MICROSCOPY RESEARCH AND TECHNIQUE 75:586–595 (2012)

Azadirachta indica Exhibits Chemopreventive Action Against

Hepatic Cancer: Studies on Associated Histopathological
and Ultrastructural Changes
SANJAY BHARATI,1 PRAVEEN RISHI,2 AND ASHWANI KOUL1*
1
Department of Biophysics, Basic Medical Sciences Block, Panjab University, Chandigarh 160014, India
2
Department of Microbiology, Panjab University, Chandigarh 160014, India

KEY WORDS hepatocellular carcinoma; transmission electron microscopy; scanning electron

microscopy
ABSTRACT The present study was designed to evaluate the anticarcinogenic potential of Aza-
dirachta indica against N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis. Further,
the associated histopathological and ultrastructural changes were also analyzed. Hepatic cancer
model was developed by the intraperitoneal administration of NDEA to mice at weekly intervals,
in successive increasing doses, for a period of 8 weeks. Aqueous A. indica leaf extract (AAILE)
was administered orally at a dosage of 100 lg/g body weight thrice a week till termination of the
study. A relationship between histopathological grading and chemopreventive effect of A. indica
had been established at various stages of carcinogenesis. Anticancer activity of A. indica was eval-
uated in terms of tumor incidence, tumor multiplicity, and survival rate. A significant reduction in
tumor incidence (33%), tumor multiplicity (42%), and increase in survival (34%) was observed
upon administration of AAILE to NDEA-abused mice. Transmission and scanning electron micro-
scopic investigations showed severe alterations in organelle organization, cellular arrangement,
degree of differentiation, cellular metabolism, and morphology of the hepatocytes. These changes
appeared to be distinctly delayed upon AAILE supplementation. The results suggest A. indica
may have anticancer potential against NDEA-induced hepatic cancer. Microsc. Res. Tech. 75:586–
595, 2012. V 2011 Wiley Periodicals, Inc.
C

INTRODUCTION Kuriyama, 1991), thus rendering liver a target organ

Environment and cancer have a very strong relation-
ship. Environmental factors, which trigger carcinogen-
esis, include exposure to certain natural and man-
made substances, lifestyle choices like cigarette smok-
ing, low fruit and vegetable intake, physical inactivity,
unhealthy sexual practice, and exposure to certain
pathogens (Weiderpass, 2010).
N-nitrosamines are well-known group of carcinogens
which are present almost everywhere in soil, water,
air, food and are also known to be generated from the
metabolism of certain therapeutic agents. Based on
sufficient evidence of carcinogenicity in experimental
animals, among series of these compounds, NDEA is
reasonably anticipated to be a human carcinogen
(Tricker and Preussmann, 1991). The general popula-
tion may be exposed to unknown quantities of this com-
pound present in food, beverages, herbicides, pesti-
cides, and industrial pollution. Since NDEA is a light-
sensitive compound, possibility of its direct exposure is
minimal to general population, but its production
within the body by metabolism of certain food products
poses a major threat (Crew, 2010).
NDEA needs biological activation to exert its carcino-
genic action and this activation is provided by carcino-
gen biotransformation enzymes. Human liver ex-
presses CYP 2A6 and CYP2E1, both enzymes primarily
involved in NDEA activation (Kawajiri and Fujii-
for its metabolism and carcinogenicity.
Primary liver cancer is the seventh most common
cancer worldwide, and it is the third leading cause of
cancer deaths (Yang and Roberts, 2010). Despite the
immense efforts to improve the strategy and methodol-
ogy for the treatment of this disease, survival is 14% at
5 years (Jemal et al., 2010). A variety of chemopreven-
tive agents have been identified in epidemiological and
experimental studies that exhibited profound anti-
cancer activity. For example, tamoxifen, raloxifene, as-
pirin, isotertinoin, finasterid have effectively reduced
the cancer incidence, but simultaneously increased the
risk of cardiovascular and hepatic diseases (Vogel,
2010). Therefore, primary protection through modifica-
tions in life style, dietary habits, and of course chemo-
prevention by herbal agents can be the best possible
strategy for combating hepatic cancer.
The presence of a wide variety of phytochemicals in
plant extracts synergize the medicinal effects and
Conflicts of interests: None
*Correspondence to: Ashwani Koul; Department of Biophysics, Panjab Univer-
sity, Chandigarh 160014, India.
E-mail: drashwanikoul@yahoo.co.in, ashwanik@pu.ac.in
Abbreviations: NDEA, N-nitrosodiethylamine; AAILE, Azadirachta indica leaf
extract; H&E, hematoxylin and eosin
Received 27 May 2011; accepted in revised form 23 August 2011
DOI 10.1002/jemt.21095
Published online 14 October 2011 in Wiley Online Library (wileyonlinelibrary.com).

V
C 2011 WILEY PERIODICALS, INC.
 CHEMOPREVENTIVE ACTIVITY OF AZADIRACHTA
antagonize the side effects of the active principles.
Therefore, extracts from plants have emerged as a new
and promising source of anticancer remedies (Koul
et al., 2009; Mehta et al., 2010).
Azadirachta indica A. Juss (neem) is well known in
India since time immemorial as one of the most versa-
tile medicinal plant having a wide spectrum of biologi-
cal activity. Chemopreventive effects of neem leaf
extract against experimental oral and gastric carcino-
genesis have been reported by several investigators
(Atawodi and Atawodi, 2009). Neem leaf glycoprotein
is shown to enhance carcinoembronic antigen presenta-
tion of dendritic cells to T and B cells for induction of
antitumor immunity (Sarkar et al., 2010). We have pre-
viously reported the anticancer activities of A. indica
leaf extract against benzo[a]pyrene-induced gastric
and DMBA-induced skin carcinogenesis (Gangar and
Koul 2007; Koul et al., 2006). Various underlying mech-
anisms have been proposed by various laboratories
including ours showing promises of anticarcinogenic
activity of A. indica (Arora et al., 2011 b; Kumar et al.,
2009).
Therefore, the present study was designed to analyze
the activity of A. indica against NDEA-induced hepato-
carcinogenesis in terms of histological changes at vari-
ous stages of cancer development and ultrastructural
alterations at the termination of the study.
MATERIALS AND METHODS
Chemicals and Reagents
NDEA, glutaraldehyde, and paraformaldehyde were
obtained from Sigma Chemical Co. (St. Louis, MO). All
other chemicals utilized were obtained from local In-
dian firms and were of highest purity grade.
A. indica Leaf Extract. A. indica leaf extract
(AAILE) was prepared according to the method
described by Arora et al. (2011 b). Briefly, fresh A. ind-
ica leaves were obtained from Panjab University cam-
pus. The leaves were rinsed with water, blot dry, and
then macerated to obtain the crude extract. The mix-
ture was freeze dried and stored at 48C for further use.
The standard qualitative analysis screening of vari-
ous phytochemicals present in the dried powder was
carried out for the presence of phytochemicals. Tan-
nins, alkaloids, proteins, steriods/triterpenoids, glyco-
sides, flavonoids, and carbohydrates were evaluated
according to the methods described by Koul et al.
(2009).
Animal Experiments
Male Balb/c mice in the weight range of 25–30 g pro-
cured from Central Animal House, Panjab University,
Chandigarh, India, were housed in polypropylene
cages bedded with sterilized rice husk. Mice in all the
groups had free access to standard animal pellet diet
(Ashirwad Industries, Ropar, Punjab, India) and tap
water throughout the experiment. The temperature of
the animal room was maintained at 21 6 18C, humidity
50–60%, and a 12 h dark and light cycle was main-
tained. All the experimental protocols were approved
by the Institutional Ethics Committee (Panjab Univer-
sity, Chandigarh, India. 1-12/IAEC dated 3-09-2009)
and conducted according to the Indian National Sci-
ence Academy Guidelines for the use and care of exper-
imental animals. Before starting the various treat-
Microscopy Research and Technique
INDICA 587
ments, the animals were allowed to acclimatize to ex-
perimental conditions for 1 week and final day of their
acclimatization period was considered as day 0 of the
experiment.
Mice were randomly divided into four groups (n 5
25–30), control, NDEA, AAILE, and NDEA 1 AAILE.
NDEA was administered intraperitoneally at a weekly
interval for 8 weeks, starting with 10 lg/g body weight
in the first week. Body weight of individual animal was
measured once a week and concentration of NDEA was
thus adjusted in such a way that each animal received
a cumulative dose of 200 mg/kg body weight. AAILE
was administered orally at a dose level of 100 lg/g body
weight thrice a week on alternate days until the termi-
nation of the experiment. NDEA treatment was com-
menced after 2 weeks of pre-treatment with AAILE.
Three mice randomly selected from each group were
sacrificed at 8th, 12th, and 16th week for histopatho-
logical evaluation. At the end of the 16th week, all ani-
mals were sacrificed and liver tissues were removed for
further investigations.
Histopathological Studies
Livers from three randomly selected animals at 8th,
12th, and 16th week from each group were fixed in for-
malin and were processed for hematoxylin and eosin
(H&E) staining using conventional laboratory proce-
dure. Briefly, the tissues were dehydrated through
ascending grades of alcohol, cleared in benzene, and
embedded in low melting point paraffin wax. Sections 5
mm thick were cut and placed serially on clean glass
slides. Three contiguous sections were made from each
tissue and stained with H&E for evaluation under light
microscope (LEICA DM 3000). Similar processing was
also carried out for animals that died post 8 weeks of
NDEA first dose. Tissue samples were graded as
G1. well differentiated
G2. moderately differentiated
G3. poorly differentiated
G4. undifferentiated
Tumor Analysis
All tumors developed post 12 weeks of first dose of
NDEA were morphologically analyzed. Tumors were di-
vided into two categories on the basis of size: Big
tumors ( 3 mm) and small tumors (<3 mm). Hepato-
somatic index was calculated as the ratio of liver
weight to body weight. The chemopreventive response
was assessed on the basis of tumor incidence: percent-
age number of animals having tumors, tumor multi-
plicity: total number of tumors counted/total number of
animals having tumors, and survival rate: expressed
as number of animals survived to the total number of
animals.
Ultrastructural Investigations
Transmission Electron Microscopy. Liver tissues
from NDEA and NDEA 1 AAILE groups were first
fixed for 2 h in 4% glutaraldehyde solution prepared in
200 mmol/L phosphate buffer (pH 7.2) and post fixed in
1% cacodylate buffer (pH 7.2) for 3 h. Ultrathin sec-
tions were obtained using Leica Ultracut (UCT) after
588 S. BHARATI ET AL.

graded dehydration in acetone and embedding in epon sections were then viewed under LEO 435 VP scanning
resin. Sections were then mounted on colloidon–car- electron microscope.
bon-coated grids and examined with Philips CM-10
electron microscope.
Scanning Electron Microscopy. Liver tissues were RESULTS
fixed in 4% glutaraldehyde solution prepared in 200
mmol/L phosphate buffer (pH 7.2) for 2 h. Critical point Various phytochemicals were screened and their
drying was carried to remove any traces of water after results are presented in Table 1.
graded dehydration of tissue in acetone. Sections were After 8 weeks, majority of the animals started devel-
then trimmed, so as to expose the required surface and oping ascites and food intake was significantly reduced
kept inside sputter coater for gold coating. Gold-coated (data not shown); an irregular pattern of body weights
was observed during the whole period of the study (Fig.
1a). Hepatosomatic index was found to be significantly
higher in NDEA group as compared to control, AAILE
TABLE 1. Phytochemical screening of AAILE group, and AAILE 1 NDEA group. No significant
Phytoconstituents and change was observed in the hepatosomatic index of
S. No. qualitative tests AAILE AAILE 1 NDEA group when compared to control and
AAILE groups (Fig. 1b).
1. Tannins 1
2. Alkaloids 2 Control mice had normal and distinct liver lobes,
3. Proteins 1 whereas NDEA group mice exhibited altered gross
4. Steroid/triterpenoids 2 morphology, with nondistinguishable and swollen liver
5. Flavonoids 1 lobes. The livers obtained from AAILE 1 NDEA group
6. Carbohydrates 1
also exhibited abnormal morphology. Lobes of some

Fig. 1. Clinical observations. a: Body weights of mice at weekly analyzed by one-way ANOVA followed by post hoc test. *#P  0.05]; c:
intervals; b: liver weight/body weight (hepatosomatic index) was Survival at weekly intervals; d: Cumulative fraction of animals in G3
found significantly higher in NDEA-treated group than those in con- and G4 stage of histopathological grading by the end of 16 weeks.
trol and AAILE 1 NDEA group [data expressed as mean 6 SD and

Microscopy Research and Technique

 CHEMOPREVENTIVE ACTIVITY OF AZADIRACHTA INDICA 589

Fig. 2. a: 1. Normal animal; 2. Animal showing ascites. Gross morphology of liver (b) normal
(c) AAILE 1 NDEA (d) NDEA mice. Arrows indicate HCC nodules. [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]

TABLE 2. Chemopreventive effects of AAILE on hepatocellular carcinoma (HCC)

Parameter/group Control NDEA AAILE AAILE 1 NDEA
Big tumor ( 3 mm) — 24 None 12
Small tumor (< 3 mm) — 11 None 04
Tumor incidence 0% (0/12) 86% (13/15) 0% (0/12) 53% (8/15)
Tumor multiplicity — 3.4 6 1.14 — 1.45 6 0.60a
a
Statistical significance of difference for tumor multiplicity was analyzed using Students’s t-test (P < 0.05 wrt NDEA-treated group).

animals were not distinct instead appeared to be
shrunken (Figs. 2c and 2d).
Tumor multiplicity, tumor incidence, and survival
rate were taken as an index of chemopreventive
response to tumorigenesis (Table 2). It was observed
that mice from NDEA and AAILE 1 NDEA groups
developed hepatic tumors, whereas none of the mice
from control and AAILE-treated groups developed
tumors. Tumor incidence was observed to be 86% in
NDEA-treated group and 53% in AAILE 1 NDEA-
treated group. This observation clearly demonstrated
that AAILE treatment potentially reduced the tumor
incidence by 33%. AAILE administration to NDEA-
treated animals caused a significant decrease in tumor
multiplicity when compared to the NDEA-treated ani-
mals. The total number of tumors in NDEA group and

Microscopy Research and Technique

590 S. BHARATI ET AL.
AAILE 1 NDEA group were 35 and 16, respectively.
Histopathological investigations revealed that liver
from control animals exhibited normal histoarchitech-
ture, clear cut hepatic lobules, separated by interlobu-
lar septa, and traversed by portal veins. The hepato-
cytes were polyhedral in shape. Central vein and radi-
ally arranged hepatocytes were seen clearly in livers of
control group at all stages of investigation (Fig. 3a).
NDEA treatment to the animals resulted in progres-
sion from well-differentiated HCC characterized by
microtrabecular structure, cell plate thickening of 3–4
cells and irregular sinusoids with vascular invasion at
12 weeks to poorly differentiated HCC at the end of 16
weeks (Figs. 3d and 3f). NDEA 1 AAILE group had
low- to high-grade dysplastic nodules at a period of 12
weeks showing nuclear crowding and irregular
arrangement of nuclear cords (Fig. 3e) with no visible
stromal invasion, which subsequently developed to
well-differentiated HCC by 16 weeks (Fig. 3g).
Histopathological assessment demonstrated that
84% animals in case of NDEA and 50% in case of
NDEA 1 AAILE group had moved to poorly differenti-
ated stage by 16 weeks.
Transmission electron microscopy revealed smooth
and regular nuclear membrane of hepatocytes of con-
trol group. The cytoplasm was granular with numerous
RER around nuclear membrane and mitochondria,
which were scattered in the cytoplasm along with
smooth endoplasmic reticulum (Figs. 4a and 4b). How-
ever, in NDEA-treated mice, nuclear membrane was
irregular, bent, and indented. The nucleolus appeared
to be irregular, huge, or ring shaped. The cytoplasm
was rarefied and had few RER and scanty mitochon-
dria. Glycogen stores of the cell had been depleted and
SER were prominent (Figs. 4c and 4d). NDEA 1
AAILE-treated animals showed rounded to oval regu-
lar nuclear membrane with prominent nucleoli.
Increase in the number of free ribosomes, glycogen
granules, and mitochondria were also observed with
respect to NDEA-treated animals (Figs. 4e and 4f).
Scanning electron microscopic study of control ani-
mals showed liver to be comprised of polyhedral hepa-
tocytes that formed 1–2 cell thick hepatocellular cords
(Fig. 5a). Hepatocytes were loosely attached to each
other and along the margins of the bile canaliculi (Fig.
5a, arrow).
Treatment of NDEA led to various cell surface
changes with decrease in overall cell size. Cells lost
their normal polyhedral structure and appeared to be
rounded with decreased intercellular contacts. A multi-
ple cell plate thick liver cell cords were also observed
with loss of normal radiating pattern (Fig. 5b). Hepato-
cytes observed in NDEA 1 AAILE mice were of vari-
able shapes and sizes. Normal radiating pattern was
lost. Numerous apoptotic cells were seen (Fig. 5c).
DISCUSSION
Epidemiological studies indicate a causal relation-
ship between exposure to nitrosamines and cancer in
humans. The general population is exposed to both ex-
ogenously and endogenously formed nitrosamines. In
association with other contributing factors, i.e., genetic
susceptibility to nitroso carcinogens, intake of other
modifying chemicals, infection of certain viruses and
micronutrient deficiency, the intake levels of precur-
sors that are considered to be normal is sufficient to
cause cancer (Bartsch and Montesano, 1984). For all
these reasons, prevention of induction of cancer is con-
sidered to be of prime importance.
In the present study, A. indica has been indicated as
an effective chemopreventive agent against NDEA-
induced hepatocarcinogenesis. The development of as-
cites after 8 weeks of NDEA administration might
have contributed to the irregular pattern of body
weight changes observed in both NDEA and NDEA 1
AAILE groups. The increase in liver to body weight,
i.e., hepatosomatic index in NDEA group could be cor-
related to the other morphological and histological
observations indicating increase in size of liver lobes,
hyperproliferation of cells, and tumor development.
Delay in the development and progression of tumor
from well-differentiated HCC to poorly/undifferenti-
ated HCC as assessed by histological investigations at
8, 12, and 16 weeks of study demonstrated the protec-
tive role of A. indica against NDEA. Tumor size along
with histological grading is considered to be a very im-
portant prognostic parameter. Large-sized tumors
along with advanced histological grading decrease the
overall survival (Kojiro, 2005; Tamura et al., 2001).
The decreased percentage of animals in G3/G4 stage at
16 weeks further indicated that A. indica might have
played an important role in the process of tumor induc-
tion and progression. These findings along with tumor
incidence, tumor multiplicity, and survival rate demon-
strated chemopreventive potential of A. indica against
hepatocarcinogenesis.
Ultrastructural investigations using TEM revealed
that hepatocytes of mice treated with NDEA exhibited
various abnormalities. The nucleus had irregular,
bent, and indented nuclear membrane. This observa-
tion could be correlated to the changes in the cytoskele-
ton of cancerous cells (Guido et al., 2010). Damage
induced by free radicals generated in the process of me-
tabolism of NDEA might have resulted in changes in
cytoskeleton of cells leading to change in the overall
structure. Moreover, malignant cells required more
surface area for the exchange of materials, indentation
of membrane might have provided the extra area
needed for nuclear cytoplasmic exchange of substances.
The nucleoli of normal cells is generally regular, round,
and solid (Poojari et al., 2010; Zhizhi et al., 1999) but
nucleoli of NDEA-treated group exhibited various
irregular shapes, indicating disturbance in protein me-
tabolism. It was also observed that NDEA treatment to
mice resulted in depletion of glycogen, decrease in the
number of RER and mitochondria, and increase in
SER. Transformed cells rely on glycolysis for their
energy production, which might be due to disturbance
in mitochondrial respiration or unavailability of oxy-
gen. As SER is involved in glycogenolysis, its increase
is expected in transformed cells (Ismail et al., 2009).
However, presence of large number of mitochondria in
NDEA 1 AAILE-treated mice hepatocytes suggested
that substantial fraction of energy production is aero-
bic in nature. Amount and structure of organelle can
indicate the differentiation and metabolism function of
tumor cells. A correlation exists for number of organ-
elle present in a cell and its degree of differentiation,
amount of organelle has the tendency to decrease when
differentiation declines (O’Conor et al., 1972). NDEA
Microscopy Research and Technique
 CHEMOPREVENTIVE ACTIVITY OF AZADIRACHTA INDICA 591

Fig. 3. Histopathological assessment at different weeks. a: control
mice liver; b: NDEA mice at 8 weeks, high-grade dysplastic nodule
with distortion in liver architecture (arrow); c: AAILE 1 NDEA at 8
weeks, presence of differential staining cells (arrow); d: 12 weeks
NDEA mice, well-differentiated HCC characterized by microtrabecu-
lar pattern, pseudoglandular arrangement, and vascular invasion; e:
AAILE 1 NDEA mice liver at 12 weeks representing high-grade dys-
plasia with loss of normal architecture but no vascular invasion; f: un-
differentiated and poorly differentiated cells (arrow) in NDEA group
at 16 weeks; g: AAILE 1 NDEA at 16 weeks indicating well-differen-
tiated microtrabecular HCC. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]

Microscopy Research and Technique

592 S. BHARATI ET AL.
Fig. 4. Transmission electron micrograph of mice liver. a–b: Nor-
mal hepatocyte showing nucleus (N) with densely and lightly stained
chromatin, nucleolus (Nu), and cytoplasmic organelle {mitochondria
(M), rough endoplasmic reticulum (R), smooth endoplasmic reticulum
(S), and glycogen particles (Gly)}; c–d: NDEA mice with irregular-
shaped nucleus (N), lightly stained chromatin, large nucleolus (Nu)
treatment to mice resulted in overall rarefaction of
cytoplasm, indicating decreased level of differentiation
in these cells. However, AAILE supplementation to
NDEA-challenged mice had decreased the rarefaction
having different shapes, rarefied cytoplasm lesser mitochondria (M),
and rough endoplasmic reticulum (R); e–f: NDEA 1 AAILE mice
showing normal rounded to oval appearing nucleus (N), large number
of mitochondria (M), few rough endoplasmic reticulum (R), and
smooth endoplasmic reticulum (S).
of cytoplasm indicating comparatively higher degree of
differentiation. Moreover, the lower the degree of dif-
ferentiation, the higher the degree of HCC malignancy
(Ho et al., 2003). The nuclear–cytoplasmic exchange
Microscopy Research and Technique
 CHEMOPREVENTIVE ACTIVITY OF AZADIRACHTA
Fig. 5. Scanning electron micrograph after 16 weeks of NDEA
treatment. a: Normal liver composed of polyhedral hepatocytes
arranged in one cell thick plates with bile canaliculi along the midline
(Bc). Sinusoids (S) are lined by endothelial cells and perforated by
many small fenestrations. b: NDEA, SEM, hepatocytes lost their poly-
could be considered normal in these hepatocytes as
they had round regular nuclear membrane with nor-
mal appearing nucleoli. Poorly differentiated tumor
cells have higher nuclear to cytoplasm (N/C) ratio. Sim-
ilar results were obtained in the present study. N/C ra-
tio of mice treated with NDEA was higher than normal
and AAILE 1 NDEA-treated group.
This observation was further supported by scanning
electron microscopy for surface morphological evalua-
tion. Hepatocytes in normal adult mammalian liver are
arranged predominantly in single cell thick plate in
which anastomosing bile canaliculi are enclosed (Holz
et al., 2010; Ogawa et al., 1979). In the present study,
decrease in the cell size, rounding of cells along with
increase in cell cord thickness and distortion of liver
cell plates might represent high proliferative rate and
carcinogenic transformation of the cells. Such observa-
Microscopy Research and Technique
INDICA 593
hedral structure and appeared to be more rounded and smaller in
size. Note cells in upper half are smaller (arrow) than in lower half,
liver plates are multiple cell thick. c: AAILE 1 NDEA, SEM, irregular
to rounded hepatocytes with apoptotic bodies (arrow).
tions regarding the morphological characteristics of
the tumor cells have been reported from our laboratory
earlier (Gangar and Koul, 2008). AAILE pre-protection
thus decreased the extent of degenerative changes
observed after NDEA administration.
Some of the major phytochemicals present in various
parts of A. indica are nimbin, nimbolide, nimbidin,
quercetin, azadirachtin, gallic acid, catechin, gedunin,
etc. Other constituents include proteins, polysaccha-
rides, sulfurous compounds, polyphenolics such as fla-
vonoids, tannins, etc. The qualitative analysis of the
A. indica leaf extract used by us indicated the presence
of flavonoids, tannins, carbohydrates, and polysaccha-
rides. These phytochemicals have been documented to
retard carcinogenesis at initiation as well as promotion
stages of carcinogenesis by virtue of their radical scav-
enging properties (Makita et al., 1996; Rice-Evans
594 S. BHARATI ET AL.
et al., 1996). Recently, Kumar et al. (2011) has reported
that a carbohydrate, glucosamine, which is the active
component of neem leaf is responsible for its immunos-
timulatory activity. Two water soluble polysaccharides
GIa and GIb demonstrated antitumor effect with com-
plete regression of tumor in mice after subcutaneous
inoculation of sarcoma-180 cells. Other polysaccha-
rides, GIIa and GIIIa, are known to be effective anti-
inflammatory components in neem (Biswas et al.,
2002). Triterpenoids like azadirachtin, nimbolide, and
salannin have been documented as inhibitors of cell
proliferation and inducers of apoptosis in cancerous
cells. Nimbolide, a limonoid present in various parts of
A. indica, exhibited cytotoxic effects on leukemic cell
lines (Roy et al., 2007).
Reports from our laboratory and several others have
indicated that AAILE modulates initiation phase of
murine forestomach tumorigenesis by altering the ac-
tivity of carcinogen biotransformation enzymes in liver
and forestomach (Gangar and Koul, 2007). Gangar and
Koul (2008) in their study on murine forestomach tu-
morigenesis have reported that intervention with A.
indica during the tumorigenesis process mediated the
induction of apoptosis in the forestomach tumors.
Recent investigations carried out in our laboratory
have revealed that intervention of skin carcinogenesis
using AAILE causes an inhibition in skin tumor inci-
dence and mean tumor burden (Arora et al., 2011 b).
This was accompanied by alterations in the expression
of proteins (bax, bcl-2, caspase 3, and caspase 9) that
form the apoptotic apparatus of the cell. Expression of
transcription factors like NF-kappa B, STAT 1, and AP-
1 which regulate the transcription of genes involved in
cell proliferation, survival, differentiation, apoptosis,
etc., were also modulated in skin tumors of mice treated
with AAILE (Arora et al., 2011 a). Manikandan et al.
(2008) have demonstrated anticancer and antioxidant
activities in various fractions of A. indica leaf extract.
The ethanolic extract of A. indica modulated apoptosis,
proliferation, and differentiation during hamster buc-
cal pouch carcinogenesis (Subapriya et al., 2005, 2006).
Together with the reports from literature and obser-
vations of the present study, it may be suggested that
A. indica has anticancer activity against NDEA-
induced hepatic tumorigenesis in mice.
ACKNOWLEDGMENTS
The financial assistance for carrying out the present
research work provided by University Grants Commis-
sion, New Delhi, India, is gratefully acknowledged.
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