Toxicology in Vitro 24 (2010) 898–904

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Toxicology in Vitro

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Evodiamine-induced human melanoma A375-S2 cell death was mediated byPI3K/Akt/caspase and Fas-L/NF-jB signaling pathways and augmented byubiquitin–proteasome inhibition

Che Wang a,*, Song Li b, Min-wei Wang c

a Department of Pharmacy, College of Chemistry and Chemical Engineering, Liaoning Normal University, Dalian 116029, Chinab Institute of Neuroinformatics, Dalian University of Technology, Dalian 116024, Chinac Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 30 July 2009Accepted 26 November 2009Available online 11 December 2009

Keywords:EvodiaminePI3K/AktNF-jBIjBUbiquitin–proteasome

0887-2333/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tiv.2009.11.019

* Corresponding author. Tel.: +86 411 84223126.E-mail address: wangche126@yahoo.com.cn (C. W

Evodiamine, a major alkaloidal component of Evodiae fructus exhibits anti-tumor activities. We have pre-viously reported that evodiamine has a marked inhibitory effect on IL-1 sensitive human melanomaA375-S2 cells proliferation, and this action might be through inactivation of PI3K signaling. However,the detailed molecular mechanisms of evodiamine-induced cell death remains poorly understood. Inpresent study, we further confirmed that Akt is the main effector molecule involved in this pathway.Evodiamine also led to IjBa phosphorylation and degradation that reflect translocation of NF-jB. Pre-treatment of A375-S2 cells with ubiquitin–proteasome inhibitor MG132 was shown to aggregate theevodiamine caused cell death at 24 h. In addition, MG132 reduced ERK phosphorylation, increased cas-pase-3 activation, Fas-L expression and Bcl-2 cleavage in evodiamine-treated A375-S2 cells. These resultssuggested the PI3K/Akt/caspase and Fas-L/NF-jB signaling pathways might account for the responses ofA375-S2 cell death induced by evodiamine, and these signals could be augmented by ubiquitin–protea-some pathway.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Evodiamine is one of the main constituents of Evodiae fructus(Shoji et al., 1986) and has been shown to exhibit anti-tumor, bron-chocontractive, anti-nociceptive, catecholamine secretory, vasore-laxant and nitric oxide inductive properties (Fei et al., 2003;Kobayashi et al., 2000; Matsuda et al., 1997; Yoshizumi et al.,1997; Chiou et al., 1992, 1996; Yang et al., 2008). It was reportedthat evodiamine reduced the phosphorylation of Akt, inhibitedthe augmentation of phosphorylated ERK by blocking the MAPKsignaling pathway (Wang et al., 2008). It was also reported thatevodiamine remarkably inhibited the invasion and migration of tu-mor cells in vitro (Ogasawara et al., 2001). Our previous study hasdemonstrated the molecular mechanism by which evodiamine in-creases the proapoptotic Bax and decreases anti-apoptotic Bcl-2expression, which amplified the activation of caspase cascade, trig-gering consequent responses and cell death (Fei et al., 2003; Zhanget al., 2003, 2004b). PI3K and PKC also exhibited essential regula-tory effects on functions of SIRT1, p53 and other signaling proteinsinvolved in evodiamine-induced A375-S2 cell death (Wang et al.,2005a).

ll rights reserved.

ang).

The ubiquitin–proteasome system is the principal pathway forselective protein degradation in eukaryotic cells. This system tar-gets both short-lived, normal proteins and misfolded, abnormalproteins (Kornitzer and Ciechanover, 2000). Since various signalingmolecules are degraded via the ubiquitin–proteasome pathway,proteasome inhibitors have been used as useful tools in cell biologyto modulate cell proliferation, gene regulation and apoptosis (Leeand Goldberg, 1998; Ciechanover, 1998).

Proteasome inhibitors prevent activation of NF-jB (nuclear fac-tor of the j-enhancer in B cells). NF-jB is sequestered in the cyto-plasm as an inactive complex bound by an inhibitor known as IjB(Baldwin, 1996). In response to various signaling events, phosphor-ylation of IjB targets itself for degradation by the proteasome(Zandi and Karin, 1999), liberating the transcription factor NF-jBto translocate into the nucleus and activate its target genes. NF-jB has mainly been described to induce expression of anti-apopto-tic genes and protects from apoptosis (Wang et al., 1998; Bargouet al., 1997; Cao et al., 2003). In contrast, apoptosis induced byultraviolet-B (UVB) radiation was shown to be significantly en-hanced upon IL-1-mediated NF-jB activation (Kothny-Wilkeset al., 1999; Pöppelmann et al., 2005). Previous research also re-ported that PI3K/Akt and NF-jB signaling pathway were involvedin apoptosis induced by evodiamine or other natural products(Shih et al., 2007; Kim et al., 2007). These findings suggest that

C. Wang et al. / Toxicology in Vitro 24 (2010) 898–904 899

NF-jB in regulating cell growth and differentiation is controversialfor cell growth and apoptosis, depending on cell type and condition(Raffoul et al., 2006; Woods et al., 2002).

PI3K family was described to play important roles in humanmelanoma A375-S2 cell death caused by natural compounds(Zhang et al., 2004a; Wang et al., 2005a). PI3K works as an up-stream activator of PKB/Akt and modulates its activity. In many sit-uations, some anti-apoptotic effect is mediated by the PI3K-induced activation of PKB/Akt (Songyang et al., 1997; Dudeket al., 1997) and the translocation of NF-jB is mediated by PI3K/Akt or TNF/Fas-L signaling pathway (Ma et al., 2004; Kim et al.,2005, 2007; Shih et al., 2007; Imamura et al., 2004). Extracellularsignal-regulated kinase (ERK), a member of mitogen-activated pro-tein kinase (MAPK) family, is also activated by PI3K survival path-way, resulting in protection against cell death (Li et al., 2001). Inour previous study, the inhibition of ERK that was dependent oninactivation of PI3K might contribute to evodiamine-induced celldeath (Wang et al., 2005a). In addition, another major signal trans-duction molecule Fas ligand (Fas-L) that was modulated by up-stream PI3K/Akt was known to play a pivotal role in cancer cellproliferation or apoptosis (Watanabe et al., 2003).

Bcl-2 family proteins that trigger mitochondrial membranedepolarization and caspase cascade activation were involved inapoptosis (Zhang et al., 2003; Renvoize et al., 1997), and the inhi-bition of PI3K could partially block Bcl-2 expression (Wang et al.,2005a). The biochemical relationship between Bcl-2 and caspaseshas been controversial. Bcl-2 functions upstream of caspase activa-tion and prevents apoptosis by suppressing caspase activity (Ren-voize et al., 1997). On the other hand, some other investigatorsreported that once apoptosis was initiated, Bcl-2 was cleaved bycaspase-3 to attenuate its anti-apoptotic effects (Kirsch et al.,1999). And our previous results showed that Bcl-2 cleavage was re-versed by caspase-3 inhibitor (Zhang et al., 2003).

Although the roles of ERK, Fas-L, caspases and Bcl-2 in apoptosishas been investigated extensively, much less is known about theubiquitin–proteasome system and its potential role in regulatingthese molecules. In the present study, we seek to explore the pos-sibility whether the ubiquitin–proteasome pathway is involved inregulating evodiamine-induced cell death.

2. Materials and methods

2.1. Reagents

Evodiamine was purchased from Beijing Institute of BiologicalProducts (Lot: 0802–9702, Beijing, China); and its purity was deter-mined to be about 98% by HPLC measurement. Evodiamine was dis-solved in dimethylsulfoxide (DMSO) to make a stock solution anddiluted by RPMI-1640 (Gibco, Grand Island, NY, USA) before theexperiments. DMSO concentration in all cell culture was kept below0.01%, which had no detectable effect on cell growth or death.

Wortmannin, benzyloxycarbonyl-L-leucyl-L-leucyl-L-leucinal(MG132) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zoli-um bromide (MTT) were purchased from Sigma (St. Louis, MO,USA), ERK inhibitor (PD98059) was from Calbiochem (La Jolla,CA, USA). Rabbit polyclonal antibodies against Akt, p-Akt, IjBa,p-IjBa, ERK, Fas-L, caspase-3, mouse polyclonal antibody againstp-ERK, Bcl-2 and horseradish peroxidase-conjugated secondaryantibodies were obtained from Santa Cruz Biotechnology (SantaCruz, CA, USA). The chemical structures of Evodiamine, Wortman-nin and MG132 were presented in Fig. 5.

2.2. Cell culture and treatment

Human melanoma A375-S2 cells, obtained from AmericanType Culture Collection (ATCC, #CRL, 1872, Rockville, MD,

USA) were cultured in RPMI-1640 medium supplemented with10% heat inactivated (56 �C, 30 min) fetal calf serum (BeijingYuanheng Shengma Research Institution of Biotechnology, Bei-jing, China), 2 mM l-glutamin (Gibco, Grand Island, NY), 100kU/L penicillin and 100 mg/L streptomycin (Gibco) at 37 �Cin humidified 5% CO2/95% air. The cells in the exponentialphase of growth were pretreated with or without 200 nMWortmannin, 10 lM PD98059 or 1 lM MG132, and then werefurther treated with 15 lM evodiamine for 24 h. The treatedcells were subjected to a cytotoxicity assay and Western blotanalyses.

2.3. Cytotoxicity assay

We performed MTT assay as an index of surviving cells. In liv-ing cells, MTT is converted to formazan, which has a specificabsorption maximum. A375-S2 cells were dispensed in 96-wellflat bottom microtiter plates (NUNC, Roskilde, Denmark) at a den-sity of 5 � 104 cells per well. After the cells were treated withevodiamine in the presence or the absence of MG132, 15 lLMTT solution (5.0 mg/L) was added to each well followed by 4 hcoincubation, resulting crystals were dissolved in DMSO. Absor-bance was measured with a plate reader (Bio-Rad, Hercules, CA,USA) at 570 nm. The percentage of cell growth inhibition was cal-culated as follows: Cell death (%) = [A570 (control) – A570] / A570

(control) � 100.

2.4. Western blot analysis

A375-S2 cells were treated with 15 lM evodiamine for 0, 6,12, 24, and 48 h or pre-incubated with or without the giveninhibitors for 1 h, followed by treatment with evodiamine foradditional 24 h. Both adherent and floating cells were collected.Then Western blot analysis was carried out with some modifica-tion (Suzuki et al., 2001). Briefly, the cell pellets were resus-pended in lyses buffer, including 50 mM Hepes, pH 7.4, 1%Triton-X100, 2 mM sodium orthovanadate, 100 mM sodium fluo-ride, 1 mM edetic acid, 1 mM PMSF (Sigma), 10 lg/mL aprotinin(Sigma), 10 lg/mL leupeptin (Sigma), and lysed on ice for60 min. After centrifugation of the cell suspension at 13,000� gfor 15 min, the protein content of supernatant was determinedby Bio-Rad protein assay reagent (Bio-Rad). The protein lysateswere separated by electrophoresis in 12% SDS polyacrylamidegel electrophoresis and blotted onto nitrocellulose membrane(Amersham Biosciences, Piscataway, NJ, USA). After blocking withTween 20–Tris-buffer saline [50 mM Tris–HCl (pH 7.5), 150 mMNaCl, and 0.02% Tween 20] containing 5% nonfat milk at roomtemperature, the membrane was incubated with the indicatedantibodies in blotting buffer. Primary antibodies and diluted con-centration used were: Akt and p-Akt (1:500), IjBa and p-IjBa(1:200), ERK and p-ERK (1:1000), Fas-L (1:500), caspase-3(1:200), Bcl-2 (1:200). The secondary antibodies used for theseproteins were goat anti-rabbit IgG (1:500), goat anti-mouse IgG(1:500) conjugated with horseradish peroxidase (HRP), respec-tively, and 3,3-diaminobenzidine tetrahydrochloride as the sub-strate of HRP. Western blots were scanned, and band intensitieswere measured using Scion Image software Alpha 4.0.3.2 (ScionCo., Frederick, MD, USA).

2.5. Statistical evaluation

The results are presented as Mean ± S.D. Significant changeswere determined using Student’s t-test for unpaired data, and p-values <0.05 were considered statistically significant.

900 C. Wang et al. / Toxicology in Vitro 24 (2010) 898–904

3. Results

3.1. Inactivation of PKB/Akt signal transduction pathway on A375-S2cell death induced by evodiamine

We have reported that the inhibition of PI3K survival pathwaymight be involved in evodiamine-induced A375-S2 cell death(Wang et al., 2005a). To confirm the downstream effector mole-cules of PI3K pathway, the expression of Akt was evaluated byWestern blotting. As shown in Fig. 1A , 15 lM evodiamine reducedAkt phosphorylation at 6 h and this effect was remained detectablefor 48 h. PI3K inhibitor Wortmannin further intensified the evodi-amine’s inhibitory effect at 24 h (P < 0.05). In contrast, non-phos-phorylated Akt and b-actin remained unchanged regardless ofincubation time. The results indicated that Akt protected A375-S2 cells from evodiamine’s cytotoxic effects, and the inactivationof PI3K/Akt pathway was responsible for evodiamine-induced celldeath.

3.2. Activation of NF-jB in response to evodiamine stimulation

To investigate the intracellular signaling pathway activated byevodiamine, we further examined the levels of IjBa and phosphor-ylated IjBa proteins in cytoplasm that can reflect the translocationof NF-jB. After treatment with evodiamine for 0, 6, 12, 24, and48 h, phosphorylation of IjBa was increased and reached a peakat 24 h (P < 0.05), whereas IjBa expression was down-regulated(Fig. 1B, P < 0.05). Western blot analyses was also performed to de-tect the changes of IjB and phosphorylated IjB expression aftercoincubation with Wortmannin and evodiamine. As shown inFig. 2A , 200 nM Wortmannin attenuated the increased expressionof IjB phosphorylation and inhibited the degradation of IjB in-duced by evodiamine (P < 0.05 when compared with Evodiaminetreatment group). These results indicate that NF-jB involved in

actin

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p

Fig. 1. PI3K/Akt/NF-jB signaling involved in evodiamine-induced A375-S2 cell death. (A)were treated with 15 lM evodiamine (Evo) for the indicated time periods in the presenceAkt proteins were measured by Western blot analysis. (B) Activation of NF-jB involved i12, 24, and 48 h, cell lysates were separated by 12% SDS–PAGE, and the protein bands w

the main intracellular signaling pathway mediated evodiamine-in-duced cell death and inhibition of PI3K/Akt could reverse NF-jBactivation.

3.3. The proteasome inhibitor MG132 that inhibits activation of NF-jBenhanced evodiamine-induced cell death

MG132 blocked the degradation of IjB, which bound to NF-jBin the cytoplasm and prevented its translocation to the nucleusand thereby prevented activation of NF-jB-induced gene expres-sion (Hellerbrand et al., 1998). In present study, as expected, IjBadegradation and its phosphorylation were inhibited by 1 lMMG132 efficiently (Fig. 2A, P < 0.05). Our previous research resultshave shown that Wortmannin significantly augmented the cyto-toxic effect of evodiamine in A375-S2 cells (Wang et al., 2005a).Present results indicated that the treatment with MG132 promotedthe evodiamine-induced A375-S2 cell death from 53.1% to 63.7% at24 h (P < 0.01), however, MG132 did not show detectable enhance-ment of cell death at 12 h (Fig. 2B). These results suggested that theblockage of NF-jB activation could augment A375-S2 cell deathafter the administration of evodiamine in a certain time-window.

3.4. Effects of MG132 on the inhibition of ERK and the production ofFas-L induced by evodiamine

Our previous results showed that ERK signaling pathway pro-tected A375-S2 cells from death induced by evodiamine (Zhanget al., 2003; Wang et al., 2005a). As shown in Fig. 3A, phosphoryla-tion of ERK and total ERK were down-regulated by evodiamine. Inthe presence of 1 lM MG132, the inhibition of ERK was further en-hanced 24 h after evodiamine treatment, suggesting that the inhi-bition of NF-jB translocation by MG132 might contribute toevodiamine-induced inhibition of ERK pathway.

I B

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Inactivation of phosphorylated Akt in evodiamine-induced cell death. A375-S2 cellsor absence of 200 nM Wortmannin (Wort). Expressions of phosphorylated and totaln evodiamine-induced cell death. The cells treated with 15 lM evodiamine for 0, 6,ere detected by Western blot analysis.

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Fig. 2. Effects of PI3K and proteasome inhibitor on evodiamine-induced A375-S2 cell death. (A) Effects of PI3K and proteasome inhibitor on degradation of IjBa and itsphosphorylation. The cells were pretreated with 200 nM Wortmannin, 1 lM MG132 for 1 h, followed by the addition of 15 lM evodiamine. After 24 h, expressions of IjBaand its phosphorylation were detected by Western blot analysis. (B) Proteasome inhibitor enhanced evodiamine-induced cytotoxic effect on A375-S2 cells. The cells werepretreated with 1 lM MG132, 1 h prior to the administration of 15 lM evodiamine for additional 12, 24 h. n = 3. Mean ± S.D. **P < 0.01 vs. evodiamine alone group.

-actin

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Fig. 3. Effect of MG132 on Erk and Fas-L expression in evodiamine-induced A375-S2 cell death. (A) Effect of MG132 on evodiamine-induced Erk inhibition. The cells weretreated with 15 lM evodiamine for indicated time point in the presence or absence of 1 lM MG132. Then cell lysates were separated by 12% SDS–PAGE, and the expressionsof Erk and phosphorylated Erk were detected by Western blot analysis. (B) Effects of PI3K, Erk and proteasome inhibitor on Fas-L expression induced by evodiamine. The cellswere pretreated with 200 nM Wortmannin, 20 lM PD98059 and 1 lM MG132 for 1 h, followed by the addition of 15 lM evodiamine for 24 h. Then the expression of Fas-Lwas detected by Western blot analysis.

C. Wang et al. / Toxicology in Vitro 24 (2010) 898–904 901

902 C. Wang et al. / Toxicology in Vitro 24 (2010) 898–904

Fas-L is also involved in host defense mechanism against tu-mors (Watanabe et al., 2003). Evodiamine was capable of inducingthe production of Fas-L in a time dependent manner (Wang et al.,2005b). To study the influences of PI3K, ERK and NF-jB pathwayon Fas-L expression, the effects of their inhibitors were examined,respectively. Pretreatment with 200 nM Wortmannin, 10 lMPD98059 or 1 lM MG132 partially reduced Fas-L induction byevodiamine to various extents at 24 h (Fig. 3B, P < 0.05 comparedwith Evodiamine treatment group). Our results indicated thatFas-L induction mediated by evodiamine might be through activa-tion of PI3K, ERK and NF-jB pathway.

3.5. Inhibition of ubiquitin–proteasome pathway by MG132 enhancedcaspase-3 activation and Bcl-2 cleavage induced by evodiamine

To further investigate the potential role of ubiquitin–protea-some system in evodiamine stimulated cell death, the effects ofproteasome inhibitor MG132 on expressions of caspase-3 andBcl-2 were examined. Western blot analyses indicated that controlcultures of A375-S2 cells expressed higher levels of the 32 kDa cas-pase-3 precursor After evodiamine-induced activation of caspase-3, the minor 17 kDa fragment appeared and a proportion of cas-pase-3 precursor gradually decreased. Pretreatment with MG132markedly accelerated the cleavage of the precursor and activationof caspase-3 (Fig. 4A). Furthermore, the process of Bcl-2 cleavage inevodiamine-induced cell death was also enhanced by MG132(Fig. 4B). These results suggested that ubiquitin–proteasome path-way might participate in regulating caspase-3 and Bcl-2, both ofwhich played essential roles in A375-S2 cell death induced byevodiamine.

Fig. 5. Chemical structures of Wortmannin, MG132 and evodiamine.

4. Discussion

The ubiquitin–proteasome system plays crucial roles in the deg-radation of various cellular proteins, including signal transductionmolecules (Lee et al., 1998). Proteasome inhibitors have been

Pro-caspase-3Cle-caspase-3

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Fig. 4. Proteasome inhibitor accelerated caspase-3 activation and cleavage of Bcl-2 in evofor indicated time points in the presence or absence of 1 lM MG132. Cell lysates were sdetected by Western blot analysis. Similar results were obtained in three independent e

widely used as NF-jB inhibitors. In this study, we have investi-gated the effects of proteasome inhibitor MG132 on PI3K/Akt/NF-jB pathway and other signaling molecules involved in human mel-anoma A375-S2 cell death induced by evodiamine.

PI3K is required for cell survival via activation of PKB/Akt inneuronal survival (Songyang et al., 1997; Dudek et al., 1997) andinduction of ERK in human fibroblasts (Ahn et al., 2001). The roleof PI3K in regulating NF-jB transactivation remains controversial.Previous studies showed that the inhibitor of PI3K Wortmannincould further reduce ERK expression and phosphorylation, accord-ingly potentiating evodiamine-induced cell death (Zhang et al.,2003; Wang et al., 2005a). In present report, we demonstrated thatAkt phosphorylation was down-regulated by evodiamine in a timedependent manner. Correspondingly, phosphorylation and degra-dation of IjBa that reflects the translocation of NF-jB were en-

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diamine-induced A375-S2 cell death. The cells were treated with 15 lM evodiamineeparated by 15% SDS–PAGE, and the expressions of caspase-3 (A) and Bcl-2 (B) wasxperiments.

C. Wang et al. / Toxicology in Vitro 24 (2010) 898–904 903

hanced in this process. Therefore, enhancement of NF-jB translo-cation caused by evodiamine seemed to be associated with PI3Kinhibition, resulting in the loss of Akt activity and the phosphory-lation of ERK.

Pretreatment of proteasome inhibitor MG132 that inhibits NF-jB translocation augmented evodiamine-induced cell death at24 h. Furthermore, the inhibition of PI3K by Wortmannin at 24 hreversed IjBa phosphorylation and degradation, and the inhibitionof NF-jB by MG132 accelerated ERK inactivation. Remarkably,evodiamine-induced production of Fas-L was partially reduced at24 h by pretreatment with Wortmannin, PD98059 or MG132, sug-gesting that Fas-L existed at downstream of PI3K and consequentERK pathway. The detailed mechanism remains to be elucidated.

Anti-apoptotic protein Bcl-2 is known to regulate stress-in-duced cell death (Pastorino et al., 1999). In an attempt to furtheranalyse the mechanism of Bcl-2 cleavage in evodiamine-inducedcell death, we found that blockage of the ubiquitin–proteasomepathway by MG132 markedly promoted the cleavage of Bcl-2 inA375-S2 cells. Study with specific caspase inhibitor revealed thatthe cleavage of Bcl-2 was suppressed by the inhibitor of caspase-3 z-DEVD-fmk (Zhang et al., 2003). In addition, our results showedthat, caspase-3 was further activated by MG132 in A375-S2 cells.Taken together, Bcl-2 might act as a downstream substrate of cas-pase-3 or caspase-3-like protease in evodiamine-induced celldeath. Whereas the detailed mechanisms responsible for the acti-vation caspase-3 by MG132 are still unclear, it is possible thatthe inhibition of proteasome may promote multiple pathwaysincluding caspase-3.

In summary, we have examined the PI3K/Akt/NF-jB signalingand the potential role of ubiquitin–proteasome pathway in evodi-amine-induced A375-S2 cell death. Activation of NF-jB contrib-uted to cell death caused by evodiamine and inhibition ofubiquitin–proteasome pathway by MG132 accelerated cell deathat that time point. Therefore, these results suggest that PI3K/Akt/NF-jB signaling was involved in cell death induced by evodiamineand proteasome inhibitors may be useful adjuvants in therapy withevodiamine against human melanoma.

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