activation of p53/p21/puma alliance and disruption of pi-3/akt in multimodal targeting of apoptotic...

Activation of p53/p21/PUMA Alliance andDisruption of PI-3/Akt in Multimodal Targeting ofApoptotic Signaling Cascades in Cervical CancerCells by a Pentacyclic Triterpenediol FromBoswellia serrata

Shashi Bhushan,1 Fayaz Malik,1 Ajay Kumar,1 Harpreet Kaur Isher,2 Indu Pal Kaur,3

Subhash Chandra Taneja,1 and Jaswant Singh1*1Indian Institute of Integrative Medicine, Canal Road, Jammu, India2Department of Obstetric and Gynecology, Gyan Sagar Medical College and Hospital, Banur, Punjab, India3University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

Cervical carcinoma is a growing menace to women health worldwide. This study reports the apoptotic cell death in

human cervical cancer HeLa and SiHa cells by a pentacyclic triterpenediol (TPD) from Boswellia serrata by a mechanismdifferent from reported in HL-60 cells. It caused oxidative stress by early generation of nitric oxide and reactive oxygenspecies that robustly up regulated time-dependent expression of p53/p21/PUMA while conversely abrogating

phosphatidylinositol-3-kinase (PI3K)/Akt pathways in parallel. TPD also decreased the expression of PI3K/pAkt, ERK1/2,NF-kB/Akt signaling cascades which coordinately contribute to cancer cell survival through these distinct pathways.The tumor suppressor p53 pathway predominantly activated by TPD further up-regulated PUMA, which concomitantly

decreased the Bcl-2 level, caused mitochondrial membrane potential loss with attendant translocation of Bax and drp1to mitochondria and release of pro-apoptotic factors such as cytochrome c and Smac/Diablo to cytosol leading tocaspases-3 and -9 activation. In addition both the phospho-p53 and p21 were found to accumulate heavily in thenuclear fraction with attendant decrease in topoisomarase II and survivin levels. On the contrary, TPD did not affect

the extrinsic signaling transduction pathway effectively through apical death receptors. Interestingly, N-acetyl cysteine,ascorbate and s-methylisothiourea (sMIT) rescued cells significantly from TPD induced DNA damage and caspasesactivation. TPD may thus find usefulness in managing and treating cervical cancer. � 2009 Wiley-Liss, Inc.

Key words: triterpenediol; ROS; p53; PUMA; Akt

INTRODUCTION

Cervical cancer, next to breast cancer, is the secondcommon and fifth most deadly cancer in women.The urgency to manage the disease is reflected fromthe fact that more than 500 000 new cases of cervicalcancer are diagnosed yearly of which nearly 80%account from developing countries resulting in morethan 250 000 deaths [1]. Most cervical cancerpatients receive radiation as well as chemotherapyas a part of their treatment regimen [2]. Thecombined therapy decreases the risk by 30–50%where the prognosis in advanced cervical cancerremains very poor [2]. With the currently availablechemotherapeutics, only modest increase in 5-yrsurvival rate can be achieved in patients withadvanced cervical cancer because of poor chemo-sensitivity of cervical cancer cells to chemothera-peutics [3]. Therefore, there is a growing need todevelop novel therapeutics useful to manage theprogression of cervical cancer and improve thequality of life more effectively. Currently more than

50% of drugs are derived from medicinal plants,which still continue to provide a novel source of anti-cancer therapeutics [4]. The gum resin of Boswelliaserrata, a tree grown in the dry parts of China andIndia, contains potent anti-inflammatory constitu-ents called boswellic acids, which chemically resem-ble steroids [5–7]. In addition, the boswellic acidsand their analogs such as 11-keto-b-boswellic acid,

MOLECULAR CARCINOGENESIS 48:1093–1108 (2009)

� 2009 WILEY-LISS, INC.

Abbreviations: PUMA, p53 up-regulated modulator of apoptosis;DAF-2-DA, diaminofluoresceine-2-diacetate; Rh-123, rhodamine-123; sMIT, s-methylisothiourea; PI, propidium iodide; NAC, N-acetyl-cysteine; PMSF, phenylmethanesulfonyl fluoride; PAK, p21 activatedkinase; NO, nitric oxide; ROS, reactive oxygen species; Cmt,mitochondrial membrane potential; PI3K, phosphatidylinositol-3-kinase.

*Correspondence to: Indian Institute of Integrative Medicine,Canal Road, Jammu 180001, India.

Received 25 November 2008; Revised 8 May 2009; Accepted 12May 2009

DOI 10.1002/mc.20559

Published online 18 June 2009 in Wiley InterScience(www.interscience.wiley.com)

3-O-acetyl-b-boswellic acid and 3-O-acetyl-11-keto-b-boswellic acid also exhibit strong anti-canceractivities against brain tumors and leukemia cells[7]. We recently reported for the first time thecytotoxicity and pro-apoptotic activity of anotherchemical constituent pentacyclic triterpenediol(TPD) isolated from Boswellia serrata resin in humanleukemia HL-60 cells [8]. This chemical entity in fact,comprises of an isomeric mixture of 3a, 24-dihy-droxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene,although one of its isomers 3a, 24-dihydroxyurs-12-ene was identified long back [9,10]. We reported thatTPD induces apoptosis in HL-60 cells throughactivation of apical death receptors as well asmitochondrial mediated intrinsic signaling path-ways [8]. We further asked if TPD is able to induceapoptosis in cervical carcinoma derived HeLa andSiHa cell lines, then it can be proposed to representan important class of new cancer chemotherapeuticdrug to treat this cancer disease. It may be mentionedthat high risk HPV genes are found in all cervicalcancer cells with subsequent constant expression oftwo main viral oncogenes E6 and E7 which accountfor the development of carcinoma. E6 binds to p53tumor suppressor protein and constantly targets it toubiquitin-proteosome mediated degradation whilecervical cancer cells, unlike most tumors, rarely carrythe mutated p53 gene [3,11]. Another interestingfeature is that these cells express elevated level ofcytokeratin-19 during advanced stages of cervicalcancer which confers chemo resistance to the anti-cancer drugs treatment [12,13]. These cells as suchare also known to express multidrug resistanceprotein [14]. Therefore, drugs that induce overexpression of p53 levels in cervical cancer cells arethe drugs of choice as these may specifically targetcervical cancer cells which invariably express E6human papiloma viral oncogene. The most con-served function of the p53 protein is the tumorsuppression through the induction of apoptosis [15]while mutation of p53 is frequently associated withvarious malignancies other than cervical cancer.Elevated expression of pro-apoptotic genes withpromoters containing the p53 responsive elementrepresents one mechanism whereby p53-dependentapoptosis is induced. Intracellular concentrations ofp53 are robustly increased by several stresses andexposure to various cytotoxic agents [16]. Overexpression of p53 induces higher p53 up-regulatedmodulator of apoptosis (PUMA) levels that binds toBcl-2 and localizes to mitochondria thereby trans-locating Bax to mitochondrial membrane leadingto cytochrome c release and finally the onset ofapoptosis [17].Another very important pathwaywhich has a pivotal role in cancer prevention andtreatment is Akt pathway. Akt is a Ser/Thr kinase alsocalled protein kinase B (PKB) or RAC (related toprotein kinase A and C) is cellular homologue of theviral oncoprotein v-Akt, which is known to be

responsible for a type of leukemia in mice, and istherefore referred to as c-Akt or Akt [18,19]. Overexpression of Akt has an anti-apoptotic effect inmany cell types, resulting in a resistance to or delay ofcell death [20]. Akt signaling pathway regulatesmany normal cellular processes including cell pro-liferation, survival, growth, and motility processesthat are critical for tumorigenesis. Aberrant activa-tion of the Akt pathway has been widely implicatedin many cancers. In recent years, it has been reportedthat alterations to the Akt signaling pathway arefrequent in human cancer. Akt pathway thuspresents a promising target for cancer therapeuticintervention for several reasons. First, this pathwayserves to inhibit many ‘‘tumor suppressor-like’’proteins that negatively regulate cell survival, pro-liferation, and growth. Blocking this pathway couldtherefore simultaneously inhibit the proliferation oftumor cells and sensitize them toward apoptosis.Second, many components in the Akt pathway arekinases, one of the most ‘‘drug-like’’ classes ofintracellular targets. Third, as hyperactivation ofthe Akt pathway is found in a wide range of tumors,drugs inhibiting this pathway are likely to havebroad applications for treating different types ofcancer [21]. Several molecules designed to specifi-cally target Akt have been developed, and inducedcell cycle arrest or apoptosis in human cancer cellsin vitro and in vivo. Moreover, the combination ofan inhibitor with various cytotoxic agents enhancesthe anti-tumor efficacy. Therefore, specific inhibi-tion of the activation of Akt may be a valid approachto treating human malignancies and overcoming theresistance of cancer cells to radiation or chemo-therapy. Akt pathway is also a key regulator of cellsurvival by indirectly activating the pro-survivaltranscription factor nuclear factor-kB (NF-kB)through the phosphorylation of I-kB kinase (IKK)[22,23]. Finally, there was a reciprocal regulationbetween the Akt pathway and the tumor suppressorprotein p53 [24]. Akt can promote p53 degradationby phosphorylating and activating its negativeregulator Mdm2 [25,26]. Therefore up regulation ofp53 pathway and inhibition of Akt activation is anattractive strategy for cervical cancer treatment.

We used two cervical carcinoma cell lines in thisstudy, HeLa cells with integrated HPV18 and SiHacells with integrated HPV16 sequences, becauseHPV16 and HPV18 are the two human papillomavirus infections almost found in all cervical cancertissues [27]. Moreover, HPV positive cells have overexpression of E6/E6AP (E6-associated protein) onco-protein, which is responsible for ubiquitination ofp53 [27]. Results of the present study show that TPDinduces apoptosis of cervical cancer cells by targetingseveral anti-cancer therapeutic targets of signalingcascades. Therefore it is proposed that this com-pound may find a useful application for the treat-ment of cervical cancer.

1094 BHUSHAN ET AL.

Molecular Carcinogenesis

MATERIALS AND METHODS

Chemicals and Antibodies

RPMI-1640, diaminofluoresceine-2-diacetate (DAF-2DA), 2,7-dichlorofluoresceine diacetate (DCF-DA),Rhodamine-123 (Rh-123), s-methylisothiourea(sMIT), propidium iodide (PI), DNase-free RNase,proteinase-K, 3-(4,5, -dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ascorbate,eukaryotic protease inhibitor cocktail, N-acetyl cys-teine (NAC), penicillin, streptomycin, L-glutamine,pyruvic acid, and phenylmethanesulfonyl fluor-ide (PMSF) were purchased from Sigma-Aldrich(Bangalore, India). Fetal bovine serum was obtainedfrom GIBCO Invitrogen corporation (Carlsbad, CA)(#16000-044, lot No. 1237517). AnnexinV-FITCapoptosis detection kit and Apoalert caspases-3,-8, -9 fluorescent assay kits were from B.D.Clontech Laboratories (Mountain View, CA). Mouseanti-human antibodies to TNF-R1 (#sc-8436), Fas(#sc-8009), p53 (#sc-17846), Bcl-2 (#sc-7382), Bcl-xL(#sc-8392) Bax (#sc-20067), survivin (#sc-17779),PARP-1 (#sc-8007), b-Actin (#sc-47778), p21 (#sc-817), rabbit anti-human antibody to phospho-p53 (#sc-18079, Ser-20), PUMA (#sc-28226), p-Akt(# sc-33437-R, Ser-473), p-a-p21 activated kinase(p-a-PAK) (#sc-12925, Thr-423), goat anti-humanantibodies to ICAD (#sc-6866),DR4 (#sc-6824),p-ERK1/2 (#sc-16982), DRP1 (#sc-21804), IkB-a(sc-1643), goat anti-rabbit IgG-HRP (#sc-2030), goatanti-mouse IgG-HRP (#sc-2031) and caspase-9 inhib-itor(#sc-3085) were from Santa Cruz Biotechnology(Santa Cruz, CA). Rabbit anti human antibodies ofBid (#AB1735) and cIAP-1 (#AB3614) were fromChemicon International Inc. (Billerica, MA). Rabbitanti human PI-3 (#06-496) antibody was fromUpstate Biotechnology (Lake Placid, NY) (MilliporeCorporate, Billerica, MA). Rabbit anti-goat IgG-HRP(#401504) and Smac/Diablo (# PC-574) was fromCalbiochem (Gibbstown, NJ). Mouse anti-humanantibodies to cytochrome c (#556433), Topo IIa(#611326) and NF-kB (#554184) were from BDBiosciences, Pharmingen (San Diego, CA). Electro-phoresis reagents, Protein estimation kit and proteinmarker were from Bio-Rad Laboratories (Hercules,CA). Hyper film and ECL Plus Western blottingdetection kit were from Amersham Biosciences (GEHealthcare) Buckinghamshire, UK.

Synthesis of TPD

The source of TPD was same as described earlier [8].

Cell Culture, Growth Conditions, and Treatment

Human cervical carcinoma cell line HeLa, SiHaand monkey kidney cell line CV-1 were obtainedfrom National Centre for Cell Sciences (NCCS),Pune, India. Normal Human gingival fibroblast(hGF) cell line was a gift from Dr. Anil Ballapure,CDRI (Lucknow, India). Cells were grown in Eagles

MEM containing 10% FCS, 100 U pencillin/100 mgstreptomycin per mL medium. Cells were grownin CO2 incubator (Thermocon Electron Corporation,Houston, TX) at 378C with 98% humidity and 5%CO2 gas environment. Cells grown in monolayercultures were trypsinized with trypsin (0.1% w/v)/EDTA (1 mM) solution. Soon after cells were ready todetach, the trypsin/EDTA solution was removed.Cells were dispersed gently by pipetting in completegrowth medium, centrifuged at 200g, for 5 min. Cellswere dispersed in complete medium in culture flasksand incubated in CO2 incubator. Cells grown insemi-confluent stage (�70% confluent) were treatedwith TPD dissolved in DMSO while the untreatedcontrol cultures received only the vehicle (DMSO,<0.2%).

Cell Proliferation Assay

Cells (1�104/200 mL medium) were delivered into96-well plates and exposed to indicate concentra-tions of TPD for 6, 12, 24, and 48 h. The cells wereincubated with 20 mL of MTT solution (2.5 mg/mLPBS) for 2 h and MTT-formazon crystals formed weredissolved in DMSO; OD measured at l 570 nm withreference wavelength of 620 nm [28].

DNA Fragmentation

Cells (2�106/6 mL medium/60 mm tissue cultureplate) were treated with TPD for 24 h. The cell pelletswere lysed and extracted with phenol/chloroform/isoamyl alcohol. The genomic DNA was isolated andelectrophoresed [29].

Flow Cytometric Analysis of Apoptosis

Externalization of membrane phospholipid phos-phatidylserine is an early event during the initialonset of apoptosis, which has a very high affinity forAnnexinV antibody binding [30]. Cells (1�106/3 mLmedium/6 well plate) were treated with differentdoses of TPD for indicated time periods, washed andstained with AnnexinV-FITC antibody and PI asper the instructions given by the manufacturer(Clontech # 630110).

Flow Cytometric Analysis of Intracellular Nitric Oxide

Intracellular nitric oxide (NO) was measured bylow molecular weight and cell permeable fluorescentprobe DAF-2DA. NO generated binds rapidly withthe probe to yield strong fluorescent signal [31]. Cells(1� 106/3 mL medium/6 well plate) were pre-incubated for 30 min with DAF-2DA (10mM) beforeexposure to TPD for 6 h. Cells were collected, washedin PBS and analyzed on BD-LSR flow cytometer inFL-1 channel for estimation of NO positive cells [28].

Flow Cytometric Analysis of Intracellular Peroxides (ROS)

The level of intracellular peroxides was deter-mined by using fluorescent probe 2,7-dichlorofluor-esceindiacetate. Cells (1� 106/3 mL medium/6 well

TARGETING OF APOPTOTIC SIGNALING CASCADES 1095


plate) were incubated with TPD for 6 h and exposedto DCFH-DA (5 mM) for 30 min. Cells were washed inPBS, incubated with PI (5mg/mL) for 15 min andanalyzed on flow cytometer in FL-1 channel forreactive oxygen species (ROS) positive cells [32].

Measurement of Mitochondrial Membrane Potential

Rh-123 is often used as a fluorescent probe forestimation of loss of mitochondrial membranepotential (Cmt) [28]. Cells (1�106/3 mL medium/6well plate) were treated with indicated doses of TPDfor 6 h. Rh-123 (5 mM) was added 1 h before termi-nation. Cells were collected washed in PBS, incu-bated with PI (5 mg/mL) for 15 min and fluorescenceintensity of Cmt was analyzed in FL-1 channel onflow cytometer [28].

Caspases Assays

Cells (2�106/6 mL medium/60 mm tissue cultureplate) were incubated with indicated concentrationsof TPD in the presence and absence of ascorbate,sMIT, and NAC for 6 h. Activities of caspase-3, -8, -9were determined using BD ApoAlert caspase fluore-scent assay kits as per the manufacturer’s instruc-tions [28]. Caspase-9 inhibitor (z-LEHD-fmk, 30 mM)was used wherever required.

Cell Cycle Analysis

Cells (1� 106/3 mL medium/6 well plate) weretreated with indicated doses of TPD, in the presenceand absence of ascorbate, sMIT and NAC for 24 h.Cells were washed with PBS fixed overnight in 70%alcohol at 48C then subjected to RNase digestion(400 mg/mL) at 378C for 45 min. Finally, cells wereincubated with PI (10mg/mL) for 30 min and ana-lyzed immediately on flow cytometer (BD Bioscien-ces, San Jose, CA). The subG0/G1 peak represents theapoptotic cells population [28].

Preparation of Whole Cell Lysate for Immunoblotting

Cells (2�106/6 mL medium/60 mm tissue cultureplate) were exposed to TPD for different timeintervals. Cell were collected and incubated in coldlysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mMEDTA, 1% v/v Nonidet P-40, 1 mM PMSF and 1%(v/v) eukaryotic protease inhibitor cocktail) for30 min on ice [8]. The lysates were centrifuged at12 000g, 10 min, 48C and supernatant collected aswhole cell lysate.

Preparation of Cytosolic and Mitochondrial Fractionfor Immunoblotting

Cells (2�106/6 mL medium/60 mm tissue cultureplate) were treated with TPD for indicated timeintervals and incubated with lysis buffer containing75 mM NaCl, 8 mM Na2HPO4, 1 mM NaH2PO4, 1 mMEDTA, 350 mg/mL digitonin and 1% (v/v) eukaryoticprotease inhibitor cocktail. The lysates were centri-fuged at 12 000g for 1 min, and supernatant was

collected as cytosolic fraction [8]. Residual pellet waslysed with whole cell lysis buffer, centrifuged at12 000g for 10 min at 48C and supernatant used asmitochondrial fraction [8].

Preparation of Nuclear Fraction

Cells (6�106/18 mL medium/T-75 tissue cultureflask) were incubated in 400 mL ice-cold hypotonicbuffer (10 mM HEPES/KOH pH7.9, 2 mM MgCl2,0.1 mM EDTA, 10 mM KCl, 1 mM DTT, 0.5 mMPMSF, 1% (v/v) eukaryotic protease inhibitor cock-tail) for 10 min on ice. Suspension was vortexedand centrifuged at 15 000g for 30 s and supernatantdiscarded, the nuclear pellet was resuspended in to100 mL of ice cold saline buffer (50 mM HEPES/KOHpH7.9, 50 mM KCL, 300 mM NaCl, 0.1 mM EDTA,10% glycerol, 1 mM DTT, 0.5 mM PMSF, 1% (v/v)eukaryotic protease inhibitor cocktail) on ice for20 min. The suspension was vortexed and centri-fuged at 15 000g for 5 min at 48C and the supernatantused as nuclear fraction [8].

Protein Measurement

Protein was measured employing Bio-Rad proteinassay kit using bovine serum albumin as standard.

Western Blots Analysis

Proteins samples (50mg) were subjected to discon-tinuous SDS–PAGE at 60 V and then electro trans-ferred to PVDF membrane overnight at 48C, 30 V.Non-specific proteins binding was blocked with 5%non-fat milk in Tris-buffered saline containing 0.1%Tween-20 (TBST) for 1 h at room temperature. Theblots were probed with respective primary mouse/rabbit/goat anti human antibodies for 2 h andwashed three times with TBST. The blots were thenincubated with horseradish peroxidase conjugatedsecondary antibodies for 1 h, washed again threetimes with TBST and signals were detected using ECLplus chemiluminescence’s kit on X-ray film [8].

Statistical Analysis

Data are expressed as mean� SD of three experi-ments unless otherwise indicated. Comparisonswere made between control and treated groups usingunpaired Student’s t-test, and P values <0.01 wereconsidered highly significant.

RESULTS

Cytotoxicity of TPD

The TPD produced concentration and timedependent cytotoxicity in both HeLa and SiHa cells.The concentration of TPD required for inhibiting50% cell proliferation (IC50) of HeLa and SiHa cellswere approximately the same after different timeintervals (Figure 1). It required four to five times

1096 BHUSHAN ET AL.


higher concentration of TPD to induce comparablecytotoxicity in normal hGF and monkey kidneyCV-1 cells after 48 h treatment with TPD (Figure 1).

TPD Induced DNA Fragmentation

Apoptosis typically involves intra-nucleosomalchromatin cleavage by endonucleases in multiplesof 180 bp leading to a typical DNA ladder. GenomicDNA was isolated, after 24 h treatments of cellscultures and electrophoresed on 1.8% agarose gel.TPD treated cells exhibited typical DNA ladder,which is a hallmark of apoptosis. DNA ladder washighly distinct at concentrations above 10 mg/mL inboth the cell line (Figure 2). Exposure of cells to 70and 100 mg/mL indicated a diffused pattern of DNAladder with smear suggesting some larger fragments

possibly due to some post-apoptotic necrosis. DNAisolated from untreated and treated normal humanhGF cells did not show any DNA ladder (Figure 2).

Flow Cytometric Analysis of TPD Induced

Apoptosis/Necrosis

TPD produced dose and time dependant increasein apoptotic cell population of HeLa cells (Figure 3A).However, at higher concentration and time,both apoptotic and post apoptotic cells increased

Figure 1. Influence of TPD on proliferation of HeLa, Siha, CV-1,and hGF cells. The cells (0.02� 106/200 mL medium) grown in96-well culture plate were treated with different concentration ofTPD for 6, 12, 24, and 48 h followed by incubation with MTT. Otherconditions were same as described in Materials and MethodsSection. Data are mean� SD (n¼ 8 wells) and representative ofthree similar experiments.

Figure 2. Effect of TPD on DNA fragmentation of HeLa, SiHa, andnormal hGF cells. Genomic DNA was extracted from cells treatedwith different concentrations of TPD for 24 h. Other conditionsare described in Materials and Methods Section. The data arerepresentative one of three similar experiments.



(Figure 3A). TPD also produced almost a similarpattern in SiHa cells in terms of apoptotic cellpopulation (Figure 3B). Apoptosis thus appeared tobe the primary mode of cell death induced by TPD asno necrotic (PI fluorescence) cells were observed.

TPD Caused Early Intracellular Nitric Oxide Generation

To find out the early events which might beinvolved in the induction of apoptosis, we postu-lated that the TPD may be inducing oxidative stress.We analyzed the extent of NO generation by flow

cytometry after staining the cells with DAF-2DA(FL-1). Exposure of both HeLa and SiHa cells to TPD(50mg) for a brief period of 6 h induced earlygeneration of NO by 64% and 60%, respectively(Figure 4A and D).

TPD Mediated Intracellular Generation of ROS

Besides NO, TPD was also able to produce a parallelincrease in peroxides simultaneously as analyzed byflow cytometry after double staining with DCFH-DAand PI. There was hardly any DCF fluorescence in the

Figure 3. Flow cytometric analysis of apoptosis and necrotic cell population. (A) HeLa cells (1� 106/3 mLmedium/6 well plate) were incubated with indicated concentrations of TPD for 6, 24, and 48 h and analyzed forannexinV-FITC/PI staining to determine apoptotic and necrotic cell populations as described in Materials andMethods Section. (B) Similar studies were carried out with in SiHa cells after treatment with indicated doses of TPDfor 6 h. Data are representative of one of three similar experiments.

1098 BHUSHAN ET AL.


Figure 4. Flow cytometric analysis of various parameters relatedto apoptosis induced by TPD in HeLa and SiHa cells. Cells (1�106/3 mL medium/6 well plate) were incubated with indicated concen-trations of TPD for 6 h and analyzed for NO, ROS, and loss ofmitochondrial membrane potential as described in Materials andMethods Section. (A) TPD induced early intracellular nitric oxidegeneration in HeLa cells. The cells were analyzed by flow-cytometeryfor NO fluorescence in FL-1 channel as described in Materials andMethods Section. (B) TPD mediated early generation of peroxides in

HeLa cells. Cells were analyzed for DCF-fluorescence in the FL-1(DCF-Fluorescence) versus FL-2 (PI-Fluorescence). (C) TPD inducedloss of mitochondrial membrane potential (Dcm). Cells were stainedwith Rhodamine-123 (5mg/mL) for 1 h and PI (5 mg) and analyzed inFL-1 versus FL-2 channels of flow cytometer. Data in all figures arerepresentative of one of three similar experiments. (D) Similar studieswere carried out with in SiHa cells after treatment with indicateddoses of TPD for 6 h. Data are Mean� SD from three similarexperiments. P values: *<0.01 compared to untreated control.



cells. Both the cell lines were equally sensitive inproducing NO, that is, �80% at 50 mg/mL of TPD(Figure 4B and D).

Loss of Mitochondrial Membrane Potential by TPD

Rh-123 uptake into mitochondria is driven bymitochondrial transmembrane potential (Cmt) thatallows the determination of cell population withactive integrated mitochondrial functions (lowerright quadrant). In parallel to ROS and NO gener-ation HeLa and SiHa cells exposed to TPD wereanalyzed for Rh-123 uptake by flow cytometery. Inthe untreated control cells, almost all cells werebioenergetically active as evidenced by high Rh-123fluorescence. TPD effectively targeted mitochondrialfunctions equipotently in both cell types as it causedsignificant loss of Cmt by �50% in cultures exposedto 50 mg of TPD (Figure 4C and D).

TPD Mediated Stimulation of Caspase Activities

Both the cells were exposed to TPD and evaluatedfor caspase-3, -8, and-9 activities. The triterpenediolpredominately induced dose-dependant increasein caspase-3 and -9 activities in HeLa cells; bothregistering a six-fold increase at the highest concen-tration of TPD used (Figure 5A). While it inducedboth these caspases by four to six fold in SiHa cells(Figure 5C). It is interesting to note that activation ofcaspase-9 by TPD was very selective as there was nosignificant increase in caspase-8 activity in either ofthe cell line used. Interestingly, the stimulation ofthe caspase-3 and -9 activities were equally inhibitedby three antioxidants, viz. ascorbate, NAC and sMIT,by less than 50% of maximal stimulated activity(Figure 5A). These findings suggested that both ROSand NO are key players for caspase-3 and-9 activa-tion. Nevertheless, caspase-9 inhibitor (LEHD-fmk,30 mM) completely inhibited TPD induced caspase-3and -9 activities in HeLa cells (Figure 5B). In the lightof TPD induction of apoptosis these finding amplydemonstrate the role of caspase-9 activation in up-regulating down stream events leading to caspase-3activation, and as such further ruled out the involve-ment of caspase-8 vis-a-vis apical death receptorsin helping cervical cancer cells death.

TPD Increased Sub-G0 DNA Fraction of Cell CyclePhase Distribution

Both cell types exposed to TPD for 24 h exhibitedan increase in hypo diploid sub-G0 DNA fraction(<2n DNA) in a dose-dependent manner (Figure 6).In contrast to caspases inhibition by ascorbate, NACand sMIT, the inhibitors reduced significantly thesub-G0 fraction from 58% to 18%, 15% and 19%,respectively in HeLa cells. Also caspase-9 inhibitorwas able to inhibit the increase in sub-G0 DNAfraction significantly in both HeLa and SiHa cells(Figure 6). The studies provide evidence that both

ROS and NO are early events that might beresponsible for activation of the mitochondrialdependent apoptotic machinery in cervical carci-noma HeLa and SiHa cells.

Effect of TPD on Extrinsic Apoptotic Signaling

The immunobloting data indicated that TPD doesnot alter significantly the constitutive expression ofapical death receptors such as TNF-R1, Fas and DR4 inthe TPD treated cells thereby ruling out any involve-ment of extrinsic apoptotic signaling cascade in theinduction of apoptosis (Figure 7A). This evidencecorroborated with no alteration in the Bid andprocaspase-8 expression in TPD treated cells(Figure 7A). Hence, TPD has no prominent role inactivating extrinsic apoptotic signaling cascade inthe killing of cervical carcinoma cells.

TPD Robustly Up-Regulates the Expression ofp53/PUMA/p21

TPD increased the expression of transcriptionfactors p53 and p21 in a time-dependant mannerin both the cell types, which correlated with theincreased activity of PUMA in both HeLa and SiHacells (Figure 7B). All these proteins are poorlyexpressed in these cells. Besides increased expressionin cytosol, both phospho p53 and p21 were accu-mulated in the nucleus of cells (Figure 7C), whichmight have important implication in overcomingresistance and imparting higher sensitivity to TPD.Simultaneously, TPD significantly down regulatedthe expression of phospho-Akt through the inhi-bition of phosphatidylinositol-3-kinase (PI3K)(Figure 8A). Akt is known to trigger downstreamexpression of a cascade of responses, from cellgrowth and proliferation to survival and motility,which regulate the survival of cancer cells [20]. Aktalso activates the degradation of transcription factorp53 [25,26] and thereby act as a physiologicalantagonist to apoptosis. Therefore by inhibiting theAkt expression, as in the case of TPD, would furtherreinforce the p53 activation by itself throughsuppression of Akt activity. Once the p53 level waselevated it may enforce the up regulation of PUMAand hence alteration of various Bcl-2 family proteins.Further p21 is now recognized as a master regulatorof cell fate in response to oxidative stress and variousstimuli. It is a potent inhibitor of cyclin dependantkinases and its expression is tightly controlled byp53. TPD robustly increased the expression of p21,concomitant with p53 and PUMA, suggestingthereby an effective together alliance in convergingcancer cells to apoptotic death. The ubiquitouslyexpressed transcription factor NF-kB and the Ser/Thrprotein kinase Akt are involved in the promotion ofcancer cell growth. Down regulated expression ofAkt is known to decrease the expression of nucleartranscription factor NF-kB, IkB, and protein of IAPfamily [22,23,33]. TPD significantly reduced the

1100 BHUSHAN ET AL.


expression of Akt together with NF-kB, IkB, cIAP-1,and survivin proteins in a time dependant manner inboth HeLa and SiHa cells (Figure 8A and B).Furthermore, TPD also down regulated the expres-sion of Erk-1/2 (mitogen activated kinases, MAPKs)and a-PAK in a time dependant manner which all

gear up pro-apoptotic machinery by antagonizinganti-apoptotic Bcl-2 family protein Bcl-xL in HeLaand SiHa cells [34,35] (Figures 8A and 9A). Elevatedexpression of p53 was also responsible for theinhibition of topoisomerase-II activity [36] andTPD significantly inhibit its activity (Figure 8A).

Figure 5. (A,B) TPD induced activation of caspase-8,-9, and -3 inHeLa cells (2� 106/6 mL medium/60 mm tissue culture plate). Thecells in culture were exposed to 30 mg/mL of TPD for 6 h in thepresence and absence of ascorbate (5 mM), NAC (5 mM) sMIT(1 mM)and caspase-9 inhibitor (z-LEHD-fmk, 30 mM). All the inhibitors wereadded 1 h before TPD treatment. The caspases activities weredetermined fluorometrically in the cell lysate of HeLa cells using BDApoalert caspase assay kits as described in Materials and MethodsSection. All assays were performed according to the instructionsprovided by the supplier. Data are Mean� SD from three similar

experiments. P values: *<0.01 compared to untreated control,@<0.01 compared to TPD treated cells. (C) TPD induced activation ofcaspase-8, -9, and -3 in SiHa cells (2�106/6 mL medium/60 mmtissue culture plate). The cells in culture were exposed to 30 mg/mL ofTPD for 6 h and caspases activities were determined fluorometricallyin the cell lysate, using BD Apoalert caspase assay kits as describedin Materials and Methods Section. Data are Mean� SD from threesimilar experiments. P values: *<0.01 compared to untreatedcontrol.



TPD Alters the Ratio of Bcl-2 Family Member Proteins andActivates Intrinsic Signaling Pathway of Apoptosis

Treatment of cells with TPD resulted in alteredexpression of Bcl-2 family proteins. TPD inducedPUMA also inhibited the Bcl-2 level, produced Cmt

loss, induced translocation of Bax and dynaminrelated protein-1 (DRP1) in to mitochondria andconsequently released cytochrome c and Smac/Diablo to cytosol, events necessary to activate

caspase-9 and caspase-3 (Figure 9A). TPD alsoinhibited the anti-apoptotic protein Bcl-xL throughAkt and Akt-PAK pathway, which further enabledthe expression and translocation of Bax, cytochromec and caspase-9 in both SiHa and HeLa cells(Figure 9A). In this context, TPD drastically reducedthe Bcl-2/Bax ratio from 9.7 and 4.5 to 0.25 in SiHaand HeLa cells, respectively (Figure 9B). This wouldenable activated caspase-3 to use poly-ADP ribosepolymerase (PARP) as a substrate and release CAD

Figure 6. (A) DNA Cell cycle analyses in TPD treated HeLa and SiHa cells and influence of RNOS and caspase-9inhibitors (ascorbate, 5 mM; sMIT, 1 mM; NAC, 5 mM; z-LEHD-fmk, 30 mM). Cells (1� 106/3 mL medium/6 wellplate) were exposed to different concentrations of TPD for 24 h in the presence and absence of various inhibitors.All the inhibitors were added 1 h before TPD treatment. Both the cells were stained with PI to determine DNAfluorescence and cell cycle phase distribution as described in Materials and Methods Section. Data arerepresentative of one of three similar experiments.

1102 BHUSHAN ET AL.


Figure 7. (A) Influence of TPD on the expression of apical deathreceptors related proteins. Both HeLa and SiHa cells (2� 106/6 mLmedium/60 mm tissue culture plate) were treated with 30 mg/mL ofTPD for indicated time periods. b-actin was used as internal controlto represent the same amount of proteins applied for SDS–PAGE.Western blot analyses of indicated proteins were performed in wholecell lysate. Data are representative of one of three similar experi-ments. (B) Western blot analysis of p53 and its related apoptotic keyproteins in TPD treated HeLa and SiHa cells. Cells were treated with30mg/mL of TPD for indicated time periods and equal amount ofprotein samples were loaded on SDS–PAGE gel for Western blot

analysis as described in Materials and Methods Section. (C) Effect ofanti-oxidant on TPD induced p21 and p53 level. HeLa cells (2� 106/6 mL medium/60 mm tissue culture plate) were exposed to 30 mg/mLof TPD for 24 h in the presence and absence of NAC (5 mM). NACwas added 1 h before TPD treatment. Nuclear and cytosolic fractionsof cells were prepared and immunoblots as described in Materialsand Methods Section. Relative density of each band was measured asarbitrary units by Quantity One software of Bio-RAD gel documen-tation system. Data are representative of one of three similarexperiments. P values: *<0.01 compared to untreated control,@<0.01 compared to TPD treated cells.



from ICAD, which appears to cause DNA fragmenta-tion in the nucleus of the cells [37,38]. TPD inhibitedthe expression of ICAD and consequently inducedcleavage of PARP, from 116 to 89 kDa (Figure 9A).

DISCUSSION

Results of the present study describe the anti-cancer activity of pentacyclic triterpenediol (TPD)against human cervical carcinoma HeLa and SiHacells. All human cancer cells integrate HPV oncopro-teins which interact with the wild-type p53 andsuppress its functions. These cells consequentlyevade apoptosis because of their poor expression ofp53 and PUMA proteins [11]. The present studieshave demonstrated the cytotoxic potential of TPD incervical carcinoma cells by mechanisms disruptingapoptotic signaling pathways meant for survival andproliferation of these cells. The extent of apoptosiswas measured by various stereotype end-points andwas similar in both the cell types used. TPD wasfound to induce a condition mimicking hypoxia

responsible for p53/PUMA/p21 induction contraryto the inhibition of PI3K/AKT pathway in both thesecell types gearing up cell machinery to undergoapoptotic death. The studies demonstrate that TPDinduced early generation of reactive oxygen andnitrogen species, which altered the levels of apopto-sis related proteins leading to DNA fragmentationtypical of apoptosis. Nevertheless, the prolongedproduction of ROS and NO may bring about post-translational modifications of critical moleculesleading to mitochondrial dysfunctions. Several ear-lier studies have shown the role of environmentalstresses such as UV, ionizing radiation, etc. that arecapable of inducing apoptosis by down regulatingPI3K/Akt pathway [39,40], and curcumin is one suchexample of ROS mediated inhibition of PI-3/Aktpathway [41]. On the other hand ROS are potentactivators of p53 function and indeed the generationof ROS is believed to be a key factor in the activationof p53 where they play a role in mediating apoptosis[42]. Therefore targeting differently but simultane-

Figure 8. (A) Effect of TPD on the PI-3/Akt and other apoptotic related proteins. Cells (2� 106/6 mL medium/60 mm tissue culture plate) were treated with 30mg/mL of TPD for indicated time periods and 50 mg of cells proteinwas run on SDS–PAGE gel for western blot analysis. b-actin was used as internal control to represent the sameamount of proteins applied for SDS–PAGE. Data are representative of one of three similar experiments. (B) Effect ofTPD on NF-kB translocation. Nuclear and cytosolic fractions of cells were prepared and immunoblots as described inMaterials and Methods Section.

1104 BHUSHAN ET AL.


Figure 9. (A) Influence of TPD on the mitochondrial related apoptotic key proteins in the cell lysates of HeLa andSiHa cells. Western blot analyses of indicated proteins were performed as described in Materials and MethodsSection. b-actin was used as internal control to represent the same amount of proteins applied for SDS–PAGE. Dataare representative of one of three similar experiments. (B) Influence of TPD on the Bcl-2/Bax ratio in the HeLa andSiHa cells. Relative density of each band was measured as arbitrary units by Quantity One software of Bio-RAD geldocumentation system. Data are representative of one of three similar experiments.



ously both these pathways in cancer cells is a usefulapproach for development of anti-cancer therapeu-tics, and TPD indeed is a promising agent in thisconcern. Akt plays a pivotal role in fundamentalcellular processes such as cell proliferation andsurvival by phosphorylating a variety of key sub-strates and inactivating pro-apoptotic proteins[18,19]. Activation of Akt is again a complex processwhich essentially requires translocation to plasmamembrane before its phosphorylation mediated byPI3K [43]. These proteins are usually over expressedin cancer cells and TPD in the present studiesrobustly down regulated their expression.

On the other hand p53 is a tumor suppressortranscription factor that plays a pivotal role incontrolling cell cycle checkpoint regulation, DNArepair, transcription, and induction of apoptosis.The transcription factor is frequently mutated incancer cells and therefore, lack of p53 functionimpairs these cellular processes as well as increasesresistance to chemotherapeutic regimens [44]. Theconstitutive expression of p53 is very low in HeLaand SiHa cells for the reasons discussed earlier.Interestingly exposure to TPD resulted in a remark-able increase in its expression of p53 by more thanthreefold over the basal level in 24 h suggesting thatTPD might be interfering with the HPV oncoproteinsmediated transcription regulation of pro-apoptoticproteins which, warrants further studies. This effectof TPD on induction of p53 equipotently correlatedwith the enhanced expression of apoptosis coordi-nating proteins PUMA and p21 in cervical cancercells indicating that induction of p53 is certainlyimportant in directing these cells to undergo apop-tosis. Current evidences have indicated that thiseffect is regulated by various p53-regulated genesamong which PUMA and p21 are important [45–47]while the expression of both these proteins wasenhanced profoundly by TPD in cervical carcinomacells. Not only TPD enhanced the nuclear accumu-lation of active p53 and p21 besides increasedexpression in the cytosol. This is considered impor-tant to overcome any resistance and increasedsensitivity to TPD and multiple chemotherapyregimens during treatment of chronic malignancy.PUMA is a BH3-only member of the Bcl-2 family thatis a potent inducer of apoptosis mediated by p53.PUMA also acts to fine tune and coordinate theapoptotic network mediated by p53. It interacts withBcl-2 and Bcl-XL through a BH3-domain therebymodulating Bax activity to facilitate release ofapoptogenic proteins such as cytochrome c andothers from mitochondria; knock out of PUMArecapitulates virtually all apoptotic deficiency inp53 knockout mice [17,47,48]. TPD also enhancedthe expression level of p53 phosphorylation at Ser20and that phosphorylation of Ser20 of wild-type p53has been shown to correlate with the DNA damage-mediated transactivation of p53 and its down stream

target gene p21 [49]. Further, the time dependentincrease in expression of wt p53 may be responsiblefor corresponding decrease in topoisomerse-II activ-ity in cells treated with TPD because topoisomerase-IIa promoter activity is efficiently repressed by wtp53 as mutant p53 do not bind DNA [36]. Thus theincreased expression of wt p53 negatively regulatesthe transcription of topo IIa gene promoter activityand that loss of wt p53 as a transcriptional suppressor[36] may lead to unregulated topoisomerase-IIactivity leading to increased cell proliferation as hasbeen observed in cervical cancer cells. In addition,p53 is also involved in inducing senescence throughtranscriptional activation of the cyclin-dependentkinase inhibitor p21. Though there was remarkableinduction of p21 by TPD, yet it did not produce anycell cycle arrest in our studies. This could also be dueto the oxidative stress by TPD which can induce p21independent of p53, and through inhibition of Aktactivation observed earlier by others [44]. This iscorroborated by findings from others that activationof p21 increases intracellular levels of ROS [50,51].Since there was no arrest of cell cycle by TPD, the upregulation of p21 may have been one of thecontributory factors in the robust increase of ROSby TPD in HeLa and SiHa cells causing p53-depend-ent apoptosis through PUMA activation.

Moreover p53/PUMA appeared to mediate Bcl-2inhibition and release of cytochrome c which werealso directly checked by the Akt. Both p53 and Aktpathways exhibit negative complementarily to eachother [25]. Down regulation of Akt may haveenhanced the lethality of p53 pathway induced byTPD in several ways by down playing several keyproteins such as Bcl-xL, PAK, and mitogen activatedkinases Erk1/2 [34,35]. TPD induced such eventscontributed to activation of apoptosis by trans-location of Bax to mitochondria, release of cyto-chrome c, Smac/Diablo, DRP-1 and activation ofcaspase-9. Many studies have also implicated theincreased expression of DRP-1 in mitochondrialfission and consequent induction of apoptosis,whereas Smac/Diablo enhances the caspase-9 and-3 activities by inhibiting their physiological modu-lators IAP and survivin [8,52]. The elevated level ofcaspase-3 could utilize PARP as a substrate andprevent any repair of damaged DNA. Activatedcaspase-3 releases CAD from ICAD, which appearsto cause DNA fragmentation [37]. All these eventswere supported by TPD to activate apoptotic machi-nery. The down stream activation of caspase-3 wasstrictly mediated by caspase-9 because the caspase-9inhibitor decisively impaired the activity of caspse-3.Moreover, caspase-9 inhibitor significantly inhibitthe DNA damaged induce by TPD in both type ofcervical carcinoma cells.

The activities of other anti-apoptotic proteins suchas IAP, survivin, NF-kB, and ICAD were drasticallyreduced paving way for cells to undergo apoptosis.

1106 BHUSHAN ET AL.


Furthermore downstream Akt also inhibit the NF-kBpathway and IAP proteins like survivin [33]. Thedown-regulation of NF-kB has been related withROS/NO formation in cancer cells attenuatingthereby Bcl-2/Bax [53,54]. TPD did not inhibit thetranslocation of NF-kB from cytoplasm to nucleus,but it significantly inhibit it expression in nucleus ofboth HeLa and SiHa cells. Therefore, TPD drivenactivation of p53 and inhibition of Akt pathwaysattack the mitochondria with double sword andfinally induce the mitochondrial dependent apop-tosis in cervical cancer SiHa and HeLa cells. Thoughwe used two different cell types, the expression andaltered phenotypes profiles were almost similar inboth the cells.

Interestingly, TPD did not produce any significanteffect on the expression of apical death receptorssuch as TNF-R1, Fas and DR4 vis-a-vis caspase-8 andBid levels, the later form a bridge between extrinsicand intrinsic apoptotic pathways. This is in contrastto the extrinsic and intrinsic apoptosis signalingcascades induced by TPD in human leukemia HL-60cells we reported earlier [8]. These apical deathreceptors are prominently expressed in HeLa andSiHa cells but TPD was not able to alter theirconstitutive expression significantly. This suggestedthat TPD neither acts like a ligand nor does itstimulate ligands for the apical death receptors toactivate caspase-8 dependent apoptosis in these cells.Not only this, caspase-3 and -9 activities wereinhibited significantly by antioxidants ascorbate,NAC and iNOS inhibitor sMIT indicating again thatTPD induced oxidative stress by both reactive oxygenand nitrogen species may be important for activatingapoptotic machinery.

In conclusion, TPD, a natural pentacyclic triterpe-nediol from Boswellia serrata was found to induceapoptosis in cervical carcinoma HeLa and SiHa cellsby targeting several critical proteins of various signaltransduction pathways preparing cervical carcinomacells to commit self demise. TPD has thus emerged abroad-based multi-focal anticancer agent. In cervicalcarcinoma cells it selectively up-regulated robustlythe expression of pro-apoptotic p53/PUMA/p21pathways while antagonized Akt/PI3K survival path-way. This altered the ratio of Bcl-2 family membersproteins facilitating prominently the mitochondrialdependent apoptosis apposed to extrinsic pathway.The activities of other anti-apoptotic and cellsurvival proteins such as Erk1/2, IAP, survivin, NF-kB, and ICAD were significantly reduced paving wayfor cells to undergo apoptosis. Unlike HL-60 cells,these studies highlight the usefulness of TPD intreating cervical carcinoma cells which do notcompromise apoptotic death because of poor expres-sion of p53 and higher expression of Akt in cervicalcancer cells. Moreover, it requires four to fivefoldhigher concentrations of TPD to kill normal humancells than cervical cancer cells as has been observed in

this study. TPD may thus find usefulness either aloneor in combination with other anticancer agentsor radiation therapy to treat as well as overcomeresistance to chemotherapy of cervical cancer.

REFERENCES

1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics2002, CA-A. Cancer J Clin 2005;55:74–108.

2. Thomas GM. Improved treatment for cervical cancer—Concurrent chemotherapy and radiotherapy. N Engl J Med1999;340:1198–1200.

3. Sultana H, Kigawa J, Kanamori Y, et al. Chemosensitivity andp53-Bax pathway-mediated apoptosis in patients withuterine cervical cancer. Ann Oncol 2003;14:214–219.

4. Lee KH. Anti-cancer drug design based on plant-derivednatural products. J Biomed Sci 1999;6:236–250.

5. Han R. Highlight on the studies of anti-cancer drugs derivedfrom plants in China. Stem Cells 1994;12:53–63.

6. Safayhi H, Mack T, Sabieraj J, Anazodo MI, Subramanian LR,Ammon HP. Boswellic acids: Novel, specific, nonredox inhi-bitors of 5-lipoxygenase. J Pharmacol Exp Ther 1992;261:1143–1146.

7. Hoernlein RF, Orlikowsky T, Zehrer C, et al. Ammon Acetyl-11-keto-beta-boswellic acid induces apoptosis in HL-60 andCCRF-CEM cells and inhibits topoisomerase I. J PharmacolExp Ther 1999;288:613–619.

8. Bhushan S, Kumar A, Malik F, et al. A triterpenediol fromBoswellia serrata induces apoptosis through both theintrinsic and extrinsic apoptotic pathways in human leukemiaHL-60 cells. Apoptosis 2007;12:1911–1926.

9. Mahajan B, Taneja SC, Sethi VK, Dhar KL. Two triterpenoidsfrom Boswellia serrata gum resin. Phytochemistry 1995;39:453–455.

10. Allan GG. The stereochemistry of the boswellic acids. Phyto-chemistry 1968;7:963–973.

11. Koivusalo R, Krausz E, Helenius H, Hietanen S. Chemo-therapy compounds in cervical cancer cells primed byreconstitution of p53 function after short interfering RNAmediated degradation of human papillomavirus 18 E6mRNA: Opposite effect of siRNA in combination withdifferent drugs. Mol Pharmacol 2005;68:372–382.

12. Yuan CC, Huang HC, Tsai LC, Ng HT, Huang TS. Cytokeratin-19 associated with apoptosis and chemosensitivity in humancervical cancer cells. Apoptosis 1997;2:101–105.

13. Yuan CC, Huang TS, Ng HT, Liu RS, Hung MW, Tsai LC.Elevated cytokeratin-19 expression associated with apop-totic resistance and malignant progression of human cervicalcarcinoma. Apoptosis 1998;3:161–169.

14. Takara K, Obata Y, Yoshikawa E, et al. Molecular changes toHeLa cells on continuous exposure to cisplatin or paclitaxel.Cancer Chemother Pharmacol 2006;58:785–793.

15. Clarke AR, Purdie CA, Harrison DJ, et al. Thymocyteapoptosis induced by p53-dependent and independentpathways. Nature 1993;362:849–852.

16. Vousden KH, Lu X. Live or let die: The cell’s response to p53.Nat Rev Cancer 2002;2:594–604.

17. Yamaguchi H, Chen JD, Bhalla K, Wang HG. Regulationof Bax activation and apoptotic response to microtubuledamaging agents by p53 transcription-dependent and-independent pathways. J Biol Chem 2004;279:39431–39437.

18. Datta SR, Brunet A, Greenberg ME. Cellular survival: A play inthree Akts. Genes Dev 1999;13:2905–2927.

19. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase AKTpathway in human cancer. Nat Rev Cancer 2002;2:489–501.

20. Fresnovara JA, Casado E, Castro J, Cejas P, Belda-Iniesta C,Gonzalez-Baron M. PI3K/Akt signaling pathway and cancer.Cancer Treat Rev 2004;30:193–204.

21. Luo J, Manning BD, Cantley LC. Targeting the PI3K-Aktpathway in human cancer: Rationale and promise. CancerCell 2003;4:257–262.



22. Ozes ON, Mayo LD, Gustin JA, Pfeffer SR, Pfeffer LM, DonnerDB. NF-kappaB activation by tumour necrosis factor requiresthe Akt serine-threonine kinase. Nature 1999;401:82–85.

23. Romashkova JA, Makarov SS. NF-kappaB is a target of AKT inanti-apoptotic PDGF signalling. Nature 1999;401:86–90.

24. Trotman LC, Pandolfi PP. PTEN and p53: Who will get theupper hand? Cancer Cell 2003;3:97–99.

25. Mayo LD, Donner DB. A phosphatidylinositol 3-kinase/Aktpathway promotes translocation of Mdm2 from thecytoplasm to the nucleus. Proc Natl Acad Sci USA 2001;98:11598–11603.

26. Zhou BP, Liao Y, Xia W, Zou Y, Spohn B, Hung MC. HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2phosphorylation. Nat Cell Biol 2001;3:973–982.

27. Naniwa J, Kigawa H, Akeshima R, et al. Leptomycin Benhances CDDP-sensitivity via nuclear accumulation of p53protein in HPV-positive cells. Cancer Sci 2003;94:1099–1103.

28. Bhushan S, Singh J, Rao MJ, Saxena AK, Qazi GN. A novellignan composition from Cedrus deodara induces apoptosisand early nitric oxide generation in human leukemia Molt -4and HL-60 cells. Nitric Oxide 2006;14:72–88.

29. Kumar A, Malik F, Bhushan S, et al. An essential oil and itsmajor constituent isointermedeol induce apoptosis byincreased expression of mitochondrial cytochrome c andapical death receptors in human leukaemia HL-60 cells.Chem Biol Interact 2008;171:332–347.

30. Vermes I, Haanen C, Nakken HS, Reutellingsperger C. Anovel assay for apoptosis, flow cytometric detection ofphosphatidylserine expression on early apoptotic cells usingfluorescein labeled AnnexinV. J Immunol Methods 1995;184:39–51.

31. Kojima H, Sakurai K, Kikuchi K, et al. Development of afluorescent indicator for the nitric oxide based on thefluorescein chomophore. Chem Pharm Bull 1998;46:373–375.

32. Malik F, Kumar A, Bhushan S, et al. Reactive oxygen speciesgeneration and mitochondrial dysfunction in the apoptoticcell death of human myeloid leukemia HL-60 cells by a dietarycompound withaferin A with concomitant protection by N-acetyl cysteine. Apoptosis 2007;12:2115–2133.

33. Johnson NC, Dan HC, Cheng JQ, Kruk PA. BRCA1 185delAGmutation inhibits Akt-dependent, IAP-mediated caspase 3inactivation in human ovarian surface epithelial cells. Exp CellRes 2004;298:9–16.

34. Kinkade CW, Castillo-Martin M, Puzio-Kuter A, et al.Targeting AKT/mTOR and ERK MAPK signaling inhibitshormone-refractory prostate cancer in a preclinical mousemodel. J Clin Invest 2008;118:3003–3006.

35. Tang Y, Zhou H, Chen A, Pittman RN, Field J. The Akt proto-oncogene links Ras to Pak and cell survival signals. J BiolChem 2000;275:9106–9109.

36. Wang Q, Zambetti GP, Suttle DP. Inhibition of DNAtopoisomerase II alpha gene expression by the p53 tumorsuppressor. Mol Cell Biol 1997;17:389–397.

37. Sakahira H, Enari M, Nagata S. Functional differences of twoforms of the inhibitor of caspase-activated DNase, ICAD-L,and ICAD-S. J Biol Chem 1999;274:15740–15744.

38. Yingchang M, Thomas SD, Xiaohua X, Casson LK, Miller DM,Bates PJ. Apoptosis in leukemia cell is accompanied byalterations in the level and localization of nucleolin. J BiolChem 2003;278:8572–8579.

39. Meier R, Hemmings BA. Regulation of protein kinase B.J Recept Signal Transduct Res 1999;19:121–128.

40. Zundel W, Giaccia A. Inhibition of the anti-apoptotic PI (3) K/Akt/Bad pathwayby stress. Genes Dev 1998;12:1941–1946.

41. Woo JH, Kim YH, Choi YJ, et al. Molecular mechanisms ofcurcumin-induced cytotoxicity: Induction of apoptosisthrough generation of reactive oxygen species, down-regulation of Bcl-XL and IAP, the release of cytochrome cand inhibition of Akt. Carcinogenesis 2006;24:1199–1208.

42. Minamino T, Yujiri T, Papst PJ, Chan ED, Johnson GL, TeradaN. MEKK1 suppresses oxidative stress-induced apoptosis ofembryonic stem cell-derived cardiac myocytes. Proc NatlAcad Sci USA 1999;96:15127–15132.

43. Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: Its functionsand alterations in human cancer. Apoptosis 2004;9:667–676.

44. Wang X, Li M, Wang J, et al. The BH3-only protein, PUMA, isinvolved in oxaliplatin-induced apoptosis in colon cancercells. Biochem Pharmacol 2006;71:1540–1550.

45. Jeffers JR, Parganas E, Lee Y, et al. Puma is an essentialmediator of p53-dependent and -independent apoptoticpathways. Cancer Cell 2003;4:321–328.

46. Nakano K, Vousden KH. PUMA, a novel proapoptotic gene, isinduced by p53. Mol Cell 2001;7:683–694.

47. Yu J, Zhang L, Hwang PM, Kinzler KW, Vogelstein B. PUMAinduces the rapid apoptosis of colorectal cancer cells. MolCell 2001;7:673–682.

48. Yu J, Zhang L. No PUMA, no death: Implications for p53-dependent apoptosis. Cancer Cell 2003;4:248–249.

49. Jabbur JR, Huang P, Zhang W. DNA damage inducedphosphorylation of p53 at Ser20 correlates with p21 andMdm-2 induction in vivo. Oncogene 2000;19:6203–6208.

50. Ghanem L, Steinman R. A proapoptotic function of p21 indifferentiating granulocytes. Leuk Res 2005;29:1315–1323.

51. Macip S, Igarashi M, Fang L. Inhibition of p21-mediated ROSaccumulation can rescue p21-induced senescence. EMBO J2002;21:2180–2188.

52. Sah NK, Khan Z, Khan GJ, Bisen PS. Structural, functional andtherapeutic biology of survivin. Cancer Lett 2006;244:164–171.

53. Matthews JR, Botting CH, Panico M, Morris HR, Hay RT.Inhibition of NF-kappa B DNA binding by nitric oxide. NucleicAcids Res 1996;24:2236–2242.

54. Chae IH, Park KW, Kim HS, Oh BH. Nitric oxide inducedapoptosis is mediated by Bax/Bcl-2 gene expression,transition of cytochrome c, and activation of caspase-3 inrat vascular smooth muscle cells. Clin Chim Acta 2004;341:83–91.

1108 BHUSHAN ET AL.


activation of p53/p21/puma alliance and disruption of pi-3/akt in multimodal targeting of apoptotic...

Documents