Taxifolin possesses anti-cancer activity on the 7,12-Dimethylbenz(a)anthracene-Induced breast cancer in the sprague dawley rats by remodeling nuclear factor Erythroid 2- Kelch-Like ECH-Associated Protein 1-Heme Oxygenase 1 and anti-oxidant pathways
Md. Wasimul Haque1, Mohd. Usman Mohd Siddique2, Pritha Bose1, Shakti Prasad Pattanayak1
1 Department of Pharmaceutical Sciences and Technology, Division of Advanced Pharmacology, BIT Mesra, Ranchi, Jharkhand, India
2 Department of Pharmaceutical Sciences and Technology, Division of Pharmaceutical Chemistry, BIT Mesra, Ranchi, Jharkhand, India
|Date of Submission||19-Dec-2017|
|Date of Acceptance||28-Feb-2018|
|Date of Web Publication||28-Jun-2018|
Shakti Prasad Pattanayak
Department of Pharmaceutical Sciences and Technology, Division of Advanced Pharmacology, BIT Mesra, Ranchi - 835 215, Jharkhand
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: In mammary cancer, alterations in various gene expressions and signaling pathways occurs due to the secondary effects of oxidative stress that facilitates cancer by causing genomic instability and mutagenic alterations. Several phenolic compounds are active against various malignancies. Taxifolin (TAX) exhibits diverse bioactivity profile that also contributes toward its anticancer efficacy. Objective: The present study has been designed for estimation of the anticancer potential of TAX on 7,12-dimethylbenz(a)anthracene (DMBA)-induced breast cancer in Sprague Dawley (SD) rats. Materials and Methods: Molecular docking analysis of Kelch-like ECH-associated protein 1 (Keap-1) and heme oxygenase-1 (HO-1) was carried out using Maestro tool to rationalize the activity of TAX based on their binding potential. This was followed by DMBA administration in air pouch to induce mammary cancer in female SD rats (50–55 days old). After 90 days of cancer induction, the chemotherapeutic potential of TAX was evaluated by the administration of TAX at doses 10, 20, and 40 mg/kg/day. Besides this, the effect of TAX on Keap-1-nuclear factor erythroid-2 (Nrf-2) pathway associated with HO-1 and NADPH:quinoneoxidoreductase 1 (NQO1) expressions and their effect on the anti-oxidative and anti-proliferative activity was also evaluated through immunofluorescence analysis, real-time quantitative polymerase chain reaction, and biochemical estimations. Results: TAX revealed protective effect against lipid peroxidation, enzymatic (superoxide dismutase [SOD], manganese-containing SOD, copper- and zinc-containing SOD, catalase, and glutathione peroxidase), and nonenzymatic (reduced glutathione, α-tocopherol, and ascorbic acid) anti-oxidative markers in serum, liver, kidney, and breast tissue of both control and experimental groups. The study revealed upregulation of protective Nrf-2, HO-1, and NQO1 expressions with consequent suppression in Keap-1 mRNA expression. Conclusion: This study revealed the potency of TAX in the inhibition of mammary carcinogenesis through Nrf-2-Keap-1-HO-1 and antioxidant pathway.
Abbreviations used: RT-qPCR: Real-time quantitative polymerase chain reaction; ODC: Ornithin decarboxylase; HO-1; Heme oxygenase-1; PAHs: Polyaromatic hydrocarbons; TBARS: Thiobarbituric acid reactive species; NQO1: NADPH:quinoneoxidoreductase 1; Keap-1: Kelch-like ECH associated protein 1; Nrf2: Nuclear factor erythroid 2; SD: Sprague Dawley.
Keywords: 7,12-dimethylbenz(a)anthracene, antioxidant, breast cancer, Kelch-like ECH-associated protein 1, nuclear factor erythroid 2, taxifolin
|How to cite this article:|
Haque MW, Mohd Siddique MU, Bose P, Pattanayak SP. Taxifolin possesses anti-cancer activity on the 7,12-Dimethylbenz(a)anthracene-Induced breast cancer in the sprague dawley rats by remodeling nuclear factor Erythroid 2- Kelch-Like ECH-Associated Protein 1-Heme Oxygenase 1 and anti-oxidant pathways. Phcog Mag 2018;14, Suppl S1:110-7
|How to cite this URL:|
Haque MW, Mohd Siddique MU, Bose P, Pattanayak SP. Taxifolin possesses anti-cancer activity on the 7,12-Dimethylbenz(a)anthracene-Induced breast cancer in the sprague dawley rats by remodeling nuclear factor Erythroid 2- Kelch-Like ECH-Associated Protein 1-Heme Oxygenase 1 and anti-oxidant pathways. Phcog Mag [serial online] 2018 [cited 2022 Jan 25];14, Suppl S1:110-7. Available from: http://www.phcog.com/text.asp?2018/14/55/110/235287
- Mechanism of Action of Taxifolin for it's anti-cancer activity on Keap-1-Nrf2-HO-1 Axis.
| Introduction|| |
Cancer chemotherapeutics, utilizing naturally occurring components is thrust in cancer research worldwide. Diversity in molecular mechanisms involved in cancer progression leads to continuous and progressive discoveries of newer approaches in cancer therapy. Polycyclic aromatic hydrocarbons (PAH) are a family of structurally related chemicals that comprises major class of environmental carcinogens. These carcinogens along with certain related halogenated compounds result in mammary cancer by epidemiological and laboratory studies. 7, 12 dimethylbenz(a) anthracene (DMBA) which is a prototypical PAH is utilized to promote tumors in laboratory animals. It is also involved in the development of cancers with progressive stages, in various parts of the body mainly breast,, skin, cervical, renal, etc.
DMBA is known to produce DNA adducts., In rodents, breast tumors can be induced by DMBA administration which upregulates cellular cytosolic receptor for DMBA. In the initial stage of tumorigenesis, cytochrome P450 enzymes are upregulated, which are responsible for metabolizing DMBA into an epoxide intermediate that readily forms DNA adducts. These adducts play a key role in DNA mutations and malignant transformation which is considered to be associated with PAH-mediated carcinogenesis.,
Molecular oxygen and free radicals give rise to highly proactive molecules called reactive oxygen species (ROS). Prooxidants and antioxidants imbalance results in oxidative stress. Endogenous oxidative stress may be triggered due to impairment of cellular antioxidant defense system. Oxidative stress-induced by ROS plays a critical role in the pathogenesis of cancer. Nuclear factor-erythroid 2 (Nrf2) is a key transcription factor that stimulates cytoprotective genes transcription in response to oxidative stress, which in turn is controlled by Kelch-like ECH associated protein 1 (Keap-1) (adaptor protein). As a consequence of stress conditions, there is disruption of Keap-1 and Nrf2 interaction leading to Nrf2 accumulation in the cell nucleus. This is followed by Nrf2 binding to the antioxidant response element (ARE) in the promoter region of some phase II enzyme genes leading to expression of enzymes downstream to Nrf2. These enzymes including heme oxygenase-1 (HO-1) and NADPH: quinoneoxidoreductase 1 (NQO-1) provide protection against oxidation. Normally, controlled oxidative stress of body shifts to a progressive neoplasm after activation of certain carcinogenic pathways. Antioxidant molecular components such as Nrf-2, HO-1, hypoxia-inducible factor 1α, and NQO-1 fails to overcome the intracellular free radical loads. DMBA initiate progressive oxidative stress causing failure of antioxidant mechanisms. These changes further activate (upregulate and downregulate) specialized molecular genes/pathways such as nuclear factor-kappa B (NF-κB) and Keap-1 expression causing neoplastic changes in individual or group of cells.
One of the primary markers of carcinogenesis, the endogenous antioxidants perform a pivotal role defense against ROS-induced oxidative damage. Free radicals play an important role in promoting tumors either by direct chemical reaction or by alterating cellular metabolic process, and their scavengers (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx]) represent inhibitors at different stages of carcinogenesis. The enzymes located in mitochondrial and cytosolic functions are mainly involved in the biotransformation and detoxification of carcinogens. Superoxide radical is sequentially converted into hydrogen peroxide by SOD and CAT and subsequently to molecular oxygen and water, respectively. Manganese-containing SOD (MnSOD) and copper- and zinc-containing SOD (CuZnSOD) that comprise total SOD content of cell are predominantly situated in the cytosol and mitochondrial matrix, respectively. Elevation of superoxide radical level is associated with a reduction in cellular SOD and CAT antioxidants activity. Carcinogenesis which results in an excessive increase in cellular superoxide and peroxide levels, also elevates SOD and CAT utilization further depleting enzyme levels. Stimulation of oxidative stress due to increase in the cellular level of free radical brings enhances oxidative stress leading to critical changes of cellular antioxidant enzymes.
Epidemiologically, naturally occurring polyphenolic components are evident to possess outstanding antioxidant, anticancer, anti-inflammatory, anti-angiogenic, and apoptotic potentials. These potentials attract different researchers for their uses against various progressive disorders such as neoplasm. Among all, flavonoids are a group of natural products including flavones, flavanones, and isoflavones, and several beneficial biological activities of flavonoids including antioxidant, antitumor, and anti-inflammation properties have been identified in several previous studies.,,,, Flavonoids have proven their anticancer potentials against different cancer cell lines and experimental models. Taxifolin (TAX) is a flavone derivative having antiproliferative, anti-inflammatory, antidiabetic, and antioxidant activities. TAX has anticancer activity against colon cancer by modulating various molecular pathways such as NF-κB, β-catenin, and tumor necrosis factor-α. We performed in silico docking studies by taking few antioxidant and other proteins such as Keap-1 and HO-1. Based on the in silico results and previous literature, the present study was designed to elucidate the anticancer potential of TAX against DMBA-induced mammary carcinogenesis in SD rats and elucidating the molecular mechanism involved.
| Materials and Methods|| |
DMBA and TAX were procured from SigmaAldrich (78666-100MG-F) (St. Louis, MO, USA). All other reagents used were of high purity analytical grade.
Molecular docking of taxifolin on heme oxygenase 1 and Kelch-like ECH-associated protein 1
The co-crystal structures of proteins HO-1 (PDB ID: 3HOK); Keap-1 (PDB ID: 5FNQ); were downloaded from www.rcsb.org. Maestro-9.2, Protein preparation wizard, Ligprep, and Glide module of Schrodinger LLC suite was used for protein preparation, ligand preparation, receptor grid generation, and molecular docking running on the RHEL5 operating system installed on DELL Precision T3400 machine (n-series, Intel Core 2 Quad processor, 8 GB RAM, 500 GB. The software validation was done by extracting internal ligand and redocked into the active site. Protein preparation was carried out using protein preparation wizard and grid was generated through grid preparation wizard picking ligand to specify the binding site. Docking was done using GLIDE 5.0 with XP protocol. The docked conformers were analyzed through XP visualizer. Default parameters were employed during all the computational studies.
Female Sprague Dawley (SD) rats (50–55 days old) were used for this study and were issued from Central Animal Facility, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, India (Register No. 1968/PO/Re/S/17/CPCSEA). All the studies on animals were performed after approval no. 1972/PH/BIT/17/17/IAEC from the Institutional Animal Ethics Committee. All the experimental rats were maintained with standard laboratory conditions (relative humidity 60% ± 5%, temperature 25°C ± 1°C, and12 h dark and light cycle) throughout the experimental period. These animals were supplied with pellet diet and were provided water ad libitum. All the experimental animals were subjected to acclimatization for a week before starting the experiment.
In vivo analysis
Five experimental groups consisting of 8 SD rats (50–55 days old) in each group were used for the study. The total treatment protocol is of 118 days (90 days of tumor promotional stage and 28 days of treatment phase). Cancer was induced using DMBA suspended uniformly in olive oil (20 mg in 0.5 ml) and administered once on day 1 by air-pouch technique.,,, The normal control group (Group I) animals were administered with 0.5 ml olive oil in air pouch, the induced control group (Group II) animals were administered with DMBA only, TAX treatment groups (Group III, IV, and V) were treated with 10, 20 and 40 mg/kg b.w.(i.p.) doses, respectively , after the promotional stage (90 days after DMBA induction). Tumor location was checked and monitored by palpation ,,, each week after the induction process. After 28 days of treatment with TAX, blood samples were collected from all groups of experimental animals by retro-orbital puncture and serum/plasma was separated for biochemical assessments. Later, animals were sacrificed by cervical decapitation ,, and perfused. Liver, kidney, and mammary tissues were isolated and homogenized in phosphate buffer saline at 4°C and used for the biochemical estimations. Fractions of breast tissues were kept after washing into the liquid nitrogen for protein and mRNA preparations using standard protocols. A slice (2–4 mm) of the breast tissue was also processed for the immunofluorescence assay.
Biochemical analysis for lipid peroxidation, enzymatic and nonenzymatic antioxidants in all treatment groups
To estimate the effect of DMBA and TAX treatment on lipid peroxidation (LPO), enzymatic and nonenzymatic antioxidants biochemical analysis was carried out using previously defined well-established procedures. LPO was estimated on the principle of simple thiobarbituric acid reactive species assay. The sample aliquots were treated with KCN to deactivate CuZnSOD to estimate MnSOD. Total SOD and MnSOD activities were determined on the principle of reduction of nitro blue tetrazolium. CuZnSOD was estimated by calculating the difference between total SOD and MnSOD. Based on the principle that H2O2 reduces dichromate in acetic acid to chromic acetate, CAT activity in the test samples was determined. GPx determination in the samples was carried out based on the method described by Rotruck et al. Protein content was estimated by the method of Lowry. Reduced glutathione (GSH) was estimated by the principle and method described by Ellman.
Immunofluorescence analysis of nuclear factor-erythroid 2 and Kelch-like ECH-associated protein 1 on mammary tissue
To evaluate the effect of TAX treatment on the expressions of Nrf-2 and Keap-1, immunofluorescence analysis was carried out using standard protocols. In brief, mammary tissues (2–4 mm) after isolation were embedded into ornithine decarboxylase media and kept in liquid nitrogen for few minutes. Cryosectioning was carried out (8–10 μ), and tissue sections (5 μm) were then treated with 4% buffered formalin (cold; 10 min) and OPTIMAX (×1, 1 min). The sections were then stained with primary antibodies (Santa Cruz Biotech, USA), for Nrf-2 and Keap-1 at a concentration of 1 mg/ml incubated for 1 h at room temperature. After washing the slides twice with OPTIMAX (×1) for 1 min, the sections were then incubated with the respective secondary antibodies (Bangalore Genei, India) and incubated for 30 min on ice in the dark. Sections were then washed twice with OPTIMAX (×1, 1 min). Cleaned coverslips were applied over the tissue sections using one drop of flow mount and the slides were analyzed under a confocal microscope (FLOWVIEW, Olympus).
RNA isolation, cDNA synthesis and real time quantitative polymerase chain reaction analysis
Total RNA was extracted from cells by TRI reagent ® (Sigma-Aldrich) and purified by adding a mixture containing 150 μL DDW, 8 μL glycogen, 20 μL sodium acetate (3M), and 600 μL of absolute ethanol. The RNA mixture was kept in −80°C for 2 h and centrifuged at 13,000 rpm for 15 min at 4°C to obtain the purified RNA for cDNA synthesis. First-stranded cDNA was synthesized by cDNA Synthesis Kit (Bio-Rad) with 1 μg of RNA and oligo dTTP according to the manufacturer's instructions. The equivalent amount of cDNA was subsequently amplified in 20 μl reaction using the 2x SYBR qPCR Master Mix (Kapa-Biosystems) and specific primers. The qPCR of all gene was performed using the following sense and antisense primers: sense, 5′-TGCCCCTGTGGTCAAAGTG-3′ antisense, 5′-GGTTCGGTTACCGTCCTGC-3′ for Keap-1, sense: 5′-TAGATGACCATGAGTCGCTTGC-3′ antisense: 5′-GCCAAACTTGCTCCATGTCC-3′ for Nrf-2 and sense 5′-AAGCCGAGAATGCTGAGTTCA-3′ and antisense 5′-GCCGTGTAGATATGGTACAAGGA-3′ for HO-1, sense 5′-AGAGAGTGCTCGTAGCAGGAT-3′ and antisense 5′-GTGGTGATAGAAAGCAAGGTCTT-3′ for NQO1. The cycling conditions for Keap-1, Nrf2, HO-1, NQO1 were as follows: one cycle at 95°C for 180s; 39 cycles at 95°C for 10s, and 58°C for 30s. The validation of PCR amplification was verified through the presence of a single peak during melting curve analyses (60.0°C–95.0°C, increment 0.5°C, for 0.05). Each RT-qPCR experiment was repeated three times. β-Actin was used as internal control.
Statistical comparisons between control and treatment mean values (± standard deviation) were analyzed using one-way ANOVA and Dunnett's t-test. The level of significance was considered as P < 0.05.
| Results|| |
Molecular docking and binding pattern analysis
The results of molecular docking of reference molecule with HO-1 [Figure 1]a and molecular docking of TAX with HO-1 [Figure 1]b revealed that TAX actively fitted at active site of HO-1 (3HOK) in the similar fashion of reference molecule. The molecule was positioned in such a way that C phenyl ring exhibit Π-Π interaction with Hem300. As Hem is the active catalytic part of HO enzyme, if any molecule interacts with heme, probably it could have the potent inhibitory activity. It also displayed the polar H Bonding with GLU32, GLY139, and ASP140 residues. We suppose these interactions determine the potent HO inhibitory effect of TAX. The results of molecular docking of reference molecule with Keap-1 [Figure 1]c and molecular docking of TAX with Keap-1 [Figure 1]d revealed that the TAX molecule get occupied at the active site of Keap-1 (PDB ID: 5FNQ) in the similar fashion of reference molecule. The phenyl C ring of TAX was positioned at the place of 4-chlorophenyl ring of reference molecule and displayed the interaction with ARG415. It also exhibit two polar H bond with LEU365 and VAL463, respectively. These displayed interactions gave the good docking score compared to reference molecule and explained the inhibitory effect of Keap-1 receptor. These overall contributions of ligand amino acid interactions displayed better docking score that the reference molecule. Molecular modeling studies, docking scores (which are more than reference molecules) suggested that these overall interactions with amino acid residues at active site determine the potent inhibitory effect of TAX and could be used against various types of cancer.
|Figure 1: Molecular docking analysis of reference molecule and TAX, Where (a) overlay of reference molecule at the active pocket of HO-1; (b) overlay of TAX at the active pocket of HO-1; (c) overlay of reference molecule at the active pocket of Keap-1; (d) overlay of TAX at the active pocket of Keap-1. TAX: Taxifolin; HO-1: Heme oxygenase 1; Keap-1: Kelch-like ECH-associated protein 1|
Click here to view
Taxifolin restored lipid peroxidation, enzymatic and nonenzymatic antioxidants in all treatment groups
Restorative potential of TAX on antioxidant marker enzymes were evaluated at 10, 20, and 40 mg/kg b.w. doses in DMBA-induced mammary cancer bearing animals. DMBA-induced animals revealed statistically significant (P< 0.005) elevation in LPO [Figure 2]a and a subsequent decrease (P< 0.005) in total SOD [Figure 2]b, MnSOD [Figure 2]c, and CuZnSOD [Figure 2]d. The subsequent significant (P< 0.005) decrease in enzymatic CAT [Figure 2]e, GPx [Figure 2]f and nonenzymatic GSH [Figure 2]g, ascorbic acid [Figure 2]h, α-tocopherol [Figure 2]i antioxidant markers in serum, kidney, liver, and mammary tissues was recorded as compared to control group of animals. TAX treatment restored these enzyme levels significantly (P< 0.05) in a dose-dependent manner as compared to induced cancer-bearing animals. Hence, TAX potentially prevents peroxidation of lipids and improved antioxidant profile in animals with mammary cancer.
|Figure 2: (A) Biochemical estimations of enzymatic and nonenzymatic antioxidants in all treatment groups. Where (a) estimation of LPO, (b) total SOD, (c) MnSOD, (d) CuZnSOD. Where each value is represented as mean ± SEM; n = 8 Group I: Normal control; Group II: Induced control group; Group III: TAX (10 mg/kg/day); Group IV: TAX (20 mg/kg/day) and Group V: TAX (40 mg/kg/day). Comparisons: a = Group II, III, IV, V with Group I; b = Group III, IV and V with Group II. Significant: NSP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. (B) Biochemical estimations of enzymatic and nonenzymatic antioxidants in all treatment groups. Where (e) CAT, (f) GPx, (g) GSH (h) ascorbic acid and (i) α-tocopherol, Where each value is represented as mean ± SEM; n = 8 Group I: Normal control; Group II: Induced control group; Group III: TAX (10 mg/kg/day); Group IV: TAX (20 mg/kg/day) and Group V: TAX (40 mg/kg/day). Comparisons: a = Group II, III, IV, V with Group I; b = Group III, IV and V with Group II. Significant: NSP > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001. TAX: Taxifolin; LPO: Lipid peroxidation; SOD: Superoxide dismutase; MnSOD: Manganese-containing SOD; CuZnSOD: Copper and zinc containing SOD; SEM: Standard error of mean; CAT: Catalase; GPx: Glutathione peroxidase; GSH: Reduced glutathione|
Click here to view
Taxifolin altered the expressions of nuclear factor erythroid 2 and Kelch-like ECH-associated protein 1 during immunofluorescence analysis of mammary carcinoma tissue
To determine the TAX mediated changes in cytoplasmic expression of Keap-1 and nuclear translocation of Nrf-2, Immunofluorescence assay was carried out. The results corroborate with earlier studies showing up-regulation of Keap-1 in cancer-bearing untreated animals which on treatment with TAX was down-regulated in a dose-dependent manner [Figure 3]a. Further, Nrf2 upregulation and increased nuclear translocation was observed after TAX treatment which is represented in [Figure 3]b. These results reveal that TAX causes alteration in the expression of Keap-1 and it was involved in the nuclear translocation of Nrf-2 at the tested dose (40 mg/kg b.w).
|Figure 3: Immunofluorescence analysis of Keap-1 and Nrf-2 in mammary tissue of all experimental animals. n = 8. DAPI: florescent-blue (nucleus); Cy3: red; Cy5: dark blue. (a) Degradation of Keap-1 after TAX treatment to DMBA induced mammary cancer tissue. Keap-1 degradation is represented in a closer view (small extended slides). For the analysis immunofluorescence staining (×160) was used. (b) TAX treated experimental groups revealed Keap-1 dependent Nrf2 nuclear translocation (×170). The translocation of Nrf2 from cytosol to nucleus is represented in a closer view (small extended slides). DMBA: 7,12-dimethylbenz (a) anthracene; TAX: Taxifolin; Keap-1: Kelch-like ECH-associated protein 1; Nrf2: Nuclear factor erythroid 2|
Click here to view
Taxifolin restored the mRNA expressions of Kelch-like ECH-associated protein 1, nuclear factor erythroid 2, heme oxygenase-1, and NADPH: quinoneoxidoreductase 1 in mammary carcinogenesis
To define the effect of TAX on Keap-1, Nrf-2, HO-1, and NQO1, mRNA analysis was carried out [Figure 4]. DMBA-induced group animals revealed mRNA over expression of Keap-1 signaling proteins (P< 00.5), while the expression of Nrf-2, HO-1 and NQO1 was significantly reduced as compared to control group. TAX treatment caused dose-dependent restoration of the mRNA levels. Group III (TAX 10 mg/kg/day) revealed significant (P< 0.05) decrease while group IV (TAX 20 mg/kg/day) and group V (TAX 40 mg/kg/day) showed highly significant (P< 0.005) reduction in Keap-1 mRNA expression in comparison to the induced control group. On the other hand, treatment with TAX significantly (P< 0.005) increased the RNA expressions of Nrf-2, HO-1 and NQO1 with respect to the DMBA induced cancer group of animals.
|Figure 4: Relative mRNA expression of Keap-1, Nrf-2, HO-1, NQO1. Where each value is represented as mean ± SEM; n = 8. Group I: Normal control; Group II: Induced control group; Group III: TAX (10 mg/kg/day); Group IV: TAX (20 mg/kg/day) and Group V: TAX (40 mg/kg/day). Comparisons: a = Group II, III, IV, V with Group I; b = Group III, IV and V with Group II. Significant: NSP > 0.05; *P < 0.05; ***P < 0.001. TAX: Taxifolin; Keap-1: Kelch-like ECH-associated protein 1; Nrf2: Nuclear factor erythroid 2; SEM: Standard error of mean; HO-1: Heme oxygenase 1; NQO1: NADPH: quinoneoxidoreductase 1|
Click here to view
| Discussion|| |
We used DMBA-induced rat mammary carcinogenesis for the investigation of the underlying mechanisms of various compounds behind their antioxidant and chemotherapeutic potentials because of its high human relevance. Many reactive intermediates (O2−, H2O2, OH) are produced by DMBA after its metabolism primarily binds to nucleophilic sites of cellular macromolecules potentiating oxidative stress leading to human breast carcinoma. The pro-oxidant-antioxidant balance is disrupted which impedes protective mechanisms of endogenous antioxidant causing pathophysiological and biochemical changes leading to loss of cellular integrity. This study was performed to evaluate the underlying molecular mechanism of Nrf-2-Keap-1-HO-1 and antioxidant pathways.
Endogenous metabolism of DMBA produces oxidative radicals causing serious damage to cellular components that triggers cancer-promoting signals. Primarily cellular and extracellular antioxidant enzymes prevent free radical-mediated mutagenesis or tumorigenesis. Increased production of reactive free radicals causes failure of available antioxidant defense leading to carcinogenesis. This could be correlated with progressive carcinogenesis as previous. Overproduction of free radical formation accounts for excessive peroxidation leading to increased formation MDA. This can be associated with proliferation and metastasis of breast cancer cells. Reduction of cellular SOD and CAT antioxidant activities is associated with increased level of superoxide radical. The clearance of xenobiotics, carcinogens, cellular peroxides is mainly mediated by GPx, and its depletion causes accumulation of intracellular peroxide in body potentiating polyunsaturated fatty acid degradation, cross-linking of lipids, proteins, and nucleic acid. Depression of GSH level in DMBA-induced animals might be associated with free radical-mediated increased utilization of and in protecting “SH” containing proteins from lipid peroxides. In our study also, MDA level was found to increase in DMBA treated animals which decreased on TAX treatment. On the other hand, the induced control groups revealed depression in various enzymatic and anti-enzymatic antioxidant levels which increased dose-dependently on TAX treatment which is also in accordance with other studies.
After cancer induction, various intermediate components increase ROS causing cancer progression. Nrf2 is a cytosolic, Keap-1 conjugated redox-sensitive transcription factors that evident to initiate ARE-mediated protective responses after oxidative stress initiated Keap-1-Nrf2 complex cleavage and further nuclear translocation of Nrf2. In carcinogenesis, upregulation of Keap-1 and simultaneous downregulation of Nrf-2 expression was evident that enhances the cellular susceptibility toward ROS  leading to further carcinogenesis progression. DMBA-treated animals revealed elevated Keap-1 levels inhibiting Nrf2 nuclear translocation and mediated antioxidant protect. The increased nuclear translocation of Nrf-2 after TAX treatment to mammary cancer-bearing animal further increased intracellular antioxidant levels which were reflected in the biochemical analysis of enzymatic and nonenzymatic antioxidants in all treatment groups.
The activation of Keap-1-Nrf2 pathway due to oxidative stress leads to the expression of cytoprotective enzymes, such as NQO1 and HO-1. Similarly, TAX-treated animals revealed increased mRNA expression of Nrf2 which resulted in increased mRNA expression of antioxidant proteins HO-1, NQO1 as represented in [Figure 5]. This proposes antioxidant promoting activity of TAX.
|Figure 5: Molecular docking analysis of reference molecule and TAX, Where (a) 2D interactions of reference with HO-1 (b) 2D interactions of TAX with HO-1; (c) 2D interactions of reference molecule with Keap-1 and (d) 2D interactions of TAX with Keap-1 (pose view). TAX: Taxifolin; HO-1: Heme oxygenase 1; Keap-1: Kelch-like ECH-associated protein 1; 2D: Two-dimensional|
Click here to view
| Conclusion|| |
TAX revealed a high binding affinity toward Keap-1, HO-1. Further TAX potentially down-regulate Keap-1 and subsequently up-regulated the expressions of Nrf-2 and associated HO-1 and NQO1 proteins in cancer-bearing animals. Thus, TAX efficiently increased the antioxidant competence in the cancer cells. These molecular changes are well reflected in the biochemical analysis of endogenous antioxidant profile that revealed a significant increase in protective enzymatic and nonenzymatic antioxidants after TAX treatment in cancer bearing animals. In brief, TAX can potentially inhibit mammary cancer growth in DMBA-induced human resembling mammary carcinoma by inducing antioxidant, apoptosis, and inhibiting cell survival and growth.
The authors would like to thank the Birla Institute of Technology for providing the necessary facilities for this work.
Financial support and sponsorship
The authors are grateful to the University Grant Commission (Basic Scientific Research [BSR]), Government of India for giving financial support for this work (UGC Grant number: No. F.736/2007 [BSR]).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Medina D. Mammary tumorigenesis in chemical carcinogen-treated mice. I. Incidence in BALB-c and C57BL mice. J Natl Cancer Inst 1974;53:213-21.
Lai H, Singh NP. Oral artemisinin prevents and delays the development of 7,12-dimethylbenz[a] anthracene (DMBA)-induced breast cancer in the rat. Cancer Lett 2006;231:43-8.
Elegbede JA, Elson CE, Qureshi A, Tanner MA, Gould MN. Inhibition of DMBA-induced mammary cancer by the monoterpene d-limonene. Carcinogenesis 1984;5:661-4.
Bhattacharyya SS, Paul S, Mandal SK, Banerjee A, Boujedaini N, Khuda-Bukhsh AR, et al.
A synthetic coumarin (4-methyl-7 hydroxy coumarin) has anti-cancer potentials against DMBA-induced skin cancer in mice. Eur J Pharmacol 2009;614:128-36.
Baird WM, Hooven LA, Mahadevan B. Carcinogenic polycyclic aromatic hydrocarbon-DNA adducts and mechanism of action. Environ Mol Mutagen 2005;45:106-14.
Mannan R, Arora R, Bhushan S, Sharma S, Bhasin T, Arora S, et al.
Bioprotective efficacy of erucin against 7,12-dimethylbenz(α) anthracene-induced microstructural changes in male wistar rats. Turk Patoloji Derg 2017;33:150-6.
Trombino AF, Near RI, Matulka RA, Yang S, Hafer LJ, Toselli PA, et al.
Expression of the aryl hydrocarbon receptor/transcription factor (AhR) and AhR-regulated CYP1 gene transcripts in a rat model of mammary tumorigenesis. Breast Cancer Res Treat 2000;63:117-31.
Nebert DW, Petersen DD, Fornace AJ Jr. Cellular responses to oxidative stress: The [Ah] gene battery as a paradigm. Environ Health Perspect 1990;88:13-25.
Rundle A, Tang D, Hibshoosh H, Estabrook A, Schnabel F, Cao W, et al.
The relationship between genetic damage from polycyclic aromatic hydrocarbons in breast tissue and breast cancer. Carcinogenesis 2000;21:1281-9.
Reczek CR, Chandel NS. ROS-dependent signal transduction. Curr Opin Cell Biol 2015;33:8-13.
Dhalla NS, Temsah RM, Netticadan T. Role of oxidative stress in cardiovascular diseases. J Hypertens 2000;18:655-73.
Narendhirakannan RT, Hannah MA. Oxidative stress and skin cancer: An overview. Indian J Clin Biochem 2013;28:110-5.
Wu KC, McDonald PR, Liu J, Klaassen CD. Screening of natural compounds as activators of the keap1-Nrf2 pathway. Planta Med 2014;80:97-104.
Kobayashi M, Li L, Iwamoto N, Nakajima-Takagi Y, Kaneko H, Nakayama Y, et al.
The antioxidant defense system keap1-Nrf2 comprises a multiple sensing mechanism for responding to a wide range of chemical compounds. Mol Cell Biol 2009;29:493-502.
Pattanayak SP, Mazumder PM. Therapeutic potential of Dendrophthoe falcata
(L.f) Ettingsh on 7,12-dimethylbenz(a)anthracene-induced mammary tumorigenesis in female rats: Effect on antioxidant system, lipid peroxidation, and hepatic marker enzymes. Comp Clin Pathol 2010;20:381-92.
Cerutti PA. Prooxidant states and tumor promotion. Science 1985;227:375-81.
Sun Y. Free radicals, antioxidant enzymes, and carcinogenesis. Free Radic Biol Med 1990;8:583-99.
Wilkinson J 4th
, Clapper ML. Detoxication enzymes and chemoprevention. Proc Soc Exp Biol Med 1997;216:192-200.
Rajadurai M, Stanely Mainzen Prince P. Preventive effect of naringin on lipid peroxides and antioxidants in isoproterenol-induced cardiotoxicity in wistar rats: Biochemical and histopathological evidences. Toxicology 2006;228:259-68.
Gönenç A, Erten D, Aslan S, Akinci M, Simşek B, Torun M, et al.
Lipid peroxidation and antioxidant status in blood and tissue of malignant breast tumor and benign breast disease. Cell Biol Int 2006;30:376-80.
Shen SC, Ko CH, Tseng SW, Tsai SH, Chen YC. Structurally related antitumor effects of flavanones in vitro
and in vivo
: Involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production. Toxicol Appl Pharmacol 2004;197:84-95.
Chen Y, Yang L, Lee TJ. Oroxylin A inhibition of lipopolysaccharide-induced iNOS and COX-2 gene expression via suppression of nuclear factor-kappaB activation. Biochem Pharmacol 2000;59:1445-57.
Chen YC, Shen SC, Lee WR, Hou WC, Yang LL, Lee TJ, et al.
Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages. J Cell Biochem 2001;82:537-48.
Chen YC, Shen SC, Lee WR, Lin HY, Ko CH, Shih CM, et al.
Wogonin and fisetin induction of apoptosis through activation of caspase 3 cascade and alternative expression of p21 protein in hepatocellular carcinoma cells SK-HEP-1. Arch Toxicol 2002;76:351-9.
Lin HY, Juan SH, Shen SC, Hsu FL, Chen YC. Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1. Biochem Pharmacol 2003;66:1821-32.
Arun B, Udayachander M, Meenakshi A 7,12-dimethylbenzanthracene induced mammary tumours in wistar rats by 'air pouch' technique – A new approach. Cancer Lett 1984;25:187-94.
Kumar A, Jha S, Pattanayak SP. Daphnetin ameliorates 7,12-dimethylbenz[a] anthracene-induced mammary carcinogenesis through Nrf-2-keap1 and NF-κB pathways. Biomed Pharmacother 2016;82:439-48.
Pattanayak SP, Sunita P, Mazumder PM. Restorative effect of Dendrophthoe falcata
(L.f.) Ettingsh on lipids, lipoproteins, and lipid-metabolizing enzymes in DMBA-induced mammary gland carcinogenesis in Wistar female rats. Comp Clin Pathol 2014;23:1013-22.
Lakshmi A, Subramanian SP. Tangeretin ameliorates oxidative stress in the renal tissues of rats with experimental breast cancer induced by 7,12-dimethylbenz[a] anthracene. Toxicol Lett 2014;229:333-48.
Sun X, Chen RC, Yang ZH, Sun GB, Wang M, Ma XJ, et al.
Taxifolin prevents diabetic cardiomyopathy in vivo
and in vitro
by inhibition of oxidative stress and cell apoptosis. Food Chem Toxicol 2014;63:221-32.
Haque MW, Pattanayak SP. Taxifolin inhibits 7,12-dimethylbenz(a)anthracene-induced breast carcinogenesis by regulating AhR/CYP1A1 signaling pathway. Pharmacogn Mag 2018;13:S749-55.
Mundhe NA, Kumar P, Ahmed S, Jamdade V, Mundhe S, Lahkar M, et al.
Nordihydroguaiaretic acid ameliorates cisplatin induced nephrotoxicity and potentiates its anti-tumor activity in DMBA induced breast cancer in female sprague-dawley rats. Int Immunopharmacol 2015;28:634-42.
Kumar A, Sunita P, Jha S, Pattanayak SP. 7,8-dihydroxycoumarin exerts antitumor potential on DMBA-induced mammary carcinogenesis by inhibiting ERα, PR, EGFR, and IGF1R: Involvement of MAPK1/2-JNK1/2-Akt pathway. J Physiol Biochem 2018. Available from: https://doi.org/10.1007/s13105-018-0608-2
Devasagayam TP, Boloor KK, Ramasarma T. Methods for estimating lipid peroxidation: An analysis of merits and demerits. Indian J Biochem Biophys 2003;40:300-8.
Stajn A, Zikić RV, Ognjanović B, Saicić ZS, Pavlović SZ, Kostić MM, et al.
Effect of cadmium and selenium on the antioxidant defense system in rat kidneys. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 1997;117:167-72.
Cand F, Verdetti J. Superoxide dismutase, glutathione peroxidase, catalase, and lipid peroxidation in the major organs of the aging rats. Free Radic Biol Med 1989;7:59-63.
Sinha AK. Colorimetric assay of catalase. Anal Biochem 1972;47:389-94.
Rotruck JT, Pope AL, Ganther HE, Swanson AB, Hafeman DG, Hoekstra WG, et al.
Selenium: Biochemical role as a component of glutathione peroxidase. Science 1973;179:588-90.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 1951;193:265-75.
Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70-7.
Anbuselvam C, Vijayavel K, Balasubramanian MP. Protective effect of Operculina turpethum
against 7,12-dimethyl benz(a)anthracene induced oxidative stress with reference to breast cancer in experimental rats. Chem Biol Interact 2007;168:229-36.
Batcioglu K, Uyumlu AB, Satilmis B, Yildirim B, Yucel N, Demirtas H, et al.
Oxidative stress in the in vivo
DMBA rat model of breast cancer: Suppression by a voltage-gated sodium channel inhibitor (RS100642). Basic Clin Pharmacol Toxicol 2012;111:137-41.
Khanzode SS, Muddeshwar MG, Khanzode SD, Dakhale GN. Antioxidant enzymes and lipid peroxidation in different stages of breast cancer. Free Radic Res 2004;38:81-5.
Tanigawa S, Fujii M, Hou DX. Action of Nrf2 and keap1 in ARE-mediated NQO1 expression by quercetin. Free Radic Biol Med 2007;42:1690-703.
Taguchi K, Yamamoto M. The KEAP1-NRF2 system in cancer. Front Oncol 2017;7:85.
Li L, Dong H, Song E, Xu X, Liu L, Song Y, et al.
Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/AKT signaling. Chem Biol Interact 2014;209:56-67.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
|This article has been cited by|
||LC-MS characterized methanolic extract of zanthoxylum armatum possess anti-breast cancer activity through Nrf2-Keap1 pathway: An in-silico, in-vitro and in-vivo evaluation
| ||Roja Sahu, Rajiv Kumar Kar, Priyashree Sunita, Pritha Bose, Puja Kumari, Salona Bharti, Sharad Srivastava, Shakti P. Pattanayak |
| ||Journal of Ethnopharmacology. 2021; 269: 113758 |
|[Pubmed] | [DOI]|
||Decreasing glycerol content by co-supplementation of trehalose and taxifolin hydrate in ram semen extender: Microscopic, oxidative stress, and gene expression analyses
| ||Mustafa Numan Bucak, Nazan Keskin, Pinar Ili, Mustafa Bodu, Pinar Peker Akalin, Ali Erdem Öztürk, Hüseyin Özkan, Tohid Rezaei Topraggaleh, Fikret Sari, Nuri Baspinar, Sükrü Dursun |
| ||Cryobiology. 2020; 96: 19 |
|[Pubmed] | [DOI]|