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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.

578 M. Nigam et al. / Life Sciences 82 (2008) 577590


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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.

579M. Nigam et al. / Life Sciences 82 (2008) 577590


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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.)



11/14

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.



12/14

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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