Evaluation of in vitro and in vivo anticancer potential of two 5-acetamido chalcones against breast cancer

Article abstract of DOI:10.17179/excli2017-624

Two 5’acetamido chalcones, C1 and C2 were synthesized by Claisen-Schmidt condensation method and characterized by IR, LC-MS, ¹H NMR and ¹³C NMR. These compounds were evaluated for anticancer activity in vitro in breast cancer cell lines (MCF-7 and MDA-MB-231) using MTT assay, anti-metastatic assay, apoptotic screening by AO/EB staining and in vivo in N-Methyl-N-nitrosourea (MNU) induced breast carcinoma model. Sprague-Dawley rats with developed tumors (50 mg/kg MNU i.p.) were grouped in four, namely MNU control (0.25 % of CMC p.o.), standard group (doxorubicin 2 mg/kg once in 4 days, i.p.), C1 and C2 groups (50 mg/kg p.o. each). After 21 days of treatments, tumor volume and weight were assessed. Excised tumors were subjected to DNA fragmentation study. MTT assay showed IC₅₀ values of 62.56 and 37.8 μM by for C1 and C2. Both compounds increased apoptotic bodies more than 3 fold compared to normal control in AO/EB staining. Antimetastatic (scratch wound) assay showed a significant (p<0.05) reduction in cell migration after 24 h and 48 h treatments compared to normal control. In in vivo studies, tumor weight and tumor volume were significantly (p<0.05) reduced in the doxorubicin group as well as in test groups compared to MNU control. DNA fragmentation assay showed an increase in the number of bands formed in C1 and C2 compared to normal control. Results obtained from in vitro and in vivo studies demonstrated the significant anticancer potentials of C1 and C2.

Full text of DOI:10.17179/excli2017-624

EXCLI Journal 2017;16:1150-1163 – ISSN 1611-2156
Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
Original article:
EVALUATION OF IN VITRO AND IN VIVO ANTICANCER
POTENTIAL OF TWO 5-ACETAMIDO CHALCONES AGAINST
BREAST CANCER
1
1
2
1
1
Sonal Wankhede , Nitesh Kumar , Lalitha Simon , Subhankar Biswas , Karthik Gourishetti ,
1
1
1*
Grandhi Venkata Ramalingayya , Mit Joshi , C. Mallikarjuna Rao
1
Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal
University, Manipal, Karnataka-576104, India
Department of Chemistry, Manipal Institute of Technology, Manipal University, Manipal,
Karnataka-576104, India
2
*
Corresponding author: Dr. C. Mallikarjuna Rao, Principal & Professor of Pharmacology,
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka-
5
76104, India. Email- mallikin123@gmail.com; mallik.rao@manipal.edu
http://dx.doi.org/10.17179/excli2017-624
This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/).
ABSTRACT
Two 5’acetamido chalcones, C1 and C2 were synthesized by Claisen-Schmidt condensation method and
1
13
characterized by IR, LC-MS, H NMR and C NMR. These compounds were evaluated for anticancer activity in
vitro in breast cancer cell lines (MCF-7 and MDA-MB-231) using MTT assay, anti-metastatic assay, apoptotic
screening by AO/EB staining and in vivo in N-Methyl-N-nitrosourea (MNU) induced breast carcinoma model.
Sprague-Dawley rats with developed tumors (50 mg/kg MNU i.p.) were grouped in four, namely MNU control
(
0.25 % of CMC p.o.), standard group (doxorubicin 2 mg/kg once in 4 days, i.p.), C1 and C2 groups (50 mg/kg
p.o. each). After 21 days of treatments, tumor volume and weight were assessed. Excised tumors were subjected
to DNA fragmentation study. MTT assay showed IC50 values of 62.56 and 37.8 µM by for C1 and C2. Both
compounds increased apoptotic bodies more than 3 fold compared to normal control in AO/EB staining. Antimeta-
static (scratch wound) assay showed a significant (p<0.05) reduction in cell migration after 24 h and 48 h treat-
ments compared to normal control. In in vivo studies, tumor weight and tumor volume were significantly (p<0.05)
reduced in the doxorubicin group as well as in test groups compared to MNU control. DNA fragmentation assay
showed an increase in the number of bands formed in C1 and C2 compared to normal control. Results obtained
from in vitro and in vivo studies demonstrated the significant anticancer potentials of C1 and C2.
Keywords: chalcones, breast cancer, N-methyl-N-nitrosourea
INTRODUCTION
normal cell behavior characterized by uncon-
trolled proliferation, dedifferentiation, inva-
siveness, metastasis and alteration in several
other biological processes that maintain the
normal cycle of life. Among the various types
of cancers breast cancer is the most common
type of cancer in women. Current medication
st
Cancer is a threat of 21 century and is the
2nd most common cause of death after cardio-
vascular diseases (Cooper and Hausman
2
2
000; Global Burden of Disease Cancer
015). It is an outcome of abnormalities in
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Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
for breast cancer includes either alone or com- (MCOPS), Manipal University (MU) from
bination of approaches such as surgery, radi- designated manufacturers/suppliers. 2,2’az-
ation therapy, chemotherapy, hormone ther- ino-bis(3-ethylbenzothiazoline-6-sulphonic
apy, targeted therapy etc. Except for surgical acid (ABTS), 2,2-Diphenyl-1-picrylhydrazyl
therapy, all the above-mentioned approaches (DPPH),
(5,5'-dithio-bis-[2-nitrobenzoic
of treatments have their own shortcomings acid]) DTNB, Dulbecco’s Modified Eagle’s
such as mouth soreness, nausea, vomiting, Medium (DMEM), Dimethyl sulfoxide
weakness, loss of appetite, fatigue, hair loss (DMSO) were all procured from Sigma-Al-
etc. (Carelle et al. 2002). These side effects drich, Acetic acid, Hydrochloric acid (HCL),
not only alter the quality of life of survivors Methanol, MTT reagent, propidium iodide
but sometimes lead to their mortality.
Along with higher efficacy, chemothera- ries, Sodium hydroxide (NaOH), Sodium
peutic drugs have well established side effects chloride (NaCl), Sulphuric acid (H SO ) was
were all purchased from HiMedia laborato-
2
4
in breast cancer. Thus, extensive research is obtained from SD Fine Chemicals, Foetal bo-
required in this area to hunt for newer mole- vine serum (FBS) was purchased from Invi-
cules that can provide better therapy without trogen. Cell Lines: MCF-7 and MDAMB-231
compromising the quality of life. Chalcones were obtained from National Center for Cell
are one such category of compounds, which Sciences (NCCS) Pune.
can be thought in this perspective. They are
Synthesis of chalcones (different functional
the precursors of flavonoids and flavonol and
group substituted)
are also present in edible plants. Being a nat-
Claisen-Schmidt condensation reaction
urally occurring component, chalcones are
was followed for the synthesis of required
considered to have fewer side effects like gen-
chalcones (Simon et al. 2016). Briefly, para-
otoxicity as seen with other chemotherapeutic
cetamol was weighed and transferred into a
agents (Kumar et al. 2003) Several chalcones
1
00 mL round bottom flask. Anhydrous
have been synthesized which showed very po-
tent cytotoxicity against several cancer cell
lines like MDA-MB-231, MCF-7 etc. with es-
tablished mechanism of action like induction
of cell cycle arrest and apoptosis (Kumar et al.
aluminium chloride and nitrobenzene were
added to the flask followed by gradual addi-
tion of acetyl chloride. The temperature was
maintained at 130 °C for three hours. After
the reaction is complete, the reaction mixture
was poured into a mixture of crushed ice and
concentrated hydrochloric acid with vigorous
stirring and filtered. The crude product thus
obtained was first washed with water and then
with toluene and crystallized following which
light brown needle-shaped crystals were ob-
tained.
The product from the above step was dis-
solved in ethanol and 4-methoxy/3,4-meth-
ylenedioxy benzaldehyde was added to it. To
this, 10 % aqueous KOH solution was added
and the reaction mixture was stirred around
2
003). However, there are very few studies
available on acetamido chalcones as an
antitumor agent. In view of above scenarios in
breast cancer and related therapies, our study
was focused on evaluating in vitro & in vivo
anti-cancer activity of 5’-acetamido chalcon-
es against breast cancer. The study was fo-
cused on the synthesis and characterization of
compounds, evaluation of its in vitro and in
vivo anticancer potential in breast cancer
model and assessment of its antioxidant prop-
erty.
3
0 °C for about 12 h, and poured into crushed
MATERIALS AND METHODS
ice containing 5N HCl. The precipitated prod-
uct was washed with cold water, dried and re-
Materials
All the chemicals required for different crystallized from ethanol to yield dark yellow
experiments were procured through Manipal crystals (Figure 1).
College
of
Pharmaceutical
Sciences
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Figure 1: Synthesis of 5’-acetamido-2’-hydroxychalcones
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13
Thin layer chromatography (TLC) tech- protons ( H) and carbon atoms ( C) present
nique was applied using hexane and acetone in the compound to confirm its molecular
as a solvent system in a ratio of 6:4 for moni- structure. The instrument used was Bruker
toring of the completion of the reaction. The NMR by Bruker Corporation, USA.
retention factor was calculated to check the
In vitro cytotoxicity screening
reaction completion as well as for establishing
MTT assay is a colorimetric assay for as-
the purity of the synthesized compound. After
sessing cell viability. Cells were seeded in
the reaction completion % yield was calcu-
sterile 96-well tissue culture plate. Cells were
lated. The melting point for each compound
allowed to attach for 24 h. The compound was
was determined using melting point
prepared before the beginning of the experi-
apparatus. Mass spectroscopy was used to de-
ment and serially diluted with suitable me-
termine the molecular weight of the com-
dium to get the different concentrations (50-
pounds. The instruments used in the preseTnMt
5
1
00 μM). After 24 h, cells were treated with
00 μl of test compounds from respective top
study was Thermo ScientificTM Q Exactive
Focus hybrid quadrupole-Orbitrap MS, by
Thermo Fischer Scientific Corporation, USA.
IR spectroscopy was used for structural con-
firmation in the infrared region of the electro-
magnetic spectrum using Shimadzu FTIR, by
Shimadzu Corporation, Japan. NMR spec-
troscopy was used to determine the number of
stocks for 48 h. After 48 h 50 μl of MTT rea-
gent (Stock: 2 mg/ml in PBS) was added in
each well and incubated for 3 h at 37 °C. The
cells in the control group did not receive treat-
ment. Each treatment was performed in tripli-
cates. After 3 h of incubation medium along
with MTT was aspirated and 200 μl of 100 %
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DMSO was added to each well to solubilize 96 well plates, 50 μl of various concentrations
formazan crystals. The Optical density (O.D) of the compound was added in duplicates.
was measured by an ELISA plate reader at Followed by 50 μl solution of DPPH (0.1mM)
5
40 nm. Percentage viability of each com- was added using a multichannel pipette.
pound was calculated (Gerlier and Thomas- Ascorbic acid was used as the standard for
set, 1986).
comparison. Control included an equal
amount of ethanol and DPPH. After
Acridine orange/ethidium bromide staining
2
0 minutes incubation in the dark, absorbance
(
AO/EB staining)
was recorded at 540 nm. The experiment was
performed in triplicate (Bishayee et al., 2013).
Chromatin condensation and nuclear frag-
mentation are one of the important apoptotic
hallmarks. Therefore, examination of the
morphology of the cells is very important.
%
scavenging= [(ABS control – Abs test)] /
Abs control) x 100
(
4
Cells were harvested and (5 ꢀ10 ) cells were
seeded in 12 well plate and allowed to attach
overnight. Cells were treated with selected
In vivo study
test compound at their IC50 (determined from Animals
MTT assay) for 48 h incubation. Media was
Sprague-Dawley rats were maintained as
aspirated and each well was washed with per the guideline laid by Committee for the
PBS. Cells in each well were fixed with ice- Purpose of Control and Supervision of Exper-
cold ethanol for 20 min. Next, ethanol was as- iments on Animals (CPCSEA), Govt. of In-
pirated from each well. Acridine orange dia. Briefly, animals were acclimatized in an
(
10 mg/ml) and ethidium bromide (10 mg/ml) experimental room at temperature 23 ± 2 °C,
stain was prepared in PBS and mixed in the controlled humidity and 12:12 hour light and
ratio of 3:2 and added to each well. After dark cycle. They were provided with food and
2
0 min, unbound stain was washed using PBS water ad libitum. Study was conducted after
solution and observed under fluorescent mi- obtaining ethical committee clearance from
croscope (NikonTS100F) with an excitation the Institutional Animal Ethics Committee of
of 450-490 nm (Ribble et al. 2005).
KMC, Manipal (Clearance No. IAEC/KMC/
3/2015).
7
In vitro anti-metastasis assay
Cells, grown in DMEM containing 10 % Acute toxicity study
FBS, were seeded into 6 well plate for 24 h to
Acute toxicity study will be conducted ac-
a 70-80 % confluent monolayer. Using a ster- cording to OECD 425 guideline with 5 female
ile 200 μl pipette tip, a monolayer was mice in each group. 2000 mg/kg dose was
scratched across the center of the well. The given orally to each mouse and the animals
well was washed twice with medium to re- were observed for 14 days. No motility was
move detached cells and replenished with observed with the test compound at a dose of
fresh medium containing test compounds. 2000 mg/kg (Botham, 2004).
The cells were washed twice with PBS. Im-
ages were taken at 0 h (immediately after
In vivo antitumor activity in MNU-induced
breast cancer model
wounding), 24 h and 48 h of treatment at 40X
Breast tumor was induced in Sprague-
magnification using an inverted microscope.
Dawley rats by injecting 50 mg/kg MNU in-
(
Bishayee et al., 2013).
traperitoneally (McCormick et al., 1981).
After the development of tumor i.e., 90 days
Antioxidant assay by DPPH assay method
The antioxidants react with 1,1-diphenyl- from the injection, the animals were grouped
-picrylhydrazyl radical (DPPH), a stable free in the followings: MNU control group:
2
radical and convert it to 1,1-diphenyl-2-picryl animals were administered with 0.25 % of
hydrazine. The ability to scavenge DPPH was CMC (p.o) & 10 ml/kg saline (i.p). Standard
measured at an absorbance of 517 nm. To the group: animals were treated with doxorubicin
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at a dose of 2 mg/kg once in 4 days (i.p.). C1 12000 g for 10 min at RT. The supernatant
group: animals were treated with C1 at a dose was collected in a new tube; the DNA was
of 50 mg/kg (p.o.). C2 group: animals were then precipitated by adding chilled absolute
treated with C2 at a dose of 50 mg/kg p.o. ethanol and 0.1 M NaCl. The precipitated
Animals were administered with the aforesaid DNA was collected by centrifugation and
compounds for 21 days. Tumor volume was washed with 70 % ethanol. The DNA pellet
monitored every 6 to 7 days using a digital was dried and dissolved in distilled water. The
Vernier caliper. The length (a) and width (b) DNA samples were prepared in a loading so-
of tumor were measured at right angles. The lution (0.25 % bromophenol blue, 0.25 % xy-
tumor volume was estimated using Carlson’s lene cyanol FF and 30 % glycerol) in the ratio
2
formula V=4/3Π a b, where ‘a’ and ‘b’ 1:5. 10 µL of the DNA sample was loaded
represent the longest and shortest tumor radii into each well of 1.8 % agarose gel containing
respectively (Kumar et al., 2014). On day 0.5 mg/ml ethidium bromide. The electropho-
nd
2
2 , blood was withdrawn, the tumor was resis was carried out in TAE buffer for
isolated and various organs were isolated. The 1.5 hours. The DNA bands in the gel were ob-
isolated tumor was subjected to DNA frag- served and photographed in a Syngene G-Box
mentation study, histopathological study and Chemi XRQ Gel documentation system (Kuo
endogenous antioxidant estimation.
et al., 1996).
Organ index
Histopathological examination
Organs namely, liver, kidney, heart and
All the animals of MNU control group,
spleen were isolated and weighed in grams. standard group, C1 and C2 treated groups
The organ index was calculated by the follow- were sacrificed on day 24 from the initial day
ing formula (Fidler, 2003):
of sensitization by light ether anesthesia fol-
lowed by carotid bleeding. A small portion of
the tumor was selected for histopathology.
The specimens were stored in 10 % neutral-
ized buffered formalin, and processed for his-
topathological findings.
Organ index
=
weight of organ in grams
body weight of animals in grams
Estimation of endogenous antioxidants
Hematological parameters
Liver and tumor homogenate (10 %w/v)
were prepared with ice-cold phosphate buffer
using Teflon-glass homogenizer. This ho-
mogenate was centrifuged at 10,000 rpm for
Following parameters were monitored in
breast cancer-bearing rats to detect the
influence of chalcones: White blood cell
count, Red blood cell count Haemoglobin
count, Platelets count, Percentage of lympho-
cytes, monocytes and granulocytes using
ERMA veterinary blood cell counter, Japan.
10 min, the pellet was discarded and the su-
pernatant was used for the estimation of Total
protein: (Goa, 1953), Superoxide dismutase
estimation (SOD) (Hamberg et al., 1968), Li-
pid peroxidation (TBARS) estimation
DNA ladder assay
For the apoptotic study, the electropho-
retic analysis of fragmented DNA was done;
DNA was isolated from the tumor samples.
(
Devasagayam et al., 2003), and Estimation
of reduced glutathione (GSH) (Anderson,
985). Serum was used for evaluation of the
1
1
00 mg of tumor samples were homogenized
in 1 ml of lysis buffer [20 mM Tris (pH 7.5),
.15 M NaCl,1 mM EDTA, 1 % SDS] then
safety parameters on the liver by estimation of
ALT, AST and on the kidney by creatinine es-
timation using the semi-automatic auto ana-
lyzer.
0
centrifugation at 3000 g for 10 min was done
at RT and a supernatant was aspirated out.
2
0 µL Proteinase K (stock 20 µg/ml) was
added to the supernatant and incubated for
hours at 37 °C. This was then centrifuged at
2
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RESULTS
7 cells and 21.19 μM and 12 μM on MDA-
MB-231 respectively (Table 1).
Synthesis and characterization of
compounds
The method of synthesis for the com-
pounds was Claisen Schmidt condensation re-
Table 1: In vitro antioxidant assay
Compound
DPPH Assay
(IC50 value in μM)
action. The R
C2 were found to be 0.388, 0.347 and 81.5 %,
2 % respectively. The purity of the com-
f
value and % yield for C1 and
Ascorbic acid
32.42
9
C1
C2
125.3
83.94
pound was assessed by preliminary methods
such as melting point and TLC. Further, the
structural confirmation of purified C1 and C2
were performed by IR spectroscopy, LC-MS,
1
13
Acridine orange/ethidium bromide staining
H and C NMR which are as follows:
(
AO/EB staining)
C1: Percentage yield 81.5%, Rf value-
Apoptotic index significantly (p<0.05)
0
.388, melting point160 ± 2 °C, IR (KBr
-
1
increased in all treatment groups as compared
to normal control. Doxorubicin, C1 and C2
showed apoptotic indices 17.41 ± 3.342,
cm ) 3443 (OH), 3250 (N-H stretch), 3053
C-H aromatic), 1641 (C=O), 1616 (N-H
(
.
bend), 1166 (C-O ), Mass-m/z (M+1) =
1
1
5.79 ± 2.72520, 64 ± 0.95 respectively. The
311.17; HNMR (400 MHz, DMSO) (D6) δ
2.039 (s, 3H, CH ), 3.835 (s, 3H, O-CH ),
6 ), 9.90 (s, NH),
1
difference between apoptotic index of
doxorubicin (2 mg/ml), C1 (50 mg/ml), and
C2 (50 mg/ml), were not significant (p<0.05)
compared to each other (Figure 2).
3
3
.94-8 (9H, Ar-H and H
1.96 (s, OH).
α
, H
β
C2: Percentage yield 92 %, Rf value-
.347, Melting point 198 ± 2 °C, IR (KBr
0
Scratch wound assay
Scratch wound assay was performed in
metastatic human breast cancer cell line
-
1
cm ) 3443.05 (OH), 3342 (N-H stretch),
650 (C=O), 1614 (N-H bend), 1150 (C-O),
1
1
Mass-m/z (M+1) = 324.83; H-NMR (400
MHz, DMSO-D6) δ2.032 (s, 3H, CH ), 6.13
), 6.94-8.14 (8H, Ar-H and H
(
MCF-7) to evaluate the potential of C1 and
3
C2 to inhibit cell migration, metastasis, and
anti-proliferative effect. Percentage of cell
migration after treatment with molecules
were evaluated by image J software. Two-
time interval viz., 24 h and 48 h were selected
to evaluate antiproliferative effect of drugs.
Doxorubicin, C1 and C2 significantly
(
s, 2H, O-CH
2
α
,
13
H
β
), 9.88 (s, NH), 11.96 (s, OH); C-NMR:
4
1
1
1
1
1
00 MHz, (DMSO-D6) δppm: 24.13 (CH3),
02.28 (O-CH ), 107.47, 109.19, 118.14,
2
20.74, 121.39, 121.78, 126.43, 128.87,
29.39, 131.34, 145.05, 148.67, 150.41,
57.62, 168.55 (14C, Ar-C and C
93.45 (C=O).
α
,C ),
β
(
p<0.05) prevented the proliferation of cells
compared to normal control at both time point
24 h, 48 h). However, no significant differ-
(
In vitro evaluation
ence was observed among doxorubicin, C1
and C2 (Figure 3).
Preliminary in vitro cytotoxicity screening in
human breast cancer cell lines
In vitro cytotoxic effect of synthesized
compounds in MCF-7 & MDA-MB-231 by
MTT assay after 48 h of exposure showed that
molecules produced dose-dependent decrease
in the percentage viability of the cells. IC50
values for C1 and C2 were found to be
6
2.56 μM and 37.8 μM respectively on MCF-
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Figure 3: Effect of treatment C1 and C2 (50 µg/ml
and 24 h and 48 h) on the healing of wound
scratched in a monolayer of MCF -7 cells.
All the values are mean ± SEM of three readings.
Data are analyzed by Two-way ANOVA followed
by Bonferroni post-test method of analysis using
Graph Pad Prism, where *p < 0. 05 compared to
normal control
Figure 2: Acridine Orange/Ethidium Bromide
staining of MCF-7.
Antioxidant assay by DPPH assay method
The in vitro antioxidant activity of stand-
ard (Ascorbic acid), C1 and C2 were evalu-
ated using DPPH assay method. All the tested
compounds showed a dose-dependent de-
crease in the absorbance of the color of DPPH
solution. IC50 values of C1, C2 and standard
using DPPH assay was found to be 125.3,
These arrows indicate the presence of an
apoptotic cell. All the values are Mean ± SEM of
three readings. Data are analyzed by One-way
ANOVA followed by Tukey’s post hoc method of
analysis using GraphPad Prism, where *p< 0.05
compared to normal control
83.94 and 32.42 μM respectively. C2 showed
an IC50 value less than 100 μM, which could
be considered as moderate antioxidant poten-
tial (Table 2).
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Table 2: Effect of C1 & C2 on organ index
Groups
Spleen index
Liver Index
Heart Index
Kidney Index
MNU control
0.34±0.016
0.33±0.022
3.17± 0.15
3.68±0.12*
0.38±0.015
0.35±0.010
0.67±0.027
0.67±0.022
Standard (2 mg/kg)
C1 (50 mg/kg)
C2 (50 mg/kg)
0.31±0.027
0.37±0.027
3.032 ±0.10
3.25 ± 0.16
0.35±0.022
0.34±0.011
0.63±0.029
0.63±0.029
All values are Mean ± SEM of six samples. Data are analyzed by One-way ANOVA followed by Tukey’s
post hoc method of analysis using Graph Pad Prism, where *p< 0.05 compared to MNU control
In vivo evaluation
Acute Toxicity Study
Compounds were found safe up to
2
000 mg/kg p.o. following OECD 425 guide-
line of acute toxicity assessment.
In vivo antitumor activity in MNU-induced
breast cancer model
Effect of C1 & C2 on tumor volume
Tumor volume is a potential biomarker
for breast cancer development. Day 4 onward,
the doxorubicin-treated group showed signif-
icant (p<0.05) reduction in tumor volume as
compared to MNU control. Day 8 onwards,
treatment with C1 showed significant
Figure 4: The effect of treatment C1 and C2 on
tumor volume of rats in MNU induced breast can-
cer model.
All values are mean ± SEM of six samples. Data
are analyzed by Two-way ANOVA followed by
Bonferroni post-test method of analysis using
Graph Pad Prism, where *p < 0. 05 compared to
MNU control
(
p<0.05) reduction in tumor volume as com-
pared to MNU control. Day 16 onwards treat-
ment with C2 showed significant (p<0.05) re-
duction in tumor volume as compared to
MNU control. On day 21, all the treatment
group showed significant (p<0.05) reduction
in tumor volume as compared to MNU con- Percentage reduction in tumor volume
3
trol. Tumor volume was 243.67 ± 154.52 mm
Tumor volume is a potential biomarker
3
for standard, 1412.52 ± 309.55 mm for C1 for breast cancer development. The maximum
and 2760.99 ± 884.38 mm for C2, which reduction in tumor volume was seen in MNU
3
were significantly (p<0.05) low as compared control group. Treatment with C1 and C2
3
to MNU control (7890.69 ± 2707.97 mm ) on showed significant (p<0.05) percentage re-
Day 21 (Figure 4).
duction in tumor volume as compared to
MNU control. Doxorubicin showed signifi-
cant (p<0.05) percentage reduction in tumor
volume as compared to MNU control. Per-
centage reduction in tumor volume was 84.58
±
9.167 for standard, 72.93 ± 5.646 for C1 and
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63.47 ± 63.47 for C2, which were signifi- RBC, WBC, and concentration of Hb, while a
cantly (p<0.05) high compared to MNU con- significant increase in platelets counts was
trol (-145.1 ± 12.91) (Figure 5).
observed when compared to MNU control.
The differential leucocyte counts were
significantly (p<0.05) altered in the standard
treatment group as compared to MNU con-
trol, except the % granulocyte level which
was unaltered. In C1 and C2 treatment
groups, no significant difference was ob-
served as compared to MNU control group
(
Table 3).
Figure 5: Effect of treatment C1 and C2 on
percentage reduction in tumor volume on day 21
in comparison to day 1.
All values are Mean ± SEM of six samples. Data
are analyzed by One-way ANOVA followed by
Tukey’s post hoc method of analysis using Graph
Pad Prism, where *p< 0.05 compared to MNU
control
Figure 6: Effect of treatment C1 and C2 on tumor
weight (g) of rats in MNU induced breast cancer
model.
All values are Mean ± SEM of six samples. Data
are analyzed by One-way ANOVA followed by
Tukey’s post hoc method of analysis using Graph
Pad Prism, where *p< 0.05 compared to MNU
control
Tumor weight
Tumor progression can be determined by
measuring tumor weight. All the treatment
showed significant (p<0.05) reduction in tu-
mor weight as compared to MNU control. Tu-
mor weight was 9.87 ± 1.96 g for MNU con-
trol, 1.06 ± 0.49 g for standard, 4.90 ± 0.52 g
for C1 and 4.70 ± 1.67 g for C2 (Figure 6).
Effect of C1 & C2 on endogenous
antioxidants
Organ index
There was no significant difference ob-
served in organ indices of liver, spleen, heart Estimation of endogenous antioxidants in
and kidney in any treatment group as com- tumor
pared to MNU control except doxorubicin
Standard), which showed a significant in- (p<0.05) increase in SOD levels compared to
crease in the liver index as compared to MNU MNU control in tumor samples. Glutathione
Standard, C1 and C2 showed significant
(
control (Table 2).
levels were significantly increased in C2
treated group when compared with MNU con-
trol. There was a significant increase in MDA
levels in standard (2 mg/kg) and C1 (50 mg/
kg) treated group when compared to MNU
control group, while C2 did not produce any
significant change (Table 4).
Hematological parameters
There was no significant difference ob-
served in hematological parameters by the
treatment of C1 and C2 as compared to MNU
control. Doxorubicin treatment showed sig-
nificant (p<0.05) decrease in the count of total
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Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
Table 3: Effect of treatment C1 and C2 on the blood cell count in MNU induced breast cancer model
Hematology Parameters (Mean ± SEM)
Groups
RBC
X10 /µl)
WBC
(X10 /µl)
Platelets
(X10 /µl)
Hb (g/dl)
%LY
%MO
%GR
6
3
3
(
MNU
control
Stand-
ard
8.28±0.17 11.04±0.39
436.3±34.29
14.03±0.26 69.92±1.98 1.41±0.86 13.15±0.71
6.69±0.46*
5.7±0.81*
621.1±61.41* 11.50±0.85* 59.62±4.1* 0.86±0.15* 14.88±0.81
C1
C2
7.85±0.09
7.51±0.15
8.8±1.4
10.2±0.9
401.2±32.22
379.0±53.71
13.30±0.12 67.80±1.85 1.05±0.16 11.97±0.45
12.92±0.21 67.12±2.08 1.25±0.07 12.28±0.49
All values are Mean ± SEM of six samples. Data are analyzed by One-way ANOVA followed by Tukey’s
post hoc method of analysis using Graph Pad Prism, where *p< 0.05 compared to MNU control
Table 4: Tumor endogenous antioxidant status
Groups
SOD
U/mg of protein)
GSH
(µM/mg of protein)
TBARS
[MDA (nM/mg of
protein)]
(
MNU control
284.2±20.79
6.742±0.5983
91.19±24.62
Standard
C1
1215±37.23*
917.7±90.42*
5.498±0.8512
7.408±0.8321
290.3±99.78*
271.4±33.32*
C2
603.3±84.36*
10.51±0.1930*
140.1±18.53
All values are Mean ± SEM of six samples. Data are analyzed by One-way ANOVA followed by Tukey’s
post hoc method of analysis using Graph Pad Prism, where *p< 0.05 compared to MNU control
extraction from the tumors. Thus it may be
Estimation of endogenous antioxidants in
stated that both C1 and C2 showed the
Liver
incidence of apoptosis in breast tumors in-
duced by MNU (Figure 7).
Standard (Doxorubicin) showed signifi-
cant (p<0.05) increase in SOD level as com-
pared to MNU control in liver samples.
Whereas no statistical difference was ob-
served in C1 and C2 treatment groups as com-
pared to MNU control. Glutathione levels
were significantly (p<0.05) increased in
Standard and C2 treated group compared with
MNU control. MDA levels were significantly
(
p<0.05) increased by doxorubicin while sig-
nificantly decreased by C1 treatment as com-
pared to MNU control (Table 5).
DNA ladder assay
It was observed that intensity/no. of bands
formed by C1 and C2 were more as compared
to MNU control group in the excavated breast
tumors. This is a direct evidence for the
increased incidence of apoptosis in response
to the treatments of C1 and C2. Standard did
not show any change as compared to MNU
control which might be due to improper DNA
Figure 7: Effect of treatment on DNA ladder as-
say
a= MNU control, b= std, c= C1, d= C2,e= C1
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Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
Histology
pattern. Treatment with C1 was not signifi-
Histopathology of tumor in MNU control cant in comparison with the standard as ob-
group exhibited a distortion in the normal ar- served by the distorted architecture of the ep-
chitecture of rat mammary gland. Loss of ithelial cells. Moreover, C2 treatment was not
tubuloalveolar pattern was observed along able to restore the altered shape of the alveolar
with the presence of large epithelial cells. In cells (Figure 8).
contrast, histopathology of the standard group
displayed a restoration in the tubuloalveolar
Table 5: Liver endogenous antioxidant status
Groups
SOD
U/mg of protein)
GSH
(µM/mg of protein)
TBARS
[MDA (nM/mg of
protein)]
(
MNU control
271.9±4.514
0.427±0.1048
232.2±22.21
Standard
C1
604.8±85.07*
227.1±9.597
1.129±0.3277
1.074±0.1254
376.1±50.49*
140.6±28.45
C2
205.2±16.60
1.320±0.2389*
92.22±5.324*
All values are Mean ± SEM of six samples. Data are analyzed by One-way ANOVA followed by Tukey’s
post hoc method of analysis using Graph Pad Prism, where *p< 0.05 compared to MNU control
Figure 8: Histology of breast tumor
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EXCLI Journal 2017;16:1150-1163 – ISSN 1611-2156
Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
DISCUSSION
MNU (N-methyl-N-nitrosourea) induced
breast carcinoma is an in vivo model which
mimics ER+ve breast cancer (McCormick et
al., 1981). In the present study, we evaluated
the synthesized compounds C1 and C2
against MNU induced breast cancer. A quan-
titative method of estimating tumor progres-
sion in breast cancer model involves the de-
termination of tumor volume (Faustino-
Rocha et al., 2013). Change in tumor volume
was monitored weekly using a digital Vernier
caliper. We observed a decrease in tumor vol-
ume in the treatment group compared to
MNU control suggesting their ability to halt
tumor progression. Organ indices are markers
of toxicity (Vaijayanthimala et al., 2012),
which were calculated for liver, kidney, heart,
and spleen. In all treatment groups, there was
no significant difference in liver, spleen, heart
and kidney index suggesting the safety of the
compounds towards the major organs. Hema-
tological estimations were performed to eval-
uate the effect of C1 & C2 on blood parame-
ters. No significant changes were seen in the
treatment groups as compared to MNU con-
trol group on the blood parameters.
Liver function test (AST/ALT) and kid-
ney function test (Creatinine) were also per-
formed to check the toxicity of selected chal-
cones on organ functions. As per the results,
doxorubicin and C1 treatment showed a
significant decrease in AST level compared to
MNU control group which is not considered
as toxic sign clinically. No other changes
were observed in evaluated parameters. Thus
it can be concluded that C1 and C2 did not
have any toxic effect on liver and kidney
function.
The present study was aimed to evaluate
the anticancer potential of two 5-acetamido
chalcones derivatives C1 and C2. They were
tested for their cytotoxic potential by per-
forming MTT assay (directly measures mito-
chondrial activity hence viability of cells) on
MCF-7 and MDA-MB-231 with an
incubation period of 48 h. C1 and C2 showed
cytotoxicity with IC50 ≤ 100 µM against both
cancer cell lines. C2 was highly active
whereas C1 was moderately active against
MCF-7. Both the compounds were highly ac-
tive in MDA-MB-231 cell line when com-
pared to MCF-7 cells at micromole
concentration. MCF-7 was selected for fur-
ther study to correlate the mechanism of the
synthesized compounds in MNU induced tu-
mors, an ER+ve breast carcinoma model.
In vitro mechanistic study for the determi-
nation of apoptosis in MCF-7 cells was per-
formed by dual staining (AO/EB) to find out
the induction of nucleomorphological chang-
es by the synthesized compounds. Tumor-
selective cell death is the goal of cancer ther-
apeutics and apoptosis as a mechanism of cell
death offers therapeutic advantages (Sellers
and Fisher, 1999). Nucleomorphological
changes significantly (p<0.05) increased in
all treatment groups as compared to normal
control. In MCF-7 cells treated with C1 and
C2, a change in cellular morphology, includ-
ing chromatin condensation and nuclear frag-
mentation which is a characteristic feature of
apoptotic cell death was observed suggesting
the apoptosis-inducing potential of the
compounds.
Metastatic spread of cancer cells from the
primary site to distant organs is a major con-
cern in cancer therapeutics (Khan and
Mukhtar, 2010). Scratch wound assay was
performed to determine the anti-migratory
potential of the test compounds. A significant
reduction in the migration of MCF-7 cells was
observed in C1 and C2 groups after 24 h and
In pathological conditions like cancer, an
increase in endogenous antioxidant level can
prevent the damage associated with free radi-
cal generation (Pham-Huy et al. 2008). Effect
of C1 & C2 on endogenous antioxidants were
evaluated in tumor and liver homogenate. In
tumor samples, significant (p<0.05) increase
was observed in SOD levels for all treatment
groups, an increase in GSH level only in C2
treatment group while an increase in MDA
level in Doxorubicin and C1 treatment
4
8 h of treatment as compared to control. Our
observation suggests the anti-migratory po-
tential of the test compounds which is a prop-
erty of anti-cancer therapies.
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EXCLI Journal 2017;16:1150-1163 – ISSN 1611-2156
Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
Carelle N, Piotto E, Bellanger A, Germanaud J,
Thuillier A, Khayat D. Changing patient perceptions of
the side effects of cancer chemotherapy. Cancer.
groups. Thus overall effect showed C2 pos-
sessed better antioxidant potential compared
to other treatment groups suggesting its po-
tential in altering the endogenous antioxidant
levels. Furthermore, we also observed an in-
crease in the endogenous antioxidant levels in
liver homogenate, where SOD levels were in-
creased in doxorubicin treatment group, in-
crease in GSH with C2 treatment, and in-
crease in MDA level by doxorubicin, whereas
a decrease in the level of MDA in the C2 treat-
ment group was observed suggesting a protec-
tive effect against lipid peroxidation.
Moreover, an induction of apoptosis is ev-
ident with the formation of a small fragment
of DNA which can be observed as a ladder by
gel electrophoresis. In tumor homogenate, we
observed the formation of DNA ladder by this
technique suggesting the induction of apopto-
sis by C1 and C2 treatment.
2
002;95:155-63.
Cooper GM, Hausman RE. The cell. 2nd ed. Sunder-
land, MA: Sinauer Assoc., 2000.
Devasagayam T, Boloor K, Ramasarma T. Methods for
estimating lipid peroxidation: an analysis of merits and
demerits. Indian J Biochem Biophys. 2003;40:300-8.
Faustino-Rocha A, Oliveira PA, Pinho-Oliveira J,
Teixeira-Guedes C, Soares-Maia R, Da Costa RG, et
al. Estimation of rat mammary tumor volume using cal-
iper and ultrasonography measurements. Lab Animal.
2
013;42:217.
Fidler IJ. The pathogenesis of cancer metastasis:
the'seed and soil'hypothesis revisited. Nature Rev Can-
cer. 2003;3:453-8.
Gerlier D, Thomasset N. Use of MTT colorimetric as-
say to measure cell activation. J Immunol. Meth.
1
986;94:57-63.
Global Burden of Disease Cancer. The global burden
of cancer 2013. JAMA Oncology. 2015;1:505-27.
CONCLUSION
In the present study, two 5’ acetamido
chalcone derivatives were synthesized and
evaluated for their anticancer potential in
breast cancer. Based on in vitro studies such
as cytotoxicity study, anti-oxidant study and
in vivo studies in MNU model such as evalu-
ation of tumor volume, tumor weight and en-
dogenous anti-oxidant we can conclude that
C1 and C2 possess anti-cancer potential with
anti-oxidant activity.
Goa J. A micro biuret method for protein determination
Determination of total protein in cerebrospinal fluid.
Scand J Clin Lab Invest. 1953;5:218-22.
Hamberg M, Niehaus WG, Samuelsson B. Preparation,
isolation, and characterization of a derivative of malo-
naldehyde. Anal Biochem. 1968;22:145-53.
Khan N, Mukhtar H. Cancer and metastasis: prevention
and treatment by green tea. Cancer Metastasis Rev.
2010;29:435-45.
Kumar N, Dhamija I, Raj PV, Jayashree B, Parihar V,
Manjula S, et al. Preliminary investigation of cytotoxic
potential of 2-quinolone derivatives using in vitro and
in vivo (solid tumor and liquid tumor) models of can-
cer. Arab J Chem. 2014;7:409-17.
REFERENCES
Anderson ME. Determination of glutathione and gluta-
thione disulfide in biological samples. Meth Enzymol.
985;113:548-55.
1
Kumar SK, Hager E, Pettit C, Gurulingappa H, David-
son NE, Khan SR. Design, synthesis, and evaluation of
novel boronic-chalcone derivatives as antitumor
agents. J Medicinal Chem. 2003;46:2813-5.
Bishayee K, Ghosh S, Mukherjee A, Sadhukhan R,
Mondal J, Khuda‐Bukhsh A. Quercetin induces cyto-
chrome‐c release and ROS accumulation to promote
apoptosis and arrest the cell cycle in G2/M, in cervical
carcinoma: signal cascade and drug‐DNA interaction.
Cell Prolif. 2013;46:153-63.
Kuo M-L, Huang T-S, Lin J-K. Curcumin, an antioxi-
dant and anti-tumor promoter, induces apoptosis in hu-
man leukemia cells. Biochim Biophys Acta. 1996;
Botham P. Acute systemic toxicity - prospects for
tiered testing strategies. Toxicol in Vitro. 2004;18:227-
1
317:95-100.
3
0.
McCormick DL, Adamowski CB, Fiks A, Moon RC.
Lifetime dose-response relationships for mammary tu-
mor induction by a single administration of N-methyl-
N-nitrosourea. Cancer Res. 1981;41:1690-4.
1
162

EXCLI Journal 2017;16:1150-1163 – ISSN 1611-2156
Received: July 08, 2017, accepted: October 09, 2017, published: October 19, 2017
Pham-Huy LA, He H, Pham-Huy C. Free radicals, an-
tioxidants in disease and health. Int J Biomed Sci.
008;4:89-96.
Simon L, Srinivasan K, Kumar N, Reddy ND, Biswas
S, Rao CM, et al. Selected novel 5'-amino-2'-hydroxy-
1, 3-diaryl-2-propen-1-ones arrest cell cycle of HCT-
2
1
16 in G0/G1 phase. EXCLI J. 2016;15:21.
Ribble D, Goldstein NB, Norris DA, Shellman YG. A
simple technique for quantifying apoptosis in 96-well
plates. BMC Biotechnology. 2005;5:12.
Vaijayanthimala V, Cheng P-Y, Yeh S-H, Liu K-K,
Hsiao C-H, Chao J-I, et al. The long-term stability and
biocompatibility of fluorescent nanodiamond as an in
vivo contrast agent. Biomaterials 2012;33:7794-802.
Sellers WR, Fisher DE. Apoptosis and cancer drug tar-
geting J Clin Invest. 1999;104:1655-61.
1
163