Baicalin triggers apoptosis, inhibits migration, and enhances anti-tumor immunity in colorectal cancer via TLR4/NF-κB signaling pathway

2.1 Reagents and antibodies

Baicalin (free of endotoxin) as a 98% purity that detected by HPLC was provided by Chengdu Biopurify Phytochemicals Ltd. In vitro experiments, Baicalin was initially prepared as stock solution (80 mM) dissolved in DMSO and kept at −20°C in dark place for further use. LPS was purchased from Beyotime Biotechnology. For animal studies, Baicalin was dissolved in 5.5% DMSO in corn oil, and 5.5% DMSO in corn oil was used as a vehicle control. The primary antibodies against Bax (#2772), Bcl-2 (#3498), caspase 3 (#9662), cleaved caspase 3 (#9664), MMP-2 (#87809), MMP-9 (#13667), TIMP2 (#5738), p-IκBα (#2859), IκBα (#4814), TLR4 (#14358), NF-κB p65 (#8242), PD-L1 (#60365), Ki-67 (#12202), and β-actin (#4970) were purchased from Cell Signaling Technology.

2.2 Cell viability assays

HCT-116 and CT26 cell lines were bought from Shanghai Cell Bank, the Chinese Academy of Sciences. RPMI1640 medium (Gibco) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (streptomycin and penicillin) was exploited to culture CRC cells in atmosphere of 5% CO₂ and 37°C.

Viability of Baicalin-treated CRC cells was measured by cck8 assay. Briefly, harvest HCT-116 and CT26 cells were planted in 96-well plates (3000–5000 cells per well) and incubated at 37°C for 6-8h. Subsequently, cells in plates were exposed to designated doses (0, 5, 10, 20, 40, and 80 μg/mL) of Baicalin for 48 and 72 hr. After different treatments, 10 μL of cck8 solution was added to each well, and cells were incubated for an additional 1–4 hr at 37°C. Finally, absorbance of each well at 450 nm was measured by a microplate reader. The experiments were performed at least in triplicates.

2.3 Colony formation assays

Single cell suspension of HCT-116 and CT26 was added to 6-well plate (200–500 cells/well), and incubate at 37°C for overnight. The next day, cells were exposed to determined concentrations of Baicalin (0, 5, 10, and 20 μM) for 10–14 days. Baicalin-contained culture medium was refreshed every 2 days. Experiment was stopped when the control group formed larger clones. The culture medium was discarded, and cells were washed with phosphate-buffered saline (PBS) and fixed with ethanol for 15–30 min. Then, cells were treated with crystal violet dye at dark for 20 min. After washed with PBS, cell colonies of different groups were photographed and countered with a light microscope (Olympus, Japan).

2.4 Cell apoptosis analysis

Apoptotic rate of CRC cells was evaluated by flow cytometry (FCM) analysis. HCT-116 and CT26 cells which were seeded in 6-well plate were exposed to different concentrations (0, 5, 10, 40 μM) of Baicalin at 37°C for 48 hr. Then, cells were harvested, washed with ice-cold PBS, and stained with apoptosis detection kits (4A Biotech Co., Ltd). Finally, stained cells were applied to flow cytometer (BD Biosciences). Cell apoptosis rate was displayed as sum of proportions of annexin V⁺/PI⁺ and annexin V⁺/PI^-. Data were showed as means ± SD of at least three independent experiments.

2.5 Measurement of cellular reactive oxygen species and mitochondrial membrane potential (Ψm)

After treated with designed concentrations of Baicalin (0, 5, 10, 20 μM) for 48 hr, cells were harvested, washed with PBS, and treated with 10 μM Rh123 at 37°C for 25 min in the dark place. After washed twice with cold PBS, cells were subjected to FCM analysis. For intracellular reactive oxygen species (ROS) detection, cells treated with BA were harvest and incubated with 10 μM DCFH-DA dye for 30 min at 37°C. Stained cells were then harvest, washed twice with cold PBS, and detected by FCM. Data were displayed as means ± SD of at least three independent experiments.

2.6 Wound-healing migration assay

Wound-healing migration assay was exploited to evaluate the migrated ability of cells after treated with Baicalin. CT26 cells were seeded to 6-well plate and incubated for additional time. When cell confluence reached about 80%, a sterile 10 μL pipette tip was used to scrap on the cell monolayer. Then, cell culture medium was replaced by fresh medium containing determined concentrations (0, 5, 10, and 20 μM) of Baicalin. For further pathway activator study, cells were pre-treated with 50 ng/ml of LPS for 2 hr. After incubated for 48 hr, cells were washed, fixed, and imaged by a light microscope (Olympus, Japan). The percentage of migrated cells in control group was considered as 100%.

2.7 Transwell assays

For the cell migration experiment, 100 μL CT26 cells suspension (5 × 10⁵) with no serum and containing different concentrations of Baicalin was seeded into the upper chamber, meanwhile, 600 μL of 10% FBS-containing medium was added to the lower chamber. After incubated for 48 hr at 37°C, the migrated cells in the lower side of membrane were washed, fixed with 95% ethanol, and stained with 0.5% crystal violet. Migrated cells were photographed and counted by using a light microscope. For cell invasion assays, matrigel (dilution: 1:3, 60 μL/well, BD Biosciences) was pre-coated in the upper chamber. The following operations were similar with that of cell migration assays. Migrated/Invasive cells were counted in six randomly selected fields by using a light microscope. Data are showed as migrated and invasive rate in comparison to the control group.

2.8 Western blot analysis

Proteins of Baicalin-treated CRC cells involved in signaling pathway were measured by Western blot experiment. Briefly, HCT-116 and CT26 cells which were exposed to various doses (0, 5, 10, and 20 μM) of Baicalin for 48 hr were harvest and washed twice with cold PBS for protein extraction. The concentrations of proteins in different groups were determined by the Lowry method. Proteins (equal amount) from each group were loaded and separated by sodium dodecyl sulfate-polyacryl amide gel electrophoresis (SDS-PAGE) gels and then transferred with polyvinylidene difluoride membranes (Amersham Bioscience, Piscataway, N.J.). Subsequently, the membranes containing target proteins were treated with 5% non-fat milk for 2 hr at 37°C and incubated with corresponding primary antibodies (dilution, 1:1,000) for overnight at 4°C. After incubated with corresponded stained secondary antibodies, targeted bands in the membrane were visualized by using an enhanced chemiluminescence kit (Amersham Bioscience.).

2.9 Anti-tumor evaluation of Baicalin in xenograft tumor mouse model

All experiments about animal in the present study were approved by the Institutional Animal Care and Use Committee of Chengdu University of Traditional Chinese Medicine. Eighteen female Balb/c mice (6 weeks old, 18–20 g) which were bought from Beijing HFK Bioscience Co, Ltd (Beijing, China) were adapted to the environment for a week prior to experiment. CT26 cells (1 × 10⁶ cells per mouse) suspension was subcutaneously injected into right flank of each mouse. When tumor size grew to about 100 mm³, mice were randomly divided into three groups. Then, all mice in each group were intraperitoneally administered with different concentrations of Baicalin (Control, 20 mg/kg and 40 mg/kg) once daily for 24 days. Mice in the control group were treated with equal amount of corn oil. Tumor volume and body weight of mice in each group were measured and recorded every 3 days. Volume of tumors was calculated as follow: tumor volume (mm³) = 0.5 × length (mm) × width (mm)². At the termination of animal experiment, all mice were euthanized, and tumors from each group were collected, photographed, and weighed.

Main organs (heart, liver, spleen, lung, and kidney) of mouse in each group were collected, fixed in 10% (v/v) neutral-buffered formalin, and sectioned (5 μm) for histopathological evaluation by hematoxylin & eosin assay.

2.10 Immunohistochemistry

At the endpoint of animal experiment, tumors were collected from each group, fixed with 10% (v/v) paraformaldehyde, embedded, and sectioned for immunohistochemistry analysis. Tumor sections were stained with Ki-67, p-IκBα, and PD-L1. Images of immunohistochemistry results were taken with a light microscope (Olympus).

2.11 Tumor immune microenvironment evaluation

After treatment with Baicalin, tumors of each group were isolated and digested to obtain single tumor cell suspension. Subsequently, tumor cells were incubated with CD3, CD4, CD8, Gr-1, and CD11b for 30 min, and subjected to FCM for detection of proportion of CD4⁺/CD8⁺ T cells and myeloid-derived suppressor cells (MDSCs) in tumors.

2.12 Statistical analysis

All experiments in this study were conducted at least in triplicate. Data are displayed as mean ± standard deviation. The software of SPSS 16.0 (SPSS) was exploited to perform statistical analysis. p-values for comparison of two groups were calculated by two-tailed Student's t-test. p-values for statistical significance were indicated as follows: ^*p < .05; ^**p < .01; ^***p < .001.

3.1 Baicalin significantly inhibits viability and proliferation of CRC cells

In order to study the potential pharmacological effect of Baicalin on CRC cells, we firstly assessed the cytotoxicity of Baicalin in HCT-116 and CT26 cells. As displayed in Figure 1b,c, Baicalin significantly suppressed viabilities of HCT-116 and CT26 cells in a dose and treated time-dependent manner. Secondly, we used colony formation experiment to further study the anti-proliferation effect of Baicalin on HCT-116 and CT26 cells. As evidenced by Figure 1d, clonogenic abilities of both HCT-116 and CT26 cells were dramatically suppressed in a dose-dependent manner after treated with BA. Taken together, these results suggested that Baicalin exhibits efficient anti-proliferation activity in CRC cells in a concentration and time-dependent manner.

3.2 Baicalin promotes mitochondria-mediated apoptosis in CRC cells

For the purpose of investigating whether cytotoxicity of Baicalin in CRC cells resulted from apoptosis, Annexin V/PI dual-staining assay was used to detect CRC cells apoptotic level after treatment of Baicalin. As displayed in Figure 2a, the apoptosis induction effects were apparently observed after Baicalin treatment for 48 hr. Compared with control group (14.12 ± 1.26%), 20 μM Baicalin induced 35.83 ± 1.65% apoptosis in HCT-116 cell. Meanwhile, 56.38 ± 1.76% apoptosis was detected in 20 μM–treated CT26 cells in comparison to control group (5.48 ± 0.63%). Results above indicated that Baicalin significantly triggers apoptosis in CRC cells.

To further verify apoptotic induction of Baicalin in CRC cells, changes in apoptosis-associated proteins were evaluated by Western blot assay after exposure to Baicalin for 48 hr. As evidenced by Figure 2b,d, treatment with Baicalin dramatically upregulated expression of cleaved caspase3 in both HCT-116 and CT26 cells, which indicts apoptosis-induced effect of Baicalin. Furthermore, with an increase in BA-treated concentration, the expression of Bax/Bcl-2 ratio elevated distinctly (Figure 2c,e), implying a hypothesis that apoptosis triggered ability of Baicalin might result from mitochondrial destruction.

To verify this hypothesis, changes in the mitochondrial membrane potential was detected by Rh123 stain assay after Baicalin treatment. It can be clearly seen from Figure 3a that treatment of CT26 cells with 20 μM Baicalin leads to a dramatical loss of mitochondrial membrane potential in comparison to control group. In order to further test destruction effect of Baicalin in mitochondrial membrane, level of cellular ROS was measured by DCFH-DA staining after Baicalin treatment. As displayed in Figure 3b, the cellular level of ROS was distinctly upregulated in the dose-dependent manner in CT26 cells by treatment with Baicalin. Taken together, Baicalin triggers apoptosis via mitochondrion-related apoptosis pathway in CT26 cell.

3.3 Baicalin represses migration and invasion of CRC cells by suppressing TLR4/NF-κB signaling pathway

To investigate whether Baicalin can influence migration and invasion of CRC cells, wound-healing, transwell migration, and invasion assays were performed in CT26 cells after treated with determined dose of Baicalin for 48 hr. As shown in Figure 4a, Baicalin treatment can significantly suppress wound-healing ability of CT26 cells. Moreover, migration and invasion abilities of CT26 were dramatically inhibited in a concentration-dependent manner after treatment with different dose of Baicalin (Figure 4b,c).

For the purpose of further investigating the suppressed effect of Baicalin on migration and invasion of CT26 cells, Western blot experiment was used to measure changes in expression of cell metastasis-associated proteins after treated with Baicalin. As displayed in Figure 4c, Baicalin-treated CT26 cells exhibited higher expression of TIMP-2, and lower expression of MMP-2 and MMP-9 in comparison to control group, indicating a good anti-migration effect. Furthermore, compared with control group, expression levels of TLR4, NF-κB p65, and p-IκBα in CT26 cells were also significantly downregulated by treatment with Baicalin, suggesting an impairment of TLR4/NF-κB signaling pathway in CT26 cells (Figure 4d). Because of important role of NF-κB signaling pathway in transcription of PD-L1, we also measured whether Baicalin inhibits PD-L1 expression in CRC cells. As displayed in Figure 4d, Baicalin treatment remarkably inhibited expression level of PD-L1 in CT26 cells, which suggests that inhibition of NF-κB signaling pathway by Baicalin might ameliorate tumor immunosuppressive environment in vivo. As shown in Figure S1, mobility of CT26 cells was suppressed by treatment of 20 μM of Baicalin. However, inhibited effect of Baicalin was attenuated by pre-treated with LPS, an activator of TLR4. Taken together, these data suggested that Baicalin suppressed CRC cells migration and invasion by suppressing TLR4/NF-κB signaling pathway.

3.4 Baicalin exerts efficient antitumor efficacy by enhancing antitumor immunity via NF-κB signaling pathway

Because of the good apoptosis-induced effect of Baicalin in CRC cells in vitro, subcutaneously xenograft tumor mouse model of CT26 cells was established to assess anti-tumor activity of Baicalin in vivo. We can find from Figure 5a that administration of Baicalin significantly retarded tumor growth rate of CT26 cells in comparison to control group. At the endpoint of in vivo experiment, tumors of mice in each group were collected, and tumor size and weight were found to be much lower in Baicalin-treated groups (Figure 5b,c) than that of control group. Immunohistochemistry analysis of tumor sections of mice in each group was conducted to investigate anti-tumor mechanism of Baicalin. As shown in Figure 5d, Ki-67 positively stained cells in Baicalin-treated groups were fewer than that in control group, suggesting Baicalin-inhibited proliferative ability of colorectal tumor cells. Moreover, expression level of TLR4 and p-IκBα, key proteins of NF-κB signaling pathway, was dramatically downregulated in tumor section of Baicalin-treated groups, indicating that activation of NF-κB was inhibited by treating with Baicalin.

Inspired by the inhibited effect of Baicalin on expression of PD-L1 in CT26 cells in vivo, we further investigated whether Baicalin could suppress PD-L1 expression in vivo. As displayed in Figure 5d, significantly downregulated expression level of PD-L1 was observed in tumor section of tumors in 20 and 40 mg/kg treatment groups, implying remission of tumor immunosuppressive microenvironment. Therefore, we collected and digested isolated tumors of mice in each group to test variation of immune cells. As indicated in Figure 5f,g, proportion of myeloid-derived suppressor cells (MDSCs), which could negatively regulate system immunity, was remarkably downregulated in BA-treated groups. Furthermore, proportion of CD4⁺ and CD8⁺ T cells was significantly upregulated after Baicalin treatment (Figure 5e,h), indicating an improvement of anti-tumor immunity. Above all, our data suggested that Baicalin exerts efficient antitumor efficacy in colorectal cancer by improving antitumor immunity via NF-κB signaling pathway.

3.5 Safety evaluation of Baicalin in xenograft tumor mouse model

Systematic safety of Baicalin was evaluated in the treatment process in tumor mouse model of CT26. Compared with control group, the body weight of the mice in BA-treated groups did not display any abnormal variation during the therapy process (Figure 6a). All tumor-bearing mice were killed at the end of the experiment, and major organs (heart, liver, spleen, lung, and kidney) were collected and subjected to histopathological examination. No significant pathological changes were found following administration of Baicalin in comparison to control group (Figure 6b). Above all, Baicalin may be a safe agent for clinical therapy.