Roles of JNK/Nrf2 Pathway on Hemin-Induced Heme Oxygenase-1 Activation in MCF-7 Human Breast Cancer Cells

Heme oxygenase-1 (HO-1) is highly induced in various human disease states, including cancer, indicating that HO-1 is an emerging target of cancer therapy. In this study, we investigated that the mechanisms of hemin-induced HO-1 expression and its signaling pathways in human breast cancer cell. We used MCF-7 cells, a human breast cancer cell line. Hemin increased HO-1 expression in MCF-7 cells in a dose- and time-dependent manner. Hemin enhanced HO-1 expression through the activation of c-Jun N-terminal kinases (JNK) signaling pathway. Hemin also induced activation of Nrf2, a major transcription factor of HO-1 expression. These responses in MCF-7 cells were completely blocked by pretreatment with brazilin, a HO-1 regulator. These results indicated that brazilin inhibits hemin-induced HO-1 expressions through inactivation of JNK/Nrf2 in MCF-7 cells. Thus, our findings suggest that HO-1 is an important anticancer-target of brazilin in human breast cancer.


Introduction
Heme oxygenase-1 (HO-1) was identified by Maines [1], as a liver microsomal protein with degradation activity of heme to bilirubin. HO-1 catalyzes the degradation of heme to form the open-chain tetrapyrrole biliverdin-Ixα, carbon monoxide (CO), and free iron, which play crucial roles in the adaptation to defense against oxidative stress and cellular stress [2,3]. HO-1 is induced in cells by cellular stress including oxidants [4], hypoxia condition [5], cytokine [6,7], and ultraviolet light irradiation [5,8]. These findings suggest that HO-1 has cytoprotective actions. Therefore, studies focused on HO-1 to find potential managements for various diseases, including cancer [9,10], inflammatory diseases [11], respiratory disease [12], and circulation system [13,14]. It is known that expression of high level of HO-1 occurs in various tumors including in renal cancer [15], acute T cell leukemia [16], prostate cancer [17], lung [18], and squamous cell carcinoma in head and neck [19]. Many studies suggested the HO-1 were implicated in tumorigenesis and cancer prognosis like chemotherapeutic sensitivity [20], cancer invasion [21,22], aggressiveness [23,24] and survival rate prediction [25]. Taken together, these findings indicate that the investigating of the role of HO-1 and its targets with molecular mechanism for cancer therapy seem to be important [26].

Cell Viability Assay
The cell viability (and/or growth rate) of MCF-7 was assayed using an MTT assay. Briefly, cells were inoculated in a microtiter plate 96 well (3 × 10 3 cells/well, confluent less than 60%), and then incubated at CO 2 incubator for 12 h. Cells were either untreated or treated with 2.5, 5, 10, 20, 50 µM brazilin and 25, 50, 100 µM hemin at 37 • C and further incubated for 24 h. The cells were washed with PBS prior to the addition of 0.5 mg/mL MTT (100 µL per well), and then incubated at 37 • C for 30 min. Insoluble formazan were solubilized with DMSO (100 µL per well) and measured at 570 nm using a microplate reader (Model 3550, Bio-Rad, Hercules, CA, USA).

Western Blot Analysis
MCF-7 cells (7.0 × 10 5 cells/sample) were treated with 2.5, 5, 10, 20 µM brazilin for 60 min, and then incubated with 50 µM hemin at 37 • C for 24 h. Cells were lysed with commercial ice-cold lysis reagents (M-PER Mammalian Protein Extraction Reagent, Pierce Biotechnology, Rockford, IL, USA). Protein extracts (10 µg per well) were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Membrane was blocked for 1 to 2 h with 5% skim milk or 3% immunoglobulin-free bovine serum albumin (BSA), and then incubated 15 h at 4 • C with 1.0 µg/mL of primary antibody (1:1000 to 2000). Horseradish peroxidase-conjugated anti-IgG monoclonal antibody (1:1000 to 2000) was used as the secondary antibody. Specific band were analyzed with LAS-1000 (Fuji Film, Tokyo, Japan). The images were quantified using a computer image analysis software, Image Reader Pro (Fuji Film, Tokyo, Japan) and ImageJ (National Institutes of Health, Bethesda, MD, USA).

Quantitative Real-Time Polymerase Chain Reaction
Total RNA was isolated using the commercial kits (FastPure TM RNA Kit, Takara Bio Inc., Kusatsu, Shiga Japan). The RNA concentration and purities were determined by absorbance at 260/280 nm. cDNA was synthesized from 1 µg total RNA using a PrimeScript TM RT reagent Kit (Takara, Japan). HO-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA expression were determined by real-time qPCR using the sequence detection system (ABI PRISM 7900, Applied Biosystems, Waltham, MA, USA), and SYBR Green. The primers were used to amplify cDNA of HO-1 (NM002133) (sense: 5 -GCC AGC AAC AAA GTG CAA G-3 ; antisense: 5 -GGC ATA AAG CCC TAC AGC AA-3 ) and GAPDH (NM002046) (sense: 5 -ATG GAA ATC CCA TCA CCA TCT T; antisense: CGC CCC ACT TGA TTT TGG-3 ) (Genotech, Daejeon, Korea). The variation in mRNA concentration of all genes were normalized to the GAPDH housekeeping gene. Relative quantitation was performed using the comparative Ct method according to the manufacturer's instructions. Data were presented the mean ± S.E.M of three independent experiments.

Isolation Extracts of Nuclear Fraction
MCF-7 cells (2 × 10 6 cells/sample) were treated with brazilin in the presence or absence of hemin (50 µM) for 4 h. Cells were washed twice and carefully detached by scrapping into 1.5 mL of ice-cold PBS (pH 7.5) and then pelleted (4000× g at 4 • C for 5 min). Cytoplasmic and nuclear fraction were extracted from cells using commercial kit (NE-PER)(Pierce Biotechnology, IL, USA).

Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extract of cells was prepared as described above. An oligonucleotide containing the Nrf2 (5 -TGG GGA ACC TGT GCT GAG TCA CTG GAG-3 ) were purchased from Genotech (Daejeon, Korea). Electrophoretic mobility shift assay was performed as described in Hellman and Fried [37]. Specific binding was controlled by competition with a 50-fold excess of cold Nrf2 oligonucleotide.

Statistical Analysis
All tests were performed at least in three independent experiments. To compare means within groups we performed student t-test. Values of p < 0.05 were considered statistically significant.

Endogenous Cytotoxicity of Brazilin and Hemin on MCF-7 Cells
To examine the endogenous cytotoxicity of brazilin ( Figure 1A) and hemin ( Figure 1B) on MCF-7 cells, we treated cells with brazilin (0-50 µM) and hemin (0-100 µM) for 24 h. The influence of brazilin and hemin on MCF-7 cellular toxicity was then analyzed using MTT assay. Treatment of MCF-7 cells with indicated dosage of brazilin and hemin shown no significant change in cell viability (Figure 2A,B). Therefore, we performed further experiments in a non-toxic dose of brazilin and hemin.

Effect of Hemin on HO-1 Expression in MCF-7 Cells
To investigate the effect of hemin on HO-1 expression in MCF-7 cells, we performed Western blotting. Western blot analysis revealed that hemin increased HO-1 expression in a dose-and timedependent manner ( Figure 3A,B).

Effect of Hemin on HO-1 Expression in MCF-7 Cells
To investigate the effect of hemin on HO-1 expression in MCF-7 cells, we performed Western blotting. Western blot analysis revealed that hemin increased HO-1 expression in a dose-and timedependent manner ( Figure 3A,B).

Effect of Hemin on HO-1 Expression in MCF-7 Cells
To investigate the effect of hemin on HO-1 expression in MCF-7 cells, we performed Western blotting. Western blot analysis revealed that hemin increased HO-1 expression in a dose-and time-dependent manner ( Figure 3A,B).

Effect of Hemin on HO-1 Expression in MCF-7 Cells
To investigate the effect of hemin on HO-1 expression in MCF-7 cells, we performed Western blotting. Western blot analysis revealed that hemin increased HO-1 expression in a dose-and timedependent manner ( Figure 3A,B).

Effect of Brazilin on Hemin-Induced HO-1 Expression in MCF-7 Cells
To investigate the effect of brazilin on hemin-induced HO-1 expression in MCF-7 cells, we performed Western blotting and real-time PCR. Western blot analysis and real-time PCR revealed that brazilin-suppressed hemin-induced HO-1 expression in a dose-dependent manner ( Figure 4A,B).

Effect of Brazilin on Hemin-Induced HO-1 Expression in MCF-7 Cells
To investigate the effect of brazilin on hemin-induced HO-1 expression in MCF-7 cells, we performed Western blotting and real-time PCR. Western blot analysis and real-time PCR revealed that brazilin-suppressed hemin-induced HO-1 expression in a dose-dependent manner ( Figure 4A,B).

Effects of Hemin on MAPK Activation in MCF-7 Cells
The effect of hemin on JNK/ERK/p38 activities was investigated using Western blotting. MCF-7 cells pretreated with inhibitors of ERK (PD98059), JNK (SP600125), and p38 (SB203580) for 1 h were further incubated with hemin (50 μM) for 24 h. As shown in Figure 5A,B, inhibition of JNK only blocked hemin-induced HO-1 protein expression in MCF-7 cells. These results suggest that hemininduced HO-1 expression were mediated by JNK signal pathway.

Effects of Hemin on MAPK Activation in MCF-7 Cells
The effect of hemin on JNK/ERK/p38 activities was investigated using Western blotting. MCF-7 cells pretreated with inhibitors of ERK (PD98059), JNK (SP600125), and p38 (SB203580) for 1 h were further incubated with hemin (50 µM) for 24 h. As shown in Figure 5A,B, inhibition of JNK only blocked hemin-induced HO-1 protein expression in MCF-7 cells. These results suggest that hemin-induced HO-1 expression were mediated by JNK signal pathway.

Effects of Brazilin on Hemin-Induced JNK Activation in MCF-7 Cells
To investigate the effect of brazilin on hemin-induced JNK activation, we performed Western blotting. As shown in Figure 6A-D, hemin significantly induced phosphorylation of JNK only.

Effects of Brazilin on Hemin-Induced JNK Activation in MCF-7 Cells
To investigate the effect of brazilin on hemin-induced JNK activation, we performed Western blotting. As shown in Figure 6A-D, hemin significantly induced phosphorylation of JNK only. Interestingly, brazilin also only significantly blocked phosphorylation of JNK by hemin in MCF-7 cells ( Figure 6A,D).

Effects of Brazilin on Hemin-Induced JNK Activation in MCF-7 Cells
To investigate the effect of brazilin on hemin-induced JNK activation, we performed Western blotting. As shown in Figure 6A-D, hemin significantly induced phosphorylation of JNK only. Interestingly, brazilin also only significantly blocked phosphorylation of JNK by hemin in MCF-7 cells (Figure 6A,D).

Effect of Brazilin on Hemin-Induced Nrf2 Activation in MCF-7 Cells
To further understand the inhibitory mechanism of brazilin on HO-1 transcriptional regulation, we determined whether brazilin inhibits Nrf2 activation in MCF-7 cell after stimulation with hemin using Western blotting and EMSA. Brazilin caused inhibitory actions on nuclear translocation of Nrf2 ( Figure 7A-C). Furthermore, DNA binding activity of hemin-induced Nrf2 was markedly inhibited by treatment of brazilin in a dose-dependent manner ( Figure 8A,B). These results indicate that Nrf2 nuclear translocation is an important mechanism on hemin-induced HO-1 expression and that brazilin regulates Nrf2 translocation.
we determined whether brazilin inhibits Nrf2 activation in MCF-7 cell after stimulation with hemin using Western blotting and EMSA. Brazilin caused inhibitory actions on nuclear translocation of Nrf2 ( Figure 7A-C). Furthermore, DNA binding activity of hemin-induced Nrf2 was markedly inhibited by treatment of brazilin in a dose-dependent manner (Figure 8A,B). These results indicate that Nrf2 nuclear translocation is an important mechanism on hemin-induced HO-1 expression and that brazilin regulates Nrf2 translocation.   using Western blotting and EMSA. Brazilin caused inhibitory actions on nuclear translocation of Nrf2 ( Figure 7A-C). Furthermore, DNA binding activity of hemin-induced Nrf2 was markedly inhibited by treatment of brazilin in a dose-dependent manner ( Figure 8A,B). These results indicate that Nrf2 nuclear translocation is an important mechanism on hemin-induced HO-1 expression and that brazilin regulates Nrf2 translocation.

Discussion
This study provides evidence that brazilin, a major component of Caesalpinia sappan, blocks hemin-induced HO-1 expression in MCF-7 cells and JNK/Nrf2-mediated HO-1 expression. Thus, HO-1 may be an important target in the screening of novel pharmaceuticals to treatment of breast cancer. In human cells, two isoforms of HO are present. HO-1 is a stress-inducible enzyme, while HO-2 is a constitutive enzyme as genetically and enzymatically [38]. Hemin induces the expression of HO-1 in a variety of cell types including endothelium [39], aortic tissues [40], embryonic fibroblast and adipocytes [41].
In this study, hemin increased HO-1 expression in MCF-7 cells in a dose-and time-dependent manner, and brazilin substantially inhibited hemin-induced HO-1 expression in human breast cancer cells. It has been known that MAPK and ROS control HO-1 expression through Nrf2 activation in various cells such as pulmonary aortic endothelial cells (PAEC) [42], hepatic and hematopoietic system [43], and hepatic carcinoma cells [44]. Indeed, increases of HO-1 expression were supported anti-apoptotic effects in monocyte [45], tumor necrosis [46], then many trials were suggested with previously reported inhibitors [45]. Therefore, this study reveals that an increase in the level of hemin-induced HO-1 markedly reduces the signaling mechanism of MCH-7 cells by brazilin.
In this study, hemin increased HO-1 expression (protein and mRNA level) in MCF-7 cells in a doseand time-dependent manner. Among MAPK signaling molecules, hemin induced only JNK activation in MCF-7 cells. Our results also showed that hemin induces HO-1 expression through Nrf2 activation in MCF-7 cells. Interestingly, these responses were completely blocked by pretreatment with brazilin, indicating that brazilin inhibits hemin-induced HO-1 expressions through inactivation of JNK/Nrf2 in MCF-7 cells. In cancer cells, elevation of HO-1 expression increased in various tumors [15][16][17][18][19] and hemin, an iron-containing porphyrin with chlorine, is available HO-1 inducer in various cell types including normal and cancer cells [47,48]. In this study, we observed hemin-induced increases of HO-1 expression in MCF-7 cells and brazilin is potent regulator through HO-1 expression. On the other hand, we investigated whether brazilin-induced HO-1 expression, but it was not observed (data not shown). Moreover, novel HO-1 inhibition tools were reported such as arylethanolimidazole derivatives in cancer [49], caffeic acid phenethyl ester in diabetes [50] and fumarate hydratase as target genes in cancer [51].
Taken together, these data and previous publications indicates that hemin induced HO-1expression and JNK/Nrf2 signaling pathway plays a regulatory role in MCF-7 cells indicates that cytoprotective role in MCF cells. Therefore, blockage effects of brazilin against hemin-induced HO-1 expression in MCF-7 cells suggest useful therapeutic/prevention implication. However, the brazilin growth-inhibitory effect on human breast cancer cells still need to be studied.
This study was concluded that HO-1 is an important anti-cancer target of brazilin in human breast cancer via inhibit hemin-induced HO-1 expression through inactivation of JNK/Nrf2 in MCF-7 cells [52]. Therefore, brazilin or other HO-1 regulators may be of use in the treatment breast cancer and required further studies.