European Journal of Pharmacology 649 (2010) 64–73

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European Journal of Pharmacology

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Molecular and Cellular Pharmacology

Harmine inhibits tumour specific neo-vessel formation by regulating VEGF, MMP,

TIMP and pro-inflammatory mediators both in vivo and in vitro
T.P. Hamsa, Girija Kuttan ⁎
Amala Cancer Research Centre, Amala Nagar, Thrissur, 680 555, Kerala, India

a r t i c l e i n f o a b s t r a c t

Article history: Harmine is a beta-carboline alkaloid present in medicinal plants such as Peganum harmala that have been used as
Received 15 April 2010 folk medicine in anticancer therapy. In this study, we demonstrated the anti-angiogenic activity of harmine using
Received in revised form 21 August 2010 in vivo and in vitro assay systems. In vivo anti-angiogenic activity was studied using B16F-10 melanoma cells
Accepted 9 September 2010
which induced capillary formation in C57BL/6 mice. Intraperitoneal administration of harmine at 10 mg/kg body
Available online 19 September 2010
weight significantly decreased tumour directed capillary formation. A drastic elevation in serum pro-angiogenic
Keywords:
factors such as vascular endothelial growth factor (VEGF), nitric oxide (NO) and pro-inflammatory cytokines in
Harmine angiogenesis induced animals was significantly decreased by harmine treatment. At the same time harmine
Anti-angiogenesis increased anti-tumour factors like interleukin-2 (IL-2) and tissue inhibitor metalloprotease (TIMP). Moreover
VEGF nuclear factor (NF)-κB and other transcription factors like CREB, ATF-2 involved in tumour development and
B16F-10 melanoma angiogenesis were also inhibited by harmine. Various in vitro assays also supported the anti-angiogenic activity of
Pro-inflammatory cytokine harmine. It reduced proliferation, migration and tube formation of human umbilical vein endothelial cells
Transcription factor (HUVEC). Direct treatment of the harmine also inhibited microvessel outgrowth from the rat aortic ring.
Production of other factors by tumour cells which are involved in angiogenesis like cyclooxygenase (COX-2),
inducible nitric oxide synthase (iNOS) and matrix metalloproteases (MMPs) were also decrease by the treatment
with harmine. Our data suggest that harmine may be a strong angiogenic inhibitor with the ability to decrease the
proliferation of vascular endothelial cells and to reduce expression of various pro-angiogenic factors.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction cellular matrix (ECM). Local degradation of the vascular basement

Angiogenesis, the formation of new blood vessels from pre-
existing vasculature, is prevalent both during normal mammalian
development and in certain pathological conditions such as tumour
growth. Angiogenesis involves a sequence of coordinated events that
is initiated by the expression of angiogenic factors such as vascular
endothelial growth factor (VEGF). VEGF increases vascular perme-
ability, which leads to extravasation of plasma proteins and
dissociation of pericyte coverage (Dvorak, 2005; Roberts and Palade,
1997). VEGF promotes endothelial proliferation via the activation of
the MAPK, Akt and inducible nitric oxide synthase (iNOS) pathways
(Lal et al., 2001; Zhu et al., 2000). One of the first events to take place
during the angiogenic process involves the formation of a vascular
sprout. This begins with the conversion of endothelial cells into an
endothelial tip cell which transforms the cell from a quiescent into an
actively migrating state. Endothelial tip cells undergo extensive
migration and proliferation under the influence of VEGF and extra
⁎ Corresponding author. Department of Immunology, Amala Cancer Research Centre,
Amala Nagar, Thrissur. 680 555, Kerala, India. Tel.: + 91 487 2307968; fax: + 91 487
2307868.
E-mail addresses: amalacanceresearch@gmail.com, girijakuttan@gmail.com
(G. Kuttan).
membrane and ECM occur simultaneously by matrix metallopro-
teases (MMP) released by tip cells paving the way for newly sprouting
vessels (Adams and Alitalo, 2007).
Inflammation has been found as one of the important processes in
mediating angiogenesis. For example, many tumour cell types and
tumour-infiltrating macrophages express tumour necrosis factor (TNF-
α) and interleukins (IL). These molecules promote and sustain the
production of the classical angiogenic factors VEGF-A and basic fibroblast
growth factor (bFGF) and mediates the ECM remodeling through the
induction of metalloproteases, such as MMP-9 (Sethi et al., 2008).
Inhibiting the angiogenic process or targeting existing tumour vessels
can be used for the treatment of tumours as an alternative or in parallel
with conventional chemotherapy. The therapeutic strategy to develop
anti-angiogenic molecules involve various targets including angiogenic
molecules from tumour cells and inflammatory cells such as VEGF, bFGF,
TNF-α, IL-8, etc. and their respective receptors on endothelial cells,
endothelial cells itself, MMPs, COXs etc. (Bhat and Singh, 2008).
Endogenous angiogenesis inhibitors such as interferons (IFN), tissue
inhibitor metalloproteases (TIMP), and proteolytic fragments also act as
target for anti-angiogenic therapy (Feldman and Libutti, 2000). TIMP
inhibits the activities of MMP-1, MMP-2 and MMP-9, which leads to
reduced ECM remodeling and suppression of endothelial cell migration
and invasion (Gomez et al., 1997a).

0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2010.09.010
 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73
Beta-carboline alkaloids such as harmine (Fig. 1) are present in
medicinal plants such as Peganum harmala that have been used as folk
medicine in anticancer therapy (Berrougui et al., 2006). It is known
that these alkaloids have a wide spectrum of neuropharmacological,
psychopharmacological (Abe and Yamada, 2009), anti-tumour, anti-
bacterial and anti-plasmodial effect (Arshad et al., 2008). Therefore,
they have been traditionally used in oriental medicine for the
treatment of various diseases including cancers. It was reported that
harmine showed significant tumour inhibition in mice bearing Lewis
Lung Cancer, sarcoma180 or HepA tumour (Chen et al., 2005), and
showed broad cytotoxicity spectrum against KB, SaOS-2, A549, U-87-
MG, and MCF-7 cells(Ishida et al., 1999) and human lung carcinoma
cell lines (Abe and Yamada, 2009). Studies have demonstrated that
these alkaloids can also act as scavengers of reactive oxygen species
(Moura et al., 2007). On the basis of these potential anticancer
activities of harmine, in this study we analyzed its effect on tumour
specific angiogenesis and its mechanism of action in both in vivo as
well as in vitro models. Various in vitro assays such as endothelial cell
matrix degradation, migration, proliferation and in vivo studies like
rat aortic ring assay, tumour directed capillary formation analysis are
used to assess the anti-angiogenic activity of harmine. Various pro and
anti-angiogenic factors in the presence and absence of harmine are
also analyzed by this experiment.
2. Materials and methods
2.1. Animals
Four to six week old male C57BL/6 mice were purchased from
National Institute of Nutrition, Hyderabad, India. The animals were fed
with normal mouse chow (Sai Feeds, India) and water ad libitum. All
animal experiments were conducted according to the rules and
regulations of Animal Ethics Committee, Govt. of India. Human
umbilical vein endothelial cell (HUVEC) experiments were conducted
according to the guidelines of institutional ethical committee.
2.2. Cells
HUVECs were isolated from human umbilical cord vein by
collagenase treatment as described by Jaffe et al. (Jaffe et al., 1973).
The cells were grown in medium 199, supplemented with 20% fetal
Bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin
and 2 ng/ml VEGF and FGF at 37 °C in 5% CO2 atmosphere. B16F-10
melanoma cells were purchased from the National Centre for Cell
Science, Pune, India and maintained in culture using DMEM with 10%
FBS and antibiotics.
2.3. ELISA kits
Highly specific quantitative ‘sand witch’ Elisa kits for mouse IL-1β,
IL-6, TNF-α, GM-CSF and IL-2 were purchased from Pierce Biotech-
nology USA and the ELISA kits for VEGF and TIMP-1 were purchased
from R&D Systems, USA.
Fig. 1. Structure of harmine.
65
2.4. Chemicals
MTT (3-4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-
mide), DMEM, and oligonucleotide primer sequences were purchased
from Sigma, St. Louis, USA. All the other reagents and chemicals used
were of analytical reagent grade.
2.5. Drug administration
Harmine was dissolved in sterile water. A non toxic dose of these
compounds were administered intraperitoneally (i.p.) at a dosage of
10 mg/kg body weight of animal and for in vitro experiments harmine
was dissolved in dimethyl sulfoxide (DMSO) (0.1%) and resuspended
in culture medium. TNP-470 (from Dr. Ravivarma, NCI, USA) also
dissolved in DMSO (0.1%), resuspended in sterile water and
administered intraperitoneally as a reference compound.
2.6. Effect of harmine on tumour specific capillary formation
Angiogenesis was induced in three groups of mice (n = 8) by
injecting B16F-10 melanoma cells (106 cells/animal) intradermally on
the shaven ventral skin of each mouse and were treated simultaneously
(i.p.) with 10 mg harmine per kg body weight or 30 mg TNP-470 per kg
body weight for five consecutive days. Control animals received B16F-10
cells alone. After 24 h and 9 days of tumour challenge, blood was
collected in a sterile manner from the caudal vein of each mouse. Serum
was separated and used for the estimation of cytokines using ELISA kits
according to the manufacturers' instructions. After 9 days, the animals
were sacrificed and the skin from the ventral side was dissected out,
washed with PBS and the number of capillaries growing towards the site
of tumour induction from the pre-existing vessels were counted using a
dissection microscope at 20× magnification and compared with the
untreated control animals (Leyon and Kuttan, 2004).
2.7. Effect of harmine on the production of IL-1β, IL-6, TNF-α, GM-CSF,
IL-2, VEGF and TIMP-1 during angiogenesis
Blood obtained from the above experiment at two time intervals
was centrifuged and serum was separated. This serum was used for
the estimation of IL-1β, IL-6, TNF-α, GM-CSF, IL-2, VEGF and TIMP-1
using ELISA kits according to manufacturer's protocol.
2.8. Effect of harmine on serum nitrite levels during angiogenesis
Serum obtained from the above experiment at two time intervals
was also used for the estimation of nitrite level by Griess reaction
(Green et al., 1982). Briefly a 100 μl sample was incubated with an
equal amount of Griess reagent (one part of 0.1% N(1-naphthyl)-
diamine dihydrochloride in distilled water and one part 1% sulfanil-
amide in 5% concentrated H3PO4) and incubated for 10 min at room
temperature. Absorbance was estimated at 540 nm. The amount of
nitrite was calculated from the NaNO2 standard curve.
2.9. Cell viability by MTT assay
The viability of cultured cells was determined by assaying for the
reduction of MTT to formazan (Campling et al., 1991; Cole, 1986).
HUVECs were seeded (5000 cells/well) in titre plate and incubated for
24 h at 37 °C in 5% CO2 atmosphere. Different concentrations of
harmine (1 μg–50 μg/ml) were added and incubated further for 48 h.
Before the 4 h completion of incubation, 20 μl MTT (5 mg/ml) was
added. The percentage of viable cells was determined using an ELISA
plate reader set to record absorbance at 570 nm.
66 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73
2.10. Effect of harmine on endothelial cell proliferation (3H-thymidine
incorporation assay)
HUVECs (5000 cells/well) were plated in a 96-well culture plate and
incubated at 37 °C in 5% CO2 atmosphere. After 24 h, various
concentrations of harmine (0.5, 1 and 2 μg/ml) were added along with
2 ng/ml VEGF and further incubated for 48 h. 3H-thymidine was added
to each well (1 μCi/well) and incubation was continued for additional
18 h. After completing incubation, the plates were centrifuged and the
culture supernatant was removed, the cells were washed three times
with PBS and then treated with ice cold percholric acid for 15 min. The
resulting precipitate was dissolved in 0.5 N NaOH and was added to the
scintillation fluid and the radioactivity was counted using a Rack Beta
liquid scintillation counter.
2.11. Effect of harmine on endothelial cell migration/motility
HUVECs were seeded into wells of collagen coated 96-well flat
bottom plates at a density of 2 × 105 cells/well and incubated for 24 h
at 37 °C in 5% CO2 atmosphere. A clear area was scraped in the
monolayer with a narrow tip by applying suction and washed with
serum free medium. Different concentrations of harmine (0.5, 1 and
2 μg/ml) were added along with 2 ng/ml VEGF and further incubated
for 24 h. After incubation the cells were fixed in formalin and stained
with crystal violet and photographed (200×) (Guo et al., 2002).
2.12. Effect of harmine on endothelial cell invasion
The invasion assay was carried out in modified Boyden chambers as
described by Albini et al. (1987). The lower compartment of the
chamber was filled with serum free medium 199 and a polycarbonate
filter membrane coated with 25 μg Type I collagen was placed above
this. HUVECs (105 cells/150 μl medium 199) were then seeded on to
the upper chamber in the presence and absence of harmine (0.5, 1 and
2 μg/ml) along with 2 ng/ml VEGF and FGF and incubated at 37 °C in 5%
CO2 atmosphere for 10 h. After incubation, the filters were removed,
fixed with methanol and stained with crystal violet. Cells migrating to
the lower surface of the polycarbonate filters were counted under a
microscope. The results were expressed as percentage inhibition of
invasion.
2.13. Effect of harmine on endothelial cell tube formation
Tube formation was carried out according to the protocol of Gupta
et al. (2002). Matrigel was allowed to thaw on ice at 4 °C. A thirty
microlitre ice cold matrigel was pipetted into a 96-well flat bottomed
titre plate without introducing air bubbles and kept for 30 min at
37 °C to allow gelling of matrigel. HUVECs in medium 199 were
seeded onto the layer of matrigel at a density of 1 × 103 cells/well
along with 2 ng/ml VEGF and FGF. Various concentrations of harmine
(0.5, 1 and 2 μg/ml) were added into the wells and incubated for 48 h
at 37 °C in 5% CO2 atmosphere. After incubation, the cells were fixed
with Diff Quick fixative and stained with Diff Quick stain solutions.
Tube formation was examined and photographed using an inverted
microscope (200×).
2.14. Effect of harmine on the microvessel outgrowth from the rat aortic
ring (rat aortic ring assay)
The rat aortic ring assay was used as the in vitro angiogenesis study
model (Zhu and Nicosia, 2002). Dorsal aorta from a freshly sacrificed
Sprague–Dawley rat was taken out in a sterile manner and rinsed in ice
cold PBS. It was then cut into ∼1 mm long pieces using surgical blades.
Each ring was placed in a collagen pre-coated 96-well plate. The rings
were incubated for 24 h at 37 °C in complete medium and then replaced
with conditioned medium (CM) from B16F-10 melanoma cells and
incubated with or without different concentrations of harmine (0.5, 1
and 2 μg/ml). In a second set of experiment, the aortic rings were
incubated for 24 h in complete medium initially, and then replaced with
CM from harmine treated (pretreated CM) and non-treated B16F-10
melanoma cells. On day 6, the rings were analyzed for microvessel
outgrowth and photographed.
2.15. Quantitation of gene specific mRNA of VEGF in B16F-10 melanoma
cells
Quantikine mRNA is a novel method, which can be used to
quantitate gene specific mRNA at lower levels. Briefly, B16F-10 cells
(106 cells) were plated in 30 mm petridish in DMEM with 10% FBS at
37 °C in 5% CO2 atmosphere. Cells were then treated with harmine
(2 μg/ml) for 4 h. After incubation, the cells were washed and mRNA
preparations were made according to the manufacturer's procedures.
mRNAs were hybridized with gene specific biotin labeled detection
probes and digoxigenin alkaline labeled detection probes in a
microplate. Hybridization solution was transferred to a streptavidin
coated microplate and the mRNA probe hybrid was captured. Fol-
lowing wash to remove the unbound conjugate, a substrate solution
was added. An amplifier solution was then added and the developed
colour was measured spectrophotometrically at 490 nm.
2.16. Effect of harmine on NF-κB, c-Fos, ATF-2 and CREB-1
2.16.1. Preparation of nuclear extracts
Nuclear extracts were prepared by the previously published
method (Dignam et al., 1983). B16F-10 cells were treated with
harmine (2 μg/ml) for 2 h at 37 °C in 5% CO2 atmosphere in serum free
medium. The cells were washed twice with PBS and incubated further
with TNF-α (10 ρg/ml) for 30 min. The cells were then lysed using
lysis buffer by incubating 15 min on ice. The cell suspension was
centrifuged and disrupted using a syringe and centrifuged at 10000–
11000 × g for 20 min. The crude nuclear pellet obtained was
suspended in nuclear extraction buffer. Nuclei were disrupted by
using a fresh syringe, centrifuged and the supernatant was collected.
Protein concentrations of the nuclear extracts were estimated using
standard Bradford method and stored at −70 °C.
2.16.2. Transcription factor profiling
Transcription factor profiling was done with BD Mercury Trans-
factor kit obtained from BD Biosciences. Using an ELISA based format,
the transfactor kit detected the DNA bound transcription factors
(Shen et al., 2002). This method is faster, easier, and more sensitive
than electrophoretic mobility shift assay and does not require
radioactivity. When nuclear extracts added to the well, DNA will
bind to their consensus sequences in the well. Bound transcription
factors in the DNA were detected by specific primary antibody
towards NF-κBp65, NF-κBp50, NF-κB c-Rel, c-Fos, ATF-2 and CREB. A
horse radish peroxidase-conjugated secondary antibody was then
used to detect the bound primary antibody. The enzymatic product
was measured with standard microtitre plate reader at 655 nm.
Percentage inhibition was calculated by the formula, 1 − ([OD of
treated/OD of control] × 100).
2.17. Determination of the effect of harmine on MMPs by
gelatin zymography
SDS-PAGE was performed with 0.1% gelatin incorporated in the
separating gel (Billings et al., 1991). HUVEC cells of sub-confluent
cultures were pretreated with harmine (0.5, 1 and 2 mg/ml) for 24 h
at 37 °C in 5% CO2 atmosphere. The medium from sub-confluent (70%)
bottles of treated and untreated cells were removed, cells were then
washed and incubated in serum free medium DMEM at 37 °C for 24 h.
The CM was then collected and subjected to zymographic analysis.
 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73
After determining the protein concentration, 50 μl of sample (equiv-
alent to 100 μg protein) was activated with 5 μl trypsin solution
(75 μg/ml) in 0.1 M Tris–HCl, 10 mM CaCl2 buffer (pH-8.0) and
incubated for 1 h at room temperature. Samples were mixed with an
equal volume of 2X sample buffer and loaded on to 11% polyacryl-
amide gels containing 0.1% gelatin. Electrophoresis was carried out at
4 °C with constant current of 2 mA/tube until the tracking dye reached
the periphery. The gels were then washed with 2% Triton X-100 in
0.1 M Tris–HCl, 10 mM CaCl2 at 37 °C for 18 h followed by staining
with Gelcode Blue stain reagent for 2 h. Gels were destained to
visualize the clear area against the dark background.
2.18. Studies on the expression of VEGF, iNOS and COX-2 by RT-PCR
Logarithmically growing B16F-10 cells (5 × 104) were incubated
for 4 h in the presence and absence of harmine at a concentration of
2 μg/ml. cDNA was synthesized using the cells to-cDNA kit (Ambion
Inc). Cells from tissue culture were washed with PBS and heated in cell
lysis buffer (provided in the kit) to release the RNA into the solution.
This was followed by a heating step to inactivate endogenous
ribonucleases. The genomic DNA was further degraded by treating
with DNase followed by inactivation of DNase by heating at 70 °C.
Reverse transcription was performed at 42 °C for 50 min using
Moloney murine leukemia virus reverse transcriptase (supplied
along with the kit). The standard PCR products were generated by
using positive control cDNA provided in the kit. cDNA was amplified
using the following RT-PCR primer sets: VEGF (453 bp; 5′-
TGCTCACTTCCAGAAACACG-3′ and 5′-GGAAGGGTAAGCCACTCACA-
3′); iNOS (231 bp: 5′-CTTCAACACCAAGGTTGTCTG CAT-3′ and 5′-
CAAGACGCGGAAACGAGTACTGTA-3′); COX-2 (256 bp; 5′-GTGGAAAA
ACCTCGTCCAGA-3′ and 5′-TGATGGTGGCTGTTTTGGTA-3′); and
GADPH (557 bp; 5′-CGTCCCGTAGACAAAATGGT-3′ and 5′-CCTTCCA-
CAATGCCAAAGTT-3′). Amplified PCR products were electrophoresed
on a 1.8% agarose gel, stained with ethidium bromide, and photo-
Fig. 2. A) Inhibitory effect of harmine and TNP-40 on tumour-directed capillary formation. B) Quantitation of capillary formation in mice treated for 9 days with harmine, TNP40 and
untreated control. C) Viability of HUVEC following 24 h treatment with the indicated concentrations of harmine and untreated control as judged by MTT assay. Similar results were
observed in two independent experiments. ⁎⁎⁎ P b 0.001 compared to control.
67
graphed under ultraviolet light. Band intensity was measured using
software provided with vilber Lourmat (Photo-Capt Version 12.3 for
Windows) gel documentation system and percentage expression was
calculated.
2.19. Statistical analysis
Results were expressed as mean ± S.D. The statistical analysis was
done using one-way ANOVA followed by Dunnett's test.
3. Results
3.1. Effect of harmine on tumour specific capillary formation
The number of tumour directed capillaries were significantly
reduced by the administration of harmine (51.32%) (Fig. 2B). The
harmine received group had an average of 15.33 ± 1.03 capillaries
whereas the control group had an average of 31.50 ± 2.81 capillaries.
Animals treated with TNP-470 (reference compound) showed 89.42%
inhibition in the microvessel formation with an average number of
3.33 ± 1.37 tumour directed blood capillaries (Fig. 2A).
3.2. Effect of harmine on the production of IL-1β, IL-6, TNF-α, GM-CSF,
IL-2, VEGF and TIMP-1 during angiogenesis
The level of serum cytokines such as IL-1β, IL-6, TNF-α, and GM-
CSF is presented in Tables 1 and 2. In control animals, the level of IL-1,
IL-6, TNF-α and GM-CSF in the serum was drastically enhanced after
tumour challenge when compared with normal levels. Harmine
treatment effectively reduced the elevated levels of these serum
cytokines. VEGF levels also showed an elevation in untreated control
after tumour induction compared with the normal level. But in
harmine treated animals, VEGF was significantly reduced showing
their activity on VEGF. The lowered levels of anti-angiogenic factors
68 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73

Table 1
Effect of harmine on VEGF and pro-inflammatory cytokine profile of angiogenesis induced animals.

Cytokines Normal Control Harmine

(pg/ml)
Day 1 Day 9 Day 1 Day 9
a
VEGF 16.01 ± 4.29 71.47 ± 2.03 139.11 ± 2.17 37.63 ± 1.41 59.99 ± 1.41a
IL-1β 18.14 ± 3.75 32.13 ± 1.10 32.59 ± 1.16 31.13 ± 0.46 17.91 ± 1.48a
IL-6 36.81 ± 5.58 40.16 ± 2.57 318.86 ± 4.01 36.70 ± 2.40 73.40 ± 2.40a
TNF-α 21.18 ± 1.81 168.64 ± 1.71 582.96 ± 6.76 137.23 ± 2.34a 128.26 ± 1.71a
GM-CSF 18.12 ± 3.11 64.62 ± 0.52 27.42 ± 1.11 34.02 ± 1.61a 20.36 ± 0.52a

Blood was collected from the angiogenesis induced animals at two time points after tumour challenge. Serum was separated by centrifugation and the cytokine level was estimated
by ELISA method. All the values are mean ± S.D. (mean of triplicate).
a
P b 0.001 compared to respective control.

like IL-2 and TIMP in the control animals after tumour inoculation
were significantly increased by the treatment with harmine.
3.3. Effect of harmine on serum nitrite levels during angiogenesis
The serum nitrite level of control animals at 24 h after angiogen-
esis induction was slightly increased to 23.54 ± 0.50 μmoles and was
again elevated to 38.95 ± 0.82 μmoles on 9th day compared to normal
level (20.76 ± 0.41 μmoles). Administration of harmine could signif-
icantly inhibit the production of the serum nitrite level to 21.77 ± 1.13
μmoles at 24 h and to 23.21 ± 0.64 μmoles on the 9th day.
3.4. Effect of harmine on cell viability
The percentage viability of HUVEC cells after the treatment with
harmine is shown in Fig. 2C. The concentration needed for 50%
inhibition (IC50) of growth of HUVEC cells was found to be 32.6 μg/ml.
The cell were 100% alive below 5 μg/ml of harmine and found to be
nontoxic at 0.5, 1 and 2 μg/ml of harmine and these concentrations
were used for further in vitro experiments.
area were found to be 211 ± 13.5, 110 ± 18.8 and 19 ± 7.2 respectively
which was very much lower than that control where 397 ± 42.6 cells
were migrated (Fig. 3C). This concentration is nontoxic, as is evident
from the MTT assay; hence, the inhibitory effect could not be
attributed to cytotoxic activity.
3.7. Effect of harmine on endothelial cell invasion
Large numbers of cells were found on the lower surface of the
polycarbonate membrane showing strong invasive property of HUVEC
through collagen in the presence of VEGF (Fig. 3B). In untreated control
1903 ± 32.5 cells were observed under the polycarbonate membrane
and it was significantly reduced to 1378.5 ± 50.2, 986.5 ± 58.7 and
335.5 ± 2.1 cells when the cell were treated with harmine at 0.5, 1, and
2 μg/ml respectively. The percentage inhibitions in the invasion of
HUVEC after the treatment with harmine at these concentrations were
found to be 27.56%, 48.16% and 82.37% respectively. This demonstrates
the dose dependent inhibitory effect of harmine on the migration of
HUVEC through collagen coated membrane which is an important step
in neo-vessel formation (Fig. 3D).

3.5. Effect of harmine on endothelial cell proliferation

3
Rate of proliferation was determined by H-thymidine incorpora-
tion by the DNA of HUVECs. Thymidine incorporation is proportional
to the potential of the cells to synthesize DNA. Proliferation was
expressed as radioactive count per minute (cpm). HUVECs showed a
very high rate of proliferation (4521 ± 155 cpm) when stimulated
with VEGF. Administration of harmine at non toxic concentration (0.5,
1, and 2 μg/ml) significantly inhibited VEGF-induced proliferation of
HUVECs in a dose dependent manner (Table 3).
3.8. Effect of harmine on endothelial cell tube formation
Treatment of HUVECs with harmine significantly inhibited tube
formation (Fig. 4A). Incubation of HUVECs on matrigel with VEGF
resulted in the formation of elongated and tube-like structures.
Harmine effectively reduced the width and length of endothelial tubes
at 2 μg/ml.
3.9. Effect of harmine on rat aortic ring assay

3.6. Effect of harmine on endothelial cell motility
The effect of harmine on the motility of HUVECs is shown in
Fig. 3A. Harmine significantly inhibited the VEGF-induced migration
of endothelial cells in a dose dependent manner compared to control
where HUVECs migrated into the clear area when stimulated with
VEGF. When the cells were treated with harmine at concentrations of
0.5, 1 and 2 μg/ml, the number of cells migrated towards the wound
The vessels that grow out from aortic rings are anatomically
similar to neo-vessels. Harmine at a concentration of 2 μg/ml could
significantly inhibit the microvessel outgrowth from the rat aorta ring
induced by the CM from the B16F10 melanoma cells (Fig. 4B-c). The
aortic rings incubated with CM from the harmine treated B16F10 cells
(pretreated CM) also showed a marked reduction in vessel out growth
(Fig. 4B-b) when compared with rings incubated with CM from
untreated B16F-10 cells (Fig. 4B-a).

Table 2
Effect of harmine on TIMP and IL-2 levels of angiogenesis induced animals.

Cytokines Normal Control Harmine

(pg/ml)
Day 1 Day 9 Day 1 Day 9
a
TIMP 600.65 ± 35.63 354.26 ± 5.26 371.34 ± 6.86 947.41 ± 4.56 1052.31 ± 61.19a
IL-2 23.21 ± 3.16 16.96 ± 0.34 18.30 ± 0.76 26.19 ± 9.52a 54.83 ± 0.41a

Blood was collected from the angiogenesis induced animals at two time points after tumour challenge. Serum was separated by centrifugation, and levels of TIMP and IL-2 were
estimated by ELISA method. All the values are mean ± S.D. (mean of triplicate).
a
P b 0.001 compared to respective control.
 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73 69

Table 3 significantly reduced this elevated level of VEGF mRNA to 9.77 ±

Effect of harmine on proliferation of HUVECs. 0.18 attomoles.
Treatment Radioactive count/min (CPM) % Inhibition

Untreated control 4521 ± 155

3.11. Effect of harmine on transcription factors
Harmine μg/ml
0.5 3614 ± 40a 20.06 Harmine significantly inhibited the translocation/activation of NF-
1 2755 ± 83a 39.06 κB subunits such as p65, p50, and c-Rel. We also found that harmine
2 1591 ± 91a 64.80
inhibited the nuclear translocation of AP-1 factors as c-fos, ATF-2, and
HUVECs (5 × 103 cells/well) were grown in 96 well flat bottom plates. After 24 h various CREB (Fig. 5A).
concentrations of harmine (0.5, 1 and 2 μg/ml) were added and incubation was
continued for 48 h. After incubation, 3H-thymidine was added to each well (1 μ Ci/well)
and incubation was continued for 18 h. Cells were lysed and radioactivity was counted
3.12. Effect of harmine on matrix metalloproteases
by using Rack Beta liquid scintillation counter. Values are mean ± S.D. (mean of
triplicate). MMP activity can be assessed using gelatin zymographic assays.
a
P b 0.001 compared to control. MMPs are generally secreted as zymogens which are activated by the
proteolytic cleavage by trypsin. As shown in Fig. 4B, harmine inhibited
the production of MMPs by HUVEC cells. Gels loaded with CM without
3.10. Quantitation of gene specific mRNA of VEGF in B16F-10 melanoma trypsin activation, did not show clear degradative areas, indicating the
cells inactive form of the enzyme (Fig. 5B-a). CM from the untreated B16F-
10 melanoma cells after the trypsin activation gave the functional
Considerable inhibition of VEGF mRNA synthesis was also observed degradative activity identical to MMP-9 (92 kDa) and MMP-2
in harmine treated melanoma cells. Untreated B16F-10 melanoma cells (72 kDa) (Fig. 5B-b). Trypsin activated CM from the untreated B16F-
showed high level of VEGF mRNA expression (26.43 ± 0.37 attomoles). 10 cells loaded gels did not show clear degradative areas when it was
Treatment with harmine at a concentration of 2 μg/ml for 4 h incubated with 10 mM EDTA (Fig. 5B-c), indicating that the enzyme

Fig. 3. Effect of harmine on in vitro angiogenesis. (A) Harmine inhibited motility of HUVEC cells toward wound area in a dose dependent manner. B) Harmine inhibited VEGF-induced
migration of HUVECs through ECM coated membrane. C) Migration was quantified by counting the cell migrated towards wound area. D) Invasion was quantified by counting the
cells that invaded to the lower side of the filter after fixation and staining and photographed at 200× magnification and% inhibiton was calculated. All the values are mean ± S.D.
(mean of triplicate). ⁎⁎⁎ P b 0.001, ⁎⁎ P b 0.01 compared to control.
70 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73
A pre-requisite for the growth of solid tumours, vascular targeting has
Control 2 µg/mL
B a
b c
Fig. 4. Effect of harmine on in vitro angiogenesis. A) Harmine inhibited VEGF-induced
tube formation of HUVECs. Photographs were taken at 200× magnification. B) Harmine
inhibited VEGF-induced vessel sprouting from rat aortic ring. a) Conditioned medium
(CM) alone b) CM + harmine (2 μg/ml). c) CM pretreated with harmine (2 μg/ml).
Experiments were repeated three times.
responsible for the degradation was metalloproteinase. CM from the
harmine treated (1 and 2 μg/ml) HUVEC cells after the trypsin
activation did not give any clear degraded areas (Fig. 5B-d, e),
indicating that harmine treatment could inhibit the production of
matrix metalloproteases. CM from the harmine treated (0.5 μg/ml)
HUVEC cells after the trypsin activation gave partially degraded areas
(Fig. 5B-f), indicating that a lower dose of harmine treatment partially
inhibited the production of matrix metalloproteases.
3.13. Effect of harmine on the expression of VEGF, iNOS, and COX-2
Harmine treatment significantly down-regulated the expression of
VEGF, iNOS and COX-2 transcript levels by B16F-10 melanoma cells
compared with the untreated control (Fig. 5C). In untreated control, the
percentage expression of VEGF, iNOS and COX-2 were found to be 91.7%,
79.2% and 52.2% respectively compared to an internal control GAPDH
(100%). Harmine treatment down-regulated the level of expression of
VEGF, iNOS and COX-2, mRNA and percentage expression were 45.2%,
52.3% and 15.3%, respectively compared to GAPDH (100%) of treated
group (Fig. 5D). These results suggest that the anti-angiogenic effect of
harmine is associated with a down-regulation of pro angiogenic factors.
4. Discussion
Tumour angiogenesis is a complex process and involves tight
interplay of tumour cells, endothelial cells, phagocytes and their
secreted factors which may act as promoters or inhibitors of
angiogenesis (Ferrara, 2000). VEGF is the most important mitogenic
and survival factor for vascular endothelial cells. Since angiogenesis is
been explored as a potential strategy to suppress tumour growth and
metastasis. Several angiogenic inhibitors have been developed for
targeting angiogenic factors or their receptors, extracellular matrix
compounds, and vascular endothelial cell proliferation. Among them,
thalidomide and TNP-470 are known to inhibit the growth of
endothelial cells (D'Amato et al., 1994). In addition several phytochem-
icals have been evaluated for inhibiting various components of the
mitogenic, cell survival and inflammatory signaling in tumour cells
associated with the production of angiogenic factors (Fotsis et al., 1997).
Soybean phytochemicals (Zhou et al., 1999), resveratrol from grapes
(Brakenhielm et al., 2001) and Andrographaloide from Andrographis
paniculata (Sheeja et al., 2007) are some of the examples.
The various steps in angiogenesis including degradation of the
extracellular matrix surrounding the parent vessel, migration and
proliferation of the endothelial cells and mural cells to assemble the
new vessel, lumen formation, can be studied using both in vitro and in
vivo assays. In our study when B16F-10 melanoma cells were
introduced intradermally to the mouse, tumour directed capillary
formation was increased as a tumour grows indicating a direct
correlation between the tumour survival and vessel count (Weidner
et al., 1991) Administration of harmine significantly inhibited the
tumour specific new blood vessel formation in C57BL/6 mice when
they were induced angiogenesis with B16F-10 melanoma cells.
The serum VEGF level was highly elevated as quickly as 24 h after
tumour challenge, while treatment with harmine significantly inhibited
this elevation of serum VEGF level. This anti-VEGF activity of these
compounds may be due to their down regulatory action on the VEGF
mRNA expression, as is evident from the inhibition in the expression of
VEGF mRNA by B16F-10 melanoma cells upon treatment with harmine.
There is considerable evidence to suggest that angiogenesis and
inflammation occur in an interactive and overlapping manner
(Reinders et al., 2003). Inflammatory signals regulate the expression
and secretion of various angiogenic factors (Ono et al., 1999). In
pathological angiogenesis like tumour angiogenesis the autocrine,
paracrine and amphicrine interactions of the vascular endothelium
with the pro-angiogenic and angiostatic cytokines and growth factors
are irregularly regulated (Hanahan, 1997). Pro inflammatory cyto-
kines such as IL-1β, IL-6, TNF-α and GM-CSF contribute in the process
of neovascularization (Kofler et al., 2005). IL-1β has been proposed as
the primary driver of cell growth, proliferation, matrix degradation
and angiogenesis in invasive melanoma (Apte and Voronov, 2002;
Dinarello, 1996). In our experiment, harmine treatment was found to
inhibit IL-1β production in angiogenesis induced animals. Thus,
harmine mediated inhibition of IL-1β may contribute to diminished
tumour angiogenesis, probably because secretable IL-1β activates, by
autocrine/paracrine mechanisms, an inflammatory cascade in the
tumour's micro-environment. IL-6 induces transcriptional activation
of VEGF and regulates VEGF promoter activity via direct interaction
with STAT3. Serum IL-6 and VEGF levels were positively correlated in
advanced cancers (Maruno et al., 1997). TNF-α enhances VEGF, IL-
8 and bFGF production in human microvascular endothelial cells and
induces tubular morphogenesis at the site of inflammation/tumour
induction. GM-CSF is expressed in solid tumours and potentially
enhances tumour cell proliferation and migration as well as tumour
angiogenesis (Bussolino et al., 1991). In this study it was found that
the serum levels of IL-6, TNF-α, and GM-CSF in control animals were
highly elevated after tumour induction indicating angiogenesis
induced inflammation. But harmine treatment inhibited the produc-
tion of these cytokines and the inhibition in these pro-inflammatory
cytokines might be the cause for the reduction of serum VEGF levels of
treated animals, since most of these pro-inflammatory cytokines are
good inducers of VEGF.
IL-2 is of clinical value in natural immunity by stimulating a natural
killer cell and cytotoxic T-lymphocyte production (Neville et al.,
2001). It was reported that IL-2 presurgical immunotherapy may
 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73 71

Fig. 5. A) Effect of harmine on transcription factors: B16F-10 melanoma cells were treated with harmine (2 μg/ml) for 2 h and then incubated with TNF-α (10 pg/ml) for 30 min.
Percentage inhibition in various transcription factors were measured by ELISA method. B) Inhibitory effect of harmine on the production of matrix metalloproteinases. (a) CM from
untreated HUVECs without trypsin activation. (b) CM from untreated HUVECs with trypsin activation (c) CM from untreated HUVECs with trypsin activation + EDTA. (d) CM from
untreated HUVECs with trypsin activation + harmine (2 μg/ml). (e) CM from untreated HUVECs with trypsin activation + harmine (1 μg/ml). f) CM from untreated HUVECs with
trypsin activation + harmine (0.5 μg/ml). C) Effect of harmine VEGF-α, iNOS, and COX-2 m RNA expression by B16F-10 melanoma cells. The mRNA expression levels in each group
were normalized with GAPDH (house-keeping gene). D) Band intensity was measured using software provided with vilber Lourmat gel documentation system and percentage
expression of mRNA was calculated.

counteract surgery-induced stimulation of the angiogenesis, by either
opposing the decline in blood levels of the anti-angiogenetic cytokine
IL-12, or reducing the increase in those of the angiogenic factor VEGF.
Therefore, the control of VEGF secretion could influence the efficacy of
IL-2 (Brivio et al., 2002). In the present investigation, we found that
administration of harmine significantly increased IL-2 levels, which
may contribute to the stimulation of the immune system against the
tumour growth by VEGF-induced angiogenesis. TIMP inhibits the
activities of MMP-1, MMP-2 and MMP-9, which leads to reduced ECM
remodeling and suppression of endothelial cell migration and
invasion (Gomez et al., 1997b). TIMP-1 was increased in the harmine
administered group suggesting that increased endogenous TIMP-1
could inhibit MMP activity and consequently inhibit ECM remodeling.
Similar to other pro-angiogenic factors, intradermal introduction of
highly metastatic B16F-10 melanoma cells resulted in drastic eleva-
tion of serum nitrite level of untreated angiogenesis induced animals,
which may be the reason for increased tumour directed capillary
formation in them. But the administration of harmine significantly
reduced the elevated serum nitrite level after tumour challenge,
which in turn resulted in decreased tumour directed capillary
formation.
Inflammation-driven expression of an angiogenic factor can also
be mediated by the activation of NF- B signaling (Bhat and Singh,
2008). VEGF is one of the most potent angiogenic factors that can be
regulated by activated NF- B, because its genes have a B binding site
in its promoter region (Kang et al., 2005; Karin, 2006). Inhibition of
NF- B activity leads to decreased expression of VEGF and IL-8 in
several types of cancer cells and is associated with the suppression of
angiogenesis, invasion and metastasis in cancer (Huang et al., 1999).
One of the genes expressed as a result of NFκB signaling in
angiogenesis is the prostaglandin synthesis enzyme, COX-2. Impor-
tantly, the COX-2 enzyme is involved in the induction of VEGF and
FGF-2 growth factors that stimulate angiogenesis (Boosani et al.,
2007). COX-2 expression is also induced by pro inflammatory and
mitogenic stimuli such as growth factors (VEGF) and cytokines (TNF-
α and IL1-β). COX-2 has also been implicated as playing roles in
endothelial cells spreading and migration (Dormond et al., 2002; Zaric
and Ruegg, 2005). Vasodilation, by smooth muscle relaxation,
mediated by nitric oxide is a pre-requisite for endothelial cells to
enter angiogenic cascade (Griffioen and Molema, 2000). Since COX-2
and iNOS play important roles in the progression of various types of
cancer by stimulating angiogenesis, invasion, and apoptosis (Cuezva
et al., 2002; Gasparini et al., 2003), we tested whether harmine blocks
the activity of these molecules. Expression analysis clearly demon-
strated that treatment of B16F-10 cells with harmine could signifi-
cantly down-regulate the production of iNOS and COX-2.
To test the anti-angiogenic activity effects of harmine in vitro, we
treated HUVEC cells with harmine and analyzed various steps in
angiogenesis. Assays that allow measurement of endothelial cell
motility in response to added factors include the proliferation, migration
assay (Scrape wound), invasion assay (transwell), etc. (Lampugnani,
1999). Effects of harmine on cell survival and proliferation were
measured by direct cell counting MTT assay and thymidine incorpora-
tion assay. It was found that the rate of proliferation was decreased in a
72 T.P. Hamsa, G. Kuttan / European Journal of Pharmacology 649 (2010) 64–73
dose dependent manner. Because of the prime role of MMP in
endothelial cell invasion and migration, the inhibition of the activation
of MMPs by harmine resulted in the significant reduction of endothelial
cell motility and invasion though collagen matrix in response to VEGF in
a dose dependent manner. The tube formation stage of angiogenesis can
be modeled in vitro by plating endothelial cells with matrigel (Kubota et
al., 1988). The formation of tubes on matrigel involves endothelial cell
attachment, migration and production of enzymes capable of remodel-
ing ECM. It was shown that a reduction in tube formation by harmine
treated endothelial cells was associated with a decrease in the
proteolytic activities of both MMP-2 and MMP-9. The aortic ring assay
is an organ culture model in which angiogenic vessels grow from a
segment of the aorta (Masson et al., 2002). The vessels that grow out
from aortic rings are anatomically similar to neovessels in vivo
(Goodwin, 2007). A marked decrease in vessel out growth from the
rat aortic ring was observed in harmine treated condition medium. This
may be due to the absence of pro-angiogenic factors in CM. The effect of
harmine on MMPs was confirmed by gelatin zymographic analysis and
it was clearly revealed that harmine inhibited the production of MMP by
HUVEC cells in vitro.
In conclusion, we present experimental evidence suggesting that
harmine exerts multiple effects on the inhibition of tumour
angiogenesis and it's mechanism of anti-angiogenesis is pleotropic.
Our data clearly shows that harmine considerably inhibits tumour
directed neo-vessel formation by inhibiting pro-angiogenic and pro-
inflammatory factors in vivo and in vitro at relatively lower
concentrations so harmine may be used as anti-tumour agent by
targeting angiogenesis and inflammation.
Acknowledgment
We acknowledge Dr. Ramadasan Kuttan, Research Director, Amala
Cancer Research Centre, for his valuable suggestions and support in
this study.
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