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Nutrition and Cancer
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Combinatorial Antitumor Effect of Naringenin and
Curcumin Elicit Angioinhibitory Activities In Vivo
Kushi Anand a , Aditi Sarkar a , Anup Kumar a , Rashmi K. Ambasta a & Pravir Kumar a b
a Functional Genomics and Cancer Biology Laboratory, Center for Medical Engineering,
Vellore Institute of Technology, Vellore, Tamil Nadu, India
b Tufts University School of Medicine, Boston, Massachusetts, USA
Available online: 29 May 2012
To cite this article: Kushi Anand, Aditi Sarkar, Anup Kumar, Rashmi K. Ambasta & Pravir Kumar (2012): Combinatorial
Antitumor Effect of Naringenin and Curcumin Elicit Angioinhibitory Activities In Vivo, Nutrition and Cancer,
DOI:10.1080/01635581.2012.686648
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Nutrition and Cancer, 1–11, 2012
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Taylor & Francis Group, LLC
ISSN: 0163-5581 print / 1532-7914 online
DOI: 10.1080/01635581.2012.686648
Combinatorial Antitumor Effect of Naringenin and
Curcumin Elicit Angioinhibitory Activities In Vivo
Kushi Anand, Aditi Sarkar, Anup Kumar, and Rashmi K. Ambasta
Functional Genomics and Cancer Biology Laboratory, Center for Medical Engineering,
Vellore Institute of Technology, Vellore, Tamil Nadu, India
Pravir Kumar
Functional Genomics and Cancer Biology Laboratory, Center for Medical Engineering, Vellore Institute
of Technology, Vellore, Tamil Nadu, India, and Tufts University School of Medicine, Boston,
Massachusetts, USA
Curcumin has long been used as an antioxidative, antiinflamma-
tory, and modulator of pathological angiogenesis, whereas narin-
genin is a well-known immunomodulator. In this report, we in-
vestigated the effect of curcumin and naringenin on the growth
of Ehrlich ascites carcinoma tumor model. To achieve this, Swiss
albino mice were implanted intraperitoneally with 1 ×106Ehrlich
ascites carcinoma cells followed by the administration of oral doses
of naringenin and curcumin either individually (50 mg/kg body
weight) or in combination (20 mg/kg body weight each). A marked
reduction has been seen in the total number of cells (80%) and
accumulation of ascetic fluid (55%) when these drugs were ad-
ministered together. These drugs proved to be an effective angio-
inhibitory compound and confirmed by different in vivo assay
systems, viz. peritoneal/skin angiogenesis and chorioallantoic
membrane assay. Antiangiogenic and antiproliferative effect of
these compounds alone or in combination was further corrobo-
rated with immunoblot results where we confirmed the downregu-
lation of vascular endothelial growth factor, Hif1α, heat shock pro-
tein 90, and p-Akt. Furthermore, treatment with naringenin and
curcumin alone or in combination substantially improved hepato-
cellular architecture and no noticeable neoplastic lesions or cellular
alteration were reported. These outcomes put forward a plausi-
ble clinical application of these diet-derived compounds, as both
angioinhibitory and antitumor in association with conventional
therapy.
INTRODUCTION
The seminal work by Folkman et al. in 1971 hypothesized
that the development of tumor is angiogenesis dependent (1).
Submitted 24 November 2011; accepted in final form 16 April 2012.
Address correspondence to Pravir Kumar, Ph.D., Assistant Direc-
tor, Centre for Medical Engineering, 206-Center for Biomedical Re-
search, SBST, VIT University Vellore, India. Phone: +91416-2202513;
+919003386752. E-mail: pravirkumar@vit.ac.in or pravir.kumar@
tufts.edu
This physiological process can be compared with embryonic
development, wound healing, and arteriosclerosis (2,3). Angio-
genesis plays a major role in tumor progression that has drawn
the attention of researchers for antiangiogenic mediated cancer
therapy that have generally low toxicity (2). The neovascular-
ization process is regulated by various growth factors such as
vascular endothelial growth factor (VEGF), interleukin 8 (IL8),
transforming growth factor β(TGF β), and basic fibroblast-
like growth factor (3,4). Therefore, it is necessary to screen
suitable anticancerous agents that have nontoxic, multitarget-
ing properties with high efficacy in cancer treatment. There are
many reports advocating the usage of naturally occurring herbal
medicines and plant products such as flavanoids and polyphe-
nolic compounds effective in reducing the risk of cancer (5,6).
There are several reports suggesting the role of alkaloids in
antitumor activities with a minimal negative side effect. Cur-
cumin (diferuloyl methane) has been used due to its versatile
effect on tumorigenesis, peritoneal antiangiogenesis, apoptosis,
and signal transduction pathway (7–12), whereas naringenin
(5,7,4-trihydroxyflavanone; NGEN) is a flavonoid abundantly
present in citrus fruits and tomato (13). Both curcumin (CUR)
and NGEN have antiproliferative, antimutagenic, and anticar-
cinogenic properties (8,14,15). Several mechanisms have been
proposed to understand the action of CUR that inhibits the pro-
duction of reactive oxygen species (ROS), Bcl-2, Bcl-xl, NF-kB
c-Jun N-terminal kinase, cyclin-D, and COX-2 (8,16–21). Cur-
cumin is known for the induction of apoptosis in cancerous cells
(9,10,19,21,22) and the inhibition of neoplastic transformation
via downregulation of AP-1 and/or NF-kB transcriptional ac-
tivation and COX-2 suppression (17). On the contrary, little is
known about the antiproliferative and anticancerous properties
of NGEN.
In the last decades, several reports have strengthened the im-
portance of antiangiogenesis and apoptosis-induced destruction
of preneoplastic or cancerous cells where CUR and NGEN play
1
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2K. ANAND ET AL.
an important role in apoptotic induction in cancer of colon, liver,
and breast and in leukemia (9,11,12,15,17,19,23,24). Mounting
evidence highlights the involvement of combined drug action
to treat the cancer. The combined action of polyphenols and
sulforaphane of green tea have inhibitory effect in human colon
carcinoma cells development (25). Several studies proved the
combined action of isoflavone, CUR, and other biomolecules to
treat the cancer more effectively than the higher doses of one
drug alone (18,26,23,28).
In continuation with the above studies, molecular chaper-
ones are also playing a major role in cancer progression. For
instance, heat shock protein 90 (Hsp90) expression is higher
in tumors than in the normal tissues (29), and it is critically
involved in maintaining the stability, integrity, and function of
key oncogenic proteins that are responsible for angiogenesis
and apoptosis (28–31). In spite of the rigorous efforts made in
cancer research using in vitro assays system, there is a lack of
an effective and reproducible in vivo model system to evaluate
new therapeutic approaches in preclinical settings. Therefore,
screening and evaluation of different drugs on an in vivo tumor
model is essential to understand the dynamic process of human
cancer development and identification of suitable therapeutic
targets. In clinical manifestation, the formation of ascites is
often observed in patients with an advanced stage of cancer, es-
pecially in ovarian cancer. Once ascites fluids start accumulating
in the intraperitoneal cavity, diagnosis becomes more challeng-
ing, and in that situation, abdominal paracentesis is performed
to relieve the patient’s symptoms. Contrary to this clinical in-
tervention, reaccumulation of ascites fluid is quite rapid, where
a sufficient amount of antitumor drugs cannot be administered
because of the deteriorated health conditions of the patient (32).
Thus, it is necessary to develop cancer model systems to better
understand the etiology and complications related to the disease
and, consequently, the discovery of therapeutic molecules to
rescue or minimize the progression of cancer.
In the present study, we determined the antiangiogenic ef-
fect of various drugs and checked the antiangiogenic capacity
of CUR and NGEN alone and in combination using chorioal-
lantoic membrane (CAM) assay and in vivo Ehrlich ascites
carcinoma (EAC) model. We observed that NGEN and CUR
potentiate the antitumor effects in in vivo conditions of EAC
animal model as well as in cells. We further observed a sig-
nificant downregulation of proliferation and angiogenic mark-
ers such as VEGF, p-AKT, and Hif1αwhen cells were treated
with these drugs compared to untreated groups. Moreover, these
drugs have shown the antimetastatic property in hepatocytes.
MATERIALS AND METHODS
Reagents, Chemicals, Antibodies, and Drugs
Curcumin (CUR), NGEN, apigenin (API), DMSO, and other
chemicals were purchased from Sigma and Merck (highest grade
and purity) (Bangalore, India). Anti-p-Akt and anti-Akt antibod-
ies were purchased from Cell Signaling (Danvers, MA), whereas
anti-Hif1α, anti-VEGF, anti-Hsp90, and anti-βactin antibodies
were purchased from Santa Cruz Biotech (Santa Cruz, CA).
For secondary antibody, HRP conjugated antirabbit and anti-
mouse polyclonal immunoglobulin was purchased from DAKO
(Glostrup, Denmark).
Preparation of Drug Solution
The biomolecules that have been used in this study were
dissolved at a concentration of 2 mg/mL in cell culture grade
DMSO for CAM assay. For oral drug administration in mice,
CUR and NGEN were dissolved in cell culture grade DMSO
(0.01%). Required dilutions were made in PBS as per the assay.
Chicken CAM Assay
Chick In Ovo CAM Assay
Angiogenic activity was assayed using the chick embryo
CAM assay as described previously (7) with slight modification.
Fertilized eggs were incubated at 37◦C in a humidified and
sterile atmosphere for 3 days (eggs were rotated 5–6 times a
day). On the 4th day, a thin groove of approximately 1.5 ×
1.5 cm was made on the surface of the egg and sealed using
sterile surgical tape. The eggs were then further incubated until
the 6th day, and after that, a window was made to check for
the normal development of the embryo. Eggs possessing 80%
similarities in their vasculature were selected for the experiment.
Biomolecules (NGEN, CUR, API, and NGEN plus CUR)
were applied onto the CAM at the concentration of 5 µg/CAM
using a sterile filter disk. Apart from negative (solvent only)
and positive controls (retinoic acid, 1 µg/CAM), a sham control
(without solvent or drug) was also included in this experiment.
The CAM was inspected after 24 h for changes in the density of
blood vessels and photographed under a dissecting microscope
attached with a digital camera. For each drug, 10–30 eggs were
used.
Chick Ex Ovo CAM Assay
After incubation (until 72 h, as mentioned), the eggs were
cracked, and contents were transferred to sterile Petri plates
as per standard protocol (33). Only embryos that showed no
bleeding or deformities were selected on the 5th day. A sterile
filter disc was placed over the CAM, and test materials were
applied. The ex ovo cultures were kept in the incubator at 37◦C
with 60–65% humidity. On the 7th day, the vascular pattern was
observed as mentioned.
Animals and Maintenance of the EAC Cell Line:
Ethics Statement
The study and the number of animals used were ap-
proved by the Institutional Animal Ethics Committee (VIT/
SBST/IAEC/IIIrd/09). All experiments were strictly performed
as per the guidance set up by the institutional animal ethical
committee.
Swiss albino mice (female; 6–8 wk; 20–25 g) were accli-
matized to an experimental room having controlled standard
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ANTIANGIOGENIC ACTION OF NARINGENIN AND CURCUMIN IN CARCINOMA MODEL 3
environmental conditions. A set of animals (1 group consists of
8 mice) was housed in a sterile polypropylene cage with a ster-
ile paddy husk. Autoclaved standard pellet food and water ad
libitum was provided under an extreme hygienic environment.
EAC tumor cells were maintained in female Swiss albino mice
through serial intraperitoneal inoculation in an ascitic form at
an interval of 9–10 days as mentioned previously (26).
Antitumor Activity (In Vivo): Grouping and Treatment
For the in vivo model, exponentially grown EAC cells were
aspirated aseptically with the help of a 18-gauge needle. The
cells were washed and centrifuged (1,000 rpm; 10 min; 4◦C).
Erythrocytes, if any, were removed using RBC lysis buffer, and
after centrifugation, the cells were suspended in PBS. Suspen-
sion was incubated in Petri plates for 1 h to remove macrophage
(34,35). The cells that were free from blood and other contam-
inants were used for further study after checking the viability
using trypan blue. Viability less than 85% was not considered
for the study. Cells were counted under the microscope using
a Neubauer hemocytometer, and 1 ×106/mouse EAC cells in
0.2 mL of PBS were injected intraperitoneally (i.p.). Mice were
divided into 5 groups (n=8 each): 1) Group A: non-tumor-
bearing normal mice; 2) Group B: untreated tumor-bearing con-
trol mice; 3) Group C: NGEN-treated (50 mg/kg body weight)
tumor-bearing mice; 4) Group D: CUR-treated (50 mg/kg body
weight) tumor-bearing mice; and 5) Group E: NGEN (20 mg/kg
body weight) plus CUR-treated (20 mg/kg body weight) tumor-
bearing mice.
All the drugs were directly administered into the oral cavity
either alone or in combinations, 24 h of posttumor implantation.
The control mice were given same volume of solvent used to
dissolve the drugs. The above doses of the drugs were used for
13 sequential days. On day 15, all the mice were sacrificed.
Oral Drug Administration to the Animals
All drugs were administered using the appropriate size
(18–20 gauge) feeding tube according to the prescribed pro-
tocol by institutional animal care and institutional animal ethics
committee. In brief, the skin over the shoulder of the mouse
was grasped, and the gavage tube was placed in the diastema of
the mouth after measuring the length of the feeding tube from
the tip of the nose to the last rib. The tube was then gently and
smoothly advanced along the upper palate until the esophagus
was reached. After proper placement was verified, the drugs
were administered by a syringe attached to the end of the tube.
After dosing, the tube was gently removed following the same
angle as insertion. Following drug administration, animals were
returned to the cage and monitored for 5–10 min, looking for
any sign of labored breathing or distress.
Peritoneal and Skin Angiogenesis
All the animals were sacrificed on day 15, and a small in-
cision was made in the abdominal region. The outer skin was
removed very carefully without damaging the lining of the peri-
toneal cavity. Further, the lining of the peritoneal cavity and the
inner lining of the skin were examined for peritoneal/skin an-
giogenesis in both control and treated groups and photographed.
Histopathology of Liver Tissue
Liver slices were taken from each lobe of the liver. Af-
ter proper fixation with 10% neutral formalin and dehydration
with graded ethanol solutions, 3-µm sections were stained with
hematoxylin and eosin (H&E).
Total Number of Cells and Volume of Ascites Fluid
in Peritoneal Cavity
Prior to the removal of the EAC and the measurement of
cell volume, animals were sacrificed by cervical dislocation
under ketamine-xylazine anesthesia according to regulations
prescribed by the Animal Ethical Committee and Committee
for Control and Supervision of Experiments on Animals, New
Delhi. Here, 1 mL of PBS was injected into the peritoneal cavity,
and ascites fluid was harvested using an 18-gauge needle into a
tube containing 2 ml PBS. The volume of fluid was calculated
after subtracting the volume of PBS added previously from the
volume of the supernatant. The total number of cells per unit
ascites fluid was calculated using a hemocytometer.
Percentage Increase in Weight
The weight of each animal was observed on days 0, 12, and 15
after inoculation of tumors. The percentage increase in weight
was calculated using the formula:
% increase in weight ={(animal’s weight on respective
days/animal’s weight on day 0) −1}
×100
Calculation of Longevity Upon Drug Administration
Healthy Swiss albino mice were divided into 4 groups (A–D),
each group consisting of 6 animals for survival study—Group A:
untreated tumor bearing control mice; Group B: NGEN-treated
(50 mg/kg body weight) tumor-bearing mice; Group C: CUR-
treated (50 mg/kg body weight) tumor-bearing mice; and Group
D: NGEN (20 mg/kg body weight) plus CUR (20 mg/kg body
weight) treated tumor-bearing mice.
The mortality of the animals in each group due to tumor
burden was noted every day, and the percentage of increase in
lifespan (%ILS) was calculated using the formula:
%ILS =T−C/C×100,
where T represents the number of survival days of treated an-
imals and C represents the number of survival days of control
animals as described elsewhere (27).
Western Blot Analysis
EAC cells were harvested from control and drug-treated
mice. Cells were centrifuged and washed with 1×cold PBS
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4K. ANAND ET AL.
along with PMSF and protease cocktails after elimination of
RBC and macrophage. Proteins (30 µg) from Triton X-100 sol-
uble fractions were denatured in 2×Laemmli sample buffer at
95◦C for 20 min. Proteins were separated on 10% SDS-PAGE
and then transferred onto a PVDF membrane (GE Healthcare,
Piscataway, NJ). Membranes were blocked in 5% (w/v) nonfat
dry milk. The membrane was incubated for 3 h at room tem-
perature with primary antibody (1:500–1,000 dilution) followed
by 3 times washing in 1×TBST buffer. After washing, mem-
branes were incubated in goat antimouse IgG or goat antirabbit
IgG HRP-conjugated secondary antibody (1:2,000 dilution) for
1 h. The autoradiography signals were visualized using ECL
advance Western blotting detection kit (RPN2135) on an X-ray
film (GE Healthcare, Piscataway, NJ).
Densitometric, Angiogenesis and Statistical Analysis
For the in vivo model, 8 animals were considered for statisti-
cal analysis. Quantification of angiogenesis (in 6 different fields)
was done using AngioQuant. Relative antiangiogenic percent-
age was calculated considering sham and DMSO control as 0%
antiangiogenic for CAM assay and peritoneal/skin angiogene-
sis, respectively. All data were expressed as mean ±SEM. The
significance was calculated using 1-way ANOVA with Bon-
feroni test and Student’s t-test using GraphPad Prism software
(InStat Software Package, GraphPad, La Jolla, CA). P<0.05
was considered statistically significant. The error bar indicates
SEM.
RESULTS
Naringenin and Curcumin Elicit Antiangiogenic
Properties on CAM In Ovo and Ex Ovo Assay
For assessment of antiangiogenic activity of NGEN and
CUR, we used the CAM assay paradigm. This assay is propor-
tionately easier to perform and has extensive application. Fur-
thermore, it is a widely used model for screening antiangiogenic
properties in ovo. Upon drug administration, a considerable re-
duction in new blood vessel formation has been observed in Fig.
1A (d, e, and g); however, an intensified inhibitory activity has
been observed when drugs are administered in combination of
NGEN and CUR in Fig. 1A (f). At the same time, in the case
of API shown in Fig. 1A (c), no significant inhibition of blood
vessels has been observed. Among all the drugs used in CAM
assay (in ovo), NGEN showed the best inhibition compared to
sham and positive control in ex ovo assay [Fig. 1B (d)]. How-
ever, in combination, these drugs almost completely restricted
the neovascularization [Fig. 1B (e)]. Based on the above infer-
ence and previous works, we have taken NGEN and CUR as a
potential drug for further studies.
Angioinhibitory Effect of Naringenin and Curcumin
in the Peritoneal Cavity and Inner Skin Linings
of EAC Mice Model
To further establish the role of NGEN and CUR in angio-
genesis, we developed an EAC-bearing mice model followed
by NGEN and CUR drug administration alone and in combina-
tion. The vascularization pattern in the peritoneal cavity lining
is an established in vivo method to check angiogenesis (23). In
EAC-bearing mice, increased peritoneal angiogenesis was con-
sistently seen in Fig. 1C (b and g) when compared to the extent of
the peritoneal and skin vasculature in non-tumor-bearing mice
(a and f). In the peritoneal and inner skin linings, CUR [Fig. 1C
(c and h)] and NGEN in [Fig. 1C (d and i)] treated mice showed
a marked reduction in new blood vessel formation, whereas the
action of these drugs in combination [Fig. 1C (e and j)] resulted
in a further reduction of blood vessel formation.
Effect of CUR and NGEN on Histopathological Changes
in the Liver of EAC Mice
H&E stained sections of liver slices revealed extensive hep-
atocellular lesions. There are numerous phenotypically altered
and scattered hepatocyte populations found with hepatocellular
and neoplastic focal lesions in the livers of the vehicle-treated
EAC bearing mice [Fig. 1D (b)], whereas no such alterations
were visualized in sham control (normal mice) [Fig. 1D (a)]. In
the CUR [Fig. 1D (c)] and NGEN [Fig. 1D (d)] treated group,
moderate neoplastic focal lesions were observed. In contrast,
the cellular architecture of hepatic lobules seemed to be like
the normal liver in NGEN plus CUR treated group [Fig 1D (e
compared with a)].
Curcumin and NGEN Administration Attenuated the
Expression of Several Pro- and Antiangiogenic Markers
Chronic hypoxia, a hallmark of many tumors, is associ-
ated with angiogenesis, tumor progression, metastasis/invasion,
metabolic adaptation, and resistance to apoptosis. Expression of
many HIF1αtarget genes, such as VEGF, is induced by hypoxia
in most cancer cell types. To examine the effect of drugs on Hif
1αand angiogenic marker expression level in solid tumors,
immunoblots were performed (Fig. 2E). Membranes were incu-
bated with anti-Hif 1αand anti-VEGF antibodies, while beta-
actin served as an internal control. Immunoblot results showed
that there was marked upregulation of Hif1αand VEGF in the
untreated EAC mice (Fig. 2E, lanes 7 and 8), whereas upon
NGEN and CUR administration, these levels were significantly
reduced (Fig. 2E, lanes 1–4). However, the combination of these
drugs further downregulated the expression of the above marker,
but not to a great extent (Fig. 2E, lanes 5 and 6).
Reduction in Total Number of Cells and Body Fluid
Volume Upon Administration of NGEN and CUR
in EAC Mice Model
The effects of NGEN and CUR were observed on the total
number of EAC cells in the peritoneal cavity of treated and un-
treated mice. Harvested EAC cells from the peritoneal cavity
were counted using the trypan blue method. The total num-
ber of cells and the number of cells per mL of ascites fluid
were significantly decreased in drug-treated mice compared to
its nontreated counterpart (Fig. 3A and 3B). We observed that
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ANTIANGIOGENIC ACTION OF NARINGENIN AND CURCUMIN IN CARCINOMA MODEL 5
FIG. 1. Angioinhibitory activity and hepatocellular profile. A: In ovo chorioallantoic membrane assay (CAM) assay: Different drugs were applied on CAM of
6-day-old chick embryo. After 24 h, vascular density was inspected and (a) sham control, (b) DMSO as vehicle or negative control, (c) apigenin (API; 5 µg/CAM),
(d) curcumin (CUR; 5 µg/CAM), (e) naringenin (NGEN) (5 µg/CAM), (f) NGEN and CUR (1.25 µg/CAM each), and (g) RA (1 µg/egg) as a positive control
were applied on the chorioallantoic membrane. B: Ex ovo CAM assay: 3-day-old fertilized egg content transfer to petri plates and cultured. Drug(s) were applied
on the 5th day. (a) Sham control, (b) DMSO as negative control, (c) CUR (5 µg/CAM), (d) NGEN (5µg/CAM), and (e) NGEN (1.25 µg/CAM) plus CUR
(1.25 µg/CAM) demonstrated maximum inhibition. C: Peritoneal/skin angiogenesis: (a, f) peritoneal/skin lining from normal mice that neither bear the tumor cells
nor treated with any drug (b, g) peritoneal/skin lining of tumor-bearing mice treated with vehicle. Angioinhibitory effect evident in (c, h) CUR, (d, i) NGEN, and
(e, j) NGEN plus CUR-treated tumor-bearing mice. The combination of CUR and NGEN significantly altered angiogenesis. D: H&E section of liver from mice
showing hepatocellular profile (a) hepatocellular architecture of normal mice (Group A), (b) aberrant hepatocellular phenotype of tumor-bearing mice (Group B,
with white arrows indicating the prominent basophilic focal lesions and the presence of neoplastic focal lesions (c and d) intraperitoneal inoculation of viable EAC
cells, followed by NGEN and CUR treatment (groups C and D, respectively). Moderate/less neoplastic focal lesions were seen (white arrow), (e), whereas almost
normal hepatocellular architecture has been observed in NGEN plus CUR-treated tumor-bearing mice.
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6K. ANAND ET AL.
FIG. 2. Angioinhibitory statistical analysis and immunoblot: Images were analyzed in AngioQuant on the basis of microvessel diameter, length, and branching.
Relative antiangiogenic percentage was calculated considering sham control as 0% antiangiogenic for chorioallantoic membrane assay (CAM) assay (AandB)and
solvent control as 100% angiogenic for peritoneal/skin angiogenesis (C and D). Each column indicates mean ±SEM. E and F: Curcumin (CUR) and naringenin
(NGEN) inhibit hypoxia-inducing factor 1α(Hif1α), vascular endothelial growth factor (VEGF), pAkt, and heat shock protein 90 (Hsp90) level. Equal amounts of
each protein (30 µg) were separated on 10% SDS-PAGE followed by blotting, performed and incubated with different antibodies for Hif1α, VEGF, Akt (Ser473),
and Hsp90, whereas β-actin served as a loading control. Combination of curcumin and naringenin significantly altered the protein involved in angiogenesis and
cell proliferation. Densitometric analysis of (G) Hif1α, VEGF, Akt (Ser473), and (H) Hsp90 was shown with respect to fold increase/β-actin (relative density).
Results are expressed as mean ±SEM.
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ANTIANGIOGENIC ACTION OF NARINGENIN AND CURCUMIN IN CARCINOMA MODEL 7
FIG. 3. Determination of cell numbers, ascites fluid volume, and measurement of body weight upon drug administration on tumor-bearing mice model. Ascites
volume, total number of Ehrlich ascites carcinoma (EAC) cells, and body weight were recoded per mice. The effect of drugs on (A) total number of EAC cells, (B)
number of cells per ml of ascites fluid, and (C) volume of ascites fluid was significantly different from corresponding values of untreated group. D: Drug-treated
mice have lesser weight than untreated and normal group of mice due to less tumor burden and less volume of ascetic fluid. E: Percentage increase in body weight
as compared to day 0 of respective group. Results are expressed as mean ±SEM (n=8). CUR =curcumin; NGEN =naringenin.
NGEN along with CUR significantly reduced the total num-
ber of cells and also lessened the tumor burden compared to
when given alone. Furthermore, we investigated the volume of
ascites fluid in treated vs. control mice. Once EAC cells were
injected intraperitoneally into the mice, ascites fluids accumu-
lated in the peritoneal cavity. We observed a significant decrease
in the volume of ascites in mice treated with NGEN (5.313 ±
0.1757 mL) and CUR (6.075 ±0.1436 mL) compared to ascites
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8K. ANAND ET AL.
volume in tumor-bearing mice without treatment (8.438 ±
0.3600 mL). The volume of ascites formed further decreased
when NGEN and CUR treatments were given in combination
(3.738 ±0.0980), indicating the combinatorial effect of these 2
drugs even at low doses compared to groups C and D (Fig. 3C).
Confirmation of Increased Cell Numbers and Body
Volume by Weight Measurement
The results obtained from the above experiments were further
validated by the body weight measurement of an individual
mouse in a group. We observed that there were no differences in
the average body weight of control mice (Group A) on days 0,
12, and 15. On day 15, there was a significant increase in average
body weight of untreated EAC-bearing mice (32.31 ±0.51 g)
with respect to NGEN (28.31 ±0.60 g), CUR (28.69 ±0.36 g),
and NGEN plus CUR (26.63 ±0.33 g) treated mice (Fig. 3D).
However, the body weight of Group B showed significant weight
gains compared to day 0. They gained a maximum weight of
42.23 ±2.84% by day 15. Whereas, EAC-bearing mice with
drugs (Group C, D, and E) gained a maximum weight of 25.17 ±
1.26%, 28.35 ±1.32%, and 17.09 ±0.81%, respectively, by day
15, when compared to the day of tumor inoculation (Fig. 3E).
These data suggest that vehicles have no effect on EAC-bearing
mice, and the increment in body weight is due to the progression
of tumors and the accumulation of fluid. At the same time, the
EAC-bearing mice that have been treated with different drugs
have been attenuating the effect on cancer progression and thus
reduction in the body weight and ascitic fluid.
Effect of NGEN and CUR on Akt
To understand the mechanism behind drug-mediated reduc-
tion in cell numbers, we checked the expression of proliferation
markers. The immunoblot of Akt (Fig. 2E) exhibited a decreased
level of pAkt in NGEN- (lanes 1 and 2 compared with lanes 7
and 8 of Fig. 2E) and CUR-treated mice (lanes 3 and 4 compared
with lanes 7 and 8 of Fig. 2E), although there was further down-
regulation of pAkt when NGEN and CUR were administered in
combination (lanes 5 and 6 compared with lanes 1–4 and lanes
7 and 8, of Fig. 2E). To interpret these results, we found NGEN
and CUR alone decreased the pAkt levels. However, in com-
bination, they further downregulated the expression compared
to vehicle control. Further, we also analyzed Akt, which was
almost the same in all lanes.
Expression of Hsp90 in EAC Cell Upon Drug Treatment
Hsp90 plays an important role in cell proliferation, angiogen-
esis, and maintaining the stability of Hif1α. Hence, we checked
the expression of Hsp90 under physiological (EAC inoculated)
and pharmacological stress (drug-treated) conditions. Compar-
ative expression analysis of Hsp90 revealed that NGEN and
CUR alone downregulated the Hsp90 level significantly (lanes
1–4, Fig. 2F) with respect to the control sample (lanes 7–8, Fig.
2F). However, it was further downregulated when mice were
treated with both NGEN and CUR (lanes 6 and 7, Fig. 2F)
when compared to NGEN alone (lanes 1 and 2, Fig. 2F) and
not to a great extent when compared to the CUR-treated group
(lanes 3 and 4, Fig. 2F). From these observations, we may con-
clude that in combination, even at lower drug doses, CUR and
NGEN (20 mg/kg body weight NGEN and CUR each) lower
the expression of Hsp90.
DISCUSSION
The present study is an attempt to investigate the natural
properties of CUR and NGEN for antiangiogenic, anticancer-
ous, and antiproliferative properties that lead to the cessation of
tumor development. Initially, we have tested these compounds
separately and in combination using in ovo and ex ovo CAM
assay to check the role of the above biomolecules where both
compounds elicit a remarkable reduction in the sprouting of
new microvessels. To replicate these results, we generated an
ascitic tumor model followed by the drug administration. It has
been proposed by several studies that the combinatorial action of
isoflavanoids, CUR, and other biomolecules treat cancer more
effectively than higher doses of one drug alone (23,25,26). The
effect of CUR in EAC has been demonstrated by Gururaj et al.
and later by Belakavadi and Salimath in CAM and peritoneal
angiogenesis assays (7,10). However, NGEN also has a role in
the reduction of various angiogenic factors, such as VEGF in
cancer cell lines (37). Here, we have postulated for the first time
in vivo studies for the collective action of NGEN and CUR in
angiogenesis, cancerous cell growth, and expression of Hsp90 in
EAC cells isolated from mice. We also showed a marked reduc-
tion in new blood vessel formation in the peritoneal and inner
skin linings due to the combined action compared to untreated
cells. Although our results are based on one type of tumor cell,
we can conclude a plausible mechanism of combined drug ac-
tion to treat patients suffering from other types of cancer as well.
For further validation of our results, we used another strategy
to prove the action of combined drug mediated antiangiogenic
theory using ex ovo and in ovo CAM assay. Inhibition of EAC
cells with the reduction in cell numbers and ascitic fluid volume
in vivo validates earlier findings that CUR (7,10) and NGEN
(38) alone are antineoplastic agents (in vivo and in vitro). It has
also been reported that tumor cells secrete a vascular perme-
ability factor that promotes accumulation of ascitic fluid (39)
and may cause metastasis. Previous studies as well demonstrate
antimetastatic properties of NGEN and CUR (17,40–42). The
presence of neoplastic focal lesions reveals the infiltration of
neoplastic cells in the livers of the control group, which was
further reduced in drug-treated groups. However, confirmation
of antimetastatic properties of these compounds needs further
clarification, as it is not possible to examine all mechanisms in
a single study.
In continuation, we also checked the expression of pro- and
angiogenic markers. Hypoxia is a well-known event in several
types of cancer (43), and it is the strongest stimulus for ex-
pression of VEGF, which is mediated primarily by Hif1α,a
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ANTIANGIOGENIC ACTION OF NARINGENIN AND CURCUMIN IN CARCINOMA MODEL 9
master regulator of angiogenesis (45). Higher expression of
VEGF has been considered to be essential for neovasculariza-
tion, tumor cell growth, and proliferation (39). It binds to VEGF
receptor-2 on the endothelial cells and phosphorylate that will,
in turn, activate the signaling pathways of Akt. These cascades
are closely associated with cell growth, proliferation, and sur-
vival (44). In our study, we have shown a significant reduction
in cell proliferation due to the downregulation of these pro-
teins upon drug administration. We also postulated that there
was no significant difference in the body weight of the control
Swiss albino mice treated with CUR and NGEN alone or in
combination (data not shown). These results indicate that drugs
per se do not have any adverse effect on the growth response
and body weight in normal mice. Previous studies confirmed
that NGEN significantly induces apoptosis in human leukemia
THP-1 cells (15) and also acts as an antitumor agent without
having cancer-promoting properties in Lewis lung carcinoma
and the 4T1 mammary cancer cell-induced mice model (40).
In addition, there are several reports indicating the role of var-
ious HSPs in cancer progression, inhibition, angiogenesis, and
apoptosis (9–12). In this report, we have shown that CUR and
NGEN together inhibit the Hsp90 expression that plays a crit-
ical role in the stabilization of several client proteins impli-
cated in the control of cell growth and malignant transformation
(29–31,46,47). Inhibiting Hsp90 elicits antiangiogenic proper-
ties (9,10) and boosts antiproliferative and proapoptotic activity
(29,31) by affecting the PI-3K/Akt/eNOS/Raf-1/surviving sig-
nal transduction pathway, as well as through downregulation
of VEGF receptor-2 expression, a crucial component of the
angiogenic process. Blocking of Hsp90 inhibits the secretion
and expression of various proangiogenic growth factors and
cytokines that lead to an indirect antiangiogenic effect (30).
Our finding that the Hsp90 level was inhibited by NGEN and
CUR, which was further downregulated in case of combined
treatment, suggest that Hsp90 plays a crucial role in angio-
genesis and hyperplasia as depicted in a hypothetical model
(Fig. 4). In addition, we observed a striking feature in the
longevity of animals treated with drugs in combination (CUR +
NEGN) than alone (Table 1). The mean survival days were sig-
nificantly increased by treatment with CUR and NGEN alone
or in combination. In the case of the simultaneous drug treat-
ment, the increase in the lifespan was 87.5% when compared
FIG. 4. A hypothetical mechanism of combined drug mediated rescue of can-
cerous growth in Ehrlich ascites carcinoma (EAC) cells. Hypothetical model
demonstrating progression of cancer upon EAC implantation in peritoneal cav-
ity resulting into pronounced angiogenesis, increased metastasis, and tumor
growth. On contrary, combinatorial effect of naringenin and curcumin restrict
the angiogenesis, and tumor progression. Regression of cancer may be due to
downregulation of important protein responsible for angiogenesis and prolifer-
ation. Hsp90 =heat shock protein 90; VEGF =vascular endothelial growth
factor; Hif1α=hypoxia-induced factor 1α.
to that of the control, which had an average lifespan of 16 ±
0.26 days.
In conclusion, this study opens up a promising avenue in can-
cer therapeutics. To date, no studies have reported any toxicity
associated with the use of NGEN and CUR in either animals or
humans. Angiogenesis plays a major role in tumor progression,
which has drawn the attention of researchers to treat cancer pa-
tients with antiangiogenic therapy generally having low toxicity.
Lack of toxicity favors further preclinical evaluation of CUR and
TABLE 1
Effect of curcumin (CUR) and naringenin (NEGN) on life span in Ehrlich ascites carcinoma mice
Group Dose (mg/kg) Mean survival (days) % ILS (increase life span)
Untreated (cancer) 16 ±0.0.26
NGEN treated 50 25.67 ±0.84a60.43
CUR treated 50 25.50 ±0.62a59.4
NGEN +CUR treated 20 +20 30 ±0.6831a, b 87.5
aP<0.001, with respect to untreated control mice. bP<0.01, with respect to CUR- or NGEN-treated mice. All data are expressed as mean
±SE.
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
10 K. ANAND ET AL.
NGEN in a defined chemical carcinogenesis model. Elucidation
of its mechanisms of action at the complicated molecular circuits
will provide a better understanding of the treatment strategy and
beginning of a new chemoprevention program that could have
a broader implication for the well-being of the society. How-
ever, there are several molecular mechanisms that need to be
crosschecked at agonist and antagonist paradigm.
ACKNOWLEDGMENTS
We would like to extend our gratitude for the encouragement
by the top management of VIT University, Vellore, especially
our chancellor Dr. G. Vishwanathan and vice chancellor Prof.
V. Raju. We further confirm that no additional external funding
was received for this study. The authors are thankful to VIT
University Animal Ethical Committee and Drs. R. Kuttan and G.
Kuttan from Amala cancer research center, Thrissure (Kerala),
for providing EAC bearing mice.
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