polyporenic acid c induces caspase-8-mediated apoptosis in human lung cancer a549 cells

Polyporenic Acid C Induces Caspase-8-MediatedApoptosis in Human Lung Cancer A549 Cells

Hui Ling,1 Liang Zhou,1 Xiaobin Jia,2 Leslie A. Gapter,1 Rajesh Agarwal,3 and Ka-yun Ng1*1Department of Pharmacy, National University of Singapore, Singapore, Republic of Singapore2Jiangsu Academy of Chinese Medicine, Nanjing People’s Republic of China3Department of Pharmaceutical Sciences, School of Pharmacy, Univeristy of Colorado at Denver and Health Sciences Center,Denver, Colorado

Lung cancer continues to be the leading cause of cancer-related mortality worldwide. This warrants the search fornew and effective agents against lung cancer. We and others have recently shown that lanostane-type triterpenoids

isolated from the fungal species Poria cocos (P. cocos) can inhibit cancer growth. However, the mechanismsresponsible for the anticancer effects of these triterpenoids remain unclear. In this study, we investigated the effect ofpolyporenic acid C (PPAC), a lanostane-type triterpenoid from P. cocos, on the growth of A549 nonsmall cell lung

cancer cells (NSCLC). The results demonstrate that PPAC significantly reduced cell proliferation via induction ofapoptosis as evidenced by sub-G1 analysis, annexin V-FITC staining, and increase in cleavage of procaspase-8, -3, andpoly(ADP-ribose)-polymerase (PARP). However, unlike our previously reported lanostane-type triterpenoid, pachymicacid, treatment of cells with PPAC was not accompanied by disruption of mitochondrial membrane potential and

increase in cleavage of procaspase-9. Further, PPC-induced apoptosis was inhibited by caspase-8 and pan caspaseinhibitors but not by a caspase-9 inhibitor. Taken together, the results suggest that PPAC induces apoptosis throughthe death receptor-mediated apoptotic pathway where the activation of caspase-8 leads to the direct cleavage of

execution caspases without the involvement of the mitochondria. Furthermore, suppressed PI3-kinase/Akt signalpathway and enhanced p53 activation after PPAC treatment suggests this to be an additional mechanism forapoptosis induction. Together, these results encourage further studies of PPAC as a promising candidate for lung

cancer therapy. � 2008 Wiley-Liss, Inc.

Key words: polyporenic acid C; Poria cocos; lung cancer; apoptosis; caspase-8; Akt; JNK; PARP; p53

INTRODUCTION

Lung cancer is the leading cause of cancer mortal-ity worldwide with an estimated 5-yr survival rate ofless than 15% [1]. NSCLC accounts for approxi-mately 75–80% of all lung cancer cases and thusrepresents the bulk of lung cancers [2]. The poorprognosis of lung cancer is largely attributed tothe frequent occurrence of metastasis, since mostmetastatic tumors are unresectable at the time ofpresentation [3]. The severe morbidity and poorprognosis of lung cancer highlight the importance ofsearching for new and effective agents against lungcancer.

Natural products are one of the main sources fordiscovery of lead drug compounds. Due to theirdiverse biological effects, naturally occurring cyclictriterpenoids such as ursolic acid, tubeimoside,abieslactone and oleanolic acid have recentlyattracted much attention in cancer research [4–7].Triterpenoids refer to a class of compounds synthe-sized by the cyclization of squalene [8]. Triterpenoidsare widely found in nature and more than 4000different triterpenoid-like substances have beenidentified to date [9]. Recently, two semisynthetictriterpenoids, 2-cyano-3,12-dioxooleana-1,9(11)-dien-

28-oic acid (CDDO) and its methyl ester (CDDO-Me),have been shown to have strong cancer therapeuticand preventive activity in a variety of in vitro and invivo studies [10–14].

The fungal species, Poria cocos, has been tradition-ally used in Chinese herbal prescriptions as diureticand sedative agents [reviewed in [15]]. Alcoholicextracts of P. cocos contain various lanostane-typetriterpenoids such as pachymic acid, 3-O-acetyl-16alpha-hydroxytrametenolic acid, poricoic acid B,and polyporenic acid C (PPAC) [16–19]. Lanostane-type triterpenoids have been shown to markedlysuppress the promoting effect of the inflammatoryinducing agent, 12-O-tetradecanoylphorbol-13-acetate(TPA), on skin tumor formation in mice followinginitiation with 7,12-dimethylbenz[a]anthracene

MOLECULAR CARCINOGENESIS 48:498–507 (2009)

� 2008 WILEY-LISS, INC.

Abbreviations: PPAC, polyporenic acid C.

*Correspondence to: Faculty of Science, Department of Phar-macy, National University of Singapore, Building S4, Rm 05-02, 18Science Drive 4, Singapore 117543, Republic of Singapore.

Received 13 May 2008; Revised 14 July 2008; Accepted 6September 2008

DOI 10.1002/mc.20487

Published online 30 October 2008 in Wiley InterScience(www.interscience.wiley.com)

(DMBA) [20]. Furthermore, lanostane-type triterpe-noids from P. cocos possess cytotoxicity in varioushuman cancer cell lines [19,21,22]. We recentlyshowed that pachymic acid (Figure 1A), the maintriterpenoid found in P. cocos, induced apoptosis inprostate cancer cells [23]. To establish whetherlanostane-type triterpenoids are potential chemo-therapeutic and preventive agents for lung cancer,we examined the anticancer activity of PPAC(Figure 1B) in a NSCLC cell line, A549. PPAC, whichis found in P. cocos, has been reported to haveantibacterial activity and inhibit Epstein-Barr virusearly antigen (EBV-EA) activation by TPA [16,24,25].However, the cytotoxicity of PPAC on cancer cellshas not been well studied. Although one reportsuggests that PPAC inhibits DNA topoisomerases[21], the anticancer potency and mechanism ofPPAC remain unclear. In this study, we providefirst time evidence to suggest that PPAC potentlysuppresses lung cancer cell proliferation by induc-tion of apoptosis. Furthermore, our data suggestthat PPAC induces apoptosis through the deathreceptor-mediated pathway. We also found thatPPAC treatment suppresses PI3-kinase/Akt signalpathway and enhances p53 activation.

MATERIALS AND METHODS

Reagents

Dried sclerotia of P. cocos Wolf (Polyporaceae)harvested in Anhui, China was purchased from alocal reputable Chinese herb store. Vybrant Apop-tosis Assay Kit and 3,30dihexyloxacarbocyanineiodide [DiOC6(3)] were purchased from MolecularProbes (Eugene, OR). Antibody for actin (I-19) was

purchased from Santa Cruz Biotechnology (SantaCruz, CA). Antibodies specific for phospho-p53Ser15,phospho-AKTSer473, cleaved caspase-3Asp175, cleavedcaspase-8Asp374, caspase-9, cleaved PARP, phospho-JNK, goat anti-rabbit IgG-conjugated to horseradishperoxidase (HRP), and goat anti-mouse IgG-conju-gated to HRP were purchased from Cell SignalingTechnology (Beverly, MA). Pan caspase inhibitor(z-VAD-fmk), caspase-8 inhibitor (z-IETD-fmk) andcaspase-9 inhibitor (z-LEHD-fmk) were purchasedfrom R&D Systems (Minneapolis, MN). JNK-specificinhibitor, SP600125, was purchased from BiomolInternational LP (Plymouth Meeting, PA). All chem-icals that were not specifically indicated werepurchased from Sigma (St. Louis, MO).

Extraction and Purification of PPAC

Pulverized sclerotia of P. cocos were extractedthree times with 95% ethanol (12 L) under refluxfor 3 h. The ethanol solution was combined andevaporated in vacuum to give a crude extract. Thecrude extract was mixed with silica gel and fractio-nated using silica column chromatography and agradient elution of CHCl3 and MeOH. Fractionswere collected, combined and subjected to furtherchromatography by step-wise gradient usingCHCl3–CH3OH. The collected fractions were com-bined on the basis of their TLC characteristics to givefour pooled fractions: PCA, PCB, PCC, and PCD,listed in increasing order of polarity. PCB wassubjected to further chromatography on an ODScolumn with step-wise gradient elution conductedwith CH3OH–H2O, which gave rise to two sub-fractions: PCB-a and PCB-b. PCB-b was subjected tochromatography on a silica gel 60 column, fromwhich PPAC (Figure 1B) was isolated with stepgradient elution of CHCl3–CH3OH. Identificationof PPAC was conducted by comparison of its physicaland spectroscopic data (1H, 13C NMR, and MS)with the corresponding compound reported in theliterature [24].

Cell Culture and Treatment

A549 human NSCLC cells were obtained fromAmerican Type Culture Collection (Rockville, MD)and cultured in F12 Ham Kaighn’s modification(F12K) medium supplemented with 10% fetal bovineserum (FBS; Hyclone Laboratories, Logan, UT),10 mM HEPES (AppliChem, Darmstadt, Germany)and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin; Invitrogen, Carlsbad, CA). Thecells were maintained at 378C in a 5% CO2 humidi-fied incubator. PPAC was reconstituted in DMSO at astock concentration of 20 mg/mL and further dilutedwith DMSO to the working concentrations for theexperimental procedures. DMSO was employed as anegative control at a final concentration �0.5%in all experiments; this dose shows no significant

HOOC

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Figure 1. Chemical structures of pachymic acid (A) and PPAC (B).

POLYPORENIC ACID C INDUCES APOPTOSIS IN NSCLC 499

Molecular Carcinogenesis

difference in A549 cell growth over a 72 h time periodwhen compared to untreated cells (data not shown).

Cell Proliferation Assay

The effect of PPAC on the proliferation of A549cells was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT)as described by the manufacturer. In brief, 5� 103

A549 cells per well were plated in 96-well cultureplates. After overnight incubation, the cells weretreated for the indicated times with fresh mediumcontaining the desired concentrations of PPAC.Absorbance was measured at 590 nm using a micro-plate reader (Tecan GENios, Salzburg, Austria). Theeffect of PPAC on cell viability was expressed aspercent cell viability compared with vehicle treatedcontrol cells set at 100%.

Propidium Iodide Staining Assay

To quantify PPAC-induced apoptosis, A549 cellswere stained with propidium iodide (PI) and sub-jected to cytofluorometric analysis. The cells weretreated with various concentrations of PPAC for theindicated time periods. The floating and trypsinizedadherent cells were collected and washed twice withPBS. The cells were spun to a pellet, resuspended inPBS, and fixed using ice-cold ethanol at �208C for atleast 30 min. The cells were pelleted by centrifuga-tion and incubated with 0.5 mL of PI stainingsolution [0.1% (v/v) Triton X-100 in PBS containing200 mg/mL of RNase and 20 mg/mL of PI] at roomtemperature for 30 min. Cell cycle distributionwas analyzed by a CYAN-LX flow cytometer (Dako-Cytomation, Fort Collins, CO) equipped withSummit v4.3 software (DakoCytomation). Cells withsub-G1 DNA (as designated by R1 in the histogram)were classified as apoptotic cells.

Apoptosis Assay

A549 cells were analyzed for apoptosis by annexinV-FITC labeling according to the manufacturer’sinstructions (Vybrant Apoptosis Assay Kit [MolecularProbes]). In brief, the cells were treated with thedesired concentrations of PPAC for the indicatedtime periods. The floating and trypsinized adherentcells were collected and washed twice with PBS. Thecell pellets were suspended in 100 mL of AnnexinBinding Buffer and stained with 5 mL of Alexa Fluor1

488 annexin V and 1 mL of 100 mg/mL PI. Afterincubation at room temperature for 15 min, the cellswere gently mixed with 400 mL of Annexin BindingBuffer and analyzed immediately by flow cytometryas described above. The early apoptotic cells, whichbind to annexin V-FITC and give a green fluores-cence, are represented in the lower right quadrant ofthe histogram. The late apoptotic cells, which bindto both FITC and PI, have a red-green fluorescenceand are represented in the upper right quadrant of

the histogram. A549 cells were pretreated with thedesired inhibitor for 1 h in the experiments usingcaspase inhibitors.

Analysis of Mitochondrial Membrane Potential

The mitochondrial membrane potential (DCm)was analyzed by staining A549 cells with DiOC6(3)[26] (40 nM, Molecular Probes) during the final30 min of PPAC treatment. To detect membranedepolarization, the cells were washed twice inPBS containing 0.1% BSA (w/v), spun to a pellet,suspended in 500 mL of PBS/0.1% BSA, and analyzedby flow cytometry (DakoCytomation).

Protein Extraction and Immunoblotting

A549 cells were harvested following PPAC treat-ment as described above (PI staining and apoptosisassay) and washed with cold PBS. The cells wereincubated in ice-cold lysis buffer (Cell SignalingTechnology) containing freshly added 1 mM phenyl-methylsulfonyl fluoride, 10 mg/mL aprotinin, 5 mg/mL pepstatin A, and 100 mM leupeptin. After briefsonication on ice, the protein content was quantifiedby the bicinchoninic acid (BCA) colorimetric detec-tion method according to the manufacturer’s proto-col (Pierce, Rockford, IL). Lysate containing 40 mgprotein was then resolved through a 10% sodiumdodecyl sulfate (SDS)–polyacrylamide gel andtransferred to polyvinylidene fluoride membrane(Millipore, Bedford, MA). The transblotted mem-brane was washed, blocked, and incubated with theprimary antibody and appropriate secondary anti-body according to the manufacturer’s directions.The antibody/protein complexes were visualizedby enhanced chemiluminescence (Perkin-Elmer,Boston, MA) and digital images were acquired usinga FluorChemTM 9900 (Alpha Innotech, San Leandro,CA).

Statistical Analysis

Statistical significance between treatment andcontrol groups was analyzed using a two-tailedStudent’s t-test. Values of P< 0.05 were consideredstatistically significant.

RESULTS

PPAC Inhibits NSCLC Cell Proliferation

We first examined the growth-inhibitory effect ofPPAC on A549 cells using MTT assay. The treatmentof A549 cells with 0–200 mM of PPAC resulted in adose- and time-dependent inhibition of cell growth(Figure 2). After 72 h, the proliferation of A549 cellswas significantly inhibited by PPAC at concentra-tions as low as 6 mM (84% of A549 cells remainedviable relative to DMSO control). The relative cellviability was reduced more than 50% when A549cells were treated with 60 mM of PPAC, while at

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200 mM, PPAC inhibited cancer cell growth by morethan 90%. These data suggest that PPAC effectivelyinhibited A549 cell proliferation.

PPAC Induces NSCLC Cell Apoptosis

To determine whether PPAC inhibited A549 cellproliferation through the induction of apoptosis, weexamined apoptosis using two approaches. First,PPAC-treated cells were incubated with PI andanalyzed for sub-G1 staining. The DNA content ofapoptotic cells is low due to loss of small fragmentedDNA during the apoptotic process [27]. The sub-G1population represents cells with decreased PI stain-ing and thus represents apoptotic cells. As shownin Figure 3A, the cell population in sub-G1 (R1)increased time-dependently from 2% to 47% within48 h following 60 mM PPAC treatment. PPAC alsoinduced apoptosis after 48 h treatment in a dose-dependent manner (Figure 3B), as shown by theincreased fraction of sub-G1 cells from 5% to 51% by20 and 80 mM PPAC treatment, respectively. Toconfirm that PPAC induced apoptosis, we also usedan alternative method whereby A549 cells werestained with annexin V-FITC and PI using anapoptosis assay kit. In apoptotic cells, the phospho-lipid asymmetry of the plasma membrane is lost andphosphatidylserine (PS) is transcolated from theinner to the outer cell membrane [28]. The exposedPS is recognized and bound by FITC-conjugatedannexin V [29]. Apoptotic cells were counted as earlyor late apoptotic cells, which are shown in the lowerright region (R5) and upper right region (R3) ofthe histograms presented in Figure 3C and D. After24 h, PPAC treatment resulted in a dose-dependentincrease in the number of apoptotic cells in both theearly and late stages of apoptosis (Figure 3C): 0 mM(vehicle control, 5.6%), 60 mM (20.8%), 100 mM(33.8%). With prolonged treatment, the apoptoticfraction increased to 30.3% and 93.8% by 60 and100 mM of PPAC, respectively (Figure 3D). Takentogether, these results indicate that induction ofapoptosis was responsible for growth inhibition ofA549 cells after PPAC treatment.

PPAC Activates Caspase-8-Mediated ApoptoticSignaling Pathway

To investigate the involvement of caspases inPPAC-induced apoptosis, we examined whetherPPAC treatment activated initiator caspases-8 and-9, and the executioner, caspase-3. As shown inFigure 4A, treatment of A549 cells with 60 mM ofPPAC resulted in caspase-8 and caspase-3 cleavage,but not that of caspase-9. Cleavage of the caspase-3substrate, PARP, also correlated with caspase-3activation (Figure 4A). These results indicate thatPPAC may induce apoptosis in A549 cells throughactivation of caspase-8.

To further confirm the requirement of caspaseactivation in PPAC-induced apoptosis, we usedcaspase inhibitors to see if cells were protected fromapoptosis. The results presented in Figure 4B lendsupport to a caspase-8-dependent apoptotic signal-ing pathway: the apoptotic fractions (including earlyand late apoptosis) were 4.3% and 25.4% in thecontrol and PPAC-treated cells, respectively, com-pared with 5.1% and 6.4% in the cells coincubatedwith PPAC and pan caspase inhibitor, z-VAD-fmk, orcaspase-8 inhibitor, z-IETD-fmk. In contrast, thecaspase-9 inhibitor, z-LEHD-fmk, failed to blockPPAC-induced apoptosis (Figure 4B). Consistentwith the flow cytometry data, pretreatment withthe caspase-8 but not the caspase-9 inhibitorprevented PARP cleavage after PPAC treatment(Figure 4C). Collectively, these findings suggest thatinduction of apoptosis by PPAC is dependent on, andinitiated by, caspase-8.

Mitochondrial Membrane Potential (DCm) Is NotDisrupted by PPAC

Caspase-8 has been demonstrated to activatecaspase-3 either directly or indirectly through amitochondria-mediated pathway [30]. In the lattercase, Bid is truncated by caspase-8 which leads to thetranslocation of Bid from the cytosol to the mito-chondria where it disrupts DCm via binding toother Bcl-2 family members and triggers cytochromec release into the cytosol [30,31]. The release ofcytochrome c activates caspase-9 and caspase-3 byforming apoptosomes containing cytochrome c,Apaf-1 and procaspase-9 [30,31]. To determine ifPPAC induced apoptosis indirectly through themitochondria following caspase-8 activation, theeffect of PPAC on theDCm was evaluated. We labeledA549 cells with the cationic lipophilic dye DiOC6(3),which accumulates within mitochondria in a poten-tial-dependent manner. Upon disruption of DCm,the DiOC6(3) fluorescence signal inside the cells isdecreased due to the impaired function of cells toretain DiOC6(3). Treatment of A549 cells with PPAC(20, 60, and 100 mM) for 24 h did not result in anydecrease in DCm (Figure 5). DCm was not disruptedeven with prolonged treatment with PPAC for 48 h

Figure 2. PPAC decreases the cell viability of human NSCLC A549cells in a dose- and time-dependent manner. After treatment, A549cell viability was determined by MTT assay as detailed in Materialsand Methods Section. Columns, mean of three independentexperiments repeated in triplicate; bars, SD. *P< 0.05; **P< 0.01.



(Figure 5). Interestingly, pachymic acid, a structur-ally related lanostane-type triterpenoid from P. cocos,decreased the DiOC6(3) fluorescence intensity inA549 cells in a dose- and time-dependent manner(Figure 5). These results suggest that unlike pachymicacid, PPAC does not induce apoptosis through themitochondria and disruption of DCm. This finding is

consistent with the observations that PPAC did notactivate caspase-9 and inhibition of caspase-9 failedto protect A549 cells from PPAC-induced apoptosis(Figure 4A and B). Taken together, our data indicatethat PPAC induces apoptosis in NSCLC throughcaspase-8 driven proteolysis and direct activation ofexecution caspases.

Figure 3. PPAC induces apoptosis in A549 cells. A549 cells werestained with PI and analyzed by flow cytometry after treatment with60 mM of PPAC for 0, 10, 24, or 48 h (A) or with 0, 20, 60, or 80 mMof PPAC for 48 h (B). Sub-G1 value in (A) and (B) corresponds to thepercentage of cells in R1 region. For apoptosis assay, cells wereexposed to 0, 60, or 100 mM of PPAC for 24 h (C) or 48 h (D) and

stained with the annexin V Apoptosis Vybrant Assay before flowcytometric analysis. The percentage of early apoptotic or lateapoptotic cells is designated by the value at the lower right or upperright quadrant. Data are representative of a single experimentrepeated in three different occasions with similar results.


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Figure 4. Activation of caspase-8 is required for PPAC-inducedapoptosis. (A) Immunoblot analysis for the effect of PPAC on thecleavage of caspase-3, -8 and PARP in A549 cells after 24 htreatment with 60 mM PPAC or vehicle control. (B) Representativeflow cytometry analysis of A549 cells by annexin V-FITC assay after24 h exposure of cells to vehicle control, 60 mM of PPAC, orpretreated with 50 mM of pan caspase inhibitor z-VAD-fmk, 50 mMof caspase-8 inhibitor z-IETD-fmk or 50 mM of caspase-9 inhibitorz-LEHD-fmk for 1 h before treatment with 60 mM PPAC for 24 h. Thepercentage of early apoptotic or late apoptotic cells is designated by

the value at the lower right or upper right quadrant. (C) Immunoblotanalysis of the effect of caspase-inhibitors (pretreatment with 50 mMof pan caspase inhibitor z-VAD-fmk, 50 mM of caspase-8 inhibitorz-IETD-fmk or 50 mM of caspase-9 inhibitor z-LEHD-fmk for 1 h) orJNK inhibitor SP600125 (20 mM) on the cleavage of PARP by 24 htreatment with 60 mM PPAC. Total cellular protein was extracted,resolved using SDS-PAGE, and immunoblotted with the indicatedantibodies. The analysis was repeated three times with similar resultsand a representative immunoblot is shown.



JNK Inhibitor SP600125 Does Not Inhibit ApoptosisInduced By PPAC

Recent studies suggest that activation of c-JunNH2-terminal kinase (JNK) may lead to caspase-8activation [32,33]. We observed that PPAC treatmentled to sustained activation of JNK in A549 cells(Figure 6A). To investigate whether JNK activation isrequired for PPAC-induced apoptosis, we examinedthe effects of PPAC on PARP cleavage in the presenceof the JNK-specific inhibitor, SP600125. As shown inFigure 4C, SP600125 did not inhibit PPAC-inducedcleavage of PARP. This result suggests that inhibitionof JNK activity does not prevent PPAC-inducedapoptosis in A549 cells.

PPAC Inhibits Akt Activation

The PI3-kinase/Akt signal pathway plays a criticalrole in controlling cell survival and apoptosis.Activation of Akt may promote cancer cell survivalby inhibiting apoptosis through its ability to phos-phorylate downstream targets [34]. The tumorsuppressor, PTEN, has been shown to negativelyregulate the PI3-kinase/Akt signal pathway [34,35].To determine the potential involvement of the Aktpathway in PPAC-induced apoptosis, we evaluatedthe phosphorylation status of Akt in treated A549cells. As shown in Figure 6B, Akt phosphorylation

levels were dramatically reduced by PPAC treatment,whereas total Akt protein expression remainedunchanged. Furthermore, PPAC treatment enhanc-ed the phosphorylation of PTEN (Figure 6B), which isbelieved to stabilize PTEN and increase its biologicalactivity [36]. These results indicate that PPAC mayinduce apoptosis in A549 cells through downregula-tion of Akt activity.

PPAC Modulates p53 Activation

Tumor suppressor protein p53 is activated bycellular stress induced by a variety of stimuliincluding chemotherapeutic drugs [37]. To deter-mine whether exposure of A549 NSCLC cells toPPAC triggered p53 activation, we analyzed thephosphorylation state of this protein at Ser15 usingA549 cells which express wild-type p53 [38]. Asshown in Figure 6B, PPAC enhanced p53 activationin A549 cells as evidenced by the increased bandintensity of phosphorylated p53. These resultssuggest that PPAC treatment activates the p53 stressresponse program in A549 cells. The increasedactivation of p53 may also contribute to PPACinduced apoptosis.

DISCUSSION

The screening of active compounds from naturalproducts may provide opportunities for the manage-ment of lung cancer, which remains the leadingcause of cancer-related death among both men andwomen [1]. In the present study, we demonstratethat PPAC, a naturally occurring lanostane-typetriterpenoid from P. cocos, is a potent inhibitor ofhuman NSCLC proliferation. PPAC significantly

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Figure 5. PPAC does not change DCm while pachymic aciddisrupts DCm in a dose- and time-dependent manner. A549 cellswere stained with DiOC6(3) and subjected to flow cytometric analysisafter exposure to 0, 20, 60, or 100 mM of PPAC or pachymic acid,respectively for 24 and 48 h as detailed in Materials and MethodsSection. Relative DiOC6(3) dye intensity in A549 cells after treatmentwith PPAC or pachymic acid, respectively for 24 and 48 h wassummarized in the corresponding bar diagram. Columns, mean ofthree independent experiments; bars, S.D. *P< 0.05 versus vehicle-treated control group.

Figure 6. PPAC treatment activates JNK, suppresses Akt activationand increases the activation of p53. (A) A549 cells were treated with60 mM PPAC for 0, 3, 6, and 24 h and immunoblotted with antibodyspecific for phosphorylated JNK. (B) A549 cells were treated with60 mM PPAC for 24 h and immunoblotted with antibodies specific forphosphorylated PTEN, total Akt, phosphorylated Akt and phosphory-lated p53, respectively. The analysis was repeated three times withsimilar results, and a representative immunoblot for each protein isshown.

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reduced the viability of A549 cells in a dose- andtime-dependent manner (Figure 2). This findingis consistent with previous reports showing thatlanostane-type triterpenoids from P. cocos possessanticancer activity [19,21–23].

Resistance to apoptosis is implicated in theetiology of many cancer types, including lung cancer[39]. Drugs which promote apoptosis in cancer cellsmay be effective for cancer treatment [40]. In thepresent study, we observed that PPAC caused cellshrinkage, nuclei condensation and fragmentation,which are typical features of cell apoptosis (notshown) [31]. Furthermore, PPAC treatment increas-ed the fraction of sub-G1 cells and binding ofannexin V to treated cells (Figure 3). Collectively,these results strongly implied that PPAC-inducedgrowth inhibition of A549 cells was mainly due toenhanced apoptosis.

The activation of the caspase family plays animportant role in apoptosis triggered by pro-apoptotic stimuli [41]. To investigate if apoptosiswas indeed responsible for PPAC-induced growthinhibition, we examined the activation of caspasesby monitoring their cleavage using immunoblotanalysis. In our study, PPAC treatment cleavedcaspase-3 into the active enzyme fragments (19 and17 kDa) (Figure 4A). As a critical executioner ofapoptosis, caspase-3 is either partially or totallyresponsible for the proteolytic cleavage of manykey proteins such as the nuclear enzyme PARP[42,43]. The appearance of a typical 89 kDa PARPcleavage product (Figure 4A), a hallmark of apoptosis[44], further confirmed the proteolytic activity ofcaspase-3. To further confirm the dependence ofcaspase activation in PPAC-induced apoptosis, weincubated cells with a pan-caspase inhibitor beforetreatment with PPAC. The pan-caspase inhibitor,z-VAD-fmk, effectively suppressed PPAC-inducedapoptosis (Figure 4B). These data confirmed thatPPAC induces growth inhibition of A549 cells viainduction of apoptosis.

Two pathways are involved in activation ofapoptosis: the death receptor-mediated extrinsicpathway and the mitochondria-mediated intrinsicpathway [31]. The extrinsic pathway is initiated bythe stimulation of death receptors such as CD95 orTRAIL receptors [45]. This leads to receptor aggrega-tion and recruitment of procaspase-8 through adap-tor proteins, resulting in cleavage and activation ofcaspase-8 [45]. Activated caspase-8 initiates apopto-sis by direct proteolytic cleavage and activation ofexecution caspases, such as caspase-3, or throughmitochondrial damage via the cleavage of Bid [45].In contrast, the intrinsic pathway is initiated bydisruption of DCm and subsequent release of pro-apoptotic factors (e.g., cytochrome c) from themitochondria to the cytosol. The release of cyto-chrome c triggers caspase-3 activation throughformation of the cytochrome-c/Apaf-1/caspase-9-

containing apoptosome complex [31]. In our study,flow cytometric analysis and immunoblot datashowed that PPAC induced apoptosis throughactivation of caspase-8 (Figure 4), indicating thatPPAC induced apoptosis through the death receptor-mediated extrinsic pathway. Activation of the deathreceptor-mediated apoptotic signaling pathway bytriterpenoids is not uncommon as demonstrated bythe naturally occurring triterpenoid, acetyl-keto-b-boswellic acid, and the synthetic triterpenoid,methyl-2-cyano-3,21-dioxooleana-1,9-dien-28-oate[33,46].

Caspase-8 promotes apoptosis by direct proteo-lytic cleavage of execution caspases or indirectmediation of mitochondrial damage [30]. Based onthe finding that pachymic acid, a lanostane-typetriterpenoid from P. cocos, induced apoptosis inprostate cancer cells through the mitochondria-mediated pathway [23], we speculated that mito-chondrial damage might be involved followingactivation of caspase-8 by PPAC in A549 cells. Incontrast to pachymic acid, PPAC surprisingly did notdisrupt DCm (Figure 5). Moreover, our data indicatethat PPAC-induced activation of caspase-8 directlyleads to the activation of execution caspases such ascaspase-3 without the relay of the mitochondria.These findings clearly show that pachymic acid andPPAC prevent cell growth and induce apoptosis bydistinct mechanisms despite their similar chemicalstructures. Due to current limited understanding ofstructure–activity relationship (SAR) involving thisclass of triterpenoids, we are unclear how PPAC,which differs from pachymic acid by having an extradouble bond in the planar ring system and an oxo-instead of an acetyloxy group at the C3 position,can elicit such striking difference in mechanism ofinduction of apoptosis. Clearly, more efforts areneeded to develop a more systematic understandingof SAR if we are to elucidate the potential mech-anism of action and increase the potency of thisclass of triterpenoids in the future. Studies towardsthese laudable goals are currently underway in ourlaboratory.

The JNK cascade is activated in response to avariety of cellular stresses including ultravioletradiation, cytotoxic drugs and pro-inflammatorycytokines [32]. The role of JNK in apoptosis remainscontroversial. Several reports indicate that activationof JNK contributes to death receptor-mediatedapoptosis [32,33,46], whereas other studies suggestthat JNK is dispensable [47,48]. Given that sometriterpenoids might induce apoptosis in humancancer cells through JNK-mediated up-regulation ofdeath receptors [33,46], we questioned whetherJNK was activated by PPAC and contributed toapoptosis. Although JNK was activated followingPPAC treatment (Figure 6A), the specific JNK inhib-itor, SP600125, neither prevented the cleavage ofcaspase-8 (data not shown) nor apoptosis induced by



PPAC (Figure 4C). It therefore appears that JNKactivation is not required for PPAC-induced apopto-sis in A549 cells.

Apoptosis is tightly controlled by anti-apoptoticand pro-apoptotic molecules. PI3-kinase/Akt signal-ing is a critical pathway in regulating cancer cellsurvival [49]. Activation of Akt promotes cell survivaland inhibits apoptosis by phosphorylation of down-stream substrates, such as BAD and Bcl-2, which areinvolved in the regulation of the intrinsic apoptoticpathway [50]. Moreover, Akt also inhibits the deathreceptor-mediated apoptosis pathway through up-regulation of cellular FLICE-inhibitory protein(c-FLIP) expression [51]. c-FLIP blocks apoptosisinduced by the oligomerization of the adapterprotein and therefore functions as a caspase-8dominant negative [51]. Targeting the PI3-kinase/Akt signaling pathway has been reported to be aneffective strategy for the treatment of lung cancer[52]. In our experiments, PPAC treatment effectivelydecreased activation of Akt and increased thestability of PTEN in A549 cells (Figure 6B). Wespeculate that the down-regulation of Akt activitymight decrease c-FLIP expression, which leads to orsensitizes the activation of caspase-8. The suppres-sion of Akt activity by PPAC might be one of theessential mechanisms of PPAC-induced apoptosis.However, it remains to be elucidated whether Aktinhibition is sufficient or only one factor contribu-ting to PPAC-induced apoptosis.

Apart from the down-regulation of pro-survivalAkt, we also observed an up-regulation of pro-apoptotic p53 by PPAC. The p53 tumor suppressorprotein mediates the response to various stresssignals to suppress cell growth, either through cellcycle arrest or induction of apoptosis [37]. Weobserved an enhancement of phosphorylated p53(Ser15) protein by PPAC treatment in A549 cellswhich express wild-type p53 (Figure 6B). Phosphor-ylation of p53 at Ser15 is known to up-regulate thelevel of p53 by reducing the interaction between p53and its negative regulator, MDM2 [53]. It has beenreported that p53 may influence death receptor-induced apoptosis by mediating the transport of Fasreceptors from the Golgi complex to the cell surface[54]. However, further studies are needed to deter-mine the precise contribution of p53 in PPAC-induced apoptosis.

In conclusion, the results of the present studyprovide the first solid evidence that PPAC suppressesA549 cell proliferation by the induction of caspase-8-mediated apoptosis. The inhibition of Akt activationand enhancement of p53 function may also contrib-ute to apoptosis induced by PPAC. This work, incombination with previous reports, will enhanceour knowledge regarding the anticancer activity oflanostane-type triterpenoids and ultimately contrib-ute to the development of novel cancer therapeuticor preventive agents.

ACKNOWLEDGMENTS

This work was partially supported by Grant #5R21CA115269 (K. Ng) from the NIH and a NationalUniversity of Singapore Academic Research Fund(R148-050-068-101 and R148-050-068-133) (K. Ng).

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polyporenic acid c induces caspase-8-mediated apoptosis in human lung cancer a549 cells

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