ORIGINAL PAPER

A triterpenediol from Boswellia serrata induces apoptosis throughboth the intrinsic and extrinsic apoptotic pathways in humanleukemia HL-60 cells

Shashi Bhushan Æ Ajay Kumar Æ Fayaz Malik ÆSamar Singh Andotra Æ Vijay Kumar Sethi ÆIndu Pal Kaur Æ Subhash Chandra Taneja ÆGhulam Nabi Qazi Æ Jaswant Singh

� Springer Science+Business Media, LLC 2007

Abstract A triterpenediol (TPD) comprising of isomeric

mixture of 3a, 24-dihydroxyurs-12-ene and 3a, 24-di-

hydroxyolean-12-ene from Boswellia serrata induces

apoptosis in cancer cells. An attempt was made in this

study to investigate the mechanism of cell death by TPD in

human leukemia HL-60 cells. It inhibited cell proliferation

with IC50 ~ 12 lg/ml and produced apoptosis as measured

by various biological end points e.g. increased sub-G0

DNA fraction, DNA ladder formation, enhanced Annex-

inV-FITC binding of the cells. Further, initial events

involved massive reactive oxygen species (ROS) and nitric

oxide (NO) formation, which were significantly inhibited

by their respective inhibitors. Persistent high levels of NO

and ROS caused Bcl-2 cleavage and translocation of Bax to

mitochondria, which lead to loss of mitochondrial mem-

brane potential (Dwm) and release of cytochrome c, AIF,

Smac/DIABLO to the cytosol. These events were associ-

ated with decreased expression of survivin and ICAD with

attendant activation of caspases leading to PARP cleavage.

Furthermore, TPD up regulated the expression of cell death

receptors DR4 and TNF-R1 level, leading to caspase-8

activation. These studies thus demonstrate that TPD

produces oxidative stress in cancer cells that triggers self-

demise by ROS and NO regulated activation of both the

intrinsic and extrinsic signaling cascades.

Keywords Boswellia serrata � Triterpenediol �Apoptosis � HL-60 cells � RNOS � cytochrome c

Abbreviations

DAF-2-DA Diaminofluoresceine-2-diacetate

DCFH-DA 2,7-Dichlorofluoresceine diacetate

LPS Lipopolysaccharide

Dwm Mitochondrial membrane potential

MTT 3-(4,5-Dimethylthiazole-2-yl)-2,5-

diphenyltetrazolium bromide

PBMC Peripheral blood mononuclear cell

NAC N-acetyl-cysteine

iNOS Inducible nitric oxide synthase

PI Propidium iodide

PTP Mitochondrial permeability transition pore

NO Nitric oxide

Rh-123 Rhodamine-123

sMIT s-Methylisothiourea

AIF Apoptosis inducing factor

PMSF Phenylmethanesulfonyl fluoride

ICAD Inhibitor of caspase activating DNAse

Introduction

More than 50% of anti-cancer drugs are directly or indi-

rectly derived from medicinal plants, which still continue

to provide essential source of novel discovery leads [1].

The gum resin of Boswellia serrata, a kind of deciduous

tree grown in the dry part of China and India, has tradi-

tionally been used for the treatment of inflammatory and

arthritic diseases [2]. The pentacyclic triterpenic acids

S. Bhushan � A. Kumar � F. Malik � S. S. Andotra �V. K. Sethi � S. C. Taneja � G. N. Qazi � J. Singh (&)

Division of Pharmacology, Indian Institute of Integrative

Medicine, Canal Road, Jammu 180001, India

e-mail: jsishar1@yahoo.com

I. P. Kaur

University Institute of Pharmaceutical Sciences, Panjab

University, Chandigarh 160014, India

123

Apoptosis

DOI 10.1007/s10495-007-0105-5

called boswellic acid such as b-boswellic acid, 11-keto-b-

boswellic acid, 3-O-acetyl-b-boswellic acid and 3-O-ace-

tyl-11-keto-b-boswellic acid in the gum resin are the major

chemical constituents reported for their anti-inflammatory,

anti-proliferative and anticancer activities [3–4]. Structure

activity relationship indicated that pentacyclic ring skele-

ton of boswellic acid is important for anti-topoisomerase

activity [4]. We further isolated another pentacyclic tri-

terpenediol from Boswellia serrata, which exists in nature

as an isomeric mixture of 3a, 24-dihydroxyurs-12-ene and

3a, 24-dihydroxyolean-12-ene (TPD). One of the isomers

3a, 24-dihydroxyurs-12-ene has been earlier isolated and

reported as natural triterpenediol from the gum resin of

Boswellia serrata [5], the same when chemically synthe-

sized was found to contain an isomeric mixture of 3a, 24-

dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-12-ene

(TPD) as it exists in nature in the gum resin of Boswellia

serrata [6]. Some of our preliminary studies have indicated

that TPD is able to inhibit proliferation of a large number

of human cancer cell lines. It was therefore our interest to

find out in-depth the mechanism of action of cell cyto-

toxicity by TPD, particularly in the induction of pro-

grammed cell death (apoptosis). Because deregulation of

apoptosis is the hallmark of all cancer cells and the agents

that activate programmed cell death in cancer cells could

be valuable anticancer therapeutics [7]. Anti-cancer drugs

act through different pathways converging ultimately into

activation of apoptosis in cancer cells leading to cell

cytotoxicity. Recent studies have amply documented that

two major pathways are involved in the regulation of

apoptosis. The drugs may kill cells either by activation of

extrinsic or intrinsic apoptotic pathways. The extrinsic

apoptotic pathway involves cell surface death receptors,

such as Fas/CD95 and TNFR1, which upon activation up

regulate down stream signaling cascade leading to the

activation of caspase-8 [8]. The intrinsic pathway is

dependent on various cell stress stimuli leading to altered

ratio of Bcl-2 family members affecting cytochrome c and

apoptotic protease activating factor-1 (Apaf-1) release that

leads to caspase-9 activation [9]. The active forms of

caspase-8 and caspase-9 may activate downstream effec-

tors caspases-3, -6, and 7. Thus, enable the cleavage of

several intracellular polypeptides as well as activation of

DNase that leads to DNA fragmentation.

Growing evidence suggests that cancer cells exhibit

increased intrinsic ROS stress, due in part to oncogenic

stimulation, increased metabolic activity, and mitochon-

drial malfunction [10]. Since the mitochondrial respiratory

chain (electron transport complexes) is a major source of

ROS generation in the cells, the vulnerability of the

mitochondrial DNA to ROS-mediated damage appears to

be a mechanism to amplify ROS stress in cancer cells

[11]. The escalated ROS generation in cancer cells serves

as an endogenous source of DNA-damaging agents that

promote genetic instability. Many drugs would act as pro-

oxidant, which initially may involve generation of free

radicals such as reactive oxygen/nitrogen species eventu-

ally leading to the activation of apoptosis [12–13]. Several

anti-cancer drugs doxorubicin, mitomycin c, etoposide and

cisplatin are superoxide generating agents [14]. The anti-

estrogen tamoxifen, increasingly used alongside the other

breast cancer therapies, has also been shown to induce

oxidative stress within carcinoma cells in vitro [15]. We

thus became interested to know the mechanism of cancer

cells killing by TPD so that the lead might be developed

into a novel antineoplastic therapeutic. We here report for

the first time the cell cytotoxicity by TPD in human leu-

kemia HL-60 cells and the molecular mechanisms involved

therein. Our studies provide a deeper insight into the events

leading to TPD induced apoptosis in HL-60 cells. The

observed apoptotic activity of TPD is associated with the

early generation of ROS and NO leading simultaneously to

loss of mitochondrial membrane potential, release of pro-

apoptotic factors and activation of caspases, which alto-

gether account for apoptotic cell death.

Materials and methods

Chemicals and antibodies

RPMI-1640 medium, diaminofluoresceine-2-diacetate

(DCF-DA), 2¢, 7¢-dichlorofluoresceine diacetate (DCF-DA),

rhodamine-123 (Rh-123), s-methylisothiourea (sMIT),

propidium iodide (PI), DNase-free RNase, proteinaseK,

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bro-

mide (MTT), Hoechst 33258, diaminofluorescine 2-diace-

tate (DAF-2DA), ascorbate, PMSF, cyclosporine A, LPS,

eukaryotic protease inhibitor cocktail, N-acetyl cysteine

(NAC), tiron, penicillin, streptomycin, L-glutamine, pyruvic

acid, camptothecin, histopaque-1077 and-1119 were pur-

chased from Sigma chemical Co. Fetal bovine serum was

obtained from GIBCO Invitrogen Corporation (#16000-044,

lot No. 1237517) USA. AnnexinV-FITC apoptosis detection

kit, Caspase-8, -9 inhibitors and ApoAlert caspases assay

kits were from B.D. Clontech, USA. Mouse anti-human

antibodies to Bax (#SC20067), PARP-1 (#SC8007), Bcl-2

(#SC7382), b-Actin (#SC-47778), TNF-R1 (#SC8436),

Survivin (#SC-17779), p21 (#SC-817), goat anti-human

antibodies to DR4 (#SC-6824), ICAD (#SC-6866), goat

anti-rabbit IgG-HRP (#SC2301) and goat anti-mouse IgG-

HRP (#SC2031) were from Santa Cruz Biotechnology,

USA. Rabbit anti-human antibodies to AIF (#PC536),

Smac/DIABLO (#PC-574) and rabbit anti-goat IgG-HRP

(#401504) were from Calbiochem, USA. Mouse anti-human

Apoptosis

123

antibodies to cytochrome c (#556433, clone 7H8.2C12) and

rabbit anti-mouse antibody to iNOS (#N32030-150) were

from BD Biosciences, Pharmingen, USA. Electrophoresis

reagents, Protein estimation kit and protein markers were

from Bio-Rad Laboratories, USA. Hyper film and ECL Plus

western blotting detection kit were from Amersham Bio-

sciences, UK.

Isolation of bioactive triterpenediol 3a, 24-

dihydroxyurs-12-ene and 3a, 24-dihydroxyolean-

12-ene (TPD)

Boswellia serrata gum resin (1 kg) collected from the

forests of Madhya Pradesh, India was cleansed and made

free of extraneous impurities. The gum was extracted with

3 · 3L of ethyl alcohol (95%) at room temperature. The

combined extract after filtration was concentrated under

reduced pressure. The concentrated extract (530 g) was

diluted with 3% potassium hydroxide solution (3 L) and

the mixture stirred for one hour. The aqueous extract thus

obtained was extracted with (2 · 2 L) hexane and then

with 10% ethyl acetate in hexane (1 · 2 L). The combined

organic solvents were washed with water, dried over

anhydrous sodium sulphate and concentrated under re-

duced pressure to give 130 g of viscous liquid, comprising

the neutral components of the gum resin. A portion of the

concentrated extract (20 g) was subjected to repeated col-

umn chromatography over silica gel. Gradient elution with

hexane/ethyl acetate furnished besides other compounds, a

compound with a single spot on thin layer chromatography

(400 mg) mp 182�C, [a] D + 63 (0.26% yield). It was as an

inseparable mixture of two isomers identified by 13CNMR

and 1 HNMR [5]. The ratio of the isomeric mixture was

found to be (80:20), as determined by reverse phase HPLC

on C-18 column (acetonitrile: water; 95:5) and detection at

210 nm. The isomeric mixture may also be obtained by

semi-synthetic route from boswellic acids as described

below.

Preparation of 3a, 24-dihydroxyurs-12-ene and 3a,

24-dihydroxyolean-12-ene (TPD) from boswellic acid

The natural isomeric mixture of (a + b) boswellic acid

(R = H) was first converted to methyl ester (R = Me,

Scheme 1) by diazomethane generated in situ. Thus a

mixture of (a + b) of boswellic acid (230 mg) was dis-

solved in dry solvent ether and diazomethane solution in

diethyl ether was slowly added till completion of reaction

as monitored by TLC. After the removal of the solvent and

excess of diazomethane, the mixture comprising the methyl

esters was re-dissolved in dry solvent ether at room tem-

perature and 20 mg of lithium aluminum hydride (LiAlH4)

was added to the stirring solution under nitrogen atmo-

sphere and stirred for one hour. The excess of LiAlH4 was

neutralized by adding ethyl acetate and the organic layer

washed with water. Further concentrating the solvent gave

the desired product 3a, 24-dihydroxyurs-12-ene and 3a,

24-dihydroxyolean-12-ene in almost quantitative yield

(98%). It was crystallized from hexane-ethyl acetate to get

white crystals of TPD. The product gave similar physico-

chemical and spectra as of the one obtained directly from

the gum resin described above.

Cell culture, growth conditions and treatment

Human promyelocytic leukemia cell line HL-60, human

acute lymphoblastic leukemia cell line MOLT-4, normal

monkey kidney cell line CV-1, human breast cancer cell

line MCF-7 were procured from National Centre for Cell

Sciences (NCCS), Pune, India. Human lung carcinoma cell

line A549, human colon cancer cell line HCT-15, human

cervix carcinoma cell line HeLa and human colon cancer

cell line SW-620 procured from National Cancer Institute,

Frederick, U.S.A. Human PBMC were prepared from the

blood of normal human by using Sigma’s Histopaque-1077

and 1119 solution [16]. Cells were grown in RPMI-1640/

MEM medium containing 10% FCS, 100 unit pencillin/

100 lg streptomycin per ml medium. Cells were grown in

CO2 incubator (Thermocon Electron Corporation, USA) at

37�C with 98% humidity and 5% CO2 gas environment.

Cells were treated with TPD dissolved in DMSO while the

untreated control cultures received only the vehicle

(DMSO, <0.2%).

Preparation of mice peritoneal macrophages

Mice peritoneal macrophages were isolated and cultured as

described earlier [17]. Mice were sacrificed and peritoneal

Scheme 1

Apoptosis

123

exudates cells isolated. The cells were washed twice with

RPMI and finally adjusted to 3 · 106 cells/ml of complete

RPMI medium. Cells were allowed to adhere for 3 h and

non-adherent cells were washed off with PBS. The adher-

ent cells were then treated with the TPD and LPS in the

presence and absence of sMIT (1 mM) for 24 h and the

lysate analyzed for the iNOS expression.

Cell proliferation assay

Cells from suspension and adherent SW-620/MCF-7 cul-

tures grown in 96-well plates were exposed to indicate

concentrations of TPD for 48 h thereafter, 20 ll of MTT

solution (2.5 mg/ml) was added to each well and incubated

at 37�C for 2 h. The plates were centrifuged and the

supernatant was discarded while the MTT-formazon crys-

tals were dissolved in 100 ll DMSO. The OD measured at

570 nm with reference wavelength of 620 nm [18].

DNA fragmentation

Cells (2 · 106) in culture were treated with different con-

centrations of TPD. Cells were lysed, genomic DNA ex-

tracted and electrophoresed as described earlier [18].

Hoechst 33258 staining of cells for nuclear morphology

HL-60 cells (2 · 106 cells/3 ml) were grown in 6 well

plates and treated with TPD for 6 h. Cells were collected,

washed, fixed and stained with Hoechst-33258 and

observed under inverted fluorescence microscope

(Olympus1X70) [18].

Flow cytometric analysis of apoptosis and necrosis

The extent of apoptosis was determined using FITC-la-

beled AnnexinV antibody by flow cytometry [19]. Cells

(1 · 106) were treated with different concentrations of

TPD for 6 h, washed and stained with AnnexinV-FITC

antibody and PI as per the instructions given by the man-

ufacturer. The cells were scanned for fluorescence intensity

in FL-1 (FITC) and FL-2 (PI) channels. The fraction of cell

population in different quadrants was analyzed using

quadrant statistics. Cells in the lower right quadrant rep-

resented apoptosis and in the upper right quadrant repre-

sented necrosis or post apoptotic necrosis [18].

Flow cytometric analysis of intracellular nitric oxide

using DAF-2-DA

Intracellular nitric oxide was measured by employing a low

molecular weight fluorescent probe diaminofluoresceine 2-

diacetate (DAF-2-DA), which is membrane permeable and

usually serves as a reporter of nitric oxide synthase activity.

Immediately after NO is generated inside the cells, it binds

with the chromophore to yield strong fluorescent signal that

can be measured in the green (FL-1) channel of the flow

cytometer [20]. Cells (1 · 106/2 ml/well of 12-well plate)

were pre-incubated for 30 min with DAF-2-DA (10 lM),

and then incubated together with TPD in the presence and

absence of sMIT (1 mM). Cells were collected, washed in

PBS and analyzed on flow cytometer in FL-1 channel for

evaluation of NO positive cell population [18].

Flow cytometric measurement of intracellular

peroxides (ROS) in HL-60 cells

The level of intracellular peroxides was determined by using

2,7-dichlorofluoresceindiacetate [21, 22]. Cells (1 · 106/

2 ml/12 well plate) were incubated with TPD in the pres-

ence and absence of anti-oxidant for 6 h and finally treated

with DCFH-DA (5 lM) for 30 min. Cells were washed in

PBS and incubated with propidium iodide (5 lg/ml) for

15 min. The green DCF-fluorescence was analyzed in the

FL-1 channel (Excitation k 488 nm; Emission k 535 nm)

and PI fluorescence was analyzed in the FL-2 channel.

Flow cytometric determination of mitochondrial

membrane potential

Changes in mitochondrial transmembrane potential (Dwm)

as a result of mitochondrial perturbation were measured

after staining with Rhodamine-123 [18]. Cells (1 · 106/

2 ml/12 well plates) were treated with TPD in the presence

and absence of different ROS/NO inhibitors for 6 h. Rho-

damine-123 (5 lM) was added 1 h before the termination

of experiment. Cells were collected, washed in PBS and

incubated with propidium iodide (5 lg/ml) for 15 min. The

decrease in fluorescence intensity because of mitochondrial

membrane potential loss was analyzed in FL-1 channel.

Cell cycle analysis by flow cytometry

Cells (1 · 106/ml) were treated with different concentra-

tions of TPD for 24 h, fixed in cold 70% alcohol in PBS,

washed, digested with DNase free RNase (400 lg/ml) at

37�C for 45 min and stained with propidium iodide [18].

Cells were analyzed immediately on a SLR flow-cytometer

(Becton Dickinson, USA). The fluorescence intensity of

sub-G0 cell fraction represents the apoptotic cell popula-

tion [23].

Caspase assays

Cells were incubated with TPD in the presence and absence

of ascorbate (5 mM), sMIT (1 mM) and NAC (5 mM) for

Apoptosis

123

indicated time period. Cells were collected, washed in PBS

and lysed in cell lysis buffer. Activities of caspase-3, -8,

and -9/6 in the cell lysates were determined fluorimetrically

using BD Apoalert caspase assay kits as per instructions of

the manufacturer. Specific inhibitors were used as negative

control to determine whether fluorescence intensity chan-

ges were specific for the activity of caspases. The peptide

based inhibitors used were DEVD-CHO for caspase-3,

IETD-fmk for caspase-8 and LEHD-CHO for caspase-9

[18].

Preparation of total cell lysates for immunoblotting

Cells (3 · 106) were collected, washed with cold PBS and

incubated with cold lysis buffer (50 mM Tris pH8.0,

150 mM NaCl, 5 mM EDTA, 1% v/v Nonidet P-40, 1 mM

PMSF and 1% (v/v) eukaryotic protease inhibitor cocktail)

for 30 min on ice [24]. Cells were centrifuged at 12000 · g

for10 min at 4�C and the supernatant was collected as

whole cell lysates for western blot analysis of various

proteins.

Preparation of cytosolic and mitochondrial lysates

Cytosolic fractions for analysis of cytochrome c, Smac/

DIABLO and Bcl-2 immunoblotting were obtained by

selective plasma membrane permeabilization with digito-

nin [25]. Briefly, 2 · 106 cells were lysed for 1–2 min in

lysis buffer (75 mM NaCl, 8 mM Na2HPO4, 1 mM

NaH2PO4, 1 mM EDTA, and 350 lg/ml digitonin and 1%

(v/v) eukaryotic protease inhibitor cocktail). The lysates

were centrifuged at 12,000 · g for 1 min, and the super-

natant collected as cytosolic fraction and stored at –70�C.

Residual pellet was lysed with whole cell lysis buffer by

incubating on ice for 30 min [24]. After centrifugation at

12,000 · g for 10 min at 4�C, cell lysates were transferred

to fresh tubes and stored at –70�C as mitochondrial frac-

tion.

Preparation of nuclear fraction for AIF immunoblotting

HL-60 cells (1 · 107) pellet were washed in cold PBS and

suspended in 400 ll ice-cold hypotonic buffer (10 mM

HEPES/KOH pH 7.9, 2 mM MgCl2, 0.1 mM EDTA,

10 mM KCl, 1 mM dithiotheritol, 0.5 mM PMSF, 1% (v/

v) eukaryotic protease inhibitor cocktail) for 10 min on ice.

Suspension was vortexes and centrifuged at 15,000 · g for

30 s at 4�C. The supernatant was discarded and the cell

pellet was gently resuspended in 100 ll of ice cold saline

buffer (50 mM HEPES/KOH pH7.9, 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 for 20 min. Cells suspension was vortexes

and centrifuged at 15000 · g for 5 min at 4�C [26]. The

supernatant was stored at –70�C as nuclear lysate.

Western blots analysis

The protein lysates prepared were subjected to discontin-

uous SDS-PAGE analysis. Proteins aliquots (50 lg) were

resolved on SDS-PAGE and then electro transferred to

PVDF membrane overnight at 4�C at 30 V. Non-specific

binding was blocked by incubation with 5% non-fat milk in

Tris-buffered saline containing 0.1% Tween-20 (TBST) for

1 h at room temperature. The blots were probed with

respective primary antibodies for 2 h and washed three

times with TBST [27]. The blots were then incubated with

horseradish peroxidase conjugated mouse or rabbit sec-

ondary antibodies for 1 h, washed again three times with

TBST and signals detected using ECL plus chemilumi-

nescence’s kit on X-ray film.

Protein measurement

Protein was measured employing Bio-Rad protein assay kit

using bovine serum albumin as standard.

Statistical analysis

Data expressed as Mean ± SD, unless otherwise indicated.

Comparisons were made between control and treated

groups unless otherwise indicated using unpaired Student’s

t-test and p-values < 0.01 was considered significant.

Results

Inhibition of cell proliferation by TPD

Using a conventional tetrazolium-based colorimetric cell

proliferation assay, we evaluated the cytotoxicity of TPD in

different cancer cells as described earlier [18]. Treatment

of cells with TPD for 48 h produced concentration

dependent inhibition of cell proliferation of HL-60, Molt-4

and SW-620 with IC50 value of approx. 12 lg/ml while

MCF-7 cells were relatively more sensitive with IC50 of

5 lg/ml (Fig. 1A).

TPD induces DNA fragmentation typical of apoptosis

To investigate whether TPD treatment induced DNA

fragmentation, genomic DNA was isolated from TPD

treated HL-60 cells. Apoptosis typically involves intra-

nucleosomal chromatin cleavage by endonucleases in

multiples of 180 bp leading to DNA fragmentation result-

ing in a typical DNA laddering. Cells treated with TPD for

Apoptosis

123

18 h exhibited typical DNA ladder. At low concentration,

DNA ladder was barely visible and this became increas-

ingly prominent in cells treated with higher concentration

of TPD (Fig. 1B). The DNA ladder was diffused inter-

spersed with smear indicative of some post-apoptotic

necrosis in both HL-60 and SW-620 cells treated with

higher concentration of TPD. DNA isolated from untreated

and treated human PBMC or normal monkey kidney CV-1

cells did not show any DNA ladder (Fig. 1B).

TPD induced altered nuclear morphology is rescued by

NAC

Nuclei of untreated HL-60 cells appeared round in shape,

while treatment with TPD resulted in nuclear condensation

and formation of apoptotic bodies. The morphological

changes were accompanied by increase in number of

scattered apoptotic bodies. Pretreatment of cells with

sMIT, an inhibitor of iNOS, and NAC an ROS scavenger

Fig. 1 (A) Influence of TPD on proliferation of various human

cancer cell lines. The cells grown in 96-well culture plate were treated

with different concentration of TPD for 48 h, thereafter cultures

incubated with MTT for 2 h at 37�C. Other conditions are described

in Material and Method. Data are Mean ± SD (n = 8 wells) and

representative of two similar experiments. (B) TPD induced DNA

fragmentation in human cancer and normal cell lines. Genomic DNA

was extracted from cells treated with different concentrations of TPD

for 18 h. (i) HL-60 cells (ii) normal human PBMC and normal

monkey kidney CV-1 cells (iii) SW-620 cells. Other conditions are

described in Materials and Methods. The data are representative one

of two similar experiments. (C) Influence of TPD on nuclear

morphology of HL-60 cells. The cells were treated with 30 lg/ml

TPD for 6 h and stained with Hoechst 33258 and visualized for

nuclear morphology and apoptotic bodies. (a) Untreated control show

normal nuclear morphology; (b) Cells treated with TPD indicate

condensed nuclei, scattered and typical apoptotic bodies formation

(arrows); The TPD induced morphological changes were returned

back by incubation with (c) 5 mM NAC and (d) 1 mM sMIT

Apoptosis

123

rescued the formation of apoptotic bodies in TPD treated

cells (Fig. 1C).

Flow cytometric estimation of TPD induced apoptosis

and necrosis

Exposure of phosphotidyl serine on the surface of cells is

an early event in the onset of apoptosis, which has strong

binding affinity for AnnexinV in the presence of calcium.

HL-60 and Molt-4 cells were incubated with different

concentration of TPD and cells were stained with Annex-

inV-FITC and PI to assess the apoptotic and necrotic cell

population (Fig. 2). TPD produced dose dependant in-

crease in the apoptotic cell population. The basal apoptotic

population in the untreated culture was 3%, which in-

creased to 23% at 50 lg/ml within a short period of 6 h.

Camptothecin, used as positive control, produced about

14% apoptotic population during the same exposure period.

Apoptosis thus appeared to be the primary mode of cell

death induced by TPD.

TPD induces early generation of intracellular nitric

oxide

The extent of NO generation in cells was analyzed by flow

cytometry after staining with DAF-2DA (FL-1). It was

observed that NO levels increased significantly with

increasing concentrations of TPD. Inducible nitric oxide

Fig. 2 Flow cytometric

analysis of TPD induced

apoptosis in HL-60 and MOLT-

4 cells using AnnexinV-FITC/

PI. Cells were incubated with

indicated concentrations of TPD

for 6 h and stained with

annexinV-FITC/PI to analyze

apoptotic and necrotic cell

populations as described in

Materials and Methods. Data are

representative of one of two

similar experiments

Apoptosis

123

synthase (iNOS) inhibitor, sMIT, suppressed remarkably

the NO levels from 84% to 38% (Fig. 3A). The increase in

NO level was also observed in Molt-4 and MCF-7 cells,

which were again protected by sMIT. Whether the increase

in NO is because of iNOS induction, the western blot

analysis was carried out using rabbit anti-mouse antibody

to iNOS, which is also supposed to detect human form,

could not detect the iNOS expression in HL-60 cells [28].

However, when the same antibody was used against TPD

treated mouse macrophages, it was found that TPD induced

the expression of iNOs several fold (Fig. 3B), thereby

indirectly suggesting that NO is derived from iNOS

expression in HL-60 cells.

TPD mediated early ROS generation and influence of

inhibitors

Cells were incubated with TPD for 6 h and analyzed by

flow cytometry after double staining with DCFH-DA and

PI [29]. There was hardly any DCF fluorescence in

the untreated HL-60 cells. However, cells treated with

30 lg/ml of TPD, observed a significant increase in DCF

Fig. 3 (A) TPD induces early

intracellular nitric oxide

generation as shown in cancer

cell lines HL-60, MOLT-4 and

MCF-7. Cells (1 · 106) were

exposed to TPD and NO probe

DAF-2DA for 6 h. The cells

were analyzed by flow-

cytometery for NO fluorescence

in FL-1 channel. Data are

representative of one of two

similar experiments. (B)

Immunoblot analysis of iNOS in

mice peritoneal macrophages

treated with TPD (10, 30 lg)

and LPS (1, 5 lg) with or

without sMIT (1 mM) for 24 h.

Total cell lysates were prepared

and 50 lg protein samples were

loaded on SDS-PAGE gel for

western blot analysis of iNOS

using anti-mouse antibody as

described in Material and

Methods. Data are

representative of one of two

similar experiments

Apoptosis

123

positive cell population by about 75% (Fig. 4A). Ascor-

bate, NAC, tiron and trolox significantly suppressed the

pro-oxidant effect of TPD (30 lg/ml) from 75% to 38, 28,

51 and 38%, respectively. Oxidative burst is a character-

istic event produced by TPD not only in HL-60 cells but

also in other tumor cell lines such as A-549, HCT-15,

Molt-4 and MCF-7 cells examined (Fig. 4B).

Loss of mitochondrial membrane potential by TPD

HL-60 cells exposed to TPD for 6 h were analyzed for

mitochondrial membrane potential (Dwm) loss employing

Rh-123 uptake by flow cytometery. In the untreated control

cells, almost all cells were functionally active with high

Rh-123 fluorescence (Fig. 5A). TPD at 30 lg caused

mitochondrial damage and hence the decrease of mito-

chondrial membrane potential by about 40%. The loss of

Dwm is largely due to the opening of mitochondrial per-

meability transition pores (PTP), which conduit the leakage

of cytochrome c and pro-apoptotic proteins from mito-

chondria to the cytosol. Cyclosporine A (CsA) is a potent

inhibitor of PTP where it inhibits the opening of PTP [30].

Cells incubated with CsA (2 lM) restored the energized

state by about 45% while other ROS inhibitors sparingly

protected cells.

RNOS generation is an early event elicited by TPD

We asked if RNOS generation is an early event in the

induction of apoptosis. In fact, NO generation appeared to

be an early event with parallel ROS production. Loss of

mitochondrial membrane potential (Dwm) and onset of

apoptosis happened to be late subsequent events (Fig. 5B).

The relationship between these important critical events

was determined in the gated cell population exposed to

TPD for different time periods and measured by flow

cytometery. At 30 min there was a significant increase in

NO level and ROS generation while loss of mitochondrial

Fig. 4 (A) TPD mediated early

generation of peroxides in HL-

60 cells and modulation by

various anti-oxidants. Cells

(1 · 106) were treated with

30 lg/ml TPD in the presence

and absence of various ROS

inhibitors for 6 h, followed by

incubation with DCFH-DA

(5 lM) for 30 min. Cells were

analyzed for DCF-fluorescence

on flow-cytometer in the FL-1

(DCF-Fluorescence) vs. FL-2

(PI-Fluorescence) channels.

Data are representative of one of

two similar experiments. (B)

Sensitivity of various human

cancer cell lines towards TPD

mediated generation of

peroxides. Cells (1 · 106) after

various treatments with TPD in

the presence and absence of

NAC (5 mM) for 6 h were

incubated with DCFH-DA

(5 lM) for 30 min. Cells were

analyzed for DCF-fluorescence

as described above. Data are

Mean ± S.D. from three similar

experiments. P-value: **<0.001

compared to untreated control;

@ < 0.01 compared to TPD,

30 lg treated cells;

@@ < 0.001 compared to TPD,

30 lg treated cells

Apoptosis

123

membrane potential (Dwm) and apoptotic cell death were at

basal minimal levels. However after 6 h of treatment the

extent of ROS and NO formation were almost similar

(approx. 85%) that may be responsible for loss of mito-

chondrial membrane potential, the later corresponded with

high degree of apoptotic population, when almost 50% of

the cells had lost their Dwm (Fig. 5B).

TPD increases sub-G0 DNA fraction of cell cycle phase

distribution: protection by various inhibitors

HL-60 cells treated with TPD for 24 h exhibited concen-

tration dependent increase in hypo diploid sub-G0 DNA

fraction (<2nDNA) (Fig. 6). The sub-G0 fraction was <5%

in control cells, which increased to ~73% after 30 lg/ml of

TPD treatment. The ROS scavenger ascorbate, NAC and

tiron significantly suppressed the sub-G0 fraction from

73% to 21, 54 and 26% respectively while inhibitor of

iNOS, sMIT strongly lowered the DNA fraction from 73%

to 15%. These results suggested the involvement of both

reactive nitrogen and oxygen species (RNOS) in the

induction of apoptosis. Also both caspase-8 and -9 inhibi-

tors were able to inhibit the increase in sub-G0 DNA

fraction significantly. Further, the cell cycle G2/M phase

was not affected indicating that TPD does not produce any

mitotic block or caused delay in cell cycle. In contrast, the

sub-G0 fraction in normal human PBMC cells treated with

same concentration of TPD was poorly affected.

TPD stimulates caspase activities in HL-60 cells

TPD produced optimal caspases activation through 12 h

incubation while the prolonged incubation did not further

enhance the activity any more (Fig. 7A). The triterpenediol

produced a dose dependant increase in caspase-8 and -9

activities in HL-60 cells, incubated with it for 6 h leads to

activation of caspase-8 by 2-fold and that of caspase-9 by

10-fold. Camptothecin, a topoisomerase I inhibitor used as

positive control however, produced 2-fold activation of

caspase-8. When cells were incubated with TPD with or

without ascorbate, NAC and sMIT, it was interesting to

find that all the three inhibitors antagonized the activation

Fig. 5 (A) TPD induced loss of

mitochondrial membrane

potential (Dwm). HL-60 cells

(1 · 106/2 ml/12 well plates)

were incubated with 30 lg/ml

of TPD for 6 h. Various RNOS

inhibitors were added 1 h before

treatment with TPD. Thereafter,

cells were stained with

Rhodamine-123 (5 lg/ml) for

1 h and PI (5 lg/ml) and

analyzed in FL-1 vs. FL-2

channels of flow cytometer. (B)

Relationship between early

events of RNOS generation,

Dwm loss and extent of

apoptosis. HL-60 cells (1 · 106/

ml) grown in the 24 well culture

plates were exposed to the

30 lg/ml of TPD for indicated

time periods. Cells were stained

with DCFH-DA, DAF-2DA,

Rh-123 and AnnexinV-FITC

and analyzed by flow

cytometery for their respective

fluorochromes fluorescence as

described in Material and

Methods. Data are Mean ± S.D.

from three similar experiments.

P value: **<0.001 compared to

untreated control

Apoptosis

123

of caspase-8 and -9 equipotently. This again confirms that

pro-oxidant environment induced by TPD is responsible for

sustained stimulation of caspases in leukemia cells. Spe-

cific inhibitors for caspases were utilized in the assay

system to corroborate the specificity of caspase activities

investigated (Fig. 7B).

TPD induced mitochondrial dysfunction is associated

with translocation of pro-apoptotic molecules with

concomitant cleavage of Bcl-2

Members of Bcl-2 family of proteins are known to control

the release of cytochrome c from mitochondria [31].

Therefore we investigated the effect of TPD on Bcl-2

levels in HL-60 cells and it was interesting to find out that a

truncated 23 kDa form of Bcl-2 appeared in cytosolic

fraction (Fig. 8A). The appearance of cleaved Bcl-2 pro-

tein may represent a caspase dependant transformation of

28 kDa (anti-apoptotic) to 23 kDa (pro-apoptotic) Bcl-2

[32, 33]. The altered Bcl-2 level by TPD was significantly

reversed by NAC and sMIT. Further TPD increased the

release of Smac/DIABLO and cytochrome c in to the

cytosol with simultaneous translocation of Bax to mito-

chondria. It is mentioned that Smac/DIABLO is a proa-

poptotic protein, which is released from the mitochondria

during the intrinsic pathway of apoptosis. Translocation of

cytochrome c, Bax, Smac/DIABLO and Bcl-2 cleavage

were both time and concentration dependant and this effect

was significantly attenuated by the NAC and sMIT. TPD

significantly decrease the Bcl-2/Bax ratio (82%) in the

mitochondria from 2.51 to 0.47, which was significantly

reverted back by NAC and sMIT (Fig. 8B).

TPD activates extrinsic signaling cascade by up

regulation of apical death receptors

HL-60 cells treated with TPD exhibited increased expres-

sion of death receptor DR4 and TNF-R1 levels (Fig. 9A, B).

TNF-R1 and DR4 activation seems to be the early events

however, as postulated that NO might be responsible for

TNF-R1 over expression [33], this effect on the contrary

was not inhibited by the sMIT, but in the presence of both

sMIT and NAC, its over expression was inhibited signifi-

cantly (Fig. 9B). Nevertheless, induction of TNF-R1, DR4

and caspase-8 activity indicated that TPD might also be

contributing substantially to the apoptotic death machinery

by activation of extrinsic signaling cascade.

Effect of TPD on other apoptotic related genes

expression

The triterpenediol at 30 lg also increased the release of

apoptosis inducing factor (AIF) into the nuclei of HL-60

cells (Fig. 10A). AIF is a mitochondrial flavoprotein that is

capable of inducing chromatin condensation following an

apoptotic stimulus. Our studies also demonstrate that TPD

induces p21 over expression in HL-60 cells (Fig. 10B). p21

is a cyclin-dependent kinase (CDK) inhibitor, which is best

Fig. 6 DNA Cell cycle

analyses in TPD treated HL-60

cells and influence of RNOS

inhibitors. Cells 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. Cells were stained

with PI to determine DNA

fluorescence and cell cycle

phase distribution as described

in Materials and Methods.

Fraction of cells for hypo-

diploid (sub-G0, <2n DNA)

population analyzed from FL2-

A vs. cell counts is shown (%).

Data are representative of one of

two similar experiments

Apoptosis

123

known for its ability to regulate cell cycle, besides its ef-

fects on apoptosis and differentiation [34]. The CDK

inhibitor p21 increases intracellular levels of ROS both in

normal fibroblasts and in p53-negative cancer cells [35].

Over expression of p21 was impaired by NAC in the

present studies (Fig. 10B) suggesting that induction of p21

and ROS by TPD are complementary to each other.

Regulation of Survivin, ICAD and PARP levels in HL-

60 cells during TPD treatment

Survivin is a cell cycle regulated inhibitor of apoptosis

protein (IAP), mostly over expressed in most of tumors. It

is highly expressed in HL-60 cells, which TPD decreased

over a period of treatment. TPD mediated release of

SMAC/DIABLO from mitochondria might be expected to

down-regulate the expression of survivin and this was

observed in our studies. However, after exposure of 24 h,

the protein marginally increased, which was rescued effi-

ciently by NAC and sMIT (Fig. 11A). Another important

protein related to DNA fragmentation is caspase-activated

DNase (CAD). CAD complexes with an inhibitor of CAD,

(ICAD) also called DFF-45, in non-apoptotic growing

cells. TPD produced time related decrease in its expression,

which was protected by RNOS inhibitors. ICAD is an

acidic protein, with two recognition sites for caspase 3.

When caspase 3 is activated by apoptotic stimuli, ICAD is

cleaved, resulting in the release of CAD. This released

CAD appears to cause DNA fragmentation in the nucleus

of the cell [36]. Activated caspase-3 also uses poly ADP

ribose polymerase (PARP) as a substrate during DNA re-

pair [37]. TPD at 10 lg and 30 lg induced cleavage of

PARP in HL-60 cells (Fig. 11B). Cleavage of PARP,

116 kDa into 89 kDa, started in less than 3 h and the

uncleaved 116 kDa protein was completely cleaved into

89 kDa through 24 h treatment. Co-treatment with NAC or

sMIT resulted in a significant protection against TPD in-

duced PARP cleavage and ICAD down-regulation

(Fig. 11B).

Discussion

The results of the present study describe the pro-apoptotic

activity of a novel triterpenediol isomeric mixture (TPD),

isolated from the Boswellia serrata on human leukemia

HL-60 cells, for the first time. This is evidenced from the

fact that TPD inhibited cell proliferation and induced

apoptosis measured by various biological end points such

as DNA fragmentation, apoptotic body’s formation and

annexin-V binding in HL-60 cells. All these biological end-

points indicated that the cancer cells death is due to the

induction of apoptosis by TPD while no such effect in

terms of DNA fragmentation was observed in human

PBMC and monkey kidney normal cell line CV-1. Con-

sidering the potential that TPD offers in its development as

anticancer agent, we further sought to understand early

events associated with apoptotic cell death. We observed

Fig. 7 (A) TPD induced differential activation of various caspases in

HL-60 cells. The cells in culture were exposed to 30 lg/ml of TPD

for indicated time periods. The caspases activities were determined

fluorometrically in the cell lysate of HL-60 cells using BD ApoAlert

caspase assay kits as described in Materials and Methods. All assays

were performed according to the instructions provided by the

supplier. Specific inhibitors were used for negative control as

described in Materials and Methods. Data are Mean ± S.D. from

three similar experiments. P values: **<0.001 compared to untreated

control, @@<0.001 compared to TPD treated cells. (B) Influence of

RNOS inhibitors on TDP stimulated caspase activities. HL-60 cells

(2 · 106/2 ml) were exposed to different concentration of TPD for

6 h for assays of caspase-8 and -9 activities. Cultures were pre-

exposed for 1 h to sMIT, NAC and ascorbate prior to TPD treatment.

Camptothecin was used as positive control. Other conditions were

same as described in Fig. 7A

Apoptosis

123

that TPD induced early generation of reactive nitrogen or

oxygen species (RNOS), which may account for various

consequences leading to cell death. Though confronting

Fig. 8 (A) Influence of TPD on

the expression of important

proteins involved in the

initiation of apoptosis. HL-60

cells (2 · 106) were treated with

30 lg/ml of TPD for indicated

time periods in the presence and

absence of NAC and sMIT. b-

actin was used as internal

control to represent the same

amount of proteins applied for

SDS-PAGE. Specific antibodies

were used for detection of

cytochrome c, Smac/DIABLO,

Bax and Bcl-2. (B) Influence of

TPD on the Bcl-2/Bax ratio in

the mitochondrial fraction of

HL-60 cells. Data are

representative of one of two

similar experiments. P values:

**<0.001 compared to untreated

control, @@ < 0.001 compared

to TPD treated cells

Fig. 9 Western blot analysis of TNF-R1 and DR4 were performed in

total cell lysates of TPD treated HL-60 cells. Cells (3 · 106) were

treated with 30 lg/ml of TPD for indicated time periods (A) and for

24 h in the presence and absence of indicated inhibitors (B) Other

conditions were same as described in Fig. 8

Fig. 10 (A) Immunoblot analysis of AIF in nuclear fraction of TPD

treated HL-60 cells. Nuclear fraction was prepared and protein

(50 lg) was resolved on 10% SDS-PAGE gel for western blot

analysis as described in Fig.8. (B) Western blot analysis of p21 was

performed in total cell lysate of HL-60 cells. Cells (2 · 106) were

treated with 30 lg/ml of TPD for indicated time periods in the

presence and absence of NAC and sMIT. Other conditions were same

as described in Fig. 8

Apoptosis

123

opinions have been advanced about the role of NO in cells,

current research however, has shown that NO plays an

important role in regulating electron transport chain [12],

while its overproduction would stimulate formation of a

variety of free oxidant radicals by mitochondria leading to

inhibition of electron transport chain and hence electron

leakage. This would result in mitochondrial oxidative stress

[9, 11], which may lead to apoptosis and cell cytotoxicity.

The production of NO in leukemia cells is presumably

through iNOS induction because several human myeloid

and T lymphoblast leukemia cells are known to generate

NO, under different stimuli [38]. Further, early NO for-

mation is known to stimulate mitochondrial superoxide

generation, which we observed simultaneously in TPD

treated HL-60 cells. Interestingly both the ROS and iNOS

inhibitors had a protective effect on the DNA damage in-

duced by TPD, suggesting that TPD is triggering a pro-

oxidant effect in cancer cells.

The biological consequences of such interactions i.e.

both NO and ROS production can produce deleterious ef-

fects on mitochondrial membrane potential, Dwm [9, 39–

40]. TPD induces loss of mitochondrial membrane poten-

tial more prominently after 6 h treatment that corroborated

with increase in apoptosis. The loss of Dwm by TPD was

inhibited substantially by CsA [41] indicating involvement

of mitochondrial PTP in the release of proteins. Several

studies in the past have suggested that generation of RNOS

and abrogation of mitochondrial membrane potential lead

to the activation of caspases. TPD induces the activation of

caspase-8 and 9 in HL-60 cells, which were inhibited

significantly by ascorbate, NAC and sMIT. It may be

mentioned that TPD increased remarkably caspase -9

activity and its activation is solely regulated by mito-

chondrial release of apoptotic protease activating factor-1

(Apaf-1) and cytochrome c, which is a part of mitochon-

drial dependant apoptosis pathway [7]. TPD also induced

the caspase-8 activation in HL-60 cells because of up-

stream over-expression of cell surface receptors such as

TNF-R1 and DR-4 [42]. TPD induced over-expression of

TNF-R1 and DR4, was significantly inhibited by NAC and

sMIT, suggesting again the involvement of pro-oxidant

effect of TPD in the extrinsic signaling pathway of apop-

tosis in HL-60 cells.

In view of caspase-9 activation by TPD, we further

investigated the role of members of Bcl-2 family of pro-

teins and release of proapoptotic proteins from mitochon-

dria. Bcl-2 family proteins play both pro-apoptotic and

anti-apoptotic role in cancer cells. Normally Bcl-2 level is

much higher in cancer cell. We observed cleavage of the

Bcl-2 in cytosolic fraction of TPD treated HL-60 cells

suggesting that RNOS might have oxidatively modified

Bcl-2 protein rendering it susceptible to proteolytic

cleavage, probably by caspase-3 [43]. This is evident from

the fact that both NAC and sMIT rescued TPD modified

Bcl-2 cleavage in HL-60 cells. The cleavage of Bcl-2

obviously would shift the balance in the direction of

apoptosis. Because Bcl-2 is a substrate for activated cas-

pase-3 and the cleaved product is a 23 kDa Bax like pro-

tein, which in fact is pro-apoptotic in nature. Moreover,

cleavage of Bcl-2 is an early event in apoptosis cascade and

it is an important signal for cytochrome c release upon

specific cellular insults [32, 33]. The drugs enabling Bcl-2

cleavage also are known to accelerate apoptosis in various

cell types [44, 45]. Cleavage of Bcl-2 by TPD changes the

symmetry of mitochondria, which causes the opening of

mitochondrial transpermeable pore and brings about Bax

translocation from cytosol to mitochondria and consequent

release of small molecules like AIF, Smac/DIABLO and

cytochrome c from mitochondria to cytosol. Apoptosis

inducing factor (AIF), a 57 kDa mitochondrial flavoprotein

is released from the mitochondria and translocated to the

nucleus after an apoptotic signal where it is capable of

inducing nuclear chromatin condensation and large scale

DNA fragmentation, associated with increase in ROS for-

mation [46, 47]. Besides AIF translocation, TPD also in-

creased both cytochrome c and Bax levels in cytosolic and

mitochondrial fraction, respectively. Translocation of these

proteins impairs mitochondrial functions and brings the

cells to a ‘‘point of no return’’ to enter apoptosis via acti-

vation of caspases. Along with cytochrome c there is

simultaneous release of Smac/DIABLO in high amounts in

TPD treated cells, which not only promotes the proteolytic

activation of procaspase-3 by neutralizing the activity of

IAP such as survivin [48] but also down regulates the

Fig. 11 (A) Western blot analysis of survivin, ICAD and PARP were

performed in total cell lysates of HL-60 cells for indicated time

periods in the presence and absence of NAC and sMIT. Immuno-

blotting analysis was performed as described in Fig. 8. Data are

representative of one of two similar experiments. (B) Effect of low

concentration of TPD (10 lg/ml) on the PARP cleavage and

protection by RNOS inhibitors. Cells (2 · 106) were treated with of

TPD for indicated time periods in the presence and absence of NAC

and sMIT. Other conditions were same as described in Fig. 8

Apoptosis

123

expression of survivin. Elevated levels of survivin are

reported in cancer cells, where it inhibits the pro-caspases

from becoming functionally active [49]. So, by inhibiting

the survivin, TPD reinforces the caspase activity and finally

enhances apoptosis. It is also reported that p21 increases

intracellular levels of ROS both in normal fibroblasts and

in p53-negative cancer cells [35]. The p21 protein is a

cyclin-dependent kinase (CDK) inhibitor, which is best

known for its ability to regulate the cell cycle [34]. TPD

up-regulation of p21 level in HL-60 cells, suggests that the

triterpenediol may be inhibiting CDKs through induced

level of p21. NAC significantly inhibited the effects of

TPD on p21.

The elevated level of caspase-3 could utilize poly-ADP

ribose polymerase (PARP, 116 kDa), a DNA repair

enzyme as its substrate [37]. As a result a cleaved product

(85 kDa) of PARP was observed in our studies. When

caspase-3 was activated by apoptotic stimuli, ICAD got

cleaved, resulting in the release of CAD. This released

CAD appears to cause DNA fragmentation in nuclei [36].

TPD induced the PARP cleavage and inhibited the over

expression of ICAD in HL-60 cells and this effect again

was substantially protected by the NAC and sMIT. So these

finding suggest that TPD caused early NO and ROS pro-

duction are responsible for the induction of apoptosis

through both intrinsic and extrinsic apoptotic pathway.

Moreover TPD appeared to accelerate apoptosis more

efficiently through intrinsic pathway than the extrinsic one

because factors related to the intrinsic pathway were rela-

tively, strongly affected by sMIT and NAC. Though we

have analyzed the expression of various pro- and anti-

apoptotic proteins altered by TPD, these studies neverthe-

less provide important information about the pro apoptotic

nature of TDP prospecting this candidate for developing

into a potential anti-cancer therapeutic.

Conclusion

In conclusion, TPD, a novel and natural triterpenediol from

Boswellia serrata was found to induce early endogenous

NO and peroxide formation in leukemia HL-60 cells. The

oxidative stress produced by TPD, because of massive

ROS and NO generation, caused activation of the TNF

family proteins (TNF-R1, DR4), leads to caspase-8 acti-

vation and induces apoptosis through extrinsic pathway.

Secondly, TPD caused disruption of mitochondrial mem-

brane potential, rendered Bcl-2 cleavage, Bax transloca-

tion, decrease Bcl-2/Bax ratio, release of AIF, Smac/

DIABLO and cytochrome c from the mitochondria. These

events are accompanied by down regulation of survivin and

activation of caspases -3, 8, -9, which cleave the ICAD and

PARP and finally induce apoptosis through intrinsic path-

way. It was so interesting that TPD did not exert any

cytotoxic effect on the normal cells as confirmed by the

DNA fragmentation and cell cycle analysis in human

PBMC. These results provide the basis for further in-depth

drug targeted studies, while the pro-apoptotic feature of

triterpenediol raises the potential use of TPD as an anti-

tumor agent.

Acknowledgements We are highly thankful to Dr. Sarang Bani and

Sheikh Fayaz (SRF, CSIR) for their help in the use of Flow

Cytometery.

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