induction ofapoptosis byvanilloid compounds doesnot...

Vol. 9, 277-286, March 1998 Cell Growth & Differentiation 277

Induction of Apoptosis by Vanilloid Compounds Does NotRequire de Novo Gene Transcription and ActivatorProtein I Activity1

Antonio Macho, M.-Valle Bl#{225}zquez,Pl#{225}cidoNavas,and Eduardo Mu#{241}oz�

Departamento de Fisiologia e Inmunologia, Facultad do Medicina[A. M., M-V. B., E. M.] and Departamento de Biologla Celular, Facultadde Ciencias [P. N.], Universidad de C#{243}rdoba,14071 C#{243}rdoba,Spain.

Abstract

The vanilloid compounds, capsaicin and resiniferatoxin,are quinone analogues that inhibit the NADH-plasmamembrane electron transport system and induceapoptosis in transformed cells. Because disruption ofthe mitochondrial transmembrane potential (4�’P,��) is acommon metabolic alteration in all apoptoticprocesses, we have evaluated the role of mitochondrialpermeability transition in apoptosis induced byvanilloids in Jurkat cells. Using a cytofluorimetricapproach, we have determined that DNA nuclear lossinduced by vanilloids is preceded by an increase of theproduction of reactive oxygen species (ROS) and by asubsequent � dissipation in T-ceII lines.Overexpression of Bcl-2 and pretreatment with eitherthe immunosuppressant cyclosporin A or theglutathione precursor N-acetyl-L-cysteine blocked �‘m

disruption and apoptosis, but not the generation ofROS induced by these compounds. Capsaicin andresiniferatoxin were found to activate both isoforms ofc-jun-NH2-kinase (JNK), with a maximal activity after30 mm of treatment. Despite the activation of JNK,there was no induction of activator protein I (AP-1)activity as determined by gel shift assay or of inductionof an AP-1-responsive reporter. On the other hand,vanilloids did not signal for c-Rat kinase andextracellular signal-regulated kinases I and 2. Wesuggest that ROS generation by inhibition of theNADH-dependent plasma membrane electron transportsystem resulted in the oxidation of mitochondrialmegachannel pores that allows for the disruption of

�m and apoptosis, and that AP-1 activation is notrequired for vanilloid-induced apoptosis.

Introduction

Eukaryotic cells continuously produce ROS3 as side prod-

ucts of redox reactions. Generation of ROS is governedmostly by the mitochondria, and these ROS comprise hy-drogen peroxide, hydroxyl radicals, and superoxide anions

(1). In addition to the mitochondria, the plasma membrane

contains an electron transport chain that seems to be es-sential in the control of cell growth and differentiation, stim-ulation of certain transport functions, and defense againstbacteria (2). An important compound of this system is PMOR,which transfers electrons from cytoplasmic NADH via CoQ to

external electron acceptors (3). It is accepted that the PMOR

system plays an important role in the regulation of internal

redox equilibrium in response to external stimuli (2, 4). Thus,it has been shown that PMOR inhibitors affect growth andinduce apoptosis in different tumor cell lines (5, 6).

Capsaicin (8-methyl-N-vanillyl-6-nonenamide), the pun-

gent ingredient in a wide variety of red peppers of the genusCapsicum, and RTX (a structural homologue to capsaicin), analkaloid derived from plants of the genus Euphorbia, share acommon cellular receptor that may mediate the effects ofvanilloids in some cell systems (reviewed in Ref. 7). Never-theless, both compounds are inhibitory quinone analoguesthat can inhibit the PMOR system and induce apoptosis by a

vanilloid receptor-independent pathway (6). Moreover, cap-saicin has been used in humans for topical treatment ofcluster headache and herpes zoster (8, 9), and it hasalso been shown to be immunomodulatory in animal models(10, 11).

Apoptosis, or programmed cell death, is a natural form ofcell death controlled by a constitutively expressed machinerythat induces condensation of nucleoplasm and cytoplasm,blebbing of cytoplasmic membranes, and fragmentation of

the cell into apoptotic bodies that are rapidly recognized and

eliminated by adjacent cells (1 2-14). According to the current

understanding, morphological and biochemical alterations in

nuclear and chromatin structures of cells that undergo ap-optosis are controlled mainly, if not completely, by the mi-

tochondria (1 5-1 7). Thus, a breakdown of �1’m is an invariantfeature of early apoptosis that precedes DNA fragmentation(1 8), independent of the cell type and the apoptotic stimuli

Received 9/17/97; revised 12/23/97; accepted 1/9/98.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Supported by CICYT Grant SAF95/0474 to E. M., and DGCYT GrantPB95/0560 to P. N.2 To whom requests for reprints should be addressed, at Departamentodo Fisiologla e Inmunologla, Facultad de Medicina, Avenida do Men#{233}ndezPidal s/n, 14071 C#{243}rdoba,Spain. Fax: 34-57-218229; E-mail: [email protected].

3 The abbreviations used are: ROS, reactive oxygen species; PMOR,plasma membrane NADH-oxidoreductase; �‘I’m. mitochondrial trans-membrane potential; AP-1 , activator protein 1 ; JNK, c-jun-NH2-kinase;RTX, resiniferatoxin; ERK, extracellular signal-regulated kinase; PMA,phorbol 12-myristate 13-acetate; HE, hydroethidine; Eth, ethidium; CsA,cyclosponn A; DiOC6(3), 3,3’ dihexyloxacarbocyanine iodide; MAPK,mitogen-activated protein kinase; PT, permeability transition; NAC, N-acetyl-L-cysteine; MEK, mitogen-activated proteln kinase/ERK; mAb,monoclonal antibody; PMSF, phenylmethylsulfonyl fluoride; EMSA, elec-trophoretic mobility shift assay; CHX, cycloheximide; P1, propidium iodide;CoO, coenzyme Q; NF-AT, nuclear factor of activated T cells.

278 Apoptosis Induced by Vanilloids

(19-24). �m disrupture is mediated by mitochondrialmegachannel opening (PT; Ref. 25-28), leading to the re-lease of cytochrome C and the so-called apoptotic-inducing

factor, which can mediate nuclearfragmentation (17, 29-31).After the initial �‘m dissipation, cells hyperproduce ROS

that may contribute to the apoptotic pathway (32). It has

been shown in thymocytes that these ROS are derived fromcomplex III of the mitochondrial respiratory chain (23, 32).

In addition to its possible role in apoptosis, a growing body

of evidence suggests that ROS may also regulate signaltransduction and/or the activity of some transcription factorsthat in turn regulate gene transcription in animal cells (33-35).

Among these transcription factors, AP-1 is one of the most

extensively studied, and it has been shown to be regulated

by redox in some cell systems (33, 36). The AP-1 transcrip-tion factor complex is composed of a group of proteins

encoded by thejun (c-Jun, Jun-B, and Jun-D) and fos (c-Fos,Fos-B, Fra-1 , and Fra-2) gene families, which can bind to theAP-1 consensus sequences either as Jun/Jun or Jun/Fos

dimers (37, 38). AP-1 activity is regulated by do novo syn-thesis of Jun and Fos members as well as by posttranscrip-tional modification of Jun through phosphorylation of its

transcriptional domain (39-41). The regulation of AP-1 inresponse to external stimuli is mediated by members of theMAPK family (41 , 42). The best-characterized MAPKs are the

ERKs 1 and 2 and the JNK isoforms (also known as stress-activated protein kinases). Both JNK and ERK are activated

after surface receptor-mediated GTP binding of Ras andstimulation of a cascade of downstream kinases includingRaf-1 , MEK, and MEK kinase kinases (43-45), the latter

being a potent activator of JNK (46, 47).The role of JNK and AP-1 in different apoptotic pathways

is not yet clear. Several reports indicate that JNK and AP-1are involved in ceramide- or retinoid-induced apoptosis (48,49), whereas others demonstrate that JNK and AP-1 tran-scriptional activities may be separated from tumor necrosisfactor-induced apoptotic signals (50, 51). On the other hand,

Fas union leads to apoptosis and JNK activation in Jurkat

cells, and this activation does not result in AP-1 binding toDNA and transcriptional activities (52).

In the present report, we investigated the biochemicalpathways activated by vanilloid PMOR inhibitors in T cells.We now show that both capsaicin and RTX induce apoptosis

and JNK activation. Apoptosis is preceded by an increase in

ROS production and a consecutive breakdown in ��m’ butit is independent of AP-1 activity.

Results

Induction of Apoptosis by Capsaicin and RTX IsPreceded by a Sequential ROS Generation and ��‘I’mDisruption. We studied the role of the mitochondria in the

apoptosis induced by PMOR inhibitors, capsaicin and RTX,in Jurkat cells. Cells treated with the indicated doses of

capsaicin or RTX for 1 8 h showed a loss of nuclear DNA(chromatinolysis) measured by an increase in the frequencyof subdiploid (apoptotic) cells (Fig. 1A). Induction of apop-tosis was concentration dependent, and whereas 50 p.�i

capsaicin did not result in significant apoptosis, concentra-

tions ranging from 1 00-300 �M mediated a progressive in-

crease in the percentage of subdiploid cells. Induction ofapoptosis by RTX was also dose dependent, and whereas 10.LM RTX had minimum effect on Jurkat cells (data not shown),

up to 60% of apoptotic cells were found at 40 p�M concen-tration. Differences in the doses required to induce apoptosis

by capsaicin and RTX in Jurkat cells are in agreement withprevious reports showing that RTX is approximately 10-20

orders of magnitude more potent than capsaicin (6, 7). Thereis accumulated evidence that � breakdown and ROSgeneration are invariant features of early apoptosis (21 , 23,

24, 32, 53). Thus, we investigated both events by double-

staining experiments, using HE (nonfluorescent), which be-comes Eth (red fluorescent) after its oxidation via ROS, and

DiOC6(3) (green fluorescent), a cationic probe that accumu-lates in mitochondria as a function of its potential (54). We

show in Fig. lB that treatment of Jurkat cells with increasing

concentrations of capsaicin paralleled an increase in thepercentage of (HE��+Eth)��l9h cells that reflect ROS hypergen-

eration. Interestingly, an increase in the percentage of(HE��Eth)he�h/Dioc6(3)b0w cells was only detected with theconcentrations able to induce apoptosis of either capsaicinor RTX. These results strongly suggest a close relationshipbetween �m disruption and apoptosis in vanilloid-treatedcells.

Next, we investigated the kinetics of appearance of� and DiOC6(3)b0w Jurkat cells in response to 300�M capsaicin. Capsaicin induced a clear increase in the

percentage of � cells after 1 5 mm of treatment,and this percentage of cells increased dramatically up to90% after 6 h. On the other hand, a significant percentage of

DiOC6(3)bow cells appeared after only 60 mm of treatment, a

percentage that increased to up 50% after 6 h with capsaicin(Fig. 2). It is important to note that our experiments did notshow a significant percentage of �cells, characteristic of other apoptotic models (23, 32).

Close Relationship between Mitochondrial Perturba-

tions and Apoptosis in Capsaicin-treated Jurkat Cells. Itseems likely that the mechanism of the preapoptotic �‘m

dissipation is mediated by so-called PT pores (55), a stepthat can be transiently blocked by CsA (27, 56-61). This also

holds true for the � breakdown induced by capsaicin,because, as shown in Fig. 3A, pretreatment of Jurkat cellswith CsA prevented capsaicin-induced � disruption, but

not ROS generation. This suggests that ROS induced bycapsaicin were not derived from a mitochondrial source andwas supported by the fact that rotenone, which inhibits themitochondnal electron chain at complex I and therefore thegeneration of ROS from complex III, did not prevent this ROSgeneration (Fig. 3A).

It has been recently shown that dissipation of �‘1’m is

closely paralleled with a glutathione depletion (62), whichcould potentiate the side effects of ROS. To investigate thisclose relationship in our experimental model, we preincu-

bated Jurkat cells with the glutathione precursor NAC beforecapsaicin treatment. NAC prevented �4�m breakdown andapoptosis (Fig. 3A). To study whether the apoptotic mocha-

nisms activated by capsaicin were dependent on the newsynthesis of proteins, we preincubated Jurkat cells with theprotein synthesis inhibitor CHX, and it was observed that

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Cell Growth & Differentiation 279

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Fig. 1. Induction of apoptosis in Jurkat cells treated with vanilloid compounds. A, Jurkat cells were treated with the indicated concentrations of capsaicinand RTX for 18 h, and cell cycle was analyzed by P1 staining. Bars indicate the percentage of subdiploid cells (DNA fragmented cells). B, simultaneousassessment of �m disruption and ROS generation after 6 h of treatment with capsaicin and RTX. Cells were stained with the potential sensitive dyeDiOC6(3) and the ROS-oxidable probe HE. Results are shown as the percentage of cells obtained in biparametric histograms delimited by fourcompartments, namely, �‘m”��’ (normal cells, bottom, right); L��I�mIOW (bottom, left); (HE�+Eth)hi9�� (ROS-generating cells, top, right), and (HE-+Eth)�”9�’/

� (preapoptotic cells, top, left). Results are representative of four independent experiments.

(HE_>Eth)h�/APml0��/

U (HE_>Eth)hiifh

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Fig. 2. Kinetics of capsaicin-induced ROS generation (HE-�Eth) andVmdisruption (�mt��l Jurkat cells were treated with 300 �iM capsaicinat different times, followed by two-color staining with DiOC6(3) and HE asdescribed in Fig. lB. The results are the mean � SD of three independentexperiments.

time (mm.)

neither �m breakdown, ROS generation, nor the loss of

nuclear DNA induced by capsaicin was dependent on the

new synthesis of proteins (Fig. 3A).

The antiapoptotic effect of CsA in capsaicin-treated cells

has been explained by its ability to inactivate calcineurin, a

calmodulin-dependent phosphatase (6). The activation of

calcineurin leads to the dephosphorylation and nuclear

translocation of the cytoplasmic subunit of the NF-AT tran-

scription factor (63). Thus, we studied by transient transfec-

tion experiments whether or not capsaicin could induce NF-

AT-dependent transcriptional activities as a result of

calcineurin activation. Jurkat cells were transfected with a

plasmid containing the luciferase gene reporter driven by

three sites of the NF-AT site of the interleukin 2 promoter. It

is shown in Fig. 3B that NE-AT transcription was mediated by

the signals provided by PMA and ionomycin, and that cap-

saicin, at the concentrations tested, did not activate NE-AT

transcription alone or in combination with PMA.

Overexpression of BcI-2 Prevents �‘1fm Disruption and

Apoptosis but not the ROS Generation Induced by Cap-saicin. Because of the involvement of p53 protein in the

induction of apoptosis in some systems (64, 65) and the role

of Bcl-2 as an antiapoptotic protein (66-68), we investigated

the steady-state levels of both proteins in capsaicin- and

RTX-treated Jurkat cells. We did not detect major changes inthe expression of both proteins up to 6 h of vanilloid treat-

ment (Fig. 4A), a time when the �‘�1m breakdown has been

clearly induced (Fig. 18). Nevertheless, it has been shown

that experimental overexpression of Bcl-2 protects cells from

apoptosis in a great number of different systems. To study

this point in the apoptosis induced by capsaicin, we exam-

med 9F3 cells, a stably transfected OEM-derived clone over-

expressing human BcI-2 and the wild-type OEM line that

constitutively expresses low levels of endogenous BcI-2 (Fig.

4C; Ref. 69) as compared with Jurkat and 9E3 cells. Similar

to Jurkat cells, both ROS generation and dissipation of z�’I’m


were induced in the wild-type CEM cell line by capsaicin, and

these biochemical events were followed by an important

increase in the percentage of subdiploid cells. In Fig. 4B, it is

shown that both apoptosis and �‘1’m breakdown, but not

ROS generation, were clearly prevented in capsaicin-stimu-

lated 9F3 cells. Moreover, a close correlation seems to exist

between the expression of endogenous BcI-2 and the con-

centration of capsaicin required to induce apoptosis. As seen

in Fig. 4C, a higher expression of Bcl-2 was found in Jurkat

cells when compared with CEM cells, and when apoptosis

was compared in both cell types, it was found that CEM cells

were significantly more sensitive than Jurkat cells to the

apoptotic effect of capsaicin. Lower concentrations of this

0 15 30 60 180 360 vanilloid were required to induce a 50% apoptotic CEM cells(200 MM) than Jurkat cells (300 MM).

Effects of Capsaicin and RTX in the Activation of MAPKin Jurkat Cells. JNK activation has been implicated in the

process of programmed cell death and in different systems,

and we investigated the effect of vanilloids in JNK activation

on Jurkat cells. Thus, cells were incubated with capsaicin

and RTX for the indicated times, and in-gel JNK activity was

measured as indicated above. We found that capsaicin and

RTX activated both isoforms of JNK (55 and 47 kDa) with

similar kinetics and intensity, being detectable as little as 5

mm after treatment, with the peak at 30 mm (Fig. 5A). Basal

level was recovered after 60 mm (data not shown). ERK 1 and

2 are other members of the MAPK family that can be acti-

vated by a wide range of stimuli (43, 70). Although both JNK

and ERK may be activated by different signaling cascades

(46, 47), there is evidence that at least one pathway in which

H-Ras is involved may activate both JNK and ERK (42, 71,

72). Up-regulation of ERK activity in vivo is followed by its

phosphorylation by MEK on threonine and tyrosine residues,

resulting in a slight retardation of ERK migration on SDS-polyacrylamide gels. To test the effect of vanilloids in ERK

activation, we analyzed Jurkat cell lysates by SDS-PAGE and

immunoblotting with an ERK-1 and 2-specific mAb. Fig. SB

shows that retardation of both ERK-1 and ERK-2 mobility isinduced within S mm of incubation with PMA. On the con-

trary, no changes in ERK mobility were detected in the ly-

sates of Jurkat cells treated with capsaicin. The lack of

involvement of the Ras-Raf pathway was also evident, be-

cause capsaicin cannot induce tyrosine protein phosphoryl-

ation of Raf-1 kinase (data not shown).

Capsaicin and RTX Did Not Induce AP-1 Activity. The

activation of AP-1 is governed by a complex pathway involv-

ing transcriptional and posttranscriptional mechanisms that

include the phosphorylation of c-Jun at Ser-63 and Ser-73

within its activation domain (73). This JNK-mediated phos-

phorylation enhances the ability of c-Jun to activate tran-

scription (71). Due to the activation of JNK-1 and JNK-2 by

capsaicin and RTX in Jurkat cells, we studied the possible

induction of AP-1 activation by DNA binding analysis and by

the induction of an AP-1 -responsive reporter. Nuclear ox-

tracts from PMA plus ionomycin-treated Jurkat cells exhib-

ted a strong binding to the end-labeled AP-1 probe. The

specificity of this binding was further demonstrated by cold

competition and by supershift with anti-jun and anti-fos fam-

ily antisera (data not shown). In contrast, AP-1 binding was

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Fig. 3. A, pharmacological modulation of ROS generation, � disruption, and apoptosis in capsaicin-treated cells. Jurkat cells were pretreated for 1 hwith either 10 �&M CsA, 40 mi�i NAC, 1 0 pg/mI CHX, or 1 00 �u�i rotenone and then incubated with 300 �u�i capsaicin for 6 h for either ROS generation and�m disruption detection and 18 h forcellcycle analysis(c representsthe percentage of apoptotic cells). B, capsaicin does not induce NF-AT transcriptionalactivities. Jurkat cells were transfected with a NF-AT-Luc construct and stimulated 48 h later for 6 h with PMA (50 ng/mI), PMA plus ionomycin (0.5 pg/mI),PMA plus capsaicin, or capsaicin alone at the indicated concentrations (�.u�i). Luciferase activity was measured, and the transcriptional activity wasexpressed as x-fold transactivation over the values obtained with nonstimulated cells. Values are the means ± SD of three independent experiments.

not detected in the nuclear extracts from capsaicin-stimu-lated cells (Fig. 6A). Moreover, it was found that both cap-

saicin (Fig. 6B) and RTX (data not shown) prevented the AP-1binding induced by PMA plus ionomycin. To determine theeffects of capsaicin on the binding of AP-1 to DNA, thenuclear extracts from PMA plus ionomycin-activated cellswere incubated with various concentrations of capsaicin.EMSA showed that capsaicin did not prevent AP-1 frombinding to DNA (Fig. 6C). Western blot analyses revealed that

200 MM capsaicin inhibited the expression of both c-Fos andc-Jun proteins induced by PMA plus ionomycin in Jurkatcells (Fig. 6D). To correlate these results with transcriptionalactivities, Jurkat cells were transiently transfected with aluciferase reporter construct under the control of three AP-1

binding sites. Twenty-four h after transfection, cells weretreated for 6 h with increasing concentrations of capsaicin

alone or in combination with PMA plus ionomycin, and trans-

activation was determined. In this experiment, treatment ofJurkat cells with PMA plus ionomycin led to a 10-fold in-crease of AP-1 -dependent transactivation of the luciferasereporter gene, whereas treatments with different concentra-tions of capsaicin did not result in AP-1 transactivation. Thus,the ability of capsaicin to prevent DNA binding of AP-1 inPMA plus ionomycin-treated cells was also reflected in theblocking of AP-1 -dependent transactivation (Fig. 7).

Discussion

We have shown here that apoptosis induced by the inhibitoryCoQ analogues capsaicin and RTX in transformed T cells ispreceded by a reduction of the �‘I’m and an increase in thelevels of ROS. The first event detected was an increase inROS generation that could be due to the inhibition of theCoQ-mediated transplasma membrane electron transport.This CoQ-dependent plasma membrane redox system has

been purified and characterized (74, 75) and involves the

cytochrome b5 reductase that reduces CoQ (76). Because

this reduction is inhibited by capsaicin (74), we suggest that

the interference of vanilloids with the CoQ binding site of this

system could lead to the redirection of normal electron flow

in the complex, generating an excess of ROS. The pro-

oxidative environment generated by inhibition of the PMOR

system may induce oxidation of thiol groups in mitochondrial

PT pores (77, 78), allowing its opening and, as a conse-

quence, the free distribution of solutes at both sides of theinner mitochondrial membrane. This is supported by the data

obtained with the glutathione precursor NAC, which can

prevent mitochondrial PT pore oxidation and the subsequent

�m dissipation, rather than ROS generation (Fig. 3A). It hasbeen demonstrated that substitution of thiols by different

reagents can inhibit both �4’m and nuclear DNA fragmenta-

tion (78-80). The role of PT in vanilloid-induced apoptosis is

further supported by the fact that preincubation with CsA or

overexpression of BcI-2, which are two different PT inhibitors(1 7, 29, 32, 81), prevents �1’m disruption and apoptosis, but

not the extramitochondrial generation of ROS.

Vanilloid inhibition of the plasma membrane NADH-

dependent electron transport system has been implicated in

the apoptosis of transformed cells (6), and we show here that

the oxidative stress caused by the production of extramito-

chondrial ROS by this inhibition could be the mechanism of

this apoptotic pathway, although we cannot exclude the

possibility that some ROS are generated from the inhibition

of the mitochondrial respiratory chain. Also, Wolvetang et a!.

(6) have proposed the participation of the calcineurin phos-

phatase in capsaicin-mediated apoptosis, but our results

suggest that vanilloids do not activate this phosphatase, and

that the inhibition caused by CsA can be mediated by its

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Fig. 5. Differential activation of MAPK by capsaicin and RTX. Jurkat cellswere treated with 300 p.M capsaicin or 40 �M RTX for the indicated times,and In-gel JNK was determined as described previously (A). Cellularextracts from either PMA (50 ng/ml)- or capsaicin (300 �M)-treated Jurkatcells, for the indicated time (mm), were subjected to SDS-PAGE electro-phoresis, and ERK 1 and 2 shift mobility was detected by Western blot (B).


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Fig. 4. Role of Bcl-2 on vanilloid-induced ROS generation, �‘I’m dis-ruption, and apoptosis in trans-formed T cells. A, expression ofBcl-2 and p53 proteins in capsaicinand RTX-treated cells. Jurkat cellswere treated with capsaicin (300�LM) or RTX (40 �.tp.i)forthe indicatedtimes, and the expression of Bcl-2and p53 was detected by Westernblot, as described in “Materials andMethods.” B, effect of Bcl-2 over-expression on capsaicin-inducedROS generation (HE-*Eth), �‘I’m

disruption [DiOC6(3) understain-ing], and apoptosis. CEM and 9F3cells were cultured for 6 and 1 8 h inthe presence of 300 �M capsaicin,followed by simultaneous labellingwith DiOC6(3) and HE or stainingwith P1, respectively. C, Bcl-2 ex-pression in the three cultured celllines tested. Cell lysates from Jur-kat, CEM, and 9F3 were subjectedto Westem blot analysis.

DiOC6(3)

binding to mitochondrial cyclophilin, which can regulate the

activity of PT pores (82).

Another important finding in the present work is that

capsaicin and RTX induce a rapid and strong activation of

both isoforms of JNK. Although several reports have im-

plicated JNK and AP-1 activity in apoptosis (48, 49, 83), ithas been recently shown that cross-linking of Fas results

in JNK activation but not MEK activation (52, 84), and,

more importantly, that JNK activation in Jurkat cells is not

required for the Eas-mediated apoptotic program (52).

Several lines of evidence suggest that in vanilloid-stimu-lated transformed cells, the activation of JNK may be

independent of the apoptotic pathway: (a) the time of JNK

activation is much faster than the appearance of PT that isrequired for the process of DNA fragmentation; (b) the

induction of apoptosis by capsaicin is not affected by

either CHX or actinomycin D; and (c) vanilloids do notinduce AP-1 DNA binding or AP-1 -dependent transactiva-

tion but also inhibit the AP-1 activation mediated by PMA.

The ability of capsaicin to block PMA-induced AP-1 acti-

vation can be explained by the inhibition of do novo syn-

thesis of Jun and Fos proteins, and the role of ROS in this

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Fig. 7. Capsaicin blocks AP-1 -dependent gene transcription. Jurkatcells were transfected with an AP-1 -dependent reporter luciferase con-struct and stimulated 48 h later for 6 h, as indicated. Luciferase activitywas measured, and transcriptional activity was expressed as x-fold trans-activation over the values obtained with nonstimulated cells. Values arethe means ± SD of three independent experiments.

inhibition remains to be studied. Nevertheless, the results

of in vitro gel shift assays have shown that the DNA-

binding activity of several transcription factors, including

AP-1 , NFKB, and Myb, is regulated by a redox control

mechanism (85-87). Thus, it is possible that the ROSgenerated by vanilloids may induce posttranscriptional

modifications of specific transcription factors required for

c-jun and c-fos gene transcription.

Various effects of capsaicin and RTX are mediated

through a specific cellular receptor referred to as the va-

nilloid receptor (88, 89), but the apoptotic pathway acti-

vated by capsaicin does not involve this receptor (6). It is

possible that this receptor may be linked to the Ha-Ras


B

________ PMA+I

_______ Capsaicin:,� � � ________(�iM� �

Fig. 6. Effects of capsaicin on AP-1 activities in Jurkat cells. A, nuclear extracts were prepared from Jurkat cells treated with 300 �M capsaicin for theindicated times or with PMA (50 ng/ml) plus ionomycin (0.5 �g/ml) for 3 h. B, nuclear extracts were prepared from Jurkat cells stimulated for 3 h with PMAplus ionomycin in the presence of different concentrations of capsaicin. C, effect of capsaicin on the binding of AP-1 to DNA. Nuclear extract prepared fromPMA plus ionomiycin-stimulated Jurkat cells was incubated with indicated concentrations of capsaicin and then analyzed for AP-1 binding by EMSA. D,capsaicin inhibits c-fos and c-jun protein expression in Jurkat cells. The cells were stimulated for 3 h as indicated, and c-Fos and c-Jun protein expressionwas measured by Westem blot.

pathway, leading to JNK activation. The other pathway

would be activated by PMOR inhibition, inducing high

levels of ROS that affect the mitochondrial PT, leading to

nuclear DNA fragmentation. Alternatively, it is also possi-

ble that JNK activation may depend on ROS generation,similar to other stimuli such as UV-O rays (90) and tumor

necrosis factor-a (51). The further identification of these

pathways may be an important clue to understand the

mechanism of capsaicin in the selective destruction of

nociceptive neurons during embryonic development and

the analgesic properties of vanilloids.

Materials and MethodsCell Lines and Reagents. Jurkat cells (American Type Culture Collec-

tion, Rockville, MD) and the CEM cell line, kindly provided by Dr. S.

Geley (Institute for General and Experimental Pathology, University oflnnsbruck, Austria), were maintained in exponential growth in RPMI

1 640 (Bio-Whittaker, VerViers, Belgium) supplemented with 10% heat-

inactivated FCS, 2 mM L-glutamine, 1 m�i HEPES, and antibiotics (Life

Technologies, Inc., Paisley, Scotland). The 9F3 cell line is a CEM-

derived clone stably transfected with an expression vector encoding

the complete human Bcl-2 cDNA (69) and was maintained in completemedium containing 1 00 j.�g/ml hygromicin. [y-32P]ATP (6000 Ci/mmol)

was purchased from ICN (Costa Mesa, CA). The anti-Bcl-2 mAb, Ab-1,

was obtained from Calbiochem (Cambridge, MA), the anti-p53 mAb,

DO-7, was from Master Diagnostica (Granada, Spain), the Raf-1 anti-

serum was from Serotec (Oxford, United Kingdom), the anti-ERK1/2mAb was from Zymed (San Francisco, CA), and the rabbit antisera

anti-c-Fos and c-Jun were from Santa Cruz Biotechnology, Inc. (Santa

Cruz, CA). All other reagents were from Sigma Chemical Co. (Madrid,

Spain).

Cytofluorimetric Analysis of �‘Vm� ROS Generation, and NuclearDNA Loss. To evaluate �‘I’m and the superoxide anion generation (ROS),

treated or untreated cells (5 x 1 o� cells/mi) were incubated in PBS with

DiOC6(3) (green fluorescence; 20 nM; Molecular Probes, Eugene, OR) and

dihydroethidine (HE; red fluorescence after oxidation; 2 ; Sigma Chem-ical Co.) for 20 mm at 37C, followed by analysis on an Epics Profile II


Analyzer cytofluorimeter (Coulter, Hialeah, FL). The percentage of cells

undergoing chromatinolysis (subdiploid cells) was determined by ethanol

fixation followed by RNA digestion and P1 staining and was analyzed as

described previously (91).

Isolation of Nuclear Extracts and Mobility Shift Assays. Jurkat cellswere cultured at 2 x 1 06 cells/mI and stimulated with capsaicin, RTX,

and/or PMA in complete medium, as indicated. Cells were then washed

twice with cold PBS and resuspended in 100 �l of buffer A [20 m�i HEPES

(pH 8.0), 10 mM KCI, 0.15 mr�i EGTA, 0.15 mp�i EDTA, 0.15 m,�i spermidine,0.15 m�i spermine, 1 m�i DTT, 0.5 m� PMSF, and 0.5 pg/mI each of

leupeptin, pepstatin, and apronitin] containing 0.25% NP4O. After lysis at

4#{176}Cduring 5 mm, 20 �.tl of sucrose restore buffer [50 m� HEPES (pH 7.0),

0.25 m� EDTA, 1 0 mM KCI, and 70% sucrose] were added. Lysates were

spun at 5,000 rpm in an Eppendorf centrifuge at 4�C for 5 mm, and the

resulting pellets were resuspended in 100 �.tl of buffer B [20 mt�i HEPES

(pH 8.0), 50 mM NaCI, 25% glycerol, 0.15 mi�i EGTA, 0.25 mp�i EDTA, 1.5

mM MgCI2, 1 mM DTT, and the protease inhibitors as described above] and

centrifuged at 5,000 rpm at 4�C for 5 mm. Nuclei were resuspended in 50

I.Ll of buffer C (the same as buffer B, except that the concentration of NaCIwas 400 mM). After rotation at 4�C for 30 mm, nuclei were centrifuged at

1 0,000 rpm for 1 0 mm, and nucleoproteins were recovered from the

supematant. Protein concentration was determined by the Bradfordmethod (Bio-Rad, Richmond, CA). For the EMSA assay, a double-

stranded oligonucleotide containing the AP-1 site of the metallothionine

promoter was used. The binding reaction mixture contained 3 �.tg of

nuclear extract, 0.5 �g of poly(deoxyinosinic-deoxycytidylic acid) (Phar-

macia Fine Chemical, Piscataway, NJ), 20 m� HEPES (pH 7), 70 m� NaCI,

2 m� DTT, 0.01 % NP4O, 100 �g/ml BSA, 4% Ficoll, and 100,000 cpm of

end-labeled DNA fragments in a total volume of 20 s.d. When indicated, the

binding reaction was incubated in the presence of various concentrations

of capsaicin. After a 20-mm incubation at 4#{176}C,the mixture was electro-

phoresed through a native 6% polyacrylamide gel containing 89 m�

Tris-borate, 89 mM boric acid, and 1 m� EDTA. Gels were preelectro-

phoresed for 30 mm at 225 V and then for 2 h after loading the samples.

These gels were dried and exposed to XAR film at -70#{176}C.

Transient Transfections and Luciferase Activity. Two x 1 O� Jurkatcells were transfected by electroporation at 126 V/1700 �Fa/72fl in aBTX-600 electroporator (BTX, Inc., San Diego, CA) with 20 �g of the

following plasmids: (a) The AP-1 -Luc plasmid constructed by insertingthree copies of a SV4O Api binding site into the Xho site of pGL-2

promoter vector (Promega, Madison, WI); and (b) the NF-AT-Luc plasmid

that contains three copies of the NF-AT binding site of the interleukin 2

promoter inserted in the pGL-2 vector. Twenty-four h after transfection,

cells were stimulated as indicated for 6 h and then lysed in 25 mu�i

Tris-phosphate (pH 7.8), 8 m� MgCI2, 1 m� DTT, 1 % Triton X-100, and

1 5% glycerol. Luciferase activity was measured in a luminometer

(Berthold). The background obtained with the lysis buffer was subtracted

in each experimental value. Experiments were performed in duplicates,

and the mean relative light units value was calculated. All the experiments

were repeated at least three times.Cellular Stimulation and Western Blots. To detect ERK 1/2, Bcl-2,

p53, c-Fos, and c-Jun protein expression, Jurkat cells (1 x 106 cells/mI)

were stimulated with capsaicin or RTX for the indicated times at 37#{176}C.

Then the cells were spun, and the resulting pellet was resuspended in 50

�Ll of lysis buffer [20 mM HEPES (pH 8.0), 10 mi�i KCI, 0.15 m� EGTA, 0.15

mM EDTA, 0.5 mM Na3VO4, 5 m� NaFI, 1 m� DTT, 1 �g/ml leupeptin, 0.5

�g/ml pepstatin, 0.5 �g/ml apronitin, and 1 m� PMSF) containing 0.5%

NP4O. After 5 mm on ice, cytoplasmic proteins were obtained by centrif-

ugation, boiled in Laemmli buffer, and electrophoresed in 10% SDS-

polyacrylamide gels. Separated proteins were transferred to nitrocellulose

membranes, and immunodetection of specific proteins was carried out

with primary antibodies using an enhanced chemiluminescence system

(Amersham, Buckinghamshire, United Kingdom).

In-Gel JNK Enzymatic Assay. Stimulation of Jurkat cells was

stopped by washing cells in ice-cold PBS followed by addition of 100 �.Jof lysis buffer [25 mM HEPES (pH 7.5), 0.3 PA NaCI, 1 .5 m�i MgCI2, 0.2 mr�i

EDTA, 0.5 mM DTT, 20 mM glycerolphosphate, 0.1 mp�i Na3VO4, 0.1%

Triton X-100, 2 �tg/ml leupeptin, and 2 mi�i PMSF). After rotating samples

at 4#{176}Cfor 15 mm, they were centrifuged at 10,000 x g for 10 mm, the

supematants were recovered, and the protein concentration was deter-

mined by the Bradford method (Bio-Rad). Cell lysate proteins (100 �i.g/

lane) were denatured by boiling in SDS-containing sample buffer and

resolved by SDS-PAGE on a 10% gel containing 50 �.i-g/ml of glutathione

S-transferase-Jun fusion protein. Gels then underwent denaturation and

renaturation treatments as described previously (72), followed by incuba-

tion in 10 ml of kinase buffer [20 m� HEPES (pH 7.6), 20 m� MgCl2, 2 mi�iDir, 5 mM j3-glycerolphosphate, 0.1 m� Na3VO4, and 20 mi�i Al?] con-

taming ioo �.iCi of[-y-32P]ATP (6,000 Ci/mmol) at 30#{176}Cfor 1 h. The reaction

was achieved by washing the gel in a solution containing 5% trichloro-acetic acid and 1 % pyrophosphate at 22#{176}Cfollowed by gel drying and

autoradiography.

AcknowledgmentsWe thank Dr. S. Gutkind (NIH, Bethesda, MD) for the glutathione 5-transferase-Jun(1-73) plasmid and Dr. S. Gelley (Institute for General

and Experimental Pathology, University of lnnsbruck, Austria) forthe CEM

and 9F3 cells.

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induction ofapoptosis byvanilloid compounds doesnot...

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