Higher expression of high-mobility group box 1 in cholangiocarcinoma and association with cell growth,in vitro migration and invasion,and chemo-drug sensitivity

Abstract

Objective

Cholangiocarcinoma is a lethal malignancy with an increasing incidence and mortality worldwide. Numerous studies have indicated that high-mobility group box 1 is associated with cancer progression. The modulation of subcellular high-mobility group box 1 expression is a major cause of chemotherapeutic drug resistance. This study aimed to demonstrate the role of high-mobility group box 1 in cholangiocarcinoma, including proving the concept of high-mobility group box 1 expression in patients with cholangiocarcinoma, cancer cell growth, migration, invasion, and the modulation of chemotherapeutic drug sensitivity in cholangiocarcinoma cells.

Methods

Twenty paraffin-embedded tissue samples were analyzed for high-mobility group box 1 expression using immunohistochemistry. High-mobility group box 1 expression was silenced in KKU-213AL5 cells through siRNA transfection, followed by in vitro assays to assess cell proliferation, migration, invasion, and drug sensitivity. Furthermore, the role of high-mobility group box 1 in regulating growth and metastasis-related signaling pathways was investigated using immunoblotting and protein–protein interaction analysis.

Results

The results showed that high-mobility group box 1 was highly expressed in cholangiocarcinoma tissues compared to adjacent tissues (p-value < 0.001), as well as the HMGB1 expression analysis from the TCGA database. Silencing high-mobility group box 1 by siRNA transfection resulted in the reduction of cholangiocarcinoma cell growth, migration, and invasion. Interestingly, high-mobility group box 1 silencing enhanced sensitivity to Gemcitabine and Cisplatin by increasing cell cytotoxicity compared to transfection control (p-value = 0.0002 and 0.0258, respectively). We further demonstrated that reduction of high-mobility group box 1 expression attenuates the essential signaling proteins, including Akt, Erk, and cyclin D1, which are crucial in cancer cell growth and metastasis signaling pathways.

Conclusions

This study demonstrates that high-mobility group box 1 plays a critical role in cholangiocarcinoma proliferation, migration and invasion, and may serve as a diagnostic biomarker. Targeting high-mobility group box 1 could enhance therapeutic outcomes, particularly in overcoming drug resistance.

Keywords

HMGB1 cholangiocarcinoma antimetastatic gemcitabine cisplatin oxaliplatin

Introduction

Cholangiocarcinoma (CCA) is a fatal malignancy of the biliary duct. Although consumption of undercooked and raw foods has been discouraged to prevent liver fluke infection-induced CCA, the worldwide incidence and mortality of CCA have increased over the decades, with primary sclerosing cholangitis being the most common risk factor in the West.^1,2 Liver resection is a favorable treatment option for CCA. Conversely, transarterial chemotherapy (TACE) and systemic chemotherapy remain adjuvant treatments in cases of unresectability and inoperability.³ Gemcitabine, cisplatin, and oxaliplatin are the first- and second-line drugs used in standard systemic chemotherapy. However, poor outcomes in terms of progression-free and overall survival have been reported in patients with advanced biliary tract cancer.⁴ These poor progression-free survival and overall survival rates are suggested to be a consequence of drug resistance in CCA.⁵ Therefore, investigation of the cellular mechanisms resulting in poor prognosis and modulation of drug sensitivity in CCA is important.

High-mobility group box 1 (HMGB1) is a nonhistone protein that plays a role in DNA architecture and gene regulation. Modulation of HMGB1 has been suggested to cause cellular alterations in various cell types. In CCA, overexpression of subcellular HMGB1 correlated with the metastatic status of a Clonorchis sinensis CCA patient.⁶ HMGB1 has also been indicated as a mediator of apoptosis, autophagy, and proliferation in the bile duct carcinoma cell line.⁷ Furthermore, secretion of HMGB1 from perihilar CCA cell lines promotes angiogenesis, resulting in cancer progression.⁸ HMGB1 has been reported as a potential target for multiple myeloma treatment because its overexpression attenuated chemotherapeutic drug sensitivity.⁹ Modulation of HMGB1 has been proposed as a cause of CCA progression, which also provides evidence of drug sensitivity regulation in numerous cancer types.^10–12 However, there is little evidence of HMGB1-induced chemotherapeutic drug resistance in CCA,¹³ which may promote a novel treatment paradigm for patients with CCA.

This study aimed to study the expression of HMGB1 in CCA tissues and its function in metastatic activity and chemotherapeutic drug sensitivity in CCA cells. The results indicated that HMGB1 expression was associated with the tumorigenic status of the tumor. Silencing of HMGB inhibited in vitro migration and invasion through the ERK/Akt pathway. Notably, HMGB1 suppression also resulted in increased sensitivity to gemcitabine and cisplatin. Thus, HMGB1 is a promising target for the treatment and diagnosis of CCA.

Material and methods

Cell culture

The MMNK1 cholangiocyte cell line was obtained from Professor Naoya Kobayashi. KKU-100 and KKU-213AL5 cell lines, the CCA, were kindly gifted by Professor Dr Sopit Wongkham, Department of Biochemistry, Faculty of Medicine, Khon Kaen University (Khon Kaen, Thailand).^14,15 Cells were maintained in Dulbecco's Modified Eagle Medium (Gibco, Thermo Fisher Scientific, NY, USA) containing 10% fetal bovine serum (FBS), 1% of antibiotic-antimycotic (10,000 units/mL of penicillin, 10,000 µg/mL of streptomycin, and 25 µg/mL of Gibco Amphotericin B) (Gibco, Thermo Fisher Scientific, NY, USA). Cells were incubated at 37 °C with 5% CO₂.

Tumor tissues of CCA patients

Tissue samples were obtained from 20 patients diagnosed with CCA. These patients underwent initial surgical procedures at the Affiliated Naresuan University Hospital in Thapho, Phitsanulok, Thailand, between 2017 and 2023. None of the patients had undergone radiotherapy, chemotherapy, or any other neoadjuvant therapy prior to surgical intervention. Cases with incomplete identification or unclear classification of either tumor or normal tissues were excluded from this study. This study was approved for exemption by the Human Research Ethics Committee of Naresuan University, located in Phitsanulok, Thailand, under approval number IRB No. P1-0006/2567, dated 16 January 2024, and conducted in accordance with the Helsinki Declaration of 1975 as revised in 2024, CIOMS guidelines, and ICH-GCP. The requirement for written informed consent was waived by the Ethics Committee. The clinicopathological data of all patients were reviewed based on their medical records, including T-N-M staging. The characteristics and distribution of the CCA cohorts are shown in Table 1. This study adhered to the relevant guidelines and regulations established by the affiliated Naresuan University Hospital.

Table 1.

Association of HMGB1 expression with clinical pathologic parameters of patients with cholangiocarcinoma.

Variables	n (%)	Mean IRS	p values
Gender
Male	14 (70)	6.357	0.9164
Female	6 (30)	6.17
Age
<60	3 (15)	5.667	0.7487
>=60	17 (85)	6.412
T staging
T1	7 (35)	5.286	0.2019
T2	7 (35)	5.429
T3	0	0
T4	6 (30)	8.5
N staging
N0	15 (75)	5.533	0.0969
N1	5 (25)	8.6
M staging
M0	15 (75)	5.6	0.1322
M1	5 (25)	8.4

HMGB1: high-mobility group box 1; IRS: immuno-reactive score.

Immunohistochemistry staining

Paraffin-embedded tissue sections were stained using a two-step plus poly-HRP Anti-Rabbit IgG Detection System (ElabScience^®; TX, USA). Initially, paraffin sections were dewaxed and rehydrated. They were then incubated with 3% hydrogen peroxide (H₂O₂) for 10 minutes, followed by three washes with phosphate-buffered saline (PBS) for 2 minutes each. Normal goat blocking buffer was added, and the cells were incubated at 37 °C for 30 minutes. Next, rabbit anti-HMGB1; E-AB-70044 (ElabScience^®; TX, USA) was added and incubated for 2 hours, followed by three washes with PBS for 2 minutes each. Polyperoxidase-anti-rabbit IgG was applied and incubated at room temperature or 37 °C for 20 minutes, followed by washing with PBS. DAB Working Solution was added to the sections, which were then washed with distilled water to stop the chromogenic reaction and subsequently counterstained with hematoxylin.

Stained tissue sections were independently evaluated and scored by two pathologists. Staining intensity was rated on a 4-point scale: 0 (negative), 1 (weak), 2 (medium), or 3 (strong). The percentage of positive cells was assessed using a 4-point scale: 0 (1%–25%), 1 (26%–50%), 2 (51%–75%), or 3 (76%–100%). The immunoreactive score (IRS) was calculated by multiplying the staining intensity score by the percentage of positive cells.

TCGA dataset analysis

The web-based analytical tool Gene Expression Profiling Interactive Analysis 2 (GEPIA2), which utilizes data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) datasets (http://gepia.cancer-pku.cn/), was employed to assess survival associated with HMGB1 expression in CCA. In GEPIA2, CCA is denoted as CHOL. The dataset comprises 36 tumor samples and nine normal tissue samples. Box plot analysis was performed to compare HMGB1 expression (measured in transcripts per million [TPM]) between tumor (T) and normal (N) tissues. Additionally, the relationship between HMGB1 expression and the overall survival of patients with CHOL was examined.

HMGB1 knockdown by siRNA transfection

Transfection was conducted to inhibit the expression of HMGB1 in KKU-213AL5 cells. The KKU213L5 cell line was seeded onto a cell culture plate and incubated overnight until the cells reached 70%–90%. HMGB1 knocked down was carried out using Silencer^® pre-designed siRNA (siHMGB1) (Thermo Fisher Scientific, Life Technologies Corporation, CA, USA) compared with Silencer Negative Control siRNA as the negative control (siControl) (Thermo Fisher Scientific, Life Technologies Corporation, CA, USA) using Lipofectamine™ 3000 Reagent (Invitrogen, Thermo Fisher Scientific, NY, USA) according to the manufacturer's instructions. The expression of HMGB1 at 24, 48, and 72 hours was determined by western blotting.

Immunoblotting analysis

Following treatment, cells were washed with ice-cold PBS and lysed using NP40 lysis buffer containing a protease inhibitor cocktail. The resulting protein lysates were collected by centrifugation, and their concentrations were measured using Pierce™ BCA Protein Assay Kits (Thermo Scientific, MA, USA). Immunoblotting was performed using a luminescence separation module (BioTechne). In brief, protein lysates were mixed with a master mix and denatured for 5 minutes at 95 °C. A biotinylated ladder marker, samples, antibody diluent, specific primary antibodies dilution for 1:300 (including anti-HMGB1; 6893, anti-pAkt; 4060, anti-Akt; 9272, anti-pERK; 4370, anti-ERK; 4695, anti-cyclin D1; 9278, and anti-β-actin; 4970, all from Cell Signaling, MA, USA), secondary antibody, and a luminol-peroxide mixture were loaded into the detection module and analyzed using the Jess Automated Western Blot System (Bio-Techne, MN, USA).

Scratch-wound cell migration assays

The cells were seeded into 6-well plates at a density of 4 × 10⁵ cells/well and incubated overnight at 37 °C with 5% CO₂ until they reached 90%–100%. Cells were transfected with siHMGM1 or siControl and incubated for 6 hours. After that, the cells were scratched at the middle of the well using a 200 μL-micropipette tip and were then incubated at 37 °C with 5% CO₂. The migration ability of cells was visualized and captured at 0, 6, 12, and 24 hours using Celloger^® Mini Plus.

Cell invasion assay

On the day prior experiment, the upper chamber of transwell inserts in a 24-well plate was coated with Corning^® Matrigel^® Basement Membrane Matrix (Corning, Life Sciences, MA, USA) and incubated overnight at 37 °C with 5% CO₂. The cells were then seeded into the upper chamber of transwell inserts at a density of 5 × 10⁴ cells/well, and 700 μL of completed DMEM medium was added into the lower chamber and incubated for a further 24 hours at 37 °C, 5% CO₂. The cell culture medium was then removed, and the upper chamber was gently cleaned using a cotton swab. The invading cells were fixed with 4% formaldehyde for 10 minutes and stained with 0.5% crystal violet in 20% methanol for 15 minutes. The remaining stain was removed using tap water, and the sample was allowed to dry. The invading cells were visualized and counted using an inverted microscope. The % invasion was calculated using the following formula:

% Cell invasion = (\frac{Number of cells in knockdown HMGB 1 group}{Number of cells in negative control group}) \times 100

Drug challenge assay

Gemcitabine, cisplatin, and oxaliplatin were used as the chemotherapeutic drugs in these experiments. MMNK1, KKU100, and KKU213L5 were seeded at a density of 5 × 10³ cells/well into a 96-well plate and then incubated overnight at 37 °C with 5% CO₂. The cells were treated with different concentrations of chemotherapeutic drugs and incubated for 24 and 48 hours. The IC50 values of gemcitabine, cisplatin, and oxaliplatin were determined using the MTT assay. The IC₅₀ values of chemotherapy drugs treated with KKU213L5 were used for further challenges in HMGB1 knockdown in KKU-213AL5 cells compared with the negative control group to determine the drug resistance of the cells.

Statistical analysis

Statistical analyses were performed using GraphPad Prism version 10, and the results are presented as mean ± standard deviation. Differences between the groups were assessed using unpaired t-tests and analysis of variance (ANOVA). Statistical significance was defined as a p-value of < 0.05.

Results

HMGB1 is predominated expressed in tumors and cancer cell lines

To investigate the importance of HMGB1 in CCA, we measured the expression of HMGB1 in parafilm-embedded tissues using immunohistochemistry. Twenty tumor tissues were stained with anti-HMGB1 antibody and counterstained with hematoxylin to highlight the nuclei. The tumor and surrounding normal tissues were independently examined by two broadly certified pathologists independently and evaluated for expression of HMGB1 as an immuno-reactive score (IRS). The results showed that HMGB1 was located within cholangiocytes lining the cells of the bile duct. Interestingly, HMGB1 was significantly overexpressed in tumor tissues compared to adjacent normal tissues, either individually or overall (Figure 1(A) and (B)). Although the expression of HMGB1 in tumor tissues was not significantly associated with clinicopathological parameters, an increased immunoreactive score was observed in some patients with larger tumor size, lymph node involvement, and distant metastasis (Table 1). Kaplan–Meier survival analysis indicated that high HMGB1 expression was not significantly associated with decreased overall survival (p = 0.35; Figure 1(C)). To confirm the previous experiment, the gene expression of HMGB1 in tumor (T) and normal adjacent (N) tissues was obtained from the TCGA database and analyzed using the GEPIA online tool. Similar to protein expression, HMGB1 expression was significantly increased in CCA tissues compared to its normal counterpart. However, high expression of HMGB1 was not correlated with reduced overall survival in CCA patients (p-value = 0.76) (Figure 2(A) and (B)).

Figure 1.

HMGB1 is overexpressed in CCA tissues. (A) The expression of HMGB1 was investigated in parafilm-embedded 20 CCA tissues. (B) The immunoreactive score from the IHC method was analyzed either overall or individually in 20 patients with CCA. (C) The Kaplan-Meier overall survival was analyzed for high and low expressions of HMGB1.

Figure 2.

High expression of HMGB1 at the gene level and relevant to in vitro metastasis of cancer cell lines. (A) The expression of HMGB1 and (B) overall analysis were analyzed from web-based analytic tool Gene Expression Profiling Interactive Analysis (GEPIA), (C) the differential expression of HMGB1 was also demonstrated in cell lines, including cholangiocyte; MMNK1, low metastasis CCA cell; KKU100, and highly metastasis CCA cell; KKU213L5 by immunoblotting. Band intensity was quantified by ratio with beta-actin (n = 1). Western blots were generated using the Jess Automated Western Blot System. (D) The growth curve analysis of cell lines and doubling time that are related to the expression level of HMGB1, quantitative data are shown as the mean ± SD (n = 6). Significant differences are indicated by * p < 0.05 compared to the control group. HMGB1: high-mobility group box 1; CCA: cholangiocarcinoma; CHOL: cholangiocarcinoma; num: number; TPM: transcripts per million; HR: hazard ratio.

In agreement with the CCA cell line study, HMGB1 expression was markedly increased in KKU-100 cells and was highest in KKU-213AL5 cells, which are low-and highly metastatic CCA cell lines, respectively, compared to the normal cholangiocyte cell line MMNK1 (Figure 2(C)). Moreover, growth curve analysis of these cells showed that KKU-213AL5 exhibited the shortest doubling time (71.31 hours), followed by KKU-100 (112.5 hours) and MMNK1 (159.7 hours) (Figure 2(D)). HMGB1 expression levels were linked to differences in the growth rates of CCA cell lines, with elevated HMGB1 corresponding to faster cell growth. Taken together, these results suggested that HMGB1 is an important protein that contributes to CCA tumorigenesis and growth.

HMGB1 is involved in CCA cell growth, migration, and invasion

To strengthen the role of HMGB1 in cancer biology, the CCA cell line, KKU-213AL5, which has the highest expression of HMGB1, was suppressed by siRNA transfection. The inhibitory effect was confirmed by immunoblotting, which showed that the knockdown group had downregulated HMGB1 protein expression compared to either the parental cell or transfection control (Figure 3(A)). KKU-213AL5 cells were used to determine cell growth, in vitro migration, and invasion. Firstly, knockdown HMGB1 significantly suppressed CCA cell growth in the clonogenic assay compared to that in the transfection control (p-value <0.0001) (Figure 3(B)). Moreover, HMGB1 inhibition significantly reduced cancer cell migration in a time-dependent manner (p-value = 0.0004) and cancer cell invasion (p-value <0.0001) compared to the transfection control (Figure 3(C) and (D). Therefore, HMGB1 might contribute to CCA cell growth and metastasis.

Figure 3.

Inhibition of HMGB1 suppressed CCA cell growth, migration, and invasion. HMGB1 of KKU-213AL5 cells was suppressed using siRNA-transfection, and the efficacy was shown with immunoblotting (A). Control transfection cells and siHMGB1-treated cells were evaluated for their proliferation (B) and metastasis ability, including migration by cell scratch assay (C) and cell invasion by trans-well assay (D). All quantitative data are shown as the mean ± SD. Significant differences are indicated by * p < 0.05, *** p < 0.001, and **** p < 0.0001 compared with the control group. siControl, silencer negative control siRNA; siHMGB1’ silencer^® pre-designed siRNA to HMGB1.

Inhibition of HMGB1 increased sensitization to chemotherapeutic drugs

Previous reports have indicated that HMGB1 influences drug resistance and may lead to poor results in cancer treatment. To prove this, KKU-213AL5 transfection control and cells with HMGB1 knockdown were treated with gemcitabine, cisplatin, and oxaliplatin to assess their chemotherapeutic response. A diagram of the treatment is shown in Figure 4(A). The results revealed that siHMGB1 significantly increased the cytotoxicity of KKU-213AL5 cells compared to that of the transfection control when treated with gemcitabine and cisplatin, by reduction of IC₅₀ from uncalculated to 1289 µM and 38.23 to 24.02 µM, respectively (Figure 4(B)). However, HMGB1 silencing did not enhance oxaliplatin cytotoxicity in CCA cells. Suggesting that inhibition of HMGB1 increased the sensitivity of CCA cells to gemcitabine and cisplatin.

Figure 4.

Down-regulation of HMGB1 enhanced the sensitivity of chemotherapeutic drugs. Control transfection and HMGB1 silencing KKU-213AL5 were treated with different chemo-drugs with titrated concentration for 48 hours, and measured cell viability using MTT assay (A). The cell viability is shown as relative to the transfection control group (B). All quantitative data are shown as the mean ± SD (n = 6).

HMGB1 is associated with activation of essential growth signaling pathways

To investigate the pathways involved in HMGB1, both KKU-213AL5 and knockdown cells were cultured in a serum-free cell culture medium for 24 hours, which was then replaced with 10% FBS in the cell culture medium for 10 minutes to activate growth signals. Intracellular proteins were collected before or after stimulation, and the regulation of essential signaling proteins was investigated. The results showed that stimulation of growth signals with FBS significantly increased the phosphorylation of Akt, Erk, and the cell cycle-regulating protein cyclin D1. In contrast, HMGB1 silencing attenuated the activation of these proteins (Figure 5(A) and (B)). To provide additional explanation, HMGB1 interaction with signaling proteins involved in CCA growth and metastasis was analyzed by STRING 12.0. HMGB1 directly interacts with NF-κβ, MAPK, mTOR, EGFR, Akt, STAT3, and AGER, and is eventually involved with the transcription factor of NOTCH proteins MAML1 (Mastermind Like 1), MET, YAP1, cyclin D1, and PI3 K (Figure 5(C)). The analysis of disease—gene association revealed that these interactions are strongly associated in hepatobiliary tumors and hepatobiliary disease (Figure 5(D)). Taken together, HMGB1 plays a crucial role in signaling pathways that may contribute to CCA growth and metastasis.

Figure 5.

Inhibition of HMGB1 downregulated the growth-related signaling protein. Control transfection and HMGB1 silencing KKU-213AL5 were starved with serum-free medium and subsequently treated with 10% fetal bovine serum in cell culture medium for 10 minutes. The cellular protein was harvested, and was measured the expression of signaling protein by immunoblotting (A). Western blots were generated using the Jess Automated Western Blot System. The relative expressions of the protein of interest were quantified with actin/total form (B). All quantitative data are shown as the mean ± SD (n = 3). Significant differences are indicated by the p-value compared with unstimulated cells. Protein-protein interaction was analyzed by STRING version 12.0 (C). In addition, the disease—gene association was postulated to confirm the relation of HMGB1 and CCA growth and metastasis. ERK: extracellular signal-regulated kinase; NFKB1: nuclear factor kappa B subunit 1; MAPK1: mitogen-activated protein kinase 1; mTOR: mammalian target of rapamycin; EGFR: epidermal growth factor receptor; STAT3: signal transducer and activator of transcription 3; AGER: advanced glycation end-product specific receptor; MAML1: mastermind like transcriptional coactivator 1; MET: mesenchymal-epithelial transition; YAP1: yes-associated protein 1; CCND1: cyclin D1; PIK3CA: phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha.

Discussion

HMGB1 is a nuclear protein that plays a critical role in DNA architecture and gene regulation. Recent studies have highlighted its significant involvement in various aspects of cancer biology, including tumor development, progression, and metastasis.¹⁶ In CCA, a malignant tumor arising from the bile duct epithelium, HMGB1 has emerged as a crucial factor in tumor progression.⁸ In this study, we highlighted the significant overexpression of HMGB1 either on protein or gene expression in tumor tissues compared to adjacent tissues. Along with the expression level within cell lines, HMGB1 is predominantly expressed in cancer cells compared to normal cholangiocytes and is highly expressed in highly metastatic cells. However, survival analysis showed no statistically significant association with a poor prognosis in patients with CCA. This finding is inconsistent with previous reports showing that high levels of HMGB1 correlate with angiogenesis and poor prognosis of perihilar CCA by elevating VEGFR2 in the vessel endothelium.⁸ In addition, HMGB1 overexpression was observed in some patients with increased tumor size, lymphatic invasion, and distant metastasis (Table 1). This is consistent with a previous report that high HMGB1 expression is associated with lymph node metastasis and the metastatic status of patients with CCA.^6,17 Hence, our demonstration emphasized and proved that HMGB1 is overexpressed in patients with CCA. This expression can be used for cancer diagnosis at the gene or protein levels.

The mechanism of action of HMGB1 in CCA progression was elucidated by targeted silencing using siRNA transfection. Inhibition of HMGB1 suppressed in vitro tumor growth, migration, and invasion of KKU-213AL5. In agreement with our previous report, co-culture with cell culture medium containing HMGB1 promoted CCA cell pro-tumor activities.¹⁸ Although studies on the underlying mechanism of HMGB1 in CCA are limited, several studies on other tumors have shown that HMGB1 promotes cancer growth by interacting with various signaling pathways. For instance, HMGB1 activates the nuclear factor-kappa B pathway, which is crucial for cancer cell growth, invasion, and metastasis in hepatocellular carcinoma.¹⁹ HMGB1 promoted angiogenesis and enhanced cancer cell invasion by regulating HIF1A through the phosphatidylinositol 3 kinase/protein kinase B (PI3 K/AKT) signaling pathway.²⁰ Moreover, HMGB1 facilitates the self-renewal of cancer stem cells (CSCs) by interacting with specific receptors and signaling pathways such as the LC3II/TLR4-associated transcriptional regulator YAP1.^21,22 Hence, the in vitro modulation of HMGB1 emphasizes that HMGB1 contributes to cancer progression in CCA.

CCA is a challenging malignancy characterized by a poor prognosis and limited therapeutic options. The first-line drug for CCA treatment is gemcitabine, which is commonly combined with cisplatin, 5-fluorouracil, or oxaliplatin.²³ Several molecular mechanisms contribute to chemotherapy resistance in CCA, the most prominent of which is the overexpression of P-glycoprotein (P-gp), which has been linked to gemcitabine.^24,25 Gemcitabine is a deoxycytidine analog that incorporates into DNA, halting DNA replication and leading to apoptosis. However, overexpression of HMGB1 has been shown to activate autophagy and inhibit apoptosis, potentially counteracting gemcitabine-induced cytotoxicity.^26,27 Cisplatin and oxaliplatin are platinum-based chemotherapeutic agents that cause DNA crosslinking, which activates apoptosis via p53 and other DNA damage response pathways. HMGB1 is known to bind DNA lesions and can modulate DNA repair processes, reduce apoptotic responses, and stimulate pro-survival signaling, thereby contributing to resistance to DNA-damaging agents.^28,29 In this study, we showed that modulation of HMGB1 significantly increased the sensitivity of CCA cell lines to gemcitabine and cisplatin. HMGB1 plays an important role in chemoresistance development. HMGB1 enhances autophagy by activating RAGE/ERK signaling, which prevents cancer cell death from drug treatment.³⁰ HMGB1 activates the NF-κB signaling pathway, which leads to the upregulation of P-gp and multidrug resistance-associated protein (MRP), thereby accelerating the excretion of drugs.³¹ Moreover, elevated levels of HMGB1 have been observed in cisplatin-resistant A549/DDP cells, which are associated with the induction of IL-6 and the activation of the Janus kinase-STAT3 (JAK-STAT3) pathway.^16,32 However, research on the role of HMGB1 in chemotherapy resistance is limited owing to a lack of evidence, and its involvement in CCA has not been reported. To elucidate HMGB1 signaling in CCA cells, we investigated the induction of growth signaling proteins and HMGB1 silencing. These results indicated that the inhibition of HMGB1 attenuated the phosphorylation of Akt and ERK signaling proteins and involved decreasing cyclinD1 stimulation. This finding confirmed the relationship between HMGB1, in vitro cancer growth, invasion, and chemotherapy drug resistance. In particular, the Akt and Erk signaling pathways are involved in cancer invasion and the induction of chemoresistance.^20,30 These results indicate that the modulation of HMGB1 suppresses pro-tumor activity by restraining growth signaling. In this study, we used siRNA-mediated precise inhibition of HMGB1 by siRNA transfection to elucidate the role of HMGB1 in CCA. However, in clinical settings, using HMGB1 inhibitors may be more feasible and cost-effective than gene silencing treatment. Glycyrrhizic acid (GL), a primary active component of licorice, exhibits significant antitumor properties by directly binding to HMG boxes A and B.³³ Moreover, ethyl pyruvate (EP), the ethyl ester of pyruvic acid, has been reported to block the release of HMGB1 and attenuate inflammation by modulating the HMGB1/RAGE signaling pathway.^34,35 Preclinical studies have demonstrated that both glycyrrhizin and ethyl pyruvate exhibit potential to enhance the efficacy of chemotherapy, indicating their promise in the development of combination therapy strategies.³⁶

In this study, we focused on the concept confirmation and described the role of HMGB1 in vitro cancer growth, invasion, and chemoresistance. Although the clinical data from IHC could demonstrate essential data, a larger number of tissue samples should be included to provide more robust evidence and convincing statistical significance. Extended observational periods are necessary to assess the survival outcomes associated with low and high HMGB1 expression, which would more accurately define HMGB1's role as a potential prognostic biomarker. Moreover, intensive studies on the molecular signaling of HMGB1 in drug resistance, in vivo studies, and test verification in other cell lines should be conducted to expand this hypothesis. Lastly, for further reliability of the study, cell lines should be verified by using short tandem repeat profiling.

Conclusions

This study validated that HMGB1 is a key factor in the tumor biology of CCA, applicable both in vitro and in clinical settings. This highlights the potential of HMGB1 as a diagnostic biomarker of CCA. Given its significant roles in cancer growth, metastasis, and drug resistance. Targeting HMGB1 may improve therapeutic outcomes, especially for combination therapy development. Additionally, monitoring HMGB1 expression may help in predicting chemoresistance and personalizing treatment strategies for CCA patients.

Footnotes

Acknowledgements

We would like to express our gratitude to the patients who participated in this study. Naresuan University Hospital for access to clinical information and tumor tissue samples. We thank Associate Professor Dr Worasak Kaewkong for suggestions on the online database analysis.

ORCID iD

Worawat Songjang

Ethical considerations

The study was approved for exemption by the Human Research Ethics Committee of Naresuan University, according to the Declaration of Helsinki, Belmont report, CIOMS guidelines, and ICH-GCP (IRB No.P1-0006/2567).

Authors’ contributions

Songjang W, Jiraviriyakul A: conceived and designed the experiments, contributed reagents, and materials; all authors performed the experiments and analyzed and interpreted the data; Songjang W, Jiraviriyakul A, Promchai S, and Nensat C: wrote the paper. All authors contributed to manuscript editing and approval.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by Naresuan University (NU) and the National Science, Research, and Innovation Fund (NSRF) (grant numbers R2566B083 and R2568B087) to Songjang W, and grant number R2568B083 to Jiraviriyakul A.

Declaration of conflicting interest

The author(s) declare no competing financial interests or personal relationships that may have influenced the work reported in this study.

Data availability statement

Data will be made available on request.

References

Baria

De Toni

, et al. Worldwide incidence and mortality of biliary tract cancer. Gastro Hep Advances 2022; 1: 618–626.

Federica

Paola

Eva

, et al. Epidemiology of cholangiocarcinoma. Hepatoma Research 2022; 8: 19.

Elvevi

Laffusa

Scaravaglio

, et al. Clinical treatment of cholangiocarcinoma: an updated comprehensive review. Ann Hepatol 2022; 27: 100737.

Jung

Park

Jung

, et al. Real-world outcomes of gemcitabine, cisplatin, and nab-paclitaxel chemotherapy regimen for advanced biliary tract cancer: a propensity score-matched analysis. Gut Liver 2022; 16: 798–805.

Zheng

Zhang

, et al. Overcome drug resistance in cholangiocarcinoma: new insight into mechanisms and refining the preclinical experiment models. Front Oncol 2022; 12: 850732.

Suwannakul

Midorikawa

, et al. Subcellular localization of HMGB1 in human cholangiocarcinoma: correlation with tumor stage. Discov Oncol 2021; 12: 49.

Qin

Zhou

, et al. LncRNA HOTAIR suppresses cell apoptosis, autophagy and induces cell proliferation in cholangiocarcinoma by modulating the miR-204-5p/HMGB1 axis. Biomed Pharmacother 2020; 130: 110566.

Y-F

Liu

Z-L

Pan

, et al. HMGB1 correlates with angiogenesis and poor prognosis of perihilar cholangiocarcinoma via elevating VEGFR2 of vessel endothelium. Oncogene 2019; 38: 868–880.

Ning

Yang

Wang

, et al. HMGB1 Enhances chemotherapy resistance in multiple myeloma cells by activating the nuclear factor-κB pathway. Exp Ther Med 2021; 22: 705.

10.

Shao

L-H

Zhu

Wang

, et al. Mechanisms involved in the HMGB1 modulation of tumor multidrug resistance (review). Int J Mol Med 2023; 52: 69.

11.

Zhou

Mao

, et al. HMGB1 promotes resistance to doxorubicin in human hepatocellular carcinoma cells by inducing autophagy via the AMPK/mTOR signaling pathway. Front Oncol 2021; 11: 739145.

12.

Lei

Huang

Xie

, et al. HMGB1-mediated autophagy promotes gefitinib resistance in human non-small cell lung cancer. Acta Biochim Biophys Sin (Shanghai) 2022;54: 514.

13.

Yin

Lin

Zeng

, et al. EZH2-mediated PHF10 suppression amplifies HMGB1/NF-κB axis that confers chemotherapy resistance in cholangiocarcinoma. J Cell Mol Med 2025; 29: e70363.

14.

Sripa

Leungwattanawanit

Nitta

, et al. Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100). World J Gastroenterol 2005; 11: 3392–3397.

15.

Saentaweesuk

Araki

Vaeteewoottacharn

, et al. Activation of vimentin is critical to promote a metastatic potential of cholangiocarcinoma cells. Oncol Res 2018; 26: 605–616.

16.

Yang

Gai

, et al. Multiple functions of HMGB1 in cancer. Front Oncol 2024; 14: 3389.

17.

Han

, et al. High-mobility group box 1 expression and lymph node metastasis in intrahepatic cholangiocarcinoma. World J Gastroenterol 2015; 21: 3256–3265.

18.

Songjang

Nensat

Nernpermpisooth

, et al. Tumor-promoting activity and proteomic profiling of cisplatin/oxaliplatin-derived DAMPs in cholangiocarcinoma cells. Int J Mol Sci 2022; 23: 10540.

19.

Tang

Pang

, et al. Ozonated water inhibits hepatocellular carcinoma invasion and metastasis by regulating the HMGB1/NF-κB/STAT3 signaling pathway. J Hepatocell Carcinoma 2023; 10: 203–215.

20.

Jiang

Yang

, et al. Functional role of SIRT1-induced HMGB1 expression and acetylation in migration, invasion and angiogenesis of ovarian cancer. Eur Rev Med Pharmacol Sci 2018; 22: 4431–4439.

21.

, et al. Hypoxia-induced HMGB1 promotes glioma stem cells self-renewal and tumorigenicity via RAGE. iScience 2022; 25: 104872.

22.

Zhao

Lin

Jiang

, et al. High-mobility group box 1 released by autophagic cancer-associated fibroblasts maintains the stemness of luminal breast cancer cells. J Pathol 2017; 243: 376–389.

23.

Sasaki

Takeda

Okamoto

, et al. Chemotherapy for biliary tract cancer in 2021. J Clin Med 2021; 10: 3108.

24.

Shen

Zhang

Zeng

, et al. Inhibition of Wnt/β-catenin signaling downregulates P-glycoprotein and reverses multi-drug resistance of cholangiocarcinoma. Cancer Sci 2013; 104: 1303–1308.

25.

Wang

Zhang

, et al. Enhanced expression of ten-eleven translocation 1 reverses gemcitabine resistance in cholangiocarcinoma accompanied by a reduction in P-glycoprotein expression. Cancer Med 2019; 8: 990–1003.

26.

Tang

Kang

Zeh

, et al. High-mobility group box 1 and cancer. Biochim Biophys Acta 2010; 1799: 131–140.

27.

Liu

Yang

Kang

, et al. HMGB1-induced autophagy promotes chemotherapy resistance in leukemia cells. Leukemia 2011; 25: 23–31.

28.

Yang

Pellegrini

Napolitano

, et al. Aspirin delays mesothelioma growth by inhibiting HMGB1-mediated tumor progression. Cell Death Dis 2015; 6: e1786.

29.

Kang

Zhang

Zeh

, et al.

HMGB1 in cancer: good, bad, or both?

Clin Cancer Res 2013; 19: 4046–4057.

30.

Livesey

Kang

Vernon

, et al. P53/HMGB1 complexes regulate autophagy and apoptosis. Cancer Res 2012; 72: 1996–2005.

31.

Huang

Liu

Song

, et al. Krüppel-like factor 4 promotes high-mobility group box 1-induced chemotherapy resistance in osteosarcoma cells. Cancer Sci 2016; 107: 242–249.

32.

Zhan

, et al. Autophagy-mediated HMGB1 release antagonizes apoptosis of gastric cancer cells induced by vincristine via transcriptional regulation of Mcl-1. Autophagy 2012; 8: 109–121.

33.

Vergoten

Bailly

. Analysis of glycyrrhizin binding to protein HMGB1. Medicine in Drug Discovery 2020; 7: 100058.

34.

Mun

S-J

Yang

C-S

. Down regulation of HMGB1-RAGE pathway by ethyl pyruvate. FASEB J 2020; 34: 1.

35.

Zhang

Guan

X-W

Gribben

, et al. Blockade of HMGB1 signaling pathway by ethyl pyruvate inhibits tumor growth in diffuse large B-cell lymphoma. Cell Death Dis 2019; 10: 330.

36.

Zhou

Kuang

Huang

, et al. Potential role of high mobility group box 1 in hepatocellular carcinoma. Cell Adh Migr 2014; 8: 493–498.