Unveiling the Anticancer Potential and Molecular Mechanisms of Fangchinoline Against Cholangiocarcinoma Using FTIR Microspectroscopy,In Vitro and In Silico Approaches

Abstract

Objectives:

The limited effectiveness of traditional chemotherapeutic agents against cholangiocarcinoma (CCA) highlights the need for novel compounds. This study aimed to investigate the anticancer potential of fangchinoline (FCL) and its underlying mechanisms of action in CCA cells.

Methods:

Biomolecular changes associated with FCL-induced cell death were determined by Fourier transform infrared spectroscopy. The apoptosis-inducing effects of FCL were investigated using annexin V/7-amino-actinomycin staining, caspase-9 activity, JC-1 transmembrane potential, and reactive oxygen species (ROS) assays. Western blotting of total cell lysates was used to assess protein expression, normalized to β-actin. The Proteome Profiler™ Human Phospho-Kinase Array Kit was used to assess changes in the phosphorylation patterns of cell survival-associated proteins. Network pharmacology analysis was conducted to predict FCL targets. The interaction between the compound and its potential targets were demonstrated by molecular docking and verified by western blot analysis.

Results:

FCL induced biochemical alterations in CCA cells. A series of in vitro analyses demonstrated that FCL induced mitochondria-associated apoptotic cell death in KKU-100 cells, with ROS induction contributing to this effect. Furthermore, FCL altered the phosphorylation status of several survival-related kinases, including Yes, c-Jun, ERK1/2, HSP27, and STAT5. Network pharmacology analysis identified Src as a central hub protein with the highest degree of connectivity among the signaling proteins affected by FCL. Consistently, western blot analysis confirmed a marked reduction in Src phosphorylation following FCL treatment. Molecular docking further supported this finding, showing that FCL interacted with the catalytic residues of Src.

Conclusion:

The anticancer potential of FCL against CCA was demonstrated using several approaches. These findings indicated a potential therapeutic opportunity for FCL in CCA.

Introduction

Cholangiocarcinoma (CCA), or bile duct cancer, poses a significant threat to human health, particularly in Southeast Asia, where it exhibits a high incidence and mortality rate. ¹ The primary risk factor for CCA in this region is liver fluke infection, caused by Opisthorchis viverrini and Clonorchis sinensis, typically contracted through the consumption of raw or fermented fish. ² Liver fluke-induced chronic inflammation promotes a tumor microenvironment rich in cytokines and growth factors that disrupt intracellular signaling pathways. ² While surgical resection can be curative in early stages, most patients present with advanced disease that requires systemic chemotherapy. The standard treatment regimen for advanced CCA includes a combination of cisplatin and gemcitabine, with second-line options such as 5-fluorouracil and oxaliplatin.^3,4 However, treatment efficacy is often limited by resistance to existing drugs, highlighting the urgent need for novel therapeutic strategies.

Currently, multi-approach studies are being conducted to explore the anticancer mechanisms of compounds. Fourier transform infrared (FTIR) spectroscopy is widely used to detect molecular-level structural changes in cells based on atomic vibrations and rotations. ⁵ Each substance produces a unique fingerprint, allowing FTIR to identify biomolecular alterations in response to drugs or phytochemicals. ⁶ Network pharmacology, which integrates systems biology with network analysis, reveals compound–target interactions and key signaling pathways. ⁷ Combined with molecular docking to confirm target binding, these in silico tools complement in vitro experiments, offering a powerful strategy to investigate phytochemicals in cancer treatment and enhance drug discovery.

One potential strategy for cancer prevention and treatment is the induction of apoptosis in malignant cells. ⁸ This process involves molecular changes such as membrane alteration, protein synthesis disruption, and DNA degradation into fragments. The key apoptotic hallmarks include DNA fragmentation, caspase activation, and phosphatidylserine externalization. ⁹ Mitochondrial-mediated apoptosis, triggered by imbalance between pro- and anti-apoptotic proteins, leads to membrane disruption and cytochrome c (Cyt c) release, which activates caspase-9 and subsequently caspase-3, resulting in the degradation of nuclear and cytoskeletal components.^10,11 Cell survival and apoptosis are opposing but interconnected processes that regulate tissue homeostasis. Several intracellular signaling pathways determine whether a cell survives or undergoes death. ¹² Previous studies have shown the involvement of pathways such as MAPK, ¹³ FAK, ¹⁴ and JAK/STAT. ¹⁵ Other key regulators include Src, c-Myc proto-oncoproteins, and p53. ¹⁶ Thus, targeting dysregulated survival- and apoptosis-related pathways may be a key strategy in cancer therapy.

Phytochemicals have been recognized for their ability to modulate signaling pathways related to cancer cell survival and apoptosis, making them attractive candidates for overcoming chemotherapy resistance. Fangchinoline (FCL, Figure 1) is a major bisbenzylisoquinoline alkaloid, characterized by 2 linked benzylisoquinoline units. It is primarily isolated from the roots of Stephania tetrandra, a medicinal plant widely used in traditional Chinese medicine. ¹⁷ This compound exhibits several pharmacological properties, including anti-inflammatory, antidiabetic, and anticancer effects. ¹⁸ Notably, FCL has shown antitumor activity in various cancers, including colon and breast cancers.^19,20 Due to its broad anticancer potential, FCL has attracted interest as a candidate for drug development. However, its effects on CCA and the mechanisms involved remain poorly understood. In this study, we investigated the anticancer activity and underlying mechanisms of FCL in CCA cells for the first time. KKU-100 and KKU-213A, CCA cell lines derived from liver fluke-associated CCA patients with low sensitivity to gemcitabine, a standard chemotherapeutic drug in CCA treatment, ²¹ were employed in the study. FTIR spectroscopy was used to examine global biomolecular alterations following FCL treatment. Apoptosis assays, molecular analyses, network pharmacology, and molecular docking were conducted to explore the mode of action of FCL. Our findings provide new insights into the potential of FCL as a therapeutic candidate for CCA.

Figure 1.

Structure of FCL.

Materials

Reagents for cell culture, including Ham’s F12 medium, fetal bovine serum (FBS) and trypsin, were obtained from Gibco BRL Life Technologies (Gibco, NY, USA). FCL (99.9%) and JC-1 dye were obtained from MedChemExpress (Monmouth Junction, NJ, USA). Sigma Chemical (St. Louis, MO, USA) provided the methylthiazolyldiphenyl tetrazolium bromide (MTT), acridine orange (AO), and ethidium bromide (EB). The primary antibodies against BAX (Cat#sc-7480, RRID:AB_626729), Bcl-2 (Cat#sc-65392, RRID:AB_831019), Bcl-XL (Cat#sc-8392, RRID:AB_626739), Cyt c (Cat#sc-13560, RRID:AB_627383), β-actin (Cat#sc-47778, RRID:AB_626632), mouse anti-rabbit IgG (Cat#sc-2357, RRID:AB_628497), and m-IgGκ BP-HRP (Cat#sc-516102, RRID:AB_2687626) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Primary antibody against phospho-Src (Tyr416, Cat#2101, RRID:AB_331697) was purchased from Cell Signaling Technology. Primary antibody against Src (Cat#BF0357, RRID:AB_2833635) was purchased from Affinity Biosciences (Ohio, USA). Cozy^TM prestained protein ladder was purchased from highQu GmbH (Kraichtal, Germany). All other reagents were of analytical grade and the highest available purity.

Cell Lines and Cell Culture

The human CCA cell lines, KKU-100 (RRID:CVCL_3996) and KKU-213A (RRID: CVCL_M261) were kindly provided by Cholangiocarcinoma Research Institute, Khon Kaen University. KKU-100 cell line was derived from poorly differentiated tubular adenocarcinoma, a prevalent subtype of CCA in Southeast Asia, while KKU-213A represents mixed papillary and non-papillary CCA. Cells were grown in Ham’s F12 medium supplemented with 10% FBS, 10 mM HEPES (pH 7.3), and sodium bicarbonate. Gentamicin (100 µg/mL) and penicillin (100 U/mL) were added as antibiotics. Cells were cultured at 37°C in a humidified incubator with 5% CO₂.

Cell Viability Assay

Cell viability of KKU-100 and KKU-213A cells was assessed using the MTT assay as previously described. ²² Cells were seeded in a 96-well plate at the density of 7500 cells/well and allowed to attach overnight, then treated with vehicle or various concentrations of FCL (1-100 µM) for 24 hours. After treatment, 12 mM MTT was added and incubated for 4 hours, followed by the addition of 100 µL DMSO to dissolve the formazan crystals. Absorbance was measured at 490 nm using a microplate reader. Results were expressed as a percentage of viability relative to the control.

FTIR Microspectroscopy Analysis

The FTIR microspectroscopy analysis was conducted by previously described methods. ²³ For sample preparation, the KKU-100 and KKU-213A cells were cultured in a 6-well plate at the density of 250 000 cells/well and treated with 50 µM FCL compared to the control (untreated) for 24 hours. Cells were harvested by trypsinization and washed once with phosphate buffered saline (PBS). After washing, the cell pellets were resuspended in sterile isosmotic normal saline solution (0.9% sodium chloride) to prevent the interference of phosphate salts in the PBS formula during FTIR analysis. The cell suspension was then spotted onto a barium fluoride (BaF₂) window. The spots were dried using a vacuum desiccator. The deposited NaCl salts were repeatedly washed with deionized water until no salt was observed under a microscope. The windows were kept away from light at ambient temperature in a desiccator until FTIR analysis.

FTIR microspectroscopy was performed using a Bruker Tensor 27 spectrometer coupled to a Bruker Hyperion 3000 microscope with a 36x objective lens (Bruker Optics Inc., Ettlin Gen, Germany). The FTIR spectra were recorded in the region between 4000 and 600 cm⁻¹ at a 6 cm⁻¹ spectral resolution with 64 scans. Approximately 150 spectra (50 spectra per replicate) were recorded and used for the analysis. Spectral acquisition and instrument control were performed using OPUS software (version 7.2; Bruker Optics, Ettlinger, Germany).

For FTIR data analysis, the spectral data were subjected to Savitzky–Golay smoothing using a third-degree polynomial and 13 smoothing points. Extended multiplicative signal correction (EMSC) was applied in the regions of 3000 to 2800 and 1770 to 900 cm⁻¹ for normalization. For the second-derivative spectra, the spectral data were treated by the Savitzky–Golay derivative transform using a second-degree derivative, a third-degree polynomial, and 13 smoothing points. EMSC were used for normalization, as in the primary spectra. Biochemical changes after FCL treatment were determined using second-derivative spectra analyzed by principal component analysis (PCA) in the regions 3000 to 2800 and 1770 to 900 cm⁻¹. The data are presented as PCA score and loading plots. Data analysis was performed using Unscrambler^® X software (version 10.1; CAMO Software, Oslo, Norway).

Apoptosis Assay

Apoptotic cell death assay was performed using the Phycoerythrin (PE) Annexin V Apoptosis Detection Kit I (BD Biosciences, San Jose, CA, USA), followed by flow cytometry. The analysis was performed according to the manufacturer’s instructions. Briefly, KKU-100 cells were seeded at 250 000 cells/well in a 6-well plate and treated with FCL at various concentrations (0, 10, 20, 50, and 100 µM) for 24 hours. After incubation, the CCA cells were collected, washed, resuspended in buffer, and stained with annexin V-PE and 7-amino-actinomycin (7-AAD). The number of apoptotic cells was determined by flow cytometry (FACSCanto II; BD Biosciences).

Acridine Orange (AO)/Ethidium Bromide (EB) Staining

Detection of apoptotic cell death in CCA cells was also identified by AO/EB staining method as previously described. ²⁴ Briefly, KKU-100 cells were cultured in a 96-well plate at the density of 7500 cells/well and exposed to various concentrations of FCL for 24 hours. Thereafter, the cells were stained with 1 µg/mL AO and EB for 15 minutes in the dark. Subsequently, images were captured using a fluorescence phase-contrast inverted microscope (CKX53; Tokyo, Japan) with excitation and emission wavelengths of 480 and 535 nm, respectively

Caspase-9 Activity Assay

The detection of caspase 9 activity was performed using a commercial Caspase-Glo® 9 Assay kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Briefly, KKU-100 cells were seeded in a 96 well-white culture plate at the density of 10 000 cells/well and incubated overnight. Thereafter, the cells were treated with 50 µM FCL for designated time points. After complete treatment, the Caspase-Glo® 9 reagent was added, mixed and incubated at room temperature for 3 hours. The luminescence signal was then measured using SpectraMax^® L Microplate Reader (Molecular Devices, LLC., San Jose, CA, USA).

Mitochondrial Transmembrane Potential Assay

The JC-1 dye staining method followed by flow cytometry was used to determine the mitochondrial transmembrane potential, as previously described method. ²⁴ In brief, KKU-100 cells were cultured at the density of 250 000 cells/well in a 6-well plate and exposed to vehicle or various concentrations of FCL (20, 50, and 100 µM) for 12 hours. Thereafter, the cells were stained with JC-1 dye for 20 minutes in an incubator at 37°C. After incubation, the cells were collected, washed, suspended in PBS, and analyzed by flow cytometry.

Western Blot Analysis

Protein expression was determined using western blot analysis previously described. ²² Briefly, KKU-100 cells were seeded in a 6 well- plate at the density of 250,000 cells/well and treated with 50 µM FCL at various time points. Cell lysates were collected using RIPA lysis buffer (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Protein samples (20 µg) were subjected to western blot analysis. Proteins were loaded onto 10% gels and SDS-PAGE was performed. After a complete run, the target proteins were electrically transferred to a polyvinylidene fluoride membrane and then incubated with 5% bovine serum albumin to block non-specific binding. Subsequently, the membranes were incubated with specific primary antibodies against the target proteins at 4°C for an overnight. The membranes were then washed and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for 2 hours at room temperature. Target protein expression was detected using Luminata^TM Forte Western HRP Substrate (Merck Millipore, Watford, UK) and photographed using ChemiDoc^TM MP Imaging system (Bio-Rad, Hercules, CA, USA). The intensities of the immunoreactive bands were analyzed using Image Lab^TM Software version 5.2.1.

Reactive Oxygen Species (ROS) Assay

Intracellular ROS levels were measured using the cell-permeable fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFDA) previously described. ²⁵ Briefly, KKU-100 cells were seeded in a 6 well-plate at the density of 250 000 cells/well and treated with 50 µM FCL combined with 25 µM DCFDA for 90 minutes. After incubation, the cells were washed and resuspended in PBS for flow cytometry.

Analysis of Phosphorylation Profiles of Cell Survival-Associated Kinases

The Proteome Profiler™ Human Phospho-Kinase Array Kit (Catalog Number ARY003C; R&D Systems, Inc., Minneapolis, MN, USA) was employed to assess changes in the phosphorylation patterns of cell survival-associated proteins following the manufacturer’s instructions. Briefly, KKU-100 cells were cultured in a 6 well- plate at the density of 250 000 cells/well and treated with 50 µM FCL for 12 or 24 hours. After complete treatment, the cell lysate was collected, added to the membrane, and incubated at 4°C overnight. Thereafter, the membrane array was washed and incubated with a detection antibody cocktail at room temperature for 2 hours. The membrane was then washed, and streptavidin-HRP was added. Target protein expression was detected using Luminata^TM Forte Western HRP Substrate (Merck Millipore) and photographed using a ChemiDoc^TM MP Imaging system (Bio-Rad). The intensities of the immunoreactive bands were analyzed using Image Lab^TM Software version 5.2.1.

Network Pharmacology Analysis

Network pharmacology analysis was performed as previously described with minor modifications. ²⁶ Potential targets of FCL were retrieved from the Swiss Target Prediction database (http://www.swisstargetprediction.ch), and CCA-associated targets were obtained from the DisGeNET (https://www.disgenet.org) and GeneCards (https://www.genecards.org) databases. The datasets were combined to form a union set and their intersections were identified. The intersecting targets were then supplemented with a predefined list of markedly altered survival kinases: JUN (c-Jun), STAT5B (STAT5a/b), MAPK1 (ERK2), MAPK3 (ERK1), Yes, and heat shock protein (HSP)B1 (HSP27), to establish the final dataset. A protein-protein interaction (PPI) network was constructed using the stringApp plug-in in Cytoscape (version 3.10.3) using the “Homo sapiens” model with an interaction confidence threshold of 0.7. Core target proteins were determined using the CytoHubba plug-in by ranking the proteins based on their degree of centrality. Finally, pathway enrichment analysis was conducted using the ClueGo plug-in in Cytoscape with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, employing a Benjamini-Hochberg adjusted P-value cutoff of 0.05.

Molecular Docking Analysis

Molecular docking was performed as described in a previous report. ²⁷ First, the 3D structures of the ligands, FCL (CID 73481) and SM1-71 (CID: 134130572), were retrieved from the PubChem database (https://pubchem.ncbi.nlm.nih.gov). The ligand structures were then subjected to energy minimization using PyRx (Pyrx-Python Prescription 0.8) with the Merck molecular force field parameter (MMFF94). The 3D crystal structure of the human Src protein (PDB ID: 6ATE) was obtained from the Protein Data Bank and preprocessed by removing water molecules, solvent, and any bound ligands using PyMOL (version 2.4.1; PyMOL Molecular Graphics System, Schrödinger). Following these preparations, molecular docking between the energy-minimized ligands and the refined protein structure was performed using PyRx. The docked complexes were visually analyzed using PyMOL, and two-dimensional interactions were examined in detail using Discovery Studio Visualizer (version 21.1.0.20298; BIOVIA, San Diego, CA, USA).

Statistical Analyses

Data were analyzed using Student’s t-test or 1-way ANOVA with post hoc Student–Newman–Keuls test, where appropriate. The results were considered statistically significant at P < .05. All statistical analyses were performed using GraphPad Prism 8.0 software (GraphPad Software Inc., San Diego, CA, USA). PCA of FTIR spectral data was performed using Unscrambler^® X software (CAMO Software).

Results

FCL Decreased Cell Viability and Induced Biomolecular Changes in CCA Cells as Revealed by FTIR Microspectroscopy

The effect of FCL on the CCA cell lines, KKU-100 and KKU-213A, was initially evaluated based on cell viability. FCL reduced cell viability in a dose-dependent manner, with an IC₅₀ of 19.9 ± 2.8 and 21.3 ± 4.6 µM at 24 hours for KKU-100 and KKU-213A, respectively (Supplemental Figure S1). To investigate biomolecular changes associated with cell death, FTIR analysis was conducted on CCA cells treated with 50 µM FCL, a concentration exceeding the IC₅₀ threshold. At this dose, a substantial proportion of the cells underwent treatment-induced death, making the detected molecular alterations highly relevant to cell death mechanisms. The FTIR spectra of KKU-100 and KKU-213A cells treated with FCL are shown in Figure 2.

Figure 2.

FTIR analysis of control and FCL-treated CCA cells. (A) KKU-100 cells: Primary and second derivative FTIR spectra of untreated (control, n = 115) and FCL-treated (n = 146) cells are shown in spectral regions corresponding to lipids (3000-2800 and 1770-1725 cm⁻¹), proteins (1725-1480 cm⁻¹), and nucleic acids and carbohydrates (1280-900 cm⁻¹). Principal component analysis (PCA) score and loading plots were generated from the second derivative spectra in the 3000 to 2800 and 1770 to 900 cm⁻¹ regions. Quantitative comparisons of integrated absorbance areas for each spectral region between control and treated groups are also included. (B) FTIR analysis of KU-213A cells: FTIR spectral profiles, PCA score and loading plots, and integrated absorbance area comparisons between untreated (control, n = 145) and FCL-treated (n = 101) cells were analyzed in the same spectral regions as in (A).

For KKU-100 cells (Figure 2A), the spectral differences between untreated and FCL-treated cells were revealed in regions of lipids (3000-2800 and 1770-1725 cm⁻¹) and nucleic acids and carbohydrates (1280-900 cm⁻¹). In the lipid-related peaks, a decrease in absorbance intensity was observed at 2923 cm⁻¹ designated for the asymmetric stretching vibration of CH₂ of saturated fatty acid^{23,28 -30} and 1739 cm⁻¹ designated for stretching vibration of (C=O) of ester from phospholipids, triglycerides, and cholesterols.^{23,28 -30} For nucleic acid and carbohydrate-associated peaks, a decrease and increase in absorbance intensity were observed at 1234 cm⁻¹ designated for asymmetric stretching vibration of (PO₂) of nucleic acids^{23,28 -30} and 1155 cm⁻¹ designated for stretching vibration of (C–C), (C–O–C), and (C–OH) of ether and alcohol moieties of carbohydrates,^23,28,29 respectively. A spectral shift was also observed at 1055 cm⁻¹ designated for the stretching vibration of (C–O) of polysaccharides and glycogen.^23,28,29 For KKU-213A cells (Figure 2B), the absorbance intensity was increased in the regions of lipids and nucleic acids after FCL treatment, which is distinguished from KKU-100 cells in terms of direction. However, the effect of FCL on cellular proteins was parallel between KKU-100 and KKU-M213. These spectral variations suggest that FCL induces biochemical modifications in KKU-100 and KKU-M213 cells, potentially contributing to its anticancer effects.

PCA of secondary spectra was employed to determine the spectral similarity within groups and the difference among groups. The PCA score plot depicted the spectral similarity within untreated (control, n = 115) and FCL-treated cells (n = 146) of KKU-100 cells (Figure 2A), and untreated (control, n = 145) and FCL-treated cells (n = 101) of KKU-213A cells (Figure 2B), indicated by the clustering of spectral data. The biochemical changes were also confirmed with the FTIR spectra by distinguished clusters among groups explained by the % variance of PC-1 and PC-2. KKU-100 cells possessed 70 and 17% variance, and KKU-213A possessed 84 and 9% variance for PC-1 and PC-2, respectively. PCA loading plots also confirmed the changes in lipids and nucleic regions of KKU-100 cells, exhibited by influencing wavenumbers in the PC-1 major distinguished axis, including 2923, 2852, 1155, 1083, and 1055 cm⁻¹ (Figure 2A). PCA loading plots of KKU-M213 cells showed a predominant effect in the regions of lipids and proteins, presented by influencing wavenumbers in the PC-1, including 2921, 2852, 1656, and 1626 cm⁻¹ (Figure 2B).

The integral areas under the primary spectra demonstrate the biochemical abundance of each biomolecule under their specific region. In the KKU-100 cells (Figure 2A), the abundances of cellular lipids and proteins were significantly decreased by the FCL treatment when compared to the control (untreated cells), from 0.046 ± 0.0001 to 0.041 ± 0.0012 and 0.066 ± 0.0007 to 0.056 ± 0.002, respectively. Abundances of nucleic acid, and glycogen and carbohydrates were not significantly changed. For KKU-213A cells (Figure 2B), the abundances of cellular lipids, nucleic acids, and glycogen and carbohydrates were significantly increased by the FCL treatment from 0.053 ± 0.002 to 0.072 ± 0.002, 0.020 ± 0.0008 to 0.022 ± 0.001, and 0.012 ± 0.001 to 0.015 ± 0.0004, respectively. Though the abundance of protein was significantly decreased from 0.111 ± 0.0008 to 0.099 ± 0.001.

Since FTIR analyses revealed distinct biochemical alterations in response to FCL treatment between the KKU-100 and KKU-213A cell lines, indicating that although both CCA cells exhibited similar levels of cytotoxicity, their biochemical responses to FCL were not identical. These findings suggest potential cell-type-specific differences in the molecular mechanisms underlying FCL-induced cytotoxicity. Based on these observations, and to gain deeper insights into the mechanism of action, subsequent investigations focused on the KKU-100 cells. This CCA cell line was selected as a representative model in this study as it is derived from poorly differentiated tubular adenocarcinoma, the most common CCA subtype in Thailand,^31,32 and tubular-type CCA, including poorly differentiated forms, is more frequently observed than mixed papillary and non-papillary types in regions with a high incidence of CCA such as Southeast Asia,^33,34 making KKU-100 a clinically relevant model.

FCL Induced Apoptotic Cell Death of KKU-100 CCA Cells

Given that the biomolecular changes observed in the FTIR analysis may impact cell survival, and that apoptosis induction is a key anticancer mechanism, this study further evaluated the pro-apoptotic effect of FCL using annexin V-PE/7-AAD double dye staining followed by flow cytometric analysis. The results showed that treatment with FCL for 24 hours induced apoptosis in KKU-100 cells in a concentration-dependent manner. The percentage of apoptotic cells increased from 4.6 ± 1.6% in the untreated control to 5.6 ± 1.0%, 14.5 ± 1.0%, 23.9 ± 2.9%, and 64.9 ± 9.8% following treatment with 10, 20, 50, and 100 µM FCL, respectively (Figure 3A). The apoptosis-inducing effect of the compound was confirmed using AO/EB staining (Figure 3B). Live cells have a normal green nucleus. Cells treated with 50 and 100 µM FCL exhibited a marked increase in apoptosis, as evidenced by the presence of condensed and/or fragmented chromatin. These data indicated that the compound limited the ability of CCA cells to thrive by activating the apoptotic cell death signaling pathway.

Figure 3.

Apoptosis-inducing effect of FCL against KKU-100 CCA cells. (A) Quantification of apoptotic cells by annexin V-PE/7-AAD staining and flow cytometry. The graph shows the percentage of cells in each quadrant: live cells (lower left), early apoptotic (lower right), late apoptotic/necrotic (upper right), and debris (upper left). (B) AO/EB staining showing live cells with green nuclei and apoptotic cells (white arrows) with condensed or fragmented chromatin. (C) Caspase-9 activity increased after exposure to FCL at 12 and 24 hours. (D) JC-1 staining revealed increased JC-1 monomer, indicating depolarized mitochondria after 12 hours of FCL treatment. (E) FCL altered expression of apoptotic-related proteins. (F) ROS levels assessed by DCFDA fluorescent staining and flow cytometry. Representative images shown are from 1 of 3 independent experiments. Data are presented as mean ± SEM (n = 3).

Apoptosis-Inducing Effect of FCL was Mediated via Intrinsic Apoptosis Pathway in KKU-100 CCA Cells

Intrinsic or mitochondria-mediated apoptotic cell death is a common defense mechanism against carcinogenesis. ⁸ To evaluate whether FCL activates this pathway, several key markers were assessed. First, the activity of caspase-9, the initiator caspase in the intrinsic apoptotic pathway, was measured. FCL treatment resulted in a 2.3-fold and 4.0-fold increase in caspase-9 activity in KKU-100 cells at 12 and 24 hours, respectively (Figure 3C). Mitochondrial membrane potential was subsequently assessed using JC-1 staining and quantified by flow cytometry. As shown in Figure 3D, FCL impaired mitochondrial function, indicated by an increase in JC-1 monomer fluorescence of 16.1 ± 9.3%, 40.0 ± 6.4%, and 50.4 ± 9.7% in the control, 50 µM, and 100 µM FCL-treated groups, respectively. Consistent with these findings, FCL altered the expression of key proteins involved in the intrinsic pathway. The expression levels of the anti-apoptotic protein Bcl-2 were significantly reduced following FCL treatment, with relative levels decreasing to 0.7 at 12 hours and 0.4 at 24 hours compared to the control (normalized to 1). Similarly, Bcl-XL expression was significantly reduced to 0.7 at 12 hours and 0.6 at 24 hours. In contrast, the levels of the pro-apoptotic protein BAX and Cyt c were elevated at 12 hours post-treatment, showing 1.7- and 1.6-fold increases, respectively (Figure 3E). These results indicate that FCL induces apoptosis in KKU-100 CCA cells through activation of the intrinsic apoptotic pathway. Since reactive oxygen species (ROS) accumulation is a critical trigger of apoptosis, ⁹ ROS levels were assessed using DCFDA staining. As shown in Figure 3F, FCL treatment (50 µM) significantly increased fluorescence intensity by 3-fold, indicating elevated ROS generation. These findings suggest that FCL-induced apoptosis in CCA cells is mediated, at least in part, by ROS accumulation.

FCL Altered Phosphorylation Profiles of Cell Survival-Associated Molecules in KKU-100 CCA Cells

Apoptosis is regulated by multiple signaling cascades, many of which are closely linked to cell survival. In addition to ROS-induced apoptotic cell death, the modification of other signaling pathways may contribute to the cell death-promoting effect of FCL. Because apoptosis and survival pathways often intersect, identifying survival-associated molecules affected by FCL is important for understanding its effects in CCA cells. To explore this, the Proteome Profiler™ Human Phospho-Kinase Array Kit was employed to assess changes in the phosphorylation patterns of key survival-associated proteins. As shown in Figure 4B, phosphorylation levels of Yes, c-Jun, ERK1/2, STAT5a/b, and HSP27 were markedly altered in KKU-100 cells after 12 hours of FCL treatment. Specifically, phosphorylation of Yes, c-Jun, ERK1/2, and HSP27 increased by approximately 10-fold, 8-fold, 5-fold, and 12-fold, respectively, whereas STAT5a/b phosphorylation decreased by approximately 3-fold. Notably, the phosphorylation of c-Jun and HSP27 remained elevated at 24 hours, with increases of approximately 18-fold and 5-fold, respectively. Our results suggest that FCL modulates survival signaling pathways, potentially contributing to its ability to induce cell death in CCA cells.

Figure 4.

Antibody array analysis of phosphorylated cell survival-associated protein expression. (A) Antibody map. KKU-100 cells were exposed to 50 µM FCL for (B) 12 and (C) 24 hours, and protein lysates were subsequently collected and used to quantify the target protein expression levels. Relative protein expression levels were obtained by quantifying the pixel densities of dot blots using Gel-Pro analyzer software. Heat map illustrating increased or decreased protein expression in the treatment group compared with that in the control group. Light green denotes low relative expression level, and dark red denotes high relative expression level. Histograms show the fold change in protein expression when compared with the control. The arrows indicate the up-regulation of protein more than 3 folds or the down-regulation of protein more than 2 folds in the treatment group when compared with the control group.

Network Analysis of Potential FCL Targets Against CCA

To gain further insight into the potential molecular targets of FCL in CCA cells, a network pharmacology analysis was performed. The potential targets of FCL in CCA were identified by comparing FCL-associated targets with known CCA-related targets. The resulting shared targets were integrated into a list of significantly altered survival-associated molecules identified using antibody array analysis. A protein-protein interactions (PPI) network was constructed to further explore these interactions (Figure 5A). The network consisted of 37 nodes and 76 edges, with an average node degree of 4.3 and a clustering coefficient of 0.4, indicating a moderately connected network of interacting proteins. This structural organization suggested that the network was not randomly connected, but instead exhibited some level of modular organization, potentially reflecting regulatory interactions or signaling networks.

Figure 5.

Network pharmacology analysis of FCL targets in CCA cells. (A) Protein-protein interaction (PPI) network of potential FCL targets. Yellow nodes represent protein targets identified from in vitro survival-associated molecules significantly altered after FCL treatment. (B) Hub gene analysis based on degree centrality, where node size and color (red to yellow) indicate ranking by degree value. (C) Pathway enrichment analysis using the Reactome database. Pathways sharing >50% of proteins are color-coded to indicate redundancy. The top 3 hub genes are highlighted with orange circles.

Hub proteins within the PPI network were identified and ranked based on degree of centrality (Figure 5B). Network analysis identified Src as a central regulatory hub among the signaling proteins modulated by FCL, based on its high connectivity with other affected proteins such as Yes, c-Jun, ERK1/2, and STAT5. To further explore the functional significance of this network, KEGG pathway enrichment analysis was performed to identify associated signaling pathways. The analysis revealed 5 major pathways ranked by percentage enrichment, including focal adhesion, proteoglycans in cancer, Rap1, Ras, and MAPK signaling pathways (Figure 5C). Notably, Src serves as a critical link between these pathways, reinforcing its role as a central regulator.

FCL Suppressed Activation of Src in KKU-100 CCA Cells

As the network analysis revealed that Src is likely to be the main regulator of the anticancer action of FCL against CCA, the effect of the compound on Src activation in KKU-100 cells was subsequently investigated. Western blot analysis demonstrated that treatment with 50 μM FCL led to an approximately 50% reduction in phospho-Src levels compared to control cells (Figure 6A), indicating that FCL suppresses Src activation. In contrast, the total Src protein level remained unchanged, suggesting that the effect of FCL is specific to the phosphorylation state rather than overall Src expression.

Figure 6.

Effect of FCL on Src activation and its molecular interaction with Src kinase domain (PDB ID: 6ATE). (A) Western blot analysis for determination of the effect of FCL on the expression of phospho-Src and total Src proteins. Representative figures and quantification graphs are illustrated. Data are expressed as mean ± SEM of 3 independent experiments. (B-E) Molecular docking analysis demonstrating the interaction of FCL and Src. (B) Superposition of FCL (docking pose) and Src inhibitor SM1-71 (crystallography pose), with the ATP-binding pocket indicated by a pink dotted line. (C) 3D binding interaction analysis of FCL within the Src kinase domain. (D) 2D interaction diagram summarizing key molecular interactions. (E) Binding interaction heatmap comparing FCL and SM1-71 (docking and crystallography poses). Residues are displayed as columns, while compounds are listed as rows. Dot size and color represent interaction strength.

FCL Interacted With Src in Molecular Docking Analysis

To verify whether Src is a potential molecular target of FCL in CCA cells, molecular docking analysis was performed to evaluate their interaction. Src (PDB ID: 6ATE) was selected for docking because it corresponds to the phosphorylation stage examined in our previous experiment. Docking analysis revealed that FCL bound to the catalytic site of the Src kinase domain, overlapping with the experimental binding poses of the Src inhibitor, SM1-71 (Figure 6B and C). Further analysis of the binding interactions indicated that FCL primarily engaged Src through hydrophobic interactions (Figure 6D), suggesting a relatively weak binding affinity. To further compare the binding interactions and residue engagement between FCL and the Src inhibitor, we conducted heatmap analysis (Figure 6E). FCL interacted with fewer Src residues than SM1-71, primarily through weak hydrophobic interactions. In contrast, SM1-71 formed multiple strong interactions with the Src residues, reinforcing its high inhibitory potential. Molecular docking analysis confirmed that the binding profile of SM1-71 closely matched its experimentally determined conformation, thereby supporting the reliability of our model. The observed differences in interaction strength were aligned with their binding affinities: FCL (−6.9 kcal/mol) and SM1-71 (−9.2 kcal/mol). The weaker binding of FCL, driven primarily by hydrophobic interactions, may contribute to its lower affinity for Src than for SM1-71. Despite its weaker binding, FCL interacted with the catalytic residues of Src, indicating that it could influence Src activity and potentially contribute to the suppression of Src phosphorylation.

Discussion

Bioactive components of medicinal herbs and dietary plants are potential options for cancer therapy. Understanding the mechanisms underlying their anticancer activity is crucial for the development of effective chemotherapeutic drugs that can improve treatment outcome. In the present study, we demonstrated that CCA cells display biochemical alterations in their molecular components after treatment with FCL. Comprehensive molecular analyses demonstrated that FCL induces apoptotic cell death in KKU-100 CCA cells via a mitochondria-dependent pathway. Stimulation of ROS production leading to the induction of apoptosis and suppression of Src activation contributed, in part, to the inhibitory effect of FCL on CCA cell survival. Therefore, this phytochemical may serve as a potential complementary approach for CCA treatment.

FTIR has been applied to study the changes in cellular biomolecules, such as proteins, lipids, DNA, and RNA, in response to various substances. In this study, biochemical changes in molecular components are observed in KKU-100 and KKU-213A CCA cells after exposure to FCL. While MTT results showed comparable cytotoxic responses between both CCA cell lines, FTIR analysis revealed distinct biochemical changes, particularly in lipids, nucleic acids, and glycogen and carbohydrates. These differences imply that, although FCL exerts cytotoxic effects on both cell lines, the underlying cellular responses and mechanisms of action may differ depending on the intrinsic properties of each cell type. To explore these mechanisms in greater detail, KKU-100 was chosen for mechanistic studies as it represents a prevalent CCA subtype in Southeast Asia, thereby providing greater clinical relevance than KKU-213A.

Since the biomolecular changes observed in the FTIR analysis may impact cell survival, and given that an important strategy in the management of malignancy is to overcome the ability of cancer cells to evade apoptosis, we further investigated the effect of FCL on apoptosis induction and performed detailed mechanistic analyses in KKU-100 CCA cells. During the early stage of apoptosis, phosphatidylserine externalization occurs on the plasma membrane, which can be observed by staining with annexin V. ³⁵ In this study, FCL induced the apoptotic death of KKU-100 CCA cells. AO/EB staining confirmed the apoptosis-promoting effects of the compound. Concordance in multiple analyses suggested that FCL treatment triggered programmed cell death in KKU-100 cells, leading to chromatin condensation, DNA fragmentation, and other apoptotic morphological features. In addition to CCA, the apoptosis-inducing activity of FCL has been reported in several cancers, including colon ¹⁹ and breast cancers. ²⁰

In-depth molecular investigations were performed to understand the mechanisms underlying the apoptosis-enhancing effects of FCL in KKU-100 cells. Given that mitochondria-mediated apoptotic death is a major pathway in programmed cell death mechanism, ⁸ this pathway is emphasized herein. Proteins of the Bcl-2 family play a critical role in maintaining mitochondrial membrane integrity, thereby preventing the activation of apoptotic cascades. ¹¹ Our results demonstrated that FCL significantly altered the expression profile of key regulatory proteins involved in the intrinsic apoptotic pathway. Specifically, treatment with FCL led to an upregulation of the pro-apoptotic protein BAX at 12 hours, accompanied by a downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-XL at both 12 and 24 hours. This shift in the balance between pro- and anti-apoptotic proteins favors mitochondrial outer membrane permeabilization, resulting in the release of Cyt c into the cytosol. Consistently, Cyt c levels were elevated by following 12 hours of treatment, and caspase-9 activity was significantly increased at both 12 and 24 hours, indicating activation of the intrinsic caspase cascade. Downregulation of Bcl-2 and Bcl-XL at both time points suggests sustained inhibition of anti-apoptotic signaling, while the transient elevation of BAX and Cyt c at 12 hours may reflect an early commitment to apoptosis, potentially followed by downstream execution events or feedback regulatory mechanisms. Furthermore, FCL disrupted mitochondrial membrane potential, as shown by a dose-dependent increase in JC-1 monomer fluorescence, further supporting the role of mitochondrial dysfunction in FCL-induced apoptosis. Together, these findings clearly demonstrate that the apoptosis-inducing activity of FCL in KKU-100 cells is mediated through the intrinsic mitochondrial pathway, involving coordinated changes in the expression and activity of key apoptotic regulators. In addition to the mitochondria-mediated pathway, an earlier study found that this compound could also promote apoptosis in colorectal cancer cells through activation of the endoplasmic reticulum stress pathway, ¹⁹ suggesting that FCL may trigger multiple death signaling mechanisms depending on the cellular context.

As ROS act as essential apoptotic signals and their accumulation can stimulate the apoptotic pathway in cells, ⁹ the effect of FCL on ROS levels in KKU-100 cells was determined in the current study. The results showed that the amount of ROS in the cells exposed to FCL was significantly higher than in the control group. These data suggest that the activation of apoptosis in CCA cells induced by FCL may be, at least in part, due to its pro-oxidant properties. Consistently, it has been reported that the compound could promote oxidative stress-induced apoptosis in Jurkat T cells. ³⁶ Apart from inducing apoptosis, previous studies reported that FCL induced autophagy of colorectal ³⁷ and bladder cancer cells. ³⁸ Further investigation into the impact of this phytochemical on autophagy as well as other cell death pathways in CCA cells would be valuable.

In addition to the induction of ROS generation, which leads to apoptotic cell death, additional mechanisms that contribute to the inhibitory effect of FCL on CCA cell survival were investigated in the present study. Given that cellular kinases mainly contribute to the regulation of cell signaling cascades, analyzing the phosphorylation patterns of their molecular targets is essential for understanding how cells recognize and respond to environmental changes. In the present study, we compared the phosphorylation patterns of various cellular targets associated with cell survival in FCL-treated and control CCA cells using the Proteome Profiler^TM Human Phospho-Kinase Array Kit. An obvious alteration in the phosphorylation of Yes, c-Jun, ERK1/2, and HSP27 and STAT5 was observed following exposure to FCL. Emerging evidence underscores the role of these proteins in cell fate determination depending on the cell type and stimulus. Yes kinase (Yes1) generally acts as an oncogenic protein promoting cancer cell survival and development. ³⁹ Previous study reported the activation of this protein in response to heat stress. ⁴⁰ It is therefore conceivable that FCL-stimulated ROS production might, in part, account for the activation of Yes1 in CCA cells after exposure to the compound as early as 12 hours of treatment. However, at 24 hours, control cells also showed increased Yes1 phosphorylation, which may reflect the response of cells under starved conditions. c-Jun is a transcription factor involved in the regulation of a wide range of biological processes. ⁴¹ Phosphorylation of Ser-63 in its transactivation domain is required for its pro-apoptotic activity. ⁴² Although our findings suggest a possible association between FCL treatment and c-Jun activation, this remains speculative as no functional validation was performed. ERK, a protein in the mitogen-activated protein kinase family, plays a critical role in almost all functions of cells. ⁴³ Many studies have reported the oncogenic role of ERK in inhibiting apoptosis.^44,45 Paradoxically, a number of studies have reported that under certain conditions, the activation of ERK can stimulate apoptosis.^46,47 In this study, ERK phosphorylation increased after 12 hours of FCL treatment, possibly reflecting a pro-apoptotic response. However, similar to Yes1, ERK phosphorylation also increased in the control group at 24 hours, likely as a result of nutrient deprivation. FCL also increased HSP27 phosphorylation, a stress-responsive marker, which may reflect the oxidative cellular environment. Conversely, phosphorylated STAT5 was decreased following FCL treatment. Since STAT5 is associated with cancer cell survival, ⁴⁸ its reduced activation may contribute to the observed effects. Collectively, these findings suggest that FCL alters the phosphorylation of key signaling proteins involved in cell survival and stress responses. However, the functional relevance of these phosphorylation changes, particularly with respect to apoptosis via c-Jun and ERK activation, remains to be experimentally validated. This represents a limitation of the present study and should be addressed in future investigations to clarify the precise molecular mechanisms underlying FCL-induced cell death.

Network pharmacology is a powerful tool for identifying molecular targets and key signaling pathways. In this study, the network pharmacology analysis was conducted to predict FCL targets by integrating in vitro phospho-kinase array data, focusing on survival-related proteins altered by FCL, to enhance biological relevance beyond database-based predictions. The analysis revealed a single dominant protein cluster, with Src, an intracellular tyrosine kinase, as the central hub. Enrichment analysis identified major FCL-influenced pathways, including focal adhesion, proteoglycans in cancer, Rap1, Ras, and MAPK signaling, which regulate cell survival and apoptosis. Src acts as a critical link among these networks, reinforcing its role as a key regulatory node. Aberrant activation of Src governs cancer progression function such as cell proliferation, apoptosis and metastasis. ⁴⁹ Previous studies showed Src/FAK activation confers anti-apoptotic properties to cancer cells. ⁵⁰ Additionally, Src interaction with tyrosine kinase receptors enhances Ras/MAPK activity and cancer growth. ⁴⁹ Although the phospho-antibody array did not show a significant change in phospho-Src levels, this may be due to its low basal signal in this platform, potentially limiting the sensitivity to detect changes. Importantly, network pharmacology analysis identified Src as a key hub protein with the highest degree of connectivity among the signaling proteins affected by FCL, for example, Yes, c-Jun, ERK1/2, STAT5a/b. Given its central role in multiple signaling pathways, we prioritized Src for validation by western blotting despite the lack of change observed in the array. The results of western blot analysis confirmed a significant decrease in Src phosphorylation in KKU-100 cells following FCL treatment, while total Src protein expression remained unchanged, indicating that FCL specifically inhibits Src activity via post-translational modification. In contrast, phospho-Src expression in KKU-213A is much lower than in KKU-100, ²⁷ suggesting that Src is unlikely to be the primary target of FCL in this cell line and highlighting potential cell line-specific effects.

Molecular docking analysis was further performed to verify the molecular interplay between FCL and Src. Phosphorylation of Tyr416, a residue located in the activation loop near the active site in the catalytic domain, is required for the full kinase activity of Src. ⁴⁹ Our analysis revealed that FCL could bind within this pocket, leading to diminished phosphorylation and catalytic function, which is consistent with the results observed in western blot analysis. However, FCL primarily engages in weak hydrophobic interactions, resulting in a binding energy substantially lower than that of the potent Src inhibitor SM1-71. Moreover, FCL is mainly associated with the exterior of the catalytic site, in contrast to the interior binding of SM1-71. ⁵¹ This binding pattern is likely influenced by its bulky structure composed of 2 isoquinoline rings. Despite its comparatively modest binding interactions, FCL was able to occupy the ATP-binding pocket, indicating a viable mechanism for competitively inhibiting ATP binding. Although it may not exhibit the highest potency on its own, FCL holds promise as an ATP-competitive inhibitor of Src and merits further investigation. Additional biochemical assays, including crystallographic binding studies, are necessary to elucidate these interactions and to determine whether this binding profile aligns with our experimental observations.

In conclusion, the anticancer potential of FCL against CCA was demonstrated. It induces intrinsic apoptotic cell death, partially mediated by ROS generation. Suppression of Src activation also contributed to the inhibitory effect of FCL on CCA cell survival. These findings highlight the potential of FCL as a therapeutic agent for CCA. Although detailed mechanistic analyses were conducted using only the KKU-100 cell line, it originates from poorly differentiated tubular adenocarcinoma, the most common CCA subtype in Thailand,^31,32 and represents a clinically relevant model for CCA in regions with a high disease incidence such as Southeast Asia.^33,34 We acknowledge the potential cell-type specificity of FCL’s effects, which warrants further investigation in additional CCA models, and additional pharmacological studies are needed to confirm its in vivo efficacy. Nonetheless, our findings provide insights that may reflect the biology of the majority of CCA patients.

Supplemental Material

sj-pdf-1-ict-10.1177_15347354251396513 – Supplemental material for Unveiling the Anticancer Potential and Molecular Mechanisms of Fangchinoline Against Cholangiocarcinoma Using FTIR Microspectroscopy, In Vitro and In Silico Approaches

Supplemental material, sj-pdf-1-ict-10.1177_15347354251396513 for Unveiling the Anticancer Potential and Molecular Mechanisms of Fangchinoline Against Cholangiocarcinoma Using FTIR Microspectroscopy, In Vitro and In Silico Approaches by Piman Pocasap, Karnchanok Kaimuangpak, Krittaya Phukmee, Auemduan Prawan, Sarinya Kongpetch and Laddawan Senggunprai in Integrative Cancer Therapies