Human colorectal carcinoma cells HCT-116 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, United States). Cell culture reagents were purchased from Invitrogen (Gibco, Waltham, MA, United States). The cells were cultured in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 μg/mL streptomycin. All cells were maintained at 37°C in a humidified atmosphere of 5% CO₂.

Wogonin (purity ≥ 98%, HPLC grade, confirmed by LC/MS/MS) was purchased from Dalian Meilun Biotech, Co., Ltd. (Dalian, China). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, United States). Primary antibodies for LC3 I/II, Cleaved caspase 3, Cleaved caspase 9, IRE-1a, CHOP, PDI, Calnexin and secondary antibody Anti-rabbit IgG, HRP-linked were brought from Cell Signaling Technology, Inc. (Danvers, MA, United States). Primary antibody for p-p53(s15), p-p53(s376) and p-p53(s315) were purchased from Abcam, Inc. (Cambridge, MA, United States). Primary antibodies for GAPDH and Histone H3 and secondary antibody horseradish peroxidase-linked anti-mouse immunoglobulin G were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Western blot detection reagents were obtained from Bio-Rad Laboratories (Hercules, CA, United States).

The cell viability was measured via MTT assay. 2 × 10³ cells/well were seeded into 96-well plates and cultured with different doses (1∼100 μM) of wogonin for 72 h. At the end of treatment, 100 μL of MTT (0.5 mg/mL) was added to each sample and incubated for 4 h at 37°C. Then, the supernatants were discarded and 150 μL of DMSO was added to dissolve the formazan crystals. Then optical density was determined using a microplate reader Victor X3 at 490 nm (PerkinElmer, Waltham, MA, United States).

HCT-116 cells (300 cells/well) were seeded in 6-well plates 1 day before treatment. DMEM (2 ml) containing 10% FBS was added to each well with a vehicle (0.01% DMSO) or different concentration of wogonin (5 and 10 μM). After 2 weeks cultured, colonies with more than 50 cells were counted, the percentage of colony-forming rate was calculated to evaluate colony formation efficiency. Colony-formation rate was calculated as: (Colony counts experiment group/ Colony counts medium control group) × 100%.

HCT-116 cells were seeded in 6-well plates (3 × 10⁵ cells/well) 1 day before treatment. Then cells were treated with or without wogonin (5 and 10 μM) for 48 h. The cells were harvested and processed for cell apoptosis analysis by using FITC Annexin V Apoptosis Detection Kit (BD Biosciences). Cell apoptosis was determined by using FACS Aria III (BD Biosciences) equipped with FlowJo v7.6. Annexin-V positive PI negative (AV⁺PI^-) signals represent cells in early apoptosis, while Annexin-V positive PI positive (AV⁺PI⁺) signals indicate cells in late apoptosis.

Cells were seeded in six-well plates with density of 4 × 10⁵ cells/well, and treated with a vehicle (0.01% DMSO) or 5 and 10 μM wogonin for 72 h. Cell lysates for protein blot analysis were prepared using standard RIPA buffer (Santa Cruz Biotechnology). Western blot analysis was performed to detect the expression levels of target proteins according to the literature (Martínez-Revelles et al., 2016). Briefly, 30 μg protein was loaded on (10%) SDS-PAGE and separated. After transmembrane, PVDF membranes were stained with primary and secondary antibodies. GAPDH or Histone H3 served as a loading control. The relative intensities of the protein bands were scanned and quantified using Quantity One Program (Bio-Rad).

After wogonin treatment, total RNA was isolated using TRIzol and synthesized to cDNA by using a reverse transcription kit (TaKaRa, Shiga, Japan). SYBR Green real-time PCR amplification and detection were then performed by using an ABI 7500 system (Applied Biosystems, Foster City, CA, United States). The following primers were used: DDIT3, forward 5′-AATGAACGGCTCAAGCAGGA-3′ and reverse 5′-AGCCACTTCTGGGAAAGGTG-3′; XBP1, forward 5′-AAGTTCTGCTTCTGTCGGGG-3′ and reverse 5′-TGCACGTAGTCTGAGTGCTG-3′; GAPDH, forward 5′-GGTGTGAACCATGAGAAGTATGA-3′ and reverse 5′-GAGTCCTTCCACGATACCAAAG-3′. Relative gene expression levels were normalized to GAPDH expression. The relative mRNA expression levels were analyzed by 2^-ΔΔCt.

The immunofluorescence assays in HCT-116 cells were seeded on 15 mm confocal dish with 5 or 10 μM wogonin treating for 48 h and fixed in cold 4% paraformaldehyde. The confocal dishes were incubated with p-p53(ser15) (Abcam, 1:200). Detection of the primary antibody was performed using 1:200 Alexa Fluor^® conjugated secondary antibody. The nucleus was stained with 0.1% DAPI and microfilament was stained with 0.1% phalloidin. The images were captured and analyzed using Leica TCS SP8 confocal microscope.

All animals used in this study were obtained from Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China) and approved by Animal Care and Use Committee (IACUC) in Guangzhou University of Chinese Medicine. Colorectal cancer was induced in 4-week-old male C57BL/6 mice via the intraperitoneal injection of AOM (10 mg/kg) while the mice were maintained with a regular diet and drinking water for 14 days and then mice were subjected with three cycles of DSS treatment, with each cycle consisting of the administration of 2% DSS for 7 days, followed by a 14-day recovery period with regular water. Mice were administered with 50 and 100 mg/kg wogonin daily from 5^th to 25^th week.

All results are expressed as mean ± standard deviation (SD) of values from three independent experiments. Data were statistical analyzed by one-way ANOVA using SPSS 18.0. P-values < 0.05 (^∗), < 0.01 (^∗∗), < 0.001 (^∗∗∗) were considered as statistical significant between experiment and control groups.

The anti-cancer effects of wogonin were first investigated in vitro in human colon cancer cells. Human colon cancer HCT116 cells were treated by wogonin at various concentrations (1–100 μM) for 72 h and analyzed by MTT assay. Result showed that wogonin exhibits a dose-dependent reduction in HCT116 cell viability (Figure 1A). After treatment, wogonin significantly inhibited cell growth with the maximum inhibition ratio of 68.67 ± 2.80% at 100 μM in HCT-116 cells (Figure 1A, P < 0.001). To further analyze the influence of wogonin in cell biology, wogonin at 5 and 10 μM, with respective cell inhibition of 29.47 ± 3.34 and 36.35 ± 4.38%, were chosen for following studies. Moreover, the anti-proliferation effects of wogonin in HCT-116 cells were evaluated by colony-formation assay. The result confirmed that wogonin inhibited HCT-116 cell productive capacity in a concentration-dependent manner (Figure 1B, P < 0.001), the respective cell colony formation rate is 54.58 ± 5.60 and 26.78 ± 1.55% after wogonin treatment at 5 and 10 μM for 14 days. Collectively, our results suggest that wogonin inhibits the colon cancer HCT-116 cell viability and proliferation.

To determine whether the inhibition effects of wogonin in CRC were caused by apoptosis or autophagy, flow cytometry and western blot were chosen for further analyzed. Flow cytometry analysis of AV and PI positivity showed that wogonin treatment induced significantly apoptosis in HCT-116 cells after 48 h (Figure 1C, P < 0.001). The percentages of late apoptotic cells were significantly increased to 7.40 ± 0.26 and 21.40 ± 0.10% after wogonin (5 and 10 μM) treatment compared with 2.93 ± 0.45% in control group. Meanwhile, in order to understand the pathway involved in wogonin-induced apoptosis, expression of cleaved caspase 3 and cleaved caspase 9, central executors of intrinsic apoptotic cell death, were detected by western blot. Protein levels of cleaved caspase 3 and cleaved caspase 9 were significantly increased by 3.52 ± 0.48 (Figure 1D, P < 0.01) fold and 3.72 ± 0.15 (Figure 1D, P < 0.001) fold respectively after wogonin (10 μM) treatment for 72 h (Figure 1D). LC3-II to LC3-I conversion, the reliable autophagy markers for monitoring autophagic cell death, also were detected by western blot. As shown in Figure 1E, we found that conversion ratio of LC3-II to LC3-I was decreased 0.49 ± 0.01 fold (P < 0.01) after 10 μM wogonin treatment for 72 h in HCT-116. All these data together indicated that wogonin induce human CRC HCT-116 cell death via caspase-dependent apoptotic pathway.

It has been reported that wogonin could modulate ER stress and eventually led to autophagy and/or apoptosis (Ge et al., 2015; Hu et al., 2015). To determine whether ER stress was associated with wogonin-induced cell death, ER chaperones (CALNEXIN, PDI), ER-associated key sensors (IRE1α), downstream transcription factor CHOP and regulated genes DDIT3 and XBP1 were detected by western blotting and qRT-PCR in HCT-116 cells. As shown in Figure 2A, in HCT-116 cells, wogonin increased protein expression of PDI, CALNEXIN, IRE-1α and CHOP in a dose-dependent manner. Particularly, PDI and CALNEXIN were significantly increased by 1.76 ± 0.04 fold (P < 0.001) and 2.65 ± 0.11 fold (P < 0.001) after wogonin (10 μM) treatment. Moreover, IRE-1α showed 1.48 ± 0.03 fold increase (P < 0.01) after wogonin (10 μM) treatment. Furthermore, CHOP was significantly increased by 2.03 ± 0.16 fold (P < 0.01) after treatment with 10 μM wogonin for 72 h compared to control group. What’s more, PCR results also confirmed that mRNA levels of ER stress associated downstream gene DDIT3 and XBP1 in HCT-116 cells were both up-regulated by 42.91 ± 0.34 fold (Figure 2B, P < 0.001) and 8.10 ± 0.79 fold (Figure 2B, P < 0.001) after wogonin (10 μM) treatment, respectively.

Tauroursodeoxycholic acid is an ambiphilic bile acid and a specific inhibitor of ER stress (Fan et al., 2017; Fernandez-Bautista et al., 2017). Flow cytometry result showed that the apoptotic effect of wogonin (10 μM) caused in HCT-116 cells was completely reversed by TUDCA (500 μM) (Figure 2C). The percentages of apoptotic cells were significantly decreased from 25.36 ± 0.06 to 17.40 ± 0.26% after wogonin (10 μM) and TUDCA (500 μM) treatment. Meanwhile, cleaved caspase 3 and cleaved caspase 9 protein expressions were revealed 0.61 ± 0.02 fold (P < 0.05) and 0.52 ± 0.03 fold (P < 0.01) decrease after treating with TUDCA (500 μM) and wogonin (10 μM) for 72 h in HCT-116 cells (Figure 2D). IRE-1α and PDI were exhibited 0.57 ± 0.03 fold (P < 0.05) and 0.61 ± 0.03 fold (P < 0.05) decrease after treating with TUDCA (500 μM) and wogonin (10 μM). Besides that, the level of conversion ratio of LC3-II to LC3-I was increased by 3.46 ± 0.16 fold (P < 0.001) compare with wogonin (10 μM) treat alone for 72 h in HCT-116 cells. All these results confirmed that wogonin caused apoptotic cell death in human CRC HCT-116 cell through increased ER stress.

Given that ER stress was regulated by wogonin, this pathway could converge into transcription factor p53 by controlling its nucleocytoplamic shuffling (Qu et al., 2004; Shi and Gu, 2012). To investigate how p-p53 shuffle was affected by wogonin, we studied the cytoplasm/nucleus translocation of p-p53 by western blot and immunofluorescence. For total protein, after wogonin (10 μM) treatment for 72 h, phosphorylation at serine 315 and serine 376 of p53 both were increased by 1.98 ± 0.09 fold (P < 0.01) and 3.78 ± 0.12 fold (P < 0.001) in HCT-116 cells (Figure 3A). To further clear the distribution of p-p53, we separated the nuclear and cytoplasmic proteins to evaluate the localization of p-p53. In HCT-116 cells cytoplasm, wogonin treatment exhibits a dose-dependent protein expressions increase of phosphorylation at serine S376 of p53. Wogonin treatment at 10 μM exhibited 3.72 ± 0.06 fold increase (P < 0.001) compare to control group. While in nucleus, expressions of S376 of p53 were decreased to 0.51 ± 0.02 (P < 0.05) fold and 0.47 ± 0.04 fold (P < 0.01) after wogonin (5 and 10 μM) treatment (Figure 3A).

Immunofluorescence assay was used to further confirm whether wogonin could influence the cytoplasmic/nuclear distribution of phosphorylation of p53. In HCT-116 cells, phosphorylation at serine 15 of p53 in nucleus was dramatically decreased, and the ratio of phosphorylation at serine 15 of p53 in nucleus/cytoplasm was decreased to 0.47 ± 0.002 fold (P < 0.001) after wogonin treatment for 48 h (Figure 3B). At the same time, ER stress inhibitor, TUDCA (500 μM) completely reversed the decreased effect of the ratio of phosphorylationof p53 in nucleus/cytoplasm caused by wogonin (10 μM) (Figure 3C). These findings suggested that wogonin regulated nuclear translocation of p-p53 by increasing the ER stress response to inhibit autophagy in HCT116 colon cancer cells.

The AOM-DSS colon cancer animal model was used to evaluate the chemopreventive effect and toxicity of wogonin in vivo. As shown in Figure 4A, mice developed tumors when they were given with AOM (10 mg/kg) by i.p. injection at week 4 and fed with DSS (2%) water for three-cycles. After consecutive 21 weeks of wogonin oral administration, there was no significant body weight loss in mice (Supplementary Figure 1A). At the termination of experiment, tumor node multiplicity was calculated as tumors stratifying by size according to diameter, tumor incidence is reduced from 80.00% in model group to 54.55 and 66.67% in 50 and 100 mg/kg wogonin treatment group, tumor multiplicity with size of <2 and >4 mm were 55.55 and 11.11% in mice treated with 100 mg/kg wogonin compared with model group 33.34 and 46.67%, respectively (Figure 4B). Concurrently, wogonin also preserved colon length to normal from 6.79 ± 0.34 cm in model group to 7.06 ± 0.74 and 7.41 ± 0.56 (P < 0.05) cm in wogonin (50 and 100 mg/kg) treated groups (Figure 4C).

To determine the effect of wogonin in vivo, western blot was performed to detect apoptotic markers in wogonin treatment groups as compared with model group. Protein expression of cleaved caspase 3 and cleaved caspase 9 were significantly increased by 3.53 ± 0.13 (P < 0.001) and 1.61 ± 0.16 (P < 0.05) fold respectively in 100 mg/kg wogonin treated group. To verify the potential mechanism of wogonin in vivo, ER stress markers were measured in colon tissue of mice with AOM/DSS induced colon cancer. Results showed that, protein level of PDI was increased by 2.34 ± 0.19 (P < 0.001) fold, and IRE1α was significantly increased by 2.24 ± 0.14 (P < 0.001) fold in intestine of mice treated with 100 mg/kg wogonin as compare with model group (Figure 4D).