L-Fucose inhibits the progression of cholangiocarcinoma by causing microRNA-200b overexpression : Chinese Medical Journal

Secondary Logo

Journal Logo

Original Article

L-Fucose inhibits the progression of cholangiocarcinoma by causing microRNA-200b overexpression

Zhu, Biqiang1; Zheng, Jingjing2; Hong, Gaichao1; Bai, Tao1; Qian, Wei1; Liu, Jinsong1; Hou, Xiaohua1

Editor(s): Ji, Yuanyuan

Author Information
Chinese Medical Journal ():10.1097/CM9.0000000000002368, January 10, 2023. | DOI: 10.1097/CM9.0000000000002368
  • Open
  • PAP

Abstract

Introduction

Cholangiocarcinoma (CCA) is a highly malignant form of cancer exhibiting features of cholangiocyte differentiation.[1,2] Despite the continuous development of cancer therapy, the 5-year survival rate of patients with CCA remains at only 5% to 15%, which has not increased significantly.[3,4] L-fucose is a natural deoxyhexose sugar with a similar structure to glucose, except for its lack of a hydroxyl group on carbon 6 [Figure 1A], which has been reported to present diversified biological activities through various mechanisms.[5-7] However, the effect of L-fucose on the development of CCA has not been well studied.

F1
Figure 1:
Inhibitory effect of L-fucose on the proliferation. (A) Chemical structure of L-fucose. (B) Cell viability was determined. (C) Representative images of colonies of cells. (D) Apoptotic cells were assessed. (E) Cell cycle analysis was performed. (F) Related protein levels were assessed. Experiments were performed at least three times. P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P, ††† P, ‡‡‡ P < 0.001 vs. the control. Annexin V-FITC: annexin V-fluorescein isothiocyanate; PI: propidium iodide; PI-A: peak intensity-area; BCL2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein; CDK2: cyclin-dependent kinase 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

MicroRNAs (miRNAs), small noncoding RNAs, are related to multiple human diseases and are treated as clinical diagnostics and therapeutic targets.[8] Various miRNAs with differential expression have been identified, and numerous studies have demonstrated that miRNAs take either oncogenic or tumor-suppressive roles in the progression of CCA.[9-11] Accordingly, adjusting specific miRNAs provides distant insight into CCA therapy. A recent clinical study revealed that a total of 53 miRNAs were differentially expressed (15 upregulated, 38 downregulated) in individuals treated with fucoidan, a marine-origin sulfated polysaccharide mainly composed of L-fucose, compared to placebo-treated individuals.[12] Thus, we hypothesize that L-fucose may exert antitumor activity on CCA cells by regulating miRNAs.

This study was designed to evaluate the anti-proliferation, anti-metastasis, and anti-angiogenesis roles of L-fucose in CCA cells and to elucidate the potential molecular mechanism of drug antitumor activity.

Methods

Reagents

L-Fucose was obtained from Sigma (St. Louis, MO, USA). AX-15836 and Colivelin were purchased from MedChemExpress LLC (Shanghai, China). Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) were purchased from BD (Franklin Lakes, NJ, USA). Anti-cleaved caspase 3, anti-cleaved caspase 9, anti-B-cell lymphoma 2 (BCL2), anti-Bcl-2-associated X protein (Bax), anti-E-cadherin, anti-vimentin, anti-N-cadherin, anti-phosphorylated signal transducer and activator of transcription 3 (p-STAT3), anti- signal transducer and activator of transcription 3 (STAT3), anti-extracellular signal-regulated kinase 5 (ERK5, MAPK7), anti-cyclinD1, anti-cyclin-dependent kinase 2 (CDK2), anti-cyclinE, anti-phosphorylated protein kinase B (p-AKT), anti-protein B (AKT), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Cell Signaling Technology (Danvers, MA, USA). GAPDH was used as an internal control.

Cell culture

HIBEpiC, TFK-1, and HuCCT-1 cell lines were maintained in RPMI-1640 medium (HyClone, Logan, UT, USA) with 1% penicillin–streptomycin and 10% fetal bovine serum (Gibco BRL, Grand Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO2.

Cell proliferation assay

Cells (3000 cells/well) were seeded in 96-well plates and exposed to different concentrations of L-fucose for 24, 48, and 72 h. Subsequently, cell growth was quantified using a Cell Counting Kit-8 (CCK-8) assay (Dojindo Laboratories, Tokyo, Japan).

Colony formation assay

Cells (500 cells/well) were seeded in 35-mm culture plates and then exposed to L-fucose at concentrations of 0, 0.5, or 1 mg/mL. After 2 weeks, the cells were treated with 70% ethanol and stained with crystal violet (Sigma-Aldrich, St. Louis, MO, USA), and the colonies were then counted.

Cell apoptosis analysis

Cells (2 × 105/well) were treated with different concentrations of L-fucose in 6-well plates for 24 h, and the degree of apoptosis was then assessed using an Annexin V-FITC and PI Apoptosis Detection kit (BD Pharmingen, San Jose, CA, USA) following the manufacturer's instructions. The data were analyzed using FlowJo software (TreeStar Inc, Ashland, USA).

Cell cycle analysis

After being treated with L-fucose for 48 h, the cells were harvested and fixed with 75% ethanol overnight. The fixed cells were digested and stained using RNase and PI, respectively. The cell cycle was assessed with flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA). The experiments were repeated three times.

Wound healing assay

Cells (1 × 105 cells/well) were placed in a 6-well plate and scraped off using a sterile plastic tip. After washing with phosphate-buffered saline (PBS), the remaining cells were treated with L-fucose in serum-free media for 24 h. The degree of wound migration was measured by comparing the location of the wound at an indicated time point from that immediately after scraping. In six randomly selected fields of view, the cells that had migrated into the wound area were quantified through computer-assisted microscopy.

Cell invasion assay

Matrigel (BD Biosciences, San Diego, CA, USA) was coated onto the transwell chambers (8 μμm pore size; Corning, NY, USA). Cells (5 × 104/well) were then suspended in the upper chamber. Complete growth medium was subsequently added to the bottom chamber, and the cells were treated with L-fucose in serum-free medium for 24 h. The cells that moved to the other side of the membranes were stained with 0.1% crystal violet. For each transwell membrane, six random fields were analyzed and averaged.

Immunofluorescence assay

Cells were placed in confocal dishes (1 × 104 cells/well), fixed in 4% paraformaldehyde, and incubated in 10% goat serum and 1% Triton to increase the permeability of the cell membrane after the cell morphology had returned to normal. The sections were incubated with primary antibody at 4°C overnight, with green fluorescence-labeled goat anti-rabbit secondary antibody in the dark for 30 min and with 4′,6-diamidino-2-phenylindole (DAPI) in the dark for 15 min to stain the nuclei. The green and blue fluorescence intensities were then observed by fluorescence microscopy.

Cell transfection

The microRNA-200b (miR-200b) inhibitor was designed by and purchased from Guangzhou RiBo Biological Co., Ltd (China). Cells were incubated with miRNA inhibitors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc., USA) according to the manufacturer's guidelines. The final concentration of miRNA inhibitors was 200 nmol/L. Then, the culture medium was replaced with fresh RPMI-1640.

Tube-formation assay

Human umbilical vein endothelial cells (HUVECs) were seeded in a 96-well plate precoated with basement membrane extract (BD Biosciences). Cells were grown in HUVEC growth medium and treated with L-fucose for 24 h, and the formation of capillary-tube structures was observed under an inverted microscope.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from cells and subjected to reverse transcription to synthesize cDNA samples using a Total RNA kit (Takara, Kusatsu, Japan). SYBR Green premix (Takara), cDNA, and primers were subjected to PCR amplification. GAPDH was used as an internal control.

Immunoblot analysis

Thirty micrograms of each aliquot of lysed protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were then transferred to a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membranes were incubated with the indicated primary antibodies. The respective horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The chemiluminescence signals were visualized using an electrochemiluminescence (ECL) detection system (Thermo Fisher Scientific, Waltham, MA, USA) and calculated by densitometry using Quantity One software (Bio-Rad, Hercules, CA, USA).

Animal studies

Male athymic nude mice (aged 5 weeks) were provided by Beijing Vital River Laboratory Animal Technology (China) and housed under pathogen-free conditions. For antiproliferation investigation, HuCCT-1 cells (2 × 107 cells) were injected into the right flank of each mouse before treatment. For antimetastatic investigation, 1 × 106 cells were injected into the mouse tail vein to achieve in vivo lung metastasis. One week after cell injection, the mice were administered intraperitoneal (i.p.) injections with PBS (three times a week/200 μL), L-fucose (three times a week, 100 mg·kg−1·200 μL−1), or L-fucose (three times a week, 100 mg·kg−1·200 μL−1) + miR-200b antagomir (local injection, once a week, 10 nmol). The tumor volume and body weight were measured daily. The tumor volume was estimated according to the following formula: V = L × W2/2, where L and W represent the length and width of the tumor, respectively. At the end of the 4 weeks, the mice (n = 6 per group) were sacrificed, and the tumor tissue was excised for further analyses after the in vivo tumor status was monitored by ultrasonic imaging (USI). All animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Ultrasonic imaging

Tumor growth, angiogenesis, microangiogenesis, and degree of hardness were estimated using a Philips IU Elite Ultrasound System (Philips Health care, Amsterdam, The Netherlands).

Immunohistochemistry (IHC)

Tissues were fixed, embedded, and sectioned for preparation. Primary antibodies were incubated overnight at 4°C. Subsequently, the corresponding secondary antibody was incubated for 1 h. Finally, 3,3′-diaminobenzidine (Invitrogen, CA, USA) was used for color development, and the slides were estimated by three independent pathologists individually.

Statistical analysis

The results were expressed as the mean ± standard deviation (SD) using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA). Student's t-test or analysis of variance (ANOVA) was used to assess the statistical significance of the differences, and P < 0.05 was considered to indicate statistical significance.

Results

Effect of L-fucose on the proliferation of CCA cells

To investigate whether L-fucose can inhibit the proliferation of CCA cells, a normal bile duct epithelium cell line (HIBEpiC) and two CCA cell lines (TFK-1 and HuCCT-1) were treated with L-fucose for different durations, and CCK-8 assays were performed. Surprisingly, the data showed that although L-fucose inhibited the proliferation of TFK-1 and HuCCT-1 cells in a time- and concentration-dependent manner, there was no significant difference in HIBEpiC cells [Figure 1B]. To investigate whether L-fucose inhibits clone formation, we preincubated cells with L-fucose at different concentrations and then cultured the cells for 2 weeks in complete growth medium. As indicated in Figure 1C, CCA cell colony formation was significantly reduced by L-fucose in a concentration-dependent manner (all P < 0.05).

To investigate how L-fucose inhibits CCA cell proliferation, cell apoptosis and the cell cycle were tested. Compared with the vehicle control, L-fucose statistically significantly increased the size of the apoptotic cell populations and decreased the viable cell population in a concentration-dependent manner (all P < 0.05; Figure 1D). Cell cycle arrest is a fundamental mechanism of the infinite and rapid proliferation of cancer cells. In this study, cell cycle analysis demonstrated that L-fucose treatment resulted in G1 phase cell cycle arrest in CCA cells [Figure 1E]. To elucidate the underlying mechanism, we analyzed cell apoptosis and cell cycle regulatory proteins by immunoblotting. As shown in Figure 1F, the expression levels of cleaved caspase 3 (control (Con) vs. 0.5 mg/mL, P = 0.74 in TFK-1 cells and P < 0.05 in HuCCT-1 cells L-fucose; Con vs. 1 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.001) L-fucose and cleaved caspase 9 (Con vs. 0.5 mg/mL L-fucose, P < 0.05 in TFK-1 cells and P = 0.20 in HuCCT-1 cells; Con vs. 1 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.01 in HuCCT-1 cells), which are the key molecules of the mitochondrial apoptotic pathway,[13] were upregulated in a concentration-dependent manner. The protein expression of BCL2 (Con vs. 0.5 mg/mL L-fucose, P = 0.08 in TFK-1 cells and P < 0.05 in HuCCT-1 cells; Con vs. 1 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.01 in HuCCT-1 cells) and Bax (Con vs. 0.5 mg/mL L-fucose, P = 0.055 in TFK-1 cells and P = 0.10 in HuCCT-1 cells; Con vs. 1 mg/mL L-fucose, P < 0.05 in TFK-1 cells and P < 0.01 in HuCCT-1 cells), which are related to apoptosis,[14] was downregulated and upregulated, respectively, after L-fucose treatment. Additionally, cyclin E (Con vs. 0.5 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.001 in HuCCT-1 cells; Con vs. 1 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.001 in HuCCT-1 cells) and CDK2 (Con vs. 0.5 mg/mL L-fucose, P < 0.05 in TFK-1 cells and P = 0.62 in HuCCT-1 cells; Con vs. 1 mg/mL L-fucose, P < 0.01 in TFK-1 cells and P < 0.01 in HuCCT-1 cells) were decreased following L-fucose treatment in a dose-dependent manner, confirming G1/S cycle arrest by L-fucose. Collectively, these data demonstrate that L-fucose exerts proapoptotic effects on human CCA cells. These results show that L-fucose inhibits the growth of CCA cells by proapoptotic and cell cycle arrest effects.

Effects of L-fucose on migration, invasion, and epithelial-mesenchymal transition (EMT) phenotypes of CCA cells

To investigate the antimetastatic potential of L-fucose on CCA cells, we first examined the ability of L-fucose to influence cell invasion and migration at low concentrations. As shown in Figure 2A and 2B, the cells crossing the membrane were prominently inhibited after L-fucose treatment in Transwell migration and Matrigel invasion assays (all P < 0.05). In addition, as illustrated in Figure 2C, L-fucose also decreased the migration of cells in a concentration-dependent manner (Con vs. 0.25 mg/mL, P = 0.18 in TFK-1 cells and P < 0.05 in HuCCT-1 cells; Con vs. 0.5 mg/mL, P < 0.01 in TFK-1 cells and P < 0.01 in HuCCT-1 cells). Collectively, these data show that L-fucose suppresses the motility of CCA cells.

F2
Figure 2:
Effects of L-fucose on invasion, migration, and the EMT. Cells were treated with various concentrations of L-fucose for 24 h and then (A, B) transwell and invasion assays were performed. The cells that moved to the other side of the membranes were stained with 0.1% crystal violet. Scale bars, 200 μm. (C) Wound healing was performed. Scale bars, 200 μm. (D) Immunofluorescence analysis of E-cadherin expression in the indicated cells. Scale bars, 50 μm. (E,F) The expression of EMT-related mRNA and protein levels were determined. Experiments were performed at least three times. P, P < 0.05, ∗∗ P, †† P, ‡‡ P < 0.01, and ∗∗∗ P, ††† P, ‡‡‡ P < 0.001 vs. the control. DAPI: 4′,6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; EMT: epithelial-mesenchymal transition.

EMT is considered a key event in tumor invasion and migration.[15] To investigate the involvement of EMT in the anti-invasion and anti-migration actions of L-fucose, we assessed the expression of EMT-related proteins. As illustrated in Figure 2D, E-cadherin was detected to be significantly increased after L-fucose treatment by immunofluorescence assay. N-cadherin and vimentin are typical mesenchymal markers that were markedly reduced by L-fucose, whereas the level of the epithelial marker E-cadherin was increased in the L-fucose-treated group relative to the control group by qRT-PCR and immunoblotting [Figure 2E, F]. Thus, these data indicate that the antimetastatic activity of L-fucose in the Matrigel-based invasion assays and wound healing assays might be at least partially due to the L-fucose-induced downregulation of cell EMT.

Effects of L-fucose on miR-200b expression

After applying the published data from Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147969), miRNA expression was compared between normal and CCA patients. Twelve paired CCA tissues showed significant dysregulation of 49 miRNAs [Figure 3A]. Meanwhile, another newly published data indicated that a total of 53 miRNAs were affected among the screened 754 miRNAs after fucoidan treatment.[12] Several possible miRNAs were then identified as research targets that might be downstream after L-fucose treatment [Figure 3B]. After the qRT-PCR method, miR-200b-3p was confirmed to be increased significantly in CCA cell lines after L-fucose treatment in a concentration-dependent manner among other miRNAs, including miR-31-5b, miR-551b-5p, and miR-145-5p. In addition, this change was more significant in HuCCT-1 cells [Figure 3C]. These results show that L-fucose induced miR-200b expression in CCA cells.

F3
Figure 3:
Effects of L-fucose on miR-200b expression. (A) miRNA expression was compared between normal and CCA patients using GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE147969). (B) Several possible miRNAs as research targets were identified. (C) Related mRNA levels were assessed. Experiments were performed at least three times. ∗∗∗ P < 0.001 vs. the control. CCA: cholangiocarcinoma; miRNA: microRNA; miR-200b: microRNA-200b; miR-31: microRNA-31; miR-551b: microRNA-551b; miR-145: microRNA-145, GEO: Gene Expression Omnibus.

The inhibitory effects of L-fucose on CCA cell growth, metastasis, and angiogenesis were mediated by miR-200b suppression

To identify whether the effects of L-fucose on CCA cells were through regulation of miR-200b, miR-200b silencing was achieved by transfecting miR-200b inhibitor into HuCCT-1 cells. The data showed that the miR-200b inhibitor statistically significantly downregulated the miR-200b level (P < 0.001, t = 6.42; Figure 4A). Under L-fucose and/or transfection of inhibitor treatment, cell proliferation was significantly downregulated by L-fucose treatment, whereas transfection of inhibitor notably reversed the inhibitory effect of L-fucose [Figure 4B]. Meanwhile, transfection of the inhibitor also reversed the cell cycle arrest and cell apoptosis induced by L-fucose treatment (all P < 0.01; Figure 4C, D). After treatment with L-fucose, both cell migration and cell invasion were inhibited, whereas transfection of the inhibitor notably reversed the inhibitory effect of L-fucose [Figure 4E, F].

F4
Figure 4:
The inhibitory effects of L-fucose on CCA are mediated by miR-200b suppression. After cells were treated with L-fucose or/and miR-200b inhibitor, (A) miR-200b level was assessed. (B) Cell viability was assessed. (C) Apoptotic cells were assessed. (D) Cell cycle analysis was performed. (E) Wound healing assay was performed. Scale bars, 200 μm. (F) Transwell and invasion assays were performed. Scale bars, 200 μm. (G) Co-culture of the HuCCT-1 cells and HUVECs cells was performed, and the formation of capillary-tube structures was assessed. (H) The motility of HUVECs cells was determined. Scale bars, 200 μm. (I) VEGF-A mRNA was determined. Experiments were performed at least three times. P, P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. CCA: cholangiocarcinoma; miR-200b: microRNA-200b; NC: negative control; Annexin V-FITC: annexin V-fluorescein isothiocyanate; PI: propidium iodide; PI-A: peak intensity-area; HUVECs: human umbilical vein endothelial cells; VEGF-A: vascular endothelial growth factor-A.

To investigate the effect of CCA cells on angiogenesis after treatment with L-fucose, coculture of HuCCT-1 cells and HUVECs was performed with the apparatus shown in Figure 4G. Interestingly, the data show that L-fucose effectively suppressed HuCCT-1-elicited capillary-like structures in HUVECs. In addition, a transwell assay confirmed that tumor cell-induced migration of HUVECs was significantly impeded in the presence of L-fucose (all P < 0.05; Figure 4H). Importantly, the inhibitory effects were notably reversed by transfection of the inhibitor, indicating the indirect inhibitory effect of L-fucose on HUVECs (all P < 0.05; Figure 4G, H). Furthermore, qRT-PCR showed that L-fucose significantly inhibited the expression of vascular endothelial growth factor-A (VEGF-A), a key proangiogenic factor induced by CCA (all P < 0.05; Figure 4I). These data indicate that L-fucose-induced miR-200b upregulation impeded tumor-induced proangiogenic activity through VEGF-A.

Effect of L-fucose on the MAPK7 and STAT3 signaling pathways

Applying the miRNA target gene prediction software miRabel (University of Rouen, Normandy, France), MAPK7, which is involved in cell survival, angiogenesis, cell motility, and EMT, was found to harbor a binding site of miR-200b. As expected, MAPK7 was decreased after L-fucose treatment, whereas the transfection of the inhibitor reversed the changes in mRNA and protein levels by qRT-PCR and immunoblotting, respectively. However, the MAPK7 inhibitor (AX15836) reversed the blocking effects of the miR-200b inhibitor, indicating that L-fucose exerts anti-CCA effects partly through the miRNA/MAPK7 mechanism (all P < 0.05; Figure 5A, B). In addition, downstream-related target genes were also assessed by qRT-PCR and immunoblotting, and the data showed that L-fucose-induced cyclinD1, CDK2, cyclinE, cleaved caspase 3, and E-cadherin changes were reversed by transfection of the inhibitor. However, AX15836 reversed the blocking effects (all P < 0.05; Figure 5A, B). Intriguingly, transfection of the inhibitor did not completely reverse the above changes caused by L-fucose, indicating that there are other signaling pathways involved in the L-fucose-induced effects. Since L-fucose substantially influenced CCA cell metastasis and angiogenesis, the related signaling pathways were investigated using immunoblotting to evaluate the expression levels of phosphorylated AKT and STAT3. Figure 5C shows that L-fucose significantly inhibited STAT3 phosphorylation, whereas the change in p-AKT was not significant by immunoblotting (p-AKT vs. AKT, 0.93 vs. 0.97, P = 0.78, t = 0.30; p-STAT3 vs. STAT3, 1.09 vs. 0.46, P < 0.05, t = 4.46; Figure 5C). The same results were also found in the immunofluorescence assay [Figure 5D]. To further confirm the antitumor effect of L-fucose by inhibiting STAT3 activation, cells were treated with L-fucose + STAT3 activator (colivelin), and STAT3 and its downstream-related protein levels were assessed. The data showed that Colivelin reversed the antitumor effect of L-fucose, indicating that L-fucose also exerts anti-CCA effects by inhibiting p-STAT3 (all P < 0.05; Figure 5E). Overall, these results revealed that L-fucose regulated the miR-200b/MAPK7 and STAT3 signaling pathways, leading to the inhibition of CCA progression.

F5
Figure 5:
Effect of L-fucose on MAPK7 and STAT3 signal pathways. After the certain treatment, (A,B) cell cycle, apoptosis, and EMT-related mRNA and protein levels in CCA cells were assessed. (C) AKT and STAT3 signaling-related molecules were determined. (D) The fluorescence intensity of STAT3. Scale bars, 20 μm. (E) STAT3 and its downstream-related protein levels were assessed. Experiments were performed at least three times. P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. CCA: cholangiocarcinoma; NC: negative control; MAPK7: mitogen-activated protein kinase 7; CDK2: cyclin-dependent kinase 2; Bax: Bcl-2-associated X protein; p-AKT: phosphorylated protein kinase B; AKT: protein kinase B; p-STAT3: phosphorylated signal transducer and activator of transcription 3; STAT3: signal transducer and activator of transcription 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4’,6-diamidino-2-phenylindole; EMT: epithelial-mesenchymal transition.

Effects of L-fucose on CCA xenograft tumors in mice

The antitumor efficacy of L-fucose on CCA growth and metastasis was further investigated using HuCCT-1 xenograft tumor models. USI (B-mode, CDFI, CPA, and USE) was initially used to monitor the internal changes in the tumors after treatment. As the data showed, in comparison to the negative control group, tumor size, angiogenesis, and hardness index were all decreased in the L-fucose treatment group, whereas the status was reversed in the miR-200b antagomir treatment group (all P < 0.05; Figure 6A, B). On Day 28, the mice were euthanized, and the tumors were removed and measured. As expected, L-fucose significantly suppressed subcutaneous tumor volume and weight, whereas miR-200b antagomir treatment reversed the inhibitory effect (all P < 0.05; Figure 6C, D). The body weight of each experimental group showed that L-fucose had no significant effect on the body weight of the mice [Figure 6E]. Furthermore, qRT-PCR and immunoblot analysis revealed that E-cadherin was increased, and MAPK7 (ERK5), CDK2, BCL2, and VEGF-A were notably decreased after L-fucose treatment, whereas the status was reversed in the miR-200b antagomir treatment group (all P < 0.05; Figure 6F, G). IHC was also employed to evaluate the protein expression of representative tumor progression markers in the xenograft tumors, and the results were consistent with the above results (all P < 0.05; Figure 6H). Overall, these results revealed that L-fucose significantly inhibited CCA progression in vivo.

F6
Figure 6:
Effects of L-fucose on CCA xenograft tumors in mice. (A) Changes in tumor volumes. (B) Ultrasonography evaluation of tumors. (C) Representative images of tumors from the three groups of mice. (D) The tumor weight was statistically analyzed. (E) The body weight was statistically analyzed. (F,G) The related mRNA and protein levels were measured. (H) Related protein expression was assessed using IHC. Scale bars, 50 μm. (I) Proposed effects of L-fucose. Experiments were performed at least three times. P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the control. CCA: cholangiocarcinoma; NC: negative control; CDFI: color doppler flow imaging; USE: ultrasonic elastosonography; ns: not statistically; CDK2: cyclin-dependent kinase 2; BCL2: B-cell lymphoma 2; VEGF-A: vascular endothelial growth factor-A; MAPK7: mitogenactivated protein kinase 7; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; STAT3: signal transducer and activator of transcription 3; miR-200b: microRNA-200b; EMT: epithelial-mesenchymal transition. IHC: immunohistochemistry.

Discussion

CCA is a type of cancer that exhibits poor responses to the therapeutic options that are currently available,[16] and thus, the identification of innovative agents for the treatment of CCA is urgently needed. The present study yielded a number of novel findings. First, the cellular-level analysis revealed that L-fucose inhibited tumor cell proliferation, invasion, migration, and angiogenesis, indicating that L-fucose exerts anti-CCA effects through multiple modes of action. Second, at the molecular level, we analyzed the difference in miRNAs between normal and CCA tissues and the changes in miRNAs in human peripheral circulation after L-fucose administration and verified that the miR-200b/MAPK7 and STAT3 pathways were targets of L-fucose. This analytical approach provides a more objective representation of the mechanism of the L-fucose role in the complex human internal environment. The in vivo experiment also revealed that L-fucose elicited impressive antitumor activity to limit tumor growth, metastasis, and angiogenesis in a mouse xenograft model of CCA, which is a result that has not been obtained in previous studies [Figure 6I].

Previous studies revealed that L-fucose exhibited antitumor activity against various types of epithelial-derived cells of cancer, specifically colorectal cancer, gastric carcinoma, and hepatocellular carcinoma.[17] CCA is a typical bile duct epithelial-derived cancer that is intriguing and has been the focus of our research. Our study has once again broadened the scope of application of L-fucose to exert antitumor activity, and this study provides a good theoretical basis for L-fucose to enter clinical applications in the future. Although our in vivo experiments have validated the effectiveness of L-fucose, there is a lack of positive control group. In principle, positive control drugs should be selected to have the same structure, the same pharmacological effect, the same mechanism of effect, the same dosage form, and the same route of administration as the tested drug. Gemcitabine, as a first-line anti-CCA chemotherapeutic agent, has been widely used in the clinic; however, in this study, we could not determine the appropriate positive control group and the experimental concentration of the test group and therefore failed to verify the superiority and equivalence of L-fucose relative to gemcitabine. In the future, we will conduct a large sample of positive control trials to verify the superiority and equivalence of L-fucose.

Dysregulated expression of miRNAs contributes to the genesis of a large fraction of human tumors. Its deregulation, overexpression, or misexpression generally promotes cellular proliferation and growth and inhibits cell differentiation.[18,19] Numerous studies have demonstrated that many miRNAs promote or inhibit CCA progression. For instance, miR-92b was identified to be associated with adverse outcomes in CCA patients.[20] Ursu et al[21] demonstrated that miR-876 acts as a suppressor to target B-cell lymphoma-extra-large (Bcl-XL) and inhibit CCA progression. Han et al[22] indicated that Per1 was verified as a target of miR-34a and promoted the proliferation, migration, and invasion of CCA cells. All the evidence suggests that miRNAs may play potential therapeutic roles in CCA. Surprisingly, a recent study discovered that fucoidan, a polysaccharide rich in L-fucose, changed a series of miRNAs in healthy volunteers, illustrating that L-fucose has the potential to affect fundamental cellular processes by miRNA regulation. By comparing the changes in miRNAs in vivo after drug administration, we screened three miRNAs that may be involved in the inhibition of the progression of CCA, and miR-200b was finally identified to be involved in the processes. Since the human body is a systematic environment, this study explored the actual situation of L-fucose in the human body, which is more clinically significant than the cell study in vitro or the animal model study in vivo. Additionally, as L-fucose has no obvious toxic or side effects on the human body, we may consider combining L-fucose with anti-CCA drugs to explore whether the combination can enhance the therapeutic effect of chemotherapy drugs and verify whether miR-200b still plays a key role in it.

STAT proteins form part of a seven-member family of latent cytoplasmic transcription factors.[23,24] Emerging lines of evidence suggest that abnormal STAT signaling, particularly STAT3 signaling, promotes the development and progression of various cancers.[25,26] STAT3 contains Tyr705 and Ser727 phosphorylation sites, and of these, Tyr705 is the main phosphorylation site for STAT3 activation. Once phosphorylated, STAT3 forms a dimer and translocates from the cytosol to the nucleus, where it binds to promoter elements and functions as a pivotal transactivator for numerous oncogenic genes.[27-29] STAT3 inhibitors exhibit tumor-suppressive effects in various tumors. AG490, an important STAT3 inhibitor, can induce CCA cell apoptosis and subsequently inhibit CCA cell proliferation.[30-32] In the present study, the miR-200b inhibitor failed to completely reverse L-fucose-induced apoptosis and EMT-related protein expression. Thus, we hypothesized that there might be another signaling pathway involved in the L-fucose-induced CCA inhibition. After testing the signaling pathways that affect the proliferation and metastasis of CCA, the STAT3 signaling pathway was significantly inhibited by L-fucose treatment, indicating that L-fucose also induced dephosphorylation of STAT3 Tyr705 to yield its antitumor efficacy in CCA cells.

In conclusion, the identification of a novel anticancer agent, L-fucose, provides an enormous opportunity to improve the existing treatment approach for CCA. Based on the findings obtained in this study, the inhibitory effect of L-fucose can largely be attributed to a novel mechanism that directly targets the miR-200b/MAPK7 and STAT3 signaling pathways.

Funding

This study was supported by grants from the National Natural Science Foundation of China (Nos. 81720108006, 81974062).

Conflicts of interest

None.

References

1. Oliveira IS, Kilcoyne A, Everett JM, Mino-Kenudson M, Harisinghani MG, Ganesan K. Cholangiocarcinoma: classification, diagnosis, staging, imaging features, and management. Abdom Radiol (NY) 2017;42:1637–1649. doi: 10.1007/s00261-017-1094-7.
2. Kendall T, Verheij J, Gaudio E, Evert M, Guido M, Goeppert B, et al. Anatomical, histomorphological and molecular classification of cholangiocarcinoma. Liver Int 2019;39 (Suppl 1):7–18. doi: 10.1111/liv.14093.
3. Vignone A, Biancaniello F, Casadio M, Pesci L, Cardinale V, Ridola L, et al. Emerging therapies for advanced cholangiocarcinoma: an updated literature review. J Clin Med 2021;10:4901. doi: 10.3390/jcm10214901.
4. Thornblade LW, Wong P, Li D, Warner SG, Chang S, Raoof M, et al. Patterns of whole exome sequencing in resected cholangiocarcinoma. Cancers (Basel) 2021;13:4062. doi: 10.3390/cancers13164062.
5. Mathieu S, Gerolami R, Luis J, Carmona S, Kol O, Crescence L, et al. Introducing alpha(1,2)-linked fucose into hepatocarcinoma cells inhibits vasculogenesis and tumor growth. Int J Cancer 2007;121:1680–1689. doi: 10.1002/ijc.22797.
6. Sun J, Sun J, Song B, Zhang L, Shao Q, Liu Y, et al. Fucoidan inhibits CCL22 production through NF-kappaB pathway in M2 macrophages: a potential therapeutic strategy for cancer. Sci Rep 2016;6:35855. doi: 10.1038/srep35855.
7. Hsu WJ, Lin MH, Kuo TC, Chou CM, Mi FL, Cheng CH, et al. Fucoidan from Laminaria japonica exerts antitumor effects on angiogenesis and micrometastasis in triple-negative breast cancer cells. Int J Biol Macromol 2020;149:600–608. doi: 10.1016/j.ijbiomac.2020.01.256.
8. Tafrihi M, Hasheminasab E. MiRNAs: biology, biogenesis, their web-based tools, and databases. Microrna 2019;8:4–27. doi: 10.2174/2211536607666180827111633.
9. Howell JA, Khan SA. The role of miRNAs in cholangiocarcinoma. Hepat Oncol 2016;3:167–180. doi: 10.2217/hep-2015-0003.
10. Wang X, Hu KB, Zhang YQ, Yang CJ, Yao HH. Comprehensive analysis of aberrantly expressed profiles of lncRNAs, miRNAs and mRNAs with associated ceRNA network in cholangiocarcinoma. Cancer Biomark 2018;23:549–559. doi: 10.3233/CBM-181684.
11. Piontek K, Selaru FM. MicroRNAs in the biology and diagnosis of cholangiocarcinoma. Semin Liver Dis 2015;35:55–62. doi: 10.1055/s-0034-1397349.
12. Gueven N, Spring KJ, Holmes S, Ahuja K, Eri R, Park AY, et al. Micro RNA expression after ingestion of fucoidan: a clinical study. Mar Drugs 2020;18:143. doi: 10.3390/md18030143.
13. Budihardjo I, Oliver H, Lutter M, Luo X, Wang X. Biochemical pathways of caspase activation during apoptosis. Annu Rev Cell Dev Biol 1999;15:269–290. doi: 10.1146/annurev.cellbio.15.1.269.
14. Green DR. Life, death, BH3 profiles, and the salmon mousse. Cancer Cell 2007;12:97–99. doi: 10.1016/j.ccr.2007.07.011.
15. Puisieux A, Brabletz T, Caramel J. Oncogenic roles of EMT-inducing transcription factors. Nat Cell Biol 2014;16:488–494. doi: 10.1038/ncb2976.
16. Munugala N, Maithel SK, Shroff RT. Novel biomarkers and the future of targeted therapies in cholangiocarcinoma: a narrative review. Hepatobiliary Surg Nutr 2022;11:253–266. doi: 10.21037/hbsn-20-475.
17. Duan C, Tang X, Wang W, Qian W, Fu X, Deng X, et al. L-fucose ameliorates the carcinogenic properties of Fusobacterium nucleatum in colorectal cancer. Oncol Lett 2021;21:143. doi: 10.3892/ol.2020.12404.
18. Latini A, Borgiani P, Novelli G, Ciccacci C. miRNAs in drug response variability: potential utility as biomarkers for personalized medicine. Pharmacogenomics 2019;20:1049–1059. doi: 10.2217/pgs-2019-0089.
19. Rupaimoole R, Slack FJ. MicroRNA therapeutics: towards a new era for the management of cancer and other diseases. Nat Rev Drug Discov 2017;16:203–222. doi: 10.1038/nrd.2016.246.
20. Zhou MH, Zhou HW, Liu M, Sun JZ. The role of miR-92b in cholangiocarcinoma patients. Int J Biol Markers 2018;33:293–300. doi: 10.1177/1724600817751524.
21. Ursu S, Majid S, Garger C, de Semir D, Bezrookove V, Desprez PY, et al. Novel tumor suppressor role of miRNA-876 in cholangiocarcinoma. Oncogenesis 2019;8:42. doi: 10.1038/s41389-019-0153-z.
22. Han Y, Meng F, Venter J, Wu N, Wan Y, Standeford H, et al. miR-34a-dependent overexpression of Per1 decreases cholangiocarcinoma growth. J Hepatol 2016;64:1295–1304. doi: 10.1016/j.jhep.2016.02.024.
23. Yang J, Stark GR. Roles of unphosphorylated STATs in signaling. Cell Res 2008;18:443–451. doi: 10.1038/cr.2008.41.
24. Takeda K, Akira S. STAT family of transcription factors in cytokine-mediated biological responses. Cytokine Growth Factor Rev 2000;11:199–207. doi: 10.1016/S1359-6101(00)00005-8.
25. Sansone P, Bromberg J. Targeting the interleukin-6/Jak/stat pathway in human malignancies. J Clin Oncol 2012;30:1005–1014. doi: 10.1200/JCO.2010.31.8907.
26. Siveen KS, Nguyen AH, Lee JH, Li F, Singh SS, Kumar AP, et al. Negative regulation of signal transducer and activator of transcription-3 signalling cascade by lupeol inhibits growth and induces apoptosis in hepatocellular carcinoma cells. Br J Cancer 2014;111:1327–1337. doi: 10.1038/bjc.2014.422.
27. Benekli M, Baer MR, Baumann H, Wetzler M. Signal transducer and activator of transcription proteins in leukemias. Blood 2003;101:2940–2954. doi: 10.1182/blood-2002-04-1204.
28. Xu J, Lin H, Wu G, Zhu M, Li M. IL-6/STAT3 is a promising therapeutic target for hepatocellular carcinoma. Front Oncol 2021;11:760971. doi: 10.3389/fonc.2021.760971.
29. He G, Karin M. NF-kappaB and STAT3 – Key players in liver inflammation and cancer. Cell Res 2011;21:159–168. doi: 10.1038/cr.2010.183.
30. Lis C, Rubner S, Roatsch M, Berg A, Gilcrest T, Fu D, et al. Development of Erasin: a chromone-based STAT3 inhibitor which induces apoptosis in Erlotinib-resistant lung cancer cells. Sci Rep 2017;7:17390. doi: 10.1038/s41598-017-17600-x.
31. Liu JF, Deng WW, Chen L, Li YC, Wu L, Ma SR, et al. Inhibition of JAK2/STAT3 reduces tumor-induced angiogenesis and myeloid-derived suppressor cells in head and neck cancer. Mol Carcinog 2017;57:429–439. doi: 10.1002/mc.22767.
32. Garg M, Shanmugam MK, Bhardwaj V, Goel A, Gupta R, Sharma A, et al. The pleiotropic role of transcription factor STAT3 in oncogenesis and its targeting through natural products for cancer prevention and therapy. Med Res Rev 2020;41:1291–1336. doi: 10.1002/med.21761.
Keywords:

Cholangiocarcinoma; L-Fucose; MicroRNA-200b; Mitogen-activated protein kinase 7; Phosphorylated signal transducer and activator of transcription 3

Copyright © 2023 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.