And proliferation through the downregulation of akt Diosgenin inhibits osteoclastogenesis, invasion, and proliferation through the downregulation of akt, ib kinase activation and nfbregulated gene expression S shishodia1 and b b aggarwal1 1Cytokine Research Laboratory, Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center,
beats by dre studio cheap Houston, TX, USAReceived 2 July 2005;Revised 12 september 2005;Accepted 12 september 2005;Published online 5 december 2005. Top of pageabstractdiosgenin, a steroidal saponin present in fenugreek(Trigonella foenum graecum)And other plants, has been shown to suppress inflammation, inhibit proliferation, and induce apoptosis in a variety of tumor cells, but through a mechanism that is poorly understood.In the present study, we report that diosgenin inhibits receptoractivated nuclear factorkappab ligandinduced osteoclastogenesis, suppresses tumor necrosis factor(Tnf)Induced invasion, and blocks the proliferation of tumor cells, all activities known to be regulated by nfb.Diosgenin suppressed tnfinduced nfb activation as determined by dna binding, activation of ib kinase, ib phosphorylation, ib degradation, p65 phosphorylation, and p65 nuclear translocation through inhibition of akt activation.Nfbdependent reporter gene expression was also abrogated by diosgenin.Tnfinduced expression of nfbregulated gene products involved in cell proliferation(Cyclin d1, cox2, cmyc), antiapoptosis(Iap1, bcl2, bclxl, bfl1/a1, traf1 and cflip), and invasion(Mmp9)Were also downregulated by the saponin.Diosgenin also potentiated the apoptosis induced by tnf and chemotherapeutic agents.Overall, our results suggest that diosgenin suppresses proliferation, inhibits invasion, and suppresses osteoclastogenesis through inhibition of nfbregulated gene expression and enhances apoptosis induced by cytokines and chemotherapeutic agents. Top of pageintroductionalthough it is generally believed that traditional medicine has great value, the molecular basis for their activities is
beats by dre sale cheap for the most part lacking(Djerassi, 1992).One of the traditional medicines whose mechanisms are largely unknown, diosgenin, is a steroidal saponin found in a variety of plants(Puri et al., 1976;Djerassi, 1992)Including fenugreek(Trigonella foenum graecum), roots of wild yam(Dioscorea villosa), Solanum incanum Lloydia(Segal et al., 1977), Costus speciosus(Dasgupta and pandey, 1970), and Solanum xanthocarpum(Heble et al., 1967).Extracts from these plants have been traditionally used for the treatment of diabetes(Madar et al., 1988;Sharma et al., 1990;Gupta et al., 2001), hypercholestrolemia(Valette et al., 1984;Sauvaire et al., 1991), and gastrointestinal ailments(Pandian et al., 2002;Raju et al., 2004).Research during the last decade has shown that diosgenin suppresses proliferation and induces apoptosis in cells of human colon carcinoma(Raju et al., 2004;Wang et al., 2004), osteosarcoma(Moalic et al., 2001;Corbiere et al., 2003), leukemia(Hibasami et al., 2003;Liu et al., 2005), human erythroleukemia(Leger et al., 2004), and human rheumatoid arthritis(Liagre et al., 2004).Antiproliferative effects of diosgenin are mediated through cell cycle arrest(Moalic et al., 2001;Liu et al., 2005), disruption of Ca2+ homeostasis(Leger et al., 2004;Liu et al., 2005), the activation of p53, release of apoptosisinducing factor, and modulation of caspase3 activity(Corbiere et al., 2004).It also inhibits azoxymethaneinduced aberrant colon crypt foci(Raju et al., 2004)And has been shown to inhibit intestinal inflammation(Yamada et al., 1997)And modulate the activity of lipoxygenase(Lox) (Nappez et al., 1995)And cyclooxygenase2(Cox2) (Moalic et al., 2001).More recently, diosgenin has been shown to bind to the chemokine receptor cxcr3, which mediates inflammatory responses(Ondeykal et al., 2005). Since the transcription factor Nfb is a major mediator of inflammation, cell survival, lox, and cox2 expression, we postulated that the antiinflammatory and antiapoptotic effects of diosgenin are modulated through the suppression of Nfb.Nfb is a family of five proteins, namely crel, rela(P65), Rel B, Nfb1(P50 and p105)And Nfb2(P52) (Aggarwal, 2004)That is kept in an inactive state in the cytoplasm by the members of the inhIbitory subunit of Nfb(Ib)Family, which includes ib, ib, ib, ib, bcl3, p100, and p105(Aggarwal, 2004).The most common combination is a heterotrimer consisting of p50, p65, and ib subunits.In response to an activation signal, the ib subunit is phosphorylated, ubiquitinated, and degraded through the proteosomal pathway, thus exposing the nuclear localization signals on the p50 heterodimer.The p65 is then phosphorylated, leading to nuclear translocation and binding to a specific sequence in dna, which in turn results in gene transcription.Nfb has been shown to regulate the expression of a number of genes whose products are involved in tumorigenesis(Aggarwal, 2004;Shishodia and aggarwal, 2004b).Cyclin d1).Nfb has also been shown to mediate receptoractivated nuclear factorkappab(Nfb)Ligand(Rankl)Induced osteoclastogenesis(Aggarwal, 2003). The aim of the current study was to investigate the effect of diosgenin on tumor necrosis factor(Tnf)Induced invasion, ranklinduced osteoclastogenesis, nfb activation, and nfbregulated gene products.We found that diosgenin inhibited tnfinduced invasion of tumor cells and osteoclastogenesis induced by rankl through the inhibition of nfb and nfbregulated gene products.Diosgenin also potentiated the apoptosis induced by tnf and the chemotherapeutic drugs doxorubicin and taxol. Top of pageresultsthe aim of this study was to investigate the effect of the plant steroid diosgenin on the transcription factor nfbsignaling pathway and on nfbregulated gene products.The structure of this compound is shown in figure 1a.For most studies, human chronic myelogenous leukemia(Kbm5)Cells were used.The concentration of diosgenin used and the duration of exposure had minimal effect on the viability of kbm5 cells as determined by trypan blue dye exclusion test(Data not shown).We used tnf to examine the effect of diosgenin on the nfb activation pathway because the pathway activated by this agent is well understood. FigurE 1. (A)StruCturE of DiosgEnin. (B)DiosgEnin supprEssEs tnfinDuCED ostEoClastogEnEsis.Raw 264.7 CElls(1 104)WErE platED ovErnight, prEtrEatED with 5 m DiosgEnin for 12 h, anD thEn trEatED with 5 nm rankl.AftEr 5 Days, CElls wErE stainED for trap anD EvaluatED for ostEoClastogEnEsis.Photographs wErE takEn aftEr 5 Days inCubation with rankl. (C)ThE numbEr of trappositivE multinuclEatED ostEoclasts(>3 nuclEi)PEr wEll wErE countED. (D)DiosgEnin prEtrEatmEnt supprEssEs tnfinducEd invasivE activity.H1299 cElls(2.5 104)WErE sEEdEd into thE uppEr wElls of a matrigEl invasion chambEr ovErnight in thE absEncE of sErum, prEtrEatEd with 10 M diosgEnin for 12 h, trEatEd with 1 nM tnf for 24 h in thE prEsEncE of 1% sErum, and thEn subjEctEd to invasion assay.ThE valuE for no diosgEnin and no tnf was sEt to 1. (E)Diosgenin suppresses tnfinduced invasive activity.H1299 cElls(2.5 104)Were seeded into the upper wells of a matrigel invasion chamber overnight in the absence of serum, treated with tnf(1 nM)Alone or in combination with diosgenin(10 M)For 24 h and then subjected to invasion assay.The value for tnf alone was set to 1. Full figure and legend(126K) Diosgenin suppresses ranklinduced osteoclastogenesisrankl, a member of the tnf superfamily, induces osteoclastogenesis through the activation of nfb(Abuamer and tondravi, 1997), and inhibits the apoptosis of osteoclasts, which subsequently leads to bone loss.We first determined whether diosgenin could suppress ranklinduced osteoclastogenesis.We found that rankl induced osteoclast differentiation in raw macrophage cell lines, as indicated by the expression of trap, and that diosgenin suppressed it(Figure 1b).Rankl induced 250 osteoclasts per well, and diosgenin suppressed induction to 80 osteoclasts per well(Figure 1c). Diosgenin suppresses tnfinduced invasion activitytnf can induce tumor metastasisrelated genes such as mmp9, cox2, and icam1(Aggarwal, 2003).Tumor metastasis depends on the activity of mmps, cox2, and adhesion molecules(Liotta et al., 1982).Thus, we investigated whether diosgenin can modulate the tumor cell invasion activity induced by tnf in vitro.H1299 cells were seeded in the top chamber of the matrigel invasion chamber in the absence of serum, incubated with diosgenin, and then treated with tnf in the presence of 1% serum with diosgenin.Diosgenin suppressed tnfinduced invasion activity by 50%(Figure 1d).We also examined the effect of diosgenin posttreatment on tnfinduced invasive activity.We observed that posttreatment with diosgenin inhibited tnf induced invasive activity by over 40%(Figure 1e). Diosgenin suppresses proliferAtion, And induCes Apoptosiswe next exAmined the effeCt of diosgenin on proliferAtion ofKBm5Cells By the mtt method(Figure 2A)And [3h]thymidine inCorporAtion(Figure 2B).Diosgenin At A ConCentrAtion of 25 m inhiBited growth And proliferAtion ofKBm5Cells.Next, we exAmined the effeCt of diosgenin on the Cell CyCle.Flow CytometriC AnAlysis of the dnA from diosgenintreAted Cells showed A signifiCAnt inCreAse in the perCentAge of Cells in the suBg1 phAse, from 9 to 51%, within 48 h of diosgenin(50 M)TreAtment(Figure 2C).An ACCumulAtion of Cells in the suBg1 phAse indiCAtes thAt diosgenin induCed the Apoptosis of Cells. (A)KBm5(5000 Cells/0.1 ml)Were inCuBated at 37 with indiCated ConCentrations of diosgenin for 72 h, and the viaBle Cells were assayed using mtt reagent.From tripliCate Cultures. (B)KBm5(5000 Cells/0.1 ml)Were inCubated at 37 with indiCated ConCentrations of diosgenin for 72 h, and the viable Cells were assayed using [3h]thymidine inCorporation as desCribed in materials and methods.From tripliCate Cultures. (C)Diosgenin accumulates the cells at the subg1 phase of the cell cycle. SerumstArvedKBm5Cells (2 106 cells/ml)Were incubated in the absence or in presence of 25 m diosgenin for indicated times.Thereafter, the cells were washed, fixed, stained with propidium iodide, and analysed for dna content by flow cytometry as described in materials and methods. Full figure and legend(84K) Diosgenin suppresses the ACtivAtion of nfB in A Dose AnD timeDepenDent mAnnerour results showeD thAt Diosgenin inhiBiteD tnfinDuCeD invAsion, rAnklinDuCeD osteoClAstogenesis AnDExpression of CyClin D1 AnD suppresseD proliferAtion in tumor Cells.SinCe nfB hAs Been impliCAteD in osteoClAstogenesis, invAsion, Cell survivAl, AnD proliferAtion(AggArwAl, 2004;ShishoDiA AnD AggArwAl, 2004B), thAt Are All regulAteD By NFB, we exAmineD the effeCt of Diosgenin on TNFinDuCeD NFB ACtivAtion.Diosgenin inhiBiteD tnfmeDiAteD nfB ACtivAtion in A DoseDepenDent mAnner(Figure 3A)With signifiCAnt inhiBition oCCurring At 50 m AnD Complete ABrogAtion of nfB ACtivity oCCurring At 100 m.Diosgenin By itself DiD not ACtivAte nfB.Then, we exAmineD the time kinetiCs of nfB inhiBition By Diosgenin.The minimum time requireD for inhiBition of nfB ACtivAtion with 50 m Diosgenin wAs 24 h(Figure 3B). (A)Diosgenin inhiBits tnfDepenDent nfB aCtivation in a DoseDepenDent manner.KBm5 Cells(2 106/ml)Were preinCuBateD with the inDiCateD ConCentrations of Diosgenin for 24 h at 37 anD then treateD with 0.1 nM TNF for 30 min.NuClear extraCts were prepareD anD testeD for nfB aCtivation, as DesCriBeD in materials anD methoDs. (B)Diosgenin inhibits tnfDepenDent nfb aCtivation in a timeDepenDent manner.KBm5 Cells(2 106/ml)Were preinCubateD with 50 m Diosgenin for the inDiCateD times at 37 anD then treateD with 0.1 nM TNF for 30 min At 37 NuCleAr extrACts were prepAreD AnD then testeD for NFB ACtivAtion. (C)TnfinDuceD nfb consists of p50 anD p65 subunits. NuCleAr extrACts fromKBm5 Cells(2 106/ml)TreateD or not treateD with 0.1 nM TNF for 30 min were inCuBAteD with the AntiBoDies inDiCAteD for 30 min At room temperAture, AnD the Complex wAs AnAlyseD By supershift AssAy. (D)Diosgenin inhibits tnfinduced nfbdependent reporter gene(Seap)Expression.A293 cells were transiently transfected with an nfbcontaining plasmid linked to the seap gene and then treated with the indicated concentrations of diosgenin.After 24 h in culture with 0.1 nM TNF, cell supernatants were collected and assayed for SEAP activity as described in Materials and methods.Results are expressed as fold activity over the activity of the vector control. Full figure and legend(104K) Suppressed nfb consists of both p50 and p65since nfb is a family of proteins, various combinations of rel/nfb protein can constitute an active nfb heterodimer that binds to a specific sequence in dna(Ghosh et al., 1998).To show that the retarded band visualized by emsa in tnftreated cells was indeed the p50 and p65 subunits of nfb, we incubated nuclear extracts from tnfactivated cells with antibodies to the p50(Nfb1)And the p65(Rela)Subunit of nfb.Both antibodies shifted the band to a higher molecular mass(Figure 3c), suggesting that the TNFactivated complex consisted of p50 and p65.Preimmune serum had no effect on dna binding.Addition of excess unlabeled nfb(Cold oligo;100fold)Caused complete disappearance of the band, whereas mutated oligo had no effect on the dna binding. Diosgenin represses tnfinduced nfbdependent reporter gene expressionalthough we showed by emsa that diosgenin blocked nfb activation, dna binding alone does not always correlate with nfbdependent gene transcription, suggesting there are additional regulatory steps(Nasuhara et al., 1999).To further determine whether diosgenin inhibited nfbdependent gene transcription, we transiently transfected a293 cells with the nfbregulated secretory alkaline phosphatase(Seap)Reporter construct and then stimulated them with tnf.We found that tnf produced an almost fivefold increase in seap activity over vector control(Figure 3d), which was inhibited by dominantnegative IB, indicating specificity.When the cells were pretreated with diosgenin, tnfinduced nfbdependent seap expression was inhibited in a dosedependent manner.These results demonstrate that diosgenin inhibits nfbdependent reporter gene expression induced by tnf. Diosgenin inhibits tnfdependent ib degradationsince ib degradation is required for translocation of nfb to the nucleus(Miyamoto et al., 1994), we determined whether diosgenins' inhibition of TNFinduced NFB activation was due to inhibition of IB degradation.We pretreated cells with diosgenin and then exposed them to tnf for different times.We then examined the cells for ib in the cytoplasm by western blot analysis.We found that tnf induced ib degradation in control cells as early as 15 min, but in diosgeninpretreated cells tnf had no effect on ib degradation(Figure 4a, upper panel). FigurE 4. (A)DiosgEnin inhiBits tnfinDuCED DEgraDation anD phosphorylation of iB.KBm5 CElls(2 106/ml)WErE inCuBatED with 50 m DiosgEnin for 24 h at 37 trEatED with 0.1 nM TNF for thE inDiCatED timEs at 37 anD thEn tEstED for IB(UppEr panEl)AnD phosphorylatED iB(MiDDlE panEl)In CytosoliC fraCtions By wEstErn Blot analysis.Equal protEin loaDing was EvaluatED By aCtin(LowEr panEl). (B)DiosgEnin inhibits tnfinDuCED ikk aCtivity.KBm5 CElls(2 106/ml)WErE trEatED with 50 m DiosgEnin for 24 h anD thEn trEatED with 0.1 nM TNF for thE inDiCatED timE intErvals.WholECEll ExtraCts wErE prEparED, anD 200 g of ExtraCt was immunoprECipitatED with antiboDiEs against ikk anD ikk.ThErEaftEr immunE ComplEx kinasE assay was pErformED as DEsCribED in matErials anD mEthoDs.To ExaminE thE EffECt of DiosgEnin on thE lEvEl of ExprEssion of ikk protEins, 30 g of wholECEll ExtraCt was run on 10% sDs ElECtrotransfErrED, anD immunoblottED with inDiCatED antiboDiEs as DEsCribED in matErials anD mEthoDs. (C)DiosgEnin inhibits tnfinDucED phosphorylation of p65.KBm5 CElls(2 106/ml)WErE incubatED with 50 m DiosgEnin for 24 h anD thEn trEatED with 0.1 nM TNF for thE inDicatED timEs.ThE cytoplasmic Extracts wErE analysED by wEstErn blotting using antiboDiEs against thE phosphorylatED form of p65. (D)DiosgEnin inhibits tnfinducEd nuclEar translocation of p65.KBm5 CElls(1 106/ml)WErE EithEr untrEatEd or prEtrEatEd with 50 m diosgEnin for 24 h at 37 and thEn trEatEd with 0.1 nM TNF for thE inDicatED timEs.NuclEar Extracts wErE prEparEd and analysEd by wEstErn blotting using antibodiEs against p65. (E)Diosgenin inhibits akt phosphorylation.KBm5 CElls(2 106/ml)WErE incubatED with 50 m DiosgEnin for 24 h anD thEn trEatED with 0.1 nM TNF for thE inDicatED timEs.The wholecell extracts were analysed by western blotting using antibodies against the phosphorylated akt.Equal protein loading was evaluated by akt.Data are from a representative experiment out of the three independent ones showing similar results. Full figure and legend(126K)Diosgenin inhibits tnfinduced ib kinase(Ikk)Activationthe phosphorylation of ib is catalysed by the ikk.Ikk consists of three subunits ikk, ikk and ikk(Also called nemo).Gene deletion studies have indicated that ikk is essential for nfb activation by most agents(Aggarwal, 2004).Since diosgenin inhibits the phosphorylation and degradation of ib, we tested the effect of diosgenin
drebtshdphones on tnfinduced ikk activation, which is required for tnfinduced phosphorylation of ib(Aggarwal, 2004).As shown in figure 4b(Upper panel), diosgenin completely suppressed TNFinduced activation of IKK.Tnf or diosgenin had no direct effect on the expression of ikk proteins(Bottom panels).
