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 (Allium sativum , 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 HIF1α 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
HIF1α to an antioxidant response element and causes its transcription.
Nrf2 activation and HIF1α upregulation are found to be associated with
each other in certain selected type of cancers. A link between Nrf2 and
HIF1α led us to investigate HIF1α, 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 2nd 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-1α 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-1α 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 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.