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Centchroman induces G0/G1 arrest and Caspase-dependent Apoptosis
involving Mitochondrial Membrane Depolarization in MCF-7 and
MDA MB-231 Human Breast Cancer Cells
Manisha Nigam, Vishal Ranjan, Swasti Srivastava, Ramesh Sharma, Anil K. Balapure
Tissue and Cell Culture Unit (TCCU), Central Drug Research Institute, Lucknow, 226001, India
Received 6 July 2007; accepted 27 November 2007
Abstract
Studies with Centchroman (CC) as a candidate anti-breast cancer agent are into phase III multicentric clinical trial in stage III/IV breast cancer. We
have previously demonstrated its anti-neoplastic activity in Estrogen Receptor positive (ER+ve) MCF-7 Human Breast Cancer Cells (HBCCs). We
now present the basis for anti-neoplastic activity of CC, mediated through apoptosis in both ER+ve/ve MCF-7 and MDA MB-231 HBCCs
respectively, compared to Tamoxifen (TAM) as a positive control. All the experiments were performed with 48 h estrogen-deprived cells exposed to
CC/TAM for the subsequent 48 h. Cytotoxic potential of CC was assessed through SRB assay. Cell-cycle analysis, Time-dependent cytotoxicity,
Reactive Oxygen Species (ROS) and Mitochondrial Membrane Permeability were investigated by Flow Cytometry. Early-stage apoptosis was
detected by AnnexinPI staining. Caspases were assayed colorimetrically whereas nuclear derangements were assessed morphologically through PI
staining and finally by DNA fragmentation analysis. Cell viability studies confirmed the IC50 of CC in MCF-7 and MDA MB-231 cells to be 10 and
20 M (Pb0.001) respectively, suggesting enhanced susceptibility of the former cell type to CC. FACS data reveals CC mediated G 0/G1 arrest
(Pb0.01) along with the presence of prominent sub-G0/G1 peak (Pb0.001) in both the cell types suggesting ongoing apoptosis. Phosphatidylserine
externalization, mitochondrial events, caspase evaluation and nuclear morphology changes reveal initiation/progression of caspase-dependentapoptosis even at a dose of 1 M which eventually leads to DNA fragmentation in both the cell types. Results demonstrate that CC induces caspase-
dependent apoptosis in MCF-7 and MDA MB-231 cells irrespective of ER status similar to TAM in terms of anti-neoplastic activity.
2007 Elsevier Inc. All rights reserved.
Keywords: MCF-7; MDA MB-231; Centchroman; Tamoxifen; Apoptosis
Introduction
Apoptosis contributes to the anti-tumor activity of many
chemotherapeutic drugs (Kastan and Bartek, 2004; Zhivotovskyand Kroemer, 2004; Danial and Korsmeyer, 2004; Chipuk and
Green, 2005). Its absence activates a non-apoptotic pathway
Necroptosis (Degterev et al., 2005). Tamoxifen (TAM) widely
used to treat metastatic, hormone responsive breast cancer
(Clarke et al., 2001; Macgregor and Jordan, 1998; Shiau et al.,
1998; Muss 1992) displays its cytoproliferative/-static/-toxic
(Taylor et al., 1984; Perry et al., 1995a) effects in ER (Estrogen
Receptor) +ve/
ve Human Breast Cancer Cells (HBCCs) byER-dependent/-independent pathways (Goldenberg and Froese,
1982; Perry et al., 1995b; Obrero et al., 2002). Recently, a report
on TAM induced rapid cell death in MCF-7 cells suggests
caspase-independent mitochondria mediated cell death (Kallio
et al., 2005). However, prolonged TAM administration results
into eventual resistance to the drug and augments the risk of
endometrial carcinoma (Bergman et al., 2000) etc. Therefore,
the controversy about TAM and carcinogenesis has encouraged
search for potentially safer drug for long-term treatment.
Centchroman (CC) [67/20; INN: Ormeloxifene], a non-
steroidal anti-estrogen (AE) with mild estrogenic and strong
Available online at www.sciencedirect.com
Life Sciences 82 (2008) 577590www.elsevier.com/locate/lifescie
Corresponding author. Tel.: +91 941 506 3603; fax: +91 522 262 3405, +91
522 262 3938.
E-mail addresses: [email protected] (M. Nigam),
[email protected] (V. Ranjan), [email protected]
(S. Srivastava), [email protected] (R. Sharma),
[email protected] (A.K. Balapure), C.D.R.I Communication No. 7029.
0024-3205/$ - see front matter 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.lfs.2007.11.028
mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.lfs.2007.11.028http://dx.doi.org/10.1016/j.lfs.2007.11.028mailto:[email protected]:[email protected]:[email protected]:[email protected]:[email protected] -
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anti-estrogenic activity (Anand and Ray, 1977; World Health
Organization, 1993; Central Drug Research Institute, 1994,
1996; Kamboj et al., 1977) is one of the most extensively
documented molecules for contraceptive purposes. However, it
has also been reported to be accountable for 40.5% regression of
lesions in phase III multicentric clinical trials in stage III/IV
advanced breast cancer patients (Mishra et al., 1989; CentralDrug Research Institute, 1995). Our previous studies have
demonstrated the anti-neoplasticity of CC in ER+ve MCF-7
cells similar to TAM (Srivastava et al., 2004). Encouraged by
these studies with reported excellent therapeutic index (Singh,
2001), we have further explored the basis of its anti-
neoplasticity, in not only MCF-7 cells but in ERve MDA
MB-231 cells too with TAM as a positive control. Furthermore,
normal cell types i.e. non-tumoral cell lines (HEK 293, hGF,
MDCK) and another cancerous cell line Hep G2 were also
evaluated for cytotoxic effects of drugs. Cytotoxicity studies
reveal CC to be cytotoxic similar to TAM as determined by
parallel IC50 values for both the cell types. FACS analysissuggests that CC induces G0/G1 phase arrest and apoptosis
displayed by accumulation of cells in sub-G0/G1 fraction.
AnnexinPropidium Iodide (PI) double staining confirms CC
initiating apoptosis events even at 1 M substantiated by
mitochondrial membrane depolarization studies. Interestingly,
Reactive Oxygen Species (ROS) level imply the drug to be an
anti-oxidant except at 1 M. Activation of caspases with
apoptosis associated nuclear derangements and internucleoso-
mal DNA ladder confirmed that CC induces apoptosis in MCF-
7 and MDA MB-231 cells irrespective of ER.
Materials and methods
Materials
Tamoxifen (TAM), Dulbecco's Modified Eagle's Medium
(DMEM), N-[2-Hydroxyethyl] piperazine-N-2-ethanesulfonic
acid (HEPES), Penicillin, Streptomycin, Gentamicin sulfate,
Sulforhodamine-B (SRB), Phosphate Buffered Saline (PBS)
pH 7.4, Propidium Iodide (PI), Ethidium Bromide (EtBr),
Annexin V-FITC Apoptosis Detection Kit, Ribonuclease-A,
Rhodamine 123 (Rh 123), 3-[(3-cholamidopropyl) dimethy-
lammonio]-1-propanesulfonate (CHAPS), Caspase-8 substrate
(Z-IETD-pNA), Caspase-9 substrate (Ac-LEHD-pNA), Cas-
pase-3 substrate (Z-DEVD-pNA), Trichloroacetic acid (TCA)were purchased from Sigma Chemical Co., St. Louis, MO, USA.
2,7-Dichlorofluorescin Diacetate (DCFDA) was from Merck
Calbiochem and Fetal Calf Serum (FCS) was procured from
GIBCO BRL Laboratories, New York, USA. 100 bp DNA ladder
was sourced from Bangalore Genei, India. All other chemicals
were of analytical grade.
Cell culture
HEK 293 (Human Embryonic Kidney, Epithelial), MDCK
(MadinDarby Canine Kidney, Epithelial), Hep G2 (Human
Hepatocellular Carcinoma), MCF-7 and MDA MB-231
(Human Breast Cancer, Epithelial) cells were procured from
the National Center for Cell Sciences (NCCS), Pune, India.
These cells are routinely being cultured as reported previously
by us (Saxena et al., 1995; Shagufta et al., 2006; Gupta et al.,
2006; Srivastava et al., 2006). Briefly, the cells were cultured in
DMEM, pH 7.4 containing Penicillin (100 U/ml), Streptomycin
(100 g/ml), Gentamicin (60 g/ml) supplemented with 10%
FCS and 10 mM HEPES in a humidified 5% CO2 incubator at37 C.
The Primary Human Gingival Fibroblast (hGF) Cell Line was
developed in-house from biopsy samples obtained aseptically
after the informed consent from volunteers with clinically
healthy gingiva.
Briefly, the harvested gingival explants were transported
from the local Medical and Dental School in chilled DMEM
containing 20% fetal calf serum in minimal time on ice. They
were then chopped into small pieces of approximately 1 mm3
sizes in a petridish containing the same medium in a laminar
flow to ensure sterility. Subsequently, they were transferred into
a T-25 tissue culture flask with adequate medium for completeimmersion. The flasks were incubated in a humidified CO2incubator at 37 C with 5% CO2 and left undisturbed. By Day 5,
a mixed population of epithelial and fibroblasts emanates in an
around the explants as examined by Nikon Phase Contrast
Microscopy. By Day 15, the epithelial cells perished while the
fibroblast cells with typical oblong, flattened, spindle shaped
morphology were retained. The medium in the flask was
replaced with fresh medium as and when required. Complete
monolayer was evidenced by Day 21. DMEM containing 10%
FCS was employed to subculture the monolayer into fresh flasks
following trypsination.
For experimental purpose, the cells from a confluent flask
were trypsinated and cultured for a total of 4 days. Initially forthe first 2 days, the cells were pre-cultured in phenol red-free
DMEM containing 10% Dextran Coated Charcoal stripped FCS
(DCC/FCS) (Soto and Sonnenschein, 1985). For the subsequent
48 h, the cells were exposed to the ligands (CC/TAM). The cell
number used and the concentration of ligands that the cells were
exposed to have been described individually as below.
Cell growth and cytotoxicity assay
SRB (Shagufta et al., 2006) assay was conducted to ascertain
the cytostatic/toxic/proliferative effect(s) of CC in normal cell
types versus HBCCs and Hep G2. Briefly, 104
cells/well wereplated in a 96-welled plate and exposed to 125 M CC/TAM.
SRB assay was performed after completion of incubation with
the drugs.
Cellular morphological study
HematoxylinEosin (HE) staining of cells for morphological
studies was performed as previously reported by us ( Sharma
et al., 2002). 0.2106 cells of each type were plated on sterile
microscope cover glass in a 6-welled plate and exposed to 1
20 M CC/TAM. The HE staining was performed, cells
observed under Nikon Diaphot Phase Contrast Microscope and
photographed.
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Study of cell-cycle kinetics and time-dependent apoptosis analysis
To analyze the cell-cycle kinetics and apoptosis, 0.2106
cells were plated in a 6-welled plate and exposed to 125 M
CC/TAM. For time-dependent analysis of apoptosis, cells were
treated with IC50 doses i.e. 10 M CC/TAM for MCF-7 and
20 M CC/TAM for MDA MB-231 for varying time periods.Following trypsinization and washing with chilled Phosphate
Buffered Saline (PBS) pH 7.4, the cells were permeabilized
with chilled 70% ethanol for 30 min at 4 C. After rewashing
with chilled PBS, the cells were resuspended in 500 l of PBS
containing PI (40 g/ml) and RNase (100 g/ml). Flow
Cytometry was performed on BecktonDickinson Fluorescence
Activated CellSorter (FACS) employing the Cell Quest Software.
Cells with hypodiploid DNA (content less than that of G0/G1-
phase cells) were considered to be apoptotic (sub-G0/G1).
Annexin V-FITC and PI staining analysis
For evaluating apoptosis, 0.4106 cells of each type were
plated onto 35 mm culture dishes containing cover slips and
treated with 120 M CC/TAM. Following Annexin V-FITC
and PI staining according to manufacturer's protocol, the cells
on cover slips were analyzed on Nikon Eclipse E200
Fluorescence Microscope and photographed.
Modulation of Mitochondrial Membrane Potential (m) and
intracellular Reactive Oxygen Species (ROS) generation
Mitochondrial Membrane Potential (m) was measured by
the uptake of Rhodamine 123 (Rh 123) as a function ofm.
Rh 123 dye is readily sequestered by the functional mitochon-dria and subsequently washed out of the cells once the m is
lost resulting in decreased fluorescence. 0.2106 cells of each
type were plated in a 6-welled plate, exposed to 125 M of
CC/TAM for 48 h, washed and finally harvested in chilled PBS
containing 5 g/ml Rh 123. The samples were incubated at
37 C for 15 min in dark, washed twice with chilled PBS and
fluorescence intensities were determined on Flow Cytometer.
Reactive Oxygen Species (ROS) generated upon CC exposure
in the cells was determined through 2,7-Dichlorofluorescin
Diacetate (DCFDA) staining. 0.2106 cells of both the types
were plated in a 6-welled plate and exposed to 125 M CC/
TAM for 24 h. Subsequently, the cells were washed twice withchilled PBS and incubated with 10 M DCFDA at 37 C for
30 min in dark followed by washing twice with chilled PBS.
Cells were trypsinated and analyzed through Flow Cytometer
with excitation and emission at 490 and 530 nm respectively.
Colorimetric caspase assay
To analyze the role of Caspases, the cells were cultured and
treated with 120 M CC/TAM in T-75 flasks. Activities of
caspases were colorimetrically assayed per manufacturer's
protocol (Sigma Chemical Co, St. Louis, MO, USA). Briefly,
the cells were lysed in 250 l of lysis buffer (25 mM HEPES pH
7.5, 0.1% CHAPS, 5% Sucrose, 5 mM DTT, 2 mM EDTA) at
4 C for 30 min. 200 g of cell lysate protein was mixed in assay
buffer in a final volume of 100 l, followed by the addition of
10 l of 2 mM of the individual substrates specific for each
caspase. For Caspase-8 Z-IETD-pNA, for Caspase-9 Ac-LEHD-
pNA, and lastly for Caspase-3 Z-DEVD-pNA was respectively
employed. Following incubation of the substrate with cell lysate
at 37 C for 30 min, the liberated p-nitroaniline (pNA) was readat 405 nm on SpectraMAX 190 Microplate Reader.
Nuclear morphological study
For monitoring the drug mediated changes in nuclear
morphology, the cells were cultured and treated similarly as
for AnnexinPI staining analysis reported previously. The
protocol for staining the cells was same as for FACS analysis,
except that trypsinization was not performed in this case.
Finally, the cover slips were analyzed under Nikon Eclipse
E200 Fluorescence Microscope and photographed. The percen-
tage of apoptotic nuclei was calculated by counting theapoptotic nuclei per field (particular focused area) from ten
randomly chosen areas of the same picture.
DNA fragmentation analysis
Fragmented versus intact DNA was extracted according to
Sellins and Cohen (1987) with minor modifications. Briefly, the
cells were cultured and treated similarly as for caspase assay.
Following lysis in the lysis buffer (10 mM TrisHCl pH 7.4,
0.2% Triton X-100, 1 mM EDTA), the lysates were spun at
20,000 g for 10 min at 4 C to separate the fragmented versus
intact chromatin. Both fractions were precipitated overnight with
1 ml 25% TCA at 4 C. The pelleted DNA was hydrolyzed with160 l of 5% TCA at 90 C for 15 min and quantified
colorimetrically (Burton, 1956). Percent fragmentation refers
to the ratio of DNA in the supernatant to the total DNA recovered
in the supernatant and pellet respectively.
For qualitative DNA fragmentation studies, the cells were
cultured and treated similar to as in the caspase assay. The
DNA was isolated according to Hogquist et al. (1991) with
minor modifications. Briefly, the scraped cells were lysed in
the lysis buffer (25 mM TrisHCl pH 7.8, 25 mM EDTA,
0.5% Triton X-100) at 4 C for 2 h. After centrifugation at
10,000 g, RNase-A (50 g/ml) was added to the supernatant
for 1 h at 37 C followed by Proteinase-K (100 g/ml) for 2 hat 4 C. DNA was extracted using phenolchloroform and
precipitated with 3 M sodium acetate and chilled absolute
ethanol. The pelleted DNA was dissolved in TrisEDTA
(pH 8.0) and electrophoresed on 1.8% agarose gel pre-stained
with ethidium bromide (0.5 g/ml).
Statistical analysis
All the studies were performed using TAM as positive
control and the results are expressed as meanSE from one of
the three similar experiments each performed in triplicate.
Student's t-test was used to determine the level of significance
and a P-valueb0.05 was regarded as significant.
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Results
CC induces cytotoxicity in HBCCs
For analyzing the effect of CC on cell survival, SRB assay
(Fig. 1) was performed in Normal (HEK, hGF and MDCK)
versus Cancerous cell types (Hep G2, MCF-7 and MDA MB-231 cells) employing TAM as a positive control. The results
illustrate CC mediated dose-dependent decline in the viability
of MCF-7 and MDA MB-231 cells versus untreated controls.
However, the normal and Hep G2 cells were resistant to both
CC/TAM exposure up to 25 M dose. On the contrary, a
precipitous decline in drug treated MCF-7 cells was noticed
from 115 M beyond which the curve plateaued off for CC/
TAM. Correspondingly similar profile was observed with MDA
MB-231 cells except that the leveling off was beyond 20 M.
This suggests relatively enhanced susceptibility of MCF-7 cells
unlike MDA MB-231 with efficacy of CC comparable to TAM.
Besides, both the assays demonstrate that 1 M CC displays
insignificant anti-proliferative activity in contrast to TAM
showing estrogenic profile. In accordance with the MTT assay(data not shown), the IC50 value of CC from SRB assay for MCF-
7 and MDA MB-231 cells was 9.08M (Pb0.001) and 20.16M
(Pb0.001) respectivelysimilar to TAM. Cytotoxicity results were
in agreement with those from HE staining studies (Fig. 2).
Microscopical examination displays dose-dependent decline in
the population of both the cell types upon exposure to CC/TAM as
compared to control thereby revealed similar results.
Fig. 1. Cytotoxicity evaluation of Centchroman (CC) in Cancerous [(A) MCF-7, (B) MDA MB-231 and (C) Hep G2] versus Normal [(D) MDCK, (E) HEK, (F) hGF]
Cell Lines. 0.01 106 cells were pre-cultured for 48 h in phenol red-free DMEM (DCC treated FCS) and subsequently exposed with various doses of CC for 48 h using
Tamoxifen (TAM) as a positive control. IC50 values were calculated using SRB Assay. Percentage survival was determined as per the formula (Absorbance of drug-
treated cells/ Absorbance of Control cells) 100 and compared with Control, untreated cells regarded as 100%. Data shown are the mean S.E. of one of the threesimilar experiments each performed in triplicate. *Pb0.05; Pb0.01;#Pb0.001.
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CC induces G0/G1 arrest
Cell-cycle analysis (Table 1 and Fig. 3A and B) reveals CC
having comparable yet disparate influences on MCF-7 and MDA
MB-231cells similar to TAM.Moreover,detection of hypodiploid
(apoptotic) sub-G0/G1 population of cells confirms its role as an
inducer of apoptosis. 1 M CC initiates G0/G1 arrest whereas S-
and G2/M-phase inhibition in MCF-7 cells becomes more
pronounced at 10 and 20 M (Pb0.01). However, the G0/G1
arrested fraction decreases significantly beyond 10 M. Corre-
spondingly in MDA MB-231 cells, CC initiates significant G0/G1 phase arrest at 10 M (Pb0.01) and G2/M-phase inhibition
(Pb0.05) even at 1 M whose severity increases with the dose.
Noticeably like MCF-7 cells, higher dose of CC (2025 M)
shows a significant decline in G0/G1 arrest (Pb0.001) while
pushing these fractions into sub-G0/G1 phase. However, the S-
phase remains largely unmodulated with both the AEs except at
high dose of 25 M showing abrupt decline (Pb0.01). There is a
Fig. 2. Morphological analysis of Centchroman (CC) treated (A) MCF-7 and (B) MDA MB-231 cells after Hematoxylin and Eosin (HE) staining. Cells were pre-
cultured for 48 h in phenol red-free DMEM(DCC treated FCS)and then exposed to different doses of CC/TAM for 48 h. Subsequently, HE staining was performed and
observed under Phase Contrast Microscope (magnification 100). All pictures are typical of three independent experiments each performed under identical conditions.
Table 1
Cell-Cycle analysis of Centchroman (CC) treated MCF-7 and MDA MB-231 cells
Treatment MCF-7 MDA MB-231
Apoptosis Cell-Cycle Apoptosis Cell-Cycle
Sub-G0/G1 G0/G1 S G2/M Sub-G0/G1 G0/G1 S G2/M
Control 4.52 0.67 55.84 3.67 12.05 0.70 27.59 1.21 5.41 0.39 60.08 0.44 8.14 0.36 26.57 0.24
TAM 1 M 3.74 0.25 64.38 4.74 13.220.56 18.640.82# 6.44 0.41 62.56 0.45 8.30 0.70 22.70 0.42*
TAM 10 M 13.63 1.09# 67.215.02 8.420.66 10.760.34# 10.440.78 67.66 5.42* 7.65 0.91 14.65 0 .14#
TAM 20 M 74.70 6.02# 12.881.62# 6.010.70# 6.410.73# 12.401.14 64.21 3.33 8.61 0.56 14.66 1.03#
TAM 25 M 86.65 5.31# 2.440.14# 3.800.22# 7.010.41# 38.432.23# 46.413.15* 3.000.21 11.511.41#
CC 1 M 3.41 0.19 63.19 4.75 10.78 1.41 22.62 1.71* 8.10 0.54 61.01 0.64 7.22 0.64 23.69 1.82*
CC 10 M 12.02 1.01# 68.033.27 7.020.55 12.930.11# 11.050.88 70.580.43# 7.640.61 10.360.19#
CC 20 M 80.07 6.77# 12.340.70# 3.550.24# 4.010.12# 21.460.89# 66.53 4.21* 6.04 0.55 5.57 0.98#
CC 25 M 94.10 8.02#
1.390.12#
2.420.25#
2.080.10#
60.192.36#
29.412.32#
2.680.24 7.710.54#
Briefly, 0.5 106 cells were pre-cultured for 48 h in phenol red-free DMEM (DCC treated FCS) and then exposed to different doses of CC/TAM for 48 h. Following
incubation the cells were harvested, permeabilized, stained with Propidium Iodide (40 g/ml) and analyzed by Flow Cytometer (employing Cell Quest Software). All
phases are represented in percentage. Data shown are the meanS.E. of one of the three similar experiments each performed in triplicate.*Pb0.05; Pb0.01;#Pb0.001.
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Fig. 3. Cell-cycle analysis of Centchroman (CC) treated MCF-7 and MDA MB-231 cells. Briefly, 0.5106 (A) MCF-7 and (B) MDA MB-231 cells were pre-cultured
for 48 h in phenol red-free DMEM (DCC treated FCS) and then exposed to different doses of CC/TAM for 48 h. For time-dependent apoptosis analysis, cells were
treated with IC50 doses i.e. (C) 10 M CC/TAM for MCF-7 and (D) 20 M CC/TAM for MDA MB-231 for varying time periods. Following incubation the cells were
harvested, permeabilized, stained with Propidium Iodide (40 g/ml) and analyzed by Flow Cytometer. All pictures are typical of three independent experiments each
performed under identical conditions.
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significant yet steep decline in G0/G1, S and G2/M phase by both
the ligands at 20M in MCF-7 cells and 25M in MDA MB-231
cells. Time kinetics of CC induced apoptosis was explored by
Flow Cytometry using IC50 values of CC/TAM in MCF-7 and
MDA MB-231 cells (Fig. 3C and D). Control cells of both the type
displayed only basal apoptosis. Significant rise in apoptotic
fraction (Pb0.01) was observed after 12 h of drug treatment whichincreased with the time. In both the cell types, after 48 h,
approximately 50% of the cell population was found to be
apoptosed (Pb0.001).
CC induces apoptosis in HBCCs
This study analyses CC induced apoptosis in MCF-7 and
MDA MB-231 cells. Control, untreated cells of both the types
display rare faint green Annexin V-FITC fluorescence depicting
non-descript, early apoptosis (Fig. 4). Fair amount of Annexin
positive fraction at 1 M CC/TAM indicates onset of apoptosis
in MCF-7 and MDA MB-231 cells. The magnitude of Annexin
positive MCF-7 cells versus CC was higher than with TAM at
1 M indicating enhanced susceptibility. CC/TAM at 10 M
enhances PI positive fraction (red fluorescence) heralding cell
membrane deformation. Once again CC seems remarkably
better than TAM since more AnnexinPI positive fraction was
observed. CC at 20 M imparts intense red staining to thenuclear region with no sign of residual green stain in MCF-7
cells. Similar fluorescence profiling was noticed with MDA
MB-231 cells. However, appearance of PI positive cells began at
1M CC whose magnitude was greater at 10 and 20M than for
TAM indicating subtle differences in the response.
CC induces mitochondrial events
To explore the mitochondrial events in CC induced apoptosis,
we measured changes in m and ROS level. Control MCF-7
and MDA MB-231cells elicited maximal Rh 123 fluorescence
Fig. 4. Detection of Apoptosis in (A) MCF-7 and (B) MDA MB-231 cells by Annexin-V-FITC and Propidium Iodide (PI) staining. Briefly 0.4 106 cells were pre-
cultured for 48 h in phenol red-free DMEM (DCC treated FCS) and then exposed to different doses of CC/TAM for 48 h. Subsequently, the cells were stained withAnnexin V-FITC (green fluorescence) and PI (red fluorescence) and analyzed by fluorescence microscopy (magnification 400).
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reflecting intact, functional mitochondria (Fig. 5A). CC/TAM
treatment results in rapid dose-dependent dissipation of mas detected by consequent decrease in mean fluorescence.
Significant decrease in m starts at 1 M CC/TAM (Pb0.05)
in both cell types except MDA MB-231 with TAM. However,
abrupt decline in m begins at 10 M for both the ligands
and cell types declining further at 20 M becoming negligibleat 25 M (Pb0.001 for all). Intracellular ROS induction
consequent upon drug treatment (Fig. 5B and C) revealed
similar dose-dependent decline in both the cells where CC
accentuates ROS greater than TAM. Interestingly, MCF-7 cells
generate significant amount of ROS (Pb0.01) at 1 M CC
versus MDA MB-231 cells. However, ROS production declined
beyond this dose in both the cell types but significantly in MCF-
7 cells (Pb0.01).
CC induces caspase-dependent apoptosis
Caspase-8, -3 and -9 were assayed colorimetrically to
determine their role in CC/TAM induced apoptosis. Unlike
MCF-7 cells, MDA MB-231 cells displayed Caspase-3
activation at 1 M CC/TAM (Pb0.05) with dose-dependent
enhancement (Fig. 6). Contrarily, in both the cell types,
Caspase-8 and -9 activities increased in a dose-dependent
manner with CC/TAM. In MDA MB-231 cells like MCF-7
cells, CC was comparatively more efficient (Pb0.001 at 10 M
in MCF-7 and 20 M in MDA MB-231) than TAM (Pb0.01 at
10 M in MCF-7 and 20 M in MDA MB-231) in inducingCaspase-8. However, for Caspase-9, both the drugs and cell
types showed similar significance profile.
Analysis of nuclear morphology
This study was performed to ascertain the detailed effect(s) of
CC on nuclear morphology as compared to TAM. Control MCF-
7 and MDA MB-231 cells depict elliptical nuclei with uniform PI
staining. CC/TAM initiates chromatin condensation at 1 M,
peaking at 10 M and 20 M in either cell (Fig. 7A and B). Dark
red staining in the nucleus with higher intensity around the rim,
shrinkage (Pyknosis) along with crescenting and blebbing
(deformed nuclear morphology) was observed. Qualitativelyand quantitatively, CC scores over TAM causing nuclear
degeneration in either cells suggesting greater efficacy for the
former.
The foregoing is commensurate with the quantitative data for
percentage of apoptotic nuclei (Fig. 7C). Smooth, rounded versus
deformed nuclei were counted as normal versus apoptotic.
Control MCF-7 and MDA MB-231 cells contain 13% 1.52
and 12.9% 1.56 apoptotic nuclei respectively showing basal cell
death. 1 M CC in MCF-7 and MDA MB-231 cells resulted in
21%2.62 and 27%2.51 of the nuclei to apoptose respectively.
Conversely, TAM at same dose in either cells failed to registerany
Fig. 5. Analysis of Centchroman (CC) induced alterations in Mitochondrial
Membrane Potential (m) and Reactive Oxygen Species (ROS). Briefly,
0.2106 MCF-7 and MDA MB-231 cells were pre-cultured for 48 h in phenol
red-free DMEM (DCC treated FCS treated) and then exposed to different doses
of CC/TAM. (A) For the assessment of loss in m, trypsinized cells were
incubated with the fluorophore Rhodamine 123 (5 g/ml) for 30 min at 37 C in
dark. The unreacted dye was removed by washing the cells twice with chilled
PBS and analyzed by Flow Cytometry. For measuring ROS production, the
(B) MCF-7 and (C) MDA MB-231 cells were exposed to the ligands for 24 h,
washed twice with chilled PBS. Following this, the cells were incubated with
10M DCFDAfluorophorefor 30min at 37C in dark and subsequentlywashed
twice with chilled PBS and trypsinated. Finally the stained cells were analyzed
through Flow Cytometry. Data shown are the meanS.E. of one of the threesimilar experiments eachperformed in triplicate. *Pb0.05;Pb0.01;#Pb0.001.
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significant effect. In MCF-7 cells, 10 M CC caused a drasticincrease in apoptotic nuclei to 58% 3.01. In MDA MB-231 cells,
10 M CC significantly augments these values to 29%1.03
whereas 20M pushes these values to approximately 65% 5.02.
In brief, it can be concluded that CC causes nuclear damage
in MCF-7 and MDA MB-231 cells to varying extents.
CC induces DNA fragmentation in HBCCs
Quantitative and qualitative DNA fragmentation analysis was
carried out to substantiate the nuclear morphological studies.
Quantitatively the controls show negligible DNA fragmentation
(Table 2) due to ongoing apoptosis, 17.0%1.20 in MCF-7 and19.4%0.90 in MDA MB-231 cells. 1 M CC causes insig-
nificant fragmentation in both the cell types. However, MCF-7
unlike MDA MB-231 cells display greater sensitivity to CC
induced DNA fragmentation at 10 M [61.8%1.10 and 29.7%
2.10 in MCF-7 and MDA MB-231 cells respectively]. Subse-
quently, 20 M CC causes a slight increase to 68.0%4.20 in
MCF-7 cells and drastic rise up to 66.1%3.20 in MDA MB-231
cells. Similar fragmentation profile was obtained with TAM.
Insignificant fragmentations observed at 1 M CC in both the cell
types made us restrict our DNA laddering analysis (qualitative) to
only 10 and 20 M doses that reveal characteristic internucleo-
somal ladder (Fig. 8). Throughout the course of investigations, a
common theme that has emerged is that CC has anti-neoplastic
effect comparable to TAM and therefore affords an alternate
approach for the management of breast cancer.
Discussion
This study explores the basis of anti-neoplasticity induced by
CC in Human Breast Cancer Cells. We havealreadydemonstratedthat CC displays anti-neoplasticity in MCF-7 HBCCs (Srivastava
et al., 2004). We now report apoptosis to be the major player in
accomplishing CC induced cytotoxicity in MCF-7 (ER+ve) and
MDA MB-231 (ERve) cells respectively evaluating the role of
ER, if any, employing TAM as a positive control. Normal cell
lines (HEK 293, hGF, MDCK) were employed for evaluating the
cytotoxic potential of CC in non-tumoral cell types (Fig. 1).
Moreover like MDA MB-231, the other cancerous cell line Hep
G2, lacking functional ERs (Boix et al., 1993) was used as a
model to study and compare the cytotoxic profile of CC. This
strategy was employed to ascertain the cytotoxicity induced by
CC in the absence of ER. CC and TAM are Type-I AEs withstrong anti-estrogenic and weakestrogenic potential (Clarke et al.,
2001; World Health Organization, 1993; Kamboj et al., 1977).
Since an apoptotic cell rarely displays all the features character-
istic of cell death, hence several parameters have been examined.
Cytotoxicity (Fig. 1) and HEstaining (Fig. 2) studies reveal that in
all the cancerous cell types, CC causes a dose-dependent decline
in the percentage of surviving cells compared to corresponding
controls similar to TAM. Moreover in the Normal cell types,
both the drugs elicited cytotoxicity at comparatively higher doses
i.e. N25 M for HEK 293, Hep G2 and MDCK (data not shown)
whereas 25 M in hGF suggesting inactivity of both CC/TAM
towards Normal cells. This disparity of action of CC/TAM towards
Normal versus Cancerous cells may be attributed to differences intissue-specific levels and expression patterns of Cytochrome P450
(CYP) isoforms mediating cell-specific cytotoxicity (Sridar et al.,
2002). CYPs' is a multigene family consisting of constitutive and
inducible enzymes that may influence the response of tumors to
anti-cancer drugs. This is because several anticancer agents can be
either activated or detoxified by this enzyme system. Studies
in vitro have implicated many CYP isoforms (e.g., CYP3A,
CYP2D6, CYP2C9, CYP2C19, CYP2B6, and CYP1A2) in the
biotransformation of TAM (Crewe et al., 1997). The principle
metabolites of TAM are N-desmethyltamoxifen (formed by
CYP3A4), 4-hydroxytamoxifen and endoxifen (formed by
CYP2D6) (Crewe et al., 1997). Implication of CYP2B6 has alsobeen suggested to be involved in the metabolic activation of TAM
(Desta et al., 2004). 7-desmethylcentchroman is considered as a
possible active metabolite of CC in vivo. (Paliwal and Gupta.,
1996). In order to exert its effects via anti-estrogenic metabolites
that are more potent than the parent compound, TAM must be
activated by the Cytochrome P450 system (Coezy et al., 1982) and
therefore a similar mechanism might also be applicable for CC.
Moreover, it has been reported that CYP1B1, CYP2B6 and
CYP2D6 are overexpressed in tumors (Sridar et al., 2002). We
suggest that the differential expression of CYPs may be one of the
possible reason(s) accounting for disparity in the susceptibility of
normal versus cancerous cells to CC/TAM. However in Hep G2
cells, these enzymes have been reported to get deprived of during
Fig. 6. Assessment of caspase activities in Centchroman (CC) induced apoptosis
in (A) MCF-7 and (B) MDA MB-231cells. Cells were pre-cultured for 48 h in
phenol red-free DMEM (DCC treated FCS) in T-75 flasks and subsequently
exposed to different doses of CC/TAM for 48 h. Cell lysates were prepared and
treated with Caspase-8 substrate (Z-IETD-pNA), Caspase-9 substrate (Ac-
LEHD-pNA) and Caspase-3 substrate (Z-DEVD-pNA) individually as
described in Materials and Methods. Activities were measured as a function
of pNA released at 405 nm on SpectraMAX 190 Microplate Reader. Data shown
are the meanS.E. of one of the three similar experiments each performed in
triplicate. *Pb0.05; Pb0.01; #Pb0.001.
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in vitro cultivation (Maria et al., 2000) thereby exhibiting
insensitivity of drugs in this cell type. IC50 values for CC in
MCF-7 and MDA MB-231 cells were in accordance with TAM as
reported (Perry et al., 1995b). Disparity in the doses of CC/TAM
eliciting anti-proliferation in MCF-7 (ER+ve) and MDA MB-231
(ERve) cells corroborates the significance of ER (Perry et al.,
1995b). While ER- has been established as the foremost mediator
of proliferation, the role of ER- remains obscure. Studies have
reported high levels of ER- in normal breast tissue and its
frequent reduction during carcinogenesis (Park et al., 2003; Roger
Fig. 7. Analysis of Centchroman (CC) induced alterations in nuclear morphology of (A) MCF-7 and (B) MDA MB-231 cells by Propidium Iodide (PI) staining.
0.4106 cells were pre-cultured for 48 h in phenol red-free DMEM (DCC treated FCS) and subsequently exposed to different doses of CC/TAM for 48 h. Following
incubation, the cells were stained with PI (40 g/ml) and analyzed by fluorescence microscope (magnification 400). Arrows indicate condensed or blebbed
(apoptotic) nuclei. For quantification (C) pictures showing deformed nuclear morphology (condensed, blebbed or crescented morphology) were counted as Apoptotic
whereas rounded nuclei with uniform PI staining were counted as Normal. Data shown are the meanS.E. of one of the three similar experiments each performed in
triplicate. *Pb0.05; Pb0.01; #Pb0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
586 M. Nigam et al. / Life Sciences 82 (2008) 577590
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et al., 2001; Shaaban et al., 2003; Skliris et al., 2003). Recently, it
has been reported that ER- enhances the ability of AEs through
their mutual interaction i.e. ER-promotes TAM induced G1 arrest
by enhancing G2 arrest and expression of pro-apoptotic genes
(Gallagher et al., 2007). In MCF-7 and MDA MB-231 there are
two types of ER- (I) Wild type [wt] and (II) a product of
alternative splicing [corresponding to exon 5], lacking about
139 bp of the hormone-binding domain (Vladusic et al., 1998). In
MDAMB-231 cells unlikeMCF-7,thespliced variant is abundant,
whereas wt is barely present. This non-functional ER- could
thus lead to altered estrogen/anti-estrogen binding resulting in the
lack of anti-estrogenic response. Moreover, it has been alreadyreported that ER- also increases the potency of anti-estrogens by
shifting their dose-response curves, implying therefore that lower
concentrations of AEs may achieve the similar response if ER-
would have been present (Gallagher et al., 2007). This might
explain the higher IC50 in MDA MB-231 cells because of non-
functional ER-. Notably, the anti-estrogenicity of CC and TAM
are comparable. Flow Studies (Table 1 and Fig. 3) confirm CC
induced apoptosis with disparate effects on cell-cycle of MCF-7
and MDA MB-231 cells at any given molarity. Previous studies
demonstrate that TAM induces G0/G1 blockade in MCF-7 and
MDA MB-231 cells (Perry et al., 1995a). Our studies reveal that
at 1 and 10 M CC/TAM, both the cell types mostly display G0 /
G1 arrest with concurrent dose-dependent increase in sub-G0/G1
peak. This is possibly due to the cells under acute stress gettingample time to recover and reverting to normal status rather than
perishing. But at higher doses (i.e., beyond 20 M), the cells are
pushed into sub-G0/G1 i.e. apoptotic phase failing to undo the
drug induced damage.
In MCF-7 cells CC/TAM, besides inducing G0/G1 blockade,
also affords S- and G2/M phase inhibition with similar
consistency and level of significance. In MDA MB-231 cells,
CC/TAM inhibits only the G2/M phase leaving S-phase
unperturbed vis--vis control. Contrarily, G0/G1 arrest is
significant from 10 M for both ligands leaving S-phase
unperturbed only up to 20 M beyond which it significantly
declines. In summary, analysis of cell-cycle kinetics and disparityof CC/TAM responsiveness in the two cell types may suggest
different cell-cycle check points operable at any given time.
Moreover, time-dependent apoptosis analysis confirms that IC50dose, in both the cell types, initiates significant apoptosis post-
12 h of exposure. Despite varying IC50 doses, the time-dependent
profile of CC/TAM induced apoptosis displays similar profile.
Following 48 h incubation, approximately half of the cell
population gets apoptosed. We have already accounted for this
disparity of drug susceptibility of two cell types in question on the
basis of ER status.
Apoptotic cells undergo characteristic changes in plasma
membrane architecture e.g. externalization of Phosphatidylser-
ine (PS) which provides a recognition signal for phagocytosis ofapoptotic cells and stimulus for production of anti-inflammatory
mediators (Huynh et al., 2002; Bratton and Henson, 2005).
Apoptosis was evaluated by Annexin V-FITC and PI double
staining (Fig. 4). Acquisition of green fluorescence (Annexin V-
FITC) begins at a dose as low as 1 M with CC/TAM in either
cell whose magnitude is greater for CC, indicating early
apoptosis. Concomitant to acquisition of green fluorescence,
Table 2
Quantitative analysis of Centchroman (CC) induced DNA fragmentation in
MCF-7 and MDA MB-231 cells
Treatment Percentage DNA fragmentation
MCF-7 MDA MB-231
Control 17.0 1.20 19.4 0.90
TAM 1 M 18.0 1.28 21.1 1.10TAM 10 M 53.8 4.00 29.52.00*
TAM 20 M 65.6 5.04# 56.64.20#
CC 1 M 22.4 2.00 23.5 1.50
CC 10 M 61.8 1.10 29.72.10*
CC 20 M 68.0 4.20# 66.13.20#
The cells were pre-cultured for 48 h in phenol red-free DMEM (DCC treated
FCS) and subsequently exposed to 120 M CC/TAM in T-75 flasks for 48 h.
Following lysis in the lysis buffer, the lysates were spun at 20,000 gfor 10 min
at 4 C to separate the fragmented versus intact chromatin. Both fractions were
quantified colorimetrically as described in Materials and Methods. Percent
fragmentation refers to the ratio of DNA in the supernatant to the total DNA
recovered in the supernatant and pellet respectively. Data shown are the mean
S.E. of one of the three similar experiments each performed in triplicate.
*Pb
0.05;
Pb
0.01;#
Pb
0.001.
Fig. 8. Qualitativeanalysis of Centchroman (CC) induced DNA fragmentationin (A) MCF-7 and(B) MDAMB-231 cells. Briefly, the cells were pre-cultured for48 h in phenol
red-free DMEM (DCC treated FCS) in T-75 flasks and subsequently exposed to different doses of CC/TAM for 48 h. After culmination of incubation, DNA was isolated andsubjected to 1.8% agarose gel electrophoresis as described in Materials and Methods. The gels were stained with Ethidium bromide and visualized under UV light.
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traces of yellowish and intense red PI stained nuclear material is
observed at 10 and 20 M of CC/TAM, showing severe cell
membrane deformation at this stage as depicted by decrease in
green fluorescence.
Disruption of Mitochondrial Membrane Permeability (m) is
an early event of apoptosis leading to cell death despite removal of
stimulus before the cell actually succumbs (Kroemer, 2002). It has been reported that TAM dissipates m in MCF-7 cells
(Strohmeier et al., 2002). We analyzed that CC attenuates min both the cell types beginning at 1 M and beyond (Fig. 5A).
Cancer cells, particularlyhighly invasive or metastatic, may require
a critical level of oxidative stress to maintain a balance between
proliferation and apoptosis. Constitutively enhanced production of
H2O2 (peroxides) in some cancer cells promotes proliferation and
can induce cell-cycle arrest and/or apoptosis above a certain
threshold (Chen et al., 2005). The pro-oxidant activity of 1 M CC
in both the cell types (Fig. 5B and C) may be attributed to the
persistent sub-lethal oxidative stress promoting proliferation
in vitro (Brown and Bicknell, 2001). However, a severe declinein the ROS level beyond this dose probably suggests anti-oxidant
behavior of the ligands. These analyses thus indicate implication of
critical mitochondrial events in CC induced apoptosis.
Apoptosis is typically accompanied by the activation of
Caspases. TAM has been shown to induce Caspase-dependent
cell death in MCF-7 and MDA MB-231 cells (Mandlekar et al.,
2000). To evaluate the role of Caspases in CC mediated apoptosis,
we assayed Caspase-8, -9 and -3 (Fig. 6A and B). Results illustrate
that CC induces both Caspase-8, -9 in MCF-7 and MDA MB-231
cells along with Caspase-3 in latter cell type.Activation of Caspase-
8 suggests involvement of extrinsic pathway in CC induced
apoptosis. Caspase-3 activity was undetectable in MCF-7 cells
possibly attributed to the functional deletion of CASP-3 gene(Janicke et al., 1998). Thus, CC induces Caspase-dependent
apoptosis irrespective of ER status.
Nuclear morphological studies (Fig. 7A and B) aptly
correlated with AnnexinPI analysis. 1 M CC/TAM in each
cell type initiates apoptosis associated derangements in nuclear
morphology as condensed chromatin gets (dense red patches)
concentrated around the rim of the nucleus. Further increment in
drugdosage in either cells increase the severity illustrated by rapid
shrinkage, blebbing and crescenting and finally at maximal
dosage residual nuclei become totally deformed shrunken,
shriveled entities. Noticeably, MCF-7 cells seemingly succumb
faster to the deleterious effects of CC than TAM as displayed bygreater overall reduction in size and number of nuclei with in-
creasingdose. Similar response was observedwith the MDA MB-
231 cells. The mechanism underlying these changes can be
attributed to degradation of nuclear, cytoskeletal proteins e.g.
Lamins by Caspases resulting in chromatin condensation
(Loeffler et al., 2001; Susin et al., 1999). Besides, Apoptosis
Inducing Factor (AIF) induces Caspase-independent nuclear con-
densation (Danial and Korsmeyer, 2004).
Quantitative data for the percentage of apoptotic nuclei
(Fig. 7C) also supports the preceding. Unlike TAM, CC at
1 M in both the cell types causes a significant increase in the
percentage of apoptotic nuclei with respect to control. Exposure to
10 M CC in MCF-7 cells causes 4.5 fold increase in percent
apoptotic nuclei whereas the corresponding rise in MDA MB-231
cells was 2.2 fold. This illustrates higher vulnerability of MCF-7
cells to CC which is almost double to that in MDA MB-231cells
possibly reflecting the ER status. Interestingly, CC response peaks
at 10 M sustaining through 20 M for MCF-7 in terms
of magnitude because at latter dose thepercentage apoptotic nuclei
did not increase significantly. Correspondingly, in MDA MB-231cells at 20 M CC the increase was found to be 5.0 and 4.4 fold
showing thereby the maximal response. TAM also responded
similarly in both the cell types but the overall data confirms that
CC augments the percentage of apoptotic nuclei more than TAM.
Alterations in nuclear morphology may not necessarily
involve DNA fragmentation (Srivastava et al., 2006) leading
us to investigate drug mediated DNA fragmentation. This is
more so since our studies provide evidence for the activation of
Caspases which are principal actors of DNA fragmentation.
Apart from this, Caspase-independent apoptotic DNA degrada-
tion has been attributed to nuclear translocation of mitochondrial
proteins, Endonuclease-G and Apoptosis Inducing Factor (AIF)(Danial and Korsmeyer, 2004). TAM induced DNA fragmenta-
tion has been reported in MCF-7 and MDA MB-231 cells (Perry
et al., 1995b). Quantitatively, 1 M CC in both cell types causes
an insignificant increase in the percentage of fragmented DNA
compared to respective controls (Table 2). However, 10 M CC
in MCF-7 cells induces 3.6 fold increase in fraction of fragmented
DNA whereas the corresponding increase in MDA MB-231 cells
was 1.6 fold. This illustrates higher susceptibility of MCF-7 cells
to CC which is approximately double as compared to MDA MB-
231 cells reinforcing the role of ER. Further, 20M CC inMCF-7
cells induces 3.9 fold increase whereas the corresponding
response in MDA MB-231 cells was 3.5 fold. TAM was also
found to exhibit similar profile in both cell types but CC seems to be more competent. These results were in agreement with
qualitative analysis (Fig. 8) which reveals that 10 and 20 M CC
induces DNA laddering in both cell types. Overall analysis
suggests that CC induced apoptosis results in DNA fragmentation
of MCF-7 and MDA MB-231 cells.
In MCF-7 cells, DNA fragmentation might be contributed by
Caspase-3-independent mechanisms in agreement with Caspase
activity assay (Fig. 6). On the contrary, consistent with Caspase
activity assay, Caspase-3-dependent/-independent or both the
mechanisms might be active in MDA MB-231 cells. Apparent
in phase II clinical studies with CC treatment, patients with low
or undetectable ER responded partially thereby hypothesizingother mechanisms to be operative in the activity of AE against
breast cancer (Mishra et al., 1989).
Conclusion
Our studies clearly demonstrate that CC seems to be a potent
anti-neoplastic agent possessing remarkable similarity to TAM.
CC induces apoptosis in MCF-7 and MDA MB-231 HBCCs
irrespective of their ER status. However, further analyses of the
intrinsic/extrinsic pathways in conjunction with non-genomic
events and their regulation with CC are needed. Since
prolonged AE therapy is usually associated with several side
effects (Clarke et al., 2001, Macgregor and Jordan, 1998), CC
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seems to be a newer anti-neoplastic agent with negligible side
effects and some associated beneficial effects. The L-enantiomer
(Levormeloxifene) of CC has been shown to be anti-atherogenic
and inhibits bone resorption without stimulation of endometrial
epithelium in ovariectomized rats (Singh, 2001). Prolonged
administration of CC to rats and rhesus monkeys does not
induce toxicity and is therefore considered safe for chronicadministration (Mukerjee et al., 1997). Moreover, treatment of
CC for more than 4 years on women volunteers showed
normal hematological and biochemical parameters (Singh,
2001). DL-Centchroman and its D- and L-enantiomers lack
mutagenicity, genotoxicity and reduce toxic effects of known
mutagens (Giri et al., 2001). To our knowledge, this is the first
such report detailing the induction of apoptosis by CC in MCF-7
and MDA MB-231 HBCCs. In conjunction with previous
clinical trials in advanced cases of estrogen dependent breast
cancer (Rajan, 1996a,b), our study suggests a future prospect for
a better anti-breast cancer agent. Moreover, resolving its (CC)
other potential isomers by understanding structure
activityrelationships could increase the prospects of their potential
usage (Macgregor and Jordan, 1998).
Acknowledgements
Director, C.D.R.I. is thanked for permitting to carry out the
work. Mr. A. L. Vishwakarma is thanked for the experiments
with Flow Cytometry. Manisha Nigam (31/4(763)/2004-EMR-I)
and Vishal Ranjan (31/4(770)/2005-EMR-I) gratefully acknowl-
edge the fellowship received from C.S.I.R. New Delhi. The
work has been supported by funds from CMM-0018 and MOH,
New Delhi.
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