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.