Introduction

Gallbladder cancer (GBC), which has an incidence of approximately 2.5 cases/100,000 population, is one of the most common biliary malignancies and the seventh most common malignancy of the digestive tract1,2. The incidence of GBC presents considerable geographical differences, and it is more common in numerous developing countries3. The geographical, ethnic and cultural differences in the incidence of GBC indicate that genetic variation and environmental factors play important roles in its development and progression4,5. Because of its high degree of malignancy and poor prognosis, the exploration of new therapeutic methods, such as targeted therapy, immunotherapy and traditional Chinese medicine (TCM), as alternatives to surgery, chemotherapy and radiotherapy is urgently needed.

Berberine (BBR), which is also called Huangliansu in Chinese, is a quaternary ammonium alkaloid that is isolated from Coptis chinensis. Berberine hydrochloride (BH) is the hydrochloride salt of berberine, with the chemical name 5,6-Dihydro-9,10-dimethoxybenzo[g]−1,3-benzodioxolo[5,6-a]quinolizinium chloride and the chemical formula C20H18ClNO4. Many studies have shown that berberine hydrochloride exerts therapeutic effects on diabetes, hypertension, depression, obesity, inflammation, etc6,7,8,9,10. Recently, several in vivo and in vitro studies have shown that berberine hydrochloride can inhibit the proliferation and migration of various types of tumor cells, indicating its considerable potential for the treatment of malignancies11,12,13. Studies14 have shown that the metabolism of berberine hydrochloride in the small intestine and liver after oral absorption is the main reason for its low concentration in vivo. However, after 48 h, 22.8% of the initial berberine hydrochloride content can be recovered from bile15,16; thus, the direct administration of berberine hydrochlorideto treat biliary disease is very feasible and effective.

In addition, some studies17,18 have confirmed that the main metabolite of berberine, jatrorrhizine, is an anti-inflammatory compound. Therefore, berberine and its metabolites can reduce proinflammatory factor production, increase anti-inflammatory factor and proinflammatory mediator production, and regulate the inflammatory immune response through chemokines in different cellular inflammatory models.

Interleukin-6 (IL-6) plays major roles in inflammation, autoimmunity and cancer, with its effect mainly exerted through the IL-6 signal transducer and activator of transcription 3 (STAT3) pathway19,20. Chen21 reported that GBC-derived IL-6 activated STAT3 and thereby upregulated IL-6 expression. Meanwhile, IL-6 acted back on the GBC causing further activation of STAT3 and leading to enhanced downstream events that promote proliferation, migration, invasion and apoptosis resistance of the GBC. Therefore, the IL-6/STAT3 pathway is crucial to the pathological processes of GBC, and blocking it may inhibit GBC development.

In this study, we showed that berberine hydrochloride affected the cell cycle progression, apoptosis, metastasis and invasion of GBC cells by regulating the IL-6/STAT3 pathway and triggering an inflammatory response, ultimately leading to apoptosis. These findings suggested that berberine hydrochloride is critical for the development of GBC via the IL6/STAT3 axis, and BH may be a useful drug for the prevention and treatment of GBC.

Materials and methods

Cell culture and reagents

Two human GBC cell lines (GBC-SD and NOZ) were obtained from the Cell Bank of Fuheng Biology (Shanghai, China). GBC-SD and NOZ cells were cultured in complete RPMI-1640 medium and DMEM at 37 °C in 5% CO2.

The GBC-SD cell line was characterized through short tandem repeat profiling by Genetic Testing Biotechnology Corporation (Suzhou, China). The NOZ cell line was characterized by Fuheng Biology (Shanghai, China). The GBC-SD cell line harbors mutant p53, and the NOZ cell line harbors wild-type p53, as shown in many studies and specific databases22,23,24.

Berberine hydrochloride (020-05502, Wako, Japan, Purity: ≥97.0%) was dissolved in DMSO at a concentration of 100 mg/ml and stored in the dark at −20 °C. Owing to its sensitivity to light and short half-life, berberine hydrochloride was diluted for administration, and administration was repeated every 24 h in the following experiments.

IL-6 was purchased from Chamot (Shanghai, China). Tocilizumab was obtained from MedChemExpress (New Jersey, USA).

Cell viability assay

Cell viability was measured via a Methylthiazolyldiphenyl-tetrazolium bromide (MTT; C0009S, Beyotime Biotech, Shanghai, China) assay. Cells were seeded in 96-well plates at a density of 5 × 103 cells per well. After the cells had adhered to the plates, the medium was replaced, and the cells were incubated with medium containing berberine hydrochloride (10, 20, 50, or 100 mg/L) for 24, 48, or 72 h at 37 °C in 5% CO2. The absorbance was measured at a wavelength of 570 nm with a microplate reader (SpectraMax i3, Molecular Devices, Austria).

Colony formation assay

For colony formation assays in 2D culture, 500 cells were cultured in 10 cm dishes and treated with 50 mg/L BH, with DMSO used as the negative control (NC) for 2 weeks at 37 °C in 5% CO2. The cells were fixed using paraformaldehyde, stained with Giemsa, and observed under a microscope (Nikon, Tokyo, Japan). Colony formation rate=(number of colonies/number of seeded cells) * 100%.

Apoptosis and cell cycle analyses

Flow cytometry was used to determine the percentage of apoptotic cells after staining with the components of an Annexin V-FITC/PI Apoptosis Detection Kit (BB-4101, BestBio, Shanghai, China). 1 × 105 cells per well were seeded in 6-well plates and incubated with different treatments. Then, the cells were incubated with FITC Annexin V and PI for 15 min in the dark and analyzed with a flow cytometer (BD Biosystems, Heidelberg, Germany).

Cell cycle progression was analyzed by flow cytometry with a Cell Cycle and Apoptosis Analysis Kit (C1052; Beyotime Biotech, Shanghai, China). All the cells were incubated with different treatments in 6-well plates, harvested, incubated with PI for 15 min in the dark and analyzed with a flow cytometer (BD Biosystems, Heidelberg, Germany).

Scratch assay

Cells were cultured in 6-well plates, and the cell layer was then scratched with a sterile pipette tip to make a cell-free straight line. The cells were subsequently washed and cultured with FBS-free medium supplemented with mitomycin and subjected to the indicated treatments. After incubation for 0 h, 24–72 h, the cells were photographed under a microscope (Nikon, Tokyo, Japan).

Cell invasion and migration assays

GBC cells were treated with berberine hydrochloride (50 mg/L) for 48 h and were then trypsinized and resuspended. The cell suspensions (100 µl, 105 cells) were added to the upper chambers of Transwell plates containing a membrane with or without a Matrigel coating (Corning, NY, USA), and medium supplemented with 10% FBS was added to the lower chambers. After incubation for 24 h, the cells located on the lower surface of the membrane were stained with 4’,6-diamidino-2-phenylindole (DAPI; Beyotime Biotech, Nanjing, China) or 0.1% Crystal Violet Staining Solution (Solarbio, Beijing, China) and counted under a fluorescence microscope (Nikon, Tokyo, Japan).

Cellular Immunofluorescence staining

Cells were seeded on slides in 6-well plates. After culture for 48 h, the slides were washed twice with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% bovine serum albumin (BSA) at room temperature (RT) and then incubated at RT for 30 min with rhodamine phalloidin (PHDR1, Cytoskeleton, Denver, USA) at a dilution of 1:200. Nuclei were counterstained with DAPI. Images were acquired with a fluorescence microscope.

Human apoptosis proteome profiler array and cytokine secretion measurement

To investigate the pathways by which berberine hydrochloride induces apoptosis, a Human Apoptosis Proteome Profiler Array (AAH-APO-G1-8, RayBiotech, GA, USA) was used to measure the levels of apoptosis-related proteins according to the manufacturer’s instructions. Secreted factors were analyzed with the Quantibody Human Inflammation Array 3 Kit (GSH-INF-3-1; RayBiotech, GA, USA) following the manufacturer’s instructions.

Western blot (WB) analysis

Western blotting was performed as we described previously25. Cells were lysed in RIPA buffer (BC3710-50T, Solarbio, Beijing, China) to extract proteins. A bicinchoninic acid (BCA) assay (Thermo Fisher) was used to measure the protein concentration. After denaturation at 100 °C, the proteins were separated via 4–20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes, which were blocked for 1–2 h with 5% BSA. The membranes were incubated with primary antibodies overnight at 4 °C. Then, the PVDF membranes were washed with TBST and incubated with secondary antibodies (goat anti-rabbit/mouse Ig, diluted 1:5000) for 1 h at room temperature. After washing with TBST, enhanced chemiluminescence (ECL) solution was added to visualize the signals with ChemiDoc Imaging System (Bio-Rad, California, USA). Antibodies specific for β-Tubulin and GAPDH were used as negative controls. The primary antibodies are as follows:

caspase-3 (1:1000, AF6311, Affinity, China), caspase-8 (1:1000, AF6442, Affinity, China), XIAP (1:1000, AF6368, Affinity, China), cleaved caspase-3 (1:1000, Asp175, Cell Signaling Technology, USA), cleaved caspase-8 (1:1000, Asp384, Cell Signaling Technology, USA), β-tubulin (1:1000, 2146, Cell Signaling Technology, USA), GAPDH (1:1000, 5174, Cell Signaling Technology, USA), STAT3 (1:500, F-2, Santa Cruz Biotechnology, USA), p-STAT3 (1:500, B-7, Santa Cruz Biotechnology, USA).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

The medium was discarded from each group of cells, the cells were washed twice with PBS, and RNA was extracted with a SPARKeasy Cell RNA Rapid Extraction Kit (AC0205-B, SparkJade, Shandong, China). Reverse transcription was performed with 1 µg of total RNA. Real-time qPCR was performed with an ABS-7500 real-time PCR system (Invitrogen Life Technologies). GAPDH was used as the internal control. Primer sequences were displayed in (Supplementary Table 1).

Enzyme linked immunosorbent assay (ELISA)

The cell culture medium of each treatment group was collected and centrifuged at 3000 rpm for 20 min at 4 °C. The supernatants were collected for subsequent experiments. Cytokine concentrations were determined in accordance with the instructions of the RayBio® Human ELISA Kit (ELH-IL-6/8/11).

Liver xenograft model in nude mice

The animal experiment was conducted with the approval of the ethics committee of Linyi People’s Hospital and all methods are reported in accordance with ARRIVE guidelines. A total of 12 male BALB/c nude mice (4 weeks old) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Mice were inoculated with GBC-SD cells (1 × 107 cells/mice) in the right forelimbs, and the entire tumors were harvested, cut into pieces of the same size, and implanted into the livers of recipient nude mice. The nude mice were randomized into 2 groups (negative control, oral administration (BH, 50 mg/kg.d)). Tumor volume was assessed every 2 days for 2 weeks via ultrasound as described by Aoki S et al.26.

The tumor volume was calculated via the following formula: (short diameter2 × long diameter)/2.

Statistical analysis

All the data were processed with SPSS statistical software (version 21.0; IBM Corp., Armonk, NY, USA) and are presented as the means ± standard deviations (SDs). The chi-square test and Student’s t test were performed to compare data between two different groups. Pearson correlation analysis was used to analyze associations between gene expression levels. Differences with p < 0.05 were considered statistically significant. The graphs were generated with GraphPad Prism (version 7.0; GraphPad Software, Inc., USA), ModFit LT (version 5.0; Verity Software House, USA), FlowJo (version 10.8.1, BD Biosciences, USA) and Image J (version Fiji; National Institutes of Health; USA).

Results

Berberine hydrochloride inhibits the proliferation of GBC cells

To determine whether berberine hydrochloride exhibits suppressive effects on GBC, we first aimed to evaluate its effects on cell proliferation by viability and colony formation assays.

MTT assay was used to analyze cell viability. The degree to which berberine hydrochloride decreased the viability of GBC-SD and NOZ cells was increased at the 24 h, 48 h and 72 h time points. The half maximal inhibitory concentration (IC50) of BH at 48 h was 40.45 mg/L for GBC-SD and 61.67 mg/L for NOZ cells. We found that with increasing concentration and time, berberine hydrochloride significantly inhibited the proliferation of GBC cells, especially GBC-SD cells (p < 0.001, Fig. 1A/B). We hypothesized that the p53 mutant GBC-SD cell line would be more sensitive to berberine hydrochloride treatment than the p53 wild-type NOZ cell line. On the basis of the above experimental results, a concentration of 50 mg/L berberine hydrochloride was selected for subsequent experiments.

Fig. 1
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Berberine hydrochloride inhibits the proliferation, leads to cell cycle changes and increases apoptosis of GBC cells. A/B, GBC-SD and NOZ were treated with the indicated concentration of berberine hydrochloride for 24 h, 48 h, 72 h and examined via an MTT assay. C/D/E, few colonies were formed by GBC cells in the berberine hydrochloride-treated group: C, GBC-SD cell line; D, NOZ cell line. F/G/H/I, berberine hydrochloride induced G2/M- and S-phase arrest, with decreases in the numbers of G1-phase GBC cells in the berberine hydrochloride-treated groups compared with the NC groups: F/H, GBC-SD cell line; G/I, NOZ cell line. J/K/L, the percentages of apoptotic GBC cells in the berberine hydrochloride-treated groups were greater than those in the NC groups: J, GBC-SD cell line; K, NOZ cell line. All data represent the mean ± SD, n = 6 (A/B), n = 3 (C/D/E/F/G/H/I/J/K/L). ***P < 0.001. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride); IC50, half maximal inhibitory concentration.

Colony formation rates in 2D culture were evaluated by microscopy, and the results revealed that the numbers of colonies in the berberine hydrochloride-treated group compared with the NC group were 1.0 ± 0.89 vs. 10.6 ± 1.85 for GBC-SD cells and 2.8 ± 0.40 vs. 10.2 ± 2.23 for NOZ cells (p < 0.001 for both; Fig. 1C/D/E). In both cell lines, the number of colonies in the berberine hydrochloride-treated group was lower than that in the NC group, indicating the inhibitory effect of berberine hydrochloride and confirming the results of the MTT assay.

Berberine hydrochloride treatment leads to changes in cell cycle progression

As shown by flow cytometry, berberine hydrochloride induced G2/M- and S-phase arrest, and decreased the proportion of cells in the G1 phase compared with that in the NC group in both the GBC-SD and the NOZ cell lines (p < 0.001 and p < 0.01, Fig. 1F/J/H/I).

Berberine hydrochloride increases GBC cell apoptosis

By flow cytometry, we found that the percentages of apoptotic cells in the berberine hydrochloride group compared to the NC group were 43.85 ± 7.30% vs. 8.07 ± 0.54% and 23.14 ± 1.61% vs. 8.06 ± 0.87% for GBC-SD and NOZ cells (p < 0.001 for both, Fig. 1J/K/L). These findings indicate that berberine hydrochloride may more strongly promote apoptosis in GBC-SD cells than in NOZ cells, suggesting that p53 mutation may play an important role in berberine hydrochloride-induced apoptosis.

Berberine hydrochloride inhibits GBC cell invasion and metastasis

The results of scratch assays revealed that the migration of berberine hydrochloride-treated GBC cells was significantly decreased compared with that of negative control cells at 24 h and 72 h (p < 0.01 and p < 0.001 for GBC-SD and NOZ cells, respectively; Fig. 2A/B/D). Furthermore, cell morphology was examined, and the cell number and ruffling activity were found to be decreased in the berberine hydrochloride group compared with the NC group at 24 h; furthermore, as shown by time-lapse imaging, the invasion and metastasis of GBC-SD cells increased even further (Fig. 2C).

Fig. 2
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Berberine hydrochloride inhibits GBC cells invasion and metastasis. A/B/D, A scratch assay revealed that the migration of berberine hydrochloride-treated GBC cells was significantly decreased compared with that of negative control GBC cells: A, GBC-SD cell line; B, NOZ cell line. C, the number of cells and degree of ruffling activity were lower in the berberine hydrochloride-treated groups than in the NC groups. E/F/G/H, Transwell assays demonstrated that berberine hydrochloride reduced the migration and invasion potential of GBC cells compared with that of the corresponding control cells: E/G, migration; F/H, invasion. All data represent the mean ± SD, n = 3. **P < 0.01, ***P < 0.001. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride).

Transwell assays revealed that berberine hydrochloride reduced the migration of GBC-SD cells and NOZ cells to 50.03 ± 1.67% and 3.10 ± 0.63% of that in the corresponding NC groups, respectively, and decreased the invasion potential of GBC-SD cells and NOZ cells to 52.53 ± 3.48% and 8.56 ± 0.88%, respectively (both p < 0.001, Fig. 2E/F/G/H).

Berberine hydrochloride leads to cytoskeletal rearrangement

To determine the effect of berberine hydrochloride on cytoskeletal dynamics, actin was stained with rhodamine phalloidin. After treatment with berberine hydrochloride, actin filaments were distributed in a disordered arrangement, and no marked stress fiber formation was observed, indicating that berberine hydrochloride inhibits actin distribution (Fig. 3A/B). Furthermore, the cells contained few stress fibers at the end of mitosis (Fig. 3C), and the cells that underwent apoptosis did not contain intact cell membranes or disordered fibers (Fig. 3D). These results confirmed the morphological changes that were observed in the scratch assay described above.

Fig. 3
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Berberine hydrochloride leads to cytoskeletal rearrangement. A/B, berberine hydrochloride inhibits actin distribution in GBC cells: A, GBC-SD cell line; B, NOZ cell line. C, cells contained few stress fibers at the end of mitosis. D, apoptotic cells do not contain intact cell membranes or disordered fibers. Actin was stained with phalloidin. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride).

Mechanism underlying the effect of Berberine hydrochloride on GBC cells according to the protein arrays

To determine the mechanism by which berberine hydrochloride affects GBC cells, we used two protein arrays: an apoptosis proteome profiler array to analyze cellular proteins, and an inflammation array to analyze cell supernatants.

As shown by the apoptosis array and western blot analyses, berberine hydrochloride increased the activation of caspase-8 and caspase-3 and inhibited the expression of XIAP in GBC-SD cells (p < 0.001, adj. p < 0.05; Fig. 4A/B/C; Fig. 8A/C/D/E). These results proved that berberine hydrochloride can activate caspase-8 and in turn activate caspase-3 to induce apoptosis, while the decrease in XIAP expression attenuates the inhibition of this process. The combination of these two effects resulted in GBC-SD cell apoptosis. GO and KEGG enrichment analyses revealed that the cysteine-type endopeptidase activity-mediated apoptotic signaling pathway and the p53 signaling pathway were obviously related to the changes in the levels of these proteins (Fig. 4D/E/F/G)27,28,29. The results related to caspase-3/8 levels were not consistent between the array and western blot analyses, possibly partially because cleaved caspases are functional.

Fig. 4
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Mechanism by which berberine hydrochloride affects GBC cells according to apoptosis proteome profiler array analysis. A/B/C, berberine hydrochloride can increase the activation of caspase-8 and caspase-3 and inhibit the expression of XIAP in GBC-SD cells: A, GBC-SD cell line; B, NOZ cell line. D/E/F/G, GO and KEGG analyses revealed that the cysteine-type endopeptidase activity-related apoptotic signaling pathway and the p53 signaling pathway were obviously related to the differences in the levels of these proteins. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride).

According to the inflammation array and qPCR analysis results, berberine hydrochloride reduced the levels of IL-6, interleukin-8 (IL-8), and interleukin-11 (IL-11) in both GBC cell lines, especially in the p53 mutant GBC-SD cell line (p < 0.01, adj. p < 0.05; Fig. 5A/B/C; Fig. 6A/B/C). The GO and KEGG enrichment analysis results revealed that the cytokine-cytokine receptor interaction pathway, which includes IL-6 and its downstream JAK/STAT signaling pathway, was highly enriched (Fig. 5D/E/F/G)27,28,29. In addition, we further analyzed the relationships among IL-6, IL-8 and IL-11 expression in GBC cells. First, we confirmed the concentrations of inflammatory factors in the supernatants of the NC group and the berberine hydrochloride group via ELISA and concluded that, compared with those in the corresponding NC groups, the concentrations of IL-6, IL-8, and IL-11 in the supernatants of GBC-SD cells and NOZ cells that were treated with berberine hydrochloride were decreased to varying degrees (p < 0.05, Fig. 6D/E/F). We speculated that changes in the IL-8 and IL-11 concentrations may be induced after supplementation with IL-630. So we added IL-6 (200 ng/ml) to berberine hydrochloride-treated cells and found that the concentrations of IL-8 and IL-11 in the supernatants of GBC cells were changed (Fig. 6G/H). Then, tocilizumab31 (Anti-Human IL-6R, 65 µg/ml) was added to BH-treated cells. However, the concentrations of IL-8 and IL-11 in the supernatants of GBC cells did not change significantly (Fig. 6I/J). Tocilizumab may block the IL-6 pathway, resulting in the absence of changes in the IL-8 and IL-11 concentrations. These results suggest that IL-6 is a pivotal target of BH in the inhibition of GBC.

Fig. 5
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Mechanism by which berberine hydrochloride affects GBC cells according to the inflammation array analysis. A/B/C, berberine hydrochloride reduced the concentrations of IL-6, IL-8, and IL-11 in GBC cell supernatants: A, GBC-SD cell line; B, NOZ cell line. D/E/F/G, GO and KEGG enrichment analyses revealed that the cytokine-cytokine receptor interaction pathway, which includes IL-6 and its downstream JAK/STAT signaling pathway, was highly enriched. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride).

Fig. 6
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IL-6 might be a target of BH in GBC. A/B/C, berberine hydrochloride reduced the mRNA levels of IL-6, IL-8, and IL-11 in GBC cell lines. D/E/F, the concentrations of IL-6, IL-8, and IL-11 in the berberine hydrochloride-treated group were decreased to varying degrees. G/H, after the addition of IL-6 to the BH group, the concentrations of IL-8 and IL-11 in the supernatant of GBC cells were altered. I/J, tocilizumab was added to BH-treated GBC cells, and the concentrations of IL-8 and IL-11 in the supernatants were not significantly altered. All data represent the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.001, ns, no significance. NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride); BH + IL6, IL-6 (200 ng/ml) was added to the cells treated with berberine hydrochloride (50 mg/L); Toci, tocilizumab; BH + Toci, tocilizumab (65 µg/ml) was added to the cells treated with berberine hydrochloride (50 mg/L).

BH inhibits GBC growth through IL-6

To determine whether BH inhibits GBC by targeting IL-6, we evaluated the effects of IL-6 supplementation on apoptosis via flow cytometry and on cell migration and invasion via transwell assays. The addition of IL-6 (200 ng/ml) suppressed apoptosis in GBC cells (p < 0.05, Fig. 7A/B/C) and increased the rates of migration (p < 0.01, Fig. 7D/F) and invasion (p < 0.05, Fig. 7E/G) compared with that in the group treated with BH only. These findings suggest that BH induces growth arrest in GBC cells by targeting IL-6.

Fig. 7
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BH inhibits GBC growth through IL-6. A/B/C, the addition of IL-6 to GBC cells suppressed apoptosis compared with that in the group treated with BH only: A, GBC-SD cell line; B, NOZ cell line. D/E/F/G, the addition of IL-6 to GBC cells increased the migration and invasion rates compared with those in the group treated with BH only: D/F, migration; E/G, invasion. All data represent the mean ± SD, n = 3. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.001. BH, berberine hydrochloride (cells were treated with berberine hydrochloride); BH + IL6, IL-6 (200 ng/ml) was added to the cells treated with berberine hydrochloride (50 mg/L).

Fig. 8
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BH inhibits the IL-6/STAT3 pathway. A/C/D/E, berberine hydrochloride increased the activation of caspase-8 and caspase-3 and inhibited the expression of XIAP in GBC-SD cells. B/F, BH decreased the level of p-STAT3 in GBC cells. G/H, the decrease in tumor volume in the oral administration group was greater than that in the negative control group. All data represent the mean ± SD, n = 6 (G/H), n = 3 (A/C/D/E). *P < 0.05, **P < 0.01, ***P < 0.001, ns, no significance. GBC, gallbladder cancer; NC, negative control (cells were treated with DMSO); BH, berberine hydrochloride (cells were treated with berberine hydrochloride). Original blots/gels are presented in Supplementary Fig. 1. The samples (p-STAT3, STAT3) derive from the same experiment and that gels/blots were processed in parallel.

BH inhibits the IL-6/STAT3 pathway in vitro

In the tumor microenvironment, IL-6/STAT3 signaling drives the proliferation, survival, invasion, and metastasis of tumor cells, and IL-6 acts directly on tumor cells to induce the expression of STAT3 target genes30. To determine whether the IL-6/STAT3 pathway is a target of BH, we measured the protein levels of STAT3 and its phosphorylated form in GBC cells. GBC cells were treated with BH, with DMSO used as the negative control. BH effectively decreased the protein levels of IL-6 and p-STAT3 in GBC cells, especially in the p53 mutant GBC-SD cell line (p < 0.05, Figs. 6D and 8B/F). Thus, these findings indicate that BH significantly modulates the IL-6/STAT3 pathway.

BH inhibits GBC formation in vivo

A nude mouse xenograft model was established with GBC-SD cells. After treatment with berberine hydrochloride by oral administration, the transplanted liver tumors were smaller than those in the NC group (mice treated with DMSO by oral administration).

Discussion

In vivo, berberine can directly interact with nucleic acids and several proteins, such as telomerase, DNA topoisomerase 1 (Top1) and p53. In general, berberine can promote cell cycle arrest and increase the expression of apoptotic factors in human cancers32,33,34. In this study, we can conclude from apoptosis assay and western blot analysis results that berberine hydrochloride can significantly induce apoptosis in p53 mutant GBC-SD cells in vivo and in vitro by reducing XIAP expression and increasing caspase-8 and caspase-3 expression. XIAP is an inhibitor of apoptosis, and it represses and regulates the last step of apoptotic signal transduction; additionally, it plays an important role in the occurrence of tumors because it can protect malignant tumor cells against apoptosis35. Studies have reported that berberine hydrochloride reduces the viability of p53-deficient EU-4 and p53 mutant EU-6 acute lymphoblastic leukemia cells and induces apoptosis by downregulating XIAP36. Therefore, we speculate that berberine hydrochloride can significantly promote apoptosis by inhibiting the expression of XIAP and by directly/indirectly increasing the expression of caspase-3/8 in GBC-SD cells. In general, berberine hydrochloride can be used as a molecular inhibitor of XIAP to suppress antiapoptotic mechanisms in GBC-SD cells, and it can activate caspase-8 to increase extrinsic apoptosis and induce GBC-SD cell death.

Through inflammatory array analysis, qPCR and ELISA, we verified that berberine hydrochloride can significantly reduce the expression of IL-6 in GBC cell lines. After IL-6 or tocilizumab was added to BH-treated cells, the concentrations of IL-8 and IL-11 in the supernatants of GBC cells changed differently. Based on the above results, IL-6 was confirmed to be an essential target for BH in GBC treatment.

In some malignant tumors, IL-6 is considered a predictive biomarker for a worsening condition and poor prognosis37,38. Some studies39 have confirmed that activation of the IL-6 signaling pathway in cancer cells can induce tumorigenesis by inhibiting apoptosis and promoting survival, proliferation, angiogenesis, metastasis and invasion. To determine whether BH inhibits gallbladder cancer by targeting IL-6, we supplemented the BH-treated groups with exogenous IL-6 and co-incubated them. We found that IL-6 stimulation markedly inhibited cell apoptosis and promoted cell migration and invasion in GBC cells. Following combined IL-6 intervention, the pro-apoptotic effects induced by berberine hydrochloride in gallbladder cancer cells were partially reversed, and the addition of exogenous IL-6 partially restored the migratory and invasive capabilities of the tumor cells. These findings indicate that BH suppresses the growth of gallbladder cancer cells by targeting IL-6.

IL-6 has been shown to activate the STAT3 signaling pathway, and hyperactivation of STAT3 signaling occurs in the majority of human cancers and is correlated with poor prognosis30,40. Therefore, inhibition of the IL-6/STAT3 pathway can help to prevent tumor cell proliferation, migration and invasion as well as accelerate tumor cell apoptosis. The entry of p-STAT3 into the nucleus affects the progression and development of tumors by regulating the expression of various proteins involved in tumor proliferation, survival and differentiation and the occurrence of substantial crosstalk between inflammatory and apoptotic initiator caspases41,42. In our study, WB results demonstrated that the protein expression level of p-STAT3 decreased in the BH-treated groups, indicating that BH inhibits STAT3 phosphorylation in gallbladder cancer cells. Following BH treatment in GBC-SD and NOZ cells, the concentration of IL-6 was significantly reduced, which likely disrupted the phosphorylation process of STAT3. BH may suppress the secretion of IL-6, thereby interrupting its activation signaling to STAT3. The reduction in IL-6 levels diminished the opportunity for IL-6 to bind to its receptors, leading to decreased STAT3 phosphorylation and reduced p-STAT3 protein levels in gallbladder cancer cells. These results reveal that berberine hydrochloride exerts antitumor effects by inhibiting the IL-6/STAT3 signaling pathway. Targeting the IL-6/STAT3 pathway may serve as a potential therapeutic strategy for gallbladder cancer, while elevated expression of p-STAT3 or increased serum IL-6 levels in GBC patients could act as prognostic biomarkers for clinical evaluation.

This study has several limitations. In the present study, berberine hydrochloride had more significant inhibitory effects on malignant behaviors in the p53 mutant GBC-SD cell line than in the p53 wild-type NOZ cell line, and these findings were consistent with the results of our array analyses. Therefore, we speculate that the mechanism through which berberine hydrochloride exerts these effects is related to p53. The relationship between berberine hydrochloride and p53 mutation is still unclear. Therefore, the specific effects of berberine hydrochloride on mutant p53 and wild-type p53 still need further research.

Conclusions

Berberine hydrochloride inhibited the growth of GBC by inducing apoptosis and G2/M- and S-phase arrest and by inhibiting the migration and invasion of GBC cells, as shown in vitro, and suppressed the growth of GBC-SD xenograft tumors in vivo. Moreover, BH effectively suppressed the expression of IL-6 and the activation of STAT3 mediated by IL-6. The results of this study reveal that berberine hydrochloride inhibits the proliferation and metastasis of GBC cells by inhibiting the IL-6/STAT3 pathway. Therefore, BH may be a useful drug for the prevention and treatment of GBC.