Evaluation of the anticancer properties of the predicted hBaxBH3-mimetic compound 2-hydroxy-3,5-dinitrobenzamide in a mammary carcinogenesis-induced rat model

Page 1 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

Evaluation of the anticancer properties of the predicted hBaxBH3-mimetic compound

-hydroxy-3,5-dinitrobenzamide in a mammary carcinogenesis-induced rat model

2

a

b

Dakshinamurthy Sivakumar , Krishna Mohan Surapaneni , Ponnachipudhur Chinnaswamy

c

d

e

Prabu , Natarajan Hari , Ponnusamy Thiruvasagam , Muthu Rajasekaran and

a

Thirunavukkarasu Sivaraman *

a

Structural Biology Lab, Department of Bioinformatics, School of Chemical and Biotechnology,

SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.

b

Department of Biochemistry, Saveetha Medical College & Hospital, Saveetha University,

Chennai, Tamil Nadu, India.

c

Central Animal Facility, SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.

d

e

Department of Chemistry, Department of Biotechnology, School of Chemical and

Biotechnology, SASTRA University, Thanjavur – 613 401, Tamil Nadu, India.

*To whom correspondence should be addressed.

Tel: +91 4362 264101 Ext.2319

Fax: +91 4362 264120

Eꢀmail: sivaram@scbt.sastra.edu

RSC Advances

Page 2 of 52

View Article Online

DOI: 10.1039/C5RA23005E

Abstract

Designing small molecular prototypes having potential to disrupt binding interfaces of

proꢀapoptotic – antiꢀapoptotic/BH3ꢀonly proteins is a promising strategy in cancer

chemotherapy. Several natural and synthetic chemical compounds have been reported as

inhibitors or BH3ꢀmimetics to antiꢀapoptotic proteins of the Bclꢀ2 family playing essential roles

in apoptosis. In the present study, chemical synthesis, structural characterizations and antiꢀcancer

effect of 2ꢀhydroxyꢀ3,5ꢀdinitrobenzamide (HDNB), a chemical compound identified on the basis

of pharmacophoric features of hBaxBH3 peptide have been systematically reported.

Chemopreventive effect of the HDNB was assessed on NꢀNitrosoꢀNꢀmethylureaꢀinduced Wistar

female rats of mammary gland carcinogenesis for the study period of 18 weeks. The

carcinogenesis Wistar rats were subjected to oral administration of lowꢀdose (8 mg/kg b.w.),

mediumꢀdose (40 mg/kg b.w.) and highꢀdose (200 mg/kg b.w.) of the HDNB in three different

experimental setups. Strikingly, mediumꢀdose (40 mg/kg b.w.) of the HDNB showed appreciable

potential to reverse the abnormalities of various biochemical parameters in blood samples and as

well in breast tissues to the values as close as to that of healthy control or to that of standard

(Tamoxifen) control. The chemopreventive potential of the HDNB could be chiefly attributed to

its ability of restoring antioxidants and as well apoptosis inducing properties in Wistar animal

models.

Key words: Acute toxicities; antiꢀcancer potential; apoptotic proteins; breast cancer; chemo

preventive; HDNB.

Page 3 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

1

. Introduction

Cancer is caused by uncontrolled cell proliferations and as well by abnormally increased

1

cell life due to defects in apoptosis. The two processes are governed by quite a large number of

parallel signaling pathways, which posits great challenges on designing targetꢀbased small

2

, 3

molecular therapeutic agents to the treatments of cancers.

Fortunately, many anticancer

compounds have been shown to regulate apoptotic machinery in one way or other in order to

4

, 5

combat cancerous cells.

In these contexts, designing lead compounds depicting desirable

effects by interfering functions of Bclꢀ2 family proteins has become an attractive strategy in

6

cancer chemotherapy. Though it is well documented that Bclꢀ2 family proteins tightly govern

the apoptotic process by intrinsic or mitochondrial pathway, the mechanism by which the

proteins communicate each other and modulate the process are under debate between two key

7

ꢀ9

models: direct activation model and indirect model. The ‘direct activation model’ proposes

that proꢀapoptotic proteins (Bax and Bak) will be activated by a set of BH3ꢀonly proteins (Bim,

Bid and Puma), which may be abnormally sequestered by antiꢀapoptotic proteins (Bclꢀ2, BclꢀB,

BclꢀW, BclꢀXL, Bflꢀ1 and Mclꢀ1) in cancerous cells, whereas ‘indirect model’ proposes that

functions of proꢀapoptotic proteins will be blocked by antiꢀapoptotic proteins through heteroꢀ

complex formations between the two groups of the Bclꢀ2 family. Interestingly, both models

suggest that chemical molecules that are capable of acting as antagonists to the antiꢀapoptotic

proteins can act as efficient lead antiꢀcancer compounds, in general.

Impairment in apoptosis is one of hallmarks of cancer diseases and successively

overexpression of antiꢀapoptotic proteins (either all or a few of them) have been documented in

1

0

most types of cancers, if not all. To date, six types of antiꢀapoptotic proteins have been well

characterized in mammalian cells and all the proteins have similar threeꢀdimensional (3D)

RSC Advances

Page 4 of 52

View Article Online

DOI: 10.1039/C5RA23005E

architectures. However, biological activities and as well specificities of the antiꢀapoptotic

proteins to interact with various BH3ꢀonly proteins are not identical to each other. Many natural

products (antimycin A, chelerythrine, obatoclax, purpurogallin and gossypol) and synthetic

chemical compounds (HA14ꢀ1, BH3Iꢀ1, isooxazolidine derivatives, gossypol derivatives, ABTꢀ

737 and ABTꢀ263) have been reported as inhibitors to the antiꢀapoptotic proteins and compounds

such as ABTꢀ263, obatoclax, gossypol and isooxazolidine are right now under various levels of

4

clinical trials. Interestingly, of the many molecules mentioned above, the ABTꢀ737 and ABTꢀ

263 have only been authenticated as true antagonists/BH3ꢀmimetics to the antiꢀapoptotic proteins

as cytotoxic activities of the compounds could be well correlated with cellular concentrations of

4

, 11, 12

the antiꢀapoptotic proteins.

/BclꢀXL have also been well studied at atomic level resolutions. However, the two

compounds were found to be weak antagonists to Mclꢀ1 and as well the compounds were

Moreover, structural interactions of the compounds with Bclꢀ

11

2

1

3

inactive against cancerous cells overꢀexpressing the antiꢀapoptotic protein, Mclꢀ1. Ostensibly,

designing/identifying specific antagonists to each antiꢀapoptotic protein and likewise common

antagonists showing high affinities to all of the 6 antiꢀapoptotic proteins have been yet

challenging tasks in cancer chemotherapy.

We have recently reported 2ꢀhydroxyꢀ3,5ꢀdinitrobenzamide (HDNB) as a lead inhibitor

1

4

to human BclꢀB (hBclꢀB) by means of peptidodynmimetic method designed in our laboratory.

The method is useful to define pharmacophoric features of small peptides bound with large

protein molecules under dynamic conditions. Using the peptidodynmimetic method,

pharmacophoric residues of human BaxꢀBH3 (hBaxꢀBH3) peptide bound with hBclꢀB were

mapped out and the HDNB was identified as novel lead inhibitor to the hBclꢀB upon a database

screening followed by high throughput virtual screening on the basis of the pharmacophoric

Page 5 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

residues of the hBaxꢀBH3. Interestingly, the lead compound (HDNB) also showed comparable

binding affinities on the BH3ꢀbinding grooves of all the antiꢀapoptotic proteins but with BclꢀW,

appreciable bioavailability and desirable pharmacokinetic properties as predicted by using an

1

4, 15

array of computational strategies.

In the present study, we report chemical synthesis,

structural characterizations (using ultraviolet visible (UV) spectroscopy, Fourier transform

infrared (FTꢀIR) spectroscopy, Gas chromatography ꢀ mass spectrometry (GCꢀMS) and proton

1

13

15

(

H), carbon ( C) and nitrogen ( N) nuclear magnetic resonance (NMR) spectroscopy) and

chemopreventive potential of the HDNB in NꢀNitrosoꢀNꢀmethylurea (NMU) ꢀ induced Wistar

mammary gland carcinogenesis in detail. Comprehensive analyses of data obtained from the

experiments suggested that the HDNB is a promising lead depicting antiꢀcancer potential and the

HDNB would also be perhaps used as a seed molecule to develop highly efficient de novo

antagonists to the antiꢀapoptotic proteins.

2

. Results and Discussion

Understanding relationships between small bioꢀmolecules and macromolecules

synthesized by cells is crucial to make ‘central dogma’ of life to be entirely descriptive one.

Natural and nonꢀnatural small chemical molecules are also key elements, powerful probes and

1

6, 17

drugs in biological systems.

In these connections, designing small molecules having

potential especially to disrupt protein – protein interfaces has intrigued researchers in the past

decade. However, designing/identifying chemical molecules binding to proteinꢀprotein interfaces

1

8, 19

is an uphill task as surface areas of the interfaces are large and flat, in general.

Interestingly,

hotspots (subset of residues contributing most of the free energy of binding) of proteins

constituting interfaces could be mapped out by using siteꢀdirected mutational studies as

2

0

demonstrated for several proteins. The hotspots of the proteins could be used as ‘footholds’ for

RSC Advances

Page 6 of 52

View Article Online

DOI: 10.1039/C5RA23005E

designing of small molecules interacting on the interfaces of protein complexes. Computational

methods such as fragmentꢀbased lead designing and peptidomimetics have also been shown as

2

1

promising strategies for designing leads to interact on proteinꢀprotein interfaces. In these series

of in silico tools, a recent peptidodynmimetic method has also been shown to define

14

pharmacophoric residues of the hBaxBH3 bound with hBclꢀB under dynamic conditions. Using

the pharmacophoric residues of the peptide, MMsINC database (as represented in the original

2

article ) consisting of 17 million conformers of about 4 million diverse molecules was screened

through pepMMsMIMIC interface in order to identify lead chemical compounds mimicking the

1

4, 22

interaction of the hBaxBH3 on the BH3ꢀbinding groove of the hBclꢀB.

After subjecting a

few hundred compounds screened using the ‘peptododynmimetic strategy’ to high throughput

virtual screening followed by pharmacokinetic evaluations, the HDNB was identified as a

prototypic chemical molecular inhibitor to the hBclꢀB. Furthermore, binding affinities of the

HDNB towards the 6 antiꢀapoptotic proteins from human beings have also been recently

scrutinized by means of in silico methods and differential binding modes of the HDNB with the

antiꢀapoptotic proteins could be chiefly attributed to differences in the chemical properties of

1

5

BH3ꢀbinding grooves of the proteins.

2.1 Synthesis and structural characterizations of the HDNB

The HDNB was not commercially available during course of the present studies and even

till now. Synthetic methodology of the HDNB is outlined in Figure S1 and also described in the

‘Method’ section. Structural characterizations of the HDNB as examined by UV, FTꢀIR and GC

ꢀMS spectrometric techniques have been elaborately delineated in the supplementary material

(Figure S2 and Table S1) and comprehensive analyses of the data could consistently represent

the structure of the HDNB. In addition, elemental composition of the HDNB (C H N O ) as

7

5

3

6

Page 7 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

determined by using 2400 series CHNS Elemental analyzer (PerkinElmer, Wellesley, USA) were

in good agreement with elemental composition of the compound calculated out theoretically:

percentage compositions of C, H & N were 34.87%, 2.50% & 16.95% respectively, as

determined by the experimental method and the theoretic values were 37.02%, 2.22% & 18.50%

respectively, for the elements. Moreover, nuclear magnetic frequencies of protons, carbons and

nitrogen present in the compound were also recorded using various 1D and 2D NMR methods.

1

D H NMR spectra of the 3,5ꢀdinitiro salicylic acid (DNSA, the starting compound) and HDNB

are shown in Fig. 1A and 1B, respectively. The DNSA showed two dispersed resonances at δ

th

8

.67 and δ 8.71 for aromatic protons located at 4 and 6 positions, respectively and phenolic

OH resonance of the compound was observed as a weakly resolved peak at δ 9.0. The HDNB

also showed two peaks for its aromatic protons at δ 8.65 and δ 8.70. In addition, the compound

showed three sharp singlet resonances at δ 7.04, δ 7.21 & δ 7.38 and a weak broadened peak at δ

+

1

4.16. The former and later resonances were assigned to protonated amide group (CONH ) and

3

phenolic OH group of the compound. The resonance assignments were further authenticated by

1

acquiring 1D H NMR for the HDNB for which labile protons could be exchanged to solvent

1

deuterium and the deuterated HDNB showed two H resonances for its two nonꢀlabile aromatic

protons only (Fig. 1C). Moreover, Distortionless Enhancement by Polarization Transfer (DEPT)

1

3

experiments carried out for the HDNB is shown in Fig. 1D and the data depicted two

C

resonances at δ 125.9 and δ 131.5 indicating that the compound has only two tertiary carbon

atoms (C4 and C6, which are free of substitutions in the aromatic ring). Thus, the DEPT and 1D

1

H NMR data on the HDNB endorsed each other and confirmed the resonance assignments. We

1

5

also performed 2D Heteronuclear Single Quantum Correlation (HSQC) ꢀ N experiments for the

HDNB and the data is shown in Fig. 1E. The HSQC spectrum depicted a single cross peak at δ

RSC Advances

Page 8 of 52

View Article Online

DOI: 10.1039/C5RA23005E

1

5

1

3

58.1 of N dimension and δ 7.18 of H dimension confirming the presence of an amide group

in the compound. Taken together, the data presented above could unambiguously confirm the

structure of the HDNB as represented in the Fig. S1.

2.2 Acute oral toxicity (AOT) studies on the HDNB

The HDNB consists of two nitro groups, which could rise up skeptical concerns on

toxicity and adverse effects of the compound in human system. Since compounds consisting of

nitro groups become electrophilic in nature, it is generally considered that the nitro compounds

may cause some deleterious changes by interacting with biological nucleophilic molecules such

2

3

as nucleic acids and proteins. Unless until the nitro compounds damage healthy normal cells,

they may be of safe to use as drugs in the human system. Interestingly, several drugs with nitro

groups are already in market. For instance, drugs such as chloramphenicol (as an antibiotic),

nitrazepam (as a tranquilizer), pyrrolnitrin (as fungicide), nitridazole (as schistosonicide),

parathion (as insecticide) and azathioprine (as antineoplastic) are popular examples for the

2

3

category of the compounds. In addition, ABTꢀ737, a wellꢀknown antagonist to antiꢀapoptotic

4

proteins, has a nitro group and the compound is right now under Phase I clinical trials.

However, toxicities of small chemical molecules are not predictable in a straightforward manner,

since about 40% of lead molecules fail either in preꢀclinical or clinical trials as suggested by

2

4, 25

recent surveys on toxicities of small molecules.

In these backgrounds, irrespective of

literature evidences on nonꢀtoxic/toxic properties of compounds possessing nitro group(s), we

have thoroughly evaluated toxicity of the HDNB by using female Wistar rats.

As per OECD (Organization for Economic Cooperation and Development) 425

guidelines (OECD, 2001), ‘Main test’ (5 Wistar female rats were used in the test: first and

second rats were administrated with 175 mg/kg b.w. and 550 mg/kg b.w., respectively; other

Page 9 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

three rats were administrated with 2000 mg/kg b.w.) procedure was strictly followed to

determine ‘lethal dose’ (LD ) of the HDNB. All the 5 rats treated with various doses of the

5

0

HDNB were periodically monitored for 14 days from the treatments and the HDNBꢀtreated

animals did not develop any toxicity signs such as tremors, convulsions, salivation, diarrhea,

lethargy, sleep and coma. Only the animals administrated with 2000 mg/kg b.w. seemed to be

lethargic in the first 20 minutes of treatment periods and thereafter they become active as healthy

animals. All the five HDNBꢀtreated Wistar rats survived AOT time span of 14 days and at

completion of the treatment periods, all the 5 Wistar rats were sacrificed and subjected to

detailed necropsy studies. Fig. 2 shows histopathology of different organs (such as brain, heart,

lung, liver, kidney and intestine) of the Wistar female rats treated with 2000 mg/kg b.w. of the

HDNB. Examination on the histology of the organs suggested that there were no abnormal or

pathological changes due to oral administration of the HDNB to the test animals:

cerebral/cerebellar hemispheres of brains, myocytes of heart section and hepatocytes of liver

were characterized to be as normal as in the healthy rats; lung section showed normal alveolar /

bronchiolar epithelium with no infiltrates; kidney section showed the normal cortex and medulla

with glomeruli and tubules showing their normal microꢀarchitecture; there were no necrotic

2

6, 27

lesions of the epithelium in the intestinal section.

Thus, the AOT studies revealed that the

HDNB administrated orally to the Wistar rats was not toxic at dose level of 2000 mg/kg b.w. and

LD of the compound should also be at least greater than 2000 mg/kg b.w.

5

0

2

.3 Chemopreventive effect of the HDNB using animal models

.3.1 Cancer induction and treatment

2

Chemopreventive effect of the HDNB was tested on breast cancer animal models on the

basis of results obtained from in vitro MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]2,5ꢀdiphenyltetrazolium

RSC Advances

Page 10 of 52

View Article Online

DOI: 10.1039/C5RA23005E

bromide) assays carried out for the HDNB. Cell growth inhibition potential of the HDNB was

examined against MCFꢀ7 (human breast cancer cells), HeLa (cervical cancer cells) and MGꢀ63

(

human osteosarcoma cells) cell lines and results of the assays are shown in Fig. 3. The cell lines

were treated with various concentrations of the HDNB at constant treatment time of 48 h

Materials and Methods). By fitting the doseꢀdependent inhibition potential of the HDNB against

(

growth of the cell lines to an appropriate nonꢀlinear regression reveled that IC₅₀values of the

HDNB against MCFꢀ7, MGꢀ63 and HeLa cell lines were 574 ± 3 µM, > 2 mM and > 5 mM,

respectively. The MTT assay suggested that the HDNB strongly inhibited growth of breast

cancer cells than that of bone/cervical cancer cells used in the study. Hence, chemopreventive

effect of the HDNB on NMUꢀinduced breast cancer animal models was examined using 42

female Wistar rats for the time span of 18 weeks. The experimental animals were divided into 7

groups with 6 animals in each group (Table 1): control group, vehicle group, diseased group,

standard group and 3 HDNBꢀtreated groups (groups treated with low, medium and high doses of

the HDNB were denoted as lowꢀdose group, mediumꢀdose group and highꢀdose group,

respectively). The animals of the control group and vehicle group received only normal saline (1

ml) and corn oil (1 ml) as treatment doses, respectively, every alternative day throughout the

experimental period. The breast cancer inductions and treatments were started simultaneously by

administrating the NMU at concentration 75 mg/kg intraperitoneally (i.p) to animals of all the

groups except animals belonging to the control and vehicle groups. The NMU dose of 75 mg/kg

st

rd

i.p was given to the animals two times at 1 and 63 days of experimental timelines in the

present study. In these contexts, it should also be mentioned that dose levels (50 mg/kg –75

mg/kg), number of doses (1ꢀ3 times) and administrating time intervals (6 ꢀ 9 weeks) of the NMU

were found to be different from each other in quite a large studies on the NMUꢀinduced

Page 11 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

2

8ꢀ34

cancerous animal models as reported in literature.

After the NMU administration, treatments

regime of the groups were as follows: animals of the diseased group received only usual food

and water throughout the experiments; animals of the standard group received 20 mg/kg dose of

Tamoxifen every alternative day throughout the course of the experiments; similarly, the lowꢀ

dose, mediumꢀdose and highꢀdose groups received the HDNB at concentration of 8 mg/kg, 40

mg/kg and 200 mg/kg (the doses were calculated based on results obtained from the AOT

studies), respectively. At end of the experimental timeline, blood samples (serum and plasma)

and breast tissues from all 6 animals of each group were collected, processed and preserved as

per standard procedures. The samples were then subjected to hematology, histopathology,

biochemical and immunohistochemical analysis and outcomes of the studies have been described

in the following sections.

It is worthy of mentioning that all animals used in the chemopreventive studies were

survived for the whole period of 18 weeks and the animals did not develop any toxic symptoms

such as tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma as inferred from

protocols used to monitor the animals in a systematic manner (refer to Methods). The body

weights of the different experimental groups were also monitored in regular intervals for 18

weeks and average body weights of animals belonging to seven different groups i.e. from Group

I to VII were found to be 186 ± 3 g, 187 ± 2 g, 188 ± 4 g, 190 ± 4 g, 187 ± 3 g, 189 ± 5 g and 188

±

5 g, respectively. There were no significant differences in the body weights between the

HDNBꢀtreated groups and any other groups (control/vehicle/standard/diseased groups) as

determined from ANOVA (Analysis of Variance) statistical analysis of the data. Moreover, these

results suggested that dose levels of the HDNB used in the chemopreventive studies did not

cause any apparent behavioral changes and as well toxic symptoms to the animal models.

RSC Advances

Page 12 of 52

View Article Online

DOI: 10.1039/C5RA23005E

2.3.2 Histopathological analysis of breast tissues

All six Wistar rats of the control group showed no abnormality in cellular architectures

of their breast tissue sections (Fig. 4A). Animals of the vehicle group also showed that there

were no abnormalities in their breast tissues as compared to that of control group indicating the

vehicle (corn oil) did not cause any adverse effect to the animals. On contrary, histopathological

results clearly confirmed developments of mild dysplasia in the breast tissues of all six animals

belonging to the diseased group (Fig. 4B) and the observations are in line with the literature

3

5,36

reports on the NMUꢀinduced cancerous animal models.

rats belonging to Group IV (lowꢀdose group), Group V (mediumꢀdose group) and Group VI

highꢀdose group), respectively showed breast tissues as similar as to that of control group. It is

Interestingly, 20%, 80% and 50% of

(

also worthy of mentioning that the rest of 80% rats in Group IV were characterized to have mild

dysplasia in their breast tissues, while the rest of 20% and 50% of rats in Group V and Group VI

depicted basophilia in alveolar and ductal epithelium. In the standard group, breast tissues of

50% of rats were found to be normal and rest of them were found to have basophilia in alveolar

and ductal epithelium. Thus, the histopathological results suggested that the mediumꢀdose (40

mg/kg b.w.) of the HDNB exhibited significant chemopreventive potential by suppressing

abnormal cell proliferation occurring in the NMUꢀinduced breast carcinoma of female Wistar

rats (Fig. 4C).

2.3.3 Analyses of hematological parameters

Hematology studies were carried out to investigate alterations that may occur in standard

levels of WBC (white blood cells) count, lymphocytes, neutrophils, monocytes, eosinophil,

basophils, RBC (red blood cells) count and Hb (hemoglobin) of the animal models used in the

present studies. The results of the studies have been enumerated in Table 2. The data revealed

Page 13 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

that the diseased group showed twoꢀfold increase of WBC, lymphocytes and neutrophils as

compared to that of control group. From a review of data in Table 2, it is interesting to note that

values of the hematological parameters of the mediumꢀdose and highꢀdose groups showed no

statistically significant differences to that of control group implying levels of WBC, lymphocytes

and neutrophils could be restored in these groups as compared to the diseased group. Moreover,

there were no significant differences between the medium/high dose groups and standard group

with respect to these parameters. The same trend was also observed in the case of basophils of

animal models, while eosinophils showed no significant alterations among all the seven groups.

Concentration of hemoglobin, RBC and monocytes were found to be decreased in the diseased

group relative to that of the control group. Though there were no statistically significant

differences between all the 3 HDNBꢀtreated groups and standard group with respect to these

parameters, the mediumꢀdose group restored the hemoglobin, RBC and monocytes in a

noteworthy manner as compared to that of other 2 groups.

Taken together, remarkably decreased levels of RBC and increased levels of lymphocytes

in Wistar rats of the diseased group are indications of developing anemia and lymphocytosis,

respectively in those animal models. These types of inflammatory responses have also been

3

7, 38

reported in animals with mammary carcinoma as demonstrated in literature.

In these

contexts, the results described above are in line with those literature reports. In addition,

development of leukaemia in the NMUꢀadministered rats was also evidenced as per a few reports

3

9, 40

in the literature.

Interestingly, the present study suggested that oral administration of the

HDNB (40 mg/kg b.w.) brought back the status of WBC, RBC, lymphocytes and Hb to near

normal range in the NMUꢀtreated Wistar rats (Group V).

RSC Advances

Page 14 of 52

View Article Online

DOI: 10.1039/C5RA23005E

2.3.4 Analyses of biochemical end-products

Biochemical endꢀproducts such as total protein, albumin, creatinine, urea, alanine

transferase (ALT), aspartate transferase (AST), gluthathione reductase (GR) and glutathioneꢀSꢀ

transferase (GST) in blood serum/plasma were used to assess biological effect of the HDNB in

the NMUꢀinduced mammary carcinogenesis. Table 3 enumerates levels of these biochemical

parameters as estimated using standard assays (refer to Methods) for all seven different

experimental Wistar groups of the present study. The status of total protein and albumin were

significantly decreased in Wistar rats of the diseased group comparing to that of the control

group. Oral administration of medium dose of the HDNB (40 mg/kg b.w.) to the NMUꢀinduced

Wistar rats (Group V) significantly restored status of the total protein and albumin to near values

of the respective parameters estimated for the control group (Table 3).

Aspartate transferase (AST) and alanine transferase (ALT) are generally being used as

hepatic markers and significant alterations of these markers in blood plasma are probable

indications of liver dysfunction and/or liver damage. The levels of AST and ALT were found to

be significantly increased and decreased, respectively in the diseased group as compared to that

of the control group. Among the lowꢀ, mediumꢀ and highꢀdose groups, Wistar rats of mediumꢀ

dose group depicted AST and ALT levels, which were in good agreement with that of the

control/standard groups (Table 3). These results indicated that the HDNB could restore liver

functions by probably interfering with metabolic activations in the NMUꢀtreated Wistar rats. The

concentration of urea and creatinine, which serve as markers to examine renal functions, showed

no significant alterations among all the seven experimental Wistar groups studied in the present

study (Table 3). It should be mentioned that neither the hepatic markers nor renal markers

mentioned above were abnormally estimated in both mediumꢀdose and highꢀdose groups (Group

Page 15 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

V & VI) suggesting 18 weeks oral administration of the HDNB at the dose concentration (40/200

mg/kg b.w.) in the NMUꢀinduced mammary Wistar rats did not cause any severe adverse effects

on the liver and kidney tissues. These findings lend credence to the reports from the AOT studies

on the HDNB demonstrated in previous section of the work.

The GST and GR are phase II detoxification enzymes and they have been shown to be

4

1

significantly altered in cancerous animal models. The GST is reported to actively play critical

roles in antioxidant defense mechanisms and detoxification of electrophilic/hydrophobic

4

2

chemical compounds. The enzyme is also known to be an important preꢀneoplastic marker in

4

3

assessing the carcinogenꢀinduced free radical damage. The GR is a critical cellular antioxidant

4

enzyme which regulates the intracellular ratio of oxidized and reduced glutathione level. While

increased levels of the GST and GR facilitate excretion of carcinogenic metabolites either by

conjugation with reduced glutathione or by destroying the reactive centers of carcinogen,

activities of the enzymes are shown to be drastically decreased in cancerous animal models

4

5, 46

comparing that of healthy counterparts.

In the present study, activations of both enzymes

were significantly decreased in the diseased group as compared to that of the control group and

the results corroborate the literature reports discussed above. Oral administration of mediumꢀ

dose HDNB increased the GST and GR concentration on par with the standard drug, tamoxifen

used in the present study, for bringing back the status of the enzymes to near normal range in

blood plasma (Table 3). The data thus imply that the HDNB is capable of restoring the phase II

detoxification enzymes and consequently stimulating excretion of carcinogenic metabolites that

may be generated in the NMUꢀinduced mammary carcinogenesis.

RSC Advances

Page 16 of 52

View Article Online

DOI: 10.1039/C5RA23005E

2.3.5 Analyses of lipid peroxidation and antioxidant markers

Oxidative stress occurring due to imbalance between oxidant and antioxidant levels plays

4

7

pivotal roles in various stages of carcinogenesis. Overproduction of reactive oxygen species

ROS) under the oxidative stress causes damages to biomacromolecules such as nucleic acids,

(

proteins and lipids and subsequently altering cellular architectures and proliferations. Thus, endꢀ

products of oxidative stress, especially lipid peroxidation (oxidative degradation of lipids)

byproducts such as malondialdehyde (MDA), tumor necrosis factor – alpha (TNFꢀα) and nitic

4

8, 49

oxide are considered as markers in many inflammatory diseases and as well in cancers.

The

MDA affects DNA functions by forming DNAꢀMDA adduct and TNFꢀα plays an important role

in tumor progression by promoting expression of matrix metalloproteinases (MMP) and

5

0, 51

endothelial adhesion molecules to produce DNA damage.

While NO (Nitric Oxide) acts as

an antioxidant at low level, it is capable of damaging DNA at elevated concentrations and as well

5

2, 53

inducing alterations in cell functions to stimulate cell growth.

In contrast, antioxidants play a

major role in safeguarding the cells against the free radical induced toxicity. Hence, homeostasis

of antioxidants and proꢀoxidants are vital for normal cellular functioning and impairment in the

5

4ꢀ56

balance would trigger the pathoꢀphysiological events causing damages to vital organs.

In the present study, abnormalities of lipid peroxidation byproducts such as MDA, TNFꢀα

and NO and as well antioxidant markers such as superoxide dismutase (SOD), catalase,

glutathione peroxidase (GPx), reduced glutathione (GSH), vitamin C and vitamin E have been

analyzed in blood samples of the experimental Wistar animal models. The status of MDA, TNFꢀ

α and NO were found to be significantly elevated in the diseased group comparing to that of the

control group and the results are in conformity to lipid peroxidation effect on cancerous animal

4

9, 57

models reported in the literature.

A comparative analysis of the status of the lipid

Page 17 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

peroxidation byproducts in the HDNBꢀtreated groups and diseased group revealed that lowꢀdose

group showed no significant changes, whereas both mediumꢀdose and highꢀdose groups showed

significant variations (Table 4). Of the two groups, the mediumꢀdose group depicted better

significant variations than the highꢀdose group as compared to the diseased group with respect to

levels of the MDA, TNFꢀα and NO in those animal models. Moreover, there were no significant

differences between estimated values of the parameters from the mediumꢀdose group and the

control/standard group indicating elevated levels of the MDA, TNFꢀα and NO were reversed to

the normal levels in the NMUꢀinduced Wistar mammary carcinogenesis upon subjecting the

animal models to the HDNB mediumꢀdose (40 mg/kg b.w.) treatments for 18 weeks.

Concentration of enzymatic antioxidants such as catalase, SOD and GPx and nonꢀ

enzymatic antioxidants such as reduced glutathione and vitamin C were found to be significantly

declined in the diseased group as compared to that of the control group. However, there were no

significant changes in the status of vitamin E between the control group and other six groups

(Table 4) implying metabolic activations in the NMUꢀtreated Wistar rats had no effect on

vitamin E status of blood plasma as inferred from the present study. However, significantly

decreased level of vitamin E from the reference range was demonstrated in several animal

5

8

models possessing cancer diseases due to chemical carcinogens as reported in the literature and

further discussion on the discrepancy is beyond the scope of this present study. Comprehensive

analysis of the data on enzymatic and nonꢀenzymatic antioxidants (GSH and vitamin C) obtained

from the control and other six experimental groups revealed following information: both types of

antioxidants could not be restored in lowꢀdose HDNBꢀtreated Wistar rats; though the

antioxidants could be restored near to reference levels in both medium/highꢀdose HDNBꢀtreated

Wistar rats, efficacy of the mediumꢀdose was found to be more satisfactory on reversing the

RSC Advances

Page 18 of 52

View Article Online

DOI: 10.1039/C5RA23005E

antioxidants levels near to the standard/control ranges than that of highꢀdose group (Table 4).

Lowered levels of the antioxidants in blood plasma of the diseased group could presumably be

due to excess amount of antioxidants utilizations by cancerous tissues either to meet their

5

9

nutrient demand or to scavenge excessively generated reactive oxygen species. In general, SOD

removes the superoxide radical by converting it to hydrogen peroxide and molecular oxygen,

whereas catalase decomposes hydrogen peroxides to water and dioxygen and as well protects

60, 61

tissues from the highly reactive hydroxyl radicals.

The antioxidant glutathione peroxidase

(GPx) plays an important role in peroxyl scavenging and as well maintaining the integration of

6

2

the cell membranes. Nonꢀenzymatic antioxidants like reduced glutathione (GSH), vitamin C

and vitamin E are acting as a second line defense agents to scavenge residual free radicals

eluding from primary defense mechanisms by enzymatic antioxidants. As demonstrated above,

oral administration of the mediumꢀdose HDNB (40 mg/kg b.w.) to the NMUꢀtreated Wistar rats

was found to restore the antioxidants and lipid peroxidation byproducts to the level of the control

group suggesting the HDNB may probably prevent cancer progression in the animal models by

restoring the biomarkers scavenging the free radicals and consequently protecting cellular

architectures. Strikingly, it is also obvious to note from the studies that the capabilities of the

HDNB on restoring the antioxidants could supersede the standard drug, tamoxifen, used in the

present study (Table 4).

2.3.6 Immunohistochemical studies on cancer biomarkers

Modulating effect of the HDNB on expression pattern of cell proliferative markers (Kiꢀ

7, p53) and as well an apoptotic (Bclꢀ2) marker have been investigated and described herein.

6

Overexpression of Kiꢀ67 in all proliferating cells is reported in the literature. Moreover,

overexpression of Kiꢀ67 in more than 50% of the breast cancer cells have been shown to be at

Page 19 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

high risk of recurrent disease and thus it is also considered as a specific biomarker of breast

6

3ꢀ65

cancers.

Tumor suppressor gene, p53 plays an important role in the prevention of cancers by

inducing apoptosis, DNA repair and cellꢀcycle arrest in response to different types of cellular

6

stress. However, mutated forms of the p53 were shown to conferring a proliferative advantage

to the neoplasm. Overexpression and as well active roles of p53 mutants have been well

6

7, 68

documented in various cancer types including breast carcinoma.

Bclꢀ2, an antiꢀapoptotic

protein, promotes cell survival by inhibiting apoptosis and the protein is well characterized to be

6

9

overexpressed in various cancers, including breast cancer. In general, all six antiꢀapoptotic

proteins characterized to date are considered as crucial biomarkers as they are shown to be

overexpressed in most of cancers, if not all, though overexpression pattern of each antiꢀapoptotic

protein is unique with respect to cancer types. Overexpression of the antiꢀapoptotic proteins in

cancer is due to impairment of apoptosis regulating a delicate balance of the proꢀapoptotic and

7

0

antiꢀapoptotic proteins.

Immunohistochemical studies carried out on detecting the three molecular markers, Kiꢀ

7, p53 and Bclꢀ2 in breast tissues of female Wistar rats belonging to the diseased group

6

apparently revealed overexpression of the markers. Immunostaining of proliferative markers Kiꢀ

6

7 and p53 variants and an apoptotic marker, Bclꢀ2 for the diseased group are depicted in Fig.

B, Fig. 5E and Fig. 5H, respectively and overexpression of the molecular markers are

5

represented by ‘arrow’ symbols in the respective pictorial illustrations. Abnormal expression

patterns of the three molecular biomarkers in the female Wistar rats of the diseased group in the

present study are in line with literature reports on status of the markers in animal models of

6

5, 67, 71

different types of carcinogenesis.

Oral administration of the HDNB at a dose of 40 mg/kg

b.w. to the NMUꢀtreated Wistar rats significantly restored the three molecular markers to near

RSC Advances

Page 20 of 52

View Article Online

DOI: 10.1039/C5RA23005E

normal levels (Fig. 5C, 5F & 5I). Expression patterns of the Kiꢀ67, p53 and Bclꢀ2 in the breast

tissues of the control group are also shown for sake of comparison in Fig 5A, 5D & 5G,

respectively. This particular efficaciousness of the HDNB on restoring the expressions of the

molecular markers to normalcy justifies not only the protective effect of the HDNB in the NMUꢀ

induced Wistar mammary carcinogenesis and as well supports that the HDNB is a promising

lead molecule to be developed as an efficient antiꢀcancer drug in near future.

3

. Concluding remarks

In the present study, 2ꢀhydroxyꢀ3,5ꢀdinitro benzamide (HDNB), a chemical compound

identified by means of peptidodynmimetic computational approach, was synthesized and

structurally characterized by using molecular spectrometric techniques such as UV, FTꢀIR, GCꢀ

MS, 1D and 2D NMR. To our best knowledge, biological properties of the compound have not

yet been well documented in the literature. We have herein demonstrated the antiꢀcancer effect

of the HDNB in animal models. The AOT studies suggested that LD of the compound must be

5

0

at least greater than 2000 mg/kg b.w. The biological roles of the HDNB in NMUꢀinduced Wistar

mammary carcinogenesis were assessed by systematically analyzing the levels of various

hematological parameters (WBC, RBC, Hb, lymphocytes, monocytes, neutrophils, basophils and

eosinophils), biochemical end products (total protein, albumin, creatinine, urea ALT, AST, GST

and GR), lipid peroxidation byproducts (MDA, TNFꢀα and NO), antioxidants (vitamin C,

vitamin E, GSH, catalase, SOD and GPx), proliferative markers (Kiꢀ67 and p53) and an

apoptotic molecular marker (Bclꢀ2) by using established procedures. Mediumꢀdose (40 mg/kg

b.w.) of the HDNB showed appreciable potential to reverse the abnormalities of various

biochemical parameters in blood samples and as well in breast tissue samples to the values as

close as to that of the control/standard (Tamoxifen) groups. The chemopreventive effect of the

Page 21 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

HDNB could be primarily attributed to its ability of restoring antioxidants and as well apoptosis

inducing properties during the NMUꢀinduced mammary carcinoma in Wistar animal models. The

above results suggest that the HDNB is a promising seed molecule for developing prototypic

chemical inhibitors to the antiꢀapoptotic proteins by using computational strategies such as ‘core

1

4, 25

hopping’ and de novo drug designing approaches in near future.

4

. Materials and Methods

4.1 Chemicals

Thionyl chloride, 3,5ꢀdinitrosalicylic acid (DNSA), liquid ammonia (ammonium

hydroxide solution), tetrahydofuran (THF), dimethyl sulfoxide (DMSO), deuterium oxide (D O),

2

NꢀnitrosoꢀNꢀmethyl urea (NMU) were obtained from SigmaꢀAldrich chemicals Pvt. Ltd.,

Bangalore, Karnataka, India. Corn oil (brand name ꢀ Avita Rꢀgold) was purchased from J.S. oils,

Thanjavur, TamilNadu, India. 2,4ꢀdinitrophenylhydrazine (DNPH), FolinꢀCiocalteau reagent,

5

,5’ꢀdithiobis (2ꢀnitro benzoic acid) (DTNB), trichloroacetic acid (TCA), hydrogen peroxide,

heparin, phosphate buffer, reduced glutathione, epinephrine, ethylenediamine tetraꢀacetic acid

EDTA), 1ꢀchloroꢀ2,4 dinitro benzene (CDNB), thiobarbituric acid (TBA), diphenylꢀ1,10

(

phenonthroline, griess reagent, sodium nitroprusside were purchased from Hiꢀmedia laboratories,

Mumbai, Maharashtra, India. P53, Kiꢀ67 and Bclꢀ2 primary antibodies and corresponding

secondary antibodies conjugated with horseradish peroxidase were purchased from Dako,

Carprinteria, CA, USA. All the other chemicals/solvents used in the present study were also of

analar grade.

4.2 Synthesis of the 2-hydroxy-3,5-dinitrobenzamide (HDNB)

A mixture of 3,5ꢀdinitro salicylic acid (16.3 g, 0.05 mol) and thionyl chloride (120 ml)

was refluxed with constant stirring until the suspension mixture was converted into a clear

RSC Advances

Page 22 of 52

View Article Online

DOI: 10.1039/C5RA23005E

solution. Excess amount of thionyl chloride added to the mixture was then removed under

reduced pressure. The acid chloride of the parent compound obtained upon cooling was

immediately dissolved in dry tetrahydrofuran (THF) and the resultant solution was added

dropwise manner into a fresh ammonia solution kept in ice bath (≈ 273 K) with constant stirring.

Target compound, 2ꢀhydroxyꢀ3,5ꢀdinitro benzamide (HDNB), was formed as yellow precipitate

in the reaction mixture. The HDNB was filtered (using Grade 1 Whatman paper), excessively

washed with double distilled water to remove the acid impurity and then was dried in vacuum

oven at 353 K. The HDNB was a bright yellow solid (5.7 g, 35% yield).

4.3 Structural characterizations of the HDNB

4.3.1 UV Spectroscopy

Absorption spectra of the HDNB and DNSA were recorded at 298 K in the wavelength

range from 400 nm to 550 nm using double beam Lambda25 UV/Vis spectrophotometer

PerkinElmer, Wellesley, USA). The compounds were dissolved in DMSO and concentrations

(

were 1.5 mM for both test solutions. Scan speed and slit width were set as 100 nm/minute and 1

nm, respectively. Absorption spectra for the compounds were also appropriately corrected to

solvent effect.

4.3.2 FT-IR Spectroscopy

FTꢀIR spectra of the HDNB and DNSA were recorded on a Spectrum one FTꢀIR

spectroscopy (PerkinElmer, Wellesley, USA). Sample preparation was carried out by thoroughly

mixing HDNB/DNSA and KBr in the ratio of 1:3 (w/w) and pellet of the sample mixture was

made using hydraulic press. Percentage of transmittances of the test samples was measured at

scan speed of 0.2 cm/second and then the data were Fourierꢀtransformed to generate spectra

ꢀ1

(

covering wave number from 400 to 4000 cm ) with resolution of 2 cm . FTꢀIR (KBr, cm ) for

Page 23 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

ꢀ1

the DNSA: 824, 1534, 1677, 2442, 2516, 2596, 2854, 2925, 3104 & 3448. FTꢀIR (KBr, cm ) for

the HDNB: 805, 1564, 1602, 3135 & 3446.

4

.3.3 GC-MS analysis

Molecular mass of the HDNB was determined by using Clarus 500 GCꢀMS

PerkinElmer, Wellesley, USA). Eliteꢀ5MS capillary Column (5% Phenyl, 95%

(

dimethylpolysiloxane) of 30 meter in length was used at 453 K. 2 µL of the HDNB dissolved in

DMSO at concentration of 1mg/100 µL (about 44 mM) was used for the analysis. Helium gas

was used as carrier gas and flow rate was maintained as 1ml/minute. Sample could be

fragmented and ionized by using electron energy of 70 ev in ‘Electron Ionization’ mode. Mass

range for detecting various fragments of the HDNB upon electron bombardment was set to be

from 20 to 600 amu. Total ion chromatogram of the HDNB showed two fragment species

depicting molecular mass of 44 Da and 184 Da and the generated mass/charge data were used to

search against NIST 2005 library in order to identify the structures of the HDNB (refer to

supplementary material).

1

13

15

4

.3.4 H, C and N NMR studies

1

One dimensional H NMR (1D H NMR) studies for the DNSA and HDNB dissolved in

DMSOꢀd were carried out using a 300 MHz AVANCE II (Bruker Biospin, Fallanden,

6

Switzerland) NMR spectrometer equipped with a 5 mm BBO probe. Sample concentration was 2

1

mM in both cases. 1D H NMR spectra for the test samples were acquired using zg30 standard

pulse sequence with a relaxation delay of 1.0 sec at 298 K (temperature of the probe was

7

2

calibrated using methanol standard ) and the acquired time domain data were then processed by

1

Topspin 1.3 program. 1D H NMR data of the HDNB dissolved in DMSOꢀd :D O solvents

6

2

1

mixture (3:1, v/v) was also acquired at identical conditions as mentioned above. In all the 1D H

RSC Advances

Page 24 of 52

View Article Online

DOI: 10.1039/C5RA23005E

NMR experiments, tetramethylsilane (TMS) was used as an internal standard for referencing

chemical shifts of various protons of the DNSA and HDNB. DEPT and twoꢀdimensional HSQCꢀ

1

5

N experiments for HDNB were carried out using a 400 MHz NMR spectrometer equipped with

a 5 mm AutoSW probe (Agilent Technologies, CA, USA). Default standard parameter settings of

the programs as defined in the standard pulse sequence library of VnmrJ 2.2C were used except

the following acquisition parameters: transients and t increments were set as 4 and 512,

1

respectively, for recording the HSQC spectrum; transients and a relaxation delay were set as

1

024 and 1 second, respectively, for recording DEPT spectrum. TMS and CH NO were used as

3 2

1

3

15

internal standards for referencing chemical shifts of C and N resonances of the HDNB,

1

respectively. H NMR (300 MHz, DMSOꢀd ) of the HDNB: δ 8.65 (s, 1H, CH), 8.70 (s, 1H,

6

1

3

15

CH); DEPT C NMR (400 MHz, DMSOꢀd ) of the HDNB: δ 125.9, 131.5; HSQC ꢀ N (400

6

1

MHz, DMSOꢀ d ) of the HDNB: δ 358.1; H NMR (300 MHz, DMSOꢀd ) of the DNSA: δ 8.67

6

(s, 1H, CH), 8.71 (s, 1H, CH).

4

.4 In vitro cytotoxicity potential of the HDNB by MTT assay

The cancer cell lines (MGꢀ63, MCFꢀ7 and HeLa) were purchased from National Centre

for Cell Science (NCCS), Pune, India and were grown in Eagles Minimum Essential Medium

EMEM), supplemented with 10% (v/v) fetal bovine serum (FBS) at 37 °C in a 5% CO₂

atmosphere. The monolayer cells were detached with trypsinꢀethylene diamine tetra acetic acid

EDTA) to make single cell suspensions and viable cells were counted by trypan blue exclusion

(

assay using a hemocytometer. The cell suspensions were seeded in 96ꢀwell plates at a density of

4

1

x 10 cells/well and treated with serial concentrations (ranging from 0.2 to 1000 µM) of the

HDNB dissolved in neat DMSO. The plates were then incubated for an additional 48 h at 37 °C

in a 5% CO atmosphere. After the incubation, 15 µl of MTT (3ꢀ[4,5ꢀdimethylthiazolꢀ2ꢀyl]2,5ꢀ

2

Page 25 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

diphenyltetrazolium bromide) dissolved at concentration of 5 mg/ml in phosphate buffered saline

PBS) was added to each well and the plates were further incubated for another 4 h. Formazan

(

crystals formed were solubilized in 100 ꢁl DMSO and absorbance was measured at 570 nm using

a microplate reader. The medium containing without the HDNB was served as control and all the

experiments were performed in triplicate. The percentage of cell inhibition caused by the HDNB

7

3

was determined using standard procedure and the data were analysed by using GraphPad Prism

software 5.01 (GraphPad Software, CA, USA).

4.5 Acute oral toxicity studies

A total of 5 Wistar female rats of about 8 weeks old with average weight of 175 ± 5 g

were used as test animals for the acute toxicity studies and the animals were housed in standard

polypropylene cages. Temperature, relative humidity and lighting of animal room were

maintained at 25 ºC, 30ꢀ70% and 12/12 light/dark cycles, respectively throughout the

experiments. The rats were provided with rodent laboratory diets (M/s Provimi Pvt. Ltd.,

Bangalore, Karnataka, India) and potable water purified by reverse osmosis ad libitum. The

studies on the Wistar rats were conducted at Animal House, SASTRA University, using

protocols approved by the Institutional Animal Ethics Committee of SASTRA University

(CPCSEA – Committee for the Purpose of Control and Supervision of Experiments on Animals,

Ministry of Environment and Forest, Government of India ꢀ ethical clearance for the animal

studies is 227/SASTRA/IAEC/RPP).

Acute toxicity of the HDNB was tested in the animal models using ‘Main test’ procedure

7

4

as per the OECD guidelines 425. The HDNB was dissolved in corn oil and various doses (175

mg/kg, 550 mg/kg and 2000 mg/kg b.w.) of the compound were administrated using an oral

gavage needle to the rats. It should be mentioned that single ordered dose progression wherein

RSC Advances

Page 26 of 52

View Article Online

DOI: 10.1039/C5RA23005E

the rats were dosed one at a time, at a minimum of 48 h intervals was followed and volume of

doses was constantly maintained to be 1 ml irrespective of dose levels (dose progression factor

was 3.2) and body weights of animals. Apparent changes on the physical systems and behavior

patterns of the test animals were keenly noticed every 30 minutes for first 6 h and thereafter the

animals were periodically monitored every 12 h for the experimental time span of 14 days. After

14 days, all the experimental animals were sacrificed and gross necropsies were done to examine

any abnormalities or lesions in the internal organs such as brain, heart, lungs, liver, kidney and

intestines.

4.6 In vivo studies using Wistar rats

A total of 42 female Wistar rats of about 8 weeks old with average body weight of 180 ±

g were used as test animals for the in vivo studies and the animals were housed in standard

6

polypropylene cages. Temperature, relative humidity and lighting of animal room were

maintained at 25 ºC, 30ꢀ70% and 12/12 light/dark cycles, respectively throughout the

experiments. The rats were provided with rodent laboratory diets (M/s Provimi Pvt. Ltd.,

Bangalore, Karnataka, India) and potable water purified by reverse osmosis ad libitum. The 42

female Wistar rats were divided into seven groups (Group I – VII) and each group was consisted

of six animals. The total study period was set as 18 weeks and doses (saline/corn

oil/HDNB/Tamoxifen) were administered to animals belonging to various groups at every

alternate day throughout the study period as mentioned below herein. Wistar rats of Group I

served as ‘control group’ and the rats received only physiological saline as an oral dose

throughout the experimental period. Group II rats received only vehicle (1ml of corn oil was

orally given using stainless steel ball tipped oral intubation needle) and acted as ‘vehicle group’.

Rats in groups III to VII were subjected to induction of mammary carcinogenesis by providing

Page 27 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

st

rd

intraperitoneal injection of 75 mg of NMU at 1 and 63 days of the experiments. The

carcinogen was freshly prepared prior to administration by dissolving it in 1ml of physiological

saline. Group III rats received no other treatments throughout the experiments and hence the rats

were considered as ‘diseased group’. Group IV, V and VI rats were orally administered with the

HDNB at concentration of 8 mg/kg (lowꢀdose), 40 mg/kg (mediumꢀdose) and 200 mg/kg (highꢀ

dose), respectively. The HDNB doses were also freshly prepared by suspending the finely

grinded compound in corn oil, and the dose volume was adjusted to 1ml with plain corn oil in all

cases. The low/medium/high doses of the HDNB were orally administered to rats of Group IV, V

and VI using stainless steel ball tipped oral intubation needle. Group VII rats were orally

administered with tamoxifen citrate (20 mg/kg body weight, dissolved in 1 ml of physiological

saline) every alternate day and the dose was continued till the end of experimental timeline and

the group served as ‘standard group’.

4.6.1 Histopathology

At end of the study period, the rats were killed using CO inhalation euthanasia and

2

breast tissues and other organs of each animal were dissected out. All tissues were preserved in

0% neutral buffered formalin (NBF). The preserved breast tissues were then cut into thinner

1

section (2ꢀ3 mm thick); processed using automatic Leica tissue processor TP 1020 (Leica

Biosystems, Illinois, USA); embedded in paraffin wax using Leica EG 1150 H&C Embedding

machine (Leica Biosystems, Illinois, USA) and cut into 3ꢀ4 ꢁm sections using Leica RM 21°

RTS (Leica Biosystems, Illinois, USA), which were mounted on clean glass slides. The sections

of the tissues were stained with hematoxylin for 10 minutes followed with 0.5% eosin for

another 5 minutes. The sections were then rapidly rinsed in 95% ethanol and dehydrated in

absolute ethanol for 5 minutes using Leica ST 4040 Linear automatic stainer (Leica Biosystems,

RSC Advances

Page 28 of 52

View Article Online

DOI: 10.1039/C5RA23005E

Illinois, USA). Histological studies were carried out for the breast tissues of 6 animals from each

group. A semiꢀquantitative grading scheme was used to evaluate the extent of the lesions in the

tissues using five grades as follows: no lesion (grade 0); minimal (grade 1); mild (grade 2);

4

4, 45

moderate (grade 3); marked (grade 4) and severe (grade 5).

.6.2 Haematological studies

Prior to necropsy of Wistar rats used in the in vivo experiments, blood samples were

4

collected from the retroꢀorbital sinus of the rats in ethyleneꢀdiamineꢀtetraacetate (EDTA)ꢀcoated

vials. The blood plasma was used for estimations of various hematological parameters by using 5

7

5

Part Mythic 22 Haematology AutoAnalyzer (Orphee, Geneva, Switzerland).

.6.3 Biochemical studies

Plasma was prepared from blood samples collected from the retroꢀorbital sinus of the

experimental rats in the heparinized vials upon subjecting the blood samples to centrifugation at

500 rpm for 10 min. After the centrifugation, the buffy coat was removed and the packed cells

were washed thrice with physiological saline. The erythrocyte suspension was then prepared by

4

1

76, 77

the method of Dodge et al and modified by Quist.

Similarly, serum was prepared by

centrifuging the whole blood collected in sterile test tubes at 1500 rpm for 10 minutes. The

7

8

resultant supernatant was collected and stored separately as described elsewhere. Biochemical

analysis were carried using the plasma/serum prepared from blood samples of 6 animals from

each group and estimated values are expressed as mean ± standard error of the mean.

Lipid peroxide content was determined by thiobarbituric acid (TBA) reaction as assessed

7

9

by Okhawa et al. To the 0.5 ml of plasma, 1.5 ml of 20% acetic acid, 0.2 ml of sodium dodecyl

sulphate (SDS) and 1.5 ml of TBA were added. The mixture was made up to 4.0 ml with distilled

water and then heated for 60 minutes at 95° C using glass ball as condenser. After cooling to

Page 29 of 52

RSC Advances

View Article Online

DOI: 10.1039/C5RA23005E

room temperature, 4.0 ml of butanolꢀpyridine mixture was added, centrifuged and then

absorbance was read at 532 nm. Standard and blank were treated in a similar manner. The lipid

peroxide concentration was expressed as nmoles of malondialdehyde (MDA) formed/mg protein.

8

0

The levels of tumor necrosis factorꢀα (TNFꢀα) was assayed by the method of Konturek et al.

The wells of microtiter plates were coated with 100 µL of blood plasma appropriately diluted in

carbonate buffer. After incubating the plates at 4 °C for 12 h, 100 µL of serially diluted antibody

was added and incubated at 37 °C for another 1 h. The wells were then charged with 100 µL of

antiꢀrabbit IgGꢀconjugated with horseradish peroxidase (diluted 1:5000) at 37 °C for 1 h. The

activity of bound horseradish peroxidase was monitored by addition of 50 µL of substrate (1 µL

of hydrogen peroxide, 0.5 mg oꢀphenylenediamine (OPD) in 1 ml of citrate phosphate buffer, pH

5.0) at room temperature for 20 min in dark and the reaction was then arrested by adding 50 µL

of 2 N sulfuric acid. The TNFꢀα concentration was recorded on ELISA (EnzymeꢀLinked

Immunosorbent Assay) reader at 490 nm and expressed as ng/ml.

The extent of inhibition of nitric oxide radical generation in vitro was followed as per the

8

1

method reported by Green et al. The reaction was initiated by incubating mixture consisting of

.0 ml of sodium nitroprusside, 0.5 ml of PBS and 0.5 ml of plasma at 25 °C for 30 minutes.

2

Griess reagent (0.5 ml) was added to the mixture and incubated for another 30 minutes. The

absorbance was read at 546 nm against the reagent blank (Genesys 10ꢀS, USA) and the values

were expressed as nmole/ml. The activity of catalase, superoxide dismutase (SOD) and

Glutathione peroxidase in plasma were assayed by the method of Sinha, Misra & Fridovich and

8

2ꢀ84

Rotruck et al with some modifications, respectively.

To 0.1 ml plasma, 0.9 ml of phosphate

buffer and 0.4 ml of hydrogen peroxide were added and the reaction mixtures were kept in a

boiling water bath for 10 minutes. The colour developed was read at 530 nm and the activity of

Article Doi

Evaluation of the anticancer properties of the predicted hBaxBH3-mimetic compound 2-hydroxy-3,5-dinitrobenzamide in a mammary carcinogenesis-induced rat model

DOI: 10.1039/c5ra23005e

Source and publish data:

Authors:

Article abstract of DOI:10.1039/c5ra23005e

Full text of DOI:10.1039/c5ra23005e

Products guided by the article

R&D Labs maybe for 2912-79-0

Relevant to this article

Hot Product