research article destruxin b isolated from...

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 548929 11 pageshttpdxdoiorg1011552013548929

Research ArticleDestruxin B Isolated from Entomopathogenic FungusMetarhizium anisopliae Induces Apoptosis viaa Bcl-2 Family-Dependent Mitochondrial Pathway inHuman Nonsmall Cell Lung Cancer Cells

Chun-Chi Wu12 Tzu-Hsiu Chen3 Bing-Lan Liu4 Li-Chen Wu5 Yung-Ching Chen1

Yew-Min Tzeng4 and Shih-Lan Hsu16

1 Institute of Medicine Chung Shan Medical University Taichung Taiwan2Department of Medical Research Chung Shan Medical University Hospital Taichung Taiwan3Department of Health and Nutrition Chia Nan University of Pharmacy amp Science Tainan Taiwan4Department of Applied Chemistry Chaoyang University of Technology Taichung Taiwan5Department of Applied Chemistry National Chi Nan University Puli Nantou Taiwan6Department of Education and Research Taichung Veterans General Hospital Taichung Taiwan

Correspondence should be addressed to Yew-Min Tzeng ymtzengcyutedutw and Shih-Lan Hsu h2326vghtcgovtw

Received 20 March 2013 Accepted 20 August 2013

Academic Editor Mei Tian

Copyright copy 2013 Chun-Chi Wu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Destruxin B isolated from entomopathogenic fungus Metarhizium anisopliae is one of the cyclodepsipeptides with insecticidaland anticancer activities In this study destruxin B was extracted and purified by ion-exchange chromatography silica gelchromatography and semipreparative high-performance liquid chromatography The potential anticancer effects and molecularmechanisms of destruxin B in human nonsmall cell lung cancer cell lines were characterized Our results showed that destruxinB induced apoptotic cell death in A549 cells This event was accompanied by the activation of caspase-2 -3 and -9 Moreoverdestruxin B increased the expression level of proapoptotic molecule PUMA while decreased antiapoptotic molecule Mcl-1Additionally the translocation of Bax from cytosol to mitochondrial membrane was observed upon destruxin B treatmentKnockdown of Bax by shRNA effectively attenuated destruxin-B-triggered apoptosis inA549 cells Interestingly similar toxic effectsand underlyingmechanisms including caspase activation upregulation of PUMA and downregulation ofMcl-1 were also observedin a p53-null lung cancerH1299 cell line upon destruxin B treatment Taken together our findings suggest that destruxin-B-inducedapoptosis in human nonsmall cell lung cancer cells is via a Bcl-2 family-dependent mitochondrial pathway

1 Introduction

Lung cancer is the leading cause of death for both men andwomen inmany countries includingTaiwanwhich exhibitedthe highest rate of increase in lung cancer mortality in recentyears [1 2] The five-year survival rate of lung cancer patientis only 13ndash25 [3] Although target therapy emerged fortreatment of lung cancer patients in the past decade [4] thereis still a large amount of patients who are uncured It appearsthat the drugs with greater efficacy than existing treatmentsare urgently required for these patients

Growing evidence demonstrates that apoptosis isinvolved in many biological events including physiologicalhomeostasis and embryogenesis [5] and is also an importantdeterminant of the response of cancers to chemo- radiationor target-therapy [6 7] compounds that induce theseevents may provide potent anticancer effects for cancertreatment The regulatory mechanisms of apoptosis arequite complicated networks Two major types of apoptosispathways are mentioned one is the intrinsic pathway whichis mediated by the regulators located in the mitochondrial[8] and the other is the extrinsic pathway which is regulated

2 Evidence-Based Complementary and Alternative Medicine

by the binding of the membrane receptor to the death ligandsuch as TNF (tumor necrosis factor)TNFR and FasFasL[9] The apoptotic process is mainly executed by a class ofcysteine proteases known as caspases [10] Activation ofdifferent caspases usually presents for various upstreamapoptotic pathways For example the activation of caspase-8and -10 usually stands for the extrinsicdeath receptor-relatedpathway caspase-2 and caspase-9 are usually activated bymitochondrial pathway while caspase-12 is activated byendoplasmic reticulum pathway [10] In addition theactivation of caspase-3 -6 or -7 is usually considered as acommon executor of apoptosis by directly cleaving cellularproteins when activated [11]

Several proteins are known to be involved in the reg-ulation of apoptosis among them Bcl-2 family moleculescontribute to themitochondrial-mediated apoptotic pathwayBcl-2 is originally found in a translocation chromosomalfragment in B-cell lymphoma and is proved as a protoonco-gene [12] The members of Bcl-2 family all contain a BH(Bcl-2 homology) domain structure and are divided intotwomajor groups one composed of antiapoptotic moleculesincluding Bcl-2 Bcl-xL Bcl-w Mcl-1 BooDiva and A1Bfl-1 [13] and the other comprises proapoptotic moleculessuch as Bax Bak Bad Bim Bik PUMA and NOXA [14]Under normal conditions the Bcl-2 or Bcl-xL complexeswith Bax or Bak to form homo- or hetero dimer and suchinteraction will neutralize proapoptotic molecules to preventcell death Under apoptotic stimulation however it willchange the ratios between these two groups leading to theformation of BaxBax or BaxBak complexes followed byperforation through the membrane of mitochondria Theopened pores eventually cause the release of cytochromec from mitochondria to the cytosol and form apoptosiscomplex with Apaf-1 (apoptotic protease activation factor-1) dATP (deoxyadenosine triphosphate) and procaspase-9 to induce the caspase-9 and caspase-3 activation andirreversible apoptosis [15]

It is well known that certain compounds isolated frombacteria or fungus carry characteristic biological effectswhich have been used as traditional medicines [16 17] andseveral of these compounds have shown an antitumoralactivity [18ndash20] One group of such biological active com-pounds is depsipeptides which are the major secondarymetabolites of fungi The depsipeptides are biooligomersformed by esterification between hydroxyl groups and aminoacids Destruxin B is a cyclic depsipeptide isolated fromentomopathogenic fungus in 1961 Until recently there were39 destruxins reported [21] It has been reported that cyclicdepsipeptides exhibited strong antitumor activity in both invitro and in vivo experiments and evenly entered the clinicaltrial [22] Previous reports mentioned that destruxin B hasstrong anti-insect activity [23] inhibits hepatitis B surfaceantigen expression and induces cell death in hepatocellularcarcinoma cells [24] and leukemic cells [25] The effects ofdestruxin B on lung cancer cells and its molecular mecha-nisms however have not been examined yet In this studythe antiproliferative cytotoxic effects and underlying mecha-nisms of destruxinB (isolated andpurified from fermentationbroth of Metarhizium anisopliae) on lung cancer cell lines

were explored Our observations indicate that destruxin Binduces a Bcl-2 family-dependent mitochondrial apoptoticcell death in human nonsmall cell lung cancer A549 andH1299 cell lines

2 Material and Methods

21 Reagents The destruxin B (MW 593) was dissolved inethanol to a stock solution at concentration of 20mM for thefollowing experiments Anti-Bax Bcl-2 Bcl-xL Bak PUMAMcl-1 and 120573-actin were purchased from Santa Cruz Biotech-nology (Santa Cruz CA USA) or Calbiochem ChemicalCompany (CN Biosciences Notts UK) Anti-mouse andanti-rabbit IgG peroxidase-conjugated secondary antibodieswere purchased from Amersham (Buckinghamshire UK)Caspase activity assay kits including the fluorogenic sub-strates of caspase-2 (VDVAD-AFC) caspase-3 (DEVD-AFC)and caspase-9 (LEHD-AFC) were purchased from RampDsystems (Minneapolis MN USA) The caspase-2 inhibitor(z-VDVAD-FMK) was purchased from Santa Cruz Biotech-nology (Santa Cruz CA USA) The caspase-3 inhibitor (Z-DEVD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) werepurchased from Kamiya (Seattle WA USA)

22 Isolation and Purification of Destruxin B The destruxinB used in this study was extracted and purified from thesubmerged cultivation of fungusMetarhiziumanisopliae F061var Anisopliae according to the processes described else-where [26 27] BrieflyMetarhizium anisopliaewas cultivatedin 3maltose containing 05 bactopeptone liquid mediumat 28∘C After 14 days the culture medium was collectedand extracted three times with equal volume of methylenedichloride (CH

2Cl2) The organic layer was collected and

concentrated and further separated by two-step ion-exchangechromatography Combination of cationic (Amberlite TRA-400 CP column) and anionic (Amberlite IR 120 Plus)exchange provided the step to removing charged impuritiesexisting in the mixture and then the eluent was collectedand subjected to flash silica chromatography (Merck K-60silica gel column) as a prepurification step The columnwas eluted with a methylene dichloride-methanol mixtureFurther purification was done by loading the sample intoa semipreparative RP-C18 high-performance liquid chro-matographic (HPLC) column The which elution delivereda single peak was collected and concentrated This peakrepresented destruxin B (retention time = 176min) Thepurity of destruxin B in the sample was proved gt95 in ourearly report [28]

23 Cell Culture and Viability Assay The human lung cancerA549 and H1299 cells were obtained from CCRC (CultureCollection and Research Center Hsinchu Taiwan) TheA549-shBax cells were established by transfection of pSuper-shBax followed by antibiotics selection [29] A549 A549-shBax and H1299 cells were cultured in RPMI (GIBCOBRLMD USA) All cultured media were supplemented with10 fetal bovine serum 2mM glutamine and antibiotics(100UmL penicillin and 100 120583gmL streptomycin) at 37∘C

Evidence-Based Complementary and Alternative Medicine 3

in a humidified atmosphere of 5 CO2 The medium was

changed every 48 h For cell viability assay cells were seededin 12-well plates at a density of 1times105 cells per well After 24 hcells were treated with various concentrations of destruxin Bfor the indicated time points After treatment the numberof viable cells was determined by trypan-blue dye exclusionmethod

24 Cell Cycle Analysis Cells were seeded as a number of2 times 105 cellswell onto 6-well plates After 24 h incubationdifferent concentrations of destruxin B (0 10 20 and 30120583M)were added for another 48 h The cells were then collectedand washed with cold PBS twice followed by centrifugationat 1200 rpm at 4∘C Cell pellet was suspended with 1mL coldethanol (70) and then was incubated with buffer containingpropidium iodide (40 120583gmL) and RNase A (100120583gmL) for30min under dark conditionThe prepared cell samples weresubjected to flow cytometry (FACScan) and analyzed byModFIT LT software

25 Caspase Activity Assay Cell lysates obtained fromdestruxin-B-treated or -untreated cells were tested forcaspase-2 -3 -9 activities by addition of a caspase-specificpeptide substrate conjugated with the fluorescent reportermolecule 7-amino-4-trifluoromethyl coumarin (RampD Sys-tems Minneapolis USA) The cleavage of the peptide bythe caspase releases the fluorochrome that when excited bylight at 400 nm emits fluorescence at 505 nm The level ofcaspase enzymatic activity in the cell lysate is directly propor-tional to the fluorescence signal detected using a fluorescentmicroplate reader (Fluoroskan Ascent Labsystems Finland)

26 Protein Preparation and Immunoblotting Cells werecultured without or with destruxin B at various time pointsAfter treatment both adherent and floating cells wereharvested washed twice with ice-cold PBS and decom-posed in ice-cold modified RIPA buffer (50mM Tris-HCl (pH 74) 150mM NaCl 1 Triton X-100 025Na-deoxycholate 5mM ethylenediamine-tetra-acetic acid(pH 80) 1mM ethylene glycol-bis(2-aminoethylether)-N1015840N1015840NN1015840-tetra-acetic acid (pH 80) 1mM dithiothreitol02mM phenylmethylsulfonylfluoride 5120583gmL leupeptin5 120583gmL aprotinin and 1mM sodium orthovanadate) After30min incubation on ice cells were centrifuged at 100000 gfor 30min at 4∘C and supernatants were collected Proteinconcentration was determined using the Bradford methodFor western blot analysis equal amounts of total proteinswere loaded onto SDS-polyacrylamide gels followed byelectrophoretically transferring onto a PVDF membrane(Millipore MA USA) Membranes were incubated withspecific primary antibodies overnight After exposure tohorseradish peroxidase-conjugated secondary antibody for1 h proteins were visualized using an enhanced chemilumi-nescence detection kit (ECLKits AmershamLife Science NJUSA)

27 Indirect Immunofluorescence A549 cells were trypsinizedand seeded onto coverslips After reaching 75 confluence

cells were treated with destruxin B for 24 h Cells werewashed with PBS twice and stained with MitoTracker for15ndash30min After treatment cells were washed with PBS andfixed with ethanol at minus20∘C for 20min incubated with Baxantibody at room temperature for 4 h and then washedthree times with PBS and incubated with goat-anti-rabbit-FITC (Jackson PA USA) and 1mgmL DAPI (Sigma MOUSA) for another 1 h After washing with PBS containing01 Tween 20 the samples were mounted The fluorescenceimages of the stained cells were analyzed using a Zeissfluorescence microscope

28 RNA Extraction and Quantitative RT-PCR Total cellularRNA was extracted by RNA-Bee RNA isolation kit (TEL-TEST Friendswood TX) according to the manufacturerrsquosinstructions One microgram of total RNA was reverse-transcribed using Advantage RT for PCR Kit (Clontech CAUSA) at 42∘C for 1 hr The PCR primers were listed as followsfor Mcl-1 forward primer AAAGAGGCTGGGATGGGTTTand reverse primer CAAAAGCCAGCAGCACATTC forPUMA forward primer CCTGGAGGGTCCTGTACAATCTand reverse primer GCACCTAATTGGGCTCCATCT for120573-actin forward primer GCAAATGCTTCTAGGCGGAC-TAT and reverse primer TGCGCAAGTTAGGTTTTGTCAThe mRNA levels were determined by real-time PCR withABI PRISM 7900 Sequence Detector system according to themanufacturerrsquos instructions120573-Actin was used as endogenouscontrol PCR reaction mixture contained the SYBR PCRmaster mix (Applied Biosystems) cDNA and the primersRelative gene expression level (the amount of target normal-ized to endogenous control gene) was calculated using thecomparative Ct method formula 119864minusΔΔCt

29 Statistical Analysis Figures were obtained from at least3 independent experiments with similar patterns All dataare presented as mean plusmn SD of 9 replicates from 3 separateexperiments Statistic differences were evaluated using Stu-dentrsquos t-test with lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0001considered significant or by the calculation and groupingusing SAS program

3 Results

31 Preparation of Destruxin B Destruxin was isolatedpurified and identified from the fermentation broth ofsubmerged cultivation of fungus Metarhizium anisopliaeF061 var Anisopliae by CH

2Cl2extraction ion-exchange

chromatography silica gel chromatography andHPLC quan-tification Figure 1(a) presents a schematic diagram of extrac-tion and isolation procedures An HPLC fingerprint chro-matogram was performed for the quantification and qualitycontrol of the isolated samples (Figure 1(b)) which showedonly one major peak (retention time = 176min) The peakwas identified as destruxin B The purity of destruxin B inthe sample was proved gt95 in our early report [28] Thechemical structure of destruxin B is presented in Figure 1(c)


Metarhizium anisopliae fermentation broth

Centrifugation 8000 xg for 30 min

Supernatant was harvested and extracted with CH2Cl2

Organic layer was collected and concentrated

Two-step ion-exchange chromatography

Step 1 eluted with distilled water in an Amberlite TRA-400 CPcation-exchange chromatographic column

Step 2 eluted with distilled water in an Amberlite IR120 Plusanion-exchange chromatographic column

Silica gel chromatography eluted with methylene dichloride-methanol solventthrough a Merck K60 silica gel column

Semipreparative RP-C18 HPLC

Destruxin B (purity gt95)

(a)

176 min

0 5 10 15 20 25

0

30

60

90

120

Retention time (min)

Inte

nsity

(mV

)(b)

N

O

H

O

O

O

N O

N

N

N

H

HH

O

O

(c)

Figure 1 Isolation and purification of destruxin B from Metarhizium anisopliae fermentation broth (a) Flowchart of the extraction andpurification procedure (b) HPLC chromatographic spectrum of purified destruxin B Metarhizium anisopliae fermentation broth wascentrifuged and filtered The filtrate was extracted with CH

2

Cl2

and then the destruxin B was extracted and purified by ion-exchangechromatography silica gel chromatography and HPLC (c) The chemical structure of destruxin B

32 Destruxin B Induces a Caspase-Dependent Apoptosis Toexamine the growth inhibitory effect of destruxin B differentdoses of destruxin B (0 1 5 10 20 and 30 120583M) wereadded into lung adenocarcinoma A549 cells As shown inFigure 2(a) destruxin B induced a concentration-dependentantiproliferative effect on A549 cells with IC

50value at

49 120583M Treatment with low concentrations of destruxin Bresulted in growth inhibition but not cell death Howeverdestruxin-B-induced cytotoxicity occurred at concentrationup to 10 120583M (Figure 2(a)) Data from cell flow cytometricanalysis showed that destruxin B induced a sub-G1 popula-tion accumulation at 10sim30 120583M destruxin B administration(Figure 2(b)) indicating that destruxin B triggered the apop-totic cell death ofA549 cells To addresswhether destruxin-B-induced apoptosis was through a caspase activation pathway

fluorogenic peptide substrates were used to examine thespecific caspase activity As depicted in Figure 2(c) caspase-2 -3 and -9 were significantly activated after 48 h destruxinincubationwhile caspase-8 and caspase-12were not activatedupon destruxin B treatment (data not shown) To confirm therole of caspase activation in destruxin-B-mediated apoptosisA549 cells were pretreated with caspase inhibitors priorto destruxin B In Figure 2(d) the inhibitors of caspase-2caspase-3 and caspase-9 drastically attenuated destruxin-B-induced cell death whereas the inhibitors of caspase-8 andcaspase-12 did not affect the destruxin-induced cell death(data not shown)These observations indicate that activationof mitochondria-dependent caspase cascade might play animportant role in destruxin-B-induced apoptotic cell deathin A549 cells


A549

Control 1 5 10 20 30

0 1 5 10 20 300

20

40

60

80

100

120

Gro

wth

inhi

bitio

n(

of c

ontro

l)

IC50 = 49 120583M

Destruxin B (120583M)


(a)

Control 10 20 30

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 6000

200 Sub-G1 06G1 662S 182G2M 152

Sub-G1 80G1 687S 162G2M 79

Sub-G1 137G1 699S 117G2M 56

Sub-G1 193G1 691S 95G2M 37

0

200

0

200

0

200


(b)

ControlDestruxin B

Caspase 2 Caspase 3 Caspase 9

Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

lowastlowastlowast

0

5

10

15

20

25lowastlowastlowast lowastlowastlowast

(c)

Destruxin BCaspase-2 inhibCaspase-3 inhibCaspase-9 inhib

minus + + + +

minus minus + minus minusminus minus minus + minusminus minus minus minus +

0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowastlowast

lowastlowast

lowastlowast

(d)

Figure 2 Destruxin B induces a caspase-dependent cell death (a) Antiproliferative effect of destruxin B Human nonsmall cell lung cancerA549 cells were administrated with different concentrations of destruxin B (0 1 5 10 20 and 30 120583M) for 48 h The cellular morphology wasobserved under microscope and the cell numbers were then counted by trypan-blue dye exclusion method using hemocytometer (b) Sub-G1 population is increased by destruxin administration A549 cells were treated with different concentrations of destruxin B (0 10 20 and30 120583M) for 48 h Cells were then fixed with ethanol and subjected to flow cytometry assay (c) Destruxin B induces caspase activation A549cells were treated with 20120583M destruxin B for 48 h Cell lysates were harvested and the indicated caspase activity was measured as describedin Section 2 (d) Inhibition of caspase activity blocks destruxin-B-triggered cell death A549 cells were pretreated with indicated caspaseinhibitors for 1 h and then treated with 20 120583M destruxin B for another 48 h The cell viability was determined by trypan-blue dye exclusionmethods ( lowastlowast119875 lt 001 versus control)



12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25

lowastlowast lowastlowast

lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)

Figure 3 Regulation of Mcl-1 and PUMA by destruxin B (a) Regulation of Bcl-2 family proteins by destruxin A549 cells were treated with0 10 or 20120583M of destruxin B for 12 and 24 h The cells were collected lyzed and subjected to western blot with antibodies against PUMABax Bak Mcl-1 Bcl-xL Bcl-2 and 120573-actin respectively (b) Upregulation of PUMA but not Mcl-1 RNA expression by destruxin A549 cellswere treated with or without 20120583Mdestruxin B for 8 12 and 24 hThe cells were then collected and the total RNA was extracted followed byreal-time PCR using PUMA (upper panel) or Mcl-1 (bottom panel) primer set The actin primer set was also applied as an internal controlThe normalized relative expression folds of PUMA between untreated and treated cells were quantified ( lowast119875 lt 005 versus control lowastlowast119875 lt 001versus control lowastlowastlowast119875 lt 0001 versus control)

33 Bcl-2 Family Molecules Contribute to Destruxin-B-Mediated Apoptosis It is well documented that Bcl-2 familymolecule plays a central role in mitochondria-mediatedapoptosis To examine the mitochondrial apoptotic eventsassociated with destruxin-B-induced apoptosis we examinedthe levels of proapoptotic and antiapoptotic proteins bywestern blot analysis in whole lysates of A549 cells treatedwith 0 10 and 20120583M destruxin for 24 and 48 h It wasfound that destruxin B treatment markedly increased levelsof the proapoptotic protein PUMA and reduced levels ofantiapoptotic proteins Mcl-1 in a concentration- and time-dependent manner (Figure 3(a)) However the levels of BaxBak Bcl-2 and Bcl-xLwere not affected by destruxin To clar-ify whether destruxin B regulated the expression of PUMAand Mcl-1 at a transcriptional level the mRNA expressionwas analyzed by qPCR The results showed that additionof destruxin B drastically increased the level of PUMAmRNA (Figure 3(b) upper panel) strongly indicating thatincreasing PUMA expression by destruxin B might attributeto transcriptional regulation In contrast the mRNA level ofMcl-1 was not affected by destruxin B (Figure 3(b) bottom

panel) suggesting that destruxin-mediated Mcl-1 proteinlevel reduction might be regulated by a posttranslationalmanner

34 Mitochondrial Translocation of Bax Is Important for theDestruxin-Induced Apoptosis It has been reported that dur-ing mitochondria-mediated apoptosis Bax can translocatefrom cytosol to mitochondrial membrane and form pores todisrupt the permeability of themitochondrial membraneWethen investigated whether Bax contributed to destruxin-B-mediated mitochondrial apoptosis Data from immunoflu-orescence staining showed that Bax exhibited a diffuse dis-tribution throughout the untreated A549 cells (Figure 4(a))However administration of destruxin B resulted in a sub-cellular redistribution of Bax the immunoreactivity of Baxformed a punctate pattern and the yellow color in theoverlay of Figure 4(a) pointed out the colocalization of Baxwith mitochondrial indicating the translocation of Bax fromcytosol to mitochondria upon destruxin treatment To char-acterize the role of Bax in destruxin-B-induced apoptosis wecompared the effects of destruxin B on cell viability between


MitoTracker Bax DAPI

MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin

10 20Destruxin B (120583M)

A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)

Figure 4 Bax plays a crucial role in destruxin-B-induced apoptosis (a) A549 cells were untreated or treated with 20120583Mdestruxin B for 24 hand the cells were then fixed with 4 paraformaldehyde and subjected to indirect immunofluorescence by using anti-Bax antibody (greencolor) DAPI (blue color) was used as a nuclear staining and MitoTracker (red color) was used as a mitochondrial tracker (b c) The A549and A549-shBax cells were treated with 0 10 and 20 120583M destruxin B for 72 h (b) the cell morphology was observed under phase-contrastmicroscopy and (c) the cytotoxicity of destruxin B was determined lowastlowast119875 lt 001 versus control lowastlowastlowast119875 lt 0001 versus control

parental A549 and A549-shBax cells (which were stablytransfected with two Bax-specific shRNAs) As indicated inFigure 4(b) A549-shBax cells showed more resistance to thedestruxin-B-induced cytotoxicity than parental A549 cells(Figures 4(b) and 4(c)) Approximately 899 and 439apoptotic cells were detected inA549 andA549-shBax cells inresponse to 20 120583M destruxin B treatment respectively Theseresults clearly demonstrate that Bax is a major proapoptoticmolecule in regulation of apoptosis primed by destruxin B inA549 cells

35 Destruxin B Triggers Mitochondria-Mediated Apoptosisin H1299 Cells We expanded our study to another humanlung cancer cell line H1299 Destruxin B also inhibited

cell proliferation of H1299 cells with IC50

value at 41 120583M(Figure 5(a)) Consistently the administration of destruxinB significantly stimulated the activation of caspase-2 -3 and -9 (Figure 5(b)) Moreover destruxin B increasedthe level of PUMA while decreased Mcl-1 in H1299 cells(Figure 5(c)) These results indicate that modulation of Bcl-2 family molecules and activation of mitochondria-mediatedcaspase are required for destruxin-induced apoptosis

4 Discussion

Lung cancer has been the most common malignancy inthe world for several decades that carries poor prognosisAlthough substantial progress has been made in developingchemotherapeutic treatments for lung cancer drug efficacy is



Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)

Caspase-2 Caspase-3 Caspase-9

Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)

Destruxin B (120583M)12 24Time (h)

0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)

Figure 5 The effects of destruxin B on H1299 cells (a) Antiproliferative effect H1299 cells were untreated or treated with 1 5 10 20 and30 120583M of destruxin B for 48 h The cell number was measured (b) Activation of caspases H1299 cells were treated with 20120583M destruxinB for 48 h The cells were then lyzed and incubated with the fluorescein-conjugated substrates of caspase-2 -3 and -9 and subjected to themicroplate reader to detect caspase activity lowast119875 lt 005 versus control lowastlowast119875 lt 001 versus control lowastlowastlowast119875 lt 0001 versus control (c) H1299 cellswere treated with 0 10 or 20 120583M destruxin B for 12 and 24 h The cells were collected lyzed and subjected to western blot with antibodiesagainst PUMA Mcl-1 and 120573-actin respectively

often outweighed by undesirable side effects Nowadays lungcancer is still the first leading cause of death from cancer with14 million deaths annually Thus it is crucially importantto develop better therapeutic strategies for the managementof lung cancer In the recent years phytochemicals andmicrobial extracts isolated from various sources have shownsignificant anticancer activities [30] It is estimated thatapproximately 50 of the drugs currently used in the clinicare derived from the natural products or their syntheticanalogs which still continue to provide essential sourcesof novel discovery leads Cyclodepsipeptides the secondarymetabolites of fungi and plants [31] show a broad spec-trum of biological activity including immunosuppressant

antibiotic antifungal anti-inflammatory and anticancereffects [31] Some of these natural products and (semi-)synthetic derivatives have already been evaluated in clinicaltrials [31] Destruxin B one of the cyclodepsipeptides isisolated and purified from entomopathogenic fungi Previousstudies demonstrate that destruxin B has insecticidal andantiviral effects [24] Additionally our studies and otherstudies demonstrate that destruxin B has antitumor effectin leukemia cells [32] hepatocellular carcinoma cells [25]and colorectal cancer [33] This study firstly reported thatdestruxin B inhibits cell proliferation and induces apoptoticcell death in human nonsmall cell lung cancer cell linesA549 and H1299 Moreover destruxin-B-triggered apoptosis


N

O

H

O

OO

N O

N

N

N

H

HH

O

O

Metarhizium anisopliae fermentation and supernatant

extraction and concentration

Des

trux

in B

prep

arat

ion

Two-step ion-exchange chromatographysilica gel chromatography and semipreparative

RP-C18 HPLC

Treatment of human lung cancer cells

Caspase-2

z-VDVAD-FMKMitochondrial

translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr


Figure 6 Schematic representation of destruxin B preparation and mechanistic illustration of destruxin-B-induced apoptosis Destruxin Bisolated from Metarhizium anisopliae induces apoptosis in human nonsmall cell lung cancer cells by activating a mitochondrial-dependentcaspase activation signaling pathway

is through a Bax-dependent mitochondria-mediated caspaseactivation pathway

Our paper is the first report to conduct a mechanisticstudy to investigate how destruxin B induces apoptosis It iswell documented that apoptosis is activated mainly throughtwo pathways the mitochondria-dependent pathway (intrin-sic pathway) and the death receptor pathway (extrinsicpathway) Bcl-2 family proteins play a key role in the regula-tion of mitochondria-dependent apoptosis [34] The relativeequilibrium of various anti- and proapoptotic Bcl-2 familymembers are a critical determinant of cell apoptosis Bax ismainly localized in the cytosol of healthy cells as a solublemonomeric protein in an apoptotically inactive status [35]In response to apoptotic cues Bax undergoes conformationalchanges and then translocates to the mitochondrial outermembrane and forms large oligomeric complexes therebytriggering mitochondrial dysfunction and caspase activationto bring about apoptosis Mcl-1 can directly bind with

Bax and prevent apoptotic activation of Bax [36] In thisstudy we found that treatment with destruxin B resultedin decrease of Mcl-1 increase of PUMA and induction ofBax mitochondrial translocation Moreover silencing Baxexpression by shRNA effectively attenuated destruxin-B-triggered apoptosis This suggests that Bax plays a morecrucial role in conducting destruxin-B-mediated cytotoxicityIt is known that PUMA is a general sensor of apoptoticstimuli and a promising drug target for cancer therapy [37]Biochemical studies indicate that PUMA induces apoptosisby activating the proapoptotic protein Bax through its inter-action with antiapoptotic Bcl-2 family members includingBcl-2 Bcl-xL Mcl-1 Bcl-w and A1 [38] The interactionsof PUMA with antiapoptotic proteins cause displacementof Bax resulting in activation of the proapoptotic activityof Bax PUMA overexpression leads to a conformationalchange multimerization and mitochondrial translocationof Bax which induces apoptotic cell death [37] Based on


these data we propose that one of the mechanisms bywhich destruxin-B-induced apoptosis was by overexpressionof PUMA and decrease of Mcl-1 resulting in disruption ofMcl-1Bax complexes triggering Bax oligomerization andtranslocation to mitochondria in human nonsmall cell lungcancer cells

The apoptosis process is mediated by sequential activa-tion of caspases Caspases a family of cysteine proteasesis one of the biomarkers of apoptosis [39] Once activatedcaspases can cleave hundreds of cellular proteins resultingin the hallmark morphological characteristics of apoptosisAccumulating evidence indicates that activation of caspasecascade plays a key role in the induction of apoptosis formany cancer therapeutic drugs in many types of cancer cells[39] In particular activation of caspase-3 plays a centralrole in the execution of apoptosis Activation of caspase-3requires the activation of initiator caspases such as caspase-8 or caspase-9 in response to proapoptotic signals It iswell known that caspase-9 is the major initiator caspase ofmitochondria-mediated apoptosis while caspase-8 is the keyinitiator caspase of death receptor-triggered apoptosis Ourcurrent study clearly demonstrated that treatment of A549cells with destruxin B enhanced the activation of caspase-9and -3 Reduction of caspase cascade with specific inhibitorseffectively blocked destruxin-B-induced apoptosis Theseresults indicated that destruxin-B-induced apoptosis wasthrough a mitochondria-dependent caspase-9-3-activatedresponse Interestingly this study also showed that caspase-2 was activated in A549 and H1299 cells upon destruxin Badministration Caspase-2 is considered as both initiator andexecutor caspase in apoptosis [40] Caspase-2 has been shownto act upstream of the mitochondria and is involved in Bidcleavage and Bax translocation which results in cytochromec release during genotoxic stress [41]Therefore we proposedthat the activation of caspase-2 by destruxin B might be oneof the important regulators in inducing Bax translocationand subsequent mitochondrial dysfunction finally leading toapoptosis

In conclusion the present study supports that destruxinB isolated and purified fromMetarhizium anisopliae F061 varAnisopliae induces the intrinsic apoptotic pathway in humannonsmall cell lung cancer A549 cells and a signaling cascadeis proposed in Figure 6 Destruxin-B-mediated upregulationof PUMA and downregulation of Mcl-1 may facilitate themitochondrial translocation of Bax subsequently inducingcaspase-2 -9 and -3 activation and apoptosis Importantlyinhibition of Bax expression by shRNA significantly dimin-ishes destruxin-B-induced cytotoxicity suggesting that Bax-dependent mitochondrial pathway contributes to destruxinB activity in A549 cells Because some of these results havebeen confirmed inH1299 cells we hypothesize that destruxinB may activate some common signaling events in differenthuman nonsmall cell lung cancer cell types

Conflict of Interests

The authors declare that there is no potential conflict ofinterests

Acknowledgments

This work was supported by the National Science Councilof Taiwan (NSC 100-2113-M-324-001-MY3) the TaichungVeteran General Hospital Taichung and National Chi NanUniversity (TCVGH-NCNU1017901)

References

[1] A Jemal R Siegel E Ward Y Hao J Xu and M J ThunldquoCancer statistics 2009rdquo CA Cancer Journal for Clinicians vol59 no 4 pp 225ndash249 2009

[2] A Jemal F Bray M M Center J Ferlay E Ward and DForman ldquoGlobal cancer statisticsrdquo CA Cancer Journal forClinicians vol 61 no 2 pp 69ndash90 2011

[3] M R Stratton P J Campbell and P A Futreal ldquoThe cancergenomerdquo Nature vol 458 no 7239 pp 719ndash724 2009

[4] M G Kris R B Natale R S Herbst et al ldquoEfficacy ofgefitinib an inhibitor of the epidermal growth factor receptortyrosine kinase in symptomatic patients with non-small celllung cancer a randomized trialrdquo Journal of the AmericanMedical Association vol 290 no 16 pp 2149ndash2158 2003

[5] S Elmore ldquoApoptosis a review of programmed cell deathrdquoToxicologic Pathology vol 35 no 4 pp 495ndash516 2007

[6] T S Mok Y-L Wu S Thongprasert et al ldquoGefitinib orcarboplatin-paclitaxel in pulmonary adenocarcinomardquo TheNew England Journal of Medicine vol 361 no 10 pp 947ndash9572009

[7] T G Cotter ldquoApoptosis and cancer the genesis of a researchfieldrdquo Nature Reviews Cancer vol 9 no 7 pp 501ndash507 2009

[8] C Wang and R J Youle ldquoThe role of mitochondria in apopto-sisrdquo Annual Review of Genetics vol 43 pp 95ndash118 2009

[9] T J Sayers ldquoTargeting the extrinsic apoptosis signaling pathwayfor cancer therapyrdquo Cancer Immunology Immunotherapy vol60 no 8 pp 1173ndash1180 2011

[10] P Katiyar M Yadav and A Tiwari ldquoRole of caspase in pro-grammed cell death in multicellular organismsrdquo InternationalJournal of Biomedical Research vol 2 no 7 pp 422ndash431 2011

[11] I Budihardjo H Oliver M Lutter X Luo and X WangldquoBiochemical pathways of caspase activation during apoptosisrdquoAnnual Review of Cell and Developmental Biology vol 15 no 1pp 269ndash290 1999

[12] G Kroemer ldquoThe proto-oncogene Bcl-2 and its role in regulat-ing apoptosisrdquo Nature Medicine vol 3 no 6 pp 614ndash620 1997

[13] Y Rong and C W Distelhorst ldquoBcl-2 protein family membersversatile regulators of calcium signaling in cell survival andapoptosisrdquoAnnual Review of Physiology vol 70 pp 73ndash91 2008

[14] R J Youle andA Strasser ldquoThe BCL-2 protein family opposingactivities thatmediate cell deathrdquoNature ReviewsMolecular CellBiology vol 9 no 1 pp 47ndash59 2008

[15] Y-L P Ow D R Green Z Hao and T W Mak ldquoCytochromec functions beyond respirationrdquoNature Reviews Molecular CellBiology vol 9 no 7 pp 532ndash542 2008

[16] J W-H Li and J C Vederas ldquoDrug discovery and naturalproducts end of an era or an endless frontierrdquo Science vol 325no 5937 pp 161ndash165 2009

[17] G M Cragg P G Grothaus and D J Newman ldquoImpactof natural products on developing new anti-cancer agentsrdquoChemical Reviews vol 109 no 7 pp 3012ndash3043 2009

[18] J Kopecky V Matha and A Jegorov ldquoThe inhibitory effect ofdestruxin a on the replication of arboviruses inAedes albopictus


C636 cell linerdquo Comparative Biochemistry and Physiology Cvol 103 no 1 pp 23ndash25 1992

[19] K Adachi H Nanba and H Kuroda ldquoPotentiation of host-mediated antitumor activity in mice by 120573-glucan obtainedfromGrifola frondosa (maitake)rdquoChemical and PharmaceuticalBulletin vol 35 no 1 pp 262ndash270 1987

[20] T Ohmori K Tamura S Tsuru and K Nomoto ldquoAntitu-mor activity of protein-bound polysaccharide from Cordycepsophioglossoides in micerdquo Japanese Journal of Cancer Researchvol 77 no 12 pp 1256ndash1263 1986

[21] R E Moore ldquoCyclic peptides and depsipeptides fromcyanobacteria a reviewrdquo Journal of Industrial Microbiology vol16 no 2 pp 134ndash143 1996

[22] K L Rinehart ldquoAntitumor compounds from tunicatesrdquoMedic-inal Research Reviews vol 20 no 1 pp 1ndash27 2000

[23] G A Bradfisch and S L Harmer ldquo120596-conotoxin GVIA andnifedipine inhibit the depolarizing action of the fungal metabo-lite destruxin B on muscle from the tobacco budworm (helio-this virescens)rdquo Toxicon vol 28 no 11 pp 1249ndash1254 1990

[24] H-C Chen C-K Chou C-M Sun and S F Yeh ldquoSuppressiveeffects of destruxin B on hepatitis B virus surface antigen geneexpression in human hepatoma cellsrdquo Antiviral Research vol34 no 3 pp 137ndash144 1997

[25] F Odier A Vey and J P Bureau ldquoIn vitro effect of fungalcyclodepsipeptides on leukemic cells study of destruxins A Band Erdquo Biology of the Cell vol 74 no 3 pp 267ndash271 1992

[26] J-W Chen B-L Liu and Y-M Tzeng ldquoPurification and quan-tification of destruxins A and B from Metarhizium anisopliaerdquoJournal of Chromatography A vol 830 no 1 pp 115ndash125 1999

[27] B L Liu T M Rou Y K Rao and Y M Tzeng ldquoEffect ofpH and aeration rate on the production of destruxins A and Bfrom Metarhizium anisopliaerdquo International Journal of AppliedScience and Engineering vol 5 no 1 pp 17ndash26 2007

[28] Y P Lee C W Wang W C Liao et al ldquoIn vitro and in vivoanticancer effects of destruxin B on human colorectal cancerrdquoAnticancer Research vol 32 no 7 pp 2735ndash2745 2012

[29] G-C Chang C-T R Yu C-H Tsai et al ldquoAn epidermalgrowth factor inhibitor Gefitinib induces apoptosis througha p53-dependent upregulation of pro-apoptotic molecules anddownregulation of anti-apoptotic molecules in human lungadenocarcinoma A549 cellsrdquo European Journal of Pharmacol-ogy vol 600 no 1ndash3 pp 37ndash44 2008

[30] B S Patil G K Jayaprakasha K N Chidambara Murthyand A Vikram ldquoBioactive compounds historical perspectivesopportunities and challengesrdquo Journal of Agricultural and FoodChemistry vol 57 no 18 pp 8142ndash8160 2009

[31] R Lemmens-Gruber M R Kamyar and R DornetshuberldquoCyclodepsipeptidesmdashpotential drugs and lead compounds inthe drug development processrdquo Current Medicinal Chemistryvol 16 no 9 pp 1122ndash1137 2009

[32] E Morel M Pais M Turpin and M Guyot ldquoCytotoxicityof cyclodepsipeptides on murine lymphocytes and on L 1210leukemia cellsrdquo Biomedicine and Pharmacotherapy vol 37 no4 pp 184ndash185 1983

[33] C-T Yeh Y K Rao M Ye et al ldquoPreclinical evaluationof destruxin B as a novel Wnt signaling target suppressingproliferation and metastasis of colorectal cancer using non-invasive bioluminescence imagingrdquo Toxicology and AppliedPharmacology vol 261 no 1 pp 31ndash41 2012

[34] RMKluck E Bossy-Wetzel D R Green andDDNewmeyerldquoThe release of cytochrome c frommitochondria a primary site

for Bcl- 2 regulation of apoptosisrdquo Science vol 275 no 5303 pp1132ndash1136 1997

[35] B Leber J Lin and D W Andrews ldquoEmbedded together thelife and death consequences of interaction of the Bcl-2 familywith membranesrdquo Apoptosis vol 12 no 5 pp 897ndash911 2007

[36] A Cuconati C Mukherjee D Perez and E White ldquoDNAdamage response and MCL-1 destruction initiate apoptosis inadenovirus-infected cellsrdquo Genes and Development vol 17 no23 pp 2922ndash2932 2003

[37] J Yu and L Zhang ldquoPUMA a potent killer with or without p53rdquoOncogene vol 27 no 1 pp S71ndashS83 2008

[38] L Chen S N Willis A Wei et al ldquoDifferential targeting ofprosurvival Bcl-2 proteins by their BH3-only ligands allowscomplementary apoptotic functionrdquoMolecular Cell vol 17 no3 pp 393ndash403 2005

[39] M S Ola M Nawaz and H Ahsan ldquoRole of Bcl-2 familyproteins and caspases in the regulation of apoptosisrdquoMolecularand Cellular Biochemistry vol 351 no 1-2 pp 41ndash58 2011

[40] T-J Fan L-H Han R-S Cong and J Liang ldquoCaspase familyproteases and apoptosisrdquo Acta Biochimica et Biophysica Sinicavol 37 no 11 pp 719ndash727 2005

[41] S Kumar ldquoCaspase 2 in apoptosis the DNA damage responseand tumour suppression enigma no morerdquo Nature ReviewsCancer vol 9 no 12 pp 897ndash903 2009

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


by the binding of the membrane receptor to the death ligandsuch as TNF (tumor necrosis factor)TNFR and FasFasL[9] The apoptotic process is mainly executed by a class ofcysteine proteases known as caspases [10] Activation ofdifferent caspases usually presents for various upstreamapoptotic pathways For example the activation of caspase-8and -10 usually stands for the extrinsicdeath receptor-relatedpathway caspase-2 and caspase-9 are usually activated bymitochondrial pathway while caspase-12 is activated byendoplasmic reticulum pathway [10] In addition theactivation of caspase-3 -6 or -7 is usually considered as acommon executor of apoptosis by directly cleaving cellularproteins when activated [11]

Several proteins are known to be involved in the reg-ulation of apoptosis among them Bcl-2 family moleculescontribute to themitochondrial-mediated apoptotic pathwayBcl-2 is originally found in a translocation chromosomalfragment in B-cell lymphoma and is proved as a protoonco-gene [12] The members of Bcl-2 family all contain a BH(Bcl-2 homology) domain structure and are divided intotwomajor groups one composed of antiapoptotic moleculesincluding Bcl-2 Bcl-xL Bcl-w Mcl-1 BooDiva and A1Bfl-1 [13] and the other comprises proapoptotic moleculessuch as Bax Bak Bad Bim Bik PUMA and NOXA [14]Under normal conditions the Bcl-2 or Bcl-xL complexeswith Bax or Bak to form homo- or hetero dimer and suchinteraction will neutralize proapoptotic molecules to preventcell death Under apoptotic stimulation however it willchange the ratios between these two groups leading to theformation of BaxBax or BaxBak complexes followed byperforation through the membrane of mitochondria Theopened pores eventually cause the release of cytochromec from mitochondria to the cytosol and form apoptosiscomplex with Apaf-1 (apoptotic protease activation factor-1) dATP (deoxyadenosine triphosphate) and procaspase-9 to induce the caspase-9 and caspase-3 activation andirreversible apoptosis [15]

It is well known that certain compounds isolated frombacteria or fungus carry characteristic biological effectswhich have been used as traditional medicines [16 17] andseveral of these compounds have shown an antitumoralactivity [18ndash20] One group of such biological active com-pounds is depsipeptides which are the major secondarymetabolites of fungi The depsipeptides are biooligomersformed by esterification between hydroxyl groups and aminoacids Destruxin B is a cyclic depsipeptide isolated fromentomopathogenic fungus in 1961 Until recently there were39 destruxins reported [21] It has been reported that cyclicdepsipeptides exhibited strong antitumor activity in both invitro and in vivo experiments and evenly entered the clinicaltrial [22] Previous reports mentioned that destruxin B hasstrong anti-insect activity [23] inhibits hepatitis B surfaceantigen expression and induces cell death in hepatocellularcarcinoma cells [24] and leukemic cells [25] The effects ofdestruxin B on lung cancer cells and its molecular mecha-nisms however have not been examined yet In this studythe antiproliferative cytotoxic effects and underlying mecha-nisms of destruxinB (isolated andpurified from fermentationbroth of Metarhizium anisopliae) on lung cancer cell lines

were explored Our observations indicate that destruxin Binduces a Bcl-2 family-dependent mitochondrial apoptoticcell death in human nonsmall cell lung cancer A549 andH1299 cell lines

2 Material and Methods

21 Reagents The destruxin B (MW 593) was dissolved inethanol to a stock solution at concentration of 20mM for thefollowing experiments Anti-Bax Bcl-2 Bcl-xL Bak PUMAMcl-1 and 120573-actin were purchased from Santa Cruz Biotech-nology (Santa Cruz CA USA) or Calbiochem ChemicalCompany (CN Biosciences Notts UK) Anti-mouse andanti-rabbit IgG peroxidase-conjugated secondary antibodieswere purchased from Amersham (Buckinghamshire UK)Caspase activity assay kits including the fluorogenic sub-strates of caspase-2 (VDVAD-AFC) caspase-3 (DEVD-AFC)and caspase-9 (LEHD-AFC) were purchased from RampDsystems (Minneapolis MN USA) The caspase-2 inhibitor(z-VDVAD-FMK) was purchased from Santa Cruz Biotech-nology (Santa Cruz CA USA) The caspase-3 inhibitor (Z-DEVD-FMK) and caspase-9 inhibitor (Z-LEHD-FMK) werepurchased from Kamiya (Seattle WA USA)

22 Isolation and Purification of Destruxin B The destruxinB used in this study was extracted and purified from thesubmerged cultivation of fungusMetarhiziumanisopliae F061var Anisopliae according to the processes described else-where [26 27] BrieflyMetarhizium anisopliaewas cultivatedin 3maltose containing 05 bactopeptone liquid mediumat 28∘C After 14 days the culture medium was collectedand extracted three times with equal volume of methylenedichloride (CH

2Cl2) The organic layer was collected and

concentrated and further separated by two-step ion-exchangechromatography Combination of cationic (Amberlite TRA-400 CP column) and anionic (Amberlite IR 120 Plus)exchange provided the step to removing charged impuritiesexisting in the mixture and then the eluent was collectedand subjected to flash silica chromatography (Merck K-60silica gel column) as a prepurification step The columnwas eluted with a methylene dichloride-methanol mixtureFurther purification was done by loading the sample intoa semipreparative RP-C18 high-performance liquid chro-matographic (HPLC) column The which elution delivereda single peak was collected and concentrated This peakrepresented destruxin B (retention time = 176min) Thepurity of destruxin B in the sample was proved gt95 in ourearly report [28]

23 Cell Culture and Viability Assay The human lung cancerA549 and H1299 cells were obtained from CCRC (CultureCollection and Research Center Hsinchu Taiwan) TheA549-shBax cells were established by transfection of pSuper-shBax followed by antibiotics selection [29] A549 A549-shBax and H1299 cells were cultured in RPMI (GIBCOBRLMD USA) All cultured media were supplemented with10 fetal bovine serum 2mM glutamine and antibiotics(100UmL penicillin and 100 120583gmL streptomycin) at 37∘C











3 Results















(a)

176 min

0 5 10 15 20 25

0

30

60

90

120


Inte

nsity

(mV

)(b)

N

O

H

O

O

O

N O

N

N

N

H

HH

O

O

(c)


2

Cl2



50value at




A549

Control 1 5 10 20 30

0 1 5 10 20 300

20

40

60

80

100

120

Gro

wth

inhi

bitio

n(

of c

ontro

l)

IC50 = 49 120583M



(a)

Control 10 20 30

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 6000

200 Sub-G1 06G1 662S 182G2M 152

Sub-G1 80G1 687S 162G2M 79

Sub-G1 137G1 699S 117G2M 56

Sub-G1 193G1 691S 95G2M 37

0

200

0

200

0

200


(b)

ControlDestruxin B


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

lowastlowastlowast

0

5

10

15

20


(c)


minus + + + +


0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowastlowast

lowastlowast

lowastlowast

(d)




12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25


lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)







MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


























3 Results















(a)

176 min

0 5 10 15 20 25

0

30

60

90

120


Inte

nsity

(mV

)(b)

N

O

H

O

O

O

N O

N

N

N

H

HH

O

O

(c)


2

Cl2



50value at




A549

Control 1 5 10 20 30

0 1 5 10 20 300

20

40

60

80

100

120

Gro

wth

inhi

bitio

n(

of c

ontro

l)

IC50 = 49 120583M



(a)

Control 10 20 30

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 6000

200 Sub-G1 06G1 662S 182G2M 152

Sub-G1 80G1 687S 162G2M 79

Sub-G1 137G1 699S 117G2M 56

Sub-G1 193G1 691S 95G2M 37

0

200

0

200

0

200


(b)

ControlDestruxin B


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

lowastlowastlowast

0

5

10

15

20


(c)


minus + + + +


0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowastlowast

lowastlowast

lowastlowast

(d)




12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25


lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)







MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























(a)

176 min

0 5 10 15 20 25

0

30

60

90

120


Inte

nsity

(mV

)(b)

N

O

H

O

O

O

N O

N

N

N

H

HH

O

O

(c)


2

Cl2



50value at




A549

Control 1 5 10 20 30

0 1 5 10 20 300

20

40

60

80

100

120

Gro

wth

inhi

bitio

n(

of c

ontro

l)

IC50 = 49 120583M



(a)

Control 10 20 30

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 6000

200 Sub-G1 06G1 662S 182G2M 152

Sub-G1 80G1 687S 162G2M 79

Sub-G1 137G1 699S 117G2M 56

Sub-G1 193G1 691S 95G2M 37

0

200

0

200

0

200


(b)

ControlDestruxin B


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

lowastlowastlowast

0

5

10

15

20


(c)


minus + + + +


0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowastlowast

lowastlowast

lowastlowast

(d)




12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25


lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)







MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















A549

Control 1 5 10 20 30

0 1 5 10 20 300

20

40

60

80

100

120

Gro

wth

inhi

bitio

n(

of c

ontro

l)

IC50 = 49 120583M



(a)

Control 10 20 30

0 200 400 600 0 200 400 600 0 200 400 600 0 200 400 6000

200 Sub-G1 06G1 662S 182G2M 152

Sub-G1 80G1 687S 162G2M 79

Sub-G1 137G1 699S 117G2M 56

Sub-G1 193G1 691S 95G2M 37

0

200

0

200

0

200


(b)

ControlDestruxin B


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

lowastlowastlowast

0

5

10

15

20


(c)


minus + + + +


0

20

40

60

80

100

120

Cel

l via

bilit

y (

of c

ontro

l)

lowastlowast

lowastlowast

lowastlowast

(d)




12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25


lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)







MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















12 24Time (h)

0 10 20 0 10 20

PUMA

Bax

Bak

Mcl-1

Bcl-xL

Bcl-2

120573-Actin

(a)

ControlDestruxin B

8 12 24Time (h)

8 12 24Time (h)

0

21

3456789

0

05

1

15

2

25


lowastlowastlowast

Rela

tive q

uant

ity o

fPU

MA

mRN

A ex

pres

sions

Rela

tive q

uant

ity o

fM

cl-1

mRN

A ex

pres

sions

(b)







MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















MergeMitoTracker

Bax

MergeMitoTracker

BaxDAPI

Des

trux

in B

Con

trol

(a)


0 10 20

A549

A549-shBax

(b)

Time (h) 24 48

A549A549-shBax A549

A549-shBax

Bax

120573-Actin


A549A549-shBax

0

20

40

60

80

100

120

Cyto

toxi

city

( o

f tot

al ce

ll nu

mbe

r)

lowastlowastlowast

lowastlowast

(c)






4 Discussion




Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Control 1 5 10 20 30

H1299

0

20

40

60

80

100

120

Gro

wth

inhi

bitio

n (

of c

ontro

l)

0 1 5 10 20 30Destruxin B (120583M)

IC50 = 41 120583M

(a)


Casp

ase a

ctiv

ity (f

olds

of c

ontro

l)

0

3

6

9

12

15

18

ControlDestruxin B

lowastlowastlowast

lowastlowastlowast

lowastlowastlowast

(b)


0 10 20 0 10 20

PUMA

Mcl-1

Bax

120573-Actin

(c)





N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















N

O

H

O

OO

N O

N

N

N

H

HH

O

O



Des

trux

in B

prep

arat

ion


RP-C18 HPLC


Caspase-2


translocationof Bax

shBax

Caspase-9

Caspase-3z-LEHD-FMK

Apoptosis

Cel

lula

r act

ions

of d

estr

uxin

B

z-DEVD-FMK

darrMcl-1 PUMAuarr












Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















Acknowledgments


References


















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article destruxin b isolated from...

Documents