Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases.

0.52 MB·English

by  Lizarte, F.S.

BrazilianJournalofMedicalandBiologicalResearch(2013)46:71-78,http://dx.doi.org/10.1590/1414-431X20121423
ISSN1414-431X
Kaurene diterpene induces apoptosis in
U87 human malignant glioblastoma cells
by suppression of anti-apoptotic signals
and activation of cysteine proteases
F.S. Lizarte Neto1, D.P.C. Tirapelli1, S.R. Ambrosio2, C.R. Tirapelli3, F.M. Oliveira4,
P.C. Novais1, F.M. Peria4, H.F. Oliveira4, C.G. Carlotti Junior1 and L.F. Tirapelli1
1DepartamentodeCirurgiaeAnatomia,FaculdadedeMedicinadeRibeira˜oPreto,UniversidadedeSa˜oPaulo,
Ribeira˜oPreto,SP,Brasil
2Nu´cleodePesquisaemCieˆnciaseTecnologia,UniversidadedeFranca,Franca,SP,Brasil
3Laborato´riodeFarmacologia,DepartamentodeEnfermagemPsiquia´tricaeCieˆnciasHumanas,
EscoladeEnfermagemdeRibeira˜oPreto,UniversidadedeSa˜oPaulo,Ribeira˜oPreto,SP,Brasil
4DepartamentodeClı´nicaMe´dica,FaculdadedeMedicinadeRibeira˜oPreto,UniversidadedeSa˜oPaulo,
Ribeira˜oPreto,SP,Brasil
Abstract
Gliomasarethemostcommonandmalignantprimarybraintumorsinhumans.Studieshaveshownthatclassesofkaurene
diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human
glioblastomacelllineU87totreatmentwithent-kaur-16-en-19-oicacid(kaurenoicacid,KA).Weanalyzedcellsurvivalandthe
inductionofapoptosisusingflowcytometryandannexinVstaining.Additionally,theexpressionofanti-apoptotic(c-FLIPand
miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of
mRNAlevelsinU87cellsthatwereeitheruntreatedortreatedwithKA(30,50,or70mM)for24,48,and72h.U87cellstreated
with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The
percentageofapoptoticcellswas9%forcontrolcells,26%forcellssubmittedto48hoftreatmentwith50mMKA,and31%for
cellssubmittedto48hoftreatmentwith70mMKA.Similarly,inU87cellstreatedwithKAfor48h,weobservedanincreasein
theexpressionofapoptoticgenes(caspase-8,-3)andadecreaseintheexpressionofanti-apoptoticgenes(miR-21 andc-
FLIP).KApossessesseveralinterestingpropertiesandinducesapoptosisthroughauniquemechanism.Furtherexperiments
willbenecessarytodetermineifKAmaybeusedasaleadcompoundforthedevelopmentofnewchemotherapeuticdrugsfor
thetreatmentofprimarybraintumors.
Keywords:Kaurenoicacid;Glioblastoma;miR-21;c-FLIP;Apoptosis
Introduction
Gliomas are the most common and lethal malignant Clearly, overcoming apoptotic resistance through the
primarybraintumorsinhumans.Glioblastomamultiforme reactivation of apoptosis in malignant cells will be
is the highest grade, the most aggressive and the most important forthe development ofnovelglioma therapies.
frequent glioma (1). Because gliomas are characterized Recently,severalnaturalproductsderivedfromplants
by a diffuse infiltrative growth into the surrounding brain have been reported to prevent tumorigenesis and
tissue, the complete surgical resection of glioblastoma suppress the growth of established tumors. Research
multiforme tumors is virtually impossible (2). In addition, has intensely focused on the identification of new herbal
high-grade gliomas exhibit only limited sensitivity to medicinesandonthemechanismsbehindtheanti-tumor
multimodal treatment with radiotherapy and chemother- properties ofexisting naturalproducts(4).
apy, which in large part is caused by the inherent and Many reports have extensively shown that kaurene-
potent resistance of the tumor cells to apoptosis (3). type diterpenes exert several biological activities. One
Correspondence: L.F. Tirapelli, Departamento de Cirurgia e Anatomia, FMRP, USP, Av. Bandeirantes, 3900, 14049-900 Ribeira˜o
Preto,SP,Brasil.E-mail:[email protected]
ReceivedAugust26,2011.AcceptedSeptember14,2012.FirstpublishedonlineJanuary11,2013.
www.bjournal.com.br BrazJMedBiolRes46(1)2013
72 F.S.LizarteNetoetal.
such kaurene-type diterpene, ent-kaur-16-en-19-oic acid flow cytometric analysis of annexin V staining, miR-21
(kaurenoic acid, KA), has been reported to have gene expression and the expression of anti-apoptotic (c-
antimicrobial activity against Bacillus subtilis (5,6), FLIP) and apoptotic (Fas, caspase-8 and caspase-3)
Staphylococcus aureus, Mycobacterium smegmatis genes that are known to be associated with the extrinsic
(5,7), Saccharomyces cerevisae, Escherichia coli, pathwayof apoptosis.
Cladosporium herbarium, andCandida albicans(5).
In addition to its antimicrobial and anti-parasitic Material and Methods
activities, KA also exerts cytotoxic effects. Cytotoxic and
anti-proliferative actions on tumor cell cultures (human Procedurefor KAisolation
breast cancer, human colon cancer and leukemia) have Certified dried leaves of Mikania hirsutissima (1.0kg)
been described for this diterpene (8). Interestingly, were purchased from Nutri Come´rcio de Ervas Ltda.
Cavalcanti et al. (9) evaluated the potential genotoxicity (Brazil). The plant material was pulverized and dichlor-
ofKAagainstChinesehamsterlungfibroblast(V79)cells omethane (3.5L) was exhaustively extracted at room
in vitro. They found that KA failed to induce significant temperature to yield 42.0g crude extract. This extract
DNA damage and micronucleus frequencies at concen- was suspended in 300 mL methanol-H O (9:1) and
2
trations of 2.5, 5, and 10mg/mL; however, the exposure filtered. The soluble fraction was partitioned using n-
of V79 cells to higher concentrations of KA (30 and hexane (300mL, partitioned 4 times). The solvent was
60 mg/mL)causedsignificantincreasesinthecelldamage evaporatedunderreducedpressure,resultingin6.0 gofa
index and frequency. This study supports a dose- hexane-solublefraction.
dependent cytotoxiceffect ofKA onnon-cancercells. The n-hexane-soluble fraction was chromatographed
Apoptosis,orprogrammedcelldeath,isaformofcell overaSigel60column(0.063-0.200 mm)usingvacuum
death that is defined by a characteristic set of morpho- chromatography with n-hexane, and increasing amounts
logical and biochemical changes. Apoptosis is a highly ofethylacetatewereusedforelution(16).Thisprocedure
regulated process that involves the activation of a series yielded12fractionsof250mLeach.Thesecondfraction
of cellular events that ultimately lead to cell death. (1730.0mg)waswashedwithcoldmethanoltoyieldent-
Apoptosis plays an important role in the inhibition of kaur-16-en-19-oic acid (KA; 800.0mg; Figure 1). The
cancer development, and impaired apoptosis is now purityofKAwasestimatedbythin-layerchromatography
recognized to be a key step in tumorigenesis (10). The using different solvent systems. Moreover, KA was also
activation of apoptotic pathways is a key mechanism by submitted to 1H and 13C nuclear magnetic resonance
whichcytotoxicdrugskilltumorcells(10,11).Indeed,the spectral data analysis, which indicated that the purity
induction of apoptosis is now considered to be an grade wasbetween 95and 98%.
important method for the assessment of the clinical
effectivenessofmanyanti-tumordrugsandisasignificant Cell culture
index used for the selection of new anti-tumor drugs The U87 human glioblastoma cell line (ATCC, USA)
(12,13). was cultured in Dulbecco’s modified Eagle’s medium
MicroRNAs(miRNAs)aresmall(,20-22nucleotides) (Sigma, USA) supplemented with 10% heat-inactivated
non-codingRNAsthatbindtocomplementaryrecognition bovineserum,100 U/mLpenicillinand50mg/mLstrepto-
sequences in mRNA, causing either mRNA degradation mycin/neomycininahumidifiedincubatorat37˚Cand5%
ortheinhibitionoftranslation.Thus,miRNAsarecapable carbon dioxide. All experimental procedures were
of effectively silencing their mRNA targets. This pre- approved by the Research Ethics Committee of Hospital
translational control is estimated to affect up to one-third
of all human transcripts, including transcripts that are
involvedincancer(14).Recently,microRNA-21(miR-21)
was shown to be strongly overexpressed in glioblastoma
tumors and cell lines.In the same study,the inhibition of
miRNA-21 by a locked nucleic acid led to increased
caspase-dependent apoptosis, suggesting that miR-21
couldbeactingasanoncogenebyinhibitingapoptosisin
glioma cells(15).
Althoughstudieshaveshownthatclassesofkaurene
diterpene have anti-tumor activity due to the induction of
apoptosis,therearenodataintheliteratureregardingthe
actionofKAongliomatumorcells.Here,weaddressthis
shortcoming by evaluating the cellular responses of the
U87 human glioblastoma cell line to KA treatment. We Figure1.Structureofent-kaur-16-en-19-oicacid(kaurenoicacid,
examined cell survival, the induction of apoptosis via the KA).
BrazJMedBiolRes46(1)2013 www.bjournal.com.br
KaurenediterpeneinducesapoptosisinU87glioblastomacells 73
das Clı´nicas, Faculdade de Medicina de Ribeira˜o Preto, method was used to calculate the relative abundance of
Universidadede Sa˜oPaulo (protocol No.3506/09). miR-21comparedtob-actin(AssayIDHs99999903_m1).
To determine the expression level of miR-21, total
Cell treatments RNAwasextractedwithTrizolfromcellsthatwereeither
Experimentswerecarriedoutusingcellsgrownon6- untreated or treated with 70mM KA for 72 h (5.0ng/
well culture plates (TPP-Techno Plastic Products AG, reaction). The RNA was reverse-transcribed using
Switzerland). The U87 glioma cell line was seeded at a reagents from the High Capacity C-DNA Archive Kit
densityof16105cells/well.After24h,themediumwas (Applied Biosystems), 3.8 U RNase inhibitor (Applied
removed,and2mLof aKA-Dulbecco’smodifiedEagle’s Biosystems), and the specific looped RT primer for miR-
medium was added to each well. KA was used at 21, which was provided in the assay kit. The samples
concentrations of 30, 50, or 70mM. The treated cells were incubated for 30min at 16˚C, 30min at 42˚C, and
were returned to the incubator and kept at 37˚C with 5% 5minat85˚C.Sampleswerethenheldat4˚C.Real-time
carbon dioxide for 24, 48, or 72 h. The cells were PCR analysis of the cDNA samples was performed at
subsequently used in the assays described below. The 95˚C for 10min followed by 40 cycles in the ABI Prism
concentrations of KA used in the present study were as 7300 sequence detection system (Applied Biosystems)
previously described, and did not induce genotoxicity in according to manufacturer instructions. We used the
Chinesehamster lungfibroblast (V79)cells (9). Taqman reaction master mix (Applied Biosystems) and
thehsa-mir21(Hsa-mir21003438)primer.Thecompara-
Cell viabilityassay tive Ct method was used to calculate the relative
Cellviabilitywasassessedusingthetrypanbluedye- abundance of miR-21 compared to RNU24 (Assay ID
exclusionmethod.Briefly,cellsweretreatedwithdifferent 001001).
concentrations of KA and collected. Equal volumes of a
trypanbluesolutionwerethenaddedtothecells.Aftera Statistical analysis
15- to 20-min incubation at room temperature, the cells Data are reported as means ± SE. For statistical
werecountedusingahemocytometer.Theratioofviable comparisons, the New Instat program (Graph pad-Instat,
cells to the total number of cells was calculated and USA) wasused toperformthe t-testor one-wayANOVA
recorded. followedbytheBonferronitest.Pvaluesoflessthan0.05
were consideredtobestatistically significant.
Annexin Vand propidiumiodide (PI)staining
Apoptosis was detected using an annexin V/PI Results
detection assay (17,18). Briefly, U87 cells were treated
with different concentrations of KA. After 48h, the cells Cell viabilityassay
were analyzed by flow cytometry (FACScan, Becton U87 cells were treated with three selective doses of
Dickinson, USA) according to manufacturer instructions KAtoexaminetheefficacyofthecompoundindecreasing
(annexin V-FITC apoptosis kit, BD Biosciences, USA). cellviabilityandinducingapoptosis.Residualcellviability
DatawerefurtheranalyzedwiththeCELLQuestsoftware was determined with the trypan blue exclusion dye test
(Becton Dickinson). The experiments were performed in using light microscopy, and viability was assessed after
serum-free medium. treatment of the cells with 30, 50, or 70 mM KA at time
intervals of 24, 48, and 72h. As shown in Figure 2, KA
RNA isolation andreal-time polymerase chain significantlydecreasedcellviabilityinU87cellsinadose-
reaction(PCR) and time-dependent manner. U87 cells treated with
The Trizol reagent (Applied Biosystems, USA) was 30mM KA did not show reduced cell viability; however,
used according to manufacturer instructions to extract at 50 mM KA, a decrease in cell viability was observed
total RNA from cells that had been treated with different upon comparison of the 24 and 72 h time points (P ,
concentrations of KA. To prepare the real-time PCR, 0.05). At 70mM KA, a decrease in cell viability was
reversetranscriptionoftheRNAsampleswasperformed observed upon comparison of the 24 and 48h, 24 and
using the High-Capacity cDNA kit (Applied Biosystems). 72h, and48 and72 htimepoints (P ,0.05).
Real-time PCR analysis of the cDNA samples was
performed at 95-60˚C for 45 cycles in the ABI Prism KA-inducedapoptosis of U87cells
7300 sequence detection system (Applied Biosystems) The externalization of phosphatidylserine, which is a
according to manufacturer instructions. We used the hallmark of apoptotic cells, has also been observed in
Taqman reaction master mix (Applied Biosystems) and alternativetypesofprogrammedcelldeath.Todetermine
the following primers: FAS (Assay ID Hs00163653_m1), whether such processes were induced upon KA treat-
c-FLIP(AssayIDHs01116280_m1),caspase-8(AssayID ment,U87cellsweretreatedfor24,48,and72hwith30,
Hs01018156_m1), and caspase-3 (Assay ID 50, or 70mM KA. The annexin V/PI assay revealed that
Hs00234385_m1). The comparative cycle time (Ct) thenumberofannexinV-andannexinV/PI-positivecells
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74 F.S.LizarteNetoetal.
increasedafter48 hoftreatment with50and 70mMKA.
The percentages of apoptotic cells were 9, 26, and 31%
forcontrolcells,cellssubmittedto48 hoftreatmentwith
50mM KA, and cells submitted to 48h of treatment with
70mMKA,respectively (P ,0.05; Figure3).
Fas,caspase-3 andcaspase-8 mRNAexpressionin
glioblastomacells after treatmentwith KAfor 24,48,
and72 h
The expression of apoptotic (Fas, caspase-8, cas-
pase-3) and anti-apoptotic (c-FLIP) genes was analyzed
by quantifying the relative mRNA levels in U87 human
glioblastoma cells that had been left untreated or were
Figure2.Effectofkaurenoicacid(KA)treatmentoncellviability.
U87 cells were treated with different concentrations of KA, and treatedwithdifferentconcentrationsofKAfor24,48,and
the trypan blue dye-exclusion method was used to detect cell 72h. Among the apoptotic genes studied, no significant
viability. The y-axis shows the percentage of cell viability. The differenceinFasgeneexpressionwasobservedbetween
concentrations of KA used and the durations of the treatments
untreatedcellsandcellstreatedwithKAfor48h(datanot
are indicated. Data are reported as means ± SD of 3
shown).
determinations.*P,0.05for50mMKA:24comparedto72h;
+P,0.05for70mMKA:24comparedto48and72h;#P,0.05 As mentioned above, no significant differences were
for 70mM KA: 48 compared to 72h (Bonferroni multiple observed in the expression of apoptotic (Fas, caspase-8
comparisontest). and caspase-3) and anti-apoptotic (c-FLIP) genes
Figure 3. Kaurenoic acid (KA) induces apoptosis in U87 cells. U87 cells were treated with 30, 50, or 70mM KA for 48h.
Phosphatidylserine(PS)externalizationwasdeterminedbythecombinedannexinV/propidiumiodide(PI)assay.Cellsthatstained
positive for annexin V represented cells with intact membranes and externalized PS (percentages are indicated in the lower right
panel).CellsthatstainedpositiveforannexinV/PIrepresentcellsthathadlostmembraneintegrity(percentagesareindicatedinthe
upper right panel). Data are reported as means ± SD of 3 determinations. P , 0.05 for 70 and 50mM KA treatment vs control
(Bonferronimultiplecomparisontest).
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KaurenediterpeneinducesapoptosisinU87glioblastomacells 75
Figure4.Caspase-3andcaspase-8geneexpressioninU87cells.They-axisindicatesthefold-changeincaspase-3andcaspase-8
expression. Data are reported as means ± SD for 3 measurements. The expression levels of caspase-3 and caspase-8 were
comparedbetweencellsthathadbeentreatedwithdifferentconcentrationsofkaurenoicacid(KA)for48handuntreatedcells.#P,
0.0001 for 70mM KA treatment vs control cells for caspase-3. +P , 0.001 for 70mM KA treatment vs control cells for caspase-8
(Bonferronimultiplecomparisontest).
betweenuntreatedU87cellsandcellstreatedwithKAfor observed a decrease in miR-21 expression in U87 cells
24and72h(datanotshown);however,incomparisonto treated with 70mM KA for 48h compared to untreated
the U87 control cells, high expression of caspase-3 and cells(P , 0.01;Figure6).
caspase-8 were observedin cellstreated with 70 mM KA
for 48h (P , 0.001 for caspase-3 and P , 0.0001 for Discussion
caspase-8;Figure4).Moreover,theexpressionofc-FLIP,
a regulator of apoptosis, was decreased in U87 cells Plant-derived compounds are known to exhibit a
treated with 50 or 70 mM KA for 48h in comparison to curative potential against many types of malignancies.
untreatedcells (P ,0.05; Figure5). Kaurenoic acid has been shown to have significant
cytotoxic and anti-proliferative effects on tumor cell
miR-21levelsinglioblastomacellsafterKAtreatment cultures (human breast cancer, human colon cancer and
for 48h leukemia) (19). The present study demonstrates that KA
TherelativemiRNAlevelsofthemiR-21anti-apoptotic has considerable cytotoxic and anti-proliferative actions
gene were quantified in untreated U87 human glioblas- ontheU87humanglioblastomacellline.Interestingly,the
tomacellsandincellstreatedwith70mMKAfor48 h.We concentrations of KA used in the present investigation
Figure 5. c-FLIP gene expression in U87 cells. The y-axis Figure 6. miR-21 gene expression in U87 cells. The y-axis
indicatesthefold-changeinc-FLIPexpression.Dataarereported indicates the fold-change in miR-21 expression. Data are
asmeans±SDfor3measurements.Theexpressionofc-FLIP reportedasmeans±SDfor3measurements.Theexpression
wascomparedbetweencellsthathadbeentreatedwithdifferent of miR-21 was compared between cells that had been treated
concentrations of kaurenoic acid (KA) for 48h and untreated with 70mM kaurenoic acid (KA) for 48h and untreated cells.
cells. *P , 0.05 for 70mM KA treatment vs control cells ?P , 0.01 for 70mM KA treatment vs control cells (Bonferroni
(Bonferronimultiplecomparisontest). multiplecomparisontest).
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76 F.S.LizarteNetoetal.
werepreviouslyreportedtonotexertcytotoxiceffectson found that the treatment of U87 cells with different
fibroblasts (9). Moreover, in the present study, we concentrations of KA for 48h led to increased mRNA
investigated the involvement of pro-apoptotic signals in levelsofcaspase-3.Severalgroupsofinvestigatorshave
U87 cells that were treated with KA. Apoptotic signaling reportedthatkaurenediterpenetreatmentledtocaspase-
can occur by two different pathways: the death receptor- 3 activation and apoptosis in human glioblastoma cell
associated extrinsic pathway and the mitochondria- linesand human leukemia cells(26-28).
dependent intrinsic pathway. Fas is a cell surface death Cellular FADD-like interleukin-1b-converting enzyme
receptor and a member of the tumor necrosis factor inhibitory protein (c-FLIP), an anti-apoptotic protein, was
receptor (TNFR) family. The Fas death receptor triggers recently reported to be recruited to the DISC. Once
apoptotic signals by binding to its cognate ligand, FasL recruited,c-FLIPwasshowntointerrupttheaminoterminal
(CD95L), and recruiting the adaptor molecule Fas- effector domain-amino terminal effector domain (DED-
associated death domain protein (FADD). This subse- DED) interaction between Fas-associated death domain
quently results in the formation of the death-inducing (Fas) and caspase-8. This disruption resulted in an
signaling complex (DISC) and the activation of procas- inhibitionofFas-mediatedapoptosis.Twoc-FLIPisoforms
pase-8. There is also evidence supporting the idea that havebeendetectedinhumantissues:thelongisoform(c-
kaurene diterpene-induced apoptosis involves pathways FLIP )andtheshortform(c-FLIP ).c-FLIP isstructurally
L s L
that are dependent upon death receptors, including Fas similartocaspase-8.ItcontainstwoDEDsandacaspase-
and TNFR. Although some studies have shown that the likedomainthatlackscatalyticactivityduetoreplacement
up-regulation of Fas levels may be one of the mechan- of the active-central tyrosine with a cysteine. The short
ismsresponsiblefordrug-inducedapoptosisinavarietyof form,c-FLIP ,contains two DEDsbutlacks thecaspase-
s
cancers, no variation of Fas expression upon treatment likedomain.Bothformsofc-FLIParerecruitedtotheDISC,
was observedin thepresent study(20-22). and they inhibit apoptosis that is induced by FasL and
The caspases are a family of intracellular cysteine tumor necrosis factor-related apoptosis inducing ligand
proteases with specificity for aspartic acid residues. Two (TRAIL) (29-32). Our present research shows that the
ofthesecysteineproteases,referredtoas‘‘initiator’’ and treatmentofU87cells withdifferentconcentrationsofKA
‘‘effector’’ caspases,playimportant rolesintheapoptotic for 48h led to an increase in the mRNA level of c-FLIP.
process. Previous reports have indicated that the Therefore, c-FLIP may be involved in the resistance of
apoptotic signal is transmitted through the activation of tumorstoapoptosisbymodulatingeitherreceptor-induced
caspase-8inthedeathreceptorrouteortheactivationof apoptosis or phosphorylation events. In addition, tumor
caspase-9 in the mitochondrial route (23). Recently, the cellsmayexhibitresistancetoapoptosisduetovariations
mechanismofcaspase-8activationhasbeeninvestigated intheirexpressionofc-FLIP.Interestingly,thedrug-based
ingreaterdetail. Themechanismofcaspase-8activation targeting of phosphorylation events is considered to be a
by kaurene diterpenes is considered to involve three potent approach to restoring the susceptibility of tumor
signalingpathways.Thefirstoftheseisadeathreceptor- cellstoapoptosis.
dependentpathway,thesecondisap38MAPK-dependent The emerging significance of miRNAs in cancer has
pathway, and the third is a caspase-dependent pathway. resulted in a recent spike in interest in these unique
Schrantz et al. (24) reported that p38MAPK could activate RNAs. This increased interest has resulted in numerous
caspase-8 during transforming growth factor b-induced profilingstudiesthataimedtoassesstheroleofmiRNAs
apoptosis, and Castrillo et al. (25) reported that kaurene incancer.Thecellularmechanismsthatcontrolapoptosis,
diterpenes could modulate p38MAPK activity. In the cell growth and the cell cycle are efficient fail-safe
present study, the treatment of U87 glioblastoma cells mechanisms that prevent cancer development, despite
with different concentrations of KA for 48 h resulted in the trillions of somatic cell division events that occur
elevated mRNA levels of caspase-8. These results duringahumanlifetime.Therefore,cancercellscanonly
suggestthatKAinducesapoptosisthroughtheactivation survive if the apoptotic response is dampened or
of caspase-8 by either the death receptor route or the disrupted, cell cycle checkpoints are evaded, and unre-
mitochondrial route. Further experiments focusing on the stricted cell proliferation is allowed to occur. Recent
involvement of p38MAPK activation in kaurene diterpene- studieshaveshownthataberrantincreasedexpressionof
inducedcaspase-8activationarenecessary.Suchexperi- miR-21 in glioblastoma cell lines may result in the down-
mentswillbeusefulinincreasingourunderstandingofthe regulation of mRNAs that encode apoptosis-related
apoptotic machinery that is activated during kaurene genes. Thus, our data indicate that miR-21 also acts as
diterpene-inducedapoptosis. an anti-apoptotic factor in KA-treated glioblastoma cells.
Caspase-3 is one of the most important downstream ThesedatasuggestthatmiR-21couldactasakeyfactor
executioner caspases, and is capable of cleaving many intheresistanceofgliomacellstoapoptosisandthatKA
important cellular substrates. Caspase-3-mediated cell maybeuseful incancer therapy(15,33).
death plays an important role in both the pathogenesis Malignantgliomaswithanastrocyticlineage,including
and treatment of a variety of malignancies (12). We astrocytomas, anaplastic astrocytomas, and glioblas-
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KaurenediterpeneinducesapoptosisinU87glioblastomacells 77
tomas, are the most common human primary brain chemotherapeutic agents exhibit only pro-apoptotic
tumors. Despite intensive research over the last decade, effects. In U87 cells, KA has an effect on the regulation
the most current treatment modalities can only modestly of several genes involved in the apoptotic pathway,
improve patient survival (34). Therefore, finding new including c-FLIP, caspase 3, caspase 8, and miR-21,
drugs and effective therapies for the clinical treatment of and KA treatment results in decreased cell viability.
glioblastoma multiforme will remain an important area of Further experiments will be necessary to determine if
research. KA may be useful as a lead compound for the develop-
We found that KA possesses interesting properties mentofnewchemotherapeuticdrugsforthetreatmentof
with regard to its apoptosis-inducing mechanism. Most primarybrain tumors.
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