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Research Article

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Samrat Ghosh1, Kausik Bishayee 1, Avijit Paul 1, Avinaba Mukherjee 1, Sourav Sikdar1,
Debrup Chakraborty1, Naoual Boujedaini2, Anisur Rahman Khuda-Bukhsh1
1. Cytogenetics and Molecular Biology Laboratory, Department of Zoology, University of Kalyani, Kalyani
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2. Boiron Laboratories, Lyon 69110, France

OBJECTIVE: Preventive measures against skin melanoma like chemotherapy are useful but
suffer from chronic side effects and drug resistance. Ethanolic extract of Phytolacca decandra
(PD), used in homeopathy for the treatment of various ailments like chronic rheumatism, regular
conjunctivitis, psoriasis, and in some skin diseases was tested for its possible anticancer
potential.
METHODS:&\WRWR[LFLW\RIWKHGUXJZDVWHVWHGE\FRQGXFWLQJGLPHWK\OWKLD]RO\O
GLSKHQ\OWHWUD]ROLXPEURPLGHDVVD\RQ ERWK QRUPDOSHULSKHUDOEORRG PRQRQXFOHDUFHOOVDQG
$FHOOV)OXRUHVFHQFHPLFURVFRSLFVWXG\RIƍGLDPLGLQRSKHQ\OLQGROHGLK\GURFKORULGHVWDLQHGFHOOV
was conducted for DNA fragmentation assay, and changes in cellular morphology, if any, were
also recorded. Lactate dehydrogenase activity assay was done to evaluate the percentages of
apoptosis and necrosis. Reactive oxygen species (ROS) accumulation, if any, and expression
study of apoptotic genes also were evaluated to pin-point the actual events of apoptosis.
RESULTS: Results showed that PD administration caused a remarkable reduction in
proliferation of A375 cells, without showing much cytotoxicity on peripheral blood mononuclear
cells. Generation of ROS and DNA damage, which made the cancer cells prone to apoptosis,
were found to be enhanced in PD-treated cells. These results were duly supported by the
analytical data on expression of different cellular and nuclear proteins, as for example, by down-
regulation of Akt and Bcl-2, up-regulation of p53, Bax and caspase 3, and an increase in number
of cell deaths by apoptosis in A375 cells.
CONCLUSION: Overall results demonstrate anticancer potentials of PD on A375 cells through
activation of caspase-mediated signaling and ROS generation.
KEYWORDS: Phytolacca decandra; skin neoplasms; reactive oxygen species; apoptosis; gene expression

'2,MLQWHJUPHG
Ghosh S, Bishayee K, Paul A, Mukherjee A, Sikdar S, Chakraborty D, Boujedaini N, Khuda-Bukhsh AR.
Homeopathic mother tincture of Phytolacca decandra induces apoptosis in skin melanoma cells by activating
caspase-mediated signaling via reactive oxygen species elevation. J Integr Med
Received June 25, 2012; accepted August 9, 2012.
Open-access article copyright © 2013 Samrat Ghosh et al.
Correspondence: Anisur Rahman Khuda-Bukhsh, PhD, Professor; Tel: +91-33-25828750-315; E-mail:
SURIBDUNE#\DKRRFRLQNKXGDEXNKVKB#UHGLIIPDLOFRP

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*OUSPEVDUJPO type of skin cancer comprises just 5 % of all malignant
skin cancers, it is responsible for a 75%-80% of deaths
A dynamic increase of skin melanoma mortality rate has caused by these tumors[1]. The high rate of malignancy

March 2013, Vol.11, No.2  Journal of Integrative Medicine

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numerous early metastases and high resistance to %LRWHFQRORJ\ 86$ 'LPHWK\OWKLD]RO\O
conventional treatments involving surgery, chemotherapy GLSKHQ\OWHWUD]ROLXPEURPLGH077ZDVSXUFKDVHGIURP
and radiation. Although preventive measures like Sisco Research Laboratory, India. TRIzol and antibiotics
chemotherapy are very useful, these often result in SHQLFLOOLQVWUHSWRP\FLQQHRP\FLQ 361 ZHUH REWDLQHG
manifestation of chronic side effects[2,3]. Therefore, there IURP +L0HGLD ,QGLD 'LFKORURGLK\GURIOXRUHVFHLQ
LVDJURZLQJGHPDQGIRUWKHGHYHORSPHQWRIDOWHUQDWLYH diacetate (H2'&)'$DQGƍGLDPLGLQRSKHQ\OLQGROH
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products of various sources, particularly from plants, 86$51DVHDQGSQLWURSKHQ\OSKRVSKDWH3133ZHUH
have been regarded as a precious alternative to modern purchased from Merck (Germany). All the primers used
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anticancer potential and less side effects have opened up
QHZHUDYHQXHV[4].
In cancer treatment, one of the approaches to restrain
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in the tumor cells[5]. Apoptosis, a highly structured and
RUFKHVWUDWHGSURFHVVSHUIRUPVDVLJQL¿FDQWUROHLQUHJXODWLQJ
FHOOQXPEHUIRUWKHJURZWKDQGKRPHRVWDVLVRIWLVVXHVE\
HOLPLQDWLQJDJHGGDPDJHGDQGXQZDQWHGFHOOV[6,7].
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prepared the drug adopting the guidelines of European
Pharmacopoeia and supplied the drug in 65% ethanolic
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A375 and HeLa (National Centre for Cell Science, NCCS,
Phytolacca decandra3'FRPPRQO\NQRZQDVSRNH ,QGLDFHOOVZHUHFXOWXUHGLQ'0(0ZKHUHDV3&FHOOV
root, is an ornamental plant of the Phytolaccaceae family. 1&&6,QGLDZHUHFXOWXUHGLQ530,VXSSOHPHQWHGZLWK
Phytochemical characterisation revealed that terpenoids, KHDWLQDFWLYDWHG)%6DQG361DQGPDLQWDLQHGDW
saponins, alkaloid, resins, phytolacic acid and tannin are ž&ZLWK&22 in a humidified incubator (Thermo
the major bioactive ingredients of Phytolacca species[8-11].
)URP D ORQJ WLPHPhytolacca has been used to combat
different ailments in complementary and alternative
medicine (CAM). In homeopathy, mother tincture of
Phytolacca is used against chronic rheumatism, regular
conjunctivitis, psoriasis and some skin diseases [12] .
6FLHQWL¿F86$&HOOVZHUHKDUYHVWHGZLWKîWU\SVLQ('7$
in phosphate buffered saline (PBS) and plated at required
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studied for scientific validation, stray reports exist in isolated from the human blood sample by conventional
literature enlisting some of the ingredients like saponins ILFROO JUDGLHQW PHWKRG DQG WUHDWHG ZLWK 3' DW GLIIHUHQW
and alkaloids extracted and separated from other plant
sources to have anticancer potentials [13,14]. Since the
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no earlier study appears to have been conducted on the
anticancer effect on the basis of a reliable investigation,
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MTT assay[15].
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DFWLYLW\LIDQ\RI3'DJDLQVW$FHOOVin vitro and if and treated for 48 h on A375 cells. To calculate the
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cell death involved. measured and expressed as drug doses in concentration
XQLW—JP/The positive (vehicle) control set received


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received any drug nor ethanol. As the positive control
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86$ &HOO FXOWXUH PHGLD 'XOEHFFR¶V PRGLILHG (DJOH experiments.
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Journal of Integrative Medicine  March 2013, Vol.11, No.2

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Results of MTT assay revealed that the cell viability
gradually decreased from minimal drug concentration
to the higher ones in both the cases of 24 and 48 h of
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for the treatment of 24 and 48 h respectively on A375 cells
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Table 1The list of the sequences of the primers used

Primer name Primer sequence
Caspase 3 )RUZDUGƍ$****7&$777$7***$&$ƍ
5HYHUVHƍ7$&$&***$7&7*777&777*ƍ
Akt )RUZDUGƍ&&7**$&7$&&7*&$&7&7&**$$ƍ
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Bcl-2 )RUZDUGƍ*7*$&77&&*$7&$**$$**ƍ
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p53 )RUZDUGƍ**$$$777*7$7&&&*$*7$7&7*ƍ
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Figure 1 Results of cell viability assay

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error of mean, n=3; *P<0.05, vs—JP/

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Journal of Integrative Medicine  March 2013, Vol.11, No.2

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7KHGUXJWUHDWPHQWODVWHGIRUK7KHUHVXOWVDUHH[SUHVVHGDVPHDQ“VWDQGDUGHUURURIPHDQn=3; ǀ P<0.05, vs control group.
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March 2013, Vol.11, No.2  Journal of Integrative Medicine

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Figure 5 Analysis of accumulation of reactive oxygen species

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fluorescent dye supported the increased cellular ROS
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normal human cells possess certain level of ROS activity,
but the cancer cells contain a higher level of ROS activity
due to its higher metabolic rates. If the ROS level could
be elevated to a further extent to the threshold level, the
cells push themselves to apoptosis[24]7KLVFRXOGEHDZD\
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decrease in Bcl-2 expression and the increase in Bax and
 March 2013, Vol.11, No.2
 www.jcimjournal.com/jim

Figure 6 Expression of mRNA and protein

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Table 2 The Bcl-2/Bax ratio at the both mRNA and protein levels
mRNA expression Protein expression
Gene
Control ' ' ' Control ' ' '
Bcl-2 1 0.997 802 0.903 297 0.734 066 1 0.962 838 0.856 419 0.810 811
Bax 1 1.068 293 1.138 211 1.193 496 1 1.005 747 1.026 820 1.042 146
Bcl-2/Bax 1 0.934 016 0.793 611 0.615 055 1 0.957 336 0.834 050 0.778 021
In case of both mRNA and protein expressions of Bcl-2 and Bax, drug-treated ones are expressed as fractional values considering the
control as the unity.

March 2013, Vol.11, No.2  Journal of Integrative Medicine

 www.jcimjournal.com/jim

caspase 3 expression both at mRNA and protein levels (by the diurnal, ontogenetic and seasonal variation in the
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Bax ratio and caspase 3 activation indicate that the cells
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anticancer effect on skin melanoma by indulging the cells
to apoptosis via elevation of ROS. Our study suggested
that the ROS might activate p53 and block Akt and induce
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practitioners to use Phytolacca as a supportive medicine
as an anti-melanoma drug.


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Journal of Integrative Medicine (JIM) is a PubMed-indexed, W\SHVLQFOXGHUHYLHZVV\VWHPDWLFUHYLHZVDQGPHWDDQDO\VHV

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all aspects of integrative medicine, such as acupuncture and
traditional Chinese medicine, Ayurvedic medicine, herbal
medicine, homeopathy, nutrition, chiropractic, mind-body
medicine, Taichi, Qigong, meditation, and any other modalities
of complementary and alternative medicine (CAM). Article


randomized controlled and pragmatic trials, translational and
patient-centered effectiveness outcome studies, case series and
reports, clinical trial protocols, preclinical and basic science
studies, papers on methodology and CAM history or education,
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Editors-in-Chief: Wei-kang Zhao (China) & Lixing Lao (USA). ISSN 2095-4964. Published by Science Press, China.

March 2013, Vol.11, No.2  Journal of Integrative Medicine