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Cancer Therapies
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Inhibition of B16F-10 MelanomaInduced Lung Metastasis in C57BL/6 Mice by Aerva lanata via Induction
of Apoptosis
Kodappully Sivaraman Siveen and Girija Kuttan
Integr Cancer Ther 2013 12: 81 originally published online 26 November 2012
DOI: 10.1177/1534735412443853
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XXX10.1177/1534735412443853Siveen and KuttanIntegrative Cancer Therapies


The Author(s) 2010
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Inhibition of B16F-10 MelanomaInduced


Lung Metastasis in C57BL/6 Mice by Aerva
lanata via Induction of Apoptosis

Integrative Cancer Therapies


12(1) 8192
The Author(s) 2013
Reprints and permission:
sagepub.com/journalsPermissions.nav
DOI: 10.1177/1534735412443853
http://ict.sagepub.com

Kodappully Sivaraman Siveen, MSc, MPhil1, and Girija Kuttan, PhD1

Abstract
In this study, the antimetastatic potential of the ethanolic extract of Aerva lanata was evaluated using the B16F-10 melanoma
induced lung metastasis model. Metastasis was induced in C57BL/6 mice by injecting highly metastatic B16F-10 melanoma
cells through the lateral tail vein. Simultaneous treatment with A lanata inhibited tumor nodule formation in the lungs
(70.53%), and there was a 65.3% increase in the survival rate of metastatic tumorbearing animals. These results correlated
with biochemical parameters such as lung collagen hydroxyproline, hexosamine, and uronic acid contents; serum sialic acid
and -glutamyl transpeptidase levels; and histopathological analysis. In vitro studies using B16F-10 cells showed that A lanata
inhibited migration of tumor cells, cell invasion through type-I collagen-coated polycarbonate filter and activation of matrix
metalloproteinases. Treatment with A lanata induced apoptotic response, characterized by apoptotic morphology, a typical
ladder of DNA fragmentation, and detection of 3 hydroxyl ends in DNA by TUNEL assay. There was an increase in the
percentage of cells in the sub-G0/G1 phase indicating cell cycle arrest. A lanata treatment resulted in downregulation of bcl2 and cyclin-D1 expression and upregulation of p53, bax, caspase-9, caspase-3, p21, and p27 gene expression in B16F-10 cells.
Proinflammatory cytokine production and gene expression were also found to be downregulated in A lanatatreated cells.
Keywords
Aerva lanata, B16F-10 melanoma, metastasis, matrix metalloproteinases, caspases, cell cycle arrest

Introduction
Cancer is one of the leading causes of death worldwide.
Among cancers, melanoma is the most malignant skin cancer, and its occurrence has remarkably increased during the
past few decades.1 Worldwide, 9 million deaths result from
cancer each year, which may rise to 20 million by 2020, as
anticipated by WHO.
The hallmarks of a cancer cell are self-sufficiency of
growth signals, resistance to growth-inhibitory signals, resistance to apoptosis, sustained angiogenesis, and the acquisition of invasive and metastatic potential. Most malignancies
have defects in the apoptotic regulatory pathways, leading to
defects in apoptosis. So one of the crucial therapeutic strategies against cancer is to trigger a cellular suicide pathway and
induce tumor cell apoptosis in response to stimuli using suitable drugs.2
The most devastating aspect of cancer is metastasis,
which is often resistant to conventional therapies such as chemotherapy and radiation therapy. In the majority of cancer
patients, by the time of diagnosis of the primary tumor, metastasis to the regional lymph node and/or distant organs has
occurred.3 An important step in metastasis is the breakdown

of connective tissue barriers, which is mediated by proteolytic activity. Typically, proteases such as matrix metalloproteinases (MMPs) are overexpressed and are localized to
the tumor cell surface at the invadopodia to mediate extracellular matrix (ECM) breakdown and facilitate invasion.4
Apoptosis is a controlled physiological process that
occurs in a morphologically and biochemically distinct manner and ultimately leads to cell death. The process involves a
sequence of events, including cell shrinkage, increased cytoplasmic density, chromatic condensation and segregation
into sharply circumscribed masses, and the formation of
membrane-bound surface apoptotic bodies.5 Apoptotic cells
are phagocytized from the midst of living tissues by neighboring cells or macrophages without eliciting an inflammatory reaction. The apoptotic process is misregulated in
various diseases, including cancer, and alterations in the
1

Amala Cancer Research Centre, Thrissur, Kerala, India

Corresponding Author:
Girija Kuttan, Department of Immunology, Amala Cancer Research
Centre, Amala Nagar, Thrissur, Kerala, India
Email: girijakuttan@gmail.com

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Integrative Cancer Therapies 12(1)

function of apoptosis-related genes have a profound influence on tumor progression. Therefore, activation of the
apoptotic pathway in tumor cells is considered to be a protective mechanism against the development and progression of cancer.
Aerva lanata, belonging to the family Amaranthaceae, is
an important medicinal plant grown as a common garden
plant in India. It is used in Indian folk medicine for the treatment of diabetes mellitus, urinary calculi, hematemesis,
bronchitis, nasal bleeding, cough, and so on.6 Our previous
studies with ethanolic extract of A lanata showed that it could
enhance the total white blood cell count, the number of
plaque-forming cells in the spleen, circulating antibody titer,
and proliferation of splenocytes, thymocytes, and bone marrow cells and inhibit Daltons lymphoma ascites (DLA)induced solid tumor development in mice.7 In the present
study, the effect of ethanolic extract of A lanata on the inhibition of pulmonary metastasis induced by B16F-10 melanoma
cells in C57BL/6 mice was evaluated with special emphasis
on the mechanism of action, using in vitro models.

Materials and Methods


Animals
C57BL/6 mice (20-25 g body weight, 6- to 8-week-old
males) were purchased from the National Institute of
Nutrition, Hyderabad, India. The animals were fed with
mouse chow (Sai Feeds, India) and water ad libitum. All the
animal experiments were carried out with the prior approval
of the Institutional Animal Ethics Committee and were conducted strictly adhering to the guidelines of the Committee
for the purpose of Control and Supervision of Experiments
on Animals constituted by the Animal Welfare Division of
the Government of India.

Cell Line
B16F-10 melanoma, a highly metastatic cell line, was
obtained from the National Centre for Cell Sciences, Pune,
India. The cells were maintained in Dulbeccos modified
Eagles medium (DMEM) and supplemented with 10% fetal
calf serum (FCS) and antibiotics.

Chemicals
DMEM was obtained from Himedia (Mumbai, India). Fetal
bovine serum was procured from Life Technologies (Grand
Island, NY). Hydroxyproline and glucuronic acid lactone
were purchased from Sigma Chemicals (St Louis, MO).
N-acetyl neuraminic acid was purchased from Sisco
Research Laboratory (Mumbai, India). Oligonucleotide
primer sequences were purchased from Sigma Aldrich
(Bangalore, India; Table 1). Highly specific quantitative

sandwich ELISA kit for mouse interleukin (IL)-1, IL-2,


IL-6, tumor necrosis factor (TNF)-, and granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from Pierce Biotechnology (USA), and ELISA kits
for vascular endothelial growth factor (VEGF) and tissue
inhibitors of MMPs (TIMP-1) were purchased from R & D
system (USA). ApopTag Peroxidase in situ apoptosis detection kit was purchased from CHEMICON International Inc
(Massachusetts). Cycletest Plus DNA reagent kit was purchased from Becton-Dickson, Canada. All other reagents
were of analytical grade.

Preparation of Plant Extract


Authenticated A lanata obtained from Amala Ayurveda
Pharmacy was shade dried and powdered. Then, 100 g of
powder was subjected to 85% ethanolic extraction in a
Soxhlet apparatus for 24 hours. The solvent was evaporated
to dryness at 42C under reduced pressure using a rotary
evaporator. The yield of the extract was 16.7% (w/w).7
The extract was redissolved in a minimum amount of
dimethyl sulfoxide for in vitro studies. For animal experiments, it was resuspended in 1% gum acacia and administered intraperitoneally at a dose of 10 mg/kg body weight
(based on toxicity studies). The extract was administered
using 3 different modalities as follows;
1. Prophylactic administration: animals were treated
with 10 consecutive doses of the drug prior to
tumor inoculation.
2. Simultaneous administration: the drug was given
to the animals simultaneously with metastatic
tumor cells and continued for 10 consecutive days.
3. Developed administration: 10 days after tumor
inoculation, the drug was administered on 10 consecutive days.

Determination of Antimetastatic
Activity in the In Vivo System
Pulmonary colonization assay. C57BL/6 mice were divided
into 4 groups (n = 16). Metastasis was induced in all the
animals by injecting 106 B16F-10 melanoma cells through
the lateral tail vein. To 3 groups of animals, the ethanolic
extract of A lanata was administered intraperitoneally at a
dose of 10 mg/kg body weight for 10 consecutive days using
3 different modalities: prophylactic administration (group I),
simultaneous administration (group II), and developed
administration (group III). Group IV animals were untreated
metastatic tumorbearing controls. Eight animals from each
group were killed on the 21st day after tumor challenge; the
lungs were excised, and blood was collected. The lungs were
used for morphological examinations of metastatic tumor
nodules and for the estimation of collagen hydroxyproline,8

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Siveen and Kuttan


Table 1. Oligonucleotide Primer Sequences
Genes
MMP-2

MMP-9

TIMP-1

TIMP-2

ERK-1

ERK-2

nm-23

VEGF

p53

caspase-8

caspase-9

caspase-3

bcl-2

bid

bax

p21

p27

cyclin D1

GAPDH

Primer Sequence
Forward 5-GAGTTGGCAGTGCAATACCT-3
Reverse 5-GCCGTCCTTCTCAAAGTTGT-3
Forward 5-AGTTTGGTGTCGCGGAGCAC-3
Reverse 5-TACATGAGCGCTTCCGGCAC-3
Forward 5-CTGGCATCCTCTTGTTGCTA-3
Reverse 5-AGGGATCTCCAGGTGCACAA-3
Forward 5-AGACGTAGTGATCAGGGCCA-3
Reverse 5-GTACCACGCGCAAGAACCAT-3
Forward 5-GCACGACCACACTGGCTTTC-3
Reverse 5-GATCAACTCCTTCAGCCGCTC-3
Forward 5-ACAGGACCTCATGGAGACGG-3
Reverse 5-GATCTGCAACACGGGCAAGG-3
Forward 5-CTCAGCCTTAATTTTTTCCCCC-3
Reverse 5-TTAACTTCCGACACTGGGTGT-3
Forward 5-TGCTCACTTCCAGAAACACG-3
Reverse 5-GGAAGGGTAAGCCACTCACA-3
Forward 5-CGGAGGTCGTGAGACGCTG-3
Reverse 5-CACATGTACTTGTAGTGGATGGTGG-3
Forward 5-TGCTTGGACTACATCCCACAC-3
Reverse 5-TGCAGTCTAGGAAGTTGACCA-3
Forward 5-GACGCTCTGCTGAGTCGAG-3
Reverse 5-CTTTGCTGTGAGTCCCATTGG-3
Forward 5-GAGCACTGGAATGTCATCTCGCTCTG-3
Reverse 5-TACAGGAAGTCAGCCTCCACCGGTATC-3
Forward 5-CAGCTGCACCTGACGCCCTT-3
Reverse 5-CCCAGCCTCCGTTATTCTGGA-3
Forward 5-GCCGAGCACATCACAGACC-3
Reverse 5-TGGCAATGTTGTGGATGATTTCT-3
Forward 5-CCGGCGAATTGGAGATGAACT-3
Reverse 5-CCAGCCCATGATGGTTCTGAT 3
Forward 5-GTGGCCTTGTCGCTGTCTT-3
Reverse 5-GCGCTTGGAGTGATAGAAATCTG-3
Forward 5-GGGCAGATACGAGTGGCAG-3
Reverse 5-CCTGAGACCCAATTAAAGGCAC-3
Forward 5-GTCATCAAGTGTGACCCG-3
Reverse 5-GCACAGTCTGCCTGATGC-3
Forward-5-CGTCCCGTAGACAAAATGGT-3
Reverse-5-CCTTCCACAATGCCAAAGTT-3

Product Size (bp)


354

327

414

525

512

216

310

453

205

169

288

414

235

226

229

155

126

401

557

Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitors of MMPs; ERK, extracellular regulated kinases;VEGF, vascular endothelial growth
factor.

hexosamine,9 and uronic acid10 contents. Serum was separated from the blood and used for determining the sialic
acid11 and -glutamyl transpeptidase (GGT)12 levels. Serum
IL-2, VEGF, and TIMP-1 levels were estimated using the
ELISA kit per manufacturers protocol. A lung from 1 animal was used for histopathological analysis and from
another animal for total RNA isolation. The remaining 6
animals in each group were observed for their survival. The
mortality of the animals was observed, and the percentage
increase in life span was calculated.

Analysis of the expression of various prometastatic and


antimetastatic genes. Total RNA was isolated from the
lungs, and cDNA was synthesized using Moloney murine
leukemia virus reverse transcriptase. Amplification was
performed using specific primers of MMP-2, MMP-9,
TIMP-1, TIMP-2, extracellular regulated kinases (ERK)-1,
ERK-2, VEGF, and nm23 (Table 1). The amplified products were electrophoresed on a 1.8% agarose gel, stained
with ethidium bromide, and photographed under ultraviolet light.

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Integrative Cancer Therapies 12(1)

Histopathological analysis. Lung tissues were fixed in 10%


formalin, dehydrated in different concentrations of alcohol,
and embedded in paraffin wax. Sections (4 m) were stained
with eosin and hematoxylin.

Determination of Antimetastatic
Activity Using In Vitro Models
The following parameters were used to assess the antimetastatic action using the in vitro system.
Cell viability. B16F-10 melanoma cells were seeded (5000
cells/well) in 96-well flat-bottomed titer plates and incubated for 24 hours at 37C in 5% CO2 atmosphere. Different
concentrations of A lanata (1-500 g/mL) were added and
incubated further for 48 hours. Then, 4 hours before completion of incubation, 20 L 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (5 mg/mL) was added.13,14
The percentage of viable cells was determined using an
ELISA plate reader set to record absorbance at 570 nm.
Proliferation assay. B16F-10 melanoma cells (5000 cells/
well) were plated in a 96-well culture plate and incubated at
37C in 5% CO2 atmosphere. After 24 hours, various concentrations of A lanata (5, 10, and 25 g/mL) were added and
further incubated for 48 hours. 3H-thymidine was added to
each well (1 Ci/well) and incubation continued for 18 hours
more. After completing incubation, the radioactivity was
measured15 using a Rack Beta liquid scintillation counter.
Collagen matrix invasion assay. The invasion assay was
carried out in modified Boyden chambers as described by
Albini et al.16 The lower compartment of the chamber was
filled with serum-free DMEM, and a polycarbonate filter
coated with 25 g type I collagen was placed above this.
B16F-10 melanoma cells (105 cells/150 L DMEM) were
then seeded on to the upper chamber in the presence and
absence of A lanata (5, 10, and 25 g/mL) and incubated at
37C in 5% CO2 atmosphere for 10 hours. After incubation,
the filters were removed, and the cells were 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.
Migration assay. Analysis of the effect of A lanata on the
migration of the tumor cells was carried out using a Boyden
chamber. The polycarbonate filters were placed in Boyden
chambers. The lower compartment of the chamber was filled
with serum-free DMEM, and B16F-10 cells (105 cells/chamber)
suspended in DMEM were then seeded on to the upper
chamber in the presence and absence of A lanata (5, 10, and
25 g/mL) and incubated for 10 hours at 37C in 5% CO2.
The number of cells that migrated to the lower compartment
was determined, and results are expressed as percentage
inhibition.
Gelatin zymography. SDS-PAGE was performed with 5%
gelatin incorporated in the separating gel.17 B16F-10 melanoma

cells of subconfluent cultures were incubated with serumfree medium for 24 hours at 37C in 5% CO2 atmosphere.
The conditioned medium was then collected and subjected
to zymographic analysis. Then, 50 L of sample (equivalent
to 100 g protein) was activated with 5 L trypsin solution
(75 g/mL) in the presence and absence of A lanata (5, 10,
and 25 g/mL) in 0.1 M TrisHCl, 10 mM CaCl2 buffer (pH
8.0) and incubated for 1 hour at room temperature. Samples
were mixed with an equal volume of 2 sample buffer and
loaded on to 11% polyacrylamide gels containing 5% gelatin. Electrophoresis was carried out at 4C with a 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 TrisHCl, 10 mM CaCl2 at 37C for 18 hours followed by staining with GelCode Blue stain reagent for 2
hours. Gels were destained to visualize the clear area against
the dark background.

Determination of the Effect of


A lanata on the Apoptosis of B16F-10 Cells
The effect of A lanata on the apoptosis of B16F-10 melanoma cells were analyzed by various parameters, such as
morphological analysis, DNA fragmentation analysis,
TUNEL assay, cell cycle analysis, and expression studies of
proapoptotic and antiapoptotic genes by reverse transcription polymerase chain reaction (RT-PCR).
Morphological analysis. B16F-10 melanoma cells (5000 cells/
well) suspended in DMEM supplemented with 10% FCS, 100
g/mL streptomycin and penicillin, and 2 mmol/L glutamine
were plated in a 96-well flat-bottomed titer plate and incubated
for 24 hours at 37C in 5% CO2 atmosphere. After 24 hours,
different concentrations of A lanata (5, 10, and 25 g/mL) were
added to the cells and incubated further for 48 hours under the
same conditions. The cells were then washed with phosphatebuffered saline (PBS; pH 7.4), fixed with 5% formalin, stained
using hematoxylin and eosin, and observed under phase contrast microscopy; photographs were also taken. Apoptosis was
characterized by examining the morphological changes such as
chromatin condensation, nuclear condensation, membrane
blebbing, or presence of apoptotic bodies.
DNA fragmentation analysis. One million B16F-10 melanoma cells were treated with different concentrations of A
lanata (5, 10, and 25 g/mL) as described in the previous
experiment. After incubation, the cells were treated with 0.1
mL lysis buffer (100 mmol/L TrisHCl, pH 8.0, containing
0.2% Triton-X 100, and 1 mmol/L ethylene diamine tetraacetic acid [EDTA]) for 10 minutes at 20C. DNA was
extracted using the phenol-chloroform method, precipitated
with chilled ethanol, and resuspended in Tris/EDTA buffer
(10 mmol/L TrisHCl, pH 8.0 and 1 mmol/L EDTA). DNA
samples were separated by electrophoresis in 1% agarose
gels. DNA was stained with ethidium bromide and photographed under UV light.

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Siveen and Kuttan


TUNEL assay. TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay was done to detect
apoptosis via DNA fragmentation using ApopTag peroxidase in situ apoptosis detection kit. B16F-10 melanoma
cells (5000 cells/well) suspended in DMEM supplemented
with 10% FCS, 100 g/mL streptomycin and penicillin, and
2 mmol/L glutamine were plated in a 96-well flat-bottomed
titer plate and incubated for 24 hours at 37C in 5% CO2
atmosphere. After incubation, 25 g/mL A lanata was added
to the cells, and they were incubated for a further 48 hours
under the same conditions. The cells were washed in PBS
and stained per manufacturers protocol.
Determination of the effect of A lanata on cell cycling of
B16F-10 melanoma cells. The cell apoptotic rate was measured
by quantification of the percentage of cells with sub-G0/G1
DNA levels, as determined via flow cytometry analysis.
Briefly, 1 106 logarithmically growing B16F-10 cells suspended in DMEM with 10% FCS were seeded in a culture
flask. The cells were treated with A lanata (25 g/mL) and
incubated for 24 hours at 37C in a CO2 atmosphere. After
incubation, the cells were washed with PBS and stained
with propidium iodide. DNA content was analyzed by a
fluorescence-activated cell sorter (Becton Dickinson, FACS
Calibur) using the Cycletest Plus DNA reagent kit per the
manufacturers protocol.

Determination of the Effect of A lanata


on the Expression of p53, caspase-9,
caspase-3, caspase-8, bcl-2, bid, bax, p21,
p27, and cyclin-D1 using RT-PCR
B16F-10 melanoma cells (2 104 cells) suspended in
serum-free DMEM (250 L) were seeded in a 96-well titer
plate and incubated for 24 hours at 37C in 5% CO2 atmosphere. A lanata at a concentration of 25 g/mL was added,
and incubation was continued for another 4 hours. cDNA was
synthesized using cell to cDNA kit (Ambion Inc, California,
USA). Cells were washed with PBS and heated in cell lysis
buffer (provided in the kit) to release the RNA into the solution followed by a heating step to inactivate endogenous
RNases. The genomic DNA was further degraded by treating with DNase followed by inactivation of DNase by heating at 70C. RT was performed at 42C for 50 minutes
using Moloney murine leukemia virus RT (supplied along
with the kit). Gene expression analysis was done by PCR.
The mouse p53, caspase-9, caspase-3, caspase-8, bcl-2, bid,
bax, p21, p27, and cyclin-D1 (Table 1) were amplified
against GAPDH standard. The cycling conditions used were
as follows: 1 minute at 94C, 1 minute at 58C, and 1 minute at 72C for 40 cycles, followed by a 10-minute extension
at 72C. Amplified samples were subjected to electrophoresis in an agarose gel (1.5%) containing 0.5 g/mL ethidium
bromide and photographed under UV light.

Table 2. Effect of Aerva lanata on Lung Tumor Colony Formation


and Survival Rate1,2
Treatment
Tumor control
Prophylactic
Simultaneous
Developed

Inhibition on Lung
Colony Formation
0%
62.36%a
70.53%b
38.51%c

Percentage
ILS
0%
48.2%a
65.3%b
27.1%c

Abbreviation: ILS, increase in life span.


1
B16F-10 melanoma cells (1 106) were injected into each animal via
the lateral tail vein. Aerva lanata was administered intraperitoneally for 10
days. Animals were killed on the 21st day and lung tumor nodules counted. For the survival study, death caused by tumor burden was recorded,
and the life span was calculated.Values are mean standard deviation.
2
a, b, c: Means without a common superscript differ (P < .05).

Determination of the Effect of A lanata


on Proinflammatory Cytokines
and GM-CSF Levels
B16F-10 melanoma cells (5000 cells/well) suspended in
DMEM supplemented with 10% FCS, 100 g/mL streptomycin and penicillin, and 2 mmol/L glutamine were plated
in a 96-well flat-bottomed titer plate and incubated for 24
hours at 37C in 5% CO2 atmosphere. A lanata at a concentration of 25 g/mL was added to the cells, and they were
incubated for a further 48 hours under the same conditions.
The supernatant was used for the estimation of cytokines;
IL-1, IL-6, TNF-, and GM-CSF using specific ELISA
kits per manufacturers protocol.

Statistical Analysis
Values are expressed as mean standard deviation. The
statistical analysis was done by 1-way ANOVA followed by
Tukeys multiple comparison using Graphpad InStat software. A P value <.05 was considered to be statistically
significant.

Results
Antimetastatic Activity in the In Vivo System
Effect of A lanata on the inhibition of lung metastasis and
survival. Metastatic tumor-bearing animals treated with A
lanata showed significant reduction in tumor nodule formation (Table 2). Metastatic control animals had massive tumor
growth and were assigned an arbitrary number of 250.18 The
3 different modalities of compound administration were
found to be significantly (P < .05) effective. Of these, simultaneous mode of administration of ethanolic extract of A
lanata (10 mg/kg body weight, intraperitoneal) produced
maximum inhibition of lung nodules (70.53%) followed by

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Table 3. Effect of Aerva lanata on Lung Collagen Hydroxyproline,


Hexosamine, and Uronic Acid Levels of Metastatic Tumor
Bearing Animals1,2

Treatment
Normal
Tumor control
Prophylactic
Simultaneous
Developed

Collagen
Hexosamine
Hydroxyproline (mg/100 mg
(g/mg Protein)
Tissue)
1.86 0.07a
21.25 0.94b
10.37 0.59c
8.41 0.52d
16.31 0.94e

0.42 0.01a
3.39 0.18b
1.44 0.09c
1.28 0.07d
2.32 0.12e

Uronic Acid
(g/100 mg
tissue)
25.74 1.83a
316.72 17.29b
174.81 7.51c
158.30 9.86d
217.95 12.47e

B16F-10 melanoma cells (1 106) were injected into each animal via the
lateral tail vein. A lanata was administered intraperitoneally for 10 days.
Animals were killed on the 21st day, lungs were dissected out, and the
levels of lung hydroxyproline, hexosamine, and uronic acid were determined.Values are mean standard deviation.
2
a, b, c, d, e: Means without a common superscript differ (P < .05).

prophylactic mode of administration (62.36%) and then


administration after tumor development (38.51%).
Administration of A lanata significantly increased the
life span of metastatic tumorbearing animals (Table 2).
The life span was highly enhanced when A lanata was
administered simultaneously (65.3%). Prophylactic administration of A lanata enhanced the life span by 48.2%,
whereas administration after tumor development increased
the lifespan by 27.1%.
Effect of A lanata on lung and serum biochemical parameters of metastatic tumorbearing animals. The effect of A
lanata on lung collagen hydroxyproline, hexosamine, and
uronic acid content is shown in Table 3. Tumor-bearing
control animals showed an increased level of lung collagen
hydroxyproline (21.25 0.94 g/mg protein), which was
significantly (P < .05) reduced in animals treated with A
lanata using prophylactic (10.37 0.59 g/mg protein),
simultaneous (8.41 0.52 g/mg protein), and developed
(16.31 0.94 g/mg protein) modalities.
Tumor-bearing control animals had a high level of lung
uronic acid content (316.72 17.29 g/100 mg tissue wet
weight; P < .05) when compared with normal animals
(25.74 1.83 g/100 mg tissue wet weight). Treatment of A
lanata reduced the uronic acid content in all the 3 modalities: prophylactic (174.81 7.51 g/100 mg tissue wet
weight), simultaneous (158.30 9.86 g/100 mg tissue wet
weight), and developed (217.95 12.47 g/100 mg tissue
wet weight).
Hexosamine content was also high in the lungs of tumor
controls (3.39 0.18 mg/100 mg tissue dry weight) when
compared with normal animals (0.42 0.01 mg/100 mg tissue dry weight). A lanata treatment significantly (P < .05)
reduced the lung hexosamine content in both the prophylactic (1.44 0.09 mg/100 mg tissue dry weight) and simultaneous (1.28 0.07 mg/100 mg tissue dry weight) modality

group, whereas it was only 2.32 0.12 mg/100 mg tissue


dry weight in the developed-modality group.
The effect of A lanata on serum biochemical parameters
is presented in Table 4. The serum sialic acid level of control metastatic tumor-bearing animals was highly increased
(134.76 8.39 g/mL serum) as compared with normal values (22.64 1.03 g/mL serum). Here also the simultaneous administration of A lanata significantly (P < .05)
reduced the elevated serum sialic acid level to 62.86 4.68
g/mL serum, followed by the prophylactic modality (68.93
3.17 g/mL serum); in the developed-modality group, it
was reduced only to 96.08 5.92 g/mL serum.
Serum -GGT level was also drastically (P < .05) enhanced
in metastatic control animals (104.27 7.94 U/L) compared
with normal animals (3.93 0.24 U/L). After the administration of A lanata, the elevated -GGT level was reduced significantly (P < .05) to 63.71 3.29 U/L in the prophylactic
group and 58.25 3.65 U/L in the simultaneous-modality
group. In animals with developed tumor, the serum -GGT
level was reduced to 82.64 5.72 U/L by A lanata treatment.
Effect of A lanata on serum IL-2, TIMP-1, and VEGF levels in
metastatic tumorbearing animals.. Serum VEGF, IL-2, and
TIMP-1 levels are shown in Table 4. The normal serum VEGF
level was only 18.37 0.82 pg/mL. This was significantly
increased (P < .05) to 163.71 10.2 pg/mL in metastatic
tumorbearing mice. This elevated level of serum VEGF was
reduced significantly (P < .05) in the groups with prophylactic
(89.04 3.49 pg/mL) and simultaneous (83.53 5.64 pg/mL)
modalities. The elevated serum VEGF level was also reduced
in animals with developed tumor (107.29 8.61 pg/mL) after
A lanata administration. There was a significant reduction in
the serum levels of IL-2 (9.84 0.51 pg/mL) and TIMP-1
(384.57 21.43 pg/mL) in tumor-bearing animals. Administration of A lanata using the prophylactic (21.53 1.39 and
583.14 28.62 pg/mL), simultaneous (24.76 1.92 and 603.21
23.79 pg/mL), and developed (27.38 1.06 and 540.86
16.35 pg/mL) modalities significantly (P < .05) restored the
levels of IL-2 and TIMP-1 to almost normal levels.
Effect of A lanata on the expression of various prometastatic and
antimetastatic genes. Expression of MMP-2, MMP-9, ERK-1,
ERK-2, and VEGF genes were downregulated in the groups
where A lanata was administered prophylactically and simultaneously with tumor induction (Figure 1). But there was a diminished expression of these genes in animals treated with A lanata
after tumor development. The expression of antimetastatic
genes TIMP-1 and TIMP-2 was minimal, and nm23 was nearly
absent in tumor-bearing animals. Treatment with A lanata
resulted in the upregulation in the expression of these genes.

Histopathological Analysis of Lungs


The hematoxylin and eosin stained sections of lung tissues
are shown in Figure 2 (100 magnification). Lungs in the
control animals showed infiltration of the neoplastic cells

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Siveen and Kuttan


Table 4. Effect of Aerva lanata on Serum Sialic Acid, -GT,VEGF, IL-2, and TIMP-1 Levels in Metastatic Tumor-Bearing Animals1,2
Treatment
Normal
Tumor control
Prophylactic
Simultaneous
Developed

Sialic acid (g/mL)

-GT (U/L)

VEGF (pg/mL)

IL-2 (pg/mL)

TIMP-1 (pg/mL)

22.64 1.03a
134.76 8.39b
68.93 3.17c
62.86 4.68c
96.08 5.92d

3.93 0.24a
104.27 7.94b
63.71 3.29c
58.25 3.65c
82.64 5.72d

18.37 0.82a
163.71 10.2b
89.04 3.49c
83.53 5.64c
107.29 8.61d

23.04 1.27a
9.84 0.51b
21.53 1.39a
24.76 1.92a
27.38 1.06c

613.48 24.07a
384.57 21.43b
583.14 28.62a
603.21 23.79a
540.86 16.35c

Abbreviations: GT, glutamyl transferase;VEGF, vascular endothelial growth factor; IL, interleukin; TIMP, tissue inhibitor of matrix metalloproteinase.
1
B16F-10 melanoma cells (1 106) were injected into each animal via the lateral tail vein. A lanata was administered intraperitoneally for 10 days. Animals were killed on the 21st day, blood was collected by heart puncture and serum separated, and the levels of serum sialic acid, GT, IL-2, TIMP-1, and
VEGF were determined.Values are mean standard deviation.
2
a, b, c, d: Means without a common superscript differ (P < .05).

Figure 1. Expression of MMP-2, MMP-9, TIMP-1, TIMP-2, ERK-1,


ERK-2, nm23, and VEGF in lungs of metastatic tumorbearing
mice: Lane 1, metastatic tumor control; lane 2, prophylactic Aerva
lanata treatment; lane 3, simultaneous A lanata treatment; lane 4,
A lanata treatment 10 days after tumor induction
Abbreviations: MMP, matrix metalloproteinase; TIMP, tissue inhibitors of
MMPs; ERK, extracellular regulated kinases;VEGF, vascular endothelial
growth factor.

around the main bronchioles extended to the pleura. This


together with fibrosis reduces alveolar space, which in turn
leads to reduced vital capacity. Prophylactic and simultaneous administration of A lanata resulted in significant reduction in tumor mass. The alveoli and pleura were tumor free,
and the alveolar passage was lined with healthy ciliated
columnar epithelial cells, almost similar to the normal lung.
Considerable reduction in tumor mass was also observed in
developed modalities of administration.

Determination of Antimetastatic
Activity in the In Vitro System
Cell viability assay. Cytotoxicity of A lanata toward B16F10 melanoma cells in culture is shown in Table 5. At

Figure 2. Lungs of metastatic tumor-bearing mice: A. lung


tumor nodule formation: (a) normal; (b) tumor control with
nodules; (c) Aerva lanata, prophylactic treatment; (d) A lanata,
simultaneous treatment; (e) A lanata treatment 10 days after
tumor induction. B. Histopathology of lung of metastatic tumorbearing animals (100 magnification): (a) normal; (b) tumor
control with nodules; (c) A lanata, prophylactic treatment; (d) A
lanata, simultaneous treatment; (e) A lanata treatment 10 days
after tumor induction. C. Survival plot

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Table 5. Effect of Aerva lanata on Cell Viability1,2


Concentration (g/mL)

Percentage Viability
45.57a
65.64b
88.38c
96.45d
100e
100e

500
250
100
50
25
10
1

B16F-10 melanoma cells (5000 cells/well) were plated in a 96-well titer


plate with different concentrations of extract and incubated for 48 hours.
The percentage of viable cells was determined by 3-(4,5-dimethylthiazol2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.Values are means of
triplicate.
2
a, b, c, d, e: Means without a common superscript differ (P < .05).

Table 6. Effect of Aerva lanata on Proliferation of B16F-10


Cells1,2
Concentration
(g/mL)

Counts Per
Minute

Percentage Inhibition
in Proliferation

3981.5 73.8a
3095 49.2b
3602.5 62.8c
3938.5 57.2a

22.26
12.24
1.19

Figure 3. The effect of Aerva lanata on tumor-cell collagen


matrix invasion and migration: A. collagen matrix invasion. (A)
untreated control; (B) treatment with A Lanata, 5 g/mL; (C)
treatment with A Lanata, 10 g/mL; (D) treatment with A Lanata,
25 g/mL. B. Percentage inhibition of tumor-cell invasion and
migration by A lanata

concentrations of 10 and 25 g/mL, A lanata was found to


be nontoxic to B16F-10 melanoma cells, and these concentrations were used for further in vitro experiments.
Tumor cell proliferation. The effect of A lanata on the proliferation rate of B16F-10 melanoma cells was determined
by the 3H-thymidine incorporation assay. Thymidine incorporation is proportional to the potential of the cells to synthesize DNA. The rate of proliferation is expressed as
radioactive counts per minute (cpm; Table 6). Untreated
B16F-10 cells had very high rates of proliferation (3981.5
73.8 cpm). Administration of A lanata at a concentration of
25 g/mL significantly (P < .05) reduced the proliferation
(3095 49.2 cpm; 22.26%) of B16F-10 melanoma cells.
Considerable inhibition in the proliferation of tumor cells
was also observed when A lanata was administered at a
concentration of 10 g/mL (3602.5 62.8 cpm; 12.24%).
Collagen matrix invasion assay. Metastatic B16F-10 melanoma cells show a highly invasive property through the collagen matrix. Very high numbers of cells were found in the
lower surface of the polycarbonate membrane, but

administration of A lanata produced significant inhibition


in the invasion of the collagen matrix by the tumor cells in
a dose-dependent manner. At a concentration of 25 g/mL,
A lanata significantly inhibited the invasion of B16F-10
melanoma cells by 53.76%, whereas at concentrations of 10
and 5 g/mL, the percentage inhibition of invasion was
found to be 39.49% and 16.18%, respectively (Figure 3).
Migration. Inhibition of tumor cell migration by A lanata
is given in Figure 3. A lanata significantly inhibited the
migration of B16F-10 melanoma cells across the polycarbonate filters in a dose-dependent manner.
Gelatin zymographic analysis. A lanata inhibited the activation of MMPs produced by B16F-10 melanoma cells as
shown in Figure 4. Conditioned medium after trypsin activation showed digested clear areas at 92 and 72 kD, which
was identical to MMP-9 and MMP-2 activity (Figure 4,
Lane 2). Gels loaded with conditioned medium, without
trypsin activation, did not show any clear areas, indicating
the inactive form of the enzyme collagenase (Figure 4,
Lane 1). Trypsin-activated conditioned mediumloaded
gels, after incubation with 10 mM EDTA, did not show
clear areas, which indicates that the enzyme responsible for
degradation is metalloproteinase (Figure 4, Lane 3). When
the conditioned medium was treated with A lanata during

Control
25
10
5

B16F-10 cells (5000/well) were incubated with different concentrations


(25, 10, and 5 g/mL) of A lanata extract. 3H-thymidine was added to each
well (1 Ci/well), and incubation was continued for 18 hours. Cells were
lysed, and radioactivity was measured using a Rack Beta liquid scintillation counter.Values are mean standard deviation.
2
a, b, c: Means without a common superscript differ (P < .05).

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Figure 4. Effect of Aerva lanata on MMP-2 and MMP-9


production by B16F-10 melanoma cells: Lane 1. Conditioned
medium from untreated B16F-10 melanoma cells without trypsin
activation. Lane 2. Conditioned medium from untreated B16F-10
melanoma cells after trypsin activation. Lane 3. Conditioned
medium from untreated B16F-10 melanoma cells after trypsin
activation + EDTA. Lane 4.Conditioned medium from pretreated
B16F-10 melanoma cells (5 g/mL A lanata) after trypsin
activation. Lane 5. Conditioned medium from pretreated B16F10 melanoma cells (10 g/mL A lanata) after trypsin activation.
Lane 6. Conditioned medium from pretreated B16F-10
melanoma cells (25 g/mL A lanata) after trypsin activation

trypsin activation, no clear bands were observed (Figure 4,


Lane 4 and Lane 5), indicating that A lanata inhibited the
activation of procollagenase to active collagenase at concentrations of 25 and 10 g/mL. Conditioned medium from
cells treated with A lanata at a concentration of 5 g/mL
are shown as 2 faded bands (Figure 4, Lane 6).

Effect of A lanata on Inducing


Apoptosis of B16F-10 Cells

Figure 5. Effect of Aerva lanata on the apoptosis of B16F-10


cells: A. Morphology of B16F-10 melanoma cells: (a) control, (b)
5 g/mL A lanata, (c) 10 g/mL A lanata, (d) 25 g/mL A lanata. B.
DNA ladder formation: lane 1, molecular weight marker; lane 2,
control; lane 3, 5 g/mL A Lanata; lane 4, 10 g/mL A Lanata; lane
5, 25 g/mL A lanata. C. TUNEL assay: (a) untreated control after
48 hours of incubation, (b) treated with 25 g/mL A lanata for 48
hours

apoptotic bodies and membrane blebbing in a dosedependent manner (Figure 5). The maximum effect was on
the cells treated with 25 g/mL A lanata. The observed
dose-dependent enhancement of DNA fragmentation in A
Lanatatreated cells when compared with the untreated
controls also supports the above observation (Figure 5).
TUNEL assay. TUNEL assay has confirmed the presence
of apoptotic bodies by staining free 3-OH termini using
enzymatically labeled nucleotides, supporting the above
observation (Figure 5). These new DNA ends that are generated on DNA fragmentation are typically localized in
morphologically identifiable nuclei and apoptotic bodies. In
contrast, the normal B16F-10 cells (controls) that have relatively insignificant numbers of DNA 3-OH ends did not
stain in the above experiment.

Effect of A lanata on Cell Cycle Analysis

Apoptotic cells were characterized by cell shrinkage, DNA


fragmentation, membrane blebbing, and the presence of
apoptotic bodies. B16F-10 cells treated with different doses
of A lanata (5, 10, and 25 g/mL) showed the presence of

Figure 6 shows the effect of A lanata on the B16F-10 cell


cycle. In the untreated controls, 3.29% of cells were in the
sub-G0/G1 phase, 56.5% of the cells were in the G0/
G1-phase (M1), 14.79% of the cells were in the S-phase,
and 17.27% of the cells were in the G2/M-phase (M2).

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Figure 6. Effect of Aerva lanata on cell cycling of B16F-10


melanoma cells: (A) untreated control, (B) treated with 25 g/mL
A lanata for 24 hours. M1, G0/G1 (diploid); M2, G2/M (tetraploid);
M3, S (synthetic phase); M4, sub-G0/G1 phase. The population
of cells in the sub-G0/G1 phase consists of cellular fragments
resulting from apoptosis.

Treatment with 25 g/mL A lanata increased the percentage


of cells in the sub-G0/G1 phase to 28.49% after 24 hours of
incubation, whereas the percentage of cells in the G0/G1
phase was reduced to 45.28% in the same period.

Effect of A lanata on the Expression of p53,


caspase-9, caspase-3, caspase-8, bcl-2, bid,
bax, p21, p27, and cyclin-D1 Using RT-PCR
Agarose gel electrophoresis of the amplified samples
showed (Figure 7) that the expression of p53, caspase-9,
caspase-3, bax, p21, and p27 were upregulated in A lanata
(25 g/mL) treated cells compared with the nontreated control cells. Caspase-8 did not show any expression in control
and A Lanatatreated cells. Bcl-2 and cyclin-D1 genes
showed a diminished expression in A lanatatreated B16F10 cells.

Effect of A lanata on GM-CSF and


Proinflammatory Cytokine Levels
The effect of A lanata on GM-CSF and proinflammatory
cytokine levels is shown in Table 7. A lanata significantly
inhibited the production of TNF-, IL-1, IL-6, and GM-CSF
by B16F-10 melanoma cells in culture.

Discussion
Metastasis is the process by which cancer cells migrate
from a primary tumor to other sites in the body. It is the
leading cause of death in cancer patients, and by the time

Figure 7. Effect of Aerva lanata on the expression of p53,


caspase-9, caspase-3, caspase-8, bcl-2, bid, bax, p21, p27, and
cyclin-D1 genes: lane 1, untreated control; lane 2, treated with 25
g/mL A lanata for 4 hours

Table 7. Effect of Aerva lanata on the Release of TNF-, IL-1,


IL-6, and GM-CSF by B16F-10 Melanoma Cells1,2
Cytokines
TNF-
IL-1
IL-6
GM-CSF

Control

Treated

28.93 0.86a
94.85 4.71a
58.13 1.98a
37.52 1.54a

18.52 0.73b
65.14 3.06b
41.37 1.57b
23.84 1.04b

Abbreviations: TNF, tumor necrosis factor; IL, interleukin; GM-CSF, granulocyte macrophage colony-stimulating factor.
1
B16F-10 cells (5000 cells) were cultured in the presence of 25 g/mL A
lanata extract for 48 hours. Concentration of cytokines in the culture supernatant was estimated by ELISA.Values are mean standard deviation.
2
a, b: Means without a common superscript differ (P < .05).

most cancers are diagnosed, metastasis has already occurred,


and the presence of multiple metastases makes complete
eradication by surgery, radiation, chemotherapy, or biotherapy nearly impossible. Metastasis involves many distinct,

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Siveen and Kuttan


successive steps. Tumor cells must escape from the primary
tumor site, enter the bloodstream or the lymph system,
evade host defense systems, lodge in a spot that is conducive to growth, and establish a colony by recruiting blood
vessels for nourishment.19
Many plant-derived bioactive constituents, including paclitaxel (from Taxus brevifolia), camptothecin (from Camptotheca
acuminata), podophyllotoxin (from Podophyllum emodi), and
vinblastine (from Catharanthus roseus), have been developed
as potential sources of anticancer agents.20 Recent scientific
efforts have focused on the potential roles of extracts of traditional herbs as alternative and complementary medications for
cancer treatment.
Administration of the ethanolic extract of A lanata was
found to reduce the number of lung metastases caused by
the tail vein injection of highly metastatic malignant murine
melanoma cells, B16F-10. Inhibition of tumor nodule formation was highest when animals were treated with the A
lanata extract simultaneously with tumor administration.
This was validated during the histopathological examination of lungs from tumor control and treated animals. There
was a significant increase in the life span of tumor-bearing
animals treated with the extract when compared with that of
the untreated controls. The biochemical parameters in lungs
and serum also validated the inhibition of metastasis by A
lanata treatment.
TIMPs act as natural inhibitors of MMPs by tightly binding the MMP in a 1:1 stoichiometric ratio and are associated
with normal and pathological extracellular matrix turnover.21 The level of tissue inhibitors of metalloproteinases
(TIMP-1) was reduced in the tumor condition, which was
brought back to near normal range by treatment with A
lanata. The gene nm23 was the first metastasis suppressor
gene described and serves as a prototype for this class of
genes. In several different tumor systems, lower levels of
nm23 mRNA and protein levels have been associated with a
more aggressive/metastatic phenotype in vitro.22 There was
an increase in the expression of antimetastatic genes, nm23,
and MMP inhibitors (TIMP-1 and TIMP-2) in extracttreated animals along with downregulation of prometastatic
genes such as MMP-2, MMP-9, ERK-1, ERK-2, and VEGF.
The majority of MMPs are secreted as zymogens, in
which the latency is maintained by interaction of the cysteine residue in the prodomain sequence PRCGVPC with the
catalytic zinc ion. Activation of the enzyme in vivo is mediated through the activity of plasmin, cathepsin G, neutrophil elastase, or cellular oxidative changes. In addition to
activation of the zymogen, MMP activity is also regulated
at the level of transcription and through inhibition of the
activated enzyme. The expression of MMPs is induced by a
variety of external stimuli such as cytokines and growth
factors, including ILs, interferons, EGF, FGF, VEGF, TNF, TGF-, and so on.4 Zymographic analysis reveals the
effect of A lanata on almost complete inhibition of MMP

activity at 25 and 10 g/mL concentration and reduction in


MMP-mediated cleavage of gelatin at 5 g/mL. Effect of A
lanata on MMP production and activity is also evident from
the inhibition of collagen matrix invasion by B16F-10 cells.
Ethanolic extract of A lanata showed significant cytotoxic activity toward B16F-10 cells and inhibited the proliferation of B16F-10 melanoma cells in a dose-dependent
manner.
Apoptosis is an important way to maintain cellular
homeostasis between cell division and cell death. A complex
balance of proapoptotic and antiapoptotic factors tightly
controls the process of apoptosis. Cancer cells can exploit
these factors to bypass the normal physiological checkpoints
that would trigger defective cells to undergo apoptosis.
Apoptosis is characterized by the presence of distinct morphological features and formation of a ladder of DNA fragments.23 Our results support marked morphological changes
indicative of cell apoptosis, including membrane blebbing,
chromatin condensation, vacuole formation, nuclear and
cytoplasmic fragmentation, and appearance of a DNA ladder
in A lanatatreated cells in a dose-dependent manner.
The tumor suppressor gene p53 has been termed the
guardian of the genome, given its essential role in surveillance of DNA damage, regulation of the cell cycle, and
regulation of apoptosis. The wild-type p53 gene is essential for regulation of cell growth, and it is easy to understand that in a general sense, loss of p53 function may be
involved in the early steps of tumor formation through the
survival of cells with genetic mutations. Current evidence
suggests that p53 functions to detect DNA damage and
subsequently arrest cells in the G1 phase of the cell cycle to
allow for repair; however, if the damage cannot be repaired,
then apoptotic cell death is triggered.24 There was a significant increase in the percentage of cells in the sub-G0/G1
phase, according to cell cycle analysis by flow cytometry.
Caspases are highly selective cystine proteases that control
all steps of apoptosis. Caspases can be grouped into 2
types: initiator caspases and effector or executioner caspases. The initiator caspases (eg, caspase 8, caspase 9, and
others) act by activating other procaspases, which become
effector caspases. Caspase 3, also referred to as apopain, is
the major effector caspase. TUNEL assay of A lanata
treated B16F-10 melanoma cells confirms that these cells
have free 3 hydroxyl groups formed by DNA cleavage
during apoptosis.
The upregulation of antiapoptotic genes and the downregulation of proapoptotic genes by intervention of natural
products are possible mechanisms to induce apoptosis in
cancer cells. Alterations in the Bcl-2 family of proteins, a
major apoptosis regulatory protein family, often occur in
cancers.25 There was a decrease in the mRNA levels of bcl2 in the extract-treated cells. A lanata induced cell apoptosis
via the intrinsic pathway through the upregulation of antiapoptotic genes (p53, caspase-3, caspase-9, bid, and bax)

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Integrative Cancer Therapies 12(1)

and cell cycle regulating genes (p21 and p27), which were
not expressed in control cells.
In conclusion, this study clearly demonstrates that the
ethanol extract of A lanata strongly inhibits lung metastasis
of B16F-10 melanoma cells in vivo, which was mediated
through induction of apoptosis, via activation of p53induced caspase-3mediated proapoptotic signaling and
inducing cell cycle arrest at the sub-G0/G1 phase.
Acknowledgment
The authors sincerely thank Dr Ramadasan Kuttan, Research Director,
Amala Cancer Research Centre, for his valuable suggestions.

Declaration of Conflicting Interests


The authors declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.

Funding
The authors received no financial support for the research, authorship, and/or publication of this article.

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