The HCT116, A549, Panc-1 and FHC (normal human colorectal mucosal cells) cell lines were obtained from American Type Culture Collection (Rockville, MD, USA) and were grown in RPMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (CellMax, Beijing, USA) and penicillin- streptomycin (Beyotime Institute of Biotechnology, Nanjing, China). The cells were cultured in a humidified incubator at 37°C and 5% CO₂ and were exposed to the indicated concentrations (0, 10, 20 or 40 µM) of baicalein (Jiangsu Institute for Food and Drug Control, Nanjing, China), 1 µM gemcitabine (cat. no. G8970, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), SP600125 (cat. no. S1460, Selleck Chemicals, Houston, TX, USA), SB203580 (cat. no. S1076, Selleck Chemicals) or SCH772984 (cat. no. S7101, Selleck Chemicals).

The cell lysates were prepared with RIPA buffer containing PMSF (Beyotime Institute of Biotechnology). Protein concentration was determined by Enhanced Bicinchoninic Acid Protein assay kit (cat. no. P0010, Beyotime Institute of Biotechnology). Equal quantities (50 µg) of cellular protein were loaded onto a 12% SDS-PAGE gel and then transferred onto a PVDF membrane (cat. no. IPVH00010, EMD Millipore, Billerica, MA, USA). The membranes were blocked by 5% non-fat milk in Tris-buffered saline with 0.07% Tween-20 for 2 h and incubated with specific primary antibodies (1:1,000) in 1% bovine serum albumin (Biosharp, Hefei, China) in Tris-buffered saline with 0.07% Tween-20 at 4°C overnight. The following primary antibodies were used: Rabbit anti-DEPP (cat. no. 25833-1-AP, 1:1,000) from ProteinTech Group, Inc. (Rosemont, IL, USA); mouse anti-β-actin (cat. no. 21800, 1:1,000) from SAB Signalway Antibody (Nanjing, China); and rabbit anti-Gadd45a (cat. no. 4632, 1:1,000), rabbit anti-caspase-3 (cat. no. 14220, 1:1,000), rabbit anti-caspase-9 (cat. no. 9502, 1:1,000), rabbit anti-cleaved-capase-9 (cat. no. 9505, 1:1,000), mouse anti-JNK (cat. no. 3780, 1:1000), rabbit anti-phosphorylated (p-)JNK (cat. no. 4668, 1:1,000), rabbit anti-ERK (cat. no. 4695, 1:1,000), rabbit anti-p-ERK (cat. no. 4370, 1:1,000), rabbit anti-p38 (cat. no. 9212, 1:1,000) and rabbit anti-p-p38 (cat. no. 4511, 1:1,000) from Cell Signaling Technology, Inc. (Danvers, MA, USA). The secondary antibodies used were goat anti-rabbit IgG (cat. no. L3012-2, 1:5,000) or goat anti-mouse IgG (cat. no. L3032-2, 1:5,000) from SAB Signalway Antibody. The membranes were incubated with diluted secondary antibodies (1:5,000) in 1% bovine serum albumin at room temperature for 1.5 h. The signal was visualized using the Immobilon Western Chemiluminescent HRP Substrate (cat. no. WBKLS0500, EMD Millipore), using Image Lab 5.2.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The quantitative densitometric analysis was performed using ImageJ 1.48v with 32-bit Java 1.6.0_20 (National Institutes of Health, Bethesda, MD, USA).

Apoptosis induced by baicalein was determined using an Annexin V-FITC/PI Apoptosis Detection kit (cat. no. A211-02, Vazyme, Nanjing, China). The cells were seeded into a 12-well plate at 5×10⁴ cells/ml and treated with different concentrations of baicalein for 48 h. The cells were detached with 0.25% trypsin without EDTA, washed twice with cold PBS and resuspended in 100 µl binding buffer. The cells were then incubated with 5 µl Annexin V-FITC and 5 µl PI for 10 min at room temperature in the dark, and analyzed with the Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Total RNA from the cultured cells was extracted using TRIzol (cat. no. R401-01, Vazyme, Piscataway, NJ, USA) and chloroform/isopropanol, as specified by the manufacturer, and then reverse transcribed using a HiScript Q RT SuperMix for qPCR (+gDNA wiper) kit (cat. no. R223-01, Vazyme). The qPCR was subsequently performed using a ChamQ SYBR qPCR Master Mix kit (cat. no. Q311-02, Vazyme) and each sample [5 µl 2X ChamQ SYBR qPCR Master Mix, 0.2 µl Primer 1 (10 µM), 0.2 µl Primer 2 (10 µM), 0.2 µl 50X ROX Reference Dye1, 2 µl template cDNA and 2.4 µl ddH₂O] was analyzed on the StepOne™ Real-Time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Stage 1, 95°C for 3 min; Stage 2, 95°C for 10 sec, 56°C for 30 sec, 72°C for 30 sec; Stage 3, 95°C for 15 sec, 60°C for 60 sec, 95°C for 15 sec. The following PCR primers were used: GAPDH forward, 5′-GGGAAACTG TGGCGTGAT-3′ and reverse, 5′-GAGTGGGTGTCGCTGTTGA-3′; DEPP forward, 5′-ATACGTCCTGTGGTGGCATTG-3′ and reverse, 5′-CCTGATTCCCGTTCCCTGAT-3′; Gadd45a forward, 5′-TTGCAATATGACTTTGGAGGAA-3′ and reverse, 5′-CATC CCCCACCTTATCCAT-3′; p21 forward, 5′-AGCAGAGGAAGA CCATGTGGA-3′ and reverse, 5′-AATCTGTCATGCTGGTCTGCC-3′; and p53 forward, 5′-CTCTCCCCAGCCAAAGAAGAA-3′ and reverse, 5′-TCCAAGGCCTCATTCAGCTCT-3′ (Springen Biotechnology, Nanjing, China). The relative mRNA expression was normalized to that of GAPDH and was determined using the comparative Cq method (2^−ΔΔCq) (15).

siRNA targeting DEPP and Gadd45a, and non-specific siRNA (NC) were synthesized by GenScript Co., Ltd. (Nanjing, China): DEPP, 5′-GCAGUGUCCUCAGAACACU-3′; Gadd45a 5′-AAAGUCGCUACAUGGAUCAAU-3′; NC 5′-UUCUCCG AACGUGUCACGU-3′. The cells were transfected with siDEPP, siGadd45a or non-specific siRNA using Entranster™-R4000 (Engreen Biosystem, Ltd., Beijing, China) according to the manufacturer's protocol.

The HCT116 cells were transfected with 0.82 µg/µl of either empty pcDNA3.1 or pcDNA3.1 (GenScript Co., Ltd.) containing DEPP cDNA using Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific, Inc.). The cells were harvested 72 h following transfection.

The cells were treated with the indicated concentrations of Baicalein at 37°C for 48 h. Subsequently, MTT was added to reach a final concentration of 5 mg/ml for 4 h. The supernatant was removed, and the purple-colored formazan precipitate was dissolved in 150 µl DMSO and measured at 490 nm with a microplate reader (iMark; Bio-Rad Laboratories, Inc.).

All data are shown as the mean ± standard deviation of at least three independent experiments. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA), and data were analyzed using one-way analysis of variance, followed by the Dunnett's multiple comparison test. In certain cases, Student's t-test was used for comparing two groups. P<0.05 was considered to indicate a statistically significant difference.

Firstly, an MTT assay was performed to confirm that the appropriate concentration of baicalein was used. The data from the HCT116 (Fig. 1A) and FHC (Fig. 1B) cells demonstrated that 40 µM baicalein significantly inhibited the proliferation of HCT116 (~24%) and cased less damage to FHC cells (~10%). Subsequently, to detect the effect of baicalein on human cancer cells, light microscopy was used to observe the morphological changes of HCT116, A549 and Panc-1 cells following treatment with 0, 10, 20 or 40 µM baicalein with 1 µM gemcitabine serving as a positive control. As shown in Fig. 1C, cells treated with baicalein (10, 20 or 40 µM) or gemcitabine (1 µM) were in flattened, blebby and shrunken in appearance, consistent with cell death, whereas the negative control cells presented with an intact and polygonal morphology. Additionally, baicalein markedly inhibited the proliferation of HCT116 (Fig. 1D), A549 (Fig. 1E), and Panc-1 (Fig. 1F) cells in a dose-dependent manner. For example, 40 µM baicalein inhibited proliferation by up to 90%. Furthermore, the data indicated that baicalein induced apoptosis of the HCT116 cells in a dose-dependent manner as assessed by the Annexin V/PI double staining assay (Fig. 2A), and significantly increase the levels of cleaved-caspase-3 and cleaved-caspase-9 (Fig. 2B) following treatment for the indicated times. Similar results were also observed in the A549 (Fig. 2C and D) and Panc-1 (Fig. 2E and F) cells, suggesting that baicalein induced the apoptosis of HCT116, A549 and Panc-1 cells.

To verify the potential role of DEPP and Gadd45a in baicalein- induced apoptosis, the present study investigated whether the expression levels of DEPP and Gadd45a were altered in baicalein-treated cells. Western blot analysis (Fig. 3A) and RT-qPCR analysis (Fig. 3B) showed that the protein and mRNA expression levels of DEPP and Gadd45a were elevated more than two-fold in the baicalein-treated HCT116 cells. The data for protein levels in the A549 and Panc-1 cells were similar to those for the HCT116 cells. However, the mRNA expression of DEPP and Gadd45a in the A549 and Panc-1 cells had distinctly increased following 6 h of baicalein treatment, vs. 24 h of treatment (Fig. 3C–F), which may be attributable to the delay between transcription and translation. These results confirmed that baicalein upregulated DEPP and Gadd45a in three distinct human cancer cell lines.

To determine whether DEPP and Gadd45a were necessary for baicalein-induced apoptosis, siRNA against either DEPP or Gadd45a was transfected into HCT116 cells. Western blot analysis showed that the accumulation of baicalein-induced cleaved-caspase-3 and cleaved-caspase-9 were markedly reduced when DEPP and Gadd45a were silenced (Fig. 4A and B). These results were further confirmed by the Annexin V/PI double staining assay. As shown in Fig. 4C and D, there was a decrease of ~10% in the apoptotic rate of cells treated with 40 µM baicalein when transfected with either DEPP siRNA or Gadd45a siRNA, compared with cells transfected with non-specific siRNA. Taken together, these data showed that baicalein upregulated the expression of DEPP and Gadd45a, which was involved in the apoptotic response in HCT116 cells through the activation of caspase-3 and caspase-9.

To further elucidate the mechanism linking baicalein-induced apoptosis and the expression of DEPP and Gadd45a, the activation of MAPK was examined using an immunoblot assay in HCT116 cells at 0, 6, 12 and 24 h post-baicalein treatment. Notably, increases in the phosphorylation of JNK, ERK and p38 were observed in the western blot analysis (Fig. 5A). The expression of DEPP was then stably inhibited using siRNA to determine whether DEPP is required for the baicalein-induced phosphorylation of MAPKs. Following baicalein treatment, the absence of DEPP (Fig. 5B and C) had a negative effect on the baicalein-mediated protein expression of p-JNK, p-ERK, p-p38 and Gadd45a, or on the mRNA expression of p21, p53 and Gadd45a. In addition, a DEPP-overexpression plasmid was used to further validate these findings (Fig. 5D). Similarly, p-JNK, p-ERK, p-p38, p21, and p53 were induced only when the overexpression of DEPP was present (Fig. 5D and E). Furthermore, the gene expression of Gadd45a was increased by 1.60-fold. Together, these results suggested that DEPP is required for the baicalein-induced phosphorylation of MAPKs and upregulated expression of Gadd45a at the protein and mRNA levels. The present study subsequently examined whether Gadd45a also regulated the baicalein- induced phosphorylation of MAPKs, and similar results were observed in the levels of p-JNK, p-ERK and p-p38 in cells transfected with siRNA against Gadd45a (Fig. 5F). In summary, these data suggested that DEPP and Gadd45a are essential in baicalein-induced apoptosis via MAPK signaling.

The abovementioned results showed that Gadd45a upregulated the phosphorylation of JNK, ERK and p38 in the baicalein-induced apoptosis of HCT116 cells. A previous report demonstrated that MAPK signaling mediates the expression of Gadd45a (14). Therefore, to determine whether MAPK signaling was involved in the baicalein-mediated upregulation of Gadd45a, the JNK-specific inhibitor SP600125, p38 inhibitor SB203580, and ERK inhibitor SCH772084 were used to prevent the activation of JNK, p38 and ERK, respectively. The inhibition of JNK and p38 was accompanied by a marked decrease in the baicalein-induced expression of Gadd45a (Fig. 6A and B), however, no such change was observed in the presence of the ERK inhibitor SCH772084 (Fig. 6C). These results suggested that there was a positive feedback loop between Gadd45a and JNK/p38 when baicalein triggered the apoptotic response in human colon cancer cells, whereas the suppression of ERK had no effect on the expression of Gadd45a. Therefore, these data revealed the critical role of the activation of MAPK in inducting of the expression of Gadd45a in baicalein-induced apoptosis (Fig. 7).