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/streptomycin (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 from Sigma-Aldrich (St. Louis, MO, USA) and its structure is shown in Fig. 1A. The purity of baicalein was over 99% in HPLC analysis.

HeLa cells were seeded in 96-well plates at a density of 1×10⁵ cells/ml and cultured overnight. Following cell treatment with different concentrations of baicalein (10–100 µM) for 12 h, 10 µl MTT solution (5 mg/ml) was added 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.

A pNF-κB-Luc plasmid for NF-κB luciferase reporter assay was obtained from Strategene (La Jolla, CA, USA). Transfections were performed as previously described (12). NF-κB-dependent luciferase activity was measured using the Dual-luciferase reporter assay system. Briefly, HeLa cells (1×10⁵ cells/well) were seeded in a 96-well plate for 24 h. The cells were then transfected with plasmids for each well and then incubated for a transfection period of 24 h. After that, the cell culture medium was removed and replaced with fresh medium containing various concentrations of baicalein for 6 h, followed by treatment with 10 ng/ml of TNF-α for 6 h. Luciferase activity was determined in MicroLumat plus luminometer (EG&G Berthold, Bad Wildbad, Germany) by injecting 100 µl of assay buffer containing luciferin and measuring light emission for 10 sec. Co-transfection with pRL-CMV (Promega, Madison, WI, USA), which expresses Renilla luciferase, was performed to enable normalization of data for transfection efficiency.

HeLa cells were cultured in 10 cm-dishes and allowed to adhere for 24 h. After treatment with various concentrations of baicalein in the presence or absence of TNF-α (10 ng/ml), then, cells were harvested and lysed. An equal amount of protein was separated by SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat dried milk for 1 h, the membrane was incubated with the primary antibodies. Antibodies for IκBα, phosphor (Ser32)-specific IκBα, p65, PARP, caspase-8, cIAP-1, cIAP-2, phospho-ERK, phospho-JNK, phospho-p38, ERK, JNK and P38 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies for COX-2, MMP-9, VEGF, BCL-2, FLIP, and Topo-I were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibody for α-tubulin was from Sigma-Aldrich. After binding of an appropriate secondary antibody coupled to horseradish peroxidase. Then the immunoreactive bands were visualized by enhanced chemiluminescence according to the manufacturer's instructions (Amersham Pharmacia Biotech, Buckinghamshire, UK).

HeLa cells were seeded into 24-well plates at 1×10⁴ cells/well. Twenty-four hours later, cells were pretreated with baicalein (100 µM) for 12 h, followed by treatment with TNF-α (10 ng/ml) for 30 min. Cells pretreated with DMSO and TNF-α (10 ng/ml) alone were used as negative and positive controls, respectively. Subsequently, the cells were washed in PBS, fixed at room temperature with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100. Immunofluorescence staining was performed according to the standard procedures. Briefly, the treated cells were first stained with the anti-p65 antibody followed by incubation with FITC conjugated anti-rabbit IgG secondary antibody and nuclei were counterstained with DAPI. The staining was examined using a fluorescence microscope.

Apoptosis assays were performed as previously described (13). Annexin V-staining was performed using Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) following the instructions of the manufacturer. Briefly, after incubation, detached cells were collected with the supernatant, pelleted by centrifugation. The adherent cells were rinsed twice with medium before harvesting. Then cells were harvested in trypsin without EDTA. Detached and adherent cells were finally pooled and were resuspended in binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) to a final concentration of 1×l0⁵ cells/ml. The pooled cells were stained with Annexin V-FITC and 2 µg/ml propidium iodide for 15 min at 37°C in the dark. To the samples was added 400 µl binding buffer before analyzed by flow cytometry. The CellQuest software was used to analyze the data (Becton-Dickinson, Franklin Lakes, NJ, USA).

Reverse transcription-PCR (RT-PCR) was performed to determine NF-κB target gene expression as previously described (14). In brief, HeLa cells were preincubated with the indicated concentrations of baicalein at 37°C for 12 h and then followed by treatment with 10 ng/ml of TNF-α for 12 h. Cells were harvested and washed twice with ice-cold PBS, and then total RNA was isolated from cells using RNeasy Mini kits according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). Complementary DNA was synthesized from 1 µg of total RNA in a 20 µl reverse transcription reaction mixture according to the manufacturer's protocol (Takara Bio, Kyoto, Japan). The PCR primers for interleukin-8 (IL-8), 5′-TCTGCAGCTCTGTGTGAAGG-3′ and 5′-ACTTCTCCACAACCCTCTG-3′; for MCP1, 5′-CCCCAGTCACCTGCTGTTAT-3′ and 5′-AGATCTCCTTGGCCACAATG-3′; for c-Myc, 5′-CTCTCAACGACAGCAGCCCG-3′ and 5′-CCAGTCTCAGACCTAGTGGA-3′; for GAPDH, 5′-ACCAGGTGGTCTCCTCT-3′ and 5′-TGCTGTAGCCAAATTCGTTG-3′. The mRNA levels of all genes were normalized to that of GAPDH. PCR products were separated on 3% agarose gel and then stained with ethidium bromide. Stained bands were visualized under UV light and photographed.

HeLa cells were cultured in 6-well plates until 70–80% confluent. The cells were then treated with baicalein at indicated concentrations in serum-free medium. Cells were then washed with PBS, fixed in ice-cold 70% ethanol and stained with PI buffer (0.1% Triton X-100, 0.2 mg/ml RNaseA, and 0.05 mg/ml PI) for 30 min. The DNA content was measured using a FACSCalibur flow cytometer with CellQuest software (Becton-Dickinson). For all assays, 10,000 events were counted. The ModFit LT v4.0 software package (Verity Software House, Inc., Topsham, ME, USA) was used to analyze the data.

All values are expressed as mean ± SD. A comparison of the results was performed with one-way ANOVA and Tukey's multiple comparison tests (GraphPad Software, Inc., San Diego, CA, USA) and the Student's t-test. P-values of <0.05 were considered statistically significant.

We first investigated the effect of baicalein on TNF-α-induced NF-κB activation by NF-κB-dependent reporter gene assay. HeLa cells were transiently transfected with the NF-κB-regulated luciferase reporter vector. When the HeLa cells were pretreated with various concentration of baicalein, TNF-α-induced NF-κB-reporter gene expression was inhibited in a dose-dependent manner (Fig. 1B). We evaluated the cytotoxic effects of baicalein on HeLa cell survival by MTT assay. The results showed that up to 100 µM of baicalein had no cellular toxicity on HeLa cells (Fig. 1C).

Transcriptional activity of NF-κB is dependent on IκBα phosphorylation. To determine whether baicalein inhibition of TNF-α-induced NF-κB activation, total cell extracts were prepared with baicalein and then exposed to TNF-α for various time periods, phosphorylation and degradation of IκBα was analyzed by western blot analysis. The results showed that baicalein potently inhibited the TNF-α-induced phosphorylation and degradation of IκBα in a dose-dependent manner (Fig. 2A). In addition, TNF-α-induced phosphorylation and degradation of IκBα were occurred as quickly as 15 min (Fig. 2B). Next, we examined whether baicalein modulates TNF-α-induced nuclear translocation of p65. Nuclear extracts were pretreated with baicalein and then exposed to TNF-α for various time periods and analyzed p65 nuclear translocation by western blot analysis. The results showed that baicalein also potently inhibited TNF-α-induced nuclear translocation of p65 in a dose-dependent manner (Fig. 2C), and the earliest inhibition also occurred within 15 min after TNF-α addition (Fig. 2D). To further confirm these results, the immunofluorescence staining assay was performed. Immunofluorescence images showed that in untreated, p65 was localized in the cytoplasm. In TNF-α alone treated, p65 was translocated to the nucleus. Followed by inhibited nuclear translocation of p65 with baicalein pretreatment (Fig. 2E).

NF-κB regulates the expression of anti-apoptotic gene products cIAP-1, cIAP-2, BCL-2 and FLIP, proliferation gene products COX-2 and cyclin D1, invasion and angiogenesis gene products MMP-9 and VEGF, which are known to be induced by TNF-α. We used western blotting to determine whether baicalein inhibits the expression of these gene products in HeLa cells. Our results showed that baicalein markedly downregulated TNF-α-induced expression of all these proteins in a dose-dependent manner (Fig. 3A). NF-κB regulates major inflammatory cytokines and proliferation, including IL-8, MCP1 and c-Myc. Thus, we also investigated whether baicalein can modulate TNF-α-induced expression of these genes by RT-PCR analysis. The results showed that baicalein blocked TNF-α-induced mRNA expression of IL-8, MCP1 and c-Myc in a dose-dependent manner (Fig. 3B).

HeLa cells were sequentially treated with baicalein and TNF-α, then stained with Annexin V-FITC and propidium iodide and analyzed using a flow cytometer. As shown in Fig. 4A, treatment of HeLa cells with vehicle only, TNF-α alone, and baicalein alone induced apoptosis of 4.5, 9.1 and 17.2% respectively. However, combined treatment of the cells with TNF-α and baicalein resulted in a significant potentiated apoptosis of HeLa cells (39.8%). To assess whether baicalein can enhance the TNF-α-induced apoptosis, the activation of caspases-8 and PARP was also investigated. Our results showed that baicalein alone had little effect on caspases-8 and PARP cleavage, However, combined treatment of TNF-α with baicalein potentiated their activation (Fig. 4B). These results together indicate that baicalein enhances the apoptotic effects of HeLa cells by TNF-α.

Next, in order to investigate the effects of baicalein on HeLa cell proliferation, the proliferation assay were performed. Indeed, as in MTT experiments, the strongest growth inhibitory effect was observed at 72 h of baicalein (100 µM) incubation (Fig. 5A). In order to elucidate if impairment of cell cycle participate in the reduction of the HeLa cell growth rate induced by baicalein, the flow cytometric analyses of cell cycle were performed. Our results showed that baicalein increased the population of G1 phase cells. These results suggest that baicalein inhibits cell proliferation through blocking cell cycle progression in G1 phase in HeLa cells.

The inflammatory response can be activated through the MAP kinase pathway. Thus, we determined whether baicalein can inhibition TNF-α-induced inflammatory responses through MAPK signaling. Since the MAPK pathway is phosphorylation-dependent, the phosphorylated proteins were easily detectable by western blot analysis. The results showed that baicalein decreased TNF-α-induced ERK1/2 and p38 by inhibiting their phosphorylation (Fig. 6).