Activation of c-Jun NH2-terminal Kinase and Subsequent CPP32/Yama duringTopoisomerase Inhibitor b-Lapachone-induced Apoptosis through anOxidation-dependent Pathway1
Institute of Toxicology, College of Medicine, National Taiwan University [S-G. S., S-C. S., M-L. K.]; Division of Cancer Research, National Health Research Institute [S-E. C.];and Institute of Anatomy, School of Life Sciences, National Yang-Ming University [Y-P. C.], Taipei, Taiwan
ABSTRACT
b-Lapachone (b-Lap) has been found to inhibit DNA topoisomerases(Topos) by a mechanism distinct from that of other commonly knownTopo inhibitors. Here, we demonstrated a pronounced elevation of H2O2
and O22 in human leukemia HL-60 cells treated with b-Lap. Treatment
with other Topo poisons, such as camptothecin (CPT), VP-16, and GL331,did not have the same effect. On the other hand, antioxidant vitamin C(Vit C) treatment effectively antagonizedb-Lap-induced apoptosis. Thissuggested that a reactive oxygen species (ROS)-related pathway wasinvolved in b-Lap-induced apoptosis program. We also found that c-JunNH2-terminal kinase (JNK) but not p38 mitogen-activated protein kinaseor extracellular signal-regulated kinase 1/2 was persistently activated inapoptosis induced byb-Lap. Overexpression of a dominant-negative mu-tant mitogen-activated protein kinase kinase kinase 1 (MEKK1-DN) ortreatment with JNK-specific antisense oligonucleotide or Vit C all pre-ventedb-Lap-induced JNK activation and the subsequent apoptosis. Onlythe expression of MEKK1-DN, not Vit C treatment, blocked the JNKactivity induced by CPT, VP-16, or GL331. These results confirm againthat ROS acts as a mediator for JNK activation during b-Lap-inducedapoptosis. Furthermore, we found thatb-Lap can stimulate CPP32/Yamaactivity, which was, however, markedly inhibited by the MEKK1-DNexpression or Vit C treatment. Again, CPT-induced CPP32/Yama activa-tion can be abolished by MEKK1-DN but not by Vit C treatment. Takentogether, these results indicate thatb-Lap but not other Topo inhibitorstriggers apoptosis signaling,i.e., JNK and subsequent CPP32/Yama acti-vation are mediated by the generation of ROS.
INTRODUCTION
b-Lap3 (3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2-b]pyran-5,6-di-one) is a naturally occurring plant quinone obtained from the lapachotree (Tabebuia avellanedae), which is native to South America (1).b-Lap possesses a variety of pharmacological effects, including anti-bacterial, antifungal, and antitrypanocidal activities (2–4). In addition,it prolongs the survival of mice infected with Rauscher leukemiavirus, probably through inhibition of reverse transcriptase (5, 6).b-Lap is also a potent DNA repair inhibitor that sensitizes tumor cellsto DNA-damaging agents (7, 8). Several lines of evidence suggest thatb-Lap can directly target Topo I (9) and Topo II (10) and inhibit theiractivity, which results in cytotoxicity. However, the inhibitory mech-anism ofb-Lap is distinct from that of other typical Topo inhibitors,
such as CPT. More recent studies have shown thatb-Lap potentiallyinduces apoptotic cell death in human leukemia and prostate cancercell lines in a p53-independent pathway (11, 12). The increase of ROSby b-Lap was mainly attributed to apoptosis in human leukemicHL-60 cells (13). The selective action ofb-Lap in inducing apoptosisin human leukemia and prostate cancer cells has implicated its poten-tial clinical utility against both cancers. However, the signaling trans-duction pathway or pathways that lead to apoptosis program in re-sponse tob-Lap in such cell system are not known completely.
Response to numerous types of extracellular signals is mediated byMAPKs, which are members of a serine/threonine kinase family (14,15). A well-defined MAPK subfamily consists of JNKs, also calledstress-activated protein kinases, which are responsible for a variety ofstresses (16). Activation of JNKs requires dual phosphorylation atconserved threonine and tyrosine residues by MAPK kinase 4 or JNKkinase, which in turn can be phosphorylated by upstream kinaseMEKK1 (17). JNK activity can be induced by diverse stimuli, such asgrowth factors, cytokines, certain protein synthesis inhibitors, UVirradiation, heat shock, H2O2, and osmotic shock (18–23). Recentemerging evidence suggest that sustained activation of JNK is arequisite for the initiation of apoptosis. The overexpression of MEKK,the JNK kinase kinase, had a lethal effect on fibroblasts (24). Inaddition, overexpression of activated JNK caused cell death in trans-fected Jurkat cells. In contrast, expression of a dominant-negativemutant of MAPK kinase 1 or JNK prevented the UV-C- andg-irra-diation-induced cell death (25). Various well-known chemotherapeu-tic drugs, such as Adriamycin, vinblastine, VP-16, and CPT, are alsocapable of activating JNK. These drugs are critical in triggeringapoptosis program in different cell lines (26, 27). These data suggestthat the JNK kinase cascade may dominantly participate in apoptosis.
ICE and related cysteine-proteases, such as CED-3, CPP32/Yama,Ich-1/Nedd2, Ich-2/ICErel-II/TX, or Mch2, are thought to be down-stream regulators of apoptosis (28, 29). Overexpression of theseproteases leads to apoptosis of various cell types (30). Although someof these proteases seem to constitute a multiple protease cascade, themolecular mechanism of the cascade triggering is poorly understood.Interestingly, a recent study indicated that JNK induction by antican-cer drugs can lead to the activation of CED-3-like protease and,ultimately, cell death (27). This finding provided a relevance todelineate the signaling flow from upstream JNK to downstream ex-ecutor cysteine protease in apoptotic cell death program.
Here, we examined whether the JNK signaling pathway is involvedin b-Lap-induced apoptosis in human leukemic HL-60 cells. Our datashowed thatb-Lap treatment induced sustained activation of JNK butnot other MAPK subfamilies,i.e., ERK1/2 and p38, during the celldeath process.b-Lap was also found to induce a large amount of ROSbut not the other Topo inhibitors, such as VP-16, CPT, and GL331.Antioxidant ascorbic acid can effectively inhibit the production ofROS, JNK activation, and ultimate apoptosis induced byb-Lap;ascorbic acid failed to prevent other Topo inhibitor-induced JNKactivation and cell death. Furthermore, expression of a MEKK1-DNor treatment with JNK-specific antisense oligonucleotide can abolish
Received 5/8/98; accepted 11/13/98.The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby markedadvertisementin accordance with18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by National Science Council of Taiwan (Republic of China)Grant NSC88-2314B-002-094.
2 To whom requests for reprints should be addressed, at Laboratory of Molecular andCellular Toxicology, Institute of Toxicology, College of Medicine, National TaiwanUniversity, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan. Phone: 886-2-3970800 ext.8607; Fax: 886-2-3410217.
JNK activity and subsequent CPP32/Yama activity. These resultsindicate that, unlike other Topo inhibitors,b-Lap induces JNK/CPP32-associated apoptosis mediated by an oxidation-dependentfashion.
MATERIALS AND METHODS
Cell Culture and Chemicals. HL-60 cells, a human promyelocytic leuke-mia cell line, were obtained from American Type Culture Collection (Rock-ville, MD). Cells were maintained in a humidified 5% CO2 atmosphere andcultured in RPMI supplemented with 10% FCS, 2 mM L-glutamine, 100units/ml penicillin, and streptomycin.b-Lap was prepared according to theprocedures described by Schaffner-Sabbaet al. (1). b-Lap was dissolved inice-cold absolute alcohol as a stock solution at 10 mM concentration and storedin aliquots at220°C. DCFH-DA and HE were obtained from MolecularProbes. Ascorbic acid, CPT, etoposide, and propidium iodide were obtainedfrom Sigma Chemical Co.
Intracellular Hydrogen Peroxide Determination by Fluorescence Mi-croscopy. Intracellular hydrogen peroxide production was monitored by flu-orescence microscopy using DCFH-DA. Briefly, cells (23 105) were pre-treated with 25mM Vit C for 1 h and then coincubated with 50mM DCFH-DAin the absence or presence of anticancer drugs at 37°C for 1 h. After incuba-tion, cells were washed and resuspended in ice-cold PBS and placed oncoverglass at dark for fluorescence microscopy (Nikon) analysis.
Superoxide Anion Determination by Flow Cytometric Analysis. Thefluorochrome HE was used to measure superoxide anion generation as de-scribed (31). Briefly, after drugs treatment, cells were incubated at 37°C for 15min in the presence of 2mM HE, followed by immediate analysis of fluoro-chrome incorporation in a FACScan flow cytometry (Becton Dickinson).
Hypodiploid Cell Assay. Cells were harvested and washed with PBS, andhypodiploid cells were analyzed by flow cytometer as described previously(13).
DNA Fragmentation Assay. HL-60 cells were treated with various anti-cancer drugs or combined with antioxidants for 4 h (indicated in Fig. 1C).After that, treated cells were harvested and washed with PBS, and DNAfragmentation was analyzed by agarose gel electrophoresis as described pre-viously (13).
Establishment of HL-60/MEKK1-DN Clones. Transfection was createdby electroporation (model T800; BTX, San Diego, CA) of HL-60 cells with theglucocorticoid-inducible pSRa-MEKK(K432M) vector (a gift from Dr.Michael Karin of the Department of Pharmacology, School of Medicine,University of California, San Diego, La Jolla, CA). Briefly, cells were sus-pended in 1 ml of HEPES-buffered saline containing plasmid DNA and thenreceived electric treatment as follows: electric amplitude, 900 V; pulse width,99 ms. After 10 min on ice, the cells were transferred to fresh completemedium and cultured for 24 h before addition of hygromycin. To avoidproblems with clonal variation, the transfected cells were selected for hygro-mycin for 4 weeks, and all of the clones were pooled.
Immunoprecipitation and Kinase Activity Assays. Cell lysis and im-mune complex kinase assays were performed as described (32). HL-60 cellswere treated with different drugs, washed twice with ice cold PBS, and lysedin buffer containing 20 mM HEPES (pH 7.4), 50 mM b-glycerophosphate, 1%Triton X-100, 10% glycerol, 2 mM EGTA, 1 mM DTT, 10 mM sodium fluoride,1 mM sodium orthovanadate, 1mg/ml leupeptin, 1mg/ml aprotinin, and 1 mMphenylmethylsulfonyl fluoride. The soluble extracts were prepared by centrif-ugation at 14,500 rpm for 15 min at 4°C. Following normalization of proteinconcentration, equal amounts of protein were incubated with protein A-Sepha-rose and anti-JNK1 (1mg; C17; Santa Cruz Biotechnology, Santa Cruz, CA),anti-ERK1 (1mg; C16; Santa Cruz Biotechnology), or anti-p38 (1mg; N20;Santa Cruz Biotechnology) for 3 h at 4°C. The immune complexes werewashed twice with lysis buffer and then once with kinase assay buffer [20 mM
MOPS (pH 7.2), 2 mM EGTA, 20 mM MgCl2, 1 mM DTT, and 0.1% TritonX-100], following which they were resuspended in 20ml of kinase assay buffercontaining 5mCi of [g-32P]ATP, 30mM cold ATP, and 2mg of substrate andincubated for 20 min at 30°C. Reactions were terminated by the addition of theSDS sample buffer and boiling for 5 min. The phosphorylated proteins wereresolved by SDS-PAGE and visualized by autoradiography. GST-c-jun(1/79)was used as a substrate for JNK1, myelin basic protein was used for assayingERK1, and ATF-2 was used as a substrate for p38.
Antisense Oligonucleotides Treatment.The rationale of JNK1 antisenseoligonucleotide design and treatment is based on the report by Seimiyaet al.(27). The JNK1-specific antisense (59-GTCACGCTTGCTTCTGCTCAT-GAT-39) and sense (59-ATCATGAGCAGAAGCAAGCGTGAC-39) phospho-rothioates were synthesized and purified by high-performance liquid chroma-tography (Genset Co.). These sequences represent amino acids21 to 17 ofJNK1. The oligonucleotides were dissolved in distilled and sterilized water andadded into culture medium. After treatment with the oligonucleotides for 16 h,cells were analyzed the JNK1 activity and JNK1 protein level.
Western Blot Analysis. Western blot was measured by the method de-scribed previously (13). Briefly, cell lysates were prepared, electrotransferred,and then immunoblotted with anti-JNK1, anti-ERK1, anti-p38, anti-PARP, andanti-MEKK1 (C22) antibody (Santa Cruz Biotechnology). Detection was per-formed with Western blotting reagent ECL (Amersham), and chemilumines-cence was exposed by the filters of Kodak X-Omat films.
Yama/CPP32 Activity Assay. CPP32 protease activity was measured bythe method described previously (30). In brief, cytosolic extracts were pre-pared by repeated cycles of freezing and thawing in 300ml of extraction buffer[12.5 mM Tris (pH 7.0), 1 mM DTT, 0.125 mM EDTA, 5% glycerol, 1 mM
phenylmethylsulfonyl fluoride, 1mg/ml leupeptin, and 1mg/ml aprotinin]. Celllysates (100mg) were then diluted with 1 ml of assay buffer [50 mM Tris, 1 mM
EDTA, and 10 mM EGTA (pH 7.0)] and incubated at 37°C for 30 min in darkwith 10 mM fluorescence substrate, Ac-DEVD-AMC. The fluorescence of thecleaved substrate was determined using a spectrofluorometer (Hitachi F-3000)set at an excitation wavelength of 380 nm and an emission wavelength of 460nm.
RESULTS
Effect of b-Lap on ROS-related Apoptosis.To verify whetherb-Lap induces ROS generation, we used DCFH-DA fluorescent dyeto detect intracellular peroxides level and HE dye to detect superoxideanion (O2
2). Under examination by fluorescence microscope, wefound that;94 6 1.01% of b-Lap (1 mM)-treated leukemia cellsdisplayed evident DCFH fluorescence after 30 min treatment (Fig.1A). No such fluorescence was observed in the control or other Topoinhibitor-treated cells. Ascorbic acid (Vit C) treatment completelyprevented the DCFH fluorescence induced byb-Lap. We furtherexamined the O22 level using HE detection and quantitated by flowcytometry. Fig. 1B shows that;19.776 0.86% HL-60 cells producedsignificant amount of O2
2 following b-Lap (1 mM) treatment. Simi-larly, we did not detect the production of O2
2 in the control orVP-16-, CPT-, or GL331-treated HL-60 cells (Fig. 1B). We alsoobserved that Vit C almost blocked the elevation of O2
2 by b-Lap.We then examined whether ROS generation was involved in
b-Lap-mediated apoptosis in HL-60 cells. As Fig. 1C shows, Vit Ctreatment effectively preventedb-Lap-induced internucleosomalDNA fragmentation, a hallmark of apoptosis, whereas Vit C failed toinhibit CPT-, VP-16-, or GL331-induced DNA fragmentation. Theinhibitory effect of Vit C onb-Lap-induced apoptosis was also seenin morphological characteristics by Hoechst 33258 staining (data notshown). Our previous study has indicate thatb-Lap induced;70%apoptosis of HL-60 cells at 1mM for 24 h (13).
Effect of b-Lap on MAPKs Activity. To explore whether theapoptosis-related signaling pathway(s) are activated in HL-60 cells inresponse tob-Lap, we examined JNK, ERK1/2, and p38 activities byusing immunocomplex kinase assay. Fig. 2Ashows that, following 1mM b-Lap treatment, JNK activity was detectable and increased at 15min and was sustained up to 2 h. Western blot analysis showed thatthese JNK activations were not due to enhanced expressions of JNKprotein (Fig. 2A,bottom). However, under the same dose, we did notdetect any significant p38 or ERK1/2 activation during the apoptosisprocess (Fig. 2,B and C). A kinetic study on DNA fragmentationshows that an initial DNA fragmentation occurred at 2 h after 1mM
Fig. 1.b-Lap- and other Topo inhibitor-induced ROS generation and DNA fragmentation in HL-60 cells.A, intracellular peroxide generation in HL-60 cells was detected by fluorescencemicroscopy using a peroxide-sensitive dye, DCFH-DA. Cells were pretreated with 25mM Vit C for 1 h and then exposed to 1mM b-Lap or other Topo inhibitors for 30 min in the presenceof 50 mM DCFH-DA. The quantities of DCFH fluorescence intensity were detected by using flow cytometry (13). Briefly, cells were coincubated with 50mM DCFH-DA in the absence orpresence ofb-Lap or other Topo inhibitors at 37°C for indicated time. After incubation, cells were resuspended in ice-cold PBS and placed on ice for flow cytometry analysis.B, superoxidegeneration was quantitated by flow cytometry using a dye, HE. After drug treatment, cells were washed twice with PBS and incubated with 2mM HE for 15 min at 37°C.C, DNA fragmentationwas assayed by agarose gel electrophoresis. Cells were pretreated with 25mM Vit C for 1 h and then exposed to 1mM b-Lap or other Topo inhibitors (CPT, 1mM; VP-16, 1mM; and GL331,1 mM) for 4 h. Cellular DNA was extracted and subjected to 2% agarose gel as described in “Materials and Methods.”
b-Lap treatment (Fig. 2D). These observations indicated that JNKactivation preceded the occurrence of DNA fragmentation (apoptosis).
A dose-response experiment for these kinases was also studied.Treatment of HL-60 cells with 0.5, 1, and 2mM b-Lap induced 1.61-,7.5-, and 7.1-fold increase of JNK activity, respectively (Fig. 3A). Wedid not observe any change in ERK1/2 or p38 activity in HL-60 cellseven when the dose was increased up to 2mM (Fig. 3,B andC). Thissuggests that JNK but not ERK1/2 or p38 is persistently activatedduring b-Lap-induced apoptosis.
Role of JNK Activation in b-Lap-induced Apoptosis. To verifythe role of JNK in b-Lap-induced apoptosis, we transfected andexpressed a MEKK1-DN plasmid [pSRa-MEKK1 (K432M)], whichwas under control by a glucocortcoid-inducible promoter, in HL-60cells. We subsequently treated the MEKK1-DN-transfected HL-60(HL-60/MEKK-DN) and parental HL-60 cells with 1mM b-Lap andthen examined the JNK activity and apoptosis. Under the presence ofDex, theb-Lap-stimulated JNK activation was inhibited down to thebasal level in HL-60/MEKK-DN cells (Fig. 4A, top). To confirm theexpression of the MEKK1-DN protein, the transfectants were ana-lyzed their MEKK1 protein level under the presence or absence ofDex by Western blotting with an antibody specific to MEKK1. In-deed, HL60/MEKK-DN cells expressed a truncated form of MEKK1(MEKKD) protein in the presence of Dex. (Fig. 4A, bottom). Theseresults indicate that the MEKK1-DN expression vector is functional insuppressing JNK activity.
Fig. 2. Effects ofb-Lap on the activation of JNK, ERK1/2, and p38. HL-60 cells weretreated with 1mM b-Lap for the indicated times, after which cells were harvested and thecytosolic fraction analyzed for JNK1 (A), ERK1/2 (B), or p38 (C). The kinases activitieswere determined by immune complex kinase assay (top), and the protein levels weredetermined by Western blot (bottom) as described in “Materials and Methods.” GST-c-jun, myelin basic protein, and ATF-2 were used as a substrate for JNK1, ERK1/2, and p38,respectively.Numbers below lanes, folds of kinase activity.D, time-dependent DNAfragmentation induced byb-Lap in HL-60 cells. Cells were treated with 1mM b-Lap for0–8 h. After treatment, cellular DNA was extracted and subjected to 2% agarose gelelectrophoresis.
Fig. 3. Dose-response relationship forb-Lap-induced JNK1, ERK1/2, and p38 activity.HL-60 cells were treated with the indicated concentrations ofb-Lap for 1 h. Cells wereharvested, and JNK1, ERK1/2, and p38 activities in the lysates were analyzed usingimmune complex kinase assays.A, effect ofb-Lap on JNK1 activity.B, effect ofb-Lapon ERK1/2 activity.C, effect ofb-Lap on p38 activity.
In the presence of Dex, HL-60/MEKK-DN cells became resistant toDNA fragmentation (apoptosis) induced byb-Lap (Fig. 4B). In con-trast, in the absence of Dex, HL-60/MEKK-DN cells were equallysensitive tob-Lap compared with parental HL-60 cells either with orwithout Dex (Fig. 4B). Consistent with other reports (9), we alsoobserved that HL-60/MEKK-DN cells, under the presence of Dex,were resistant to CPT-, VP-16-, and GL331-induced apoptosis (datanot shown). Given direct evidence to prove the importance of JNK inb-Lap-induced apoptosis, we treated HL-60 cells with JNK1-specificantisense oligonucleotide. HL-60 cells were treated with 25mM
JNK1-specific antisense oligonucleotide for 16 h before the additionof 1 mM b-Lap for another 4 h. Upon treatment with JNK1 antisenseoligonucleotide for up to 16 h, cellular amounts of JNK1 proteindecreased in HL-60 cells. Control sense oligonucleotide marginallyaffected the JNK1 contents (data not shown). Under this condition, theb-Lap induction of apoptosis could obviously be prevented by theJNK1-specific antisense oligonucleotide phosphorothioate but not byits sense oligonucleotide (Fig. 5A). The antisense oligonucleotidetreatment was nontoxic to the HL-60 cells because antisense-treatedcells retained membrane integrity and normal proliferation rate for upto 24 h (data not shown). This observation strongly suggests that thereduction of JNK activity by antisense oligonucleotide is not anartifact of nonspecifically toxic to cells. Immunocomplex kinase assayshows again thatb-Lap-elicited JNK activity could be blocked byJNK1 antisense oligonucleotide but not by its sense oligonucleotide(Fig. 5B).
These results showed that interfering with JNK signaling can pre-ventb-Lap-induced cell death, an indication that JNK kinase cascadeis required for cell death signaling.
Effect of ROS on b-Lap-elicited JNK Activation. The resultsobtained thus far in this study led us to propose that ROS generation
Fig. 4. Effect of inducible MEKK1-DN onb-Lap-induced apoptosis.A: top, inhibitionof b-Lap-induced JNK1 activation by expression of MEKK1-DN;bottom, immunoblotanalysis of MEKK1 protein level in MEKK1-DN transfectants and parental HL-60 cells.Transfected MEKK1-DN HL-60 cells were incubated with or without 1mM Dex for 16 h.Cells were then treated with 1mM b-Lap for 1 h. Cells were collected, and cell lysateswere assayed for JNK1 activity with an immune complex kinase assay and for MEKKDprotein level with a MEKK1-specific antibody.B, inhibition of b-Lap-induced DNAfragmentation by expression of MEKK1-DN. Transfected MEKK1-DN and controlHL-60 cells were incubated with 1mM Dex for 16 h. The cells were then treated with 1mM b-Lap for 4 h. After treatment, cellular DNA was extracted and subjected to 2%agarose gel electrophoresis.
Fig. 5. Effect of JNK1-specific antisense oligonucleotide onb-Lap-induced apoptosisand JNK activity in HL-60 cells.A, inhibition of b-Lap-induced apoptosis by JNK-specific antisense oligonucleotide. Cells were treated with 25mM JNK-specific antisenseor sense oligonucleotide phosphorothioates for 16 h before the addition of 1mM b-Lap foranother 4 h. The apoptosis assay was performed by FACScan as described in “Materialsand Methods.”B, inhibition of b-Lap-elicited JNK activity by JNK-specific antisenseoligonucleotide. Cells were treated as described inA. JNK kinase assay was performed asdescribed in “Materials and Methods.”
may initiate JNK activation. To resolve this issue, we examined theeffect of Vit C on JNK activity in HL-60 cells treated withb-Lap orother drugs. As Fig. 6A shows,b-Lap induced JNK activation wassuppressed by the addition of Vit C. However, Vit C did not affectCPT-, VP-16-, or GL331-stimulated JNK activation. Quantitation ofsub-G1 cells by flow cytometer also revealed that Vit C preventedb-Lap-mediated apoptosis but failed against other Topo inhibitors-induced apoptosis (Fig. 6B). These data suggested thatb-Lap initiallyinduces the generation of ROS that can activate JNK, which, in turn,triggered the apoptosis program.
CPP32/Yama as a Downstream Target for JNK.BecauseCPP32/Yama protease plays an important role in various drug-induced apoptoses, we examined the role of CPP32/Yama in theb-Lap-mediated apoptotic process. To address this issue, we deter-mined the cleavage of PARP protein and CPP32/Yama activity byusing Western blotting and spectrofluorometry, respectively. WhenHL-60 cells were treated with 1mM b-Lap or CPT, cleavage of PARPprotein was obviously detected after exposure to both drugs. How-ever, onlyb-Lap-mediated PARP cleavage was prevented by Vit C(Fig. 7A). The anti-PARP antibodies used in this study recognized anepitope at the NH2 terminus of the PARP polypeptides; thus theNH2-terminal Mr 30,000 fragment can be detected by immunoblot-ting. Consistent with the immunoblotting, we found thatb-Lap-induced increase of CPP32/Yama activity, which is determined byusing a fluorogenic tetrapeptide substrate Ac-DEVD-AMC, was com-pletely inhibited by Vit C. Vit C treatment did not affect CPT-inducedCPP32/Yama activation (Fig. 7B). However, when JNK activity wassuppressed by expression of MEKK-DN, bothb-Lap- and CPT-induced CPP32/Yama activation were effectively diminished (Fig.
Fig. 6. Effect of the antioxidant Vit C on the activation of JNK1 byb-Lap and otherTopo inhibitors.A, HL-60 cells were incubated with 25mM Vit C for 1 h prior to 1mM
b-Lap or other Topo inhibitors (CPT, 1mM; VP-16, 1mM; GL331, 1mM) for 1 h. Cellswere harvested, and JNK1, ERK1, and p38 activities were analyzed by immune complexkinase assay.B, cell death was determined after 4 h of treatment with various drugs(b-Lap, 1 mM; CPT, 1mM; VP-16, 1mM; and GL331, 1mM) by flow cytometry assay.Columns, means of three independent experiments;bars, SD.
Fig. 7. Effect of Vit C on PARP cleavage and CPP32/Yama activation byb-Lap andCPT.A, effect of Vit C on PARP cleavage induced byb-Lap and 1mM CPT. HL-60 cellswere incubated with 25mM Vit C for 1 h prior to 1mM b-Lap or 1mM CPT for 4 h. Theproteolytic cleavage of PARP was detected by anti-PARP antibody (Santa Cruz Biotech-nology) using Western blot analysis.B, effect of Vit C on CPP32/Yama activity. Thecytosolic fraction was prepared from cells that were treated as described inA and theCPP32/Yama activity was measured as described in “Materials and Methods.”C, theCPP32/Yama activity was measured in transfected MEKK1-DN HL-60 cells. TransfectedMEKK1-DN HL-60 cells were incubated with or without 1mM Dex for 16 h. The cellswere then treated with 1mM b-Lap for 4 h. Cells were collected and cell lysates wereassayed for CPP32/Yama activity.Columns, means of three independent experiments;bars, SD.
7C). These data suggest that CPP32/Yama protease acts as a down-stream executor of JNK signaling triggered byb-Lap.
DISCUSSION
In this study, the capability of inducing ROS generation byb-Lapis clearly demonstrated in human leukemia HL-60 cells. In contrast,we did not detect any significant ROS generation in same cell systemby treatment with other commonly known Topo inhibitors. Thisindicates that the mechanism of action ofb-Lap is somehow differentfrom those of other Topo inhibitors. According to previous studies,the inhibitory mechanism ofb-Lap on Topo I and II is possibly dueto directly chemical modification of these enzymes. This is charac-teristically different from other Topo poisons, which inhibit Topo inan ATP-dependent manner (10). Other findings raised by Liet al.(12)showed that the cytotoxic effect of Topo poisons is always associatedwith G2-M arrest. On the one hand, cell death induced byb-Lap inhuman prostate PC-3 cancer cells was associated with increases in thefractions of G1, sub-G1, and cells with higher DNA ploidy. Thestructure ofb-Lap is relatively similar to that of menadione. Frydmanet al. (10) did not rule out the possibility that free radical formation isinvolved in the proposed chemical modification of the Topo-DNAcomplex. They also found thatb-Lap possesses a thiol reactivityunderin vitro conditions. These findings seemed to indicate that thecharacteristic of ROS production is likely one of major differentmechanisms betweenb-Lap and other commonly used Topo poisons.It is also relevant that Topo may be one of targets attacked byb-Lap-induced ROS, which ultimately leads to cell death. Whetherthe inhibition of Topo byb-lap is through ROS modification remainsunknown and need further investigation.
Antioxidant Vit C treatment effectively blockedb-Lap-inducedapoptosis but failed to prevent cell death induced by other Topopoisons. Another antioxidant,N-acetyl-L-cysteine, also strongly an-
tagonized the apoptosis induced byb-Lap (data not shown). ROSgeneration may specifically be involved inb-Lap- but not in otherTopo poison-mediated apoptosis programs. Supportive of this finding,many drugs, such as 1-b-D-arabinofuranosylcytosine, ceramide, Ad-riamycin, and methylprednisolone, induced apoptosis, which is ac-companied with an increase of intracellular oxidants and elevatedlevel of lipid peroxidation (22, 26, 33, 34).
Recent studies have suggested thatb-Lap is capable of accepting asingle electron to form semiquinone radical. Quinone radicals undergooxidation in the presence of quinone reductase, such as NADH,NADPH, or SOD, to produce H2O2 and O2
2 in mitochondria ormicrosomes (35, 36). A decrease of mitochondrial membrane poten-tial was correlated with an elevation of ROS in HL-60 cells followingtreatment withb-Lap.4 These observations strongly indicate thatb-Lap may interfere with the mitochondrial function (i.e., electrontransport chain) through its thiol reactivity and subsequently lead tothe generation of ROS.
The role of JNK activation in apoptosis has been extensivelydiscussed (37). However, this study is the first to demonstrate that thenew drugb-Lap can stimulate a rapid and pronounced activation ofJNK during the onset of apoptosis. Our data further showed that ROSwas fully attributed tob-Lap-induced JNK activation, as evidenced bythe blocking effect of Vit C on JNK activity. Although ROS has beenreported to activate ERK1/2 or p38 kinase under certain experimentalconditions (38), we found in our study that only JNK, not otherMAPKs, was stimulated byb-Lap-generated ROS. Because the ac-tivities of these three MAPK subfamilies are tightly regulated by theirrespective upstream kinases (39), an initial molecule responsible forthe JNK-related signaling may have been highly reactive tob-Lap-generated ROS. A similar finding by Loet al. (40) showed that ROSacted as a mediator for cytokine-stimulated JNK activation in bovinechondrocytes. These and our data suggest that overstimulation of JNKactivity by excessive production of ROS may have to do with path-ological conditions in different cell types in response to exogenousstimuli. Expression of MEKK1-DN significantly abolishedb-Lap andother Topo poison-induced JNK activation and apoptosis. This findingis consistent with other previous studies, which suggested that JNKactivation plays a prominent role in apoptosis process induced by adiverse type of drugs (41). On the other hand, many studies (42, 43)have recently shown that JNK activation is involved in tumor necrosisfactor receptor-associated protein-mediated antiapoptotic signals inlymphocytes upon TNF treatment. These contrary findings suggestthat JNK exerts multifaceted roles in regulation of cellular survivaland death. ICE/CED-3-like proteases, such as ICE and CPP32, arefunctionally involved in apoptosis of many cell types (44). Consistentwith other studies, we demonstrated that CPP32 is activated duringb-Lap-induced apoptosis. Again, expression of MEKK1-DN or treat-ment with Vit C effectively abolished theb-Lap-induced CPP32activity in HL-60 cells. Only MEKK1-DN overexpression, not Vit C,was able to antagonize the Topo 1 inhibitor CPT-induced CPP32activity. This implies a commonly downstream role of CPP32 in JNKsignaling. However, a recent study showed that caspase can induceapoptosis by activating MEKK1, which, in turn, activates morecaspase activity, comprising a positive feedback loop (45). Whetherthis feedback regulation mechanism is involved inb-Lap-inducedcaspase activity and apoptosis remain elusive. Here, we found that theCPP32-specific tetrapeptide inhibitor, Ac-DEVD-CHO, only partiallyreducedb-Lap-induced apoptosis (data not shown). Thus, it is likelythat other ICE-like proteases may also be involved in such cell deathprocesses. Studies by Davis (46) showed that ICE-like cysteine pro-
4 S-G. Shiah and M-L. Kuo, unpublished data.
Fig. 8. A possible signaling pathway duringb-Lap-induced apoptosis. Initially,b-Lap,but not other Topo inhibitors, can elevate the intracellular ROS, which, in turn, triggersJNK activation, subsequent downstream CPP32/Yama activation, and, ultimately, celldeath. However, it does not exclude the possibility that theb-Lap-generated ROS maypoison Topo that also caused the activation of JNK signaling.
tease does not have a proline-directed serine/threonine residue that ispreferentially phosphorylated by ERK/JNK family. Therefore, itseems unlikely that JNK directly phosphorylated and activated theprotease. There must be additional transducers that connected the twoevents, JNK activation and the protease activation. These transducersmust be elucidated in a future study.
In conclusion, here we present the first evidence demonstrating thatb-Lap induces a novel ROS-dependent apoptosis program. In thisstudy, we delineated theb-Lap-induced apoptosis signaling pathwayin which ROS initially generated and, in turn, activated JNK that leadto triggering the CPP32 protease and facilitated apoptosis of HL-60cells (Fig. 8). However, other Topo inhibitors activated JNK, CPP32,and apoptosis (i.e., not through ROS generation).
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