Research ArticleExperimental Studies

Eriodictyol Attenuates Cholangiocarcinoma Malignancy by Regulating HMOX1 Expression: An In Vitro Study

JUNGWHOI LEE, WOOGWANG SIM, SONG-I HAN, JI-HUN BYEON, SOO-BEOM JIN, NUR AZIZAH ARRYANIE BINTI MORSHIDI, YE-YOUNG HONG, YUBIN JUNG and JAE HOON KIM
Anticancer Research August 2022, 42 (8) 3789-3798; DOI: https://doi.org/10.21873/anticanres.15869

Abstract

Background/Aim: Cholangiocarcinoma remains one of the most dangerous types of cancer. Eriodictyol is a well-known flavonoid having effective bioactivity against various malignant tumor types. However, the anticancer effect of eriodictyol against cholangiocarcinoma remains ambiguous. Thus, the aim of the present study was to investigate the effects of eriodictyol on human cholangiocarcinoma. Materials and Methods: The biological effects of eriodictyol were validated by viability assay, colony formation and western blot analysis. The significance of heme oxygenase 1 (HMOX1) expression in cholangio-carcinoma was demonstrated using bioinformatics analysis and knockdown of HMOX1 by transfection with short interfering (si)-RNA. Results: Eriodictyol highly reduced the in vitro viability of SNU-308, SNU-478, SNU-1079, and SNU-1196 cholangiocarcinoma cells compared with that of 293T cells, in a dose-dependent manner. The anticancer effect of eriodictyol was achieved by caspase-3-mediated apoptosis. In particular, eriodictyol increased HMOX1 expression, which resulted in attenuation of cholangiocarcinoma cell proliferation. In contrast, ablating HMOX1 expression by si-RNA transfection against HMOX1 made cholangiocarcinoma cells insensitive to the antiproliferative effect of eriodictyol treatment. Conclusion: These results collectively indicate that eriodictyol acts as an anticancer agent via regulation of HMOX1 expression against human cholangiocarcinoma.

Key Words:

Cholangiocarcinoma is a deadly type of cancer associated with bile duct epithelia. It accounts for 2% of cancer-related deaths annually in the world (1). Despite the rarity of cholangiocarcinoma, its incidence rate is continually increasing (2). This, combined with its ambiguous pathogenesis, means an effective therapy against cholangiocarcinoma is becoming increasingly important (3).

Limited chemotherapeutic agents are used singly, in combination, or in conjunction with surgery to treat cholangiocarcinoma (4). However, these agents can evoke toxicity in normal organs and cells, which can cause serious adverse reactions rather than antitumor effects (5, 6). Thus, development of safe anticancer agents with reduced harmful side-effects is urgently required.

Flavonoids are abundantly found in various plants and have tremendous pharmacological functions, including anticancer activity with potential multiple targets (7, 8). In particular, eriodictyol [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-2,3-dihydrochromen-4-one] (Figure 1A), a natural flavanone, is ubiquitous in fruits such as lemon (Citrus limon Burm. f.) (9), sutil lemon (Citrus aurantiifolia) (10), and bergamot (Citrus bergamia Risso) (11). Eriodictyol has been reported to possess bioactivities such as anti-oxidant, anti-inflammatory, and neuroprotective effects via clear intracellular signaling (12-14). In addition, eriodictyol has shown anticancer effects against various malignant tumor types such as liver, lung, glioma, and colorectal cancer (15-19). However, the anticancer effect of eriodictyol against cholangiocarcinoma and its underlying mechanism of action have not been reported.

Figure 1.
Figure 1.

Effect of eriodictyol on viability of human cholangiocarcinoma cells. A: Structure of eriodictyol. B-F: Four human cholangiocarcinoma cells SNU-308 (B), SNU-478 (C), SNU-1079 (D), and SNU-1196 (E) and human embryonic kidney cells 293T (F) were incubated with different concentrations of eriodictyol for 72 h. The viability was then measured by WST-1 assay (n=3; Tukey’s post-hoc test was applied to detect significant differences in ANOVA, with p<0.0001. Significantly different from the untreated control at: *p<0.05, **p<0.01 and ***p<0.001.

Considering the pleiotropic bioactivities of eriodictyol, the anticancer effect evoked by eriodictyol treatment should be examined with respect not only to its mode of action, but also to its potential safety as a drug against cholangiocarcinoma. In the present study, we report important in vitro evidence suggesting that eriodictyol inhibits the malignancy of cholangiocarcinoma at a dosage which is non-cytotoxic to non-tumor cells.

Materials and Methods

Data collection and gene expression analysis. Expression levels of mRNA in tumor or normal tissues were obtained from Gene Expression Omnibus public microarray database at National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/geo/), ArrayExpress (https://www.ebi.ac.uk/arrayexpress/), cBioportal (https://www.cbioportal.org/) and International Cancer Genome Consortium (https://icgc.org/).

Cell culture. Four cholangiocarcinoma cell lines, SNU-308, SNU-478, SNU-1079, and SNU-1196, were purchased from the Korean Cell Line Bank (Seoul, Korea). A normal human kidney cell line (293T) was obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured with Dulbecco’s modified Eagle’s medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL) and 100 mg/l penicillin-streptomycin (Gibco-BRL) at 37°C in a humidified atmosphere containing 5% CO2 as described previously (20).

Cell viability assay. Briefly, cholangiocarcinoma and 293T cells were seeded at a density of 2.5 × cells/well into 24-well plates (SPL Life Sciences, Seoul, Republic of Korea) and incubated for 24 h. These cells were then treated with 5, 10, 20, 50, or 100 μM of eriodictyol for an additional 72 h at 37°C. Cell viability was measured using WST-1 [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl) 2H-tetrazolium] solution (Boehringer Mannheim, Mannheim, Germany). After treatment with WST-1 solution, absorbance was measured at 450 nm on a Multiskan Spectrum microplate reader (Thermo Fisher Scientific, Vantaa, Finland).

Colony formation. SNU-478 and SNU-1196 cells were seeded into 60-mm dishes at a density of 200 cells/dish. After 24 h of incubation, these cells were treated with 0, 25, or 50 μM of eriodictyol for 14 days at 37°C in a humidified atmosphere containing 5% CO2. Colonies were then fixed with 4% paraformaldehyde solution (Sigma-Aldrich, St. Louis, MO, USA), stained with 0.1% crystal violet solution (Sigma-Aldrich) for 2 h, and then washed with sterile deionized water. Visible colonies were then counted.

Small interfering RNA (siRNA) transfection. SNU-308, SNU-478, SNU-1079, and SNU-1196 cells were transfected with siRNAs against heme oxygenase 1 (HMOX1) using Effectene (Qiagen, Hilden, Germany) as reported previously (21). Oligonucleotides specific for HMOX1 (3162-1 or sc-35554) were purchased from Bioneer (#1; Daejeon, Republic of Korea) or Santa Cruz Biotechnology (#2; Santa Cruz, CA, USA). Scrambled siRNA (sc-37007) was obtained from Santa Cruz Biotechnology. The efficacy of scrambled or HMOX1 siRNA transfection was confirmed by western blot analysis of HMOX1 protein.

Western blot analysis. To analyse profiles of endogenous HMOX1 expression in four cholangiocarcinoma and 293T cells, western blot analysis was performed as described previously (22). Antibodies against focal adhesion kinase (FAK), phospho-FAK (Y397), AKT serine/threonine kinase 1 (AKT), phospho-AKT (S473), caspase-3, cleaved caspase-3, HMOX1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA). For whole cell lysates, cells were lysed in M-PER lysis buffer (Thermo Scientific, Bonn, Germany) containing protease and phosphatase inhibitors. The total quantity of protein was determined by bicinchoninic acid quantification method (Bio-Rad, Richmond, CA, USA). Cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane (Amersham Bioscience, Little Chalfont, UK). The membrane was blocked using 5 % Bovine Serum Albumin and 2% Tween in Tris-buffered saline then incubated with diluted antibodies (1:1000 specific for HMOX1, p-FAK, p-AKT, cleaved caspase-3; and 1:3000 for FAK, AKT, caspase-3, GAPDH) overnight at 4°C. To visualize bands in an X-ray film, western blot detection reagent (iNtRON, Seoul, Korea) was used. Bands were measured by densitometry analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Enrichment analysis of selected signature genes. Enrichment analysis and protein–protein interaction network analysis were performed using Reactome Functional Interaction (FI) plugin app of Cytoscape (23). All enrichment results from Gene Ontology and the Reactome database with a false-discovery rate under 0.05 were filtered and top 15 results are presented in order of the number of genes (Reactome FI Network Version: 2019, FI Network Construction Parameters: Fetch FI annotations).

Statistical analyses. All data are presented as the mean±standard deviation and are representative of three individual experiments. Significance between two independent samples was determined by Student’s t-test. One-way analysis of variance with Tukey’s post hoc test was performed for analysis among independent groups using SPSS 12.0K for Windows (SPSS Inc., Chicago, IL, USA). Statistical significance was considered when the p-value was less than 0.05.

Results

Eriodictyol reduces the viability of cholangiocarcinoma cells by inducing apoptosis. To investigate the effect of eriodictyol on the viability of cholangiocarcinoma cells, four human cholangiocarcinoma cells, SNU-308, SNU-478, SNU-1079, and SNU-1196, were treated using eriodictyol along with human embryonic kidney 293T cells. The viability of cholangiocarcinoma cells was significantly reduced in a dose-dependent manner compared to that of 293T cells. Treatment with 100 μM eriodictyol attenuated the viability by approximately 20% for SNU-308 cells, 37% for SNU-478 cells, 25% for SNU-1079 cells, 37% for SNU-1196 cells, and 13% for 293T cells (Figure 1B-F). To validate the anti-viability effect of eriodictyol, we performed colony-formation assays using a sublethal dosage of eriodictyol in 293T cells, up to a maximum of 50 μM. Treatment with 25 μM of eriodictyol significantly inhibited the clonogenicity of SNU-478 and SNU-1196 cells (Figure 2A). To ascertain the pathways underlying the effect of eriodictyol on cell viability, we investigated its effects on the expression of cleaved caspase-3 in SNU-478 and SNU-1196 cells. Eriodictyol induced the cleavage of caspase-3, which was effectively suppressed when SNU-478 and SNU-1196 cells were pre-treated with caspase inhibitor carbobenzo-xy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (1 μM) (Figure 2B).

Figure 2.
Figure 2.

Effect of eriodictyol on colony formation by human cholangiocarcinoma cells. A: Representative images from a colony-formation assay using SNU-478 and SNU-1196 cells after treatment with 0, 25, or 50 μM of eriodictyol for 10 days. Surviving cells were measured by colony-formation assay. Data are presented as percentage±standard deviation of two individual experiments. Significantly different from the untreated control at: **p<0.01 and ***p<0.001. B: SNU-478 and SNU-1196 cells were incubated in the absence or presence of 50 μM eriodictyol with/without 1 μM caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (z-VAD) for 48 h. Cell lysates were subjected to immunoblot analysis using antibodies against total caspase-3, cleaved caspase-3, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative pixel intensities were normalized by GAPDH and measured using ImageJ software. Colony-formation and western blot images are representative of three individual experiments (values in parentheses are the variance).

Eriodictyol has anticancer effects by regulating intracellular signaling pathways in human cholangiocarcinoma. We next performed an in silico analysis after eriodictyol treatment to understand the mechanism underlying its ability to blockade the viability of human cholangiocarcinoma cells. In silico target protein network by eriodictyol was constructed using the Reactome Functional Interaction (FI) plugin app of Cytoscape (23). Regarding the protein–protein interaction network, HMOX1, nuclear factor erythroid-derived 2-like 2 (NFE2L2), and tumor necrosis factor were markedly connected with eriodictyol (Figure 3A and B).

Figure 3.
Figure 3.

Effect of eriodictyol on expression of heme oxygenase 1 (HMOX1) in human cholangiocarcinoma cells. A: Target genes network by eriodictyol in cholangiocarcinoma was constructed using The Human Protein Reference Database and Reactome database. ABCB1: ATP-binding cassette subfamily B member 1; ABCC1: ATP-binding cassette subfamily C member 1; BCL2A1: BCL2-related protein A1; CAT: catalase; CCL2: C-C motif chemokine ligand 2; CCND1: cyclin D1; CXCL10: C-X-C motif chemokine ligand 10; CXCL8: C-X-C motif chemokine ligand 8; GPT: glutamic-pyruvic transaminase; GSTP1: glutathione-S-transferase Pi 1; IL1B: interleukin 1 beta; IL6: interleukin 6; IRF3: interferon-regulatory factor 3; MAPK8: mitogen-activated protein kinase 8; MPO: myeloperoxidase; NFE2L2, NFE2-like BZIP transcript factor 2; NFKBIA: nuclear factor-Embedded ImageB inhibitor alpha; NLRP3: NLR family pyrin domain-containing 3; PTGS2: prostaglandin endoperoxide synthase 2; RELA: v-rel reticuloendotheliosis viral oncogene homolog A; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TNFAIP3: tumor necrosis factor alpha-induced protein 3, IFNG, interferon gamma; TRPM3: transient receptor potential cation channel subfamily M member 3; VEGFA: vascular endothelial growth factor A. B: The top 15 ranked genes affected by eriodictyol are displayed with reference count and organism count. Reference count and organism count indicate the number of previous reports on the function of eriodictyol and on organisms sensitive to eriodictyol, respectively. C: SNU-478 and SNU-1196 cells were incubated with eriodictyol at different concentrations for 24 h. Cell lysates were subjected to immunoblot analysis using antibodies against HMOX1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative pixel intensities were measured using ImageJ software. Data are representative of three individual experiments. Bracket indicates variance. D: SNU-478 and SNU-1196 cells were incubated with eriodictyol at different concentrations for 24 h. Cell lysates were subjected to immunoblot analysis using antibodies against focal adhesion kinase (FAK), phospho-FAK (Tyr397), AKT serine/threonine kinase 1 (AKT), phospho-AKT (Ser473), and GAPDH. Relative pixel intensities were normalized by FAK or AKT and measured using ImageJ software. Western blot data are representative of three individual experiments (values in parentheses are the variance).

According to previous reports, eriodictyol can regulate NRF2//HMOX1 in a liver injury model, endothelial cells, and retinal ganglial cells (14, 22, 24). In addition, the NRF2/HMOX1 axis has been proposed to have functional roles in malignant cancer (25, 26). Thus, we determined the effect of eriodictyol on HMOX1 expression in cholangiocarcinoma cells and 293T cells. Treatment with eriodictyol enhanced HMOX1 expression in SNU-478 and SNU-1196 cells with increasing eriodictyol dosage (Figure 3C). In addition, eriodictyol treatment reduced the phosphoylation of FAK and AKT in SNU-478 cells. On the contrary, eriodictyol treatment increased the phosphoylation of FAK and AKT in 293T cells in a dose-dependent manner (Figure 3D).

Silencing HMOX1 reduces the sensitivity of human cholangiocarcinoma cells to eriodictyol. To further investigate the functional significance of HMOX1 expression in human cholangiocarcinoma cells, four cholangiocarcinoma cell lines were transfected with different si-RNAs against HMOX1 and with scrambled si-RNA. As expected, reduced HMOX1 expression caused by siRNA transfection significantly reduced the viability of SNU-478 and SNU-1196 cells (by approximately 21% and 20%, respectively). However, the same treatment had little effect on SNU-308 and SNU-1079 cells (Figure 4A and B). We determined whether ablating HMOX1 expression altered the antiproliferatve effect of eriodictyol in SNU-308 and SNU-1079 cells that were more resistant to the effect of si-HMOX1 transfection. Treatment with eriodictyol at 50 and 100 μM significantly reduced the viability of scrambled si-RNA-transfected SNU-308 and SNU-1079 cells. At the same concentrations of eriodictyol, this effect was effectively lost in both cell lines by transfection with si-HMOX1 (#1) (Figure 4C). Similar results were obtained from transfection with another HMOX1 si-RNA (#2) in SNU-308 and SNU-1079 cells (Figure 4D). Overall, enhanced HMOX1 expression was associated with an antiproliferative effect of eriodictyol treatment on human cholangiocarcinoma cells (Figure 5).

Figure 4.
Figure 4.

Effects of silencing heme oxygenase 1 (HMOX1) expression on sensitivity of human cholangiocarcinoma cells to eriodictyol. A: SNU-308, SNU-478, SNU-1079, and SNU-1196 cells were transfected with scrambled or HMOX1-specific siRNA (#1) for 72 h. Viability was then measured using a WST-1 assay (n=3; Tukey’s post-hoc test was applied to detect significant differences in si-scrambled vs. si-HMOX1 by analysis of variance (ANOVA), with p<0.0001. Significantly different from the untreated control at: *p<0.05, **p<0.01 and ***p<0.001). B: The efficacy of HMOX1 silencing in SNU-308, SNU-478, SNU-1079 and SNU-1196 cells was validated using western blot analysis. glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Western blot data are representative of three individual experiments. C and D: SNU-308 and SNU-1079 cells were transfected with scrambled or HMOX1-specific siRNA #1 (C) or #2 (D) for 24 h and treated with eriodictyol at different concentrations (0, 5, 10, 20, 50, or 100 μM) for an additional 72 h. Cell viability was then measured by WST-1 assay (n=3; Tukey’s post-hoc test was applied to detect significant differences between cells transfected with si-scrambled vs. si-HMOX1 by ANOVA, p<0.0001. *Significantly different from scrambled si-RNA cells at p<0.05.

Figure 5.
Figure 5.

Graphical scheme of the mechanism of action of eriodictyol against human cholangiocarcinoma cells. AKT: AKT serine/threonine kinase 1; Cas-3: caspase-3; FAK: focal adhesion kinase; HMOX1: heme oxygenase 1.

Discussion

Cholangiocarcinoma is the second most common primary tumor of the liver following hepatocellular carcinoma. However, cholangiocarcinoma can randomly arise at any point in the biliary tree (1, 3). The carcinogenesis of cholangiocarcinoma has been associated with parasitic infestation, hepatitis C virus, and long-term biliary inflammation (27). However, the background of cholangiocarcinoma pathogenesis remains poorly defined (28). In the present study, eriodictyol as a natural flavanone had in vitro anticancer effects against human cholangiocarcinoma cells without showing cytotoxicity to human embryonic kidney cells, apparently through caspase-mediated induction of apoptosis. Of particular note, treatment with eriodictyol altered the expression of HMOX1, which resulted in attenuated viability of human cholangiocarcinoma cells.

Eriodictyol has well-defined bioactivities such as antioxidant, anti-inflammatory, neuroprotective, and anticancer effects (12-19). In agreement with previous reports, we found that treatment with eriodictyol selectively attenuated the viability of human cholangiocarcinoma cells through induction of the apoptotic caspase cascade. In the late 1990s, gemcitabine was approved by the US Food and Drug Administration as an anticancer drug for patients with pancreatic cancer. However, it showed no statistical benefit in clinical trials (29, 30). Moreover, gemcitabine has critical side-effects in almost all patients, such as anemia, diarrhea, vomiting, and depilation (31). Unfortunately, gemcitabine has been used in chemotherapy against cholangiocarcinoma due to the lack of a proper substitute (3, 32). In the present study, we demonstrated that eriodictyol effectively inhibited the viability of human cholangiocarcinoma cells without causing any cytotoxicity to human embryonic kidney cells. This suggests its safety as an anticancer agent against cholangiocarcinoma.

The continual development of bioinformatics for malignant cancer enables a huge amount of useful data to be handled (33). Using data relating to cholangiocarcinoma, we constructed protein–protein interaction pathways related to eriodictyol. Of interest, HMOX1 as a target of eriodictyol was the most inspiring. According to previous reports, expression of HMOX1 is correlated with cancer progression and chemoresistance in various malignant cancer types such as human melanoma, hepatoma, lymphosarcoma, prostate cancer, pancreatic cancer, renal cell carcinoma, and cholangiocarcinoma (34-39). In addition, ablating HMOX1 expression induced an anti-growth effect on pancreatic cancer cells resistant to gemcitabine treatment (40), implying that enhanced HMOX1 expression might be a convincing anticancer strategy for malignant cancer. The present study clearly revealed that up-regulating HMOX1 expression by eriodictyol treatment reduced growth of neoplastic cells by inducing caspase-3-mediated apoptosis pathway and reducing intracellular signaling pathways. Under physiological conditions, the NRF2/HMOX1 signaling pathway is associated with the maintenance of cellular homeostasis and has a critical function in adaptive response to cancer progression (26, 41, 42). Thus, enhanced expression of HMOX1 by eriodictyol treatment is quite likely to dysregulate the cellular balance enough to induce an effect on viability through induction of apoptosis in human cholangiocarcinoma cells. In contrast, silencing HMOX1 transfection made human cholangiocarcinoma cells insensitive to the antiproliferative effect of eriodictyol treatment, implying that eriodictyol treatment may evoke an anticancer effect by dysregulating HMOX1 expression in human cholangiocarcinoma. To support this scenario, the underlying mechanism should be investigated further. In addition, effective strategy to maximize the anticancer effect of eriodictyol treatment should be established against cholangiocarcinoma. Notably, we showed for the first time that eriodictyol had anticancer effects against human cholangiocarcinoma cells.

Collectively, the present study revealed that eriodictyol is a promising inhibitor of cholangiocarcinoma cells by dysregulating HMOX1 expression. Therefore, eriodictyol might be an easily available anti-cholangiocarcinoma agent.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Education (2016R1A6A1A03012862) and (2021R1I1A1A01041462).

Footnotes

  • Authors’ Contributions

    J. Lee designed the study and drafted the article. J. Lee, S.I. Han, J. Byeon, S. Jin, A. Morshidi, Y-Y. Hong, and Y. Jung performed in vitro experiments. W. Sim arranged all data-sets. J. Lee and J.H. Kim supervised the project. All Authors discussed data and read the article.

  • Conflicts of Interest

    There are no potential conflicts of interest regarding this study.

  • Received May 18, 2022.
  • Revision received June 14, 2022.
  • Accepted June 21, 2022.

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Eriodictyol Attenuates Cholangiocarcinoma Malignancy by Regulating HMOX1 Expression: An In Vitro Study
JUNGWHOI LEE, WOOGWANG SIM, SONG-I HAN, JI-HUN BYEON, SOO-BEOM JIN, NUR AZIZAH ARRYANIE BINTI MORSHIDI, YE-YOUNG HONG, YUBIN JUNG, JAE HOON KIM
Anticancer Research Aug 2022, 42 (8) 3789-3798; DOI: 10.21873/anticanres.15869
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