SCC25, CAL27 and HSC3 cells were kindly provided by Professor Bin Shi (University of Wuhan, China). All cell lines were cultured in RPMI-1640 media (HyClone; GE Healthcare Life Sciences) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C and humidified atmosphere of 5% CO₂. Baicalein (Sigma-Aldrich; Merck KGaA) was dissolved in DMSO at 100 mM as a stock solution, and diluted to a working concentration (1 mM) with PBS prior to use. For the in vivo xenograft studies, baicalein was dissolved in a solution containing 80% PBS and 20% DMSO.

For the construction of the Sp1 expression plasmid, the full-length cDNA was amplified and inserted into the EcoRI/XhoI sites of the pcDNA3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). The primers for Sp1 were as follows: Forward, 5'-CCA AAA TGC GAT CGC ATG AGC GAC CAA GAT CAC-3' and reverse, 5'-GAA TCA AGT TTA AAC TCA GAA GCC ATT GCC ACT-3'. The NF-κB activity plasmid was a gift from Professor H. Shu (University of Wuhan). The PRL-TK plasmid was a gift from Professor D. Guo.

SCC25, CAL27 and HSC3 cells (1x10⁴ cells/well) were seeded in 96-well plates and treated with DMSO control (0.01%) or increasing concentrations of baicalein (30, 60 and 120 µM) at 37°C. After incubation for 24, 48 and 72 h, the cells were treated with 10 µl Cell Counting Kit-8 reagent (Dojindo Molecular Technologies, Inc.) and the plates were incubated at 37°C for 1 h in the dark. The optical density was measured at an absorbance of 450 nm using an ELx800 microimmunoanalyser (BioTek Instruments, Inc.).

SCC25, CAL27 and HSC3 cells were seeded in 6-well plates and treated with DMSO (0.01%) or baicalein (60 µM) at 37°C for 24 h. The process was performed as described previously (24). Briefly, cells were digested and washed twice with cold PBS solution, and then resuspended in cold 75% ethanol. After fixation at -20°C for 24 h, cells were collected and resuspended in 0.5 ml cold PBS. Cells were mixed with reagent A [Multisciences (Lianke) Biotech Co., Ltd.] and incubated at 4°C for 30 min in the dark. Cell cycle analysis was performed using a Beckman Coulter system (EPICS Altra II; Beckman Coulter, Inc.).

Apoptosis of OSCC cells was detected using the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit [Multisciences (Lianke) Biotech Co., Ltd.], as instructed by the manufacturer. Briefly, following treatment with baicalein (15, 30 and 60 µM) at 37°C for 24 h, cells were digested. After three washes with PBS, 2x10⁵ cells were resuspended in 500 µl 1X binding buffer, followed by addition of 5 µl Annexin V-FITC and 10 µl PI, and incubated for 30 min at 20-28°C in the dark. Cell apoptosis was immediately analyzed using a Beckman Coulter system (EPICS Altra II; Beckman Coulter, Inc.).

At 4 h post-transfection, SCC25, CAL27 and HSC3 cells were washed with PBS and treated with DMSO (0.01%) or baicalein (30 or 60 µM). After 48 h of incubation, cells were harvested and dissolved in RIPA lysis buffer (Beyotime Institute of Biotechnology) with 0.5% cocktail protease inhibitor (Roche Diagnostics). Following incubation on ice for 10 min, cell lysates were collected and sonicated for 20 sec. Protein concentration was determined using a BCA assay (BioRad Laboratories, Inc.). Quantified proteins were mixed with 5X loading buffer [250 nM Tris-Hcl (pH 6.8), 0.5% bromophenol blue, 50% glycerol, 10% SDS and 5% β-mercaptoethanol] and boiled for 5 min. The lysates were separated by SDS-PAGE on 10% gels, then subjected to immunoblot analyses. The primary antibodies used were as follows: GAPDH (cat. no. 10494-1-AP; 1:5,000; ProteinTech Group, Inc.); cleaved caspase-3 (cat. no. 9664; 1:1,000; Cell Signaling Technology, Inc.); caspase-9 polyclonal antibody (cat. no. A2636; 1:1,000; ABclonal Biotech Co., Ltd.); cleaved poly(ADP-ribose) polymerase (PARP-1; cat. no. sc-56196; 1:500; Santa Cruz Biotechnology, Inc.); Sp1 (cat. no. 9389; 1:1,000; Cell Signaling Technology, Inc.); p65 (cat. no. ab16502; 1:1,500; Abcam); p-p65 (cat. no. ab86299; 1:1,000; Abcam); and p50 (cat. no. 3035; 1:1,000; Cell Signaling Technology, Inc.). Western blot gray values were determined by ImageJ software (National Institutes of Health).

PCR (RT-qPCR). SCC25, CAL27 and HSC3 cells (1x10⁵) were placed in 24-well plates. After attaching to the wells, the cells were washed with PBS and treated with DMSO (0.01%) or baicalein (30 or 60 µM). After incubation at 37°C for 24 h, total RNA was extracted by using TRIzol reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Subsequently, cDNA was synthesized from total RNAs (1 µg) using an RT kit (Takara Bio, Inc.) according to the manufacturer's instructions. The mRNA expression levels of Sp1, p65 and p50 were quantified using the CFX96 Real-Time PCR Detection System with an SYBR Premix Ex Taq kit (Takara Bio, Inc.). The primers were as follows: p65, forward 5'-CGG GAT GGC TTC TAT GAG G-3' and reverse 5'-CTC CAG GTC CCG CTT CTT-3'; p50, forward 5'-ACC CTG ACC TTG CCT ATT TG-3' and reverse 5'-AGC TCT TTT TCC CGA TCT CC-3'; Sp1, forward 5'-ATG GGG GCA ATG GTA ATG GTG G-3' and reverse 5'-TCA GAA CTT GCT GGT TCT GTA AG-3'; GAPDH, forward 5'-GGT GGC TTC TGA CTT CAA CA-3' and reverse 5'-GTT GCT GTA GCC AAA TTC GTT GT-3'. The mRNA levels were normalized to GAPDH as the reference gene.

A specific short hairpin RNA (shRNA) targeting Sp1 (5'-GCA TAT TTG CCA CAT CCA AGG-3', Sp1-Homo-1828; GenePharma, Co., Ltd.) or a non-specific control (NC; 5'-TTC TCC GAA CGT GTC ACG T-3'; Shanghai GenePharma Co., Ltd.) were transfected to cells using X-treme GENE HP DNA Transfection Reagent (Roche Diagnostics; 40 pmol for each shRNA) according to the manufacturer's instructions. PcDNA3.1-Sp1 or pcDNA3.1 (vehicle control) was transfected to cells using X-treme GENE HP DNA Transfection Reagent. At 4 h post-transfection, cells (SCC25) were washed with PBS and treated with DMSO (0.01%) or baicalein (60 µM). After 48 h, the cells were harvested for western blot analysis. For RT-qPCR, total RNA was collected after 12 or 24 h of incubation. For the dual luciferase reporter assay, cells were harvested after 24 or 48 h of incubation.

Cells (1x10⁵) were placed in 24-well plates and incubated at 37°C overnight prior to transfection. Plasmids were co-transfected into the cells for 24 h. After treatment with baicalein or control for 24 h, cells were collected and analyzed using the Dual Luciferase Reporter Assay system (Promega Corporation) as previously described (25).

The Medical Ethics Committee of Wuhan University approved the animal experimental protocols in the present study (G201725). A total of 10 BALB/c nude mice were obtained from the Animal Biosafety Level-III Laboratory of Wuhan University and housed in a specific pathogen-free environment. SCC25 cells (5x10⁶/100 µl) were subcutaneously injected into the flanks of the mice when they were 6-7 weeks old. When the tumors became macroscopically visible (~7 days), the mice were randomly divided into two groups (n=5/group). The control group mice were injected with PBS (0.01% DMSO) and baicalein group mice were injected with baicalein (30 mg/kg, three times a week). The mice were sacrificed after 21 days of treatment. The mouse weight and tumor size were measured. Tumor volume was calculated as 0.5 x length x width². Tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin and cut into 3-µm sections).

Immunohistochemistry was performed using antibodies against Sp1 (cat. no. 9389; 1:1,000; Cell Signaling Technology, Inc.), p65 (cat. no. ab16502; 1:1,500; Abcam) and cleaved caspase-3 (cat. no. 9664; 1:1,000; Cell Signaling Technology, Inc.) according to the manufacturer's instructions. The process was performed as described previously (24). Briefly, after deparaffinization, the tissue sections were boiled in citric acid (pH 6.0) for 20 min and immersed in 3% H₂O₂ for 10 min to quench the endogenous peroxidase activity. After blocking in goat serum for 1 h at 20-28°C, the tissue sections were incubated with primary antibodies overnight at 4°C. After washing, the tissue sections were incubated with secondary antibody (MaxVision™ Kits; MaxVision Biosciences, Inc.) conjugated to horseradish peroxidase. Subsequently, the tissue sections were incubated with diaminobenzidine for 1 min and lightly counterstained with hematoxylin.

Data are presented as the mean ± standard deviation of three independent experiments. For the comparison of two groups, Student's t-test was selected. For the comparison of multiple groups, one-way ANOVA followed by the Newman-Keuls post hoc test was carried out. All statistical data were analyzed by using GraphPad Prism for Windows, version 5.0 (GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To determine whether baicalein decreases the viability of OSCC cells, SCC25, CAL27 and HSC3 cells were treated with varying doses of baicalein and for different durations. The chemical structure of baicalein (5,6,7-trihydroxyflavone) is shown in Fig. 1A. Baicalein significantly (P<0.05) reduced the viability of SCC25, CAL27 and HSC3 cells compared with cells treated with DMSO control (Fig. 1B-D). These results suggest that baicalein effectively inhibits the proliferation of different OSCC cell lines.

To examine the apoptotic effect exerted by baicalein on OSCC cells, SCC25, CAL27 and HSC3 cells were treated with increasing doses of baicalein for 24 h. As shown in Fig. 2A and B, baicalein significantly induced apoptosis of SCC25, CAL27 and HSC3 cells. To further assess stimulation of the apoptotic pathway, expression of several apoptosis-associated proteins was detected by western blot analysis. Baicalein increased the protein levels of cleaved caspase-9, cleaved caspase-3 and cleaved PARP-1 in SCC25, CAL27 and HSC3 cells (Fig. 2C), suggesting that baicalein induces OSCC cell apoptosis via the mitochondrial apoptotic pathway.

To determine whether baicalein affects the cell cycle, SCC25, CAL27 and HSC3 cells were treated with vehicle control (0.01% DMSO) or baicalein (60 µM) for 24 h. The distribution of cells in different cell cycle phases was analyzed by flow cytometry. As shown in Fig. 2D and E, baicalein treatment induced cell cycle arrest in the G0/G1 phase. The fraction of SCC25, CAL27 and HSC3 cells in the G0/G1 phase was increased by 18.8, 29.4 and 19.5%, respectively, when treated with baicalein (60 µM). These results demonstrated that baicalein reduces the proliferation of OSCC cells by causing G0/G1 cell cycle arrest.

To determine whether baicalein alters the expression of Sp1 in OSCC cells, three different OSCC cell lines (SCC25, CAL27 and HSC3) were treated with DMSO control or baicalein. As shown in Fig. 3A–C, baicalein significantly (P<0.05) decreased Sp1 expression in SCC25, CAL27 and HSC3 cells. Sp1 has been previously demonstrated to be a transcription factor that regulates the expression of p65 and p50 (26). Therefore, the expression of p65 and p50 was measured, and the levels of p65, p-p65 and p50 were found to be decreased following treatment with baicalein (Fig. 3A-C).

The observed decrease in protein expression may be due to reduced mRNA levels; therefore, the effects of baicalein on Sp1 were also analyzed at the transcriptional level. SCC25, CAL27 and HSC3 were treated with DMSO control or baicalein, and total RNA was collected and subjected to RT-qPCR analysis. The mRNA expression of Sp1 in SCC25, CAL27 and HSC3 cells was found to be decreased by 75.7, 87.0 and 78.8%, respectively, compared with controls at 48 h (Fig. 3D). Additionally, the mRNA levels of p65 and p50 were analyzed. As shown in Fig. 3E and F, the mRNA levels of p65 and p50 were significantly decreased following treatment with baicalein. These results suggest that baicalein decreases the expression of Sp1, p65 and p50 in OSCC cell lines by reducing the mRNA expression.

The reduced expression of p65 and p50 may lead to suppression of the NF-κB pathway. To confirm this hypothesis, SCC25, CAL27 and HSC3 cells were treated with DMSO control or baicalein, and the activity of NF-κB was determined using a Dual Luciferase Reporter Assay system. As shown in Fig. 4A-C, the activity of the NF-κB pathway was downregulated to 15.3% (SCC25), 8.6% (CAL27) and 12.0% (HSC3) following treatment with baicalein (60 µM) for 24 h. These results suggest that baicalein markedly inhibits the activation of NF-κB signaling in OSCC cells.

To determine whether the decrease in p65 and p50 expression was associated with the expression of Sp1 in OSCC cells, SCC25 and CAL27 cells were transfected with a specific shRNA targeting Sp1. Total proteins were harvested and subjected to western blot analysis. As shown in Fig. 5A and B, silencing of Sp1 significantly reduced the expression of p65 and p50. Additionally, the mRNA levels of p65 were decreased to 44.3 and 29.3% following silencing of Sp1 in SCC25 and CAL27 cells, respectively (Fig. 5C). The mRNA levels of p50 in SCC25 and CAL27 cells with silenced Sp1 expression were decreased to 40.3 and 25.0%, respectively (Fig. 5D). These results indicate that downregulated expression of Sp1 reduces the expression of p65 and p50 in OSCC cells.

To determine the effect of Sp1 silencing on NF-κB activity and cell viability, SCC25 and CAL27 cells were transfected with Sp1-specific shRNA. The activity of NF-κB was determined using the Dual Luciferase Reporter Assay system. As shown in Fig. 5E, the activity of the NF-κB pathway was reduced to 35 and 39.3% by Sp1-shRNA in SCC25 and CAL27 cells, respectively. The viability of SCC25 and CAL27 cells was decreased to 63.7 and 44.7%, respectively, by specific Sp1-shRNA (Fig. 5F). These results indicate that silencing of Sp1 expression inhibits NF-κB activity and reduces the viability of OSCC cells.

To determine whether the effects of baicalein on NF-κB activity and cell viability are dependent on Sp1, SCC25 cells were transfected with shRNA-Sp1 or homo-NC, and treated with baicalein or DMSO control. As shown in Fig. 6A, combination with shRNA-Sp1 significantly enhanced the baicalein-induced suppression of Sp1, p65 and p50 expression. In addition, knockdown of Sp1 significantly contributed to the inhibitory effects of baicalein on NF-κB activity (Fig. 6B) and cell viability (Fig. 6C).

To determine whether the effects of baicalein on cell viability are primarily caused by the reduced expression of Sp1, SCC25 cells were transiently transfected with pcDNA3.1-Sp1 or vehicle control (pcDNA3.1). The cells were subsequently treated with DMSO or baicalein. As shown in Fig. 6D, pcDNA3.1-Sp1 significantly increased the expression of Sp1, p65 and p50. Compared with Sp1 overexpression alone, combined treatment with baicalein and pcDNA3.1-Sp1 transfection decreased the expression of Sp1, p65 and p50. As shown in Fig. 6E, the NF-κB activity was enhanced by 273.3 and 503.1% following transfection with pcDNA3.1-Sp1 for 12 and 24 h, respectively. Overexpression of Sp1 attenuated the effects of baicalein on NF-κB activity. In addition, Sp1 overexpression increased the viability of SCC25 cells and attenuated the inhibitory effect of baicalein on cell viability (Fig. 6F). These results suggest that baicalein reduces SCC25 cell viability and NF-κB activity, which is mediated by the Sp1 pathway.

To determine the effects of baicalein on OSCC in vivo, SCC25 cells were used to establish subcutaneous xenograft tumors in immune-deficient BALB/c nude mice. The mice were administered baicalein or DMSO at 7 days after tumor cell inoculation. After treatment with baicalein or DMSO for 21 days, the mice were sacrificed and their tumors were measured. The weight of the mice was also evaluated. As shown in Fig. 7A, tumor volumes were decreased by treatment with baicalein. Additionally, there was no significant difference in the weight of the mice between the two groups (Fig. 7B). Representative images of the tumors are shown in Fig. 7C. To further elucidate the mechanism underlying the effects of baicalein on tumor growth, the expression and distribution of p65, Sp1 and cleaved caspase-3 were determined by immunohistochemical examination of the tumor tissues. As shown in Fig. 7D, the expression p65 and Sp1 was stronger in the sections from the control group compared with the baicalein-treated group. Conversely, cleaved caspase-3 expression was weaker in the control group compared with that in the baicalein-treated group. These results suggest that baicalein reduces the growth of SCC25 OSCC cell xenografts in vivo.