Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line

  • Authors:
    • Kitti Intuyod
    • Aroonsri Priprem
    • Chawalit Pairojkul
    • Chariya Hahnvajanawong
    • Kulthida Vaeteewoottacharn
    • Porntip Pinlaor
    • Somchai Pinlaor
  • View Affiliations

  • Published online on: March 7, 2018     https://doi.org/10.3892/ijo.2018.4306
  • Pages: 1715-1726
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Abstract

Cholangiocarcinoma (CCA) is a deleterious bile duct tumor with poor prognosis and is relatively resistant to chemotherapy. Therefore, alternative or supplementary agents with anticancer and chemosensitizing activities may be useful for the treatment of CCA. A novel anthocyanin complex (AC) nanoparticle, developed from extracts of cobs of purple waxy corn and petals of blue butterfly pea, has exhibited chemopreventive potential in vivo. In the present study, the anti-CCA activities of AC and their underlying molecular mechanisms were investigated further in vitro using a CCA cell line (KKU213). The potential use of AC as a chemosensitizer was also evaluated in a gemcitabine-resistant CCA cell line (KKU214GemR). It was demonstrated that AC treatment suppressed proliferation of KKU213 CCA cells in dose- and time-dependent manners. AC treatment also induced apoptosis and mitochondrial superoxide production, decreased clonogenicity of CCA cells, and downregulated forkhead box protein M1 (FOXM1), nuclear factor-κB (NF-κB) and pro-survival protein B-cell lymphoma-2 (Bcl-2). The expression of endoplasmic reticulum (ER) stress-response proteins, including protein kinase RNA-like ER kinase, phosphorylated eIF2α, eukaryotic initiation factor 2α and activating transcription factor 4, also decreased following AC treatment. It was also identified that AC treatment inhibited KKU214GemR cell proliferation in dose- and time-dependent manners. Co-treatment of KKU214GemR cells with low doses of AC together with gemcitabine significantly enhanced efficacy of the latter against this cell line. Therefore, it is suggested that AC treatment is cytotoxic to KKU213 cells, possibly via downregulation of FOXM1, NF-κB, Bcl-2 and the ER stress response, and by induction of mitochondrial superoxide production. AC also sensitizes KKU214GemR to gemcitabine treatment, which may have potential for overcoming drug resistance of CCA.

Introduction

Cholangiocarcinoma (CCA) is a bile-duct tumor that is rare in the majority of countries (1,2), but which has a far greater incidence in the Greater Mekong sub-region of southeast Asia, particularly in northeastern Thailand, where the prevalence of the small human liver fluke Opisthorchis viverrini is high (3). O. viverrini is classified as a group I carcinogen by the International Agency for Research on Cancer (4). Infection with this fluke causes chronic inflammation, leading to periductal fibrosis and ultimately contributing to the development of CCA (5,6). In northeastern Thailand, CCA is the major primary liver cancer, with its management costing US$120 million annually (5,7). Clinical presentation is typically observed at a late stage and therefore the majority of patients with CCA cannot be cured by surgical resection (8,9). Furthermore, the disease is able to develop resistance to standard chemotherapeutic drugs over time (10). Therefore, using phytochemicals with anti-inflammatory and anticancer activities to treat cancer or to enhance the efficacy of other chemotherapeutic drugs, may be an alternative approach for the management of CCA and help to avoid drug-resistance (1113).

Anthocyanins are the water-soluble flavonoids responsible for the blue, purple and red colors in a number of fruits, flowers and leaves (14). Anthocyanins exhibit anti-inflammatory, anti-angiogenesis, antioxidant and anti-proliferative effects, and thus have a number of medical applications (1518). Among these are prevention and treatment of cancer (19,20). Previous studies have demonstrated that the consumption of anthocyanin-rich foods is associated with a decreased risk of chronic diseases, including cardiovascular disease, arthritis and diabetes mellitus, and development of esophageal, colon, lung and skin cancers (17,21).

Cyanidin and delphinidin glycosides are the most abundant and well-studied anthocyanins with potential anticancer activity (2224). However, a major obstacle in the use of anthocyanins is that they are unstable and prone to degradation (25). Previously, cyanidin and delphinidin were isolated from cobs of purple waxy corn (Zea mays L. var. ceritina Kulesh) and petals of blue butterfly pea (Clitoria ternatea L.) and were manipulated to form a complex with turmeric extract and other trace elements (26) to increase their stability and activity. This novel anthocyanin complex (AC) nanoparticle exhibited anti-inflammatory and anti-fibrotic effects in an O. viverrini-infected hamster model, establishing its potential for chemoprevention of CCA (26). However, the utility for CCA treatment has not yet been investigated.

The aim of the present study was to demonstrate the application of AC for CCA treatment. Anticancer activities of AC and the potential underlying molecular mechanisms against CCA were investigated in vitro using a CCA cell line. The effect of combined treatment of AC and gemcitabine against the gemcitabine-resistant CCA cell line (KKU214GemR) was also investigated. The results of the present study provide an insight into the promising use of AC phytochemical products for the treatment of CCA.

Materials and methods

Chemicals and reagents

AC was prepared as described previously (26). In brief, aqueous extracts of purple waxy corn cobs and blue butterfly pea petals were mixed with turmeric (Curcuma longa) extract (7:2:1) in the presence of 100 mM caffeic acid and piperine (Sigma Aldrich; Merck KGaA, Darmstadt, Germany) and 2 mM zinc sulfate (Ajax Finechem; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Thereafter, the mixture was cooled and dried to yield of AC. Only one batch of AC was used throughout the present study to avoid batch-to-batch variation. Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, trypsin-EDTA and fetal bovine serum (FBS) were purchased from Gibco; Thermo Fisher Scientific, Inc. 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), dimethyl sulfoxide (DMSO), sulforhodamine B (SRB) and the broad-spectrum caspase inhibitor quinolone-Val-Asp-difluorophenoxymethyl ketone (Q-VD-OPh) were purchased from Sigma-Aldrich; Merck KGaA. Rabbit anti-protein kinase RNA-like endoplasmic reticulum (ER) kinase (PERK; cat. no. 5683), anti-p65 (cat. no. 8242), anti-activating transcription factor 4 (ATF4; cat. no. 11815), anti-phosphorylated eukaryotic initiation factor 2α (p-eIF2α; cat. no. 3398) (Ser51), anti-eIF2α (cat. no. 9722), anti-poly(ADP-ribose) polymerase (PARP; cat. no. 9542), anti-B-cell lymphoma-2 (Bcl-2; cat. no. 2872), anti-caspase-3 (cat. no. 9662) and anti-β-tubulin (cat. no. 2128) and radioimmunoprecipitation assay (RIPA) buffer were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Rabbit anti-forkhead box M1 (FOXM1; cat. no. sc-502) (C-20) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (cat. no. 111-035-003) secondary antibody was purchased from Jackson ImmunoResearch, Inc. (West Grove, PA, USA). A dead-cell apoptosis kit [Annexin V/propidium iodide (PI)], Pierce™ bicinchoninic acid (BCA) protein assay kit, Hank's balanced salt solution (HBSS) and MitoSOX™ Red mitochondrial superoxide indicator were obtained from Thermo Fisher Scientific, Inc. ECL™ Prime western blotting detection reagent and polyvinylidene difluoride (PVDF) membrane were obtained from GE Healthcare (Chicago, IL, USA).

Human CCA cell lines

The KKU213 CCA cell line was established from Thai CCA patients as described previously (27). The gemcitabine-resistant CCA cell line KKU214GemR was established previously by exposure to stepwise increases in the concentration of gemcitabine as described previously (28). It should be noted that the parental line of KKU214GemR cells appears to be a KKU213 cell derivative (web.expasy.org/cellosaurus/CVCL_M264). However, due to the fact that KKU214GemR cells were induced to be a gemcitabine-resistant CCA cell line, it would not have any bearing on the results of the present study. The cell lines were maintained in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator containing 10% CO2. KKU214GemR cells were cultured in the presence of gemcitabine to maintain its resistant phenotype, but were cultured in a drug-free medium for one passage prior to performing experiments.

Assessment of half-maximal inhibitory concentration (IC50) of AC

The IC50 was assessed using the SRB assay. For instance, KKU213 cells were seeded at 2,000 cells/well in flat-bottomed 96-well plates (Corning Inc., Corning, NY, USA). The following day, the cells were incubated with either DMSO (diluent control) or various concentrations of AC (100–800 µg/ml) dissolved in DMSO for 12, 24, 36 and 48 h at 37°C in a humidified incubator containing 10% CO2. Cells were fixed with ice-cold 40% trichloroacetic acid at 4°C for 1 h. Following washing three times with running tap water, 0.4% (w/v) SRB solution in 1% acetic acid was added and incubated further for 1 h at room temperature. Excess SRB solution was removed by washing with 1% acetic acid and SRB was dissolved by adding 10 mM Tris buffer. Absorbance at 492 nm was determined using an ELISA plate reader (Tecan Group Ltd., Männedorf, Switzerland). The absorbance at 492 nm of DMSO-treated cells was used as control. For KKU214GemR cells, the same procedure was performed, but the cells were treated with either single agent (300 µg/ml AC; 20 or 40 µM gemcitabine) or a combination of AC (300 µg/ml) and gemcitabine (20 or 40 µM) for 24, 48 and 72 h.

Assessment of cellular apoptosis

Cellular apoptosis was determined by Annexin V/PI staining according to the manufacturer's protocol. In brief, following trypsinization and washing with sterile PBS, ~106 KKU213 cells were resuspended in 1X binding buffer. Annexin V/PI solution was added prior to incubation at room temperature in the dark for 15 min. Stained cells were detected using a BD FACSCanto II flow cytometer and analyzed using BD FACSDiva software (version 6.1.3) (both from BD Biosciences, San Jose, CA, USA).

Plasmid transfection

The pCMV4-p65 plasmid was a gift from Professor Warner Greene, Gladstone Institute of Virology and Immunology, San Francisco, CA, USA (Addgene plasmid #21966). In total ~2×106 KKU213 cells were seeded into 10-cm cell culture dishes for 24 h, prior to transfection with pCMV4-p65 plasmid using X-tremeGENE HP (Roche Diagnostics, Basel, Switzerland), according to the manufacturer's protocol. Cells were then harvested at 24 h post-transfection for further experiments.

Clonogenic assay

CCA cells were seeded into 6-well plates at a density of 1,000 cells/well overnight prior to drug treatment. Cells were treated with different concentrations of AC (100–800 µg/ml) and incubated for 48 h at 37°C in a humidified incubator containing 10% CO2. In the case of KKU214GemR cells, cells were treated with either a single agent (300 µg/ml AC; 20 or 40 µM gemcitabine) or a combination of AC (300 µg/ml) and gemcitabine (20 or 40 µM). The culture medium was changed every 2 days and cells were cultured for a further 14 days. Finally, cells were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet. Stained cells were dissolved with 33% acetic acid and absorbance at 620 nm was measured using an ELISA reader (Tecan Group Ltd.).

Measurement of mitochondrial superoxide production

Mitochondrial superoxide production was determined using MitoSOX Red mitochondrial superoxide indicator, according to the manufacturer's protocol. In brief, CCA cells were trypsinized, washed once and resuspended in HBSS with Ca2+/Mg2+. Subsequently, 4 µM MitoSOX Red solution was added and the mixture was incubated at 37°C for 30 min in the dark. CCA cells were centrifuged at 600 × g for 5 min at room temperature and washed with 1 ml HBSS/Ca2+/Mg2+. Finally, CCA cells were resuspended in HBSS/Ca2+/Mg2+ and the fluorescence intensity at 575 nm was measured using a flow cytometer (BD Biosciences).

Western blot analysis

Protein was extracted from CCA cells using RIPA buffer (50 mM Tris/HCl, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and 1 mM AEBSF) and the protein concentration was determined using the BCA assay. Subsequently, 20 µg protein was separated by SDS-PAGE (7 or 12% gels) and then transferred onto a PVDF membrane. Following blocking with 5% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20, membranes were incubated with primary antibodies against PERK, p65, ATF4, p-eIF2α (Ser51), eIF2α, PARP, caspase-3, Bcl-2 and β-tubulin (all 1:1,000) overnight at 4°C. Following washing, membranes were incubated with secondary antibody (1:3,000) and the chemiluminescent reaction was developed using ECL™ Prime blotting detection reagent. Immunoreactivity bands were captured using the ImageQuant™ LAS4000 mini imager (GE Healthcare).

Statistical analysis

Data are expressed as the mean ± standard deviation. Student's t-test and analysis of variance and Tukey's test were performed to determine differences among experimental groups using SPSS (version 13.0; SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. Non-linear regression analysis was performed to determine the IC50 values using GraphPad Prism (version 6; GraphPad Software, Inc., La Jolla, CA, USA).

Results

AC treatment inhibits proliferation and induces caspaseindependent apoptosis of the CCA cell line

Inhibition of tumor growth and induction of cellular apoptosis are important modes of action of the majority of phytochemical agents. To determine whether AC exerts proliferation-inhibitory effects on CCA cells, and to determine relevant IC50 values, KKU213 cells were treated with various concentrations of AC. The SRB assay revealed that AC inhibited cell proliferation in this cell line in a dose- and time-dependent manner (Fig. 1A). The proliferation of KKU213 cells was completely inhibited by treatment with 800 µg/ml AC after 48 h. Non-linear regression analysis identified that the IC50 value of AC for KKU213 was 620 µg/ml (Fig. 1B).

It was subsequently investigated whether suppression of CCA cell proliferation by AC treatment is associated with induction of apoptosis. KKU213 cells were treated with 300 or 600 µg/ml AC for 24 h, and cellular apoptosis was investigated using flow cytometry (Fig. 2A). The results revealed that early (1.50±0.27% for AC300 and 10.33±0.83% for AC600) (Fig. 2B) and late (5.50±0.66% for AC300 and 56.70±6.05% for AC600) (Fig. 2C) apoptosis was significantly induced dose-dependently in AC-treated groups compared with the control group. These results indicated that AC exhibited cytotoxicity against CCA cells through the induction of cellular apoptosis. Levels of cleaved caspase-3, a protein marker for cellular apoptosis, was also assayed using western blotting. Cleaved caspase-3 was not observed in AC-treated KKU213 cells, but was detected in a gemcitabine-treated group (Fig. 3A). In addition, a pan-caspase inhibitor (Q-VD-OPh) was combined with AC treatment to test whether cell death was due to a caspase-independent pathway. A clonogenic assay demonstrated that treatment with 2 and 10 µM Q-VD-OPh, identified to prevent caspase activation (29), did not affect cell viability of the KKU213 cells (Fig. 3C). Notably, Q-VD-OPh treatment was not able to prevent cell death when combined with AC treatment (Fig. 3D and E), indicating that caspase-independent cell death was occurring in AC-treated CCA cells.

AC treatment inhibits colony formation of the CCA cell line

The clonogenic assay is the method of choice to determine cell reproductive viability (ability of cells to produce progeny or ability of a single cell to form a colony) following treatment with radiation or a cytotoxic agent (30,31). This approach was employed to determine the cytotoxic effects of AC treatment on a CCA cell line (Fig. 4A). Consistent with the results of the SRB assay, the presence of AC significantly inhibited KKU213 colony formation in a dose-dependent manner compared with the control group (P<0.0001) (Fig. 4B). This result indicated that the AC-mediated decrease in reproductive viability is one of the underlying molecular mechanisms of AC against CCA.

AC treatment induces mitochondrial superoxide production partly via suppression of Bcl-2 expression

Induction of superoxide production in mitochondria is an important event in the induction of apoptosis (32). To investigate whether induction of mitochondrial superoxide production is involved in AC-induced apoptosis of CCA cells, KKU213 cells were treated with AC, and levels of mitochondria-specific superoxide were determined using flow cytometry. Minimal superoxide production was detected in the control group; however, production was slightly increased in KKU213 cells treated with 300 µg/ml AC (Fig. 5). Treatment with 600 µg/ml AC significantly induced superoxide production in the mitochondria of KKU213 cells relative to the control group (P<0.001) (Fig. 5).

Since superoxide production may be inhibited by the anti-apoptotic Bcl-2 protein (32), the expression of Bcl-2 protein was investigated further in KKU213 CCA cells. Western blot analysis revealed that the expression of Bcl-2 protein mirrored superoxide production. Expression of Bcl-2 protein decreased in the AC-treated group, particularly when 600 µg/ml AC was used, relative to the DMSO-treated control (Fig. 6). These results indicated that AC induced superoxide production-mediated apoptosis partly via the inhibition of Bcl-2 protein expression.

AC treatment targets pro-survival and endoplasmic reticulum stress (ER stress) response of the CCA cell line

FOXM1 and NF-κB are well-known oncogenic proteins involved in the survival of cancer cells (33,34). The expression of these proteins was determined in the CCA cell line following treatment with AC for 24 h. Western blot analysis revealed that expression of FOXM1 and the p65 subunit of NF-κB decreased in a dose-dependent manner in KKU213 cells treated with AC (Fig. 6). Notably, expression of these proteins was almost completely inhibited in KKU213 cells treated with 600 µg/ml AC compared with the DMSO-treated control (Fig. 6). Additionally, KKU213 cells were transfected with p65 plasmid DNA and the influence of p65 induction on AC treatment of KKU213 cells was determined using a clonogenic assay. The p65 subunit of NF-κB was successfully induced in KKU213 as identified using western blot analysis (Fig. 7A). However, the clonogenic assay revealed that p65 overexpression did not affect the viability of KKU213 cells compared with non-transfected and mock-transfected controls (Fig. 7B and C). This implies that AC is a potent inducer of apoptosis against CCA cells, and that induction of a pro-survival transcription factor was not able to protect CCA cells from cell death following AC treatment. A previous study has identified that AC treatment dramatically induced mitochondrial superoxide production; excessive superoxide production may cause protein misfolding and ultimately induce ER stress (35). Therefore, the expression of proteins in the PERK/eIF2α/ATF4 axis, which is an important ER stress-response pathway, was investigated. Western blot analysis revealed that expression of PERK, p-eIF2α (Ser51) and ATF4 in KKU213 cells decreased following AC treatment (Fig. 6). Notably, total eIF2α expression was not affected by AC treatment, suggesting that AC suppresses phosphorylation of the eIF2α protein. In addition to FOXM1, p65 and the PERK/eIF2α/ATF4 axis, it was identified that PARP expression was also suppressed by AC treatment, particularly at the highest dose used (Fig. 6).

AC treatment increases gemcitabine sensitivity of the gemcitabine-resistant KKU214GemR CCA cell line

Resistance to chemotherapeutic treatment is an important obstacle for the treatment of various types of cancer, including CCA (36). Therefore, whether co-treatment with AC was able to enhance the effect of gemcitabine was investigated. The SRB assay revealed that the IC50 of gemcitabine against the KKU214GemR CCA cell line was 32.11 µM at 72 h, whereas the IC50 of gemcitabine against the parental KKU214 cell line was 0.40 µM at 72 h (data not shown), agreeing with a previous study (28). Treatment of KKU214GemR cells with AC inhibited cell proliferation in a dose- and time-dependent manner (Fig. 8A). For gemcitabine treatment alone, KKU214GemR cell proliferation was markedly inhibited by treatment with 40 µM for 72 h (Fig. 8B). AC at a dose of 300 µg/ml, which did not exert a proliferation-inhibitory effect on KKU214GemR cells, was selected for co-treatment with 20 or 40 µM gemcitabine. As expected, the two combinations significantly enhanced gemcitabine-mediated proliferation inhibition compared with treatments with single agents (P<0.01 for 20 µM gemcitabine vs. 300 µg/ml AC+20 µM gemcitabine; P<0.001 for 40 µM gemcitabine vs. 300 µg/ml AC+40 µM gemcitabine) (Fig. 8C). Furthermore, the clonogenic assay also identified a significant enhancement of gemcitabine treatment when co-treated with AC (P<0.05) (Fig. 8D). Annexin V/PI staining coupled with flow cytometry was used to detect apoptotic cells following combination treatment of KKU214GemR cells. Administration of AC (300 µg/ml) and gemcitabine (40 µM) significantly induced early apoptosis of these cells compared with single treatments and the control group (P<0.0001) (Fig. 9). These results indicated that AC treatment significantly enhanced the efficacy of gemcitabine against the gemcitabine-resistant KKU214GemR cell line.

Discussion

Several previous studies have demonstrated that anthocyanins exert anticancer activity against various types of cancer, including HCC, melanoma, colon cancer, lung cancer and breast cancer (3739). In our previous study, we developed a novel AC, which primarily consisted of extracts of purple corn cobs, blue butterfly peas and turmeric (26). The combination of these exhibited increased antioxidant activity and increased thermal stability compared with that of individual extracts (26). In the present study, to the best of our knowledge, we have identified, for the first time, anti-CCA activity of AC, which exhibited cytotoxicity against a CCA cell line through the suppression of cell proliferation and induction of caspase-independent apoptosis probably via increased mitochondrial superoxide production. Furthermore, AC also suppressed the expression of a number of oncogenic proteins that have previously been reported to be upregulated in CCA, including FOXM1, NF-κB and ER stress-response proteins. The potential underlying molecular mechanisms of AC treatment on CCA cells are summarized in Fig. 10. AC was able to potentiate gemcitabine treatment against the gemcitabine-resistant KKU214GemR cell line. We therefore hypothesize that AC has a potential function as an alternative or supplementary treatment for CCA.

cDNA analysis of O. viverrini-associated CCA clinical samples revealed that FOXM1 was highly expressed, being the second most abundant gene in these samples (40). FOXM1 has been implicated primarily in cell proliferation and survival, as well as in cancer progression (34). In the present study, suppression of cell proliferation and induction of cellular apoptosis following AC treatment were observed alongside downregulation of FOXM1 expression. These results suggest that suppression of FOXM1 is, in part, the mode of action of AC against CCA. To the best of our knowledge, the present study is the first to identify the effects of cyanidin and delphinidin on FOXM1 expression. However, a previous study demonstrated that curcumin, an important bioactive compound in turmeric extract, was able to suppress FOXM1 (41). Therefore, suppression of FOXM1 using AC treatment is potentially mediated by either anthocyanin(s) (cyanidin and/or delphinidin) or turmeric extract or both. In addition to FOXM1, western blot analysis also demonstrated the downregulation of NF-κB in AC-treated CCA cells. NF-κB is a well-known transcription factor associated with inflammation and cancer (4244). A previous study has identified that NF-κB, particularly its p65 subunit, was overexpressed in O. viverrini-associated CCA tumor tissues. Furthermore, treatment with dehydroxymethylepoxyquin-omicin inhibited CCA cell proliferation and induced apoptosis in KKU213 cells via the suppression of NF-κB and Bcl-2 proteins (45). The results of the present study are in general agreement with this previous study, demonstrating that the p65 subunit of NF-κB and Bcl-2 were also suppressed concurrently with induction of apoptosis in the AC-treated KKU213 CCA cell line. Cyanidin and delphinidin are known to suppress the expression of NF-κB and Bcl-2 (46,47). Furthermore, the turmeric extract in AC was also identified to suppress NF-κB and Bcl-2 (48,49). Therefore, we hypothesized that cyanidin, delphinidin and turmeric extract in AC may synergistically inhibit NF-κB and Bcl-2. Curcumin, an important active ingredient in turmeric extract, is able to inhibit NF-κB expression and induce cancer cell apoptosis. AC, composed of purple corn cob, blue butterfly pea and turmeric extracts at a ratio of 7:2:1, contains ~60 µg turmeric extract per 600 µg/ml AC. In its standard form, turmeric contains 5% curcumin, implying that there is ~3 µg or 8 µM curcumin in 60 µg turmeric extract. In our previous study, we identified that significant suppression of proliferation, induction of apoptosis (4% of the cell population compared with the untreated control) and suppression of pro-survival proteins in the KKU213 cell line were only observed when treated with 50 µM curcumin (49). The synergistic effect of combining different anthocyanins was also demonstrated previously (50,51). These results support our hypothesis that the cytotoxicity of AC against CCA cells is mediated by synergism of cyanidin, delphinidin and turmeric extract. Notably, in the present study, the function of p65 on AC treatment of KKU213 cells was also determined using p65 plasmid DNA transfection coupled with a clonogenic assay. Although p65 was successfully induced in KKU213 cells, the clonogenic assay revealed that induction of pro-survival transcription factor expression was not able to protect KKU213 cells from apoptosis following AC treatment. Failure of NF-κB to prevent cell death has also been demonstrated in tumor necrosis factor α-induced apoptosis in the HeLa cell line (52). Furthermore, failure to escape apoptosis following p65 induction may be because AC treatment induces CCA cell apoptosis through multiple pathways. Therefore, AC is a potent apoptosis inducer against CCA cells.

AC treatment also markedly induced superoxide production from mitochondria in KKU213 cells. Mitochondrial super-oxide has been demonstrated as an inducer of apoptosis (32). Apoptosis induced in this manner is consistent with a previous study demonstrating that diphenyleneiodonium induces mitochondrial superoxide-mediated apoptosis (53). Notably, in the present study, cleaved PARP (data not shown) and cleaved caspase-3 were not observed after 24 h of AC treatment in CCA cell lines, suggesting that caspase-independent cell death occurred without activating Bcl-2-associated X protein, cytochrome c and caspase-3 (54). Caspase-independent cell death was also indicated by treatment with a combination of AC and pan-caspase inhibitor (Q-VD-OPh): addition of Q-VD-OPh was not able to protect CCA cells from AC-mediated cell death. The defects in the caspase activation pathway are common in cancer, leading to resistance to certain pro-apoptotic stimuli (55). Therefore, triggering caspase-independent cell death is one of the novel approaches to treat cancer (56). Therefore, in addition to CCA, the AC may be useful for the treatment of cancer, particularly that with the ability to evade caspase-dependent cell death. Decreasing PARP expression in CCA cell lines was observed following AC treatment. This result may be explained by the fact that this protein is involved in DNA damage repair (57). Induction of mitochondrial super-oxide production was able to induce DNA damage (58,59), suggesting that decreasing of PARP expression after AC treatment could lead to a decrease in DNA damage repair by PARP. Therefore, we hypothesize that AC-treated cells underwent apoptosis via DNA damage-induced caspase-independent cell death (60).

Superoxide in mitochondria may be converted into H2O2 and diffuse into the cytoplasm. H2O2 may be catalyzed further to form other highly reactive oxygen species (ROS). Under basal physiological conditions, ROS accumulation may be prevented by cellular antioxidant defense mechanisms (61). However, excessive ROS production caused by excessive mitochondrial superoxide production triggers disturbance of ER redox homeostasis, thus aggravating the accumulation of misfolded or unfolded proteins in the ER or ER stress (62). The process known as the unfolded protein response (UPR) is thus activated to restore ER homeostasis (63). However, if ER stress is severe or prolonged, it may induce cell death (64). The PERK/eIF2α/ATF4 axis is important in UPR signaling during ER stress (65). PERK activates the phosphorylation of eIF2α at Ser51, resulting in global translation inhibition (66), but induces the expression of ATF4 (67) to overcome ER stress. The results of the present study identified downregulation of PERK/eIF2α/ATF4 in an AC-treated CCA cell line. Therefore, we hypothesize that AC treatment induces ER stress and eventual cell death via the stimulation of mitochondrial superoxide production and suppression of PERK/eIF2α/ATF4 axis-mediated UPR. This hypothesis is supported by previous studies demonstrating that inhibition of PERK or eIF2α rendered cells susceptible to ER stress-mediated cell death (6870). Furthermore, a recent study has demonstrated that activation of PERK and eIF2α was inhibited in cyanidin-treated cells (71).

Drug resistance is a major barrier for chemotherapy in a number of types of cancer, including CCA. Enhancement of chemotherapeutic drug treatment and chemosensitization of cancer cells by plant polyphenols have been the focus of much work and discussion (72). In the present study, the effect of co-treatment of AC and gemcitabine against a gemcitabine-resistant KKU214GemR cell line was investigated. It was identified that KKU214GemR cells were ~80-fold more resistant to gemcitabine compared with its parental cell line. However, the combination of AC with gemcitabine significantly enhanced the efficacy of gemcitabine against KKU214GemR cells compared with single agent treatment. Although the potential of AC against a CCA cell line as well as its potential as a chemo-sensitizer in a gemcitabine-resistant CCA cell line have been demonstrated in the present study, these results have not been verified in an animal model of CCA. Therefore, the anticancer activity of AC should be investigated further in xenograft mouse or liver fluke-induced hamster CCA models. Furthermore, the anticancer potential of AC requires testing in diverse cancer cell types to support its potential use as an alternative or supplementary treatment for cancer, particularly CCA. Additionally, high-throughput approaches, including RNA sequencing, are required to explore the precise mechanisms underlying anticancer and chemosensitization activities of AC.

In conclusion, the results of the present study demonstrated that AC possesses cytotoxicity against CCA cell lines by suppression of cell proliferation and induction of caspase-independent apoptosis possibly via downregulation of FOXM1, NF-κB and the ER stress response, and by induction of mitochondrial superoxide production. AC also sensitizes KKU214GemR cells to gemcitabine treatment. Therefore, AC has potential as an alternative treatment agent and may assist in overcoming drug resistance of CCA when co-administered with other chemotherapeutic agents.

Acknowledgments

The authors thank Miss Sucharat Limsitthichaikoon for assisting with AC preparation and the Research Instrument Unit, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand, for technical and facility support.

Abbreviations:

AC

anthocyanin complex

AEBSF

4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride

ATF4

activating transcription factor 4

BCA

bicinchoninic acid

CCA

cholangiocarcinoma

DMEM

Dulbecco's modified Eagle's medium

DMSO

dimethyl sulfoxide

ER

endoplasmic reticulum

FBS

fetal bovine serum

FOXM1

forkhead box protein M1

HBSS

Hank's balanced salt solution

NF-κB

nuclear factor-κB

PERK

protein kinase RNA-like endoplasmic reticulum kinase

eIF2α

eukaryotic initiation factor 2α

PVDF

polyvinylidene difluoride

RIPA

radioimmunoprecipitation assay

SRB

sulforhodamine B.

Notes

[1] Funding

This study was supported in part by the Thailand Research Fund (TRF) and Khon Kaen University through Royal Golden Jubilee PhD joint funding program (grant no. PHD/0166/2553) and also by the TRF and Medical Research Council (MRC) UK via TRF-MRC Joint Health Research (grant no. DBG5980004) and Khon Kaen University Research Fund (grant no. KKU600701).

[2] Availability of data and materials

The data sets generated during the study are available from the corresponding author on reasonable request.

[3] Authors' contributions

SP and KI mainly designed, performed the research and wrote the manuscript. KI analyzed the data, and AP, CP, CH, KV and PP helped to prepare and provided materials, and analyzed the data. All authors have read and approved the final version of the manuscript.

[4] Ethics approval and consent to participate

Not applicable.

[5] Consent for publication

Not applicable.

[6] Competing interests

The authors declare that they have no competing interests.

References

1 

Banales JM, Cardinale V, Carpino G, Marzioni M, Andersen JB, Invernizzi P, Lind GE, Folseraas T, Forbes SJ, Fouassier L, et al: Expert consensus document: Cholangiocarcinoma: Current knowledge and future perspectives consensus statement from the European Network for the Study of Cholangiocarcinoma (ENS-CCA). Nat Rev Gastroenterol Hepatol. 13:261–280. 2016. View Article : Google Scholar : PubMed/NCBI

2 

Kirstein MM and Vogel A: Epidemiology and risk factors of cholangiocarcinoma. Visc Med. 32:395–400. 2016. View Article : Google Scholar

3 

Sripa B and Pairojkul C: Cholangiocarcinoma: Lessons from Thailand. Curr Opin Gastroenterol. 24:349–356. 2008. View Article : Google Scholar : PubMed/NCBI

4 

IARCNA: A Review of Human Carcinogens: Opisthorchis viverrini and Clonorchis sinensis. IARC Monogr Eval Carcinog Risks Hum. 100B:341–370. 2012.

5 

Sripa B, Brindley PJ, Mulvenna J, Laha T, Smout MJ, Mairiang E, Bethony JM and Loukas A: The tumorigenic liver fluke Opisthorchis viverrini - multiple pathways to cancer. Trends Parasitol. 28:395–407. 2012. View Article : Google Scholar : PubMed/NCBI

6 

Prakobwong S, Pinlaor S, Yongvanit P, Sithithaworn P, Pairojkul C and Hiraku Y: Time profiles of the expression of metalloproteinases, tissue inhibitors of metalloproteases, cytokines and collagens in hamsters infected with Opisthorchis viverrini with special reference to peribiliary fibrosis and liver injury. Int J Parasitol. 39:825–835. 2009. View Article : Google Scholar : PubMed/NCBI

7 

Loaharanu P and Sornmani S: Preliminary estimates of economic impact of liver fluke infection in Thailand and the feasibility of irradiation as a control measure. Southeast Asian J Trop Med Public Health. 22(Suppl 22): 384–390. 1991.PubMed/NCBI

8 

Khan SA, Davidson BR, Goldin R, Pereira SP, Rosenberg WM, Taylor-Robinson SD, Thillainayagam AV, Thomas HC, Thursz MR and Wasan H; British Society of Gastroenterology: Guidelines for the diagnosis and treatment of cholangiocarcinoma: Consensus document. Gut. 51(Suppl 6): VI1–VI9. 2002. View Article : Google Scholar : PubMed/NCBI

9 

Anderson CD, Pinson CW, Berlin J and Chari RS: Diagnosis and treatment of cholangiocarcinoma. Oncologist. 9:43–57. 2004. View Article : Google Scholar : PubMed/NCBI

10 

Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, Madhusudan S, Iveson T, Hughes S, Pereira SP, et al ABC-02 Trial Investigators: Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 362:1273–1281. 2010. View Article : Google Scholar : PubMed/NCBI

11 

Surh YJ: Cancer chemoprevention with dietary phytochemicals. Nat Rev Cancer. 3:768–780. 2003. View Article : Google Scholar : PubMed/NCBI

12 

Wang H, Khor TO, Shu L, Su ZY, Fuentes F, Lee JH and Kong AN: Plants vs. cancer: A review on natural phytochemicals in preventing and treating cancers and their druggability. Anticancer Agents Med Chem. 12:1281–1305. 2012. View Article : Google Scholar : PubMed/NCBI

13 

Singh CK, Siddiqui IA, El-Abd S, Mukhtar H and Ahmad N: Combination chemoprevention with grape antioxidants. Mol Nutr Food Res. 60:1406–1415. 2016. View Article : Google Scholar : PubMed/NCBI

14 

Castañeda-Ovando A, Pacheco-Hernández ML, Páez-Hernández ME, Rodríguez JA and Galán-Vidal CA, Pacheco-Hernández MdL, Páez-Hernández ME, Rodríguez JA and Galán-Vidal CA: Chemical studies of anthocyanins: A review. Food Chem. 113:859–871. 2009. View Article : Google Scholar

15 

Cerletti C, De Curtis A, Bracone F, Digesù C, Morganti AG, Iacoviello L, de Gaetano G and Donati MB: Dietary anthocyanins and health: Data from FLORA and ATHENA EU projects. Br J Clin Pharmacol. 83:103–106. 2017. View Article : Google Scholar :

16 

Lin BW, Gong CC, Song HF and Cui YY: Effects of anthocyanins on the prevention and treatment of cancer. Br J Pharmacol. 174:1226–1243. 2017. View Article : Google Scholar

17 

Li D, Wang P, Luo Y, Zhao M and Chen F: Health benefits of anthocyanins and molecular mechanisms: Update from recent decade. Crit Rev Food Sci Nutr. 57:1729–1741. 2017. View Article : Google Scholar

18 

Yousuf B, Gul K, Wani AA and Singh P: Health benefits of anthocyanins and their encapsulation for potential use in food systems: A Review. Crit Rev Food Sci Nutr. 56:2223–2230. 2016. View Article : Google Scholar

19 

Nichenametla SN, Taruscio TG, Barney DL and Exon JH: A review of the effects and mechanisms of polyphenolics in cancer. Crit Rev Food Sci Nutr. 46:161–183. 2006. View Article : Google Scholar : PubMed/NCBI

20 

Umar Lule S and Xia W: Food phenolics, pros and cons: A Review. Food Rev Int. 21:367–388. 2005. View Article : Google Scholar

21 

Wang LS and Stoner GD: Anthocyanins and their role in cancer prevention. Cancer Lett. 269:281–290. 2008. View Article : Google Scholar : PubMed/NCBI

22 

Yang X, Luo E, Liu X, Han B, Yu X and Peng X: Delphinidin-3-glucoside suppresses breast carcinogenesis by inactivating the Akt/HOTAIR signaling pathway. BMC Cancer. 16:4232016. View Article : Google Scholar : PubMed/NCBI

23 

Chen PN, Chu SC, Chiou HL, Kuo WH, Chiang CL and Hsieh YS: Mulberry anthocyanins, cyanidin 3-rutinoside and cyanidin 3-glucoside, exhibited an inhibitory effect on the migration and invasion of a human lung cancer cell line. Cancer Lett. 235:248–259. 2006. View Article : Google Scholar

24 

Kamei H, Kojima T, Hasegawa M, Koide T, Umeda T, Yukawa T and Terabe K: Suppression of tumor cell growth by anthocyanins in vitro. Cancer Invest. 13:590–594. 1995. View Article : Google Scholar : PubMed/NCBI

25 

Eiro MJ and Heinonen M: Anthocyanin color behavior and stability during storage: Effect of intermolecular copigmentation. J Agric Food Chem. 50:7461–7466. 2002. View Article : Google Scholar : PubMed/NCBI

26 

Intuyod K, Priprem A, Limphirat W, Charoensuk L, Pinlaor P, Pairojkul C, Lertrat K and Pinlaor S: Anti-inflammatory and anti-periductal fibrosis effects of an anthocyanin complex in Opisthorchis viverrini-infected hamsters. Food Chem Toxicol. 74:206–215. 2014. View Article : Google Scholar : PubMed/NCBI

27 

Tepsiri N, Chaturat L, Sripa B, Namwat W, Wongkham S, Bhudhisawasdi V and Tassaneeyakul W: Drug sensitivity and drug resistance profiles of human intrahepatic cholangiocarcinoma cell lines. World J Gastroenterol. 11:2748–2753. 2005. View Article : Google Scholar : PubMed/NCBI

28 

Wattanawongdon W, Hahnvajanawong C, Namwat N, Kanchanawat S, Boonmars T, Jearanaikoon P, Leelayuwat C, Techasen A and Seubwai W: Establishment and characterization of gemcitabine-resistant human cholangiocarcinoma cell lines with multidrug resistance and enhanced invasiveness. Int J Oncol. 47:398–410. 2015. View Article : Google Scholar : PubMed/NCBI

29 

Kuželová K, Grebeňová D and Brodská B: Dose-dependent effects of the caspase inhibitor Q-VD-OPh on different apoptosis-related processes. J Cell Biochem. 112:3334–3342. 2011. View Article : Google Scholar

30 

Franken NA, Rodermond HM, Stap J, Haveman J and van Bree C: Clonogenic assay of cells in vitro. Nat Protoc. 1:2315–2319. 2006. View Article : Google Scholar

31 

Puck TT and Marcus PI: Action of x-rays on mammalian cells. J Exp Med. 103:653–666. 1956. View Article : Google Scholar : PubMed/NCBI

32 

Cai J and Jones DP: Superoxide in apoptosis. Mitochondrial generation triggered by cytochrome c loss. J Biol Chem. 273:11401–11404. 1998. View Article : Google Scholar : PubMed/NCBI

33 

Piva R, Belardo G and Santoro MG: NF-kappaB: A stress-regulated switch for cell survival. Antioxid Redox Signal. 8:478–486. 2006. View Article : Google Scholar : PubMed/NCBI

34 

Myatt SS and Lam EW: The emerging roles of forkhead box (Fox) proteins in cancer. Nat Rev Cancer. 7:847–859. 2007. View Article : Google Scholar : PubMed/NCBI

35 

Cao SS and Kaufman RJ: Endoplasmic reticulum stress and oxidative stress in cell fate decision and human disease. Antioxid Redox Signal. 21:396–413. 2014. View Article : Google Scholar : PubMed/NCBI

36 

Holohan C, Van Schaeybroeck S, Longley DB and Johnston PG: Cancer drug resistance: An evolving paradigm. Nat Rev Cancer. 13:714–726. 2013. View Article : Google Scholar : PubMed/NCBI

37 

Longo L, Platini F, Scardino A, Alabiso O, Vasapollo G and Tessitore L: Autophagy inhibition enhances anthocyanin-induced apoptosis in hepatocellular carcinoma. Mol Cancer Ther. 7:2476–2485. 2008. View Article : Google Scholar : PubMed/NCBI

38 

Neto CC, Amoroso JW and Liberty AM: Anticancer activities of cranberry phytochemicals: An update. Mol Nutr Food Res. 52(Suppl 1): S18–S27. 2008.PubMed/NCBI

39 

Rugină D, Hanganu D, Diaconeasa Z, Tăbăran F, Coman C, Leopold L, Bunea A and Pintea A: Antiproliferative and apoptotic potential of cyanidin-based anthocyanins on melanoma cells. Int J Mol Sci. 18:949–959. 2017. View Article : Google Scholar

40 

Jinawath N, Chamgramol Y, Furukawa Y, Obama K, Tsunoda T, Sripa B, Pairojkul C and Nakamura Y: Comparison of gene expression profiles between Opisthorchis viverrini and non-Opisthorchis viverrini associated human intrahepatic cholangiocarcinoma. Hepatology. 44:1025–1038. 2006. View Article : Google Scholar : PubMed/NCBI

41 

Zhang JR, Lu F, Lu T, Dong WH, Li P, Liu N, Ma DX and Ji CY: Inactivation of FoxM1 transcription factor contributes to curcumin-induced inhibition of survival, angiogenesis, and chemosensitivity in acute myeloid leukemia cells. J Mol Med (Berl). 92:1319–1330. 2014. View Article : Google Scholar

42 

Karin M: Nuclear factor-kappaB in cancer development and progression. Nature. 441:431–436. 2006. View Article : Google Scholar : PubMed/NCBI

43 

Hoesel B and Schmid JA: The complexity of NF-κB signaling in inflammation and cancer. Mol Cancer. 12:862013. View Article : Google Scholar

44 

Baldwin AS Jr: The NF-kappa B and I kappa B proteins: New discoveries and insights. Annu Rev Immunol. 14:649–683. 1996. View Article : Google Scholar : PubMed/NCBI

45 

Seubwai W, Wongkham C, Puapairoj A, Khuntikeo N, Pugkhem A, Hahnvajanawong C, Chaiyagool J, Umezawa K, Okada S and Wongkham S: Aberrant expression of NF-κB in liver fluke associated cholangiocarcinoma: Implications for targeted therapy. PLoS One. 9:e1060562014. View Article : Google Scholar

46 

Hafeez BB, Siddiqui IA, Asim M, Malik A, Afaq F, Adhami VM, Saleem M, Din M and Mukhtar H: A dietary anthocyanidin delphinidin induces apoptosis of human prostate cancer PC3 cells in vitro and in vivo: Involvement of nuclear factor-kappaB signaling. Cancer Res. 68:8564–8572. 2008. View Article : Google Scholar : PubMed/NCBI

47 

Hecht SS, Huang C, Stoner GD, Li J, Kenney PM, Sturla SJ and Carmella SG: Identification of cyanidin glycosides as constituents of freeze-dried black raspberries which inhibit anti-benzo[a]pyrene-7,8-diol-9,10-epoxide induced NFkappaB and AP-1 activity. Carcinogenesis. 27:1617–1626. 2006. View Article : Google Scholar : PubMed/NCBI

48 

Kim JH, Gupta SC, Park B, Yadav VR and Aggarwal BB: Turmeric (Curcuma longa) inhibits inflammatory nuclear factor (NF)-κB and NF-κB-regulated gene products and induces death receptors leading to suppressed proliferation, induced chemo-sensitization, and suppressed osteoclastogenesis. Mol Nutr Food Res. 56:454–465. 2012. View Article : Google Scholar

49 

Prakobwong S, Gupta SC, Kim JH, Sung B, Pinlaor P, Hiraku Y, Wongkham S, Sripa B, Pinlaor S and Aggarwal BB: Curcumin suppresses proliferation and induces apoptosis in human biliary cancer cells through modulation of multiple cell signaling pathways. Carcinogenesis. 32:1372–1380. 2011. View Article : Google Scholar : PubMed/NCBI

50 

Rahman MM, Ichiyanagi T, Komiyama T, Hatano Y and Konishi T: Superoxide radical- and peroxynitrite-scavenging activity of anthocyanins; structure-activity relationship and their synergism. Free Radic Res. 40:993–1002. 2006. View Article : Google Scholar : PubMed/NCBI

51 

Kausar H, Jeyabalan J, Aqil F, Chabba D, Sidana J, Singh IP and Gupta RC: Berry anthocyanidins synergistically suppress growth and invasive potential of human non-small-cell lung cancer cells. Cancer Lett. 325:54–62. 2012. View Article : Google Scholar : PubMed/NCBI

52 

Casanelles E, Gozzelino R, Marqués-Fernández F, Iglesias-Guimarais V, Garcia-Belinchón M, Sánchez-Osuna M, Solé C, Moubarak RS, Comella JX and Yuste VJ: NF-κB activation fails to protect cells to TNFα-induced apoptosis in the absence of Bcl-xL, but not Mcl-1, Bcl-2 or Bcl-w. Biochim Biophys Acta. 1833:1085–1095. 2013. View Article : Google Scholar : PubMed/NCBI

53 

Li N, Ragheb K, Lawler G, Sturgis J, Rajwa B, Melendez JA and Robinson JP: DPI induces mitochondrial superoxide-mediated apoptosis. Free Radic Biol Med. 34:465–477. 2003. View Article : Google Scholar : PubMed/NCBI

54 

Kondo K, Obitsu S, Ohta S, Matsunami K, Otsuka H and Teshima R: Poly(ADP-ribose) polymerase (PARP)-1-independent apoptosis-inducing factor (AIF) release and cell death are induced by eleostearic acid and blocked by alpha-tocopherol and MEK inhibition. J Biol Chem. 285:13079–13091. 2010. View Article : Google Scholar : PubMed/NCBI

55 

Fernald K and Kurokawa M: Evading apoptosis in cancer. Trends Cell Biol. 23:620–633. 2013. View Article : Google Scholar : PubMed/NCBI

56 

Mathiasen IS and Jäättelä M: Triggering caspase-independent cell death to combat cancer. Trends Mol Med. 8:212–220. 2002. View Article : Google Scholar : PubMed/NCBI

57 

Herceg Z and Wang ZQ: Functions of poly(ADP-ribose) polymerase (PARP) in DNA repair, genomic integrity and cell death. Mutat Res. 477:97–110. 2001. View Article : Google Scholar : PubMed/NCBI

58 

Schieber M and Chandel NS: ROS function in redox signaling and oxidative stress. Curr Biol. 24:R453–R462. 2014. View Article : Google Scholar : PubMed/NCBI

59 

Dizdaroglu M, Jaruga P, Birincioglu M and Rodriguez H: Free radical-induced damage to DNA: Mechanisms and measurement. Free Radic Biol Med. 32:1102–1115. 2002. View Article : Google Scholar : PubMed/NCBI

60 

Borges HL, Linden R and Wang JY: DNA damage-induced cell death: Lessons from the central nervous system. Cell Res. 18:17–26. 2008. View Article : Google Scholar

61 

Murphy MP: How mitochondria produce reactive oxygen species. Biochem J. 417:1–13. 2009. View Article : Google Scholar

62 

Higa A and Chevet E: Redox signaling loops in the unfolded protein response. Cell Signal. 24:1548–1555. 2012. View Article : Google Scholar : PubMed/NCBI

63 

Schröder M and Kaufman RJ: ER stress and the unfolded protein response. Mutat Res. 569:29–63. 2005. View Article : Google Scholar

64 

Xu C, Bailly-Maitre B and Reed JC: Endoplasmic reticulum stress: Cell life and death decisions. J Clin Invest. 115:2656–2664. 2005. View Article : Google Scholar : PubMed/NCBI

65 

Ron D: Translational control in the endoplasmic reticulum stress response. J Clin Invest. 110:1383–1388. 2002. View Article : Google Scholar : PubMed/NCBI

66 

Shi Y, Vattem KM, Sood R, An J, Liang J, Stramm L and Wek RC: Identification and characterization of pancreatic eukaryotic initiation factor 2 alpha-subunit kinase, PEK, involved in translational control. Mol Cell Biol. 18:7499–7509. 1998. View Article : Google Scholar : PubMed/NCBI

67 

Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M and Ron D: Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol Cell. 6:1099–1108. 2000. View Article : Google Scholar : PubMed/NCBI

68 

Scheuner D, Song B, McEwen E, Liu C, Laybutt R, Gillespie P, Saunders T, Bonner-Weir S and Kaufman RJ: Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol Cell. 7:1165–1176. 2001. View Article : Google Scholar : PubMed/NCBI

69 

Harding HP, Zhang Y, Bertolotti A, Zeng H and Ron D: Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol Cell. 5:897–904. 2000. View Article : Google Scholar : PubMed/NCBI

70 

Rajesh K, Krishnamoorthy J, Kazimierczak U, Tenkerian C, Papadakis AI, Wang S, Huang S and Koromilas AE: Phosphorylation of the translation initiation factor eIF2α at serine 51 determines the cell fate decisions of Akt in response to oxidative stress. Cell Death Dis. 6:e15912015. View Article : Google Scholar

71 

Thummayot S, Tocharus C, Suksamrarn A and Tocharus J: Neuroprotective effects of cyanidin against Aβ-induced oxidative and ER stress in SK-N-SH cells. Neurochem Int. 101:15–21. 2016. View Article : Google Scholar : PubMed/NCBI

72 

Garg AK, Buchholz TA and Aggarwal BB: Chemosensitization and radiosensitization of tumors by plant polyphenols. Antioxid Redox Signal. 7:1630–1647. 2005. View Article : Google Scholar : PubMed/NCBI

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Volume 52 Issue 5

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Spandidos Publications style
Intuyod K, Priprem A, Pairojkul C, Hahnvajanawong C, Vaeteewoottacharn K, Pinlaor P and Pinlaor S: Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line. Int J Oncol 52: 1715-1726, 2018
APA
Intuyod, K., Priprem, A., Pairojkul, C., Hahnvajanawong, C., Vaeteewoottacharn, K., Pinlaor, P., & Pinlaor, S. (2018). Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line. International Journal of Oncology, 52, 1715-1726. https://doi.org/10.3892/ijo.2018.4306
MLA
Intuyod, K., Priprem, A., Pairojkul, C., Hahnvajanawong, C., Vaeteewoottacharn, K., Pinlaor, P., Pinlaor, S."Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line". International Journal of Oncology 52.5 (2018): 1715-1726.
Chicago
Intuyod, K., Priprem, A., Pairojkul, C., Hahnvajanawong, C., Vaeteewoottacharn, K., Pinlaor, P., Pinlaor, S."Anthocyanin complex exerts anti-cholangiocarcinoma activities and improves the efficacy of drug treatment in a gemcitabine-resistant cell line". International Journal of Oncology 52, no. 5 (2018): 1715-1726. https://doi.org/10.3892/ijo.2018.4306