ArticlePDF Available

Combinatorial Antitumor Effect of Naringenin and Curcumin Elicit Angioinhibitory Activities In Vivo

Authors:

Abstract and Figures

Curcumin has long been used as an antioxidative, antiinflammatory, and modulator of pathological angiogenesis, whereas naringenin is a well-known immunomodulator. In this report, we investigated 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 × 10(6) Ehrlich 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 administered 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 corroborated with immunoblot results where we confirmed the downregulation of vascular endothelial growth factor, Hif1α, heat shock protein 90, and p-Akt. Furthermore, treatment with naringenin and curcumin alone or in combination substantially improved hepatocellular architecture and no noticeable neoplastic lesions or cellular alteration were reported. These outcomes put forward a plausible clinical application of these diet-derived compounds, as both angioinhibitory and antitumor in association with conventional therapy.
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 μ g/CAM), (d) curcumin (CUR; 5 μ g/CAM), (e) naringenin (NGEN) (5 μ g/CAM), (f) NGEN and CUR (1.25 μ g/CAM each), and (g) RA (1 μ g/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 μ g/CAM), (d) NGEN (5 μ g/CAM), and (e) NGEN (1.25 μ g/CAM) plus CUR (1.25 μ g/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.
… 
Content may be subject to copyright.
This article was downloaded by: [Pravir Kumar]
On: 29 May 2012, At: 20:30
Publisher: Routledge
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK
Nutrition and Cancer
Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/hnuc20
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
To link to this article: http://dx.doi.org/10.1080/01635581.2012.686648
PLEASE SCROLL DOWN FOR ARTICLE
Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions
This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
Nutrition and Cancer, 1–11, 2012
Copyright C
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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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 37C 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 37C
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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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; 4C).
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 =TC/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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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
95C 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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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.
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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.
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
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.
REFERENCES
1. Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med
285, 1182–1186, 1971.
2. Folkman J: Angiogenesis in cancer, vascular, rheumatoid and other disease.
Nat Med 1, 27–31, 1995.
3. Ferrara N and Alitalo K: Clinical applications of angiogenic growth factors
and their inhibitors. Nat Med 5, 1359–1364, 1999.
4. CarmelietP and Jain RK: Angiogenesis in cancer and other diseases. Nature
407, 249–257, 2000.
5. Amin Ruhul ARM, Kucuk O, Khuri FR, and Shin DM: Perspectives for
cancer prevention with natural compounds. J Clin Oncol 27, 2712–2725,
2009.
6. Nobili S, Lippi D, Witort E, Donnini M, Bausi L, et al.: Natural compounds
for cancer treatment and prevention. Pharmacol Res 59, 365–378, 2009.
7. Gururaj AE, Belakavadi M, Venkatesh DA, Marm´
e D, and Salimath BP:
Molecular mechanisms of anti-angiogenic effect of curcumin. Biochem
Biophys Res Commun 297, 934–942, 2002.
8. Kunnumakkara AB, Anand P, and Aggarwal BB: Curcumin inhibits prolif-
eration, invasion, angiogenesis and metastasis of different cancers through
interaction with multiple cell signaling proteins. Cancer Lett 269, 199–225,
2008.
9. Jiang MC, Yang-Yen HF, Yen JJ, and Lin JK: Curcumin induces apoptosis
in immortalized NIH 3T3 and malignant cancer cell lines. Nutr Cancer 26,
111–120, 1996.
10. Belakavadi M and Salimath BP: Mechanism of inhibition of ascites tumor
growth in mice by curcumin is mediated by NF-kB and Caspase activated
DNase. Mol Cell Biochem 273, 57–67, 2005.
11. Simon A, Allais DP, Duroux JL, Basly JP, Fontanier SD, and Delage
C: Inhibitory effect of curcuminoids on MCF-7 cell proliferation and
structure–activity relationships. Cancer Lett 129, 111–116, 1998.
12. Kuo ML, Huang TS, and Lin JK: Curcumin, an antioxidant and anti-tumor
promoter, induces apoptosis in human leukemia cells. Biochim Biophys
Acta 1317, 95–100, 1996.
13. Guerreiro S, Monteiro R, Calhau C, Azeved I, and Soares R: Naringenin
inhibits cell growth and migration in human breast cancer cell lines. FA S E B
21, 848–845, 2007.
14. Anto RJ, George J, Dinesh Babu KV, Rajasekharan KN, and Kuttan R:
Antimutagenic and anticarcinogenic activity of natural and synthetic cur-
cuminoids. Mutat Res 370, 127–131, 1996.
15. Park JH, Jin CY, Lee BK, Kim GY, Choi YH, et al.: Naringenin induces
apoptosis through downregulation of Akt and Caspase-3 activation in hu-
man leukemia THP-1 cells. Food Chem Toxicol 46, 3684–3690, 2008.
16. Balasubramanyam M, Koteswari AA, Kumar RS, Monickaraj SF, Mah-
eswari JU, et al.: Curcumin induced inhibition of cellular reactive oxygen
species generation: novel therapeutic implications. JBiosci28, 715–721,
2003.
17. Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, et al.:
Curcumin suppresses the paclitaxel-induced nuclear factor-κB pathway in
breast cancer cells and inhibits lung metastasis of human breast cancer in
nude mice. Clin Cancer Res 11, 7490–7498, 2005.
18. Wang Z, Desmoulin S, Banerjee S, Kong D, Li, Y, et al.: Synergistic effects
of multiple natural products in pancreatic cancer cells. Life Sci 83, 293–300,
2008.
19. Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S, et al.: Cur-
cumin (diferuloylmethane) downregulates expression of cell proliferation
and antiapoptotic and metastatic gene products through suppression of
Ikappa βalphakinase and Akt activation. Mol Pharmacol 69, 195–206,
2006.
20. Chen YR and Tan TH: Inhibition of the c-Jun N-terminal kinase (JNK)
signaling pathway by curcumin. Oncogene 17, 173–178, 1998.
21. Anto RJ, Mukhopadhyay A, Denning K, and Aggarwal BB: Curcumin
(diferuloylmethane) induces apoptosis through activation of Caspase-8,
BID cleavage and cytochrome c release: its suppression by ectopic ex-
pression of Bcl-2 and Bcl-xl. Carcinogenesis 23, 143–150, 2002.
22. Pal S, Choudhuri T, Chattopadhyay S, Bhattacharya A, Goutam K, et al.:
Mechanisms of curcumin-induced apoptosis of Ehrlich’s ascites carcinoma
cells. Biochem Biophys Res Commun 288, 658–665, 2001.
23. Leonardi T, Vanamala J, Taddeo SS, Davidson LA, Murphy ME, et al.:
Apigenin and naringenin suppress colon carcinogenesis through the aber-
rant crypt stage in azoxymethane treated rats. Exp Biol Med 235, 710–717,
2010.
24. Kanno S, Tomizawa A, Hiura T, Osanai Y, Shouji A, et al.: Inhibitory
effects of naringenin on tumor growth in human cancer cell lines and
sarcoma S-180-implanted mice. Biol Pharm Bull 28, 527–530, 2005.
25. Nair S, Hebbar V, Shen G, Gopalakrishnan, A, Khor TO, et al.: Syn-
ergistic effects of a combination of dietary factors sulforaphane and
(-)epigallocatechin-3-gallate in HT-29 AP-1 human colon carcinoma cells.
Pharm Res 25, 387–399, 2007.
26. El-Azab M, Hishe H, Moustafa Y, El-Sayed E: Anti-angiogenic effect of
resveratrol or curcumin in Ehrlich ascites carcinoma-bearing mice. Eur J
Pharmacol 652, 7–14, 2011.
27. Kuttan R, Bhanumathy P, Nirmala K, and George MC: Possible anticancer
activity of turmeric. Cancer Lett 29, 197–202, 1985.
28. Shen G, Khor TO, Hu R, Yu S, Nair S, et al.: Chemoprevention of familial
adenomatous polyposis by natural dietary compounds sulforaphane and
dibenzoylmethane alone and in combination in ApcMin/+mouse. Cancer
Res 67, 9937–9944, 2007.
29. GiommarelliC, Zuco V, Favini E, Pisano C, Dal-Piaz F, et al.: The enhance-
ment of antiproliferative and proapoptotic activity of HDAC inhibitors by
curcumin is mediated by Hsp90 inhibition. Cell Mol Life Sci 67, 995–1004,
2010.
30. Staufer K and Stoeltzing O: Implication of heat shock protein 90 (HSP90)
in tumor angiogenesis: a molecular target for anti-angiogenic therapy. Curr
Cancer Drug Targets 10, 890–897, 2010.
31. Trepel J, Mollapour M, Giaccone G, and Neckers L: Targeting the dynamic
HSP90 complex in cancer. Nat Rev Cancer 10, 537–549, 2010.
32. Sun J and Liao JK: Induction of angiogenesis by heat shock protein 90
mediated by protein kinase Akt and endothelial nitric oxide synthase. Ar-
terioscler Thromb Vasc Biol 12, 2238–2244, 2004.
33. Yukita A, Asano M, Okamoto T, Mizutani S, and Suzuki H: Suppression of
ascites formation and reaccumulation associated with human ovarian cancer
by an anti-VPF monoclonal antibody in vivo. Anticancer Res 2, 155–160,
2000.
34. Majumder S, Ilayaraja M, Seerapu HR, Sinha S, Siamwala JH, et al.: Chick
embryo partial ischemia model: a new approach to study ischemia ex vivo.
PLoS One 5, e10524, 2010.
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
ANTIANGIOGENIC ACTION OF NARINGENIN AND CURCUMIN IN CARCINOMA MODEL 11
35. Natalia B, Juliana LD, Caio VR, Marcelly VP, Nelson D, et al.: Growth
inhibition and pro-apoptotic activity of violacein in Ehrlich ascites tumor.
Chemico Biological Interactions 186, 43–52, 2010.
36. Chakraborty T, Bhuniya D, Chatterjee M, Rahaman M, Singha D,
et al.: Acanthus ilicifolius plant extract prevents DNA alterations in a
transplantable Ehrlich ascites Carcinoma bearing murine model. Worl d J
Gastroenterol 13, 6538–6548, 2007.
37. Schindler R and Mentlein R: Flavonoids and vitamin E reduce the release
of the angiogenic peptide vascular endothelial growth factor from human
tumor cells. JNutr136, 1477–1482, 2006.
38. Orsoli´
c N and Basi´
c I: Water-soluble derivative of propolis and its
polyphenolic compounds enhance tumoricidal activity of macrophages.
J Ethnopharmacol 102, 37–45, 2005.
39. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, et al.: Tumor
cells secrete a vascular permeability factor that promotes accumulation of
ascites fluid. Science 219, 983–985, 1983.
40. Du G, Jin L, Han X, Song Z, Zhang H, et al.: Naringenin: a potential
immunomodulator for inhibiting lung fibrosis and metastasis. Cancer Res
69, 3205–3212, 2009.
41. Menon LG, Kuttan R, and Kuttan G: Anti-metastatic activity of curcumin
and catechin. Cancer Lett 141, 159–165, 1999.
42. Qin L, Jin L, Lu L, Lu X, Zhang C, et al.: Naringenin reduces lung
metastasis in a breast cancer resection model. Protein Cell 2, 507–516,
2011.
43. Schmalt C, Hardenbergh PH, Wells A, and Fisher DE: Regulation of
proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol
18, 2845–2854, 1998.
44. Kang MS, Gao KP, Morris T, Sharma S, and Steele VE: Comparison
of inhibition of angiogenesis and expression of VEGF and avβ3in
CAM angiogenesis mode (abstr.). Cellular, Molecular, and Tumor Biol.
2: Angiogenesis Inhibitors II. Proc. Am. Assoc. Cancer Res 45, 17,
2004.
45. Katschinski DM, Le L, Heinrich D, Wagner KF, Hofer T, et al.: Heat in-
duction of the unphosphorylated form of hypoxia-inducible factor-1alpha is
dependent on heat shock protein-90 activity. JBiolChem277, 9262–9267,
2002.
46. Isaacs JS, Jung YJ, Mimnaugh EG, Martinez A, Cuttitta F, et al.: Hsp90
regulates a von Hippel-Lindau independent hypoxia-inducible factor-1α-
degradative pathway. JBiolChem277, 29936–29944, 2002.
47. Ciocca DR and Calderwood SK: Heat shock proteins in cancer: diagnostic,
prognostic, predictive, and treatment implications. Cell Stress Chaperones
10, 86–103, 2005.
Downloaded by [Pravir Kumar] at 20:30 29 May 2012
... Moreover, biotin modified-nanoparticles of gefitinib and naringenin showed promising anti-cancer activities via down-regulation of anti-apoptotic proteins like [197] (continued on next page) MMP-9, Bcl-2 plus Bax, and caspases-9 up-regulation in the lung cancer model [203]. Other compounds showing anti-tumorigenic effects with naringenin include curcumin, denatonium, ABT-737 (Bcl-2 inhibitor), 17β-estradiol, myricetin, and copper II complexes ( Table 2) [204][205][206][207][208][209][210]. ...
Article
Naringenin is an important phytochemical which belongs to the flavanone group of polyphenols, and is found mainly in citrus fruits like grapefruits and others such as tomatoes and cherries plus medicinal plants derived food. Available evidence demonstrates that naringenin, as herbal medicine, has important pharmacological properties, including anti-inflammatory, antioxidant, neuroprotective, hepatoprotective, and anti-cancer activities. Collected data from in vitro and in vivo studies shows the inactivation of carcinogens after treatment with pure naringenin, naringenin-loaded nanoparticles, and also naringenin in combination with anticancer agents in various malignancies, such as colon cancer, lung neoplasms, breast cancer, leukemia and lymphoma, pancreatic cancer, prostate tumors, oral squamous cell carcinoma, liver cancer, brain tumors, skin cancer, cervical and ovarian cancer, bladder neoplasms, gastric cancer, and osteosarcoma. Naringenin inhibits cancer progression through multiple mechanisms, like apoptosis induction, cell cycle arrest, angiogenesis hindrance, and modification of various signaling pathways including Wnt/ß-catenin, PI3K/Akt, NF-ĸB, and TGF-β pathways. In this review, we demonstrate that naringenin is a natural product with potential for the treatment of different types of cancer, whether it is used alone, in combination with other agents, or in the form of the naringenin-loaded nanocarrier, after proper technological encapsulation.
... It also arrested the cell cycle by impairing its progression and migration (Krishnakumar et al. 2013b). Naringenin-loaded nanoparticles (NARNPs) showed anticancer properties against DMBA (7,12-dimethylbenz(a)anthracene)-mediated oral carcinogenesis; it entirely stopped the SCC formation and the biochemical status to standard assortment in oral carcinogenesis caused by DMBA (Anand et al. 2012). ...
Article
Full-text available
Though the incidence of several cancers in Western societies is regulated wisely, some cancers such as breast, lung, and colorectal cancer are currently rising in many low- and middle-income countries due to increased risk factors triggered by societal and development problems. Surgery, chemotherapy, hormone, radiation, and targeted therapies are examples of traditional cancer treatment approaches. However, multiple short- and long-term adverse effects may also significantly affect patient prognosis depending on treatment-associated clinical factors. More and more research has been carried out to find new therapeutic agents in natural products, among which the bioactive compounds derived from plants have been increasingly studied. Naringin and naringenin are abundantly found in citrus fruits, such as oranges and grapefruits. A variety of cell signaling pathways mediates their anti-carcinogenic properties. Naringin and naringenin were also documented to overcome multidrug resistance, one of the major challenges to clinical practice due to multiple defense mechanisms in cancer. The effective parameters underlying the anticancer effects of naringenin and naringin include GSK3β inactivation, suppression of the gene and protein activation of NF-kB and COX-2, JAK2/STAT3 downregulation, downregulation of intracellular adhesion molecules-1, upregulation of Notch1 and tyrocite-specific genes, and activation of p38/MAPK and caspase-3. Thus, this review outlines the potential of naringin and naringenin in managing different types of cancers.
... Administration of naringenin in combination with curcumin can impede angiogenesis activity since the combination decreases the production of new blood vessels in the peritoneal and inner skin linings of an Ehrlich Ascites Carcinoma (EAC) tumor-bearing mice model. 117 Naringenin also causes cell death via apoptosis occurring through several mechanisms against skin cancer in humans. Ahamad et al 118 reported that naringenin-induces apoptosis by 1) ROS-mediated mitochondrial membrane depolarization, 2) DNA fragmentation which signifies apoptosis that causes damage to cells, 3) induction of nuclear condensation inside human epidermoid carcinoma A431 cells, and 4) obstruction of cells in G 0 or G 1 phase of cell cycles leading to apoptosis and initiation of caspase-3 that is one of the key roles in apoptosis occurring through cellular substrate splitting. ...
Article
Full-text available
The skin is the largest organ in the human body, composed of the epidermis and the dermis. It provides protection and acts as a barrier against external menaces like allergens, chemicals, systemic toxicity, and infectious organisms. Skin disorders like cancer, dermatitis, psoriasis, wounds, skin aging, acne, and skin infection occur frequently and can impact human life. According to a growing body of evidence, several studies have reported that natural products have the potential for treating skin disorders. Building on this information, this review provides brief information about the action of the most important in vitro and in vivo research on the use of ten selected natural products in inflammatory, neoplastic, and infectious skin disorders and their mechanisms that have been reported to date. The related studies and articles were searched from several databases, including PubMed, Google, Google Scholar, and ScienceDirect. Ten natural products that have been reported widely on skin disorders were reviewed in this study, with most showing anti-inflammatory, antioxidant, anti-microbial, and anti-cancer effects as the main therapeutic actions. Overall, most of the natural products reported in this review can reduce and suppress inflammatory markers, like tumor necrosis factor-alpha (TNF-α), scavenge reactive oxygen species (ROS), induce cancer cell death through apoptosis, and prevent bacteria, fungal, and virus infections indicating their potentials. This review also highlighted the challenges and opportunities of natural products in transdermal/topical delivery systems and their safety considerations for skin disorders. Our findings indicated that natural products might be a low-cost, well-tolerated, and safe treatment for skin diseases. However, a larger number of clinical trials are required to validate these findings. Natural products in combination with modern drugs, as well as the development of novel delivery mechanisms, represent a very promising area for future drug discovery of these natural leads against skin disorders.
... This demonstrates the anti-tumor potential of the MAE in a dose-dependent fashion. Observation of the vascularization pattern in the peritoneal cavity lining is an established in-vivo method to check the angiogenesis (Anand et al. 2012;Bhattacharjee et al. 2017). The photographs of the internal linings of peritoneal epithelium that were cut open on the 14th day of the study showed a significantly subsided neovascularization and inhibited angiogenesis in groups treated with higher doses of MAE and standard drug compared to tumor control (Fig. 2f-j). ...
Article
Ulva fasciataDelile, a marine macroalga is abundantly available on the intertidal rocky surface of Visakhapatnam coasts, India, during summer. The algae belong to family Ulvaceae and division Chlorophyta is glossy green in color. Being a prolific source of bioactive compounds, this thallus allured an unprecedented concern of the author to study its anatomy, histo-chemistry and in-vivo anticancer property. Primarily, the morpho-anatomy and histo-chemistry of the thallus were studied to identify correct species and locate the active phytoconstituents in the cell saps. Besides, the in-vivo anticancer activity of the methanolic algal extract (MAE) was studied on Ehrlich's ascites carcinoma (EAC) xenografted mice. The light microscopy of the thallus divulged very thick cuticularized layers in the sectional view. A fat middle lamella was observed in between two layers of vertically oblong palisade cells. The histo-chemistry revealed the presence of starch grains, calcium oxalate crystals, tannin, protein,and lipids as major cell inclusions. The extract exhibited a profound inhibitory activity on EAC xenografted mice (p˂0.001). Increase in mean survival time (p˂0.001) and restoration of the hematological parameter in drug-treated rodents were clear indicators of anticancer potential of the MAE. It was espoused by the results garnered from various parameters like reduced ascites tumor volume, declined peritoneal cavity size, the percentage increase in life span (%ILS), declined cell viability andperitoneal angiogenesis, and the improved result obtained from histopathology of vital organs.
... This demonstrates the anti-tumor potential of the MAE in a dose-dependent fashion. Observation of the vascularization pattern in the peritoneal cavity lining is an established in-vivo method to check the angiogenesis (Anand et al. 2012;Bhattacharjee et al. 2017). The photographs of the internal linings of peritoneal epithelium that were cut open on the 14th day of the study showed a significantly subsided neovascularization and inhibited angiogenesis in groups treated with higher doses of MAE and standard drug compared to tumor control (Fig. 2f-j). ...
Article
Ulva fasciataDelile, a marine macroalga is abundantly available on the intertidal rocky surface of Visakhapatnam coasts, India, during summer. The algae belong to family Ulvaceae and division Chlorophyta is glossy green in color. Being a prolific source of bioactive compounds, this thallus allured an unprecedented concern of the author to study its anatomy, histo-chemistry and in-vivo anticancer property. Primarily, the morpho-anatomy and histo-chemistry of the thallus were studied to identify correct species and locate the active phytoconstituents in the cell saps. Besides, the in-vivo anticancer activity of the methanolic algal extract (MAE) was studied on Ehrlich’s ascites carcinoma (EAC) xenografted mice. The light microscopy of the thallus divulged very thick cuticularized layers in the sectional view. A fat middle lamella was observed in between two layers of vertically oblong palisade cells. The histo-chemistry revealed the presence of starch grains, calcium oxalate crystals, tannin, protein,and lipids as major cell inclusions. The extract exhibited a profound inhibitory activity on EAC xenografted mice (p˂0.001). Increase in mean survival time (p˂0.001) and restoration of the hematological parameter in drug-treated rodents were clear indicators of anticancer potential of the MAE. It was espoused by the results garnered from various parameters like reduced ascites tumor volume, declined peritoneal cavity size, the percentage increase in life span (%ILS), declined cell viability andperitoneal angiogenesis, and the improved result obtained from histopathology of vital organs.
... Transmission electron microscope images showed very distinct morphological changes related to apoptotic cell death without significant side effects on the animals. Oral dose of curcumin in combination with naringenin (20 mg/kg each) received by Swiss albino mice, transplanted with murine mammary Ehrlich ascites carcinoma (EAC) cells, resulted in reduction in the formation of new blood vessels in peritoneal and inner skin linings with reduction (80%) of total number of cells/mL in ascites fluid [128] . Athymic mice xenograft model of human triple-negative breast cancer MDA-MB-231 cells showed significant decrease in angiogenesis, cell proliferation and tumor size on treatment with 300 mg/kg/day intraperitoneal (i.p.) dose of curcumin [129] . ...
Article
Full-text available
Curcumin, a polyphenol, has a wide range of biological properties such as anticancer, antibacterial, antitubercular, cardioprotective and neuroprotective. Moreover, the anti-proliferative activities of Curcumin have been widely studied against several types of cancers due to its ability to target multiple pathways in cancer. Although Curcumin exhibited potent anticancer activity, its clinical use is limited due to its poor water solubility and faster metabolism. Hence, there is an immense interest among researchers to develop potent, water-soluble, and metabolically stable Curcumin analogs for cancer treatment. While drug resistance remains a major problem in cancer therapy that renders current chemotherapy ineffective, curcumin has shown promise to overcome the resistance and re-sensitize cancer to chemotherapeutic drugs in many studies. In the present review, we are summarizing the role of curcumin in controlling the proliferation of drug-resistant cancers and development of curcumin-based therapeutic applications from cell culture studies up to clinical trials.
... Flavonoids such as naringenin act as effective inhibitors of angiogenesis and hence are regarded as a good treatment for the control of malignancies [65]. Naringenin exhibits its angio-inhibitory effect by decreasing vascular endothelial growth factor (VEFG) and other related factors [66]. It has also been reported to cause downregulation of TGF-β pathway, thereby decreasing metastasis and invasion in pancreatic cells [67]. ...
Article
Full-text available
Extensive research has been carried out during the last few decades, providing a detailed account of thousands of discovered phytochemicals and their biological activities that have the potential to be exploited for a wide variety of medicinal purposes. These phytochemicals, which are pharmacologically important for clinical use, primarily consist of polyphenols, followed by terpenoids and alkaloids. There are numerous published reports indicating the primary role of phytochemicals proven to possess therapeutic potential against several diseases. However, not all phytochemicals possess significant medicinal properties, and only some of them exhibit viable biological effects. Naringenin, a flavanone found in citrus fruits, is known to improve immunity, repair DNA damage, and scavenge free radicals. Despite the very low bioavailability of naringenin, it is known to exhibit various promising biological properties of medicinal importance, including anti-inflammatory and antioxidant activities. This review focuses on the various aspects related to naringenin, particularly its physicochemical, pharmacokinetic, and pharmacodynamic properties. Furthermore, various pharmacological activities of naringenin, such as anticancer, antidiabetic, hepatoprotective, neuroprotective, cardioprotective, nephroprotective, and gastroprotective effects, have been discussed along with their mechanisms of action.
Article
Angiogenesis is a complex physiological process that cannot be treated with single agent therapy. Several edible fungi have been known to encompass bioactive compounds, and are promising sources of multi-component drugs. One such widely consumed edible fungi is Cantharellus cibarius, which has been explored for its biological activities. The present study focused on assessing the anti-angiogenic activity of petroleum ether and ethanol extracts of C. cibarius using chick chorioallantoic membrane (CAM) assay. Both the extracts showed a dose-dependent response which was compared with the anti-angiogenic activity of the positive controls silibinin, and lenalidomide. The extracts were also studied for their lipoxygenase (LOX) inhibitory potential and compared to ascorbic acid as the positive control. The IC50 values of the petroleum ether extract, ethanol extract, and ascorbic acid for LOX inhibition assay were 135.4, 113.1, and 41.5 µg/mL, respectively. Although both the extracts showed similar responses in CAM assay, ethanol extract proved to be more potent in LOX inhibition assay. Finally, the extracts were investigated for their chemical composition using GC-MS. A correlation between LOX inhibition and anti-angiogenic potential was established at the molecular level. A meticulous literature search was carried out to correlate the biochemical composition of the extracts to their anti-angiogenic activity.
Article
Background In cancer chemotherapy, conventional drugs aim to target the rapidly growing and dividing cells at the early stages. However, at an advanced stage, cancer cells become less susceptible because of the multidrug resistance and the recruitment of alternative salvage pathways for their survival. Besides, owing to target non-selectivity, healthy proliferating cells also become vulnerable to the damage. The combination therapies offered using flavonoids to cure cancer not only exert an additive effect against cancer cells by targetting supplementary cell carnage pathways but also hampers the drug resistance mechanisms. Thus, the review aims to discuss the potential and pharmacokinetic limitations of flavonoids in cancer treatment. Further successful synergistic studies reported using flavonoids to treat cancer has been described along with potential drug delivery systems. Methods A literature search was done by searching various online databases like Pubmed, Scopus, and Google Scholar with the specific keywords like “Anticancer drugs,” “flavonoids,” “oncology research,” and “pharmacokinetics.” Results Dietary phytochemicals, mainly flavonoids, hinder cell signalling responsible for multidrug resistance and cancer progression, primarily targeting cancer cells sparing normal cells. Such properties establish flavonoids as a potential candidate for synergistic therapy. However, due to low absorption and high metabolism rates, the bioavailability of flavonoids becomes a challenge. Such challenges may be overcome using novel approaches like derivatization, and single or co-delivery nano-complexes of flavonoids with conventional drugs. These new approaches may improve the pharmacokinetic and pharmacodynamic of flavonoids. Conclusion This review highlights the application of flavonoids as a potential anticancer phytochemical class in combination with known anti-cancer drugs/nanoparticles. It also discusses flavonoid’s pharmacokinetics and pharmacodynamics issues and ways to overcome such issues. Moreover, it covers successful methodologies employed to establish flavonoids as a safe and effective phytochemical class for cancer treatment.
Article
Background: Naringenin, a flavonoid present in citrus fruits has many health promoting activities. It has been reported to protect skin from UV radiation, thermal damage and atopic allergies. Despite many skin protective effects, in vivo effect of naringenin on skin cancer has not been reported so far. Objective: The present work was designed to study the chemo preventive effect of naringenin on chemically induced skin cancer in mice. Methods: Two stage model of skin papillomagenesis, using DMBA plus croton oil, was used to study the effect of naringenin in Swiss albino mice. The chemo preventive effect was evaluated using morphological, histopathological and biochemical features. Results: Oral administration of naringenin reduced the skin papilloma in both pre-treatment as well as post-treatment groups of mice. The number as well as size of papilloma was significantly reduced in the treated groups. Histopathological studies showed that naringenin treatment suppressed papillomagenesis. Biochemical studies further revealed decrease in the activity of glyoxalase-1 enzyme and an increase in carbonyl content. The effect was more pronounced in ant-initiation group. Conclusion: Naringenin exhibited anti-tumor effect in two stage carcinogenesis mouse skin tumor model. This study revealed that consumption of citrus fruits and the naringenin therein may be helpful in suppression of skin cancer.
Article
Full-text available
Curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), is a potent antioxidant and anti-inflammatory agent. It has been recently demonstrated to possess discrete chemopreventive activities. However, the molecular mechanisms underlying such anticancer properties of curcumin still remain unrealized, although it has been postulated that induction of apoptosis in cancer cells might be a probable explanation. In the current study, curcumin was found to decrease the Ehrlich's ascites carcinoma (EAC) cell number by the induction of apoptosis in the tumor cells as evident from flow-cytometric analysis of cell cycle phase distribution of nuclear DNA and oligonucleosomal fragmentation. Probing further into the molecular signals leading to apoptosis of EAC cells, we observed that curcumin is causing tumor cell death by the up-regulation of the proto-oncoprotein Bax, release of cytochrome c from the mitochondria, and activation of caspase-3. The status of Bcl-2 remains unchanged in EAC, which would signify that curcumin is bypassing the Bcl-2 checkpoint and overriding its protective effect on apoptosis.
Article
Full-text available
Metastasis is the main cause of death in cancer patients. To improve the outcomes of patients undergoing a surgery, new adjuvant therapies that can effectively inhibit metastases have to be developed. Studies have shown that flavonoid naringenin, a natural product that is mainly present in grapes and citrus, may contribute to cancer prevention. It has many advantages compared to traditional chemotherapeutic drugs, such as low toxicity. To determine whether naringenin can also inhibit metastases, a breast cancer resection model that mimics clinical situations was established. We found that orally administered naringenin significantly decreased the number of metastatic tumor cells in the lung and extended the life span of tumor resected mice. Flow cytometry analysis revealed that T cells displayed enhanced antitumor activity in naringenin treated mice, with an increased proportion of IFN-γ and IL-2 expressing T cells. In vitro studies further demonstrated that relief of immunosuppression caused by regulatory T cells might be the fundamental mechanism of metastasis inhibition by naringenin. These results indicate that orally administered naringenin can inhibit the outgrowth of metastases after surgery via regulating host immunity. Thus, naringenin can be an ideal surgical adjuvant therapy for breast cancer patients.
Article
Full-text available
Hypoxia-inducible factor (HIF)-1α is the oxygen-sensitive subunit of HIF-1, a transcriptional master regulator of oxygen homeostasis. Oxygen-dependent prolyl hydroxylation targets HIF-1α for ubiquitinylation and proteasomal degradation. Unexpectedly, we found that exposing mice to elevated temperatures resulted in a strong HIF-1α induction in kidney, liver, and spleen. To elucidate the molecular mechanisms responsible for this effect, HepG2 hepatoma cells were exposed to different temperatures (34–42 °C) under normoxic (20% O2) or hypoxic (3% O2) conditions. Heat was sufficient to stabilize mainly a phosphatase-resistant, low molecular weight form of HIF-1α (termed HIF-1αa). Heat-induced HIF-1αaaccumulated in the nucleus but neither bound to DNA nortrans-activated reporter or target gene expression, demonstrating the need for post-translational modifications for these functions. The protein banding pattern of heat-induced HIF-1α in immunoblot analyses was clearly distinct from the HIF-1α pattern after prolyl hydroxylase inhibition (by hypoxia or iron chelation/replacement) or following proteasome inhibition, suggesting that heat stabilizes HIF-1α by a novel mechanism. Inhibition of the ATP-dependent chaperone activity of HSP90 by novobiocin or geldanamycin prevented heat-induced as well as hypoxia-induced HIF-1α accumulation, indicating a common role of the HSP90 chaperone activity in HIF-1α stabilization by these two environmental parameters.
Article
Full-text available
The phytochemicals, resveratrol or curcumin, have been shown to possess many pharmacological activities including anti-inflammatory, anti-oxidant, anti-microbial and anti-cancer effects. However, the underlying mechanism for their anti-tumor activity is yet to be evaluated. The present study was carried out to investigate the anti-angiogenic effect of resveratrol or curcumin when used alone or in combination with carboplatin in Ehrlich ascites carcinoma (EAC)-bearing mice. Solid tumors were induced by intradermal injection of EAC cells. These tumors were used for the evaluation of microvessel density, plasma vascular endothelial growth factor (VEGF) and its intra-tumoral receptor type-2 (Flk-1). All parameters were determined as a time course on days 7, 14, and 21 post-inoculation. Individual treatments with resveratrol or curcumin and their combination with carboplatin produced a significant reduction in microvessel density. Plasma levels of VEGF were significantly reduced in groups treated with resveratrol or curcumin and their combination with carboplatin on day 7 post-inoculation. Treatment with resveratrol or curcumin reduced the percentage of Flk-1-rich tumors to reach 42.9% and 28.6%, respectively. Their co-administration with carboplatin has produced a further reduction in the percentage of Flk-1-rich tumors to reach 28.6% and 14.3%, respectively. Correlation studies showed strong association between plasma VEGF and microvessel density. In conclusion, resveratrol or curcumin inhibited angiogenesis as demonstrated by the reduction of microvessel density by these agents. Both proved to exert their anti-angiogenic effect by inhibition of VEGF and its receptor type-2. The results suggest the beneficial role of these phytochemicals as adjuvant to chemotherapy in the treatment of cancer.
Article
Full-text available
The molecular chaperone heat shock protein 90 (HSP90) has been used by cancer cells to facilitate the function of numerous oncoproteins, and it can be argued that cancer cells are 'addicted' to HSP90. However, although recent reports of the early clinical efficacy of HSP90 inhibitors are encouraging, the optimal use of HSP90-targeted therapeutics will depend on understanding the complexity of HSP90 regulation and the degree to which HSP90 participates in both neoplastic and normal cellular physiology.
Article
Pancreatic cancer (PC) remains the fourth most common cause of cancer related death in the United States. Therefore, novel strategies for the prevention and treatment are urgently needed. Numerous dietary and pharmacological agents have been proposed as alternative strategies for the prevention and/or treatment of PC. Isoflavone is a prominent flavonoid found in soy products and has been proposed to be responsible for lowering the incidence of PC in Asians. Similarly, curcumin, an active ingredient of turmeric, that inhibits growth of malignant neoplasms, has a promising role in the prevention and/or treatment of PC. Here we examined whether isoflavone together with curcumin could elicit a greater inhibition of growth of PC cells than either agent alone, and also sought to determine the molecular mechanism of action. We found that the inhibition of cell growth and induction of apoptosis was significantly greater in the combination group than that could be achieved by either agent alone. These changes were associated with decreased Notch-1 expression and DNA binding activity of NF-kappaB and its target genes such as Cyclin D1, Bcl-2, and Bcl-xL. Moreover, we found that the combination of four natural agents at lower concentration was much more effective. Collectively, our results suggest that diet containing multiple natural products should be preferable over single agents for the prevention and/or treatment of PC. The superior effects of the combinatorial treatment could partly be attributed to the inhibition of constitutive activation of Notch-1 and NF-kappaB signaling pathways.
Article
The inhibition of oncogenic signaling pathways has gained great interest for cancer therapy. In this context, the molecular chaperone heat shock protein 90 (HSP90) has emerged as a promising molecular target, since it is critically involved in maintaining stability, integrity and functions of key oncogenic proteins. A variety of HSP90 inhibitors have been developed in the past decade and have shown convincing anti-neoplastic activity in pre-clinical tumor models. Importantly, HSP90 inhibitors are predominantly being recognized as "tumor cell targeting" agents since cancer cells a) overexpress HSP90 protein, b) highly rely on HSP90 function for maintaining oncogenic signaling, and c) HSP90 inhibitors bind with high affinity to HSP90 in tumor cells. Nevertheless, results from recent studies also suggest that HSP90 inhibitors elicit anti-angiogenic properties by affecting the PI-3K/Akt/eNOS signal transduction pathway in endothelial cells, as well as through down-regulation of VEGFR-2 expression, a crucial component of the angiogenic process. In addition, blocking HSP90 may also diminish the secretion and expression of tumor cell-derived pro-angiogenic growth factors and cytokines, thus leading to "indirect" anti-angiogenic effects. This review article focuses on the role of HSP90 in angiogenesis and on delineating the effects of HSP90 inhibitors on angiogenic signaling pathways involved in tumor vascularization.