Biomolecules & Therapeutics 2024; 32(3): 368-378  https://doi.org/10.4062/biomolther.2023.198
Cordycepin Enhanced Therapeutic Potential of Gemcitabine against Cholangiocarcinoma via Downregulating Cancer Stem-Like Properties
Hong Kyu Lee, Yun-Jung Na, Su-Min Seong, Dohee Ahn and Kyung-Chul Choi*
Laboratory of Biochemistry and Immunology, College of Veterinary Medicine, Chungbuk National University, Cheongju 28644, Republic of Korea
*E-mail: kchoi@cbu.ac.kr
Tel: +82-43-261-3664, Fax: +82-43-267-3150
Received: November 7, 2023; Revised: December 8, 2023; Accepted: January 12, 2024; Published online: April 9, 2024.
© The Korean Society of Applied Pharmacology. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Cordycepin, a valuable bioactive component isolated from Cordyceps militaris, has been reported to possess anti-cancer potential and the property to enhance the effects of chemotherapeutic agents in various types of cancers. However, the ability of cordycepin to chemosensitize cholangiocarcinoma (CCA) cells to gemcitabine has not yet been evaluated. The current study was performed to evaluate the above, and the mechanisms associated with it. The study analyzed the effects of cordycepin in combination with gemcitabine on the cancer stem-like properties of the CCA SNU478 cell line, including its anti-apoptotic, migratory, and antioxidant effects. In addition, the combination of cordycepin and gemcitabine was evaluated in the CCA xenograft model. The cordycepin treatment significantly decreased SNU478 cell viability and, in combination with gemcitabine, additively reduced cell viability. The cordycepin and gemcitabine co-treatment significantly increased the Annexin V+ population and downregulated B-cell lymphoma 2 (Bcl-2) expression, suggesting that the decreased cell viability in the cordycepin+gemcitabine group may result from an increase in apoptotic death. In addition, the cordycepin and gemcitabine co-treatment significantly reduced the migratory ability of SNU478 cells in the wound healing and trans-well migration assays. It was observed that the cordycepin and gemcitabine cotreatment reduced the CD44highCD133high population in SNU478 cells and the expression level of sex determining region Y-box 2 (Sox-2), indicating the downregulation of the cancer stem-like population. Cordycepin also enhanced oxidative damage mediated by gemcitabine in MitoSOX staining associated with the upregulated Kelch like ECH Associated Protein 1 (Keap1)/nuclear factor erythroid 2–related factor 2 (Nrf2) expression ratio. In the SNU478 xenograft model, co-administration of cordycepin and gemcitabine additively delayed tumor growth. These results indicate that cordycepin potentiates the chemotherapeutic property of gemcitabine against CCA, which results from the downregulation of its cancer-stem-like properties. Hence, the combination therapy of cordycepin and gemcitabine may be a promising therapeutic strategy in the treatment of CCA.
Keywords: Cholangiocarcinoma, Cordycepin, Chemoresistance, Gemcitabine, Cancer stem cell
INTRODUCTION

Cholangiocarcinoma (CCA) is one of the most aggressive types of cancers of the biliary tract, which accounts for ~15% of all primary hepatic malignancies (Banales et al., 2020). Remarkable advances in CCA research over the past few decades have resulted in the development of various therapeutic interventions for CCA treatment. However, the survival rate and prognosis of CCA have not improved substantially, with the 5-year overall survival standing at 7-20% (Bertuccio et al., 2019; Banales et al., 2020). CCA is usually diagnosed only in its advanced stages, as the patient remains asymptomatic in the early stages of the disease. Thus, the therapeutic options for the treatment of advanced CCA are limited (Lindner et al., 2015). Currently, surgical resection is considered the only curative therapy for CCA, but tumor relapses post-resection result in poor prognosis (Alabraba et al., 2019; Strijker et al., 2019). A combination of cisplatin and gemcitabine is considered the standard treatment for CCA in patients with post-resection residual tumors. However, the response to these drugs is poor, and frequent recurrences are common (Lindner et al., 2015; Alabraba et al., 2019). Therefore, the development of more targeted interventions is necessary to improve the chemotherapeutic potential of these treatments.

The major challenge in the management of CCA with chemotherapeutics is the acquisition of drug resistance. Accumulating evidence indicates that the mechanism of chemoresistance is complicated and influenced by both tumor intrinsic factors and extrinsic tumor microenvironment (TME) (Pan et al., 2016; Brivio et al., 2017). For instance, tumor cells resist anti-cancer drugs by facilitating drug export systems such as the adenosine triphosphate (ATP)-binding cassette subfamily (Ferreira et al., 2015), enhancing DNA repair system (Obama et al., 2008; Asakawa et al., 2010), and upregulation of survival signals (Bordoloi et al., 2022; Dong et al., 2022). The specific cell populations in heterogenetic cancer cell types, which share these characteristics, are the cancer stem cells (CSCs). The CSCs also interconnect with various factors in the TME, including immune cells, fibroblasts, and the extracellular matrix, thereby mediating TME reconstruction (Xie et al., 2019), chemoresistance (Babu et al., 2022), and even metastasis (Babaei et al., 2020). Therefore, the identification of the CSC characteristics is essential for regulating drug resistance, and targeting CSCs can be a promising therapeutic strategy for suppressing cancer recurrence.

Cordyceps militaris is a potent nutraceutical mushroom in traditional Chinese medicine and contains a variety of bioactive compounds with anti-inflammatory, antioxidant, and anti-cancer potential (Paterson, 2008; Ashraf et al., 2020). Cordycepin, a type of nucleoside analog, is the major bioactive compound extracted from Cordyceps militaris and has various biological properties, including anti-bacterial (Jiang et al., 2019), anti-ischemic (Cheng et al., 2011), and anti-diabetic activities (Yan et al., 2023). Cordycepin showed potent anti-tumor properties against several cancer types, including liver cancer, lung cancer, and brain cancer, and the involved mechanisms include apoptosis induction, cell cycle arrest, and anti-metastasis (Hueng et al., 2017; Guo et al., 2020; Zhang et al., 2023). Specifically, cordycepin sensitizes esophageal cancer cells to cisplatin and glioma cells to temozolomide (Bi et al., 2018; Gao et al., 2020). Cordycepin has been reported to inhibit cell growth and induce apoptosis in CCA (Wang et al., 2017; Liu et al., 2020), but the ability of cordycepin to chemosensitize CCA cells to gemcitabine has not yet been evaluated.

The present study investigated the effect of cordycepin on the sensitization of CCA to gemcitabine and its related mechanisms by analyzing its anti-apoptotic, migratory, and antioxidant properties in the CCA SNU478 cell line with cancer stem-like properties. In addition, the combination therapy of cordycepin and gemcitabine was evaluated in the CCA xenograft model. The results of this study suggest that cordycepin enhances the gemcitabine-mediated antitumor potential and the combination therapy of cordycepin and gemcitabine may be a promising therapeutic option in the treatment of CCA.

MATERIALS AND METHODS

Cell cultures and reagents

The CCA cell line SNU478 was obtained from the Korea Cell Line Bank (Seoul, Korea). SNU478 cells cultured in Dulbecco’s modified eagle medium (DMEM; Gibco, Gaithersburg, MD, USA) supplied with 10% fetal bovine serum (FBS; RMBIO, Missoula, MT, USA) and 2% Antibiotic-Antimycotic solution (Gibco). SNU478 cell line was maintained in a humidified chamber with 5% CO2 at 37°C. Cordycepin (Fig. 1A) was kindly provided by Prof. Dr. Yeon Hee Seong (Laboratory of Pharmacology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Korea). Gemcitabine was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cordycepin and gemcitabine were dissolved in distilled water to prepare a stock solution at 200 mM and 5 mM, respectively, and diluted to testing concentration with medium before use. The control group received no treatment at all.

Figure 1. Inhibitory effects of cordycepin on the growth of SNU478 cells. (A) The chemical structure of cordycepin. Cell viability in SNU478 cells (B) treated with cordycepin with different concentrations for 72 h and (C) treatment of cordycepin and/or gemcitabine with different incubation times (24, 48, and 72 h) assessed using WST assay. (D) Representative images of colony formation in SNU478 cells and (E) quantitative analysis of colonies. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. **p<0.01 vs. control group; ##p<0.01 vs. cordycepin group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine; IC50, half-maximal inhibitory concentration.

Cell viability assay

Cell viability was evaluated by Quanti-MAX water-soluble tetrazolium salt (WST)-8 cell viability kit (Biomax, Seoul) by assessing the mitochondrial dehydrogenase activity according to the manufacturer’s instructions. SNU478 cells were seeded at 4×103 cells/well in a 96-well cell culture plate. After 24 h of incubation, various concentrations of cordycepin (0-320 μM) were treated for 72 h to assess the half-maximal inhibitory concentration (IC50). In addition, cordycepin (20 μM) and/or gemcitabine (500 nM) were treated for 24, 48, and 72 h. After the drug incubation period, WST-8 reagent was added to each well, followed by incubation for an additional 1 h at 37°C. The optical density (O.D.) of each well excluding blank O.D. was measured using a Neo2 Hybrid Multimode Reader (Agilent Technologies, Inc., Santa Clara, CA, USA) at 460 nm.

Clonogenic assay

The ability of single cells to form a colony was evaluated by colony formation assay as described previously with slight modifications (Kim et al., 2022). SNU478 cells were seeded at 5×102 cells/well in a 6-well cell culture plate as a single-cell suspension and incubated for 6 h to adhere. Then, cells were treated with cordycepin (20 μM) and/or gemcitabine (1 nM) for 48 h, and the medium was replaced with the treatment-free medium. After 8 days of further incubation, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) and stained with 0.5% crystal violet (Sigma-Aldrich). Stained images were captured with a digital camera, and colony-forming areas were measured by Image J software (National Institutes of Health, Bethesda, MD, USA).

AnnexinV/PI staining

Different types of cell death apoptosis or necrosis were determined by Annexin V/PI staining according to the manufacturer’s recommendations with slight modification. SNU478 cells were seeded at 2×105 cells/well in a 6-well plate. After 24 h incubation, cells were treated with cordycepin (20 μM) and/or gemcitabine (500 nM) for 48 h. After harvesting cells, FITC-conjugated Annexin V (BioLegend, San Diego, CA, USA) and propidium iodide (PI, Invitrogen Life Technologies, Carlsbad, CA, USA) was stained. Then, samples were acquired using fluorescence-activated cell sorting (FACS) Symphony A3 (BD Bioscience, San Diego, CA, USA) and data were analyzed with the FlowJo Software v. 10.8.1 (TreeStar, San Carlos, CA, USA).

Wound healing assay

Collective migration and wound healing rate were evaluated by wound healing assay, also known as scratch assay, as described previously with minor modifications (Go et al., 2022). SNU478 cells were seeded at 5×105 cells/well in a 6-well plate and incubated to confirm a confluent monolayer for 24 h. Then, cells were treated with Mitomycin C (Sigma-Aldrich) for 1.5 h and scratched mechanically using a sterile blunt plastic tip. After preparing cell-free scratches immediately treated with cordycepin (20 μM) and/or gemcitabine (100 nM), wound areas were captured with a phase-contrast microscope (Olympus, Tokyo, Japan) with a series of time-laps at 0, 24, and 48 h after treatment. Images for analysis were captured in at least five fields per well, and the wound area was measured by CellSens Dimension v. 1.13 (Olympus).

Trans-well migration assay

The ability of single cells to directional migration was evaluated by trans-well migration assay as described previously (Kim et al., 2022). SNU478 cells were seeded at 1×105 cells/well in the 24-well trans-well insert (8.0 μm membrane pore size; Corning, NY, USA) with a serum-free media treated with cordycepin (20 μM) and/or gemcitabine (100 nM), and lower chambers contained complete media with 10% FBS. After 24 h incubation, remove the trans-well insert and carefully remove the remaining cells from the top side of the membrane with a cotton swab. The migrated cells attached to the bottom side of the membrane were fixed with ethanol and stained with 0.2% crystal violet (Sigma-Aldrich). Images were captured using IX-73 inverted microscope (Olympus) and the number of migrated cells was counted by CellSens Dimension v. 1.13 (Olympus).

FACS analysis

The proportion of CD44 and CD133 positive cells in SNU478 cells was measured by FACS analysis. After 48 h of cordycepin (20 μM) and/or gemcitabine (500 nM) treatment, cells were harvested. After washing, cells were reacted with FITC-conjugated anti-human CD44 (Clone BJ18; BioLegend) and APC-conjugated anti-human CD133 (Clone clone7; BioLegend) antibody. Stained samples were detected by FACS Symphony A3 (BD Bioscience) and results were analyzed by the FlowJo Software v. 10.8.1 (TreeStar).

Mitochondrial superoxide (MitoSOX) analysis

Mitochondrial reactive oxygen species (ROS) were detected by the MitoSOXTM Red superoxide indicator kit (Invitrogen Life Technologies) according to the manufacturer’s instructions with slight modifications. SNU478 cells were seeded 6×103 cells/well in a 96-well cell culture plate, and preincubated for 24 h. Then, cells were replaced with media containing cordycepin (10 or 20 μM) and/or gemcitabine (500 nM). After 48 h of incubation, cells were stained with MitoSOXTM (5 μM) and Hoechst 33342 (Sigma-Aldrich) after washing, the cells were imaged under fluorescence microscopy using LionheartTM FX (Biotek Instruments, Inc., Winooski, VT, USA) and analyzed by Gen5 v.3.14.03 (Agilent Technoloies, Inc.).

Western blot analysis

Total protein from SNU478 cells was extracted by PRO-PREP protein extraction solution (iNtRON Biotechnology, Seongnam, Korea), and protein concentration was determined using a bicinchoninic acid protein assay kit (Sigma-Aldrich). Approximately 1.5 μg of proteins were loaded on a capillary-based Western blot system (ProteinSimple, San Jose, CA, USA) and automatically measured each protein level. Finally, each Western band images were acquired and intensities of the target protein with specific molecular size were quantified by Compass for Simple Western v. 6.1.0. (ProteinSimple). Monoclonal antibodies against B-cell lymphoma 2 (Bcl-2; clone 100; BioLegend), phosphorylated protein kinase B (p-Akt; clone D9E; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:100), Akt (clone 11E7; Cell Signaling Technology, Inc.; 1:100), Sex determining region Y-box 2 (Sox-2; clone D6D9; Cell Signaling Technology, Inc.; 1:100), nuclear factor erythroid-2 related factor 2 (Nrf2; clone D1Z9C; Cell Signaling Technology, Inc.; 1:100), Kelch-like ECH-associated protein 1 (Keap1; clone D6B12; Cell Signaling Technology, Inc.; 1:100), and polyclonal antibodies against β-actin (Cell Signaling Technology, Inc.; 1:200) were used for the analysis.

Xenograft mouse model

Five- to six-week-old female NSG-dKO (NOD.Cg-Prkdcscid H2-K1b-tm1Bpe H2-Ab1g7-em1Mvw H2-D1b-tm1Bpe Il2rgtm1Wjl/SzJ) mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA) and maintained in a specific pathogen-free area of Laboratory Animal Research Center of Chungbuk National University (Cheongju, Korea). Animal studies were conducted in compliance with the ethical policies and procedures approved by the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-1686-22-01).

The CCA xenograft model in NSG-dKO mice was generated by subcutaneously inoculating SNU478 cells (1×107 cells were suspended in 100 μL Dulbecco’s phosphate-buffered saline (DPBS)/head; this is day 0 of the in vivo experiment). Seven days post-inoculation of SNU478 cells, cordycepin (50 mg/kg, every other day) and/or gemcitabine (50 mg/kg, twice/week) were administered intraperitoneally until day 25. Saline (0.9% NaCl) was used as a vehicle for administration. Tumor volumes were measured using an electric caliper and calculated by the formula ((length)×(width)2/2). Mice were sacrificed at the study termination (day 26) and the implanted tumors were harvested and fixed in 10% neutral buffered formalin.

Immunohistochemistry (IHC)

The fixed tumors were processed, paraffin-embedded, and cut into 4 μm on silane-coated slide glass. After hydration, slides were incubated with 10 mM sodium citrate buffer (pH 6.0) at 100°C for 10 min for antigen retrieval. Then, slides were reacted with 3% hydrogen peroxide (Daejung, Siheung, Korea) in DPBS and 5% bovine serum albumin (Sigma-Aldrich) to block non-specific reactions. Tissue slides were incubated overnight with rabbit monoclonal antibodies against proliferating cell nuclear antigen (PCNA; clone PC10; Cell Signaling Technology, Inc.; 1:100) and antigen Kiel 67 (Ki-67; clone D3B5; Cell Signaling Technology, Inc.; 1:100). Slides were incubated with a biotinylated anti-rabbit secondary antibody for 1 h followed by 30 min of further incubation with avidin-biotin peroxidase complex (ABC Elite kit; Vector Labs, Burlingame, CA, USA). Visualization of the peroxidase was performed using a DAB kit (Vector Labs) and counterstained with hematoxylin (Sigma-Aldrich). Images of each slide were acquired by SLIDEVIEW VS200 digital slide scanner system (Olympus) and captured in at least four fields per slide using OlyVia 3.2 software (Olympus). The DAB intensity was semi-quantified using Image J Fiji v. 1.53c software blindly as described previously (Lee et al., 2023).

Statistical analysis

All data are presented as standard deviation (SD) or means ± standard errors of the mean (SEM). Statistical significances of data were analyzed by one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s test using the GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA). The p-values<0.05 was considered statistically significant.

RESULTS

Co-treatment of cordycepin and gemcitabine additively inhibits SNU478 cell growth

To evaluate the effect of cordycepin on CCA cell growth, a WST assay was performed on the SNU478 cancer cell line. The cordycepin treatment for 72 h inhibited cell proliferation in a dose-dependent manner, with an IC50 of 35.83 μM in SNU478 cells (Fig. 1B). We also investigated the effect of 20 μM cordycepin treatment and/or 500 nM gemcitabine treatment on time-dependent growth inhibition at 24, 48, and 72 h. As shown in Fig. 1C, the cordycepin and/or gemcitabine treatment significantly decreased cell proliferation compared with the control group and the co-treatment showed additional growth inhibitory effects at each time point.

A clonogenic assay was performed to confirm the effect of cordycepin (20 μM) and gemcitabine (1 nM) on cell survival by evaluating the ability of a single cell to proliferate into a colony. The concentrations of cordycepin and gemcitabine were set at 20 μM and 1nM, respectively, because a very high concentration could interfere with the continuation of the clonogenic assay. The cordycepin and/or gemcitabine treatment significantly inhibited colony formation (Fig. 1D, 1E). These results indicate that cordycepin and gemcitabine demonstrate remarkable anti-proliferative effects against SNU478 cells.

Cordycepin in combination with gemcitabine results in additional apoptotic cell death

To determine the type of cell death that occurs during the inhibition of cordycepin and/or gemcitabine-mediated cancer cell growth, an Annexin V/PI staining assay was performed. The results showed that the cordycepin (20 μM) and gemcitabine (500 nM) co-treatment significantly increased Annexin V+PI+ (late apoptosis) as well as Annexin V+PI- (early apoptosis) compared to all other groups (Fig. 2A, 2B).

Figure 2. Effects of cordycepin on gemcitabine-mediated apoptotic changes in SNU478 cells. The cells stained with Annexin V/PI were assessed after treatment of cordycepin and/or gemcitabine by FACS analysis and expression levels of apoptosis-related proteins were analyzed by Western blot. (A) Representative flow cytometry plot of SNU478 cells for Annexin V/PI staining and (B) percentage of apoptotic cells. (C) Representative band images of proteins and (D) the relative intensity ratio of Bcl-2/β-actin were presented. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; #p<0.05 and ##p<0.01 vs. cordycepin group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

To further confirm these results, the changes in the expression levels of the apoptosis-associated protein Bcl-2 were assessed. As shown in Fig. 2C and 2D, the Bcl-2 expression in the cordycepin (20 μM) and gemcitabine (500 nM) co-treated group was significantly lower than that of the control group, which was consistent with the results of the Annexin V/PI staining analysis. These results indicate that a significant reduction in cell viability in the cordycepin+gemcitabine group could result partly from apoptotic cell death in the SNU478 cells.

Cordycepin hampers the migratory ability of SNU478 cells

To verify the effect of cordycepin on the migratory ability of the CCA cells, a wound healing assay and a trans-well migration assay were performed. In the wound healing assay, wound closure was significantly reduced after the cordycepin (20 μM) and gemcitabine (100 nM) co-treatment compared to the control (Fig. 3A, 3B). The results of the trans-well migration assay consistently showed a marked decrease in the number of migrated cells in the cordycepin (20 μM) and gemcitabine (100 nM) co-treated group when compared with those of all the other groups (Fig. 3C, 3D). In addition, the cordycepin-alone treatment significantly reduced the number of migrated SNU478 cells when compared with that of the control, but the gemcitabine-alone treatment presented no significant differences in the migrated cells between each group. These results indicate that cordycepin inhibits the migratory ability of SNU478 cells, and this is enhanced by combining it with gemcitabine treatment.

Figure 3. Effects of cordycepin on migratory ability in SNU478 cells. Cell migration after treatment of cordycepin and/or gemcitabine was assessed by wound healing assay and trans-well migration assay. (A) Representative images of wound healing were captured with different incubation times (at 0, 24, and 48 h). (B) Wound areas of SNU478 cells were measured by image J and the percentage of wound closure was calculated. (C) Representative images of migratory cells and (D) the number of migrated cells were assessed. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; #p<0.05 vs. cordycepin group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=500 μm. Cordy, cordycepin; Gem, gemcitabine.

Cordycepin in combination with gemcitabine inhibits cancer stem-like cell properties

Since cancer stemness is known to play a pivotal role in the acquisition of chemoresistance (Babu et al., 2022), the cancer stem-like cell subpopulation in the SNU478 cells was examined. In the FACS analysis, the cordycepin (20 μM) and gemcitabine (500 nM) co-treatment significantly reduced the proportion of CD44highCD133high cells compared to all the other groups, indicating the additive inhibition of cancer stem-like cell population due to the combination treatment (Fig. 4A, 4B).

Figure 4. Effects of cordycepin treatment on cancer stem-like cell population in SNU478 cells. SNU478 cells were treated with cordycepin and/or gemcitabine, and expression of CD44/CD133 was detected by flow cytometry. (A) Representative flow cytometry plots of SNU478 cells for CD44 and CD133 and (B) percentage of CD44highCD133high cells in total cells were presented. Protein expression levels of p-Akt, Akt, and Sox-2 in SNU478 after treatment of cordycepin and/or gemcitabine were confirmed by Western blot. (C) Representative band images of each protein and the relative intensity ratio of (D) p-Akt/Akt and (E) Sox-2/β-actin were presented. The control group received no treatment at all. Data are expressed as means ± SD. from at least three independent experiments. *p<0.05 and **p<0.01 vs. control group; ##p<0.01 vs. cordycepin group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

To further verify the effects of cordycepin on the cancer stem-like properties, the expression levels of CD44 activation-related protein, including p-Akt, Akt, and Sox-2, were evaluated by the Western blot. As shown in Fig. 4C and 4D, cordycepin (20 μM) and/or gemcitabine (500 nM) treatment significantly reduced the p-Akt/Akt expression ratio compared with the control. The expression level of Sox-2 was markedly decreased in the cordycepin-treated group compared with that of the control group (Fig. 4C, 4E). Moreover, the cordycepin and gemcitabine co-treatment significantly downregulated the Sox-2 expression compared with the gemcitabine-alone treatment. These results indicate that the cordycepin and gemcitabine co-treatment downregulates the cancer stem-like properties of SNU478 cells.

Co-treatment of cordycepin and gemcitabine mediates oxidative stress

Since cancer stemness is associated with the downregulation of oxidative stress (Cadamuro et al., 2017), the effect of the combination on the formation of mitochondrial ROS was evaluated. In the MitoSOX analysis, the cordycepin (10 μM or 20 μM) and gemcitabine (500 nM) co-treatment significantly increased the MitoSOX fluorescence intensity compared with the control and the cordycepin-alone treatment (Fig. 5A, 5B).

Figure 5. Effects of co-treatment cordycepin and gemcitabine on mitochondrial ROS generation in SNU478 cells. The content of mitochondrial ROS was determined by MitoSOX staining and related protein levels were confirmed by Western blot. (A) Representative images of SNU478 cells stained with MitoSOX (red, mitochondria peroxide) and Hoechst 33342 (blue, nuclei) are shown. (B) MitoSOX fluorescence intensity was quantified. (C) Representative band images of proteins and (D) the relative intensity ratio of Keap1/Nrf2 were presented. The control group received no treatment at all. Data are expressed as means ± SD from at least three independent experiments. **p<0.01 vs. control group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=200 μm. Cordy, cordycepin; Gem, gemcitabine.

The expression levels of oxidative stress-related proteins such as Nrf2 and upregulated Keap1 were evaluated in the SNU478 cells. As shown in Fig. 5C and 5D, the cordycepin-treated group showed a significantly increased Keap1/Nrf2 expression ratio compared with the control, and the cordycepin and gemcitabine co-treatment markedly upregulated the Keap1/Nrf2 expression ratio compared with the gemcitabine-alone treatment. These results indicate that the cordycepin and gemcitabine co-treatment additively increased oxidative stress-mediated cell damage, partly associated with the upregulation of the Keap1/Nrf2 expression.

Co-treatment of cordycepin and gemcitabine additionally delays tumor growth

To evaluate the antitumor potential of cordycepin and/or gemcitabine treatment against CCA, SNU478 cells were xenografted into immunocompromised mice (Fig. 6A). Tumor volumes were measured thrice a week after the administration of cordycepin (50 mg/kg, every other day) and/or gemcitabine (50 mg/kg, thrice a week). It was found that compared to the vehicle-treated group, the cordycepin and/or gemcitabine treatment significantly delayed tumor growth from day 9 post-administration (Fig. 6B). The tumor weights were measured after animal sacrifice (day 26). There were no significant differences in the body weights between the groups during the experimental period (Fig. 6C). Consistent with the tumor volume results, the administration of cordycepin and/or gemcitabine significantly decreased tumor weights compared to those of the vehicle-treated animals (Fig. 6D). Specifically, the tumor volumes and tumor weights of the cordycepin and gemcitabine co-administered group showed a remarkable reduction when compared with all the other groups.

Figure 6. Effects of cordycepin in combination with gemcitabine on tumor growth in SNU478 xenograft mouse model. (A) Scheme of in vivo experiment. (B) The tumor volumes and (C) body weights in SNU478 xenografted mice were measured thrice a week after treatment, and (D) the tumor weights were measured at study termination (day 26). Saline (0.9% NaCl) was used as a vehicle for administration. Data are expressed as means ± SEM. *p<0.05 and **p<0.01 vs. vehicle group; #p<0.05 and ##p<0.01 vs. cordycepin group; @p<0.05 vs. gemcitabine group (Dunnet’s test). Cordy, cordycepin; Gem, gemcitabine.

The study also assessed the expression levels of the tumor proliferation index markers, including PCNA and Ki-67, in the SNU478 xenografted tumors. In the IHC analysis of the xenografted tumors, it was observed that the cordycepin-alone treatment significantly reduced the expression of PCNA when compared with the vehicle-treated group (Fig. 7A, 7B). The co-administration of cordycepin and gemcitabine markedly decreased the PCNA expression in tumors compared with the gemcitabine-alone treatment. In addition, cordycepin in combination with gemcitabine significantly reduced Ki-67 expression in tumors compared with the vehicle-treated group (Fig. 7A, 7C). These results indicate that cordycepin in combination with gemcitabine exerts an additive effect on tumor growth inhibition, partly associated with the downregulation of PCNA and Ki-67 expression by the cordycepin treatment.

Figure 7. Effects of cordycepin and/or gemcitabine treatment on PCNA and Ki-67 expression in xenografted SNU478 tumors. The expression levels of PCNA and Ki-67 in xenografted SNU478 tumors were analyzed by IHC staining. (A) Representative IHC images against PCNA and Ki-67 in tumors and the relative DAB intensity of (B) PCNA and (C) Ki-67 were analyzed. Data are expressed as means ± SEM. *p<0.05 vs. vehicle group; @@p<0.01 vs. gemcitabine group (Dunnet’s test). Bar=50 μm. Cordy, cordycepin; Gem, gemcitabine.
DISCUSSION

CSCs are a subpopulation of cancer cells that possess a capability for tumor initiation, invasiveness, and chemoresistance, and are associated with poor prognosis in most cancer types (Razi et al., 2023). CSCs account for approximately <2-3% of the total tumor cells in many solid tumors, including breast cancer, prostate cancer, and melanoma (Cardinale et al., 2015). However, the CSCs are known to comprise a surprisingly high proportion in CCA, indicating the relatively crucial role of CSCs in CCA progression (Cadamuro et al., 2017; Banales et al., 2020). The expression patterns of CSC markers specific to each cancer type are now well known. The surface markers for CSCs, including CD44, CD133, and aldehyde dehydrogenase, increase in CCA (Wu and Chu, 2019). In the current study, the combination of cordycepin with gemcitabine reduced the CD44high/CD133high subpopulations in SNU478 cells, suggesting the downregulation of cancer stemness by the cordycepin and gemcitabine co-treatment. These observations also indicate that the anti-proliferative potential of the combination of cordycepin with gemcitabine against SNU478 cells could be associated with a reduction in cancer stem-like subpopulation.

CD44 is a transmembrane glycoprotein receptor that primarily binds to hyaluronic acid (HA) and is one of the most well-known CSC markers in CCA (Suwannakul et al., 2018). When HA binds to CD44, the downstream pathways such as the phosphoinositide 3-kinase/Akt pathway and extracellular signal-regulated kinase (ERK) 1/2 are activated, resulting in the promotion of cell survival and proliferation (Kashyap et al., 2018). In this study, cordycepin and/or gemcitabine significantly downregulated the Akt pathway in SNU478 cells, which might be related to the suppression of CD44 by the combination of cordycepin with gemcitabine. These results are partially consistent with the results of previous studies wherein the Akt pathway was inhibited by cordycepin in glioma cells and esophageal cancer cells (Bi et al., 2018; Gao et al., 2020). In addition, the suppression of the Akt pathway by cordycepin is associated with sensitization to chemotherapeutics (Li et al., 2021). It has been reported that the phosphorylation of Akt is connected to Sox-2 overexpression in CSCs, with reciprocal interaction (Singh et al., 2012). In the present study, cordycepin significantly downregulated Sox-2 expression in SNU478 cells. These results suggest that the inhibition of Akt by cordycepin might be partly correlated with the downregulation of the Sox-2 mediating suppression of cancer stemness. Sox-2 promotes tumorigenesis by facilitating proliferation, invasiveness, and cancer stemness, and the overexpression of Sox-2 is associated with frequent relapse and poor prognosis in many cancer types, including breast cancer, pancreatic cancer, and CCA (Sun et al., 2014; Zhang et al., 2020). Intriguingly, gemcitabine resistance was reported to be associated with Sox-2 overexpression (Jia et al., 2019), but gemcitabine treatment had no effect on Sox-2 expression in the current study. Therefore, the downregulation of Sox-2 by cordycepin might be partly attributable to the sensitization of CCA cells to gemcitabine.

Acquisition of migratory properties is one of the critical steps in metastasis and is associated with its invasiveness and chemoresistance (Soleymani et al., 2021). The facilitated motility of cancer cells is mediated by mesenchymal traits and this promotes resistance to apoptosis in CCA cells (Vaquero et al., 2017). It has been reported that the downregulation of CD44 in the CCA cell line inhibits epithelial to mesenchymal transition (EMT) (Suwannakul et al., 2020). The current study also confirmed that cordycepin markedly suppresses the migratory ability of SNU478 cells, which is partly associated with the sensitization of CCA cells to gemcitabine. Cordycepin has been reported to suppress metastasis and EMT in CCA by regulating lipid metabolism (Zhou et al., 2023). Therefore, our results support a possible role of cordycepin in the relationship between EMT and the cancer stem-like properties in CCA.

Conventional anticancer drugs, including cisplatin and gemcitabine, induce apoptotic changes by disturbing the mitochondrial membrane permeability and activating apoptosis-associated proteins such as the Bcl-2-associated X protein (Bax), Bcl-2 associated agonist of cell death (BAD), and Bcl-2 interacting mediator of cell death (BIM) (Sjostrom et al., 2002; Vogler, 2014). However, CSCs are resistant to apoptotic responses by upregulating anti-apoptotic and/or survival-related proteins, including Bcl-2 and Bcl-xL (Mukherjee et al., 2015). It has been reported that chronic treatment with conventional anti-cancer drugs often induces the acquisition of resistance, resulting in relapses or unresponsiveness (El Amrani et al., 2019). Therefore, targeting these compensatory responses by the downregulation of cancer stemness and the recovery of the anti-apoptotic signal after conventional treatment would be a promising intervention. In the present study, the cordycepin treatment downregulated Bcl-2 levels in SNU478 cells. Moreover, we found that the proliferation index PCNA and Ki-67 were downregulated by cordycepin. These results suggest that cordycepin might sensitize CCA cells to the conventional anti-cancer drug gemcitabine by hampering anti-apoptotic and proliferative activity.

Oxidative damage caused by the excessive formation of ROS mediates apoptotic cancer cell death after conventional chemotherapeutic treatment (Pelicano et al., 2004). However, CSCs exhibit a low level of ROS via the upregulation of cellular defense proteins, such as Nrf2 (Xue et al., 2020). After dissociating from Keap1, Nrf2 translocates into the nucleus and upregulates antioxidant enzymes, including heme oxygenase-1, catalase, and superoxide dismutase-2, through the regulation of antioxidant response elements (Xue et al., 2020; Mukherjee and Gopalakrishnan, 2023). The upregulation of CD44 expression in CSCs mediates p62-associated Nrf2 activation followed by the autophagic degradation of Keap1 (Ryoo et al., 2018). Therefore, the downregulation of Nrf2 suppresses cancer stem-like properties, including proliferation, metastasis, and chemoresistance (Zhao et al., 2019). In the current study, gemcitabine monotherapy did not cause a significant increase in the mitochondrial ROS, but cordycepin in combination with gemcitabine markedly increased mitochondrial ROS formation. Upregulation of the Keap1/Nrf2 expression ratio by cordycepin might be associated with increased ROS formation by the combination of cordycepin and gemcitabine. Excessive accumulation of ROS disrupts mitochondrial function and activates intrinsic apoptotic cascades, consequently resulting in apoptosis. Bcl-2, an anti-apoptotic protein, attenuates ROS-mediated programmed cell death by downregulating ROS production and facilitating anti-apoptotic signaling (Esposti et al., 1999; Chong et al., 2014). Therefore, the downregulation of Bcl-2 by cordycepin might be attributable to the enhancement of ROS accumulation by the combination of cordycepin with gemcitabine in the current study. These results suggest that the anti-CSC potential of cordycepin mediates the sensitization of CCA cells to gemcitabine by suppressing ROS-scavenging abilities and a decrease in CD44 expression by cordycepin might partly contribute to the inactivation of Nrf2-mediated antioxidant activity.

This study confirmed the anti-CSC properties of cordycepin and the enhanced anti-cancer effect of gemcitabine in combination with cordycepin by using a SNU478 cell line. It has been reported that chronic- and low-dose gemcitabine treatment produces resistant cell lines with an increased proportion of CSC (Hagmann et al., 2010). Although establishing resistant cell lines is time-consuming and laborious, further studies involving resistant cell lines may be useful in understanding chemosensitization. The current study focused on the intrinsic effect of cordycepin on CSCs in the development of chemoresistance, but the role of CSCs in TME is more complicated and multifaceted (Brivio et al., 2017; Cadamuro et al., 2017). Therefore, it is necessary to examine the comprehensive effects of cordycepin on chemosensitization using a more sophisticated experimental model, including tumor organoids, patient-derived models, and humanized mouse models.

In conclusion, the current study demonstrated that cordycepin sensitizes SNU478 CCA cells to gemcitabine cytotoxicity both in vitro and in vivo. Mechanistic studies showed that the chemosensitizing effects of cordycepin are mediated by its anti-CSC potential, which is associated with the downregulation of anti-apoptotic, migratory, and anti-oxidative properties. These results suggest that the combination of cordycepin and gemcitabine may have an additive role in inhibiting CCA growth and proliferation, and cordycepin may be a promising agent for CCA treatment.

ACKNOWLEDGMENTS

This work was supported by the Basic Research Lab Program (2022R1A4A1025557) through the National Research Foundation (NRF) of Korea, funded by the Ministry of Science and ICT. In addition, this study was also supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE; 2021RIS-001) in 2023.

CONFLICT OF INTEREST

The authors do not have any conflict of interest to declare.

AUTHOR CONTRIBUTIONS

Hong Kyu Lee: Conceptualization, Methodology, Investigation, Writing-Original Draft; Yun-Jung Na: Investigation, Visualization; Su-Min Seong: Validation, Formal analysis; Dohee Ahn: Investigation, Data Curation; Kyung-Chul Choi: Conceptualization, Resources, Writing-Review and Editing, Supervision.

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