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Dž 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
:HVW%HQJDO,QGLD
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\FRQGXFWLQJ GLPHWK\OWKLD]RO\O 
GLSKHQ\OWHWUD]ROLXPEURPLGHDVVD\RQ ERWK QRUPDO SHULSKHUDOEORRG 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

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numerous early metastases and high resistance to %LRWHFQRORJ\ 86$   'LPHWK\OWKLD]RO\O 
conventional treatments involving surgery, chemotherapy GLSKHQ\OWHWUD]ROLXPEURPLGH 077 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\OSKRVSKDWH 3133 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 RI3'LQWKHIRUPRIKRPHRSDWKLFPRWKHUWLQFWXUHZDV
QHZHUDYHQXHV[4]. VXSSOLHG E\ %RLURQ /DERUDWRU\ /\RQ )UDQFH 7KH\
In cancer treatment, one of the approaches to restrain prepared the drug adopting the guidelines of European
WXPRU JURZWK LV E\ DFWLYDWLQJ WKH DSRSWRWLF PDFKLQHU\ Pharmacopoeia and supplied the drug in 65% ethanolic
in the tumor cells[5]. Apoptosis, a highly structured and H[WUDFW%HIRUHWKHH[SHULPHQWDOWUHDWPHQWWKHGUXJZDV
RUFKHVWUDWHGSURFHVVSHUIRUPVDVLJQL¿FDQWUROHLQUHJXODWLQJ GLOXWHGLQWKH'0(0PHGLD
FHOOQXPEHUIRUWKHJURZWKDQGKRPHRVWDVLVRIWLVVXHVE\ &HOOFXOWXUH
HOLPLQDWLQJDJHGGDPDJHGDQGXQZDQWHGFHOOV[6,7]. A375 and HeLa (National Centre for Cell Science, NCCS,
Phytolacca decandra 3' 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]. 6FLHQWL¿F86$ &HOOVZHUHKDUYHVWHGZLWKîWU\SVLQ('7$
)URP D ORQJ WLPHPhytolacca has been used to combat in phosphate buffered saline (PBS) and plated at required
different ailments in complementary and alternative FHOOQXPEHUVDQGDOORZHGWRDGKHUHEHIRUHWUHDWPHQW
medicine (CAM). In homeopathy, mother tincture of &HOOYLDELOLW\DVVD\
Phytolacca is used against chronic rheumatism, regular &HOOVZHUHGLVSHQVHGLQZHOOSODWHVDWDGHQVLW\RI
conjunctivitis, psoriasis and some skin diseases [12] . î2 FHOOVSHUZHOODQGWUHDWHGZLWKYDULRXVFRQFHQWUDWLRQV
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studied for scientific validation, stray reports exist in isolated from the human blood sample by conventional
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sources to have anticancer potentials [13,14]. Since the DQGK7KHSHUFHQWDJHRIFHOOGHDWKZDVGHWHUPLQHGE\
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cell death involved. measured and expressed as drug doses in concentration
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received any drug nor ethanol. As the positive control
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protocol (Santa Cruz Biotechnology Inc, USA) using cell to the higher ones in both the cases of 24 and 48 h of
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March 2013, Vol.11, No.2  Journal of Integrative Medicine


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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&**$$ƍ
5HYHUVHƍ77*&777&$***&7*&7&$$*$$**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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Figure 5 Analysis of accumulation of reactive oxygen species


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n=3; ǀ P<0.05, vs control group.
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A noble anticancer drug should normally be expected to drug. The accumulated ROS has the capacity to induce
have the property of initiating apoptosis (cell death) to '1$ GDPDJH [25] 7KH )$&6 DQDO\VLV ZLWK + 2'&)'$
the cancer cells[20]&RQVLVWHQWZLWKWKLVH[SHFWDWLRQ3' fluorescent dye supported the increased cellular ROS
ZDVIRXQGWRLQLWLDWHDSRSWRVLVLQ$FHOOVDVWKHPDLQ generation upon drug treatment.
cause of cell death, though it also induced necrosis in a 526 UHJXODWHV FHOO SUROLIHUDWLRQ E\ GRZQUHJXODWLQJ
IHZFHOOV5HVXOWVRI/'+DFWLYLW\DVVD\VXSSRUWHGWKLV Akt gene expression [26,27] ZKLFK LQ WXUQ UHGXFHV FHOO
contention. proliferation[28]. Expressions of mRNA and protein (by
$V RQH PDMRU ZD\ RI FRPEDWLQJ FDQFHU FHOOV LV E\ LQGLUHFW (/,6$  VKRZHG GRZQUHJXODWLRQ RI$NW7KLV
killing them, one of the approaches to restrain tumor UHVXOWZDVLQJUHDWVXSSRUWRIDQWLSUROLIHUDWLYHSRWHQWLDO
JURZWK LV E\ DFWLYDWLQJ WKH DSRSWRWLF PDFKLQHU\ LQ WKH RI3'2QWKHRWKHUKDQG526UHJXODWHVSH[SUHVVLRQ
tumor cells [21]. The changes in cellular morphology Elevation in ROS level generally up-regulates the “master
(e.g., membrane distortion, blebbing), condensation and gene” p53[29] and this master gene plays a decisive role
IUDJPHQWDWLRQRI'1$DUHWZRVLJQLILFDQWFKDUDFWHULVWLF LQ ZKHWKHU WKH GDPDJHG '1$ ZLOO XQGHUJR WKH UHSDLU
features of apoptosis[22]1XFOHRVRPDOIUDJPHQWDWLRQZDV SDWKZD\RUWKHIUDJPHQWDWLRQSDWKZD\:KHQGDPDJHLV
evident through microscopic study of cellular morphology not repairable, generally the cells are led to the apoptotic
DQG'$3,VWDLQLQJRIWKHFHOOV SDWKZD\ 2XU H[SHULPHQWDO UHVXOWV WKH HOHYDWHG S
ROS plays a prodigious role in regulating cell proliferation DFWLYLW\OHYHODQG'$3,VWDLQHGFHOOVVXJJHVWWKHDERYH
DQGUHPRYDORIXQZDQWHGFHOOVWKURXJKDSRSWRVLV[23]. The event.
normal human cells possess certain level of ROS activity, Alteration in the levels of Bax and Bcl-2, i.e., the ratio
but the cancer cells contain a higher level of ROS activity RI%FO%D[LVWKHGHFLVLYHIDFWRULQGHWHUPLQLQJZKHWKHU
due to its higher metabolic rates. If the ROS level could FHOOVZLOOXQGHUJRFDVSDVHPHGLDWHGDSRSWRVLV[30,31], or be
be elevated to a further extent to the threshold level, the GLUHFWHGWRZDUGVWKHVXUYLYDOSDWKZD\[32]. In our study, the
cells push themselves to apoptosis[24]7KLVFRXOGEHDZD\ decrease in Bcl-2 expression and the increase in Bax and

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Figure 6 Expression of mRNA and protein


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EDUGLDJUDPUHSUHVHQWVWKHH[SUHVVLRQOHYHORIP51$DQGSURWHLQ&([SUHVVLRQRI*3+'DWP51$DQGSURWHLQOHYHO*3'+VHUYHVDVWKH
KRXVHNHHSLQJJHQH7KHUHVXOWVDUHH[SUHVVHGDVPHDQ“VWDQGDUGHUURURIPHDQn=3; ǀ P<0.05, vs control group.
'—JP/'—JP/'—JP/*3'+JO\FHUDOGHK\GHSKRVSKDWHGHK\GURJHQDVH

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.

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caspase 3 expression both at mRNA and protein levels (by the diurnal, ontogenetic and seasonal variation in the
LQGLUHFW(/,6$ ZHUHREVHUYHG+HQFHWKHDOWHUHG%FO molluscicidal saponin content of Phytolacca dodecandra
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514.
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