the pathogenesis of various allergic disorders, ofhuman leukemic mast cells (HMC-1) by 68.8,51.6, and 30.2% includingvasodilatation and increased vasopermeability. In at 102 4 , 1025 , and 1026 M,respectively,at day 3 (p , 0.001). addition,mast cells and histamine are involved in thepatho- Genisteinat 10 24 Mincreasedthe histamine content per 2 3 105 physiologyof irritable bowel syndrome 7 andinterstitial cellsat day 3 from5.9 6 1.2 mg/mL to 11.1 6 1.3 mg/mL (n 5 6; cystitis,8 sterileinflammatory conditions associated with p , 0.0001).These results indicate that genistein can inhibit pain,and worsened by stress. 9 proliferationand induce maturation of HMC-1 cells. (Allergyand Flavonoidsare low molecular weight benzo- g-pyrone AsthmaProc 24:373– 377, 2003) derivativesubiquitous in plants with a widespectrum of biologicactivities, such as inflammatory and immune re- astcells are primary effector cells in immediate hy- sponses,including inhibition of mastcells. 10 Genisteinis a M persensitivityresponses and their numbers are in- naturalisoflavonoid phytoestrogen present in various plant creasedin a widespectrum of pathological conditions. 1 foods,especially soy. This isoflavone has a heterocyclic, diphenolicstructure similar to estrogen; 11 atmicromolar concentrations,genistein was foundto have an inhibitory effecton protein tyrosine kinases (PTK), presumablyat the Fromthe *Department of Hematology, University Hospital of 12 Heraklion,School of Medicine University of Crete, Greece, and levelof theadenosine triphosphate binding site. Although #Departmentof Pharmacology and Experimental Therapeutics, thebiologic effects of genistein have not been fully clari- fied,genistein could induce cell cycle arrest and apoptosis TuftsUniversity School of Medicine, Boston Massachusetts 13,14 Supportedby ThetaBiomedical Consulting and Development Co., inleukemic cells andalso affect the growth and cell Inc.,Brookline, Massachusetts cycleprogression of human leukemic MOLT-4 and HL-60 15 Addresscorrespondence and reprint requests to T.C. Theoharides, cells. Apossibleinterpretation of these observations is the Ph.D.,M.D., Department of Pharmacology and Experimental abilityof genistein to inhibitthe activities of topoisomerases Therapeutics,Tufts University School of Medicine, 136 Harrison andtyrosine kinases and to induce differentiation in the 16 Ave.,Boston, MA 02111 humanleukemia cells HL-60 and K-562. Inaddition, genisteinat 150 mMwas shownto be an inhibitor of

Allergy andAsthma Proc. 373 angiogenesis, 17 in vivo.10 Severalstudies have shown that Inhibition (%) genisteincan induce apoptosis in breast, stomach, lung, head,and neck, as well as prostate cancer cell lines by 1 2 number oflive cells in treatment group 5 3 100. modulationof cellcycle and apoptosis of regulatorymole- number oflive cells in nontreatment group cules.18–20 Genisteinhas been observed to inducethe differentiation Stainingof HMC-1 Cells ofhuman myelogenous leukemia K562 cells. 21 Other fla- ellswere grownon Lab-Tek culture chamber slides voneshave been reported to induce maturation of rat baso- (NUNK, Naperville,IL). After washingwith phos- philicleukemia cells. 22 Thiseffect, as well as the inhibition C phate-bufferedsaline, cells were fixedfor 5minuteswith ofmast cell activation has been associated with particular 10%formaldehyde in 10% acetic acid (pH 2.5).Cells were structuralrequirements, especially of the B ring. 23 stainedfor 5minuteswith 0.5% toluidine blue (pH 2.5), Inthis study, we investigatedthe effect of genistein on washedwith deionized water, and air dried. Cells were thegrowth of human leukemic mast cells (HMC-1) and photographedusing an Olympus light microscope (Olym- cellularconcentrations of histamine. These results suggest pusOptical Co., Tokyo, Japan). thatgenistein may be an effective agent in the prevention and/orfor thetreatment of human mast cell disorders. HistamineMeasurement MATERIALS AND METHODS istaminewas assayedby aluminescencespectrometer (Perkin-Elmer,Norwalk, CT) witha lowerdetection Genistein(4 * ,5,7-Trihydroxyisoflavone) H levelof 5ng/mL.HMC-1 cells were washedand harvested hiscompound was purchasedfrom Sigma(St. Louis, bycentrifugation at 400 3 g for 5minutesin 1.5-mL T MO)andwas dissolvedin propylene glycol, to make Eppendorftubes. Then, cells were resuspendendin water. a0.1M solution;then, it was filter-sterilizedand diluted Thissuspension was sonicatedin a Branson1200 ultra- 24 withculture medium to get the final concentrations 10 – sounddevice (Sonifier, Model W185D, Heat Systems — 26 10 M. Ultrasonics,Inc., Plainview, NY) for 10minutes, vortexed for 5minutes,and pelleted by 10-minute centrifugation at CellCulture 6000 3 g speedin an Eppendorf Microfuge. The total MC-124 was culturedin Iscove ’smodifiedDulbecco ’s amountof histamine in the cell pellet is expressed as mi- H medium(Gibco, Grand Island, NY) supplemented crograms/2 3 105 mastcells/ mL. with10% fetal calf serum and 1.2 mM ofmonothioglycerol 2 (Sigma)in 25-cm tissueculture flasks (Becton Dickinson Statistics Labware,Franklin Lakes, NJ). Thecells were incubatedat heresults are expressed as mean 6 SDofeither num- 37°Cina humidifiedincubator with 5% CO and 95% air. 2 berof cell or of the histamine present in the cell After 3daysof culture,cells were collectedand centrifuged T pellet.Comparisons were madeand probability was calcu- for 5minutesat 1000 rpm. Cells then were resuspendedin latedwith the nonparametric Mann-Whitney U test using culturemedium and plated in 12-well flat-bottom culture analysisof variance. Values of p , 0.05were considered plates(Costar, Cambridge, MA) ata densityof 2 3 105 statisticallysignificant. cell/mL.Genistein was addedin concentrations (10 24– 1025 M);cellscultured in mediumalone served as control. RESULTS Determinationof CellViability Effects ofGenistein on Cell Proliferation ellswith or without genistein were harvested,washed MC-1cells were culturedfor 3,4, and 5 daysin the C inphosphate-buffered saline and pelleted by centrif- H presenceor absenceof genisteinat 10 2 4, 1025, and ugationfor 5minutesat 400 3 g atroom temperature. Next, 1026 M.Aconcentration-dependentinhibition of growth cellswere resuspendedin Iscove ’smodifiedDulbecco ’s was observedon the 3rd, 4th, and 5th day of culture. Cell mediumsupplemented as aforementioned. Trypan blue viabilityon day 3 remainedhigh with lower than 10% (0.1%)exclusion tests were performedat theend of culture trypan blue–positivecells in cultures treated with any for cellviability. genisteinconcentration. On day 3 thenumber of cells (3103)was decreasedfrom 977.4 6 68.9in the untreated Determinationof Inhibitionof Cell Proliferation conditionto 304.6 6 53.9 at 1024 Mgenistein,to 472.6 6 heinhibition is expressed as the percentage of the 79.8 at 1025 M and 681.8 6 96.2 at 1026 M;thecorre- T numberof live cells counted from eachtreatment spondingdegree of inhibition on proliferation of HMC-1 groupsubtracted from thetotal number of thenontreatment cells(Tables I andII) was 68.8%( p , 0.001) at 1024 M groupand then divided by thetotal number of cellsfrom the genistein,51.6% ( p , 0.001) at 1025 M,and30.2% ( p , nontreatmentgroup, according to the formula: 0.002) at 1026 M,respectively.

374 September-October 2003, Vol.24, No. 5 TABLE I InhibitoryEffect ofGenistein on HMC-1 CellProliferation Genistein Day 3 Day 4 Day 5 Cells 3 103 Inhibition Cells 3 103 Inhibition Cells 3 103 Inhibition (% Total) (% Total) (% Total) Control 977.4 6 68.90 — 1012.8 6 97.3 — 723.3 6 37.7 — 1026 M 681.8 6 96.2# 30.2 763.0 6 92.4# 24.7 600.5 6 72.6§ 16.9 1025 M 472.6 6 79.7* 51.6 580.0 6 69.5* 42.7 470.0 6 63.9* 35.0 1024 M 304.6 6 53.9* 68.8 377.5 6 33.3* 62.6 382.0 6 39.7* 47.1 n 5 6 *p , 0.0001; #p , 0.002; §p , 0.005using Mann-Whitney U test.

cells/mL at10 25 M and to 10.1 6 4.5 mg/mL at 1024 M TABLE II (p , 0.02). Effect ofGenistein on Total Histamine Content of HMC-1 Cells DISCUSSION TotalHistamine urresults clearly show that genistein inhibits HMC-1 5 (mg/2 3 10 Mast Cells/mL) O cellproliferation and induces accumulation of hista- Day 3 Day 4 Day 5 minein a dose-dependentmanner. HMC-1 was derived 24 Genistein (n 5 6) (n 5 8) (n 5 8) from apatientwith mast cell leukemia. HMC-1cells have lesshistamine than human mast cells in general. 23 Previous Control 5.9 6 1.2 5.8 6 2.4 6.1 6 1.7 studieshave shown that HMC-1 expresses a phenotypeand 1025 M 9.5 6 2.3 9.7 6 1.8 8.3 6 2.0* severalmarkers, i.e.,tryptase,histamine, heparin, and chon- p , 0.002* p , 0.02* p , 0.05* 1024 M 11.1 6 1.3 10.1 6 4.5 10.1 6 4.5 droitinsulfate, similar to that of human mast cells, and p , 0.0001* p , 0.01* p , 0.02* releasesor expressesthe mRNA ofanumberof chemokines andcytokines that are known to besecreted by humanmast *UsingMann-Whitney U test. cells.25,26 Flavonoidsare molecules rich in plants, fruits, andseeds with potent antioxidant effects. 10 Flavonoidsare lowmolecular weight benzo- g-pyronederivatives with a Onday 4, cell viability was alsohigh at 91.4%. The widespectrum of biologicactivities including regulation of 3 numberof cells( 310 )atthe respective genistein concen- inflammatoryand immune responses, especially inhibition 24 25 26 trationsof 10 , 10 , and 10 Mwas decreasedfrom ofmastcell activation. 10,23Aninverse relationship has been 1012.8 6 97.3to 377.3 6 33.3, 580.0 6 69.5 (p , 0.001; reportedbetween the consumption of flavonoids and the n 5 6),and 763.0 6 92.4 (p , 0.002),respectively. The incidenceof establishedcoronary artery disease, 10,27 as well correspondingdegree of inhibition was 62.6,42.7, and theincidence of lung cancer. 10,28,29 24.7%.Finally, on day 5, the viability dropped to ;80%. Inthis study, we examinedthe effect of genistein, a 3 Thenumber of cells ( 310 )atthe respective genistein naturalisoflavone phytoestrogen present in various plant 24 25 concentrationsof 10 and 10 was 382.0 6 39.7 (p , foods,especially soy beans, with a heterocyclic,diphenolic 0.001),470.1 6 63.9 (p , 0.001),and 600.5 6 72.6 (p , structuresimilar to estrogen, 11 onHMC-1 growth and his- 0.005),respectively; the corresponding degree of inhibition taminecontent. Genistein is known to inhibit both PTKs 12 was 47.1,35.0, and 16.9% (Table I). andDNA topoisomeraseII. 30,31 At micromolarconcentra- tions,genistein was foundto have an inhibitory effect on Effects ofGenistein on Histamine Content PTKs, presumablyat the level of the adenosine triphosphate astcells were culturedwithout or with 10 24 and 1025 bindingsite. 12,13 Genisteincould induce cell cycle arrest M Mofgenistein. Genistein increased (Table II) his- andapoptosis in leukemiccells, 14 affectthe growth and cell taminecontent on day3 ofculturefrom 5.9 6 1.2 mg/2 3 cycleprogression of human leukemic MOLT-4 and HL-60 105 cells/mL inuntreated cells to 9.5 6 2.3 mg/2 3 105 cells15 andinduce differentiation in the human leukemia cells/mL at10 25 M and to 11.1 6 1.3 mg/2 3 105 cells/mL cellsHL-60 and K-562. 16 Otherstudies showed that some at 1024 M.Onday 4, histamine increased from 5.8 6 2.4 ofthenaturally occurring flavonoids, including genistein at mg/mL to 9.7 6 1.8 mg/2 3 105 cells/mL at10 25 M and to 10–100 mM,inhibitthe human promyelocytic leukemia cell 10.1 6 4.5 mg/2 3 105 cells/mL at10 24 M,respectively. line(HL-60). 15,32 Inaddition, genistein at 150 mM has been Onday 5, genistein increased the histamine content from shownto be an inhibitor of angiogenesis, which has a 6.1 6 1.7 mg/2 3 105 cells/mL to8.3 6 2.0 mg/2 3 105 significanteffect on tumorgrowth and metastasis. 17 Several

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