Invest New Drugs (2008) 26:425–435

DOI 10.1007/s10637-008-9116-5

PRECLINICAL STUDIES

Growth inhibition and induction of apoptosis

in MCF-7 breast cancer cells by a new series
of substituted-1,3,4-oxadiazole derivatives
Akhilesh Kumar & Saritha S. D’Souza & S. L. Gaonkar &
K. M. L. Rai & Bharathi P. Salimath

Received: 22 November 2007 / Accepted: 10 January 2008 / Published online: 29 January 2008
# Springer Science + Business Media, LLC 2008

Summary The multiple pharmacological actions of unique
synthetic compounds are a prerequisite for classifying a
drug as highly efficacious, because the multiple pharma-
cological actions offer the possibility of treating various
diseases like cancer. 1,3,4-Oxadiazoles are an important
class of heterocyclic compounds with broad spectrum of
biological activities. In this study we focused on the ability
of these derivatives to induce apoptosis in cultured MCF-7
breast cancer cells. Treatment of MCF-7 cells with varying
concentrations of the different derivatives resulted in dose
and time dependent sequence of events marked by apo-
ptosis, as shown by loss of cell viability, chromatin con-
densation, internucleosomal DNA fragmentation and sub
G0 phase accumulation. Furthermore, apoptosis in MCF-7
cell was induced by upregulation of proto-oncoprotein
Bax and activation of Caspase-3 activated DNase. Although
A. Kumar : S. S. D’Souza : B. P. Salimath
Department of Studies in Biotechnology,
University of Mysore,
Manasagangotri,
Mysore 570006 Karnataka, India
S. L. Gaonkar : K. M. L. Rai
Department of Studies in Chemistry,
University of Mysore,
Manasagangotri,
Mysore 570006 Karnataka, India
B. P. Salimath (*)
DOS in Applied Botany and Biotechnology,
University of Mysore,
Mysore 570006, India
e-mail: salimathuom@rediffmail.com
the derivatives induced apoptosis was associated with Bax
protein levels, negligible Bcl-2 reduction was observed.
Analysis of the data suggests that the substituted oxadiazole
derivatives exert antiproliferative action and growth inhibi-
tion on MCF-7 cells through apoptosis induction and that it
may have anticancer properties valuable for application in
drug products.
Keywords Apoptosis . Oxadiazole derivatives .
MCF-7 cells . Bax . Bcl-2
Introduction
Breast cancer affects one in every ten women in Western
Europe and the USA [1], and it is the second leading cause
of cancer-related deaths [2]. Treatment includes surgery,
radiation, and in some cases, drugs that are specifically
targeted such as herceptin, in the case of tumors that
overexpress EGF receptor, or tamoxifen, in the case of
estrogen-dependent tumors [3]. However, the majority of
cases, especially those that result in metastasis, are still
treated with conventional chemotherapy. The problem of
drug resistance is a major obstacle in chemotherapeutic treat-
ment. Therefore, there is a great need for the development of
new therapeutic drugs that will be more efficient or will
synergize with the existing ones.
There has been a growing interest in the use of synthetic
compounds as a potent source of new therapeutic anticancer
drugs. Compound containing oxadiazole moiety plays a
pivotal role in various pharmaceutical applications. 1,3,4-
Oxadiazoles are an important class of heterocyclic com-
pounds with broad spectrum of biological activities. Such
426 Invest New Drugs (2008) 26:425–435

chemical compounds show anticancer and tyrosinase inhibi- Materials and methods
tory activity [4]. Substituted 1,2,3-oxadiazoles have revealed
antibacterial [5, 6], antimycobacterial [7], antifungal [8, 9], Chemicals
anti-inflammatory [10, 11], analgesic [12], anticonvulsant
[13, 14] and insecticidal properties [15]. Oxadiazoles find Fetal bovine serum (FBS), DMEM, penicillin–streptomycin
use as fluorescent whiteners [16] and also act as muscle (PS) and glutamine were purchased from GIBCO laborato-
relaxants [17]. ries, Grand Island, NY. Rabbit polyclonal antibody against
Recently, the relationship between apoptosis and cancer Bcl-2, Bax, and caspase-3 were procured from Santa Cruz
has been emphasized, with increasing evidence suggesting Biotechnology, Inc., Heidelberg, Germany. All other chem-
that the related processes of neoplastic transformation, icals were of the highest grade commercially available and
progression and metastasis involve the alteration of normal supplied either by Merck or Sigma. The oxadiazole deriva-
apoptotic pathways [18]. Apoptosis provides a number of tives were synthesized as reported earlier [29]. Of the many
clues with respect to effective anticancer therapy, and many derivatives synthesized, three compounds namely compound
chemotherapeutic agents reportedly exert their antitumor 1a, 1b and 1c (Fig. 1) were used to evaluate the apoptotic
effects by inducing apoptosis in cancer cells [19]. Apo- effect on MCF-7 cells.
ptosis is a strictly regulated pathway responsible for the
ordered removal of superfluous, aged, and damaged cells. It Cell culture and assessment of cell growth and viability
not only plays an important role in the development and
maintenance of tissues homeostasis, but it also represents MCF-7 cells were grown in DMEM supplemented with
an effective mechanism by which harmful cells can be 10% heat inactivated FBS, 1% penicillin- streptomycin in a
eliminated [20, 21]. Morphologically, hallmarks of this humidified incubator (5% CO2 in air at 37oC). Cells (2×105
process included loss of cell volume, hyperactivity of the cells/ well) were seeded in six well plates prior to com-
plasma membrane, and condensation of peripheral hetero- pounds addition. The MCF-7 cells were incubated with
chromatin, followed by cleavage of the nucleus and cyto- different doses (50, 100, 200 and 300 μM) of compounds
plasm into multiple membrane-enclosed bodies containing 1a-c for 0, 12, 24 and 48 h. Cultures were harvested and
chromatin fragments [22–25]. Recently, considerable atten- monitored for cell number by counting cell suspensions
tion has been devoted to the sequence of events referred with a hemocytometer. Cell growth and viability were
to as apoptotic cell death and the role of this process in checked before and after treatment with the compounds 1a-c
mediation of the diverse antineoplastic agents. using trypan blue dye exclusion and examined using phase
In 1970, the estrogen receptor-positive MCF-7 cell line contrast microscopy.
was derived from a patient with metastasic breast cancer
[26]. Since then MCF-7 cell has become a prominent model
system for the study of breast cancer as it relates to the
susceptibility of the cells to apoptosis. Despite the fact that
many tumors initially respond to chemotherapy, breast
cancer cells can subsequently survive and gain resistance to
the treatment [27]. Further, it has become increasingly
important in the treatment of a number of major solid
tumors, particularly metastatic and drug-resistant breast
cancer [28]. In this study, the effects of the new series of
oxadiazole derivatives on cultured MCF-7 human breast
cancer cells was investigated due to the interesting bio-
logical activities reported and their potential clinical appli-
cation. The data reported herein appear to demonstrate that
oxadiazole derivatives induce massive death in the MCF-7
cells, the dying cells exhibiting the ultra-structural and
biochemical features that characterize apoptosis. We also
explored the mechanism by which they reduce proliferation
of human breast cancer cells and found that the oxadiazole
derivatives can cause accumulation of cells in the Go phases
of the cell cycle. In addition, we looked into the signaling
mechanism by which the derivatives exert their beneficial
effects in the chemoprevention of breast cancer cells. Fig. 1 Structure of the three compounds a, b and c under study
Invest New Drugs (2008) 26:425–435
3[H] Thymidine uptake assay
To verify for the in vitro effect of the compounds 1a-c on the
proliferation of MCF-7 cells, the cells (1×105) were cultured
in vitro in a 12 well plate in DMEM medium supplemented
with 10% FBS, 1mg/ml penicillin/streptomycin and were
grown in 5% CO2 atmosphere at 37oC for 2 days. 3[H] thy-
midine (1 μCi/ml of medium) was added prior to addition
of the compounds 1a-c (50, 100, 200 and 300 μM). After
2 days, the cells were processed for liquid scintillation
counting [30].
DNA fragmentation assay
MCF-7 Cells (2×105 cells/60 mm dish) were treated with
or without the compounds 1a-c for different doses (50, 100,
200 and 300 µM) and different time intervals (0, 12, 24 and
48 h). The cells were lysed in a buffer containing 50 mM
Tris–HCl pH 8.0 and 0.5% SDS, incubated for 30 min at
37oC. The cell lysate was subjected to 8M potassium
acetate precipitation and left for 1 h at 4oC. The suspension
was centrifuged at 7,000 rpm for 1 h at 4oC to remove cell
debris. The supernatant was subjected to extraction using
distilled phenol: chloroform: isoamyl alcohol (25:24:1) by
centrifugation at 3,000 rpm for 30 min. The supernatant con-
Fig. 2 Effects of the compounds on MCF-7 cell growth (a) and
viability (b). Cells were treated with 0, 50, 100, 200 and 300 μM of
the compounds for 12, 24 and 48 h. Control cells were maintained in
427
taining nucleic acids was incubated with RNase (20 µg/ml)
for 10 min at 37oC and the DNA was precipitated with ice-
cold ethanol. The precipitated DNA was dissolved in
minimum quantity of TE buffer and quantitated spectropho-
tometrically. Equal concentration of DNA (10 μg) was
resolved on 1% agarose gel, viewed under UV light and
documented using Uvp-BioDoc-ItTM system.
Ethidium bromide/acridine orange staining
Nuclear staining was performed according to the method
described by Srinivas et al. [31]. Briefly, MCF-7 cells (2×
105 cells/ 60 mm dish) either treated or untreated with the
compounds 1a-c (300 μM) for 24 h were smeared on a
clean glass slide, fixed with methanol: acetic acid (3:1) and
air-dried. The cells were hydrated with PBS and stained
with a mixture (1:1) of acridine orange/ethidium bromide
(4 μg/ml). The cells were immediately washed with PBS
and viewed under a Leitz-DIAPLAN fluorescent micro-
scope for acridine orange/ethidium bromide staining.
Western blot analysis of CAD, Bax and Bcl-2
MCF-7 cells (1×105 cells/ 60 mm dish) were treated with the
compounds 1a-c for different doses (50, 100, 200 and
the vehicle for the indicated time periods. Results are represented as
mean±SEM of three assays
428
300 µM) and time intervals (0, 12, 24 and 48 h) in vitro. Cells
were homogenized in a lysis buffer (50 mM Tris-HCl,
pH 8.0, 5 mM ethylenediaminetetraacetic acid (EDTA),
150 mM NaCl, 0.5% NP-40, 0.5 mM phenylmethylsulphonyl
fluoride, 10 μg/ml leupeptin and 0.5 mM dithiothritol) for
30 min on ice. The cell debris was pelleted by centrifugation
at 10,000×g, 4oC for 30 min. For the preparation of nuclear
extract, the cells were extracted in a buffer containing 50 mM
Tris–HCl, pH 7.5, 10 mM MgCl2, 1 mM CaCl2, 1 mM
EDTA, 0.25 M sucrose, 1 mM PMSF, 2 mg/ml leupeptin and
10 mg/ml aprotenin. About 60 µg of nuclear and/or cytosolic
proteins as estimated by Lowry’s method were separated on
12.5% sodium dodecyl sulfate polyacrylamide gel electro-
phoresis (SDS-PAGE) and transferred to nitrocellulose
membranes. After blocking with 5% non-fat skimmed milk
powder in Tris-buffered saline, the membranes were incu-
bated with antibodies against CAD (DFF-40), Bax and Bcl-2.
The blots were washed thrice with Tris-buffered saline
Tween-20 (TBST) on a shaker and incubated with secondary
antibody tagged to alkaline phosphatase for 2 h. After wash-
ing with TBST for 10 min, the CAD, Bax and Bcl-2 proteins
were detected by using the chromogenic substrate BCIP/
NBT.
Flow cytometry for analysis of apoptosis
For the determination of cell cycle phase distribution,
MCF-7 cells were grown to exponential phase, seeded at a
Fig. 3 Phase contrast micro-
graphs of the compounds
treated MCF-7 cells. Cells were
treated with 300 μM of the
compounds for 24 h. Typical
results from three independent
experiments are shown
Invest New Drugs (2008) 26:425–435
density of 2×105 cells/60 mm dish, and either untreated or
treated with the compounds 1a-c (300 μM) for different
time intervals of 0, 12, 24 and 48 h. After the incubation
period cells were collected, centrifuged and washed twice
with PBS at room temperature. The cell pellets were resus-
pended in ice-cold 70% ethanol for overnight fixation at
4oC. The suspension was centrifuged and the cell pellet was
resuspended in 0.8 ml PBS containing 1 μl of RNase
(10 mg/ml) and 20 μl of PI (1 mg/ml) and incubated for
30 min at 37oC before analysis by flow cytometry. A total
of 10,000 events were acquired and analysis of flow cyto-
metric data was performed using ModFit software. A histo-
gram of DNA content (x-axis, PI fluorescence) vs counts
(y-axis) has been displayed. Apoptosis was quantitatively
measured as the percentage of cells in sub Go region.
Statistical analysis
Unless stated otherwise, all experiments were performed
in triplicates. Wherever appropriate, the data was expressed
as the mean±standard error of the mean (S.E.M) and
means were compared using one-way analysis of variance
(ANOVA). Statistical significance of differences between
control and compound 1a-c treated tumor cells were
determined by Tuckey’s post hoc test. Statistical signifi-
cance was defined as p<0.05 for all the tests. All the
analysis was performed using GLM univariate procedure
using SPSS for Windows (version 10.0).
Invest New Drugs (2008) 26:425–435
Results
Effect of the compounds on cell growth and viability
of MCF-7 cells
To investigate the potential effects of the compounds 1a-c
on proliferation and survival of MCF-7 cells, the cells were
Fig. 4 Antiproliferative effect of the compounds. To verify for the in
vitro effect of the compounds on MCF-7 cells, 1×105 cells were
plated in a 12 well plate and processed for antiproliferation assay
using 3[H] thymidine (1 µCi/ml of medium) with 0, 50, 100, 200 and
429
exposed to 50, 100, 200 and 300 µM of the compounds
for 0, 12, 24 and 48 h. Figure 2a and b show that the
compounds induce cell death in a dose and time dependent
manner, as determined using trypan blue dye exclusion.
Further, exposure to the compounds was associated with
cell shrinkage as detected in phase contrast micrographs
(Fig. 3).
300 µM of the compounds (Compound 1a, compound 1b and
compound 1c). The data represents the mean ± SEM of three
independent experiments. Effect of the compounds is statistically
significant in comparison with control (p<0.001)
430 Invest New Drugs (2008) 26:425–435

Antiproliferative effect of the compounds

To gain insights into the antiproliferative effect of the com-

pounds 1a-c on MCF-7 cells we performed a 3[H] thymidine
incorporation assay. Significant dose dependent growth
inhibition of MCF-7 cells was observed on treatment with
the compounds. The results shown in Fig. 4 clearly indicate
that the compounds inhibit proliferation in a dose dependent
manner when compared to 100% proliferation of MCF-7
cells in vitro in the absence of the compounds. F-test
showed a significant difference between different treatments
( f=910.158; p<0.001).

Compounds induce degradation of DNA in MCF-7 cells

Biochemically, apoptosis is characterized by fragmentation

of chromosomal DNA. We verified the effect of the com-
pounds 1a-c on DNA damage of MCF-7 cells. As expected,
agarose gel electrophoresis of the compounds treated
chromosomal DNA showed a ladder like pattern of DNA
fragments consisting of multiples of approximately 180–200
base pairs. The apoptosis inducing activity of the compounds
was both dose and time dependent (Fig. 5a and b).

Compounds induce nuclear condensation of MCF-7 cells

An attempt was made to verify whether the inhibition

of proliferation of MCF-7 cells was due to the apoptosis
inducing effect of the compounds 1a-c. Acridine orange/
ethidium bromide (Fig. 6) staining of MCF-7 cells treated
with the compounds indicated nuclear condensation as
compared to the untreated MCF-7 cells. The cells on treat-
ment with the compounds showed typical apoptotic
morphology, which included condensed nuclei, membrane
blebbing and formation of apoptotic bodies. In contrast the
control cells showed intact nuclear architecture.

Compounds induce translocation of CAD

In apoptotic cells, cleavage of ICAD by the Caspase-3

protease activates CAD. Western blot analysis of nuclear
extract of the compounds treated and untreated MCF-7 cells
showed the presence of CAD in compounds treated MCF-7
cells as compared to the untreated cells (Fig. 7a and b). The
CAD protein was identified in the nuclear extract of treated
cells that increased with time and dose as compared to the
control wherein CAD was not detected.
Bcl-2 and Bax are involved in the compounds
induced apoptosis
Fig. 5 DNA fragmentation of MCF-7 cells exposed to the compounds
1a-c. a MCF-7 cells incubated with 0, 50, 100, 200 and 300 μM of the
three compounds respectively for 24 h. b MCF-7 cells treated with
300 μM of the compounds for 0, 12, 24 and 48 h. DNA ladders
reflecting the presence of DNA fragments were viewed on ethidium
bromide stained gel. Typical result from three independent experi-
ments is shown

Bcl-2 and Bax protein levels were studied in cultured MCF-

7 cells to examine the involvement of Bcl-2 and Bax in the
Invest New Drugs (2008) 26:425–435 431

Fig. 6 Effect of the compounds on morphology of MCF-7 cells. Cells cells were washed extensively with PBS and viewed under fluorescent
either untreated or treated with the compounds in vitro were stained microscope for apoptotic morphology and photographed. Typical
with 1:1 stain solution of ethidium bromide and acridine orange. The result from three independent experiments is shown

compounds mediated apoptosis. Western blot analysis of cally increased Bax protein levels in a dose and time
Bcl-2 (Fig. 8a and b) and Bax (Fig. 9a and b) proteins on dependent manner. There was no effect on the Bcl-2
treatment of cells with the compounds 1a-c was verified. protein, however. These results indicate that the compounds
Incubation of MCF-7 cells with the compounds dramati- may disturb the Bcl-2 and Bax ratio.

Fig. 7 Effect of the compounds on nuclear translocation of CAD in prepared from both control and compound treated cells, resolved on
MCF-7 cells. a MCF-7 cells treated with 0, 50, 100, 200 and 300 μM 12% SDS-PAGE and transferred to nitrocellulose membrane. CAD
of compounds 1a-c for 24 h. b MCF-7 cells treated with 300 μM of protein was detected using anti-CAD rabbit polyclonal antibody
the compounds for 0, 12, 24 and 48 h. Nuclear extracts(60 μg) were
432
Fig. 8 Western blot analysis of Bax protein levels after exposure to
the compounds. a MCF-7 cells treated with 0, 50, 100, 200 and
300 μM of compounds 1a-c for 24 h. b MCF-7 cells treated with
300 μM of the compounds for 0, 12, 24 and 48 h. Cytosolic extract
Effect of the compounds on cell cycle progression
in MCF-7 cells
To elucidate the possible mechanism of the compounds
mediated tumor inhibition, we investigated the effect of
the compounds on MCF-7 cells. We found that treatment of
the compounds1a-c on MCF-7 cells induced a significant
proportion of cells to undergo apoptosis, as determined by
the flow cytometric analysis. Cell cycle analysis by flow
cytometry was used to quantitatively estimate the number
Fig. 9 Western blot analysis of Bcl-2 protein levels after treatment
with the compounds. a MCF-7 cells treated with 0, 50, 100, 200 and
300 μM of compounds 1a-c for 24 h. b MCF-7 cells treated with
Invest New Drugs (2008) 26:425–435
(60 μg) from each sample was resolved on 12.5% SDS-PAGE and
Western blot performed. Typical result from three independent
experiments is shown
of cells in each phase of cell cycle. MCF-7 cells were
treated for 0, 12, 24 and 48 h with 300 μM of the
compounds 1a, 1b and 1c and analyzed. The untreated cells
(control) showed a typical distribution in G1, S, and G2
phases on flow cytometry. After treatment, number of cells
in sub G0 area increased by 20% at 12 h and more than
70% at 24 h (Fig. 10). These results suggested that the
fragmentation of DNA in MCF-7 cells results in tumor
killing. The obvious ramifications were the growth arrest of
MCF-7 cells.
300 μM of the compounds for 0, 12, 24 and 48 h. Cytosolic extract
(60 μg) from each compound treated cells was resolved on 12.5%
SDS-PAGE and analyzed by Western blotting
Invest New Drugs (2008) 26:425–435
Fig. 10 Flow cytometric analysis of the effect of the compounds
(a Compound 1a, b Compound 1b, c Compound 1c) on the cellular
DNA content of MCF-7 cells. Cells were grown in the absence (control)
Discussion
In the present study, we studied the inhibitory effect of
oxadiazole derivatives on breast cancer cells. The results
reported herein reveal that the compounds exert antiproli-
ferative action and growth inhibition in cultured human
breast cancer MCF-7 cells. The dying cells exhibit the
ultrastructural and biochemical features that characterize
apoptosis, as shown by loss of cell viability, chromatin
condensation, internucleosomal DNA fragmentation and
sub-Go phase accumulation. The most interesting finding in
this study was the pronounced and concentration dependent
reduction in the cell growth of MCF-7 cells by the
compounds 1a-c in the whole range of concentration used.
Treatment of the compounds 1a-c inhibited proliferation
of cancer cells MCF-7 in a time and dose dependent man-
ner. However, of the three compounds used, compound 1a
433
or the presence of the compounds (300 μM) for 12, 24 and 48 h, stained
with propidium iodide, and analyzed by flow cytometry for DNA
content. Each plot is representative of three similar experiments
and 1c showed increased apoptotic effects as compared to
compound 1b. This effect may be due to the presence of the
methyl group in the compound 1b. Despite the variation in
the tumor inhibition activity, all the three compounds were
able to induce apoptosis where the mechanism of tumor
growth inhibition was involved.
It has been shown that the bcl-2 family plays an
important regulatory role in apoptosis, either as an activator
(Bax) or as an inhibitor (Bcl-2) [32, 33, 34]. It has also
been demonstrated that the gene products of Bcl-2 and Bax
play important roles in apoptotic cell death. [35, 36, 37]. Of
the Bcl-2 family members, the Bcl-2 and Bax protein ratio
has been recognized as a key factor in regulation of the
apoptotic process [32, 33, 34]. In the present study, the
increase in the compounds induced apoptosis was asso-
ciated with an increase in the levels of Bax protein, which
heterodimerizes with, and thereby inhibits, Bcl-2. Negligible
434
Bcl-2 reduction was observed, however. Analysis of our data
indicates that the compounds 1a-c may disturb the Bcl-2/Bax
ratio and, therefore, lead to apoptosis of MCF-7 cells.
The morphological hallmarks of apoptosis such as nuclear
condensation and internucleosomal fragmentation of DNA
were clearly observed in the compound 1a-c treated MCF-7
cells. One of the nuclease primarily responsible for genomic
DNA fragmentation during apoptosis is called DNA frag-
mentation factor (DFF-40) or caspase activated DNase (CAD)
[38]. In Western blot analysis, the compounds 1a-c were
shown to activate series of proteins involved in apoptosis.
Caspases are cysteine proteases that play a critical role in
execution of apoptosis [39, 40]. Several chemotherapeutic
and chemopreventive agents have been shown to cause
apoptotic cell death through activation of caspases [39, 41].
Apoptosis signals activate a cascade of caspases including
caspase-8. The substrate for caspase-8 is BID protein, whose
cleaved C-terminal fragment triggers cytochrome C release
from mitochondria. The released cytochrome C binds to
Apaf-1, which includes auto-activation of caspase-9, which
in turn activates caspase-3. Caspase-3, a major executioner
caspase upon activation can systemically dislodge the cells
by cleaving key proteins, such as poly-ADP-ribose poly-
merase (PARP) [38, 39], lamins and inhibitor of caspase
activated DNase (ICAD) in downstream of cascade, resulting
in programmed cell death [41, 42, 43]. Upon caspase activa-
tion by external stimuli, CAD dissociates from ICAD and
translocates to nucleus to disrupt the genetic material [38].
Treatment of MCF-7 cells with the compounds 1a-c showed
the activation of CAD, which is a downstream target for
caspase-3 in MCF-7 cells.
In our study, it was found that the reduced expression of
Bcl-2 and increased expression of Bax protein, the activation
of CAD through caspase-3 in MCF-7 cells resulting in func-
tional alteration of the mitochondria, may be potentially
involved in the compounds induced apoptotic responses.
These results indicate the apoptotic-signaling pathway of the
compounds 1a-c in MCF-7 cells.
Induction of apoptosis and/or cell proliferation inhibition
is highly correlated with the activation of a variety of intra-
cellular signaling pathways to arrest the cell cycle in the
G1, S, or G2 phase. In malignant tumors, cell populations
in G1 phase appear less frequent than in normal tissue. The
damages that cause G1-check point arrest are believed to be
an irreversible process and the cell ultimately undergoes
apoptosis. Our results indicate that the compounds 1a-c cause
an increase of the cells in sub G0 region in a time dependent
manner, with the decrease of cells in G1, G2 and S-phase
regions. Thus it can be concluded that the compounds 1a-c
have the ability to induce apoptosis in proliferating cells.
In conclusion, the compounds 1a-c exhibit an antiproli-
ferative effect by induction of apoptosis that is associated
with caspase 3 activation and dysregulation of Bcl-2 and Bax
Invest New Drugs (2008) 26:425–435
in MCF-7 cells. As apoptosis has become a new therapeutic
target in cancer research, these results confirm the potential
of the compounds 1a-c as an agent of chemotherapeutic and
cytostatic activity in human breast cancer cells. However,
further investigation of its activity, in vivo, is necessary to
elaborate and exploit this nascent promise.
Acknowledgements The author (AK) thanks University Grant
Commission (UGC), New Delhi, and the author (SSD) thanks Lady
Tata Memorial Trust, Mumbai, India, for the financial support. The
authors express their sincere gratitude to University of Mysore,
Mysore, India for the laboratory facility.
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