upregulation of intrinsic apoptotic pathway in nsaids

Upregulation of intrinsic apoptotic pathway

in NSAIDs mediated chemoprevention

of experimental lung carcinogenesis

Shruti Setia, Sankar N. Sanyal

Department of Biophysics, Panjab University, Chandigarh-160014, India

Correspondence: Sankar N. Sanyal, e-mail: sanyalpu @gmail.com

Abstract:

Background: Non-steroidal anti-inflammatory drugs (NSAIDs) act by inhibition of cyclooxygenase-2 (COX-2), which is overex-

pressed in cancer. The role of COX-2 and apoptosis were evaluated in 9,10-dimethylbenz(a)anthracene (DMBA)-induced lung can-

cer in rat and chemoprevention with indomethacin, a traditional NSAID and etoricoxib, a selective COX-2 inhibitor.

Methods: The animals were divided into Control, DMBA, DMBA + indomethacin and DMBA + etoricoxib groups. They received

a single intratracheal instillation of DMBA while NSAIDs were given orally daily for 32 weeks. Besides morphology and histology

of lungs, RT-PCR, western blots and immunohistochemistry were performed for the expression of apoptotic proteins and COX en-

zymes. Apoptosis was studied by DNA fragmentation and fluorescent staining.

Results: The occurrence of tumors and lesions was noted in the DMBAanimals, besides constricted alveolar spaces and hyperplasia.

COX-1 was found to be uniformly expressed while COX-2 level was raised significantly in DMBA group. The apoptotic proteins,

apaf-1, caspase-9 and caspase-3 were highly diminished in DMBA group but restored to normal level in NSAIDs groups. Also,

apoptosis was suppressed in carcinogen group by DNA fragmentation analysis and fluorescent staining of the lung cells while co-

administration of NSAIDs along with DMBA led to the restoration of apoptosis.

Conclusion: DMBA administration to the rats led to tumorigenesis in the lungs, had no effects on COX-1 expression, while elevat-

ing the COX-2 levels and suppressing apoptosis. The treatment with NSAIDs led to the amelioration of these effects. However, etori-

coxib which is a COX-2 specific inhibitor, was found to be more effective than the traditional NSAID, indomethacin.

Key words:

COX, DMBA, etoricoxib, indomethacin, mitochondrial apoptotic pathway

Abbreviations: ANOVA – analysis of variance, Apaf-1 –

apoptotic protease-activating factor-1, BAL – bronchoalveolar

lavage, BCIP/NBT – 5-bromo-4-chloro-3-indolyl phosphate;

nitroblue tetrazolium, BSA – bovine serum albumin, caspase –

cysteine-dependent aspartate-directed protease, CMC – car-

boxymethyl cellulose, COX – cyclooxygenase, DMBA –

9,10-dimethylbenz(a)anthracene, EDTA – ethylenediaminete-

traacetic acid, JC-1 – 5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-

benzimidazolylcarbocyanine iodide, NSAIDs – non-steroidal

anti-inflammatory drugs, PAH – polycyclic aromatic hydrocar-

bon, PBS – phosphate buffer saline, DYM – mitochondrial

membrane potential

Introduction

Non-steroidal anti-inflammatory drugs (NSAIDs)

have been found to have potential chemotherapeutic

efficiency against carcinogenesis [35] by the inhibi-

tion of cyclooxygenase (COX) enzyme [1, 8] which

catalyzes the production of prostaglandins from ara-

chidonic acid and therefore plays a remarkable role in

Pharmacological Reports, 2012, 64, 615�624 615

Pharmacological Reports2012, 64, 615�624ISSN 1734-1140

Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences

inflammation and cancer [14, 28]. NSAIDs are cate-

gorized into traditional non-specific NSAIDs, which

inhibit both the isoforms of COX, COX-1 (constitu-

tively expressed) and COX-2 (inducible early re-

sponse gene product), while coxibs can selectively in-

hibit COX-2. A traditional NSAID (indomethacin)

and a recently developed coxib (etoricoxib) have been

co-administered with DMBA presently to explore

their role in lung tumorigenesis. DMBA has been

found to be a promising PAH (polyaromatic hydrocar-

bon) causing lung tumorigenesis in rodents [17, 30].

The present model is also important in that the two

NSAIDs have been pre-administered daily for 16

weeks followed by a single intratracheal deposition of

DMBA and once again followed by the post admini-

stration of the NSAIDs daily for another 16 weeks pe-

riod, although the earlier studies from our laboratory

using diclofenac, a preferential COX-2 inhibitor, stud-

ied only the post-treatment of NSAIDs in DMBA-

induced cancer at various time points [32, 33].

Dysregulation of apoptosis (programmed cell

death) is an important step in the development of can-

cer as it disrupts the equilibrium between cell growth

and cell death [5, 18]. Apoptosis is characterized by

changes in cell morphology including blebbing, chro-

matin condensation, chromosomal DNA fragmenta-

tion, loss of membrane asymmetry and eventually cell

death [4, 13]. NSAIDs have also been shown to exert

proapoptotic effects on cancer cells, apart from the in-

hibition of cyclooxygenase enzyme [26]. This led to

the investigation of therapeutic activation of apoptosis

in cancer cells as a potential anticancer strategy [7,

22]. In the present paper, bronchoalveolar lavage

(BAL) was performed to study apoptosis and altera-

tions in mitochondrial membrane potential along with

the study of the intrinsic apoptotic pathway in differ-

ent treatment groups to establish the role of NSAIDs

in the induction of apoptosis in DMBA-induced car-

cinogenesis.

Materials and Methods

Chemicals

DMBA and Bradford reagent were purchased from

Sigma-Aldrich (St. Louis, MO, USA). Etoricoxib and

indomethacin were received as a generous gift from

Ranbaxy Pharmaceuticals Ltd. (Gurgaon, India). Pri-

mary antibody against COX-1, COX-2, apaf-1,

caspase-3, caspase-9 and anti-mouse b-actin were

purchased from Santa Cruz Biotechnology Inc., CA

(USA). Alkaline phosphatase-conjugated secondary

antibodies and BCIP-NBT were purchased from Genei

(Bangalore, India). All other chemicals and reagents

used in the present study were procured from the In-

dian manufacturers and were of analytical grade.

Animals

Female Sprague-Dawley rats of body weight 100–120 g

were procured from the Central Animal House, Pan-

jab University, Chandigarh. Animals were maintained

as per principles and guidelines of the Ethics Commit-

tee on the Use of Experimental Animals of Panjab

University. They were housed in polypropylene cages

with a wire mesh top and a husk bed with a maximum

of 4 animals in each cage. The animal rooms were

maintained at ambient temperature. Rats received

food and water ad libitum and were exposed to 12 h

day/night photoperiod. They were acclimatized for

1 week following their grouping. The animals were

randomly assorted into four groups: Control, DMBA,

DMBA + indomethacin and DMBA + etoricoxib. The

Control group received vehicle for NSAIDs and

DMBA. The DMBA group received intratracheal in-

tubation of 20 mg/kg b.w. DMBA [25]. The doses

chosen for indomethacin (2 mg/kg b.w.) [27] and eto-

ricoxib (0.6 mg/kg b.w.) [24] were within the thera-

peutic anti-inflammatory limits. The study design is

given in Figure 1.

Lung tumor analysis

An excess of diethyl ether anesthesia was given to sac-

rifice the animals. The lungs were perfused with

chilled phosphate buffer saline (PBS) to remove blood

from them and the five lobes were separated. They

were closely examined for the presence of any tumor

nodule or lesion with the help of a hand-held lens.

Histology

Two different lobes of the lungs were taken and fixed

in 10% neutral buffered formalin. These were fixed in

paraffin wax according to the standard technique [20],

and five micron thick sections were cut using a hand

driven microtome and transferred to egg albumin

616 Pharmacological Reports, 2012, 64, 615�624

coated slides. Sections were then dewaxed in xylene,

stained in hematoxylin and eosin, mounted in DPX,

and viewed under a light microscope (40×) and photo-

graphed (Axioscope A1, Zeiss, Germany).

Immunohistochemistry, immunofluorescence

and western immunoblots

COX-1 and COX-2 were localized in lung paraffin

sections by immunohistochemistry while apaf-1,

caspase-9 and caspase-3 were studied by immuno-

fluorescence. Protein extracted from 10% lung ho-

mogenate was used to study the expression of the pre-

viously stated five proteins by immunoblottng. These

methods have been described in detail in earlier publi-

cations from this laboratory [12, 32, 33, 36].

Isolation of bronchoalveolar lavage

The lungs were perfused with 5 ml of PBS (9% w/v,

pH 7.4) to isolate bronchoalveolar lavage (BAL)

which was then centrifuged at 1000 rpm for 10 min at

4°C. The supernatant was discarded and the pellet

was resuspended in 500 µl of PBS buffer [19]. Neu-

bayer’s hemocytometer was used to calculate the total

number of cells as well as the fraction of neutrophils.

Differential cell count was also done using Wright

Giemsa stain.

Fluorescent staining

Ten microliters of isolated cells were mixed with

10 µl of fluorescent dye, coverslip was put on the

slide and it was observed under fluorescent micro-

scope (40×) (Axioscope A1, Carl Zeiss, Germany).

Live and apoptotic cells were counted (300 cells from

4 different slides) to calculate the percentage of apop-

totic cells from each group. The fluorescent staining

dyes used included the following:

Hoechst-33342/propidium iodide: Hoechst-33342

is a DNA intercalating dye and binds at A-T base

pairs, hence can be used to stain DNA [37]. Viable

cells appeared dark blue, necrotic cells were pink

while the apoptotic cells fluoresced blue.

JC-1: JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraeth-

ylbenzimidazolylcarbocyanine iodide) is a fluorescent

cationic dye, used to signal the loss of mitochondrial

membrane potential ( DYM) [2].

DNA fragmentation

DNA was isolated from the lung tissue and fragmenta-

tion was studied as described earlier [32, 33]. Quantita-

tion of isolated DNA was done using a spectropho-

tometer at 260 nm. DNA fragmentation was visualized

by electrophoresis on 1.5% agarose gel containing 5 µg

of ethidium bromide with the aid of a Gel Doc ma-

chine (Upland, CA, USA).


Chemoprevention of lung cancer by NSAIDsShruti Setia and Sankar N. Sanyal

Fig. 1. Experimental design to studythe effect of pre- and post-treatment ofNSAIDs on DMBA induced lung car-cinogenesis in rats for a period of 32weeks

Statistical analysis

Analysis of variance (one way ANOVA) test was per-

formed using the statistical software package ‘SPSS

v. 11 for Windows’. The post-hoc comparisons of the

means from different groups were made by the

method of least significant difference. Results corre-

sponding to a p value of 0.05 or less were considered

statistically significant.

Results

Morphological and histopathological studies

The lung surface of the different groups was observed

by a hand held lens. Morphologically, tumors were

observed in about 16 weeks after the administration of

carcinogen in the DMBA group (Fig. 2). No such fea-


Fig. 2. Gross morphology of isolated lungs from the different groups, examined after 32 weeks of treatment schedule. Arrows show the pres-ence of tumors in DMBA treated group and the encircled lesion is also visible. All the other groups show normal lung surfaces and the absenceof any malignancy

Tab. 1. Tumor/lesion burden, multiplicity and incidence after 16 weeks of pre-and post-treatment of NSAIDs in DMBA induced lung carcino-genesis in rat (n = 5)

Groups Tumorincidence

Tumormultiplicity

Tumorburden

Lesionincidence

Lesionmultiplicity

Lesionburden

Control 0 0 0 0 0 0

DMBA 100% 8.7 8.7 100% 5.6 5.6

DMBA + indomethacin 0 0 0 20% 0.2 1

DMBA + etoricoxib 0 0 0 0 0 0

Incidence: percentage of animals bearing tumor or lesion. Multiplicity: total number of tumors or lesions/number of tumor or lesion bearing rats.Burden: total number of tumors or lesions / total number of rats



Fig. 3. Photomicrographs (40´) showing the histopathological changes in the rat lungs in different treatment groups as observed by H/E stain-ing. Highly constricted alveolar spaces mark the histology of DMBA group with hyperplasia and dysplasia as compared to wide spaces in thecontrol group. In NSAIDs treatment groups, the alveolar spaces are increased although still constricted while the extent of hyperplasia is re-duced as compared to DMBA

Fig. 4. (a) Immunohitochemical localization (40´) of COX-1 (circles) and COX-2 (arrows) in the lung tissues. BCIP/NBT showed blue coloredexpression while the counterstain with methyl green stained the nuclei green. (b) Western blot for COX-1 and COX-2 (c) and its densitometricanalysis from each group (*** p < 0.001 vs. control group, ** p < 0.01 vs. control group, * p < 0.05 vs. control group, +++ p 0 <.001 vs. DMBAgroup)


Fig. 5. (a) Photomicrographs (40´) showing the immunofluorescent localization of Apaf-1, caspase-9 and caspase-3 in paraffin embeddedsections. The expression is visible as green color of FITC labelled secondary antibody (arrows) while counterstain with Hoechst-33342 givesblue color to the nuclei. (b)Histogram representing the percentage of cells which show the expression of apaf-1, caspase-9 and caspase-3, re-spectively (*** p < 0.001 vs. control group, ** p < 0.01 vs. control group, +++ p < 0.001 vs. DMBA group). (c) Western blot of these proteins and(d) its densitometric analysis from each group (*** p < 0.001 vs. control group, ** p < 0.01 vs. control group, +++ p < 0.001 vs. DMBA group,++ p < 0.01 vs. DMBA group, + p < 0.05 vs. DMBA group)

tures were seen in the other groups. Tumor and lesion

parameters such as the incidence, multiplicity and

burden are given in Table 1. There was observed

a significant reduction in tumor/lesion incidence with

indomethacin and etoricoxib in the DMBA treated

rats. Histological analysis (Fig. 3) showed large al-

veolar spaces in the control group, while the DMBA

group was marked by the absence of such structure.

Cellular proliferation in the form of hyperplasia and

dysplasia were prominent in these animals. The treat-

ment with indomethacin and etoricoxib decreased the

extent of hyperplasia, although the alveoli were still

constricted as compared to the control.

Regulation of cyclooxygenase expression

Two isoforms of cyclooxygenase enzyme, COX-1 and

COX-2, were studied using western blots and immu-

nohistochemistry (Fig. 4a–c). The protein expression

of COX-1 was similar in all the groups as seen by

densitometric analysis of the bands of immunoblots.

b-Actin was used in the blot as protein loading con-

trol. Immunohistochemical expression further sup-

ported these results where COX-1 was equally ex-

pressed in all the groups. The COX-2 levels were

upregulated by the administration of DMBA as seen

by immunohistochemistry and protein expression.

The co-administration of NSAIDs, however, lowered

the levels of COX-2 significantly.

Intrinsic apoptotic pathway

The role of three important components of mitochon-

drial apoptotic pathway viz. apaf-1, caspase-9 and

caspase-3 has been explored in the present study. Im-

munofluorescent studies (Fig. 5a) showed that there

was almost no or negligible expression of these pro-

teins in the DMBA group, while clearly visible ex-

pression was seen in the other groups as localized

with FITC-labelled secondary antibody. In the DMBA

group, only the blue color of the counterstained nuclei

was observed. The cells showing the expression of the

proteins of apoptotic pathway were calculated and ex-

pressed as a histogram (Fig. 5b), which also supported

these results. The western blots (Fig. 5c–d) showed

highly diminished levels of these proteins, indicating

suppression of apoptosis with the administration of

carcinogen. The treatment of NSAIDs reversed these

levels towards normal.

Apoptotic studies in isolated BAL cells

BAL cells from the lungs were stained with the fluo-

rescent dyes Hoechst-33342/propidium iodide and

observed under a fluorescent microscope (Fig. 6a–b).

The apoptotic cells fluoresced blue and found to be

significantly diminished in the DMBA group. The

treatment with NSAIDs raised the number of these

apoptotic cells significantly. In this staining, the ne-

crotic cells appeared pink, while the live cells were

dark blue.

Alterations in mitochondrial membrane potential

JC-1, a fluorescent cationic dye, was used to study the

loss of mitochondrial membrane potential in isolated

BAL cells by fluorescent microscopy (Fig. 6c–d).

JC-1 localizes in the mitochondrial membrane of live

cells (at higher DYM) and fluoresce red by forming

J-aggregates. In the cells with lower mitochondrial

potential, the dye is no longer present as J-aggregates,

but in monomeric form, which fluoresces green. The

present results showed high concentrations of

J-aggregates in the DMBA group as compared to the

others where cells with low DYM, present as green

monomers, were more abundant.

DNA fragmentation

DNA fragmentation is a reliable technique to study

apoptosis. The apoptotic cells contain fragments of

DNA as can be seen presently in the DMBA + indo-

methacin and DMBA + etoricoxib groups. In the

DMBA group, suppression of apoptosis is evident by

the absence of fragments (DNA ladder) in this group

(Fig. 6e).

Discussion

Both clinical and experimental studies support the an-

tineoplastic effects of NSAIDs mediated by regula-

tion of COX-2 levels and induction of apoptosis [11].

A daily intake of NSAID (aspirin and ibuprofen) for

1 or 2 years is reported to reduce 60–68% of relative

risk of lung cancer [9, 10]. Chemopreventive role of

preferential COX-2 inhibitors in DMBA induced ex-

perimental lung carcinogenesis have also been re-



ported by us earlier [32, 33]. In these studies lung tu-

mor was induced by direct deposition of a single dose

of DMBA into the lungs by intratracheal intubation

while the animals were subjected to daily oral intake

of the NSAIDs post DMBA administration. The pres-

ent study gains further insight into the molecular

mechanism of apoptosis in the DMBA induced lung

cancer and its chemoprevention by NSAIDs. The

present study also explores the property of the

NSAIDs, where a daily intake of the drugs for 16

weeks before the exposure of carcinogen was found to

be more efficient in reducing the tumor and lesion in-

cidence as compared to a different protocol, where

initial carcinogen exposure is followed by NSAIDs

treatment, as studied earlier in our laboratory [34]. Al-

though tumor incidence in both the studies remains

the same (100%) for the DMBA group, the tumor

multiplicity and burden are decreased significantly

with the pre-treatment with NSAIDs in the present re-

sults. In the previous studies, lesion incidence was

100%, which has been reduced presently to 20% for

indomethacin and zero for etoricoxib (Tab. 1). With

the co-administration of indomethacin along with the

carcinogen, the lesion incidence was reduced from

100% to 20% while no lesions were found in the

DMBA + etoricoxib group. The reduced efficiency of

indomethacin may be attributed to the fact that it is

a non-specific traditional NSAID, while etoricoxib is

a COX-2 selective NSAID.

Apoptosis or programmed cell death, which leads

to cellular destruction, is dysregulated in carcinogene-

sis [6]. Apoptosis is triggered by the release of cyto-

chrome c from mitochondria and its binding with

apaf-1 and ATP, which further binds with pro-


Fig. 6. (a) Fluorescent co-staining of isolated BAL cells as studied by Hoechst-33342/propidium iodide using fluorescent microscope (40´).The fluorescent blue cells are apoptotic; viable cells are dark blue while the pink cells are necrotic. (b) Histogram showing the percentage ofapoptotic cells in each group (*** p < 0.001, * p < 0.05 vs. control group, ++ p < 0.01 vs. DMBA group). (c) JC-1 staining showing the apoptoticcells as green monomers and live cells as green colored J-aggregates. (d) Histogram showing the percent live and apoptotic cells in variousgroups (** p < 0.01 vs. control group, + p < 0.05 vs. DMBA group). (e) Visualization of apoptosis by DNA fragmentation in various groups. Morethe extent of fragmentation more is the apoptosis. 1 � CONTROL, 2 � DMBA, 3 � DMBA + INDO, 4 � DMBA + ETO

caspase-9 to create a protein complex called apopto-

some [3]. The apoptosome activates the pro-caspase

to form caspase-9 [15], which is responsible for the

activation of caspase-3, the ultimate executioner of

apoptosis [23]. The present study explored the role

of apaf-1, caspase-9 and caspase-3 as the targets of

NSAIDs in the DMBA-induced lung cancer. DMBA

induced lung tumorigenesis resulted in a significantly

low expression of these pro-apoptotic proteins and

thereby dysregulated apoptosome formation which

was found to be corrected by the NSAIDs.

One of the confirmatory protocols for the analysis

of apoptosis is DNA fragmentation [32, 33]. It is the

endonucleosomal cleavage of DNA into < 200 bp

fragments which appear as a typical ladder in agarose

gel electrophoresis. More ladder formation reflects

higher extent of apoptosis. Our results show absence

of DNA fragments in the DMBA group whereas

NSAIDs treatment has enhanced the process of apop-

tosis as is visible by ladder formation in these groups.

The BAL cells contain a majority population of

resident as well as migrating macrophages, which

play a critical role in homeostasis, host defense, the

response to foreign substances, and tissue remodeling

[16]. Upon activation, these cells may also begin to

secrete proinflammatory cytokines and chemokines,

and thus lead to the development of inflammation,

which is directly related to the development of cancer

in lungs [31]. This is also accompanied by an increase

in the number of these cells. Cancer development was

found to be inhibited by the co-administration of

NSAIDs with DMBA by the induction of apoptosis in

these cells, which was studied using DNA binding

fluorescent dye, Hoechst-33342/propidium iodide.

The number of apoptotic BAL cells was found to de-

crease drastically in the DMBA treated animals which

was restored significantly by the treatment of the

NSAIDs. Thus, to confirm the role of NSAIDs in the

induction of apoptosis, BAL cells were isolated and

fluorescent staining was performed apart from the

study of mitochondrial apoptotic pathway.

The membrane-permeant JC-1 dye is widely used

in apoptosis studies to monitor mitochondrial poten-

tial. The dye exhibits potential-dependent accumula-

tion in mitochondria, as indicated by a fluorescence

emission shift from green (~529 nm) to red (~590 nm)

for lower and higher mitochondrial membrane poten-

tial, respectively. Consequently, mitochondrial depo-

larization is indicated by a decrease in the red/green

fluorescence intensity ratio. The potential-sensitive

color shift is due to the concentration dependent for-

mation of red fluorescent J-aggregates in the mito-

chondrial membranes of the cells at high DYM. In

case of the apoptotic cells, the dye is present as green

monomers [21]. The present study shows signifi-

cantly increased number of cells at high DYM and re-

duced number of cells with low DYM in the DMBA

group as compared to the Control while the co-

administration of NSAIDs led to the correction of

these effects to a large extent. Hence, it can be in-

ferred that DMBA treatment alters the mitochondrial

potential to a large extent and the available literature

supports the association of alteration of DYM with

apoptosis as DYM has been found to collapse before

the appearance of nuclear apoptotic changes [29].

To conclude, carcinogen administration in the rats

led to tumor formation in the lungs, had no effects on

COX-1 levels, elevated the COX-2 levels and sup-

pressed apoptosis. The treatment with NSAIDs, both

traditional NSAID as well as the coxib led to the ame-

lioration of these effects. The experimental design

also shows that both pre- and post-administration of

the NSAIDs for a length of time leads to a strong che-

moprevention of lung carcinogenesis. NSAID admini-

stration resulted in a significantly increased apoptotic

lung cells in the DMBA treated animals. Anti-

neoplastic effects of the NSAIDs could thus be due to

the induction of apoptosis, which is a dominant end

effect in the killing of cancer cells. Significantly, eto-

ricoxib, which is a COX-2 selective NSAID, was

found to be more effective than the traditional

NSAID, indomethacin.

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Received: September 9, 2011; in the revised form: January 6,2012; accepted: February 2, 2012.


upregulation of intrinsic apoptotic pathway in nsaids

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