Open Access

Digitoxin inhibits ICC cell properties via the NF‑κB/ST6GAL1 signaling pathway

  • Authors:
    • Yueping Zhan
    • Rong Wang
    • Chenjun Huang
    • Xuewen Xu
    • Xiao Xiao
    • Linlin Wu
    • Jiao Wei
    • Tian Long
    • Chunfang Gao
  • View Affiliations

  • Published online on: June 25, 2024
  • Article Number: 103
  • Copyright: © Zhan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Intrahepatic cholangiocarcinoma (ICC) is a type of liver cancer associated with poor prognosis and increased mortality; the limited treatment strategy highlights the urgent need for investigation. Traditional Chinese Medicine (TCM), used alone or in combination with other treatments, can enhance therapeutic efficacy, improve life quality of patients and extend overall survival. In total, two rounds of screening of a TCM library of 2,538 active compounds were conducted using a Cell Counting Kit‑8 assay and ICC cell lines. Cell proliferation and migration abilities were assessed through colony formation, 5‑ethynyl‑2'‑deoxyuridine, would healing and Transwell assays. The impact of digitoxin (DT) on signaling pathways was initially investigated using RNA sequencing and further validated using reverse transcription‑quantitative PCR, western blotting, lectin blotting and flow cytometry. ICC cells stably overexpressing ST6 β‑galactoside α‑2,6‑sialyltransferase 1 (ST6GAL1) were generated through lentiviral transfection. It was shown that DT emerged as a highly effective anti‑ICC candidate from two rounds high‑throughput library screening. DT could inhibit the proliferation and migration of ICC cells by suppressing NF‑κB activation and reducing nuclear phosphorylated‑NF‑κB levels, along with diminishing ST6GAL1 mRNA and protein expression. The aforementioned biological effects and signal pathways of DT could be counteracted by overexpressing ST6GAL1 in ICC cells. In conclusion, DT suppressed ICC cell proliferation and migration by targeting the NF‑κB/ST6GAL1 signaling axis. The findings of the present study indicated the promising therapeutic effects of DT in managing ICC, offering new avenues for treatment strategies.


Biliary tract cancers include intrahepatic cholangiocarcinoma (ICC), perihilar, distal cholangiocarcinoma and gall bladder carcinoma based on their location of origin. Notably, ICC stands out as a type of cancer associated with increased mortality. It ranks as the second most prevalent primary liver cancer, constituting ≤20% of liver cancers and 3% of all gastrointestinal cancers (1,2). The prognosis for patients with ICC undergoing surgery with the intent to cure is poor, with a 5-year overall survival (OS) range of 5–17% (3). Patients with unresectable ICC have a median survival of 11.7 months with palliative chemotherapy, primarily gemcitabine and cisplatin (4). Despite the potential of emerging treatments such as triplet regimens, immunotherapy and targeted therapies for specific genetic alterations (5), the persistent challenges of tumor resistance and low OS rates underscore the limited efficacy of current clinical treatments for ICC. Therefore, the development of more effective anti-ICC drugs is critical and pressing issue in medical oncology research to improve treatment outcomes and patient survival.

Natural products continue to be a vital and promising reservoir for chemotherapeutic agent discovery, exemplified by drugs such as artemisinin, an antimalarial agent (6). Between 1981 and 2014, 49% of the anticancer drugs approved by the FDA originated either directly from natural sources or as derivatives. These include notable drugs such as vinblastine and colchicine, highlighting the significant role of natural resources in cancer drug development (7,8). Digitoxin (DT), a cardiac glycoside naturally sourced from species of the genus Digitalis (9), has served as an effective Na+/K+-ATPase inhibitor. For >4 decades, it has been used in the clinical management of congestive heart failure (10). Numerous studies have investigated the anticancer effects of DT, revealing its potent antitumor efficacy in a diverse range of cancer types (1114). However, the ability and underlying mechanism of DT as an anticancer reagent against ICC, a lethal cancer without satisfactory chemotherapy, remains unknown.

In the present study, a screening of a TCM library of 2,538 unique natural compounds was conducted to discover potential lead compounds exhibiting anti-ICC activity. Among the noteworthy compounds discovered, DT emerged as a significant anti-ICC chemotherapeutic agent. The effects of DT on ICC cell proliferation and metastasis were further evaluated through cell functional assays, and the key signaling pathways involved were explored. ST6 β-galactoside α-2,6-sialyltransferase 1 (ST6GAL1), which facilitates the addition of sialic acid to N-glycosylated proteins, is substantially upregulated in various types of malignancies, playing a crucial role in the progression of cancer. It was revealed that the mRNA and protein ST6GAL1 expression in ICC cells was suppressed by DT. Overexpression of ST6GAL1 could reverse the inhibitory effect of DT on ICC. It was shown that DT inhibited ICC cell proliferation and metastasis by targeting the NF-κB/ST6GAL1 signaling pathway. The findings of the present study indicated the therapeutic potential of DT in the treatment of ICC.

Materials and methods

Cell culture

HCCC-9810, RBE and 293T cell lines were obtained from the Chinese Academy of Sciences, Shanghai Branch Cell Bank. HuCCT1 were purchased from Cyagen, and normal human intrahepatic biliary epithelial cells (HiBEpiCs) were obtained from Qingqi (Shanghai) Biotechnology development Co., Ltd. Cells were cultured in RPMI-1640 medium (cat. no. 11875119; Gibco, Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (cat. no. 10099141C; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (cat. no. 15140122; Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator (HERA cell VIOS; Themo Fisher Scientific, Inc.) with 5% CO2. 293T cells were cultured in DMEM (cat. no. 11995065; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS and 1% penicillin/streptomycin. Dimethyl sulfoxide (DMSO) was obtained from Sigma-Aldrich; Merck KGaA, and DT (cat. no. HY-B1357) was purchased from MedChemExpress.

Cell viability assay

The cell suspension (100 µl/well) was seeded in 96-well culture plates (cat. no. 3599; Corning, Inc.) at a density range of 1–2×105 cells/ml. The cells were treated and incubated with DT at serial concentrations for 24, 48 and 72 h. A total of 10 µl CCK-8 (cat. no. HY-K0301; MedChemExpress) solution was then added to each well of the plate. The plate was incubated for 3 h at 37°C and absorbance was measured at 450 nm using the Molecular Devices Spectra Max iD3 Microplate Detection System. The cell viability rate was calculated as experimental optical density (OD) value/control OD value.

High-throughput screening of TCM library

A total of 2,538 compounds in the TCM Active Compound Library (cat. no. HY-L065; MedChemExpress) were used for the screening experiment. Compounds were initially screened at a concentration of 10 µM. A further screening was conducted with the criterion that ICC cell viability should be decreased by 70% compared with the control (DMSO). Subsequently, compounds that maintained a viability rate >80% in HiBEpiCs were selected as candidates for anti-ICC drugs.

Overexpression of ST6GAL1 in ICC cells

The overexpression of ST6GAL1 plasmids and control plasmids (pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO) was carried out by Hanbio Biotechnology Co., Ltd. For the construction of the ST6GAL1-overexpression cell line, transfer plasmid (10 µg; A260/A280=1.94), envelope plasmid (pMD2G, 5 µg; A260/A280=1.93) and packaging plasmids (pSPAX2, 10 µg; A260/A280=1.92) were co-transfected into 293T packaging cells using Lipofectamine 3000 (cat. np. L3000015; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions at 37°C. The culture medium was replenished 16 h later, and the culture supernatant was collected at 2 and 3 days. The virus supernatant was centrifuged in a 50-ml centrifuge tube at 4°C and 2,000 × g for 10 min to remove cell debris. Then, the clarified viral supernatant was collected and placed into an ultracentrifuge tube to be centrifuged at 4°C and 82,700 × g for 120 min. The viral pellet was resuspended in 400 µl complete medium and finally the resuspended solution was aliquoted into sterilized viral tubes. A total of 2×105 HCCC-9810 or HuCCT1 cells were seeded in 12-well plates overnight and subsequently infected with 50 µl lentivirus expressing the ST6GAL1 gene or a control lentivirus. The transfected cells were screened using puromycin (1 µg/ml) 1 week later.

Overexpression of P65/NF-κB in 293T cells

The overexpression of P65/NF-κB plasmids and control plasmids pcDNA3.1 was carried out by Hanbio Biotechnology Co., Ltd. The P65/NF-κB overexpression plasmids (4 µg; A260/A280=1.93) were transfected into 293T cells at 37°C using Lipofectamine 3000 (cat. np. L3000015; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The medium was refreshed 6 h after transfection, 50 nM DT was added 24 h post-transfection, and WB and RT-qPCR were performed at 48 h.

Colony formation and 5-ethynyl-2′-deoxyuridine (EdU) assays

A total of 1,000 ICC cells/well treated with different concentrations of DT and the ST6GAL1-overexpressed ICC cells treated with DT were plated in 12-well plates (cat. no. 3513; Corning, Inc.) and cultured at 37°C for 7 days. Subsequently, they were fixed in 4% paraformaldehyde (w/v) for 10 min at RT. After washing three times with PBS, staining was performed using 0.1% crystal violet staining solution (cat. no. C0121; Beyotime Institute of Biotechnology) for 10 min at room temperature (RT). Images were captured using a BZ-X800 fluorescence microscope (Keyence Corporation), and the colonies with aggregates of ≥50 cells were counted.

The ICC cells treated with different concentrations of DT and the ST6GAL1-overexpressed ICC cells treated with DT were seeded into a 24-well plate (cat. no. 3524; Corning, Inc.). Cell proliferation was assessed by incorporating EdU (cat. no. C0071S; Beyotime Institute of Biotechnology) following the manufacturer's instructions. The percentage of EdU+ cells was calculated by three random images captured with the BZ-X800 microscope.

Wound healing assay

The ICC cells treated with different concentrations of DT and the ST6GAL1-overexpressing ICC cells treated with DT were seeded in 12-well plates and allowed to incubate for 1 day. When the cells reached ~90% confluence, the bottom of each well was scratched with a 10-µl pipette tip. After washing with PBS, the culture medium was replenished with serum-free medium for culture. Images were captured under an inverted light microscope (cat. no. DMi8; Leica Microsystems) at 0 and 24 h, and the migration area was computed using ImageJ software (version 1.53a; National Institutes of Health).

Cell migration

The ICC cells treated with different concentrations of DT and the ST6GAL1-overexpressing ICC cells treated with DT were collected and diluted with serum-free DMEM to a concentration of 1×105 cells/ml. The upper chamber (8 µm; cat. no. 353097; Falcon; Corning, Inc.) was filled with 200 µl cell suspension in order to measure the capacity for migration. The migratory cells were subsequently stained with 0.1% crystal violet staining solution for 10 min at RT and observed using a BZ-X800 microscope. The number of migratory cells was counted using three randomly selected images of selected fields.

RNA sequencing (RNA-seq)

RNA-seq and analysis were conducted by Shanghai Biotechnology Co., Ltd. RNA extraction was performed using the MJzol Animal RNA Isolation Kit (Majorivd;, followed by purification with the RNAClean XP Kit (Beckman Coulter, Inc.) and RNase-Free DNase Set (Qiagen, Inc.). RNA integrity and quality (260/280 nm ratio >1.8) were assessed using the Agilent 2100 Bioanalyzer/Agilent 4200 TapeStation and quantified with the Qubit 2.0® Fluorometer (Thermo Fisher Scientific, Inc.) and NanoDrop ND-2000 (Thermo Fisher Scientific, Inc.). The mRNA was isolated from the purified RNA, fragmented and synthesized into cDNA, with subsequent library construction steps including end repair, adenylation, adapter ligation and enrichment. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Inc.) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, Inc.) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq6000 platform (Illumina, Inc.) with the PE150 (Pair-end 150 bp) mode. Following data analysis at the transcript level and differential gene screening, differentially expressed genes were identified. Subsequent analyses were carried out to determine the Gene Ontology (GO) functional significance and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significance.

Reverse transcription-quantitative PCR (RT-qPCR)

The EZ-press RNA purification kit (cat. no. B0004D; EZBioscience) was used to extracted total RNA from cells. The concentration of the extracted RNA was measured by NanoDrop (Thermo Fisher Scientific, Inc.) at 260 nm. PrimeScript™ RT Reagent Kit (cat. no. RR037A; Takara Bio, Inc.) was applied to reverse transcribe RNA into cDNA according to the manufacturer's protocol. Each RT reaction contained 500 ng RNA. TB Green™ Premix Ex Taq™ (cat. no. RR420A; Takara Bio, Inc.) was used for qPCR, and data collection and analysis were carried out using an Applied Biosystems PCR System (7500 Real-Time PCR System; Thermo Fisher Scientific, Inc.). The PCR thermocycling conditions were as follows: i) Stage 1, 95°C for 30 sec; ii) Stage 2, 95°C for 5 sec, 60°C for 34 sec, 40 cycles; iii) Stage 3, 95°C for 30 sec, 60°C for 1 min, 95°C for 15 sec. The 2−ΔΔCq method (15) was used to ascertain the relative expression levels of each gene, with GAPDH serving as the internal reference. Primer sequences were as follows: ST6GAL1 forward, 5′-CCCCAATCAGCCCTTTTACATCCTC-3′ and reverse, 5′-CCTGGTCACACAGCGTCATCATG-3′; and GAPDH forward, 5′-CCAGCAAGAGCACAAGAGGAAGAG-3′ and reverse, 5′-GGTCTACATGGCAACTGTGAGGAG-3′.

Western blotting and lectin blotting

Cells treated with different concentrations of DT were lysed using cell lysis buffer (cat. no. 9803s; Cell Signaling Technology, Inc.) containing ProtLytic Protease and Phosphatase Inhibitor Cocktail (cat. no. P002; NCM Biotech). Nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (cat. no. P0028; Beyotime Institute of Biotechnology). The protein concentrations were quantified by the BCA protein assay kit (cat. no. P0011; Beyotime Institute of Biotechnology). A total of 30 µg proteins were separated by 7.5 or 10% SDS-PAGE, and the resulting proteins were then moved from the gel onto PVDF membranes (cat. no. ISEQ00010; MilliporeSigma).

After 1 h of incubation with 5% skimmed milk at RT, membranes were incubated with primary antibodies against Bcl-2 (1:1,000; cat. no. 12789-1-AP; Proteintech Group, Inc.), ST6GAL1 (1 µg/ml; cat. no. AF5924; R&D Systems, Inc.), BAX (1:1,000; cat. no. 50599-2-Ig; Proteintech Group, Inc.), phosphorylated (p)-NF-κB p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.), NF-κB p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p38 MAPK (1:1,000; cat. no. 4511; Cell Signaling Technology, Inc.), p38 MAPK (1:1,000; cat. no. 8690; Cell Signaling Technology, Inc.), GAPDH (1:5,000; cat. no. 10494-1-AP; Proteintech Group, Inc.) and biotinylated Sambucus nigra (SNA) lectin recognizing α2,6-linked sialic acid (1:2,000; cat. no. B-1305; Vector Laboratories) at 4°C overnight. The membranes were then incubated with secondary antibodies at RT, goat anti-rabbit (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.), goat anti-mouse (1:5,000; cat. no. 7076; Cell Signaling Technology, Inc.), or streptavidin-HRP (1:5,000; cat. no. 3999; Cell Signaling Technology, Inc.). Protein visualization was carried out using a chemiluminescence ECL Detection kit (NcmECL Ultra; cat. no. P10300; NCM). The software used for densitometric analysis was ImageJ software (version 1.53a; National Institutes of Health).

In situ fluorescence detection of α2,6-linked sialic acid

The ICC cells treated with varying concentrations of DT and the DT-treated ST6GAL1-overexpressing ICC cells were fixed for 10 min with 4% paraformaldehyde at RT. Following fixation, cells were blocked for 1 h using blocking buffer (cat. no. P0260; Beyotime Institute of Biotechnology) for immunological staining at RT. Next, the cells were incubated with Cy5 labeled SNA lectin (cat. no. CL-1305-1; Vector Laboratories) overnight at 4°C. Each step involved washing three times with PBS for 5 min. Finally, the slides were sealed using an antifade mounting medium containing 1 µg/ml DAPI (cat. no. A4084; UElandy;; then images were captured by a BZ-X800 microscope.

Flow cytometry

After harvesting ICC cells with ST6GAL1 overexpression treated with DT, the cells were blocked for 20 min on ice using a carbo-free blocking solution (cat. no. SP-5040; Vector Laboratories) at a volume of 0.2 ml for 1×106 cells. Subsequently, these cells were incubated with Cy5-labeled SNA lectin (1 µl per 1×106 cells). Cells were analyzed with a BD FACSCanto II Flow Cytometer (BD Biosciences). FlowJo software (version 10.8.1; Becton Dickinson and Company) was used for analysis.

Statistical analysis

GraphPad Prism (version 9; Dotmatics) was used to conduct statistical analyses. Error bars in the experiments represent SD. The legends provide information about the number of events and independent experiments. Statistical comparisons between two experimental conditions were conducted using the Mann-Whitney test or unpaired Student's t-test, and the Wilcoxon rank-sum test was used for all paired samples. A one-way ANOVA was used to compare multiple experimental groups, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.


Screening of monomeric compounds with anti-ICC activity

High-throughput drug screening was performed to evaluate the antitumor properties of TCM monomeric compounds in a TCM Active Compound Library containing 2,538 compounds. The screening aimed to assess the inhibitory effects of these compounds on the viability of ICC. In the initial screening (Fig. S1A), 83 monomers were identified that exhibited a growth inhibition rate >70% in HCCC9810 and RBE cells (Fig. S1B). Through a secondary screening, 64 monomers with consistent inhibitory activity >70% were selected (Fig. S1C). By assessing the viability of HiBEpiCs, 15 active monomers were identified that showed inhibitory effects on ICC without adverse effects on HiBEpiCs (Fig. S1D). DT emerged as a standout among the 15 active compounds tested, showcasing potent anti-ICC cell activity and promising therapeutic potential against ICC.

DT reduces the proliferation of ICC cells

To assess the inhibitory effects of DT on ICC cells, a CCK-8 assay was conducted which allowed to evaluate the impact of varying concentrations and treatment durations of DT on cell viability. The findings revealed a significant dose-dependent reduction in the viability of both HuCCT1 and HCCC9810 cell lines (Fig. 1A). More specifically, the IC50 values for HCCC9810 cells after 24, 48 and 72 h of DT treatment were 106.5±23.2, 92.4±12.1 and 77.9±10.9 nM, respectively. For HuCCT1 cells, the corresponding IC50 values were 132.3±33.8, 105.1±26.6 and 98.5±4.2 nM, respectively, indicating a progressive decrease in IC50 values, which suggests an increase in drug sensitivity over time. Subsequently, to further probe the effects of DT on cell proliferation, a series of concentration gradients were applied: 0, 1/4 IC50, 1/2 IC50, IC50 of DT at 48 h (Table SI); and the proliferation capacity of ICC cells was assessed at 24 h. Through the analysis of the colony formation and EdU incorporation assays, it was observed that DT significantly decreased the colony formation and proliferation ability of HuCCT1 and HCCC9810 cells, demonstrating a dose-dependent inhibition (Fig. 1B and C). Taken together, these findings strongly suggest that DT possesses the ability to hinder both the viability and proliferation of ICC cells.

DT suppresses the migration of ICC cells

To explore the potential suppressive impact of DT on the migration capabilities of ICC cells, the cells were exposed to varying concentrations of DT, and wound healing and Transwell assays were carried out. The Transwell assay results showed that DT significantly decreased the migration of HuCCT1 and HCCC9810 cells in a dose-dependent manner (P<0.05; Fig. 2A). Additionally, an increase in DT concentration led to a decrease in the migration areas for both HCCC9810 (Fig. 2B) and HuCCT1 (Fig. 2C) cells, underscoring a progressive impediment to cell movement. Collectively, these findings demonstrated the ability of DT to inhibit the migration of ICC cells.

The NF-κB/ST6GAL1 pathway plays a crucial role in the inhibitory effects of DT on ICC cells

Subsequently, the primary signaling pathways through which DT exerted its inhibitory effects on ICC cell functions were investigated. The aim was to delineate the in vitro molecular mechanisms underlying the suppression of ICC cell activities with DT. Firstly, the intersection of differentially expressed genes in HCCC9810 and HUCCT1 cells after treatment with DT was selected, including both upregulated and downregulated genes. Subsequently, GO and KEGG functional enrichment analyses were conducted. RNA-seq analysis unveiled significant alterations in pathways related to glycosylation, regulation of NF-κB imported into the nucleus and transcriptional mis-regulation in ICC cells following treatment with DT (Fig. 3A and B). The heatmap of differentially expressed genes suggested that DT could downregulate the transcriptional levels of ST6GAL1 (Fig. 3C). Furthermore, the RT-qPCR results confirmed that DT could effectively inhibit the mRNA expression of ST6GAL1 (Fig. 3D). Next, the effects of DT on the NF-κB, P38 and STAT3 signaling pathways, as well as ST6GAL1 expression were investigated. DT treatment resulted in inhibition of phosphorylation of p-NF-κB and p-STAT3, while it upregulated the phosphorylation of P38 and reduced the expression of the anti-apoptotic protein BCL-2 (Fig. 3E; statistical charts, Fig. S2). It was found that NF-κB interacted with the promoter region of the ST6GAL1 gene, initiating its transcription (16). To confirm the effect of NF-κB on ST6GAL1, a P65/NF-κB overexpression plasmid was constructed, transfected into 293T cells and followed by DT treatment (Fig. S3). The results indicated that overexpression of NF-κB promoted the expression of ST6GAL1, and also demonstrated the inhibitory effect of DT on ST6GAL1 in 293T cells. Finally, lectin blotting with SNA was used which selectively adheres to α2,6-sialic acid structures to evaluate the α2,6-sialylation levels in situ. A dose-dependent reduction in α2,6-sialylation, predominantly triggered by DT, was observed (Fig. 3F and G). These findings collectively revealed the involvement of the NF-κB/ST6GAL1 pathway in mediating the effects of DT in ICC cells, underscoring its significance in the modulation of ICC cell abilities.

ST6GAL1 counteracts the inhibitory effects of DT on cell proliferation and migration

The aforementioned results revealed that DT reduced the expression of ST6GAL1 in a dose-dependent manner. Consequently, the ability of overexpressing ST6GAL1 to reverse the inhibitory effects of DT on ICC cells was investigated. ST6GAL1-overexpressing-ICC cells were generated by a lentivirus vector system. The increased levels of ST6GAL1 in ST6GAL1-overexpressing HCCC9810 and HuCCT1 cells were confirmed by RT-qPCR and western blotting (Fig. S4A and B). The colony formation and EdU assays provided compelling evidence that overexpression of ST6GAL1 significantly promotes the proliferation of ICC cells. These assays highlighted that ST6GAL1 could not only enhance cell proliferation, but also counteract the suppression caused by DT (Fig. 4A and B). Further investigation into the impact of ST6GAL1 on cell migration was conducted using wound healing and Transwell migration assays. The outcomes from these assays confirmed that ST6GAL1 overexpression not only augmented the migratory capabilities of ICC cells, but also effectively neutralized the migration-inhibiting influence of DT (Fig. 4C and D). Collectively, these findings revealed that ST6GAL1 acted as a potent promoter of both proliferation and migration of ICC cells, effectively reversing the inhibitory effects imposed by DT treatment.

DT inhibits ICC progression through the NF-κB/ST6GAL1 signaling pathway

To ascertain the critical role of ST6GAL1 in the signaling pathway through which DT inhibited ICC cells, ST6GAL1-overexpressing HCCC9810 and HuCCT1 cells were treated with DT at IC50 value. Western blotting results revealed that DT inhibited the expression of the anti-apoptotic protein Bcl-2, while overexpression of ST6GAL1 enhanced Bcl-2 expression and partially reversed the inhibitory effect of DT. Moreover, DT was shown to suppress ST6GAL1 expression in both control vector cells and ST6GAL-overexpressing cells (Fig. 5A and B). The aforementioned results indicated that DT could inhibit the phosphorylation of NF-κB, STAT3 and activate p-P38. Consequently, nuclear proteins were next extracted to examine the changes in nuclear p-NF-κB, p-STAT3 and p-P38 in ICC cells treated with DT at the IC50 concentration. Western blotting results suggested that DT could activate p-P38, and inhibit p-NF-κB and p-STAT3 in whole cells (Fig. 5A and B; statistical charts, Fig. S5A and B), and further nuclear protein detection revealed an increased expression of nuclear p-P38, and a decrease in p-NF-κB and p-STAT3 (Fig. 5C and D; statistical charts, Fig. S5C and D). In addition, SNA lectin flow cytometry was employed to evaluate the α2,6 sialylation levels in cells, with the aim of exploring the enzymatic activity of ST6GAL1. The experimental findings revealed that DT could reduce the α2,6 sialylation on the cell membrane, and the overexpression of ST6GAL1 was able to counteract the reduction effect induced by DT (Fig. 5E and F). These findings further elucidated that DT can modulate the expression of ST6GAL1 by impeding NF-κB activation, diminishing the nuclear presence of p-NF-κB and ultimately suppressing ICC cell progression through the downregulation of the effector protein Bcl-2.


TCM plays an important role in cancer prevention and interception (1719). TCM has long been used to maintain health and treat ailments. Growing evidence supports the use of TCM, alone or combined with other treatments, in reducing adverse effects associated with chemotherapy, improving the quality of life of patients, lowering the risk of recurrence and prolonging OS (20). In recent years, researchers have been increasingly interested in exploring potential anticancer compounds from TCM. Nobiletin, a major component of Pericarpium Citri Reticulatae, could inhibit cholangiocarcinoma (CCA) proliferation by direct binding to GSK3β (21). β-elemene, a potent component found in turmeric from the Chinese herbal medicine Rhizoma Zedoariae, has the potential to halt the advancement of CCA through the reactivation of PCDH9 expression (22). The screening results of the present study showed that DT, bufalin, neriifolin and telocinobufagin strongly suppressed ICC cell viability. DT, bufalin, neriifolin and telocinobufagin share common characteristics as cardiotonic steroids. They inhibit the plasma membrane Na+/K+-ATPase pumps leading to increased intracellular levels of Na+ and Ca+, decreased K+ levels and enhanced cardiac contractile force (23). DT was isolated from the Digitalis species and features a steroid ring with a five-carbon unsaturated butyrolactone moiety (24,25). Bufadienolides such as bufalin, cinobufagin, telocinobufagin and others, found in Venenum Bufonis from toad species, possess a six-carbon unsaturated pyrone ring attached to the steroid ring (26,27). Among them, only DT has been approved by the FDA for specific cardiac conditions, particularly heart failure and certain heart arrhythmias. Consequently, the focus of the present study was on the possible therapeutic effects of DT on ICC.

The current study presented evidence from cellular functional assays indicating that DT effectively inhibited the proliferation and migration of ICC cells in a dose-dependent manner. Through GO and KEGG enrichment analyses, confirmed by RT-qPCR and western blotting validation, it was established that DT notably dampened the activation of the NF-κB signaling pathway, including its nuclear transcription activities, impeded the activation of p-STAT3 and activated the phosphorylation of P38. This was consistent with prior studies highlighting the ability of DT to inhibit p-STAT3 activation (28) and NF-κB (29). Additionally, while DT has been revealed to inhibit IL-1β-induced activation of p44/42-MAPK and NF-κB but not that of p38-MAPK in endothelial cells (30), the findings of the current study revealed that DT could activate p38-MAPK in ICC cells. During cellular stress, active p38 had been identified to phosphorylate and induce the degradation of BCL2 (31). Taken together, these findings revealed the role of DT as a comprehensive inhibitor of cancer progression by modulating critical signaling pathways pivotal to cancer cell growth and dissemination.

Sialylation is crucial in deciding cell destiny throughout development, reprogramming and cancer progression (32). The enzyme ST6GAL1, which catalyzes the attachment of α2,6-linked sialic acid to specific N-glycosylated proteins, either present on the cell membrane or secreted into the bloodstream, has been found to be upregulated in various cancers, where it promotes growth and invasiveness (33). Elevated levels of ST6GAL1 have also been linked to increased resistance to chemoradiotherapy in cancer models by inhibiting cell death (34). Furthermore, ST6GAL1 sourced from external environments can mimic the effects of endogenous proteins, fostering aggressive tumor proliferation and invasion (35). The present study demonstrated that ST6GAL1 overexpression could enhance ICC cell proliferation and migration, effectively countering the inhibition of proliferation and migration of DT. The engagement of NF-κB with the ST6GAL1 gene promoter initiates its transcription (16), a process inhibited by DT through the inhibition of NF-κB phosphorylation and nuclear translocation, leading to reduced ST6GAL1 mRNA and protein levels. Furthermore, it was demonstrated that overexpressing P65/NF-κB in 293T cells enhanced the expression of ST6GAL1. These findings indicated that DT impeded the progression of ICC by curtailing ST6GAL1 expression via the NF-κB pathway inhibition.

Despite DT being recognized as an essential drug by the World Health Organization, its potent activity and narrow therapeutic window pose significant challenges for clinical use (36). Adults should typically have therapeutic plasma DT value range of 10–30 ng/m (13.1–39.2 nM) (37). However, within the therapeutic concentration range of 25–40 nM, DT demonstrates no marked adverse effects on normal cells, highlighting its potential for safe application (38,39). It is crucial to monitor for possible cardiac complications associated with DT, especially at serum concentrations indicative of toxicity. Evidence showed that a serum concentration range of 108–205 ng/ml (140–270 nM) might lead to DT poisoning (10). However, the current study found that lower concentrations, specifically at half the IC50 value, were within a therapeutically effective range that demonstrated a significant inhibitory impact on ICC cells. Furthermore, previous studies have reported that DT can produce anticancer effects at concentration range of 20–33 nM without manifesting notable toxicity in patients with cardiac issues (40,41). Notably, there is a lack of research on the effects of DT on ICC. The findings of the current study indicated that a low concentration of DT, specifically at 1/4 IC50 value (23 nM for HCCC9810 and 26 nM for HuCCT1) at 48 h, fell within an effective range for exerting significant inhibitory effects on ICC cells. This observation was consistent with the previous studies of the anticancer efficacy of DT at concentrations that are not toxic. Furthermore, with a half-life range of 5–8 days in the human serum, DT provides a foundation for sustained tumor suppression (42).

In conclusion, DT, an effective anti-ICC compound, was identified from a TCM library. It was demonstrated that DT inhibited the progression of ICC by targeting ST6GAL1, a promoter of ICC growth. The findings of the current study revealed that DT downregulated ST6GAL1 at both the mRNA and protein levels in ICC cells and impeded NF-κB activation, which was crucial for the transcriptional activation of ST6GAL1. Therefore, the mechanism of DT in inhibiting ICC cell proliferation and metastasis was mediated through its interaction with the NF-κB/ST6GAL1 signaling pathway. These results not only emphasized the potential role of DT as a therapeutic agent for ICC, but also highlighted the critical function of ST6GAL1 in cancer progression. Nonetheless, a significant limitation of the present study was the lack of in vivo experiments to confirm the effectiveness of DT in treating ICC. Further investigation is necessary to comprehensively evaluate the in vivo effect.

Supplementary Material

Supporting Data
Supporting Data


Not applicable.


The present study was supported by the National Science Foundation of China (grant no. 82372321), the Shanghai Shenkang Hospital Development Center Project (grant no. SHDC2023CRT015) and the Shanghai ‘Rising Stars of Medical Talent’ Youth Development Program (grant no. 2024-70).

Availability of data and materials

The data generated in the present study are included in the figures and/or tables of this article. The RNA-seq data generated in the present study may be found in the SRA database under accession number PRJNA1112815 or at the following URL:

Authors' contributions

CG conceived and directed the project, and designed the experiments. YZ, RW, CH, XXu, XXi, LW, JW and TL carried out the experiments. YZ and RW conducted the data analysis and interpreted the results. YZ and CG wrote and edited the manuscript, and confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The requirement for the ethics approval of purchased primary human cells was waived by the Ethics Committee of Yueyang Integrated Traditional Chinese and Western Medicine Affiliated with Shanghai University of Traditional Chinese Medicine in accordance with Article 32 of the Chinese Measures for the Ethical Review of Life Science and Medical Research Using Humans.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.





intrahepatic cholangiocarcinoma


Traditional Chinese Medicine


ST6 β-galactoside α-2,6-sialyltransferase 1




dimethyl sulfoxide


optical density


intrahepatic bile duct epithelial cells






Gene Ontology


Kyoto Encyclopedia of Genes and Genomes


Sambucus nigra


Cell Counting Kit-8


overall survival





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Zhan Y, Wang R, Huang C, Xu X, Xiao X, Wu L, Wei J, Long T and Gao C: Digitoxin inhibits ICC cell properties via the NF‑&kappa;B/ST6GAL1 signaling pathway. Oncol Rep 52: 103, 2024
Zhan, Y., Wang, R., Huang, C., Xu, X., Xiao, X., Wu, L. ... Gao, C. (2024). Digitoxin inhibits ICC cell properties via the NF‑&kappa;B/ST6GAL1 signaling pathway. Oncology Reports, 52, 103.
Zhan, Y., Wang, R., Huang, C., Xu, X., Xiao, X., Wu, L., Wei, J., Long, T., Gao, C."Digitoxin inhibits ICC cell properties via the NF‑&kappa;B/ST6GAL1 signaling pathway". Oncology Reports 52.2 (2024): 103.
Zhan, Y., Wang, R., Huang, C., Xu, X., Xiao, X., Wu, L., Wei, J., Long, T., Gao, C."Digitoxin inhibits ICC cell properties via the NF‑&kappa;B/ST6GAL1 signaling pathway". Oncology Reports 52, no. 2 (2024): 103.