Baicalein Inhibits TNF-Α-Induced NF-Κb Activation and Expression of NF-Κb-Regulated Target Gene Products

ONCOLOGY REPORTS 36: 2771-2776, 2016 Baicalein inhibits TNF-α-induced NF-κB activation and expression of NF-κB-regulated target gene products JUNBO LI, JUAN MA, KE SI WANG, CHUNLIU MI, ZHE WANG, LIAN XUN PIAO, GUANG HUA XU, XUEZHENG LI, JUNG JOON LEE and XUEJUN JIN Key Laboratory of Natural Resources of Changbai Mountain and Functional Molecules, Ministry of Education, Molecular Cancer Research Center, College of Pharmacy, Yanbian University, Yanji, Jilin 133002, P.R. China Received April 19, 2016; Accepted August 17, 2016 DOI: 10.3892/or.2016.5108 Abstract. The nuclear factor-κB (NF-κB) transcription factors Introduction control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB The transcription factor NF-κB was discovered in 1986 as a inhibitors from natural resources, we identified baicalein from nuclear factor that binds to the enhancer element of the immu- Scutellaria baicalensis as an inhibitor of NF-κB activation. noglobulin kappa light-chain of activated B cells (thereby As examined by the NF-κB luciferase reporter assay, we found coining the abbreviation NF-κB) (1). NF-κB represents a family that baicalein suppressed TNF-α-induced NF-κB activation of eukaryotic transcription factors participating in the regula- in a dose-dependent manner. It also inhibited TNF-α-induced tion of various cellular genes involved in the immediate early nuclear translocation of p65 through inhibition of phosphoryla- processes of immune, acute phase and inflammatory responses tion and degradation of IκBα. Furthermore, baicalein blocked as well as genes involved in cell survival (2). A commonly the TNF-α-induced expression of NF-κB target genes involved known NF-κB consists of a RelA (p65)/p50 heterodimer and in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), prolif- RelA (p65) contains a C-terminal transactivation domain eration (COX-2, cyclin D1 and c-Myc), invasion (MMP-9), in addition to the N-terminal Rel-homology domain, thus, angiogenesis (VEGF) and major inflammatory cytokines serving as a critical transactivation subunit of NF-κB (3). In the (IL-8 and MCP1). The flow cytometric analysis indicated that resting state, the inactive NF-κB is retained in the cytoplasm baicalein potentiated TNF-α-induced apoptosis and induced by an inhibitory subunit called IκB. The phosphorylation of G1 phase arrest in HeLa cells. Moreover, baicalein signifi- IκB by the IκB-kinase (IKK) containing IKKα, IKKβ and cantly blocked activation of p38, extracellular signal-regulated the regulatory protein NF-κB essential modifier (NEMO) is kinase 1/2 (ERK1/2). Our results imply that baicalein could be a key step in NF-κB activation in response to various stimuli a lead compound for the modulation of inflammatory diseases such as tumor necrosis factor-α (TNF-α) (3,4). In response to as well as certain cancers in which inhibition of NF-κB activity stimulation, IκBs are rapidly ubiquitinated and degraded by may be desirable. 26S proteasome complex and the release of IκB unmasks the nuclear localization signal and results in the translocation of NF-κB to the nucleus where it can bind to κB sites, followed by the activation of specific target genes (5). Correspondence to: Professor Xuejun Jin, Key Laboratory of It is reported that NF-κB regulates several hundreds of Natural Resources of Changbai Mountain and Functional Molecules, genes, including those involved in immunity and inflamma- Ministry of Education, Molecular Cancer Research Center, College tion, anti-apoptosis, cell proliferation, tumorigenesis and the of Pharmacy, Yanbian University, Yanji, Jilin 133002, P.R. China negative feedback of the NF-κB signal (6). NF-κB regulates E-mail: [email protected] major inflammatory cytokines, including interleukin 8 (IL-8), monocyte chemotactic protein 1 (MCP1), many of which Abbreviations: NF-κB, nuclear factor-κB; TNF-α, tumor necrosis are potent activators for NF-κB. NF-κB has been shown to factor-α; IκBα, inhibitor of NF-κB alpha; IL-8, interleukin 8; cIAP1, cellular inhibitor of apoptosis protein 1; cIAP2, cellular inhibitor of regulate the expression of several genes whose products are apoptosis protein 2; FLIP, cellular FLICE inhibitory protein; BCL-2, involved in tumorigenesis (2), including cyclooxygenase-2 B-cell lymphoma-2; MCP1, monocyte chemotactic protein 1; (COX-2), cyclin D1, c-Myc, apoptosis suppressor proteins such COX-2, cyclooxygenase-2; MMP-9, matrix metalloproteinase-9; as cellular inhibitor of apoptosis 1 (cIAP-1), cellular inhibitor VEGF, vascular endothelial growth factor; c-Myc, cellular-myelo- of apoptosis 2 (cIAP-2), cellular FLICE inhibitory protein cytomatosis viral oncogene (FLIP), B-cell lymphoma-2 (BCL-2) and genes required for invasion and angiogenesis such as matrix metalloproteinase Key words: baicalein, nuclear factor-κB, IκBα, inflammation, (MMP-9) and vascular endothelial growth factor (VEGF). cancer Baicalein is a naturally occurring flavonoid which is an active component of Scutellaria baicalensis (7). Scutellaria baicalensis is one of the most popular traditional Chinese 2772 LI et al: INHibition of NF-κB by baicalein medicine herbal remedies used in China and several oriental countries for treatment of inflammation, bacterial and viral infections, and have been shown to possess anticancer activities in vitro and in vivo in mouse tumor models (8). Previous investigations also showed that baicalein have multiple pharmacological activities including anti-oxidant effects, chemo-preventive effects against several types of cancer and anti-inflammatory effect (9-11). However, the molecular mechanism of anti-inflammatory and anticancer effects has not been sufficiently explained. In the present study, whether baicalein exerts its anti-inflammatory and anticancer effects through suppression of the NF-κB pathway was investigated. Our data demonstrated baicalein downregulates the expression of target genes involved in antiapoptosis (cIAP-1, cIAP-2, BCL-2 and FLIP), proliferation (cyclin D1, COX-2 and c-Myc), invasion (MMP-9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). Taken together, these findings support further studies of baicalein as candidate for treatment of inflammation and cancer. Materials and methods Cell culture and reagents. HeLa cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS; (Gibco, Grand Island, NY, USA) and 1% penicillin/strepto- mycin (Invitrogen, Carlsbad, CA, USA) at 37˚C with 5% CO2 atmosphere in a humidified incubator. TNF-α was obtained from R&D Systems (Minneapolis, MN, USA). Baicalein was Figure 1. Effect of baicalein (Bcl) on the TNF-α-induced NF-κB-dependent reporter gene expression. (A) Structure of baicalein (Bcl). (B) HeLa cells were from Sigma-Aldrich (St. Louis, MO, USA) and its structure transiently transfected with an NF-κB-dependent reporter gene for 24 h and is shown in Fig. 1A. The purity of baicalein was over 99% in then pretreated for 6 h with the indicated concentrations of baicalein (Bcl) HPLC analysis. followed by stimulation for 6 h with TNF-α (10 ng/ml), and the luciferase activity was determined as described in Materials and methods. Data are presented as mean ± standard deviation of three independent experiments. MTT assay. HeLa cells were seeded in 96-well plates at a **P<0.01, ***P<0.001, significantly different when compared with TNF-α- 5 density of 1x10 cells/ml and cultured overnight. Following stimulated normal cells. (C) HeLa cells were treated with the indicated cell treatment with different concentrations of baicalein concentrations of baicalein (Bcl). After 12-h incubation, cell viability was (10-100 µM) for 12 h, 10 µl MTT solution (5 mg/ml) was added determined by MTT assays. Data are presented as mean ± standard deviation of three independent experiments. into each well and incubated with cells for 4 h at 37˚C. Then, DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was measured by Multiskan GO. Western blot analysis. HeLa cells were cultured in Plasmids, transfections and luciferase reporter assay. A 10 cm-dishes and allowed to adhere for 24 h. After treat- pNF-κB-Luc plasmid for NF-κB luciferase reporter assay was ment with various concentrations of baicalein in the presence obtained from Strategene (La Jolla, CA, USA). Transfections or absence of TNF-α (10 ng/ml), then, cells were harvested were performed as previously described (12). NF-κB-dependent and lysed. An equal amount of protein was separated by luciferase activity was measured using the Dual-luciferase SDS-polyacrylamide gels and transferred to polyvinylidene reporter assay system. Briefly, HeLa cells (1x105 cells/well) fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). were seeded in a 96-well plate for 24 h. The cells were then The membrane was blocked with 5% non-fat dried milk for transfected with plasmids for each well and then incubated for a 1 h, the membrane was incubated with the primary antibodies. transfection period of 24 h. After that, the cell culture medium Antibodies for IκBα, phosphor (Ser32)-specific κI Bα, p65, was removed and replaced with fresh medium containing PARP, caspase-8, cIAP-1, cIAP-2, phospho-ERK, phospho- various concentrations of baicalein for 6 h, followed by treat- JNK, phospho-p38, ERK, JNK and P38 were purchased from ment with 10 ng/ml of TNF-α for 6 h. Luciferase activity Cell Signaling Technology (Beverly, MA, USA). Antibodies was determined in MicroLumat plus luminometer (EG&G for COX-2, MMP-9, VEGF, BCL-2, FLIP, and Topo-Ι were Berthold, Bad Wildbad, Germany) by injecting 100 µl of assay obtained from Santa Cruz Biotechnology (Santa Cruz, CA, buffer containing luciferin and measuring light emission for USA). Antibody for α-tubulin was from Sigma-Aldrich. After 10 sec. Co-transfection with pRL-CMV (Promega, Madison, binding of an appropriate secondary antibody coupled to WI, USA), which expresses Renilla luciferase, was performed horseradish peroxidase.

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