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\begin{document}

\title{Dialyl-sulfide with chalcone prevent breast cancer promoting SULT1E1
malregulations and oxidant-stress dependant HIF1a-MMPs induction.}



\author[1]{Aarifa Nazmeen}%
\author[1]{Sayantani Maiti}%
\author[1]{Smarajit Maiti}%
\affil[1]{Affiliation not available}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: In some breast cancers, decreased estrogen-sulfotransferase
(SULT1E1) and its inactivation caused by oxidative-stress lead to
elevated E2 levels as well as hypoxia-inducible tissue-damaging factors.
Methods: Here, matrix-metalloproteases (MMP2/9) activity and
SULT1E1-HIF1\selectlanguage{greek}α \selectlanguage{english}protein/gene expression (Western-blot/RTPCR) were assessed
in human breast-cancers versus their adjacent-tissues. Oxidant-stress
neutralizer, chalcone (\selectlanguage{greek}α,β \selectlanguage{english}unsaturated ketone) and SULT1E1-inducer
dialyl-sulfide (source garlic; Allium sativum) were tested to prevent
cancer causing factors in rat, in-vitro and in-vivo model. The
antioxidant-enzymres SOD1, catalase, GPx and LDH, and
matrix-degenerating MMP2/9 activities were assessed (gel-zymogram).
Histoarchitecture (HE-staining) and tissue SULT1E1-localization
(immuno-histochemistry) were screened. Extensive statistical-analysis
were performed. Results: Human cancer-tissue expresses higher SULT1E1,
paralleling HIF1\selectlanguage{greek}α \selectlanguage{english}protein/mRNA owing to lower LDH activity. In addition,
increase of MMP2/9 activities commenced tissue damage. However, chalcone
and DAS significantly induced SULT1E1 gene/protein, and suppressed HIF1\selectlanguage{greek}α
\selectlanguage{english}expression, and MMP2/9 activities in rat tissues. Correlation of
individual parameter statistics and group statistics of t-test suggest
significant correlation of oxidative-stress (MDA) with SULT1E1
(p=0.006), HIF1\selectlanguage{greek}α \selectlanguage{english}(p=0.006) protein-expression. The NPSH showed a
negative correlation (p=0.001) with HIF1\selectlanguage{greek}α, \selectlanguage{english}These two proteins and
SULT1E1 mRNA expressions in human breast tumor were significantly higher
(p\textless{}0.05) compared to the adjacent tissues. Pearson correlation
data suggest, SULT1E1 is correlated with NPSH in different exposure
groups. Conclusions: Breast cancers associate with SULT1E1, HIF1\selectlanguage{greek}α \selectlanguage{english}and
MMPs deregulations. Higher SULT1E1-protein in advanced cancer, remain
inactive in oxidant oxidative environment and may be re-activated in
chalcone induced reducing-state. Moreover, DAS induced SULT1E1 mRNA
expression augments its protein increment. Synergistic drug-effects
commenced HIF1\selectlanguage{greek}α \selectlanguage{english}and MMPs suppression. Further studies are necessary.%
\end{abstract}%



\sloppy


\textbf{INTRODUCTION}

Breast cancer is the most common female cancer globally, representing
nearly a quarter (25\%) of all cancers. Organic carcinogens covalently
bind DNA formingstable and depurinating adducts. The compound
4-hydroxyestradiol and estradiol-3, 4-quinone (E1(E2)-3,4-Q)) produces
significantly higher levels of depurinating adduct and smaller stable
adduct (1). These estrogen-DNA adducts are quickly lost by the cleavage
of the glycosyl bond from DNA and create apurinic sites that can cause
cancer (2,3). Mounting evidences demonstrate that a few E2 metabolites
(i.e. CE-3, 4-Q) interact with DNA and form 4-OHE1(E2)-1-N3Ade and
4-OHE1(E2)-1-N7Gua that causes DNA~mutation ~and potential~cellular
transformation (4,5).

The use of human breast epithelial cell lines such as MCF-10F, an
immortalized, non-transformed estrogen receptor (ER)-\selectlanguage{greek}α-\selectlanguage{english}negative cell
line has identified the initiation of cancer by estrogen-DNA adducts.
Results indicate that beside the alteration of E2-ER signaling,
genotoxic effects via estrogen metabolite are partly responsible for the
malignant transition . These findings support the hypothesis that the
availability of the active estrogen is a critical determinant in the
treatment of some breast cancer.

A large percentage of breast cancers are sensitive to estrogen and have
a good response to endocrine therapy based on selective ER modulators
(SERM) i.e. tamoxifen (7) and fulvestrant (8). Reduction in estrogen
synthesis by aromatase-inhibitors of is also a vital strategy (9).
Beyond these strategies, estrogen metabolizing enzymes are given less
priority for research. SULT1E1, a phase II metabolizing enzyme that is
located in the cytoplasm and responsible for sulfonation of estrogen
(10). SULT1E1 polymorphisms are shown to be a risk factor for breast and
endometrial cancers, (11) suggesting that its modulation might be an
attractive strategy in the prevention, management and or treatment of
breast cancer. In contrary, the steroid sulfatases (STs) converts water
soluble sulfated-estrogen to insoluble estrogen in the target tissues.
Roughly, it sketches that activation of SULT1E1 and inhibition of STS
might work in favor of breast cancer treatment.

The induction of SULT1E1 along with anti-breast~cancer~action of TM208
and tamoxifen (ER antagonist) may bring better treatment outcomes in
hormone ER+ breast cancers. Estrogen is differentially metabolized in
healthy ovarian surface epithelium (OSE) and in epithelial ovarian
cancer (EOC) cells. Total mRNA assay by qRT-PCR revealed significantly
higher SULT1E1 mRNA expression in OSE than in EOC. Inflammatory
cytokines further augmented the local production of E2 by stimulating
STS and suppressing SULT1E1 (12). It was concluded that the local
estrogen metabolism may be a target for EOC treatment. SULT1E1 is
transcriptionaly regulated under oxidative stress, and is also a target
of NRF2. Oxidative stress responses augment Nrf-2 and HIF-1a in breast
cancer cell. Hypoxia Inducing Factor (HIF) target genes in every step of
the metastatic process. Digoxin which blocks HIF action eventually
decreases primary tumor growth, vascularization and invasive metastasis
in animal models of breast cancer (13,14). Degeneration of extracellular
matrix is implicated as the metastatic growth factor and MMPs are highly
expressed in advanced breast cancer. A high expression of MMP-2 and
MMP-9 was found in \textgreater{}75\% of breast cancer patients (15).
So, it is suggested that the restriction in HIF1\selectlanguage{greek}α \selectlanguage{english}and MMP activities may
have therapeutic prospects against breast cancer.

In our earlier study, we explored that the pre-tumorigenic condition
induced by ethylnitrosourea (ENU) and E2 showed impairment of SULT1E1
expression and E2 regulations via oxidant-stress signaling (16). Thus,
induction and activation of SULT1E1 might become cancer prevention and
treatment startegy. An earlier study demonstrated that dialylsulfide
(DAS) treatment caused the nuclear accumulation of constitutive
androstane receptor (CAR) resulting in a significant increase of SULT1E1
mRNA and protein in the liver of female mice. DAS effectively reduces
lipid peroxidation, recovering cell viability, attenuating DNA strand
breaks in cultured breast cancer MCF-10A cells (17). Despite of
significant SULT1E1 induction by DAS, the endogenous E2 level was
unaltered and no increase in estrone sulfate level was noticed.
Contrarily, the clearance of exogenously administrated E2 was
accelerated in DAS treated mice (18). This suggests that under variable
oxidative stress condition, SULT1E1 transits between gain and loss of
enzyme activity Our previous report suggests that oxidative stress
induced SULT1E1 modifications may be similar in human breast cancer
tissue and experimental animal model (16). Further study demonstrates
that transcriptional regulation of stress, promotes Nrf-2 and
proinflammatory marker NFk\selectlanguage{greek}β \selectlanguage{english}through E2 mediated signaling. These
signallings are also associated with breast cancer initiation and
severity. So, a reducing environment is required to keep SULT1E1 active
which may keep E2 levels under control, SULT1A1 also sulfoconjuagtes E2
but at higher concentration and definitely contributes to control the E2
pool. Chalcone posses oxidative stress relieving property and and act as
antioxidant in different diseases (19). Hence, we opted for Chalcone in
this study to induce cellular reducing environment to keep SULT1E1
active together with Dialylsulfide that induces mRNA/protein expression
of SULT1E1. Our present study may have some therapeutic implications in
breast cancers.

\textbf{MATERIALS AND METHODS}

\textbf{Ethical clearance and fulfillments of other regulatory affairs}

This is to state that the present study was carried out in accordance
with the National Institutes of Health, USA guidelines and the
institutional ethical concerns, relevant guidelines and regulations were
maintained throughout the investigation. This study confirms that all
experimental protocols were approved by the institutional (Oriental
Institute of Science and Technology) Ethics Committee
(oist/EC/hu/bt/16/). This is also to state that informed consent was
obtained from all participants who were at their post-menopausal age.

Female Wistar rats were purchased from a small-animal firm house (Govt.
registered) that follows all ethical norms and maintain requisite
regulatory affairs. The firm house is a Government accredited
(CPCSEA-Committee for the Purpose of Control and Supervision of
Experiments on Animals: Reg. no 1A2A/PO/BT/S/15/CPCSEA. organization
under the Dept of Animal Husbandry and Dairy, Ministry of Agriculture
and Farmer's Welfare, Govt. of India. Proper permissions for all animal
experiments, were obtained from the Institutional (Oriental Institute of
Science and Technology) Review Board.

\textbf{Inclusion and exclusion criteria}

Inclusion criteria- Patients only with breast carcinoma were included.
Tumors were collected only from patients undergoing mastectomy. Those
patients were also included who had a large gap of time between
chemotherapy and mastectomy.

Exclusion criteria- Women suffering from endometriosis, pelvic
inflammatory disease, tuberculosis, or any kind of liver and kidney
disease, ovarian cancers, Poly cystic ovarian syndrome, colon carcinoma,
lung carcinoma, pregnancy, menstruation or any other infectious disease
like HIV, HPV, HCV and Hepatitis B were excluded.

\textbf{Details of the participants}

Detailed information about randomly selected patients (maintaining
both-side anonnimity and double blind random screen) who donated their
tumor samples has been provided in S-Table 1. The S-Table 1 informs
about the patient's age (44.69 \selectlanguage{ngerman}± 7.55 years; mean ± SD) and body weight
(56.08± 6.63 Kg; mean ± SD), livelihood status, nutritional status,
grade of the disease, tumor size and the details regarding the inclusion
of the lymph nodes and metastasis.

\textbf{Breast tumor sample collection}

The study was conducted in Oriental Institute of Science and technology
and a total of 23 breast tumor samples were obtained from the local
District Medical College and Hospital with proper ethical clearance.
Breast tumors are diagnosed clinically; breast cancers were classified
on the basis of TNM (The extent of the tumor (T), the extent of spread
to the lymph nodes (N), and the presence of metastasis (M)) staging and
grades. In some cases down staging of cancer with chemotherapy was done
prior to surgery and samples were collected. In this regard, this is to
mention that tumor sample and corresponding surrounding tissues were
collected separately soon after surgery and stored at -20°C. A small
part of the tissue was also stored in formalin for histology and
immunohistochemistry. We sincerely thank Dr. Guangping Chen of
Department of Physiological Sciences of Oklahoma State University for
providing the primary antibody against rSULT1E1 and hSULT1E1. Anti-
Nrf-2 and NFk\selectlanguage{greek}β \selectlanguage{english}antibodies were used in the current study.

\textbf{Treatment of animal model (rats) with Diallyl sulfide and
Chalcone.}

The animal experiments were designed to study short term, and long term
effect of the drugs. The results may provide us with valuable
information of duration required by these drugs to affect the desired
expression and activation of SULT1E1. Three different sets of experiment
were used for evaluating the effect of diallylsulfide and chalcone. Two
of the experiment were in-vivo and one in vitro experiment.

\textbf{In- vitro (3 hour, 5 hour and overnight treatment)}

\emph{Preparation of single cell suspension of rat hepatocyte}

The animal experiments were performed in the Oriental Institute of
Science and Technology, Cell and Mol. Therap. Lab. animal facility and
surgery chamber. The fresh and laboratory acclimatized animals were
anesthetized and then euthanized by intraperitoneal injection of
pentobarbital sodium (50 mg/kg). Fifty grams of~rat liver tissue
processed to have single cell suspension. A nylon mesh was used to
scrape the tissue in DBSS Buffer. The scarpped product was centrifuged
at 120\selectlanguage{ngerman}×\emph{g} . The cellular pellet was washed with constituted L-15
media. A final suspension of the cells was made in 10~ml of constituted
L-15 media containing 1500~mg/l~D-glucose, 1\% penicillin and
streptomycin, 20\% FBS, 1\% Glutamine. 650~\selectlanguage{greek}µ\selectlanguage{english}l of media containing cell
was added to each petri plate comprising of 9350~\selectlanguage{greek}µ\selectlanguage{english}l of constituted L-15
media containing the required drug.

Three in vitro groups based on duration of exposure were considered for
this study. . Grp 1-3hr, Grp 2-5 hr and Grp 3-12 hrs exposure. Each
petri dish was provided with 650 \selectlanguage{greek}µ\selectlanguage{english}l of cell suspension (from stock) and
each group comprised of 4 plates containing a total of 10 ml solution
such as Plate-1 (control): 650\selectlanguage{greek}µ\selectlanguage{english}l of cell suspension+ 9250 \selectlanguage{greek}µ\selectlanguage{english}l buffer,
Plate-2: DAS-100 \selectlanguage{greek}µ\selectlanguage{english}M+ buffer (making up to 10 ml), Plate-3:
Chal-100\selectlanguage{greek}µ\selectlanguage{english}M+buffer (making up to 10 ml), and Plate -4: DAS+Chal 100\selectlanguage{greek}µ\selectlanguage{english}M
each + buffer (making up to 10 ml).

\textbf{In --vivo (24, 48, 72 hours treatment)}

Animals were divided in 3 groups, Group 1-control, Group 2-Diallysulfide
(DAS), 80 mg DAS/100gm b.w., Group 3- Chalcone, 4 mg/100gm b.w., Group
4- Combination of DAS (80 mg) and Chalcone (4mg). The animals were
treated with the drug or drug combination by gavages. DAS and chalcone
were solubilized in 0.1\% ethanol (Ethanol+water) to get the required
concentration. The current dose of DAS and chalcone was screened after
consultation with some previously published reports (17-19). In
addition, some dose response studies were performed in our lab to
validate the selected dose. The present dose demonstrated significant
responses on some genes expression like SULT1E1 and this dose did not
initiate any toxicity which was evaluated from animal body weight, organ
weight/body weight ratios, liver and kidney function test and
haematology or other toxicity studies. Control group was given same
amount of 0.1 \% ethanol solution as. Different groups had 3-5 animals
per group and animals were serially sacrificed at 24, 48, 72 hours after
drug administration, a second dose was given to a single animal at 72
hour and was sacrificed on the 5\textsuperscript{th} day of the
experiment i.e. at 120 hours or after 48 hours of the second dose.

\textbf{In --vivo (7 day treatment)}

Four group having four animals each was taken for this experiment. Group
1 was control, Group 2 was treated per day with 20 mg of DAS, Group 3
was treated with 8 mg of chalcone and Group 4 was treated with a
combination of DAS (20mg) + Chalcone (8mg). These animals were treated
for 7 days and sacrificed on the 8\textsuperscript{th} day. All animals
were kept under UV radiation for 2 hours a day, until sacrificed. . UV
radiation was given for the purpose of immune compromisation.

\textbf{Cytosol preparation of rat liver tissues}

Immediately after animal sacrifice the liver tissue were cleaned in
phosphate buffer and homogenized (30 \% w/v) in ice-cold phosphate
buffer (0.1 mol /L, pH 7.4) then centrifuged at 10,000 rpm at 4\selectlanguage{ngerman}ºC for
30min. The supernatant (cytosol) was collected and stored at -20\selectlanguage{english}.C in
different aliquots for further assays.

\textbf{Cytosol preparation of human breast tissue sample}

Breast tumor and the corresponding surrounding tissues were homogenized
(30\% w/v) in ice-cold phosphate buffer (0.1 mol /L, pH 7.4);
andcentrifuged at 10,000 rpm at 4\selectlanguage{ngerman}ºC for 30min. The supernatant (cytosol)
was collected and stored at -20\selectlanguage{english}.C in different aliquots for further
assays.

\selectlanguage{greek}\textbf{Western blot of HIF1α and SULT1E1}\selectlanguage{english}

Western blot was conducted as in Maiti et al. 2007 with a slight
modification. A 12 \% denaturing gel was loaded with 25 \selectlanguage{greek}μ\selectlanguage{english}g of protein
(obtained from the rat liver tissue of animals treated with a single
dose of DAS, Chalcone and DAS + Chal for 24, 48and 72 hours) and
electrophoresis carried out at 100v for 3 hours, transfer to
nitrocellulose membrane was done at 100v for 2 hours. The membrane was
washed and incubated with primary antibodies (anti rHIF1\selectlanguage{greek}α \selectlanguage{english}and anti
rSULT1E1) and secondary antibodies as mentioned in the referenced
protocol (20). The membrane were treated with Diaminobenzidine (DAB)
until the brown colored bands were developed.

\textbf{MMP 2/9 zymographic analysis in breast cancer and surrounding
tissues.}

The cytosols fromtumorand its corresponding surrounding tissue were
assayed for MMP activity. One percent type B-gelatin solution prepared
in water, 8 \% resolving gel is prepared containing 1\% B-gelatin
solution. Samples were prepared in MMP dye and loaded in gel to run at
110V for 60 minute. After completion of electrophoresis the gel
wasactivated by washing buffer (as mentioned in the referenced protocol)
and then incubated in zymographic gel development buffer pH 7.4 at 37\selectlanguage{ngerman}ºC
for 42 hours. Finally the gel was stained in amide black or Coommasie
blue solution for 1 hour at room temperature in a shaker. The gels were
then destained with solution I for 15 to 30 minutes, followed by
destaining solution II for 3 to 5 hours until clear MMP bands appeared.
After washing the destaining solution the gel was incubated in a gel
preservative solution for 15 minutes and finally scanned with a digital
scanner (21)

\textbf{Determination of SULT1A1 activity in human tissues by PNPS assay
method.}

The \selectlanguage{greek}β-\selectlanguage{english}Naphthol sulfation activity was determined from the tumor tissue
and its surrounding. This assay determines phenol sulfation activities
of different isoforms of phenol sulphating SULTs. A reaction mixture
containing 50mM Tris buffer, pH 6.2, 5mM PNPS, 20 M PAPS, and 0.1mM
-naphthol was used to measure the sulfation activity. Tumor and
surrounding tissue cytosols (50 \selectlanguage{greek}μ\selectlanguage{english}g protein) were used as the enzyme
source in a total reaction volume of 250 \selectlanguage{greek}μ\selectlanguage{english}l. After 30 min incubation at
37\selectlanguage{ngerman}\textsuperscript{º}C in a shaking water bath, the reaction was stopped
by adding 250\selectlanguage{greek}μ\selectlanguage{english}l of 0.25M Tris, pH 8.7. The reaction mixtures were read
at 401 nm in a spectrophotometer. Specific activity (SA) was expressed
as nmole/minute/mg of protein (22).

\textbf{RT-PCR of SULT1E1 from rat hepatocytes}

The RNA was isolated from hepatocyte of rats treated with DAS, Chalcone
or their combination for 24, 48 and 72 hours. 1~\selectlanguage{greek}μ\selectlanguage{english}g of whole RNA utilized
to perform reverse transcription PCR using Qiagen one step RT-PCR kit
and SULT1E1 primer following the instructions provided in the kit. Total
RNA concentration was measured by a NanoDrop Microvolume
Spectrophotometers at 260 nm (A260 reading = 40 \selectlanguage{greek}μ\selectlanguage{english}g/ ml RNA) and its
purity was tested from the ratio of A260/A280. The master mixprepared
with stipulated proportions of ingredients including enzymes, dNTPs,
template RNA and gene specific primer (both for human and rat SULT1E1)
pairs (F 5'-CTTCCAGTATCATTTTGGGAAAAG-3' and R 5'-TGGATTGTTCTTCATCTC-3',
all the primer specificity was satisfactory, that was tested by
universal nucleotide alignment). To compare with control, 500 bp cDNA of
rat \selectlanguage{greek}β-\selectlanguage{english}actin and 200 bp cDNA of human \selectlanguage{greek}β-\selectlanguage{english}actin were synthesized and loaded
in the gel. The primer pair F 5'-GATGTACGTAGCCATCCA-3'/R
5'-GTGCCAACCAGACAGCA-3' for the synthesis of rat \selectlanguage{greek}β-\selectlanguage{english}actin and F
5'-GGCGGCAACACCATGTACCCT-3'/R 5'-AGGGGA GGGACTCGTCATACT-3' for human
\selectlanguage{greek}β-\selectlanguage{english}actin were designed using the GeneFisher primer designing software and
published earlier (20, 22). PCR was carried out in Eppendorf\selectlanguage{ngerman}®
Mastercycler® and the reaction program included reverse transcription at
50ºC for 30 min, PCR activation step 95ºC for 15 min, denaturation 94ºC
for 1 min, annealing 64ºC for 1 min and extension at 72ºC for 1 min (30
cycles). Final extension was allowed for 10 min at 72ºC. PCR products
were run an agarose gel electrophoresis image was captured by a BioRad
gel doc system.

\textbf{Activities assay of SOD, catalase, GPx and LDH activities by gel
zymogram}

\emph{Super oxide dismutase.} Three different SOD activities were
assayed, first in breast tumor and corresponding surrounding tissue,
second in the serum of animal model treated with DAS, Chalcone and
DAS+Chalcone for 7 days, 24, 48 and 72 hours and thirdly in the in vitro
treated heapatocytes (3, 5 and 12 hour). A tablet of nitro blue
tetrazolium (NBT) dissolved in 30 ml of water and the non-denaturing
(10\%) acrylamide gel kept soaked in it for 30 minfollowed by 40 ml SOD
solution containing 0.028 M tetramethylethylenediamine (TEMED), 2.8 x
10-5 M riboflavin, and 0.036 M potassium phosphate at pH 7.8) for 15
minin shaking condition. Finally the gel was placed on clean acetate
sheet and illuminated for 5 to 15 min and thenunder a UV illuminator
(23)

\emph{Catalase.} A non-denaturing gel (8\%) was loaded with a 25\selectlanguage{greek}µ\selectlanguage{english}g of
cytosol (from in-vitro treated rat hepatocytes for 3, 5 and 12 hours
with DAS, Chalcone and DAS+Chalcone) and electrophoresed for 3 h at 40
mA at 4 \selectlanguage{ngerman}°C. The gel was washed 3 × 10 min in distilled water and
incubated in 0.003\% H\textsubscript{2}O\textsubscript{2} for 10
minutes, followed by staining with 2\% ferric chloride and 2\% potassium
ferricyanide. In this critical step two reagents were not mixed prior to
staining rather poured together directly on top of the gel. The gel
became greenish blue except in the position containing the catalase.
After rinsingwith distilled water the gel was scanned when the maximum
contrast between the band and the background was obtained (23).

\emph{Glutathione peroxidase.} A non-denaturing gel (8\%) was loaded
with a 150 \selectlanguage{greek}µ\selectlanguage{english}g of protein obtained from rat hepatocytes treated for
different times with DAS and/or Chalcone. Electrophoresis carried out
for 3 h at 40 mA at 4\selectlanguage{ngerman}°C. The gel was washed 3 × 10 min in distilled
water containing 1 mM GSH and incubatedin 0.008\% cumene hydroperoxide
for 10 minutes. Then 1\% ferric chloride (w/v) containing 1\% GSH and
1\% potassium ferricyanide (w/v) containing 1\% GSH were prepared
separately and poured together directly on the gel. When achromatic
bands begin to form (5 -- 15 min), the stain was poured off and the gel
was washed extensively with distilled water. The image of the bands
demonstrated GPx activity was evaluated. The cumene hydroperoxides
instead of H\textsubscript{2}O\textsubscript{2} performed as a good
substrate and the GPx (contains a selenium in the active site) can
utilize this hydroperoxide to determine total peroxidase activity
according to the established protocol as described in the referenced
article (23).

\emph{Lactate dehydrogenase (LDH).} LDH zymogram in tumour and its
corresponding surrounding tissue obtained from breast cancer
pateints,was performed by using a standard protocol (24) with slight
modification. LDH zymograms were obtained by separating 10 mg of protein
on a native PAGE gel of 8 \% for 2 h at 4 W, then soaked in water
followed by washing twice with water. The LDH activity was detected by
soaking the gel in a staining solution containing 0.1 M Tris-HCl (pH
8.0), 750 mM NAD1, 200 mM lithium lactate, 0.1 mg of NBT - nitroblue
tetrazolium per ml, and 0.02 mg of phenazine methylsulfate per ml.
Enzyme activity bands were developed from the conversion of NAD1 to
NADH, which were clearly visible. The gel was scanned under the gel doc
system.

\textbf{Determination of oxidative status and cytotoxicity parameters in
rat hepatocytes}

\textbf{Estimation of Malondialdehyde (MDA) Levels}

The MDA was estimated both from liver tissue and serum samples. Tissue
was homogenized (10 \% w/v) in the ice-cold phosphate buffer (0.1 mol
/L, pH 7.4) and the homogenate was centrifuged at 10,000 rpm at 4ºC for
10 min. The MDA assay was conducted using the supernatant following the
protocol (25). To chelate iron and reduce its interference in
peroxidation reaction of unsaturated fatty acid, 1 mM EDTA was used in
the reaction mixture. To reduce the interference caused by a
yellow-orange colour produced by some carbohydrates, the reaction
mixture was heated at 80ºC instead of 100ºC. Finally, the MDA was
measured and calculated utilizing the molar extinction coefficient of
MDA (1.56 x 105 cm2/ mmol).

\textbf{Estimation of Non Protein Soluble Thiol (NPSH)}

The NPSH in serum and liver tissue homogenates (prepared in 0.1 M
phosphate buffer, pH 7.4) were determined by the standard DTNB (5, 5'-
dithiobis-2-nitrobenzoic acid) method with a slight modification (26).
In brief, the protein was precipitated by trichloroacetic acid and clear
cytosol was added to 0.1 M sodium phosphate buffer containing 5 \selectlanguage{greek}µ\selectlanguage{english}M DTNB.
The level of NPSH was determined against a GSH standard curve.

\textbf{Evaluation of~general toxicity}

Serum glutamate pyruvate transaminase (SGPT), serum glutamate
oxaloacetate transaminase (SGOT), NPSH and MDA were measured from the
rat serum treated with DAS, Chal, and DAS + Chalcone for 24, 48 and 72
hours. SGPT and SGOT were measured by standard protocol with the assay
kits (Ranbaxy, India or other reputed company). Total protein from serum
was measured following the standard protocol (27).

\textbf{Histoarchitechture studies of cancer tissues}

Breast tumor tissue and its corresponding surrounding tissues were
embedded in paraffin, serially sectioned at 5 \selectlanguage{greek}μ\selectlanguage{english}M by an automated
cryostat slicing machine (Leica Biosystems), stained with eosin and
hematoxylin (Harris), and observed under a microscope (Nikon, Eclipse
LV100, magnification 20X) to study the tissue histoarchitecture.

\textbf{Immunohistochemistry for SULT1E1}

Tumor and its corresponding surrounding tissues were embedded in
paraffin, serially sectioned at 5\selectlanguage{greek}μ\selectlanguage{english}M by an automated cryostat slicing
machine (Leica Bio systems). Sections were deparaffinized by baking at
60 c followed by xylene treatment, downgraded alcohol and water. Slides
were washed with PBST containing 1\% casein for 10 minutes, tissue
sections were incubated with 5\% casein for 30 minutes for preventing
non-specific binding followed by overnight incubation in primary
antibody SULT1E1 and HIF1\selectlanguage{greek}α\selectlanguage{english}in 1\% casein PBST, washed with 1\% PBST and
incubated in 1\% casein PBST containing secondary antibody for 1 hour,
washed with 1\% PBST followed by water and stained with chromogenic
substrate DAB for 3 minutes and then washed with water. Slides were
fixed with mounting medium and observed under a microscope (Nikon,
Eclipse LV100, magnification 20X) to study the SULT1E1 expression and
localization.

\textbf{Statistical analysis}

For the statistical evaluations of various factors, the SPSS for Windows
statistical software suite (SPSS Inc., Chicago, IL, USA, 2016) was
employed. Data from all parameters were collected from multiple
independent set as well as replicated from a single set of experiments.
The Kolmogorov-Smirnov test was used to examine the data distribution
pattern. Students t' test analysis and multiple comparison ANOVA tests
were used to evaluate the baseline continuous variables and the results
of the analyses. The level of significance were denoted within the 95\%
confidence limit and represented as p\textless{}0.001 to
p\textless{}0.05.

\textbf{RESULTS}

\textbf{Statistical analysis of relation between Cancer manifestation
and this disease causing factors}

The protein density and mRNA density (in surrounding and tumor) from
western blot and RT-PCR data suggests that both PD and MD of 1E1 and
HIF\selectlanguage{greek}α \selectlanguage{english}are significantly increased in the tumor groups (Table 1 and Fig
1). Present correlation study suggests that tissue MDA level is
positively associated with SULT1E1 tissue expression. The 1E1 is an
adaptive enzyme that catalyze the inactivation of E2 by forming E2S.
More oxidative stress as evident in high MDA level has some initial role
in active E2 nullification. MDA is negatively associated with HIF\selectlanguage{greek}α
\selectlanguage{english}expression. At the initial phase of MDA related stress response has more
adaptive responses of lower HIF\selectlanguage{greek}α. \selectlanguage{english}But in breast cancer tissue it is
noticed that, at higher disease state with prolonged oxidative stress
exposure and high MDA level HIF\selectlanguage{greek}α \selectlanguage{english}is increased significantly and Nrf2 is
shown to positively associated with HIF\selectlanguage{greek}α \selectlanguage{english}induction. NPSH and uric acids
are regarded as the endogenous soluble antioxidants those may have some
protective role against the oxidant-stress-induced 1E1 regulations and
Nrf2, HIF\selectlanguage{greek}α \selectlanguage{english}induction. Present results and correlation data suggest that
NPSH and uric acids are negatively correlated with HIF\selectlanguage{greek}α \selectlanguage{english}expression.
Lower antioxidant level augments the HIF\selectlanguage{greek}α \selectlanguage{english}level (Table-2). The data from
this table are represented from DAS and chalcone related studies shown
in the figure from in fig 4, 5 and 6. Table 3 suggests that in control,
DAS and Chalcone groups, NPSH and 1E1 are found to be associated. When
in control and in DAS these are positively associated in Chal- group
NPSH and 1E1 are shown to be negatively associated. No significant
association was noticed in DAS and Chalcone groups.

Table 4 suggests that, in case of time-based exposure NPSH and 1E1 and
1E1 are found to be positively associated and 3 and 24 hrs of exposure.
No significant relation was noticed in 5 hrs exposure. New sample size
and internal variability may be the cause for this.

\textbf{SULT1E1 protein expression in Tumor and surrounding tissues}

Expression of SULT1E1 protein in tumour was comparatively higher than
corresponding surrounding tissues. A few surrounding tissue have shown
an increased SULT1E1 expression, whose corresponding tumor shows more
intense SULT1E1 expression. Our findings abide by earlier study that
suggests that SULT1E1 is expressed more in the tumor tissues as compared
to their surroundings, and our studydid the first attempt to explain
that, though SULT1E1 is overexpressed in tumor it remains inactive (28).
Considering both the studieswe utilized DAS and Chalcone separately as
SULT1E1 inducer and reducing agent and also in combination to mainatian
an elevated active SULT1E1 state.

\selectlanguage{greek}\textbf{HIF1α protein expression in rat liver tissue treated with DAS
and chalcone}\selectlanguage{english}

The expression of HIF1\selectlanguage{greek}α \selectlanguage{english}in tumor tissue was remarkably higher when
compared to their corresponding surrounding tissues which was noticed in
SULT1E1 expression. HIF-1 plays important roles in breast cancer
metastasis by mediating hypoxia-induced expression of mRNA-encoding
genes. HIF-1 also regulates the expression of non-coding RNAs, which are
critical regulators of migration, invasion, and metastasis (14).

\textbf{Superoxide dismutase (SOD1) and Lactate dehydrogenase (LDH)
activities in breast cancer tissue}

The antioxidant enzyme superoxide dismutase (SOD) activity was found to
be higher in the tumor as compared to its surrounding (Fig-1a, SOD1). In
essence, lactate dehydrogenase (LDH) is an enzyme necessary for the
conversion of sugar into cellular energy. An elevated total serum SOD
level is a predictor of tissue damage and inflammation (29). Another
study has shown loss of LDH-B expression as an early and frequent event
in human breast cancer (30). In our current study, we found the loss of
LDH 3, 4 and 5 in breast cancer as compared to the surrounding whereas
LDH 2 seems to be equally expressed both in the surrounding and the
tumor. LDH has 2 subunits, subunit A and subunit B in different
proportion in each type of LDH. In this study, we found that the
expression of Subunit A is reduced or almost lacking in breast tumor.
Therefore, the less is the expression of LDH 3, 4 and 5 in tumor tissue
(Fig-1a).

\textbf{Matrix metalloproteases (MMP 2 and 9) activities}

Breast tissue expressed both MMP2 and MMP9. Compared to the surrounding
tissue, the tumour tissue had a higher expression of MMP9. MMP9 was not
similarly expressed in all the tumors, some tumors had less, whereas
some had more MMP9. MMP2 expression was less in the tumor in comparison
to the corresponding surroundings. MMPs are vital in cancers. MMP-9 can
cleave many extracellular matrix (ECM) proteins to regulate ECM
remodeling. Several plasma surface proteins can also be cleaved by it,
releasing them from the cell surface. MMP-9 has been widely found to
relate to the pathology of cancers, including but not limited to
invasion, metastasis and angiogenesis (31).

\textbf{Histopathology of human breast cancer sample}

In the tissue surrounding the tumors, few normal ducts lobules in
a~fibrous stromal~along with~adipose tissue are identified (Fig-2,
Surrounding).The tumor tissue shows ductal carcinoma in-situ and micro
invasive cells. Hematoxylene-eosin stained breast cancer tissue shows
several mitosis and pleomorphic nuclei. Irregular and undifferentiated
ducts are present in the tumor tissue (Fig-2).

\textbf{Immunohistochemistry of SULT1E1 of human breast cancer sample}

Immunohistochemistry results show that the tumor and surrounding both
are positive for SUT1E1. The strength of SULT1E1 staining in tumor is
stronger than corresponding surrounding tissue. The distribution and
localization of SULT1E1 was noticed in stromal tissue, adipose lining
and nucleus in both tumor and corresponding surrounding tissues. Tumor
tissue was darkly stained for SULT1E1 compared to the surrounding
suggesting increased expression (Fig-2).

\textbf{SULT1A1 activity of human breast cancer sample}

Sulfotransferase also utilizes PAPS as sulfonate donor to catalyze the
sulfate conjugation of a wide variety of acceptor molecules bearing a
hydroxyl or an amine group including estrogen. To find whether E2 is
controlled by other E2 regulating enzymes such as SULT1A1, we did
SULT1A1 activity assay. Interestingly we found no difference in the
SULT1A1 activity between tumor and their surrounding tissue (Fig-1b).
Thus, there is no SULT1A1 interference in the final outcome of E2.

\textbf{SULT1E1 protein expression (Animal experiment)}

\textbf{In vivo experiment}

Following a single dosage of DAS on day 1, the hepatic tissue animal
group treated with DAS demonstrated a progressive increase in the
SULT1E1 expression with increasing duration. SULT1E1 expression was
comparable to DAS 48 hours after DAS 2\textsuperscript{nd} dose group.
On the other hand, in 24 and 48 hours groups of chalcone treated animals
expressed highest SULT1E1 but the expression gradually decreased with
increasing duration. The combination group (DAS + Chal) showed an
expression level less than the DAS or Chalcone alone group but greater
than the control group (Fig-4a).

\textbf{In vitro experiment}

The DAS group expressed SULT1E1 more than the control group during the
in-vitro therapy, and this expression gradually increased over time. The
chalcone group had less SUT1E1 expression than the control and gradually
decreased with increasing time. Whereas the combination group had
highest expression at the first 3 hours of incubation, decreased at 5
hours and remain undetected at 12 hours of incubation (Figure-4b).
However, 3hr DAS+Chal treated tissue showed the best result with highest
level of SULT1E1 and then gradually decreased in 5 hr and least is found
in 12 hr. Thus, DAS has direct effect on the expression of SULT1E1 and
Chalcone has some moderate effect.

\textbf{Massenger RNA (mRNA) expression studies of SULT1E1}

When administered for a period of 7 days, it was discovered that DAS and
Chalcone, both alone and collectively, were able to increase mRNA in
comparison to the animals in the control group via inducing
transcription of the SULT1E1 gene. SULT1E1 mRNA was highly expressed in
the DAS treated cells. Chalcone also expresses SULT1E1 mRNA but less
than DAS. Whereas DAS+Chal treated showed highest SULT1E1 in comparison
to DAS alone and Chalcone alone group but a single animal had low
SULT1E1 mRNA. These expressions were all higher than the control group
(Fig-4c).

\textbf{Catalase}

\textbf{In - vitro Treatment for 3, 5 and 12 hours}

The enzymatic activity of catalase was low in DAS, Chalcone and DAS +
Chalcone combination group in comparison to the untreated tissue.
Catalase Activity gradually decreased in the in the DAS treated tissues
with time, and the same was true for the Chalcone treated tissue. Among
all the treated groups the highest activity was found in the
DAS+Chalcone treated tissue for 3 and 5 hours, however no activity was
found in the DAS + Chalcone- 24 hr treated tissue (Fig-3b).

\textbf{SOD (24 to 72 hours)}

The SOD activity remained unchanged in the DAS alone, chalcone alone
group with a slight decrease of activity in DAS group. The combination
group showed a decrease in the activity in the DAS+Chalcone (48 hr) and
then markedly increased in the 72 hour group and the group treated with
the 2nd dose. DAS alone and Chalcone alone group showed comparatively
less SOD activity than the control or untreated group. The combination
group of DAS+ chalcone showed a significant increase in SOD activity on
treatment with the 2\textsuperscript{nd} dose after 72 hours of the
1\textsuperscript{st} dose. Among the 3 control animals, SOD activity
was high in 2 animals and less in the 3\textsuperscript{rd}. The DAS
treated 24, 48 and 72 hour animal showed Less SOD than first 2 controls.
Animal treated with 2\textsuperscript{nd} dose of DAS had higher SOD
activity. The chalcone treated group had low SOD activity than control
and DAS group, but the 2\textsuperscript{nd} dose had high SOD activity.
Interestingly DAS+Chal had the highest SOD activity, which gradually
increased with duration till 72 hrs (Fig-3c).

\textbf{SOD activity (7 days or 3 to 12 hours treatment)}

The SOD activity was slightly less in the DAS group as compared to the
control. The chalcone group also displayed lower SOD activity in half of
the animal as compared to the control and DAS group. The other half
shows SOD activity similar to the control group. The entire group showed
comparatively lower SOD activity compared to the control (Fig-3a).

The control group showed SOD activity, and DAS at 3 hour showed SOD
activity but 5 and 12 hours showed negligible SOD. DAS+Chalcone at 5 and
12 hour showed some activity but less than the control (Fig-3b).

\textbf{Glutathione peroxidase (GPx) activity study by gel zymogram}

The GPx activity was highest in the control group (Untreated) as
compared to other group of animal such as DAS alone, Chalcone alone and
DAS + Chalcone group. The GPx activity was significantly lower in the
DAS group than it was in the chalcone, which was likewise significantly
lower. The DAS + Chalcone group a gradual increase in the GPX activity
with duration and the highest activity among them all was found in the
DAS + Chalcone 2\textsuperscript{nd} dose group (Fig-3c).

The control group showed some GPx activity and there was no activity in
the DAS treated animal group. The Chalcone group showed minute activity.
The Das + Chal group showed high Gpx activity. The GPx activity was
found to be lower in the DAS, Chalcone group as compared to the control
group of animal. The GPx activity was stimulated in the DAS+Chalcone
group of animal's hepatic tissue (Fig-3a).

\selectlanguage{greek}\textbf{Expression of HIF1α protein}\selectlanguage{english}

HIF1 expression was high in the DAS-24 hr group and then appeared to
gradually decline with time as well as with the second dosage. Yet, in
the Chalcone-treated group, HIF1 is observed to gradually express more
with time and the second dose. In the DAS+Chalcone group of animal
hepatic tissue, the expression of HIF1 was essentially the same. This
justifies why DAS+chal have little impact on the HIF1 group (Fig-4a, b).
The band density of HIF1 expression is depicted in the bar diagram,
which also clearly shows the pattern of HIF1 expression. (Fig-4c).

\textbf{Oxidative stress related parameters; Malondialdehyde (MDA) and
Non protein soluble thiol (NPSH)}

The level of non protein thiol in vitro experiment increased after 3, 5
and 24 hrs of incubation with DAS. Das+ Chalcone incubation also causes
NPSH induction as compared to the control cells (Fig-5a). In the in vivo
experiment, NPSH was negligibly increased in the DAS alone and Chalcone
alone treatedgroup, whereas a slight increase was noticed in DAS+Chal,
48 hr, 72hr and 2\textsuperscript{nd} dose treatment (Fig-5b). Serum
NPSH was found to be reduced in the Chalcone alone group, and the other
entire group had NPSH similar to the control, showing no changes in the
redox status in terms of NPSH.

Elevated MDA was seen in DAS-treated mice, demonstrating high MDA
production with the second dose of DAS, suggesting that DAS does appear
to trigger lipid peroxidation. During 48 and 72 hours, Chalcone 2nd
dosage and DAS + Chalcone also had a higher MDA, in contrast to the
other groups, which displayed MDA similar to the control group (Fig-5c).
Serum MDA levels remain stable (Fig-5g). This means that neither DAS nor
Chalcone cause systemic lipid peroxidation.

\textbf{General toxicity}

Except for the DAS alone group, neither the Chalcone alone group nor the
DAS+Chalcone group had raised SGOT or SGPT levels. Hence, these
medications lacked such liver damage. So, these drugs can be used as
therapeutic agents (Fig-5d).

\textbf{DISCUSSIONS}

Our present result of DAS induction of SULT1E1 has been supported by a
previous finding of 1E1 induction by this drug in mouse liver. Recently,
mouse SULT1E1 was shown to be induced by many chemicals/composition
including garlic extract (\emph{Allium sativum} \textbf{,} rich with
DAS) that activate CAR (18).This suggests that use of crude garlic
extract may have some beneficial effect in E2 dependant cancer.

In this study, we sought to combine the idea of SULT1E1 induction by DAS
with the creation of a reducing environment by chalcone. An enhanced
level of SULT1E1 has been found in tumour tissues in earlier research as
part of an adaptation mechanism to control active E2. However, the 1E1
activity may be insufficient to counter the significant surge of E2 in
E2 dependent breast cancer. Limitations in 1E1 activity might be due to
its inactivation in oxidative stress in breast cancer (28), where Cys83
of SULT1E1 may remain in oxidized state and hinders E2 binding and
sulfo-conjugation by this enzyme (28,20). Eventually, it allows elevated
active estrogen leading to a carcinogenic effect. This study
hypothesized that the induction of SULT1E1 by DAS and its activation by
chalcone may significantly control E2 level and the disease pathogenesis
resulting in an increase in patients' survival.

Rat treated with DAS and Chalcone demonstrated elevated SULT1E1
expression at different treatment schedule and DAS alone also showed a
consistent higher and chalcone moderate SULT1E1 expression (Fig-2a and
2b). This abides by an earlier study that SULT1E1 is an oxidative stress
responsive gene that gender specifically affects liveri/reperfusion
injury (32, 28). Chalcone has been shown to create a reducing
environment that helps to keep SULT1E1 active in the present study.

The highest expression was noticed in DAS + Chalcone, 3hr in-vitro
experiment. Both in vivo and in vitro experiments provide us a clear
outlook that this drug combination may be an effective therapeutic
strategy against E2 dependant breast cancer. Oxidative stress is known
to induce HIF1\selectlanguage{greek}α \selectlanguage{english}via Nrf2 which has been shown to be induced in human
ovarian cancer may have possible role in breast cancer also. Role of
Nrf2 and NfkB has been demonstrated in human breast cancer tissue and in
experimental rodent model (28, 33). Nrf2 binds at 32 kb upstream of
HIF1\selectlanguage{greek}α \selectlanguage{english}to an antioxidant response element and causes its transcription.
Nrf2 activation and HIF1\selectlanguage{greek}α \selectlanguage{english}upregulation are found to be associated with
each other in certain selected type of cancers. A link between Nrf2 and
HIF1\selectlanguage{greek}α \selectlanguage{english}led us to investigate HIF1\selectlanguage{greek}α, \selectlanguage{english}since Nrf2 is completely associated
with oxidative stress management. Oxidative stress is linked with
SULT1E1 induction and activation via Nrf2 (34).

Potential experimental models of rat hepatocytes exposed to current
drugs were used to test their metabolism and their ability to regulate
hepatic gene expressions (35,36). In the current study, chalcone group
induces catalase in the rat hepatocytes in 24 to 72 hours exposure.
Beside its reducing effects, chalcone showed strong antioxidant effect
and activate the catalase and both drugs strongly activate the SOD
activity also. Catalase was moderately induced in both drugs treated
group (Fig-3). Superoxide dismutase and catalase mimetic-drugs MnTmPyP
and 134 have distinct impacts on breast cancer cell proliferation via
TNFa-induced NF-B regulations confirm our findings that proposed drugs
may have positive effects (37). This result may be linked to our
previous study on human breast tumor sample (28). Anti-invasive and
anti-angiogenic potential of chalcone derivatives acted as an HIF-1
inhibitor (38) which is also noticed in our study.

DAS and chalcone mediated induction of anti-stress responses may be via
activated Nrf2, thereby, increasing of antioxidant enzyme i.e. SOD,
catalase and GPx supporting to oxidative-stress induction and its
possible adaptations have been noticed in the current study. The novel
combination of DAS and chalcone might be invaluable, because oxidative
stress not only inactivates SULT1E1 but also augments Nrf-2 and HIF-1a
in breast cancer. The NPSH levels elevated in DAS or Chalcone alone and
in combination group (Fig-5a, 5b). This ascertains the induction of the
reducing environment and minimization of oxidative stress which might be
strongly supportive in E2 related cancers. However, there is some amount
of lipid peroxidation products (MDA) are noticed in the liver tissue of
the reats of 2\textsuperscript{nd} dose DAS and DAS+Chalgroups (48 and
72 hours). An extra amount of antioxidant (vitamin C and E) as
supportive to this combination would be able to protrct the tissue
(Fig-5c). Report suggests that some chemotherapeutic applications may
cause some level of systemic stress. Several phytomaterials have
demonstrated therapeutic efficacy against certain ailments. As for
example, cardamonin inhibited the Nrf2-dependent ROS scavenging system
in addition to suppressing HIF-1a, which led to an increase in
intracellular ROS levels. As a result of decreased glucose absorption
and the production of lactic acid, it significantly aided ROS-induced
apoptosis in breast cancer cells (39).

Cancer subclones are encouraged by intramural hypoxia in metastasis.
HIF-1 expression levels at various clinical phases of the illness
predict the outcome for several malignancies, including breast cancer.
Tumor tissue in the current study has higher HIF1 protein expression
than does surrounding tissue (Fig-1f). In the DAS + Chalcone combination
group, we observed that both DAS and Chalcone had a substantial
inhibitory effect on the HIF1 (Fig- 4a \& 4b). This finding confirms our
hypothesis that the use of these medications in combination provides a
high level of clinical benefit. An earlier work demonstrated that
histone-deacetylase (HDAC) inhibitors can target the HIF-1 protein owing
to von Hippel-Lindau (VHL) protein-dependent degradation, suggesting a
potential connection between oxidative stress-controlled HIF-1a and cell
cycle regulation. Our current study was supported by a previous study
which has shown that diallyl trisulfide (DAS) dose-dependently inhibited
HIF-1\selectlanguage{greek}α \selectlanguage{english}in hypoxic MDA-MB-231 cells thereby inhibiting hypoxia-induced
pre metastatic changes and angiogenesis~in these cells. The angiogenic
responses in high rate of tumerigenic growth may be terminated by the
combined effects of DAS and Chalconein addition to their
SULT1E1induction effects.

The MMP9 was mainly expressed in tumor tissue and less expressed in
surrounding tissue; some of those have higher and variable expressions
due to inter-individual variability. The MMP2 was expressed less in the
tumor tissues. The MMPs can cleave several plasma surface proteins
causing their release from the cell surface. The MMP has been widely
found to relate to the pathology of cancers including but not limited to
invasion, metastasis and angiogenesis (31). Under hypoxic stress, HIF-1a
rapidly accumulates and activates hundreds of genes including MMPs in
breast cancer patients (40). Our preliminary findings on the potential
control of breast cancer and metastatic status by using DAS and chalcone
combination may be significant from a therapeutic standpoint. Chalcone
also triggered the mitochondrial apoptotic signaling by increasing the
amount of Bax and Bak and reducing the level of Bcl-2 and Bcl-X (L), and
subsequently activated caspase-9 in MCF-7 and MDA-MB-231 cells (41).
Chalcone may therefore operate as an apoptotic signal inducer for
malignant cell death by activating caspase through the mitochondrial
Bcl-2/BAX pathway.

Lactate dehydrogenase is responsible for conversion of sugar into energy
in cells. Alterations in LDH may be related to anaerobic oxidation and
metabolic status that eventually occur in hypoxic tissues that have been
damaged by cancer. Earlier studies show loss of LDH-B expression as an
early event that is frequently occurs in breast cancer (30). We found
loss of LDH 3, 4 and 5 in breast cancer in comparison to the surrounding
and LDH 2 seems to be equally expressed both in the surrounding and the
tumor. In the current study expression of Subunit A is reduced or almost
lacking in breast tumor. Our findings interpret that, less is the
expression of LDH 3, 4 and 5 in tumor tissue (Fig-1a). Alterations with
inter-individual variability in LDH activity has been linked to the
catalase regulations in the breast cancer patients since oxidative
stress have dual effects on antioxidant state and hypoxia mediated
energy regulations. Once again this is also related to the HIF1a
responses noticed in the current study (42). Report showed a higher
LDH-A expression in the tumor that has been associated to metastatic
breast cancer. Supportive to our present LDH result I link may be
pointed between hypoxia induced LDH and HIF1a expressions as different
stages of breast cancer predictor (43).

According to the current study there is no alteration in the SULT1A1
activity in both breast cancer tissue and its surrounding tissues
(Fig-1b) suggesting that 1A1 mediated non-specific E2-sulafation is not
taking place which supports more important role of 1E1. Altered
regulations of 1A1 and/or its polymorphism have been linked to several
types of cancer. However, under a chemotherapeutic setting the complex
role of 1A1 cannot be easily predicted. As for example, possible
interaction of anti-cancer anti-estrogenic drug tamoxifen with the
SULT1A1 may be considered here. The anti-cancer therapeutic effect of
tamoxifen and melatonin may be somewhat influenced by 1A1's
biotransformation of these medicines. (44).

SULT1E1 mRNA was also induced in DAS, Chalcone and DAS + Chalcone groups
(Fig-2c). Figures 2a and 2b from the RT-PCR and western blot experiments
indicate that DAS and Chalcone or DAS alone acts at both the
transcriptional and translational levels (Figure 7). Therefore DAS may
become a multifunctional drug that inhibits HIF-1\selectlanguage{greek}α \selectlanguage{english}and induces SULT1E1at
the transcriptional and translational levels. Previous studies from our
lab have shown that SULT1E1 expression either increases in the tumor
with increased oxidative stress at late stages of the disease or reduces
at the initial stages of the disease to let E2 be active and gradually
increases with the disease pathogenicity (28), but remains inactive due
to elevated oxidative stress, as shown in our animal studies (ENU paper)
and another earlier studies (20). The current study shows no alterations
in the SULT1A1 activity in the breast tumor and surrounding tissue
explaining that the breast cancer may have little or no role of SULT1A1
mediated nonspecific metabolism of estrogen. Direct use of breast cancer
tissue or animal model might be better for the therapeutic study. In the
present investigation, we focused less on the patho-physiological state
but more pointed on the inter-relation of some disease related genes and
proteins expression. It is known that the liver is the port of entry and
metabolism of any exogenous and endogenous drugs including estrogen
which are significantly catabolized by the phase I and phase II enzymes.
Liver produces and circulates (to target organs like breast,
endometrium) significant amounts of estrogens and liver generates
different estrogen-metabolizing enzymes like SULT1E1, STS and others.
The mRNA and protein tested in the current investigation are highly
expressed in liver at basal level and significantly respond by a
modulator. It is shown that dexamethasone (DEX) treatment increased
hepatic and MCF-7/VEGF tumor expression of \emph{Sult1e1/SULT1E1} (43
45). Glucocorticoid receptor expression induced by DEX can also augment
SULT1E1 expression in mouse liver and MCF-7 resulting in more E2
sulfation/inactivation and tumor growth inhibition (46,16). Liver is the
competent source for study a large number of genes/proteins expression.
Other important point is that liver is an important target of breast
cancer metastasis. Report reveals that breast cancer liver metastasis
(BCLM) is linked with poor prognoses of this disease. Tumor intrinsic
subtype demonstrates preferential metastasis to liver with several types
of breast cancer (47). In this cancer, it is an adaptive strategy by
increasing SULT1E1 gene and protein expression, tissues makes an initial
attempt to get rid of extra amount of E2. But due to more oxidative
environment, SULT1E1 cannot perform properly. In the mean time, high
rate of cell division especially in the tumor region with comparison to
its surrounding, oxidative stress further increases, hypoxic environment
worsen the situation by increasing HIF1a, Nrf2, NfkB and eventually
tissue-degenerating MMPs expressions, these have been shown in our
current study and in several earlier studies. So, to counteract the fast
rate kinetics of tumor growth and adversely functioning of these
genes/proteins, application of some therapeutic measure might help. In
the current study DAS and chalcone combination has been shown to
significantly increase SULT1E1 expression/activation, and decrease
oxidative stress, HIF1a, MMPs expressions.

\textbf{CONCLUSIONS}

The current study explores the events of the redox dependent adverse
regulations of SULT1E1 and possible foul-play of estradiol in breast
cancer tissues of postmenopausal women. This has been linked to the
induction of HIF1\selectlanguage{greek}α \selectlanguage{english}and MMP2/9 activation. In an attempt towards the
therapeutic approach, we have demonstrated that dially sulfide is a good
inducer of SULT1E1 gene and protein which significantly decreased the
HIF1\selectlanguage{greek}α \selectlanguage{english}and MMPs. In addition chalcone played an ideal rectifier role of
cellular redox environment. Together, the two drugs may be applied as
effective therapeutic materials against some forms of breast cancer. To
make definitive statements, proper pharmacological testing must be
performed.

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\textbf{Figure legends}

\textbf{Figure 1.} 1a. Expression of SOD1, LDH 2, 3\&4, and MMP2, MMP9
in human breast tumor (n=3) and its corresponding surrounding tissues
(n=3): Lane distribution-1, 2, 3-surounding \& 4, 5, 6 --Tumor.

1b. Activity assay of SULT1A1 in human tumor tissue (n=3) and its
corresponding surrounding areas (n=3).

1c. SULT1E1 (protein) expression in tumor (n=6) tissue and their
corresponding surrounding (n=6) tissue are calculated and densitometric
data are presented. (blot image not shown). Level of significance in
Student's t --test is, b= p\textless{}0.01.

1d. HIF1\selectlanguage{greek}α \selectlanguage{english}(protein) expression in tumor tissue (n=6) and their
corresponding surrounding (n=4) tissue with densitometric analysis.
Molecular weight marker \selectlanguage{greek}β-\selectlanguage{english}galactosidase (120kD) was used in this study.
Human \selectlanguage{greek}β-\selectlanguage{english}actin was used to verify the control protein. Level of
significance in Student's t --test is, b= p\textless{}0.01.

1e. SULT1E1 (gene) expression RTPCR data in tumor (n=5) tissue and their
corresponding surrounding (n=3) tissue are calculated and densitometric
data are presented. Level of significance in Student's t --test is, b=
p\textless{}0.01.

\textbf{Figure 2.} Immunohistochemistry- SULT1E1 expression in tumor
tissue and adjacent surrounding tissue. HE staining- Histoarchitecture
of tumor and adjacent surrounding tissue.

\textbf{Figure 3.} 3a. Gel zymographic data of SOD activity in serum and
liver tissue and GPx activities in liver tissues of rats treated with
DAS, chalcone, or DAS+chalcone.

3b. Gel zymographic data of SOD and catalase activity in the liver
tissues of rat treated for 3, 5 or 12 hours with DAS, chalcone or
DAS+chalcone.

3c. Gel zymographic data of SOD and GPx activity in liver tissue of rats
treated for 24, 48 and 72 hours with DAS, chalcone or DAS+chalcone.

\textbf{Figure 4.} 4a. Expression of rSULT1E1 protein and its
densitometric analysis from rat treated with DAS, chalcone (Chal) or
DAS+chalcone for 24, 48 and 72 hours and in one group DAS+chalcone with
a second dose of DAS. Expression of r\selectlanguage{greek}β-\selectlanguage{english}actin was studied from all these
experimental groups. Lane distribution- details are mentioned at the
bottom of the lanes.

4b. Expression of rSULT1E1 protein and its densitometric analysis in rat
liver cells (primary culture) treated with DAS, chalcone or DAS+chalcone
for 3, 5 and 12 hours. Lane distribution- details are mentioned bottom
of the lanes. Two molecular weight marker M1 and M2 were also run to
verify SULT1E1 protein.

4c. Expression of rSULT1E1 mRNA from the liver of rat treated with DAS,
chalcone or Das+chalcone and their densitometry data are presented.
Expression of r\selectlanguage{greek}β-\selectlanguage{english}actin mRNA was also screened as a loading control.

\textbf{Figure 5.} Expression of rHIF1\selectlanguage{greek}α \selectlanguage{english}protein and its densitometric
analysis in rat model treated with DAS, chalcone (Chal) and DAS+chalcone
for 24, 48 or 72 hours and a group with chalcone second dose. Lane
distribution of -5a, As mentioned at the bottom of the blot image.

5b. Densitometric analysis and relative band densities of fig. 5a are
presented as bar in the diagram.

5c. Gel zymographic data of MMP2 and MMP9 from the liver tissues of rat
treated with DAS, chalcone or DAS + Chalcone for 7 days treatment. 5e.
Gel zymographic data of MMP2 and MMP9 from the liver tissues of rat
treated with DAS, chalcone or DAS + chalcone for 24, 48 or 72 hours.

\textbf{Figure 6.} Different parameters were measured from rat liver
tissue after in-vitro treatment with DAS, chalcone or DAS+chalcone and
data are presented as bar diagram. The representation of bars are
mentioned in the figures-Individual data from in vitro experiments are
presented in fig 6a-6c. Different groups had 3-5 animals per group as
depicted in the picture and animals were serially sacrificed at
stipulated time interval. In vivo experimental data from 7 days
treatment groups are presented in fig 6d-6g. Bars represent the means \selectlanguage{ngerman}±
SE from 4 animals in each group. Levels of significances of difference
between two groups are verified Student's t test.

\textbf{Figure 7} Schematic representation of breast cancer association
with redox-regulated SULT1E1 dysfunction and HIF1a/MMPs up-regulations.
The prominent therapeutic role of Dialylsulfide (DAS) and chalcone via
reversal of these protein dysfunctions has been summarized.

Table 1. The protein density and mRNA density (in surrounding and tumor)
from western blot and RT-PCR data\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
\textbf{Group Statistics} & \textbf{Group Statistics} & \textbf{Group
Statistics} & \textbf{Group Statistics} & \textbf{Group Statistics} &
\textbf{Group Statistics} & t & &\tabularnewline
\midrule
\endhead
Type & Type & N & Mean & SD & SE & & One-Sided p & Two-Sided
p\tabularnewline
SULT\_PD & Surr & 6 & 1.6667 & 0.81650 & 0.33333 & -3.138 & 0.005 &
0.011\tabularnewline
& Tumor & 6 & 2.9333 & 0.55737 & 0.22755 & & &\tabularnewline
HIFaPD & Surr & 4 & 1.8750 & 0.85391 & 0.42696 & -2.863 & 0.011 &
0.021\tabularnewline
& Tumor & 6 & 3.6667 & 1.03280 & 0.42164 & & &\tabularnewline
SULT1E1\_mD & Surr & 3 & 1.2000 & 0.20000 & 0.11547 & -2.857 & 0.018 &
0.036\tabularnewline
& Tumor & 4 & 3.2250 & 1.18708 & 0.59354 & & &\tabularnewline
\bottomrule
\end{longtable}

Table 2. Statistical analysis of Pearson's correlations amongst
different oxidative-stress parameters and SULT1E1 expressions in three
different diseases conditions in the tumor/surrounding tissues

\textbf{Correlations}\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
& & MDA & NPSH & URIC\_ACID & HIFa\_Expr & SULT1E1\_Expr\tabularnewline
MDA & r & 1 & 0.090 & 0.026 & -.509\textsuperscript{**} &
.486\textsuperscript{**}\tabularnewline
& P & & 0.611 & 0.888 & 0.006 & 0.006\tabularnewline
& N & 34 & 34 & 32 & 28 & 30\tabularnewline
NPSH & r & 0.090 & 1 & 0.138 & -.605\textsuperscript{**} &
0.159\tabularnewline
& P & 0.611 & & 0.452 & 0.001 & 0.402\tabularnewline
& N & 34 & 34 & 32 & 28 & 30\tabularnewline
URIC\_ACID & r & 0.026 & 0.138 & 1 & -0.364 & 0.282\tabularnewline
& P & 0.888 & 0.452 & & 0.068 & 0.145\tabularnewline
& N & 32 & 32 & 32 & 26 & 28\tabularnewline
HIFa\_Expr & r & -.509\textsuperscript{**} & -.605\textsuperscript{**} &
-0.364 & 1 & -.518\textsuperscript{**}\tabularnewline
& P & 0.006 & 0.001 & 0.068 & & 0.005\tabularnewline
& N & 28 & 28 & 26 & 28 & 28\tabularnewline
SULT1E1\_Expr & r & .486\textsuperscript{**} & 0.159 & 0.282 &
-.518\textsuperscript{**} & 1\tabularnewline
& P & 0.006 & 0.402 & 0.145 & 0.005 &\tabularnewline
& N & 30 & 30 & 28 & 28 & 30\tabularnewline
*. Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed).\tabularnewline
\bottomrule
\end{longtable}

Table 3. The correlation data suggest that in Control, DAS and Chalcone
groups, NPSH and SULT1E1 are found to be associated.

\textbf{Correlations}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Type & Type & Type & NPSH & SULT1E1\tabularnewline
CONTROL & NPSH & Pearson Correlation & 1 &
.944\textsuperscript{**}\tabularnewline
& & Sig. (2-tailed) & & 0.005\tabularnewline
& & N & 6 & 6\tabularnewline
& SULT1E1 & Pearson Correlation & .944\textsuperscript{**} &
1\tabularnewline
& & Sig. (2-tailed) & 0.005 &\tabularnewline
& & N & 6 & 6\tabularnewline
DAS & NPSH & Pearson Correlation & 1 &
.931\textsuperscript{**}\tabularnewline
& & Sig. (2-tailed) & & 0.007\tabularnewline
& & N & 6 & 6\tabularnewline
& SULT1E1 & Pearson Correlation & .931\textsuperscript{**} &
1\tabularnewline
& & Sig. (2-tailed) & 0.007 &\tabularnewline
& & N & 6 & 6\tabularnewline
CHAL & NPSH & Pearson Correlation & 1 &
-.855\textsuperscript{*}\tabularnewline
& & Sig. (2-tailed) & & 0.030\tabularnewline
& & N & 6 & 6\tabularnewline
& SULT1E1 & Pearson Correlation & -.855\textsuperscript{*} &
1\tabularnewline
& & Sig. (2-tailed) & 0.030 &\tabularnewline
& & N & 6 & 6\tabularnewline
CHAL,DAS & NPSH & Pearson Correlation & 1 & 0.159\tabularnewline
& & Sig. (2-tailed) & & 0.763\tabularnewline
& & N & 6 & 6\tabularnewline
& SULT1E1 & Pearson Correlation & 0.159 & 1\tabularnewline
& & Sig. (2-tailed) & 0.763 &\tabularnewline
& & N & 6 & 6\tabularnewline
*. Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed).\tabularnewline
. Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed).\tabularnewline
\bottomrule
\end{longtable}

Table 4. Time dependent correlation data of different groups of DAS,
Chalcone exposed rat

\textbf{Correlations}\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
Hour & Hour & Hour & NPSH & SULT1E1\tabularnewline
3.00 & NPSH & Pearson Correlation & 1 &
.895\textsuperscript{**}\tabularnewline
& & Sig. (2-tailed) & & 0.003\tabularnewline
& & N & 8 & 8\tabularnewline
& SULT1E1 & Pearson Correlation & .895\textsuperscript{**} &
1\tabularnewline
& & Sig. (2-tailed) & 0.003 &\tabularnewline
& & N & 8 & 8\tabularnewline
5.00 & NPSH & Pearson Correlation & 1 & 0.606\tabularnewline
& & Sig. (2-tailed) & & 0.111\tabularnewline
& & N & 8 & 8\tabularnewline
& SULT1E1 & Pearson Correlation & 0.606 & 1\tabularnewline
& & Sig. (2-tailed) & 0.111 &\tabularnewline
& & N & 8 & 8\tabularnewline
24.00 & NPSH & Pearson Correlation & 1 &
.828\textsuperscript{*}\tabularnewline
& & Sig. (2-tailed) & & 0.011\tabularnewline
& & N & 8 & 8\tabularnewline
& SULT1E1 & Pearson Correlation & .828\textsuperscript{*} &
1\tabularnewline
& & Sig. (2-tailed) & 0.011 &\tabularnewline
& & N & 8 & 8\tabularnewline
*. Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed). & **.
Correlation is significant at the 0.01 level (2-tailed).\tabularnewline
. Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed). & *.
Correlation is significant at the 0.05 level (2-tailed).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{english}
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Figure 1\selectlanguage{english}
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Figure 2\selectlanguage{english}
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Figure 3\selectlanguage{english}
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Figure 4\selectlanguage{english}
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Figure 5\selectlanguage{english}
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Figure 6\selectlanguage{english}
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Figure 7

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