Introduction
Cholangiocarcinoma (CCA), a type of digestive cancer derived from the biliary tract epithelium, is a devastating malignancy with a poor prognosis (1–5). CCA accounts for ~3% of malignant tumors of the gastrointestinal system and ranks second in frequency among primary liver tumors (6). Worldwide, CCA is a rare type of cancer, but the incidence and mortality rate of this disease are increasing, with notable increases reported in the US, the UK and Asia, including China (7). Approximately 90% of CCAs are adenocarcinomas and are classified as intrahepatic, perihilar or distal CCAs (7). Early diagnosis of CCA is difficult because there are no specific symptoms during the early stages of tumor development. Consequently, most CCA patients present with advanced incurable disease, and few cases are eligible for surgery in the clinic (8,9). Even for patients who have undergone complete surgical resection, recurrence is common, and the 5-year survival rate of CCA is <5% (10,11). Chemotherapy is the most common treatment for CCA patients, although this treatment has not been shown to substantially improve survival in patients with inoperable CCA. Many chemotherapeutic drugs include targeted chemotherapeutic agents that have been tested as single agents or in combination with other drugs. However, drug inefficacy and drug resistance remain major obstacles in the treatment of CCA (12,13). Thus, novel treatment strategies directed against this malignancy are urgently needed to improve patient survival.
A varied array of phytochemicals, which are obtained from vegetables, fruits, nuts and spices, has demonstrated the ability to selectively inhibit the growth of tumor cells (14–17). Virgin olive oil (VOO) is one of the traditional local foods in Mediterranean countries. VOO is an important part of the ‘Mediterranean diet’ and is considered to be responsible for the health benefits associated with this diet. In particular, individuals who consume VOO present a lower incidence of several types of cancers, and the general health benefits of olive oil have been demonstrated in recent years, particularly in the prevention of cardiovascular diseases and cancers (18–20). The polyphenols in extra VOO have been suggested to exert an anticancer effect on several human cancer cell lines by inhibiting different stages of disease (initiation, promotion or metastasis) or by inducing apoptosis (18,21). It is now generally recognized that the minor components of olive oil, including phenols, have biological relevance. Hydroxytyrosol (HT) is one of the major polyphenol compounds in olive oil (21) and has received much attention due to its anticancer ability, preferable biocompatibility and few side-effects. The cellular mechanisms by which olive oil polyphenols exert these anticancer effects remain poorly understood. Although many studies have shown that HT can inhibit the proliferation of certain types of human cancer cells, induce cell cycle arrest and promote apoptosis in a wide variety of human cancer cell lines (22–26), the feasibility of using this phytochemical in CCA treatment has not yet been evaluated. Therefore, the objective of the present study was to investigate the in vivo and in vitro anticancer potential of HT and delineate underlying changes in certain signaling pathways that play an important role in the growth and apoptosis of CCA.
Our results revealed that HT acts as a potent antitumor agent. Our experiments indicated that HT could potently inhibit proliferation of CCA and human gallbladder cancer cell lines and induce apoptosis both in vivo and in vitro. We also observed significant changes in signaling pathways, such as the inhibition of ERK1/2, upregulation of cleaved caspase-3 and cleaved caspase-9 and alteration of Bax/Bcl-2 and cyclin B1 expression, which most likely play an important role in the effect of HT on CCA and human gallbladder cancer cell lines. More importantly, our results demonstrated the therapeutic potential of HT in CCA, and we identified several molecular mechanisms that may be important in the activity of HT, which will constitute the focus of our future research.
Materials and methods
Cell lines, reagents and antibodies
The human CCA cell line TFK-1 (27) was kindly provided by Tohoku University. The human CCA cell line KMBC, the human gallbladder cancer cell line GBS-SD, and the human bile duct cell line HIBEpiC were obtained from Shanghai Bioleaf Biotech Co., Ltd. (Shanghai, China). TFK-1 cells were cultured in RPMI-1640 medium (Gibco-BRL), and the human CCA cell line KMBC and the human gallbladder cancer cell line GBS-SD were cultured in DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin in 95% air and 5% CO2 at 37ºC. HT (H4291) was purchased from Sigma-Aldrich. Primary antibodies against Bcl-2, Bax, caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, cyclin B1, actin, PARP, cleaved PARP, p44/42 MAPK (total (T)-ERK1/2), phospho-p44/42 (P-ERK1/2), p-cdc2 (Tyr15), p-cdc2 (Tyr161) and Ki-67 were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). TUNEL reagent was purchased from Roche (Beijing, China).
Cell proliferation assay
Once in the exponential growth phase, these 4 cell lines were seeded (5–10×103 cells) in 96-well microtiter plates and cultured for 24 h. After confirmation of cell adherence, HT was added to each well at a final concentration of 75 or 150 μM, and the cells were cultured for an additional 24–72 h. Cell proliferation was measured with the Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer’s protocol. To examine cell proliferative activity, proliferation was expressed as a percentage of the absorbance of treated wells relative to the absorbance of untreated (control) wells, as determined using a Model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). Three independent experiments were performed.
Cell cycle analysis
KMBC, TFK-1 and GBC-SD cells were plated at a density of 5×105/well on 6-well plates. After treatment with HT at varying concentrations, the cells were treated with trypsin, detached from the plate and centrifuged at 1,000 rpm for 5 min at room temperature. After aspiration of the medium, the cells were resuspended in cold PBS. The cells were then fixed in 70% ethanol for 1 h at 4ºC. The cells were washed twice with PBS, and 10 mg/ml RNase A was added. Propidium iodide (PI) was then added to the tubes at a final concentration of 0.05 mg/ml, and the samples were incubated at 4ºC for 30 min in the dark. Cell cycle analysis was performed with a Becton-Dickinson FACScan using an FL2 detector with a bandpass filter at specifications of 585 F 21 nm. In each analysis, 1×104 events were recorded. The results were analyzed using ModFit LT software (Verity Software House, Topsham, ME, USA).
Cell apoptosis analysis
The CCA cell lines TFK-1 and KMBC and the human gallbladder cancer cell line GBS-SD (3×105 cells/well) were cultured with HT for 48 h. Next, 1×106 cells were collected and washed twice with ice-cold PBS, resuspended in binding buffer (100 μl) (BD Biosciences, San Jose, CA, USA), treated with Annexin V and PI (BD Biosciences) and incubated in the dark for 15 min. Another 300 μl of binding buffer was then added, and flow cytometry analysis was performed within 1 h to measure apoptosis (BD FACSCalibur).
DAPI nuclear staining
To examine the apoptotic changes in CCA cells, a DAPI (4′,6′-diamidino-2-phenylindole) nuclear staining assay was performed. To form monolayer cultures, the cells (5×105/well) were plated on 6-well plates. At 80–90% confluence, the cells were treated with HT at a concentration (75 or 150 μM) calculated according to the IC50 values for 24 h. After completion of the treatment, the cells were fixed in methanol for 30 min at 4ºC in the dark. The fixed cells were washed twice with PBS, and the DAPI solution was then spread over the plates, followed by incubation for 1 h at 4ºC in the dark. The labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and then evaluated under a fluorescence microscope (Leica Microsystems, Wetzlar, Germany).
Western blotting
KMBC, TFK-1 and GBC-SD cells were plated at a density of 5×105/well on 6-well plates. After incubation with HT (75 or 150 μM) the cells were rinsed twice with ice-cold PBS and treated with 100 μl sample buffer (SDS β-ME) on ice for 30 min. The whole-cell lysate was centrifuged at 12,000 rpm for 10 min at 4ºC. Protein lysates (20 μl) were electrophoresed on a 12% SDS gel.
The proteins were transferred to a PVDF membrane (Bio-Rad Laboratories) and the membrane was blocked for 1 h with blocking solution (5% non-fat dry milk in TBS-0.5% Tween-20). The membrane was then reacted overnight at 4ºC with primary antibodies diluted 1:1,000. Primary antibodies against the following proteins were used: ERK1/2, phospho-ERK1/2, cleaved caspase-3, caspase-3, cleaved caspase-9, caspase-9, cleaved PARP, PARP, cyclin B1, p-cdc2 (Tyr15), p-cdc2 (Tyr161) and actin. A secondary antibody was then added to the membrane and reacted with the primary antibody. After rinsing, the membrane was treated with a chemiluminescence (ECL) solution for 5 min. Specific bands were detected on X-ray film following appropriate exposure in the dark room or using VersaDoc 5000 MP (Bio-Rad) for image processing. We evaluated the resulting bands by densitometric measurement with ImageJ (NIH, Bethesda, MD, USA).
Tumor xenograft experiments
All operative procedures and care were approved by the Institutional Ethics Committee at Harbin Medical University. All experiments were performed in accordance with the guidelines of the Committee on the Use of Live Animals in Teaching and Research of Harbin Medical University, Harbin, China. Male nude BALB/c mice between 6 and 8 weeks of age were obtained from Vital River Laboratories (VRL; Beijing, China). Tumors were established by the subcutaneous injection of 5×106 TFK-1 cells into the flanks of the mice. When the tumor volume reached ~120 mm3 in the treatment group, HT was dissolved in PBS and administered via intraperitoneal injection (500 mg/kg/day) every day for 3 weeks. The same volume of PBS was injected in the control group. Body weight was recorded starting from the first day of treatment, and tumor volumes were also calculated at the same time points using the following equation: Tumor volume = length × (width)2 × π/6. The mice in the treatment and control groups were sacrificed when paraffin-embedded tumor tissue blocks had been obtained.
Ki-67 immunohistochemistry
Formalin-fixed, paraffin-embedded sections (5 μm) were rinsed with PBS, blocked with 3% bovine serum albumin for 2 h and then stained with an anti-Ki-67 antibody. The sections were subsequently incubated for 30 min with the appropriate secondary antibody, and immunoreactivity was developed with SigmaFAST DAB. The sections were counterstained with hematoxylin, mounted and examined by microscopy. The results are presented as the percentage of Ki-67-positive cells in 10 random visual fields at ×400 magnification under a microscope. The values were subjected to one-way ANOVA and were later compared between groups using an unpaired Student’s t-test.
In situ detection of apoptotic cells (TUNEL)
Apoptotic cells were detected using TUNEL reagent according to the vendor’s protocol. Apoptosis was evaluated by counting the number of TUNEL-positive cells (brown-stained) and the total number of cells in 5 randomly selected fields in each sample at ×400 magnification.
Statistical analysis
All experiments were performed in triplicate. All experiments were repeated 3 times and expressed graphically as the mean ± SD. A Student’s t-test was used for statistical analysis, and P<0.05 was considered to indicate a statistically significant result. GraphPad (v5.0) software was used for analysis.
Results
HT inhibits the proliferation of the CCA cell lines
We initially assessed the proliferation of the human CCA cell lines TFK-1 and KMBC, the human gallbladder cancer cell line GBS-SD, and the human bile duct cell line HIBEpiC following HT treatment (25–200 μM) using a cell proliferation assay. The results showed that HT significantly inhibited the proliferation of all CCA and gallbladder cancer cell lines within a period of 24–48 h (P<0.05), and this inhibition persisted until 72 h post-treatment (Fig. 1A). These effects were more obvious at a dose of nearly 200 μM HT, although this dose had no significant effect on the proliferation of the human bile duct cell line HIBEpiC (Fig. 1A). In summary, significant growth inhibitory activity of HT was observed in viable CCA and gallbladder cancer cells and was dependent on the concentration and exposure time of the drug, whereas no marked proliferative inhibition was noted in the human bile duct cell line HIBEpiC (P<0.05). These results were evaluated by absorbance measurements.
It is well known that the activation of ERK may be linked to the proliferation of CCA cells. In the present study, western blot analyses of the KMBC, TFK-1 and GBC-SD cell lines indicated there was a time- and dose-dependent inhibition of ERK phosphorylation at all concentrations of HT (75 and 150 μM), although there were few changes in T-ERK, as compared to the control (Fig. 1B).
HT induces G2/M cell cycle arrest in CCA cell lines
Cell cycle analysis was performed to determine the stage at which HT arrests CCA and gallbladder cancer cell division. HT-treated (75 or 150 μM) TFK-1, KMBC and GBC-SD cells were fixed, and the cell cycle distribution was determined by flow cytometry. In the cell line KMBC, compared to the control, a 72-h exposure to 75 μM HT increased the G2/M fraction from 6.65 to 16.02% (P<0.05), and this change was more marked following treatment with 150 μM HT (45.32%) (Fig. 2A and B). Similar results were observed for TFK-1 and GBC-SD cells (Fig. 2A and B). Western blotting for G2/M cell cycle regulatory molecules demonstrated that cyclin B1 and Thr15 phosphorylation of cdc2 showed a time-dependent decrease with increasing doses of HT (75 and 150 μM), and Thr161 phosphorylation of cdc2 significantly increased following HT treatment (Fig. 2C). These results suggest that the inhibitory effect of HT on CCA cell proliferation is associated with the induction of G2/M phase arrest.
HT induces cell apoptosis in CCA cell lines
In the CCK-8 assay, we observed a marked inhibition of CCA and gallbladder cancer cell proliferation. Thus, we examined the apoptotic effect induced by HT in these cell lines using an Annexin V/PI assay, as described in Materials and methods. In KMBC, TFK-1 and GBC-SD cells, compared to the control, HT treatment resulted in concentration-dependent apoptosis, including both early and late apoptotic cell death. As shown in Fig. 3A, this result demonstrated that HT induced the apoptosis of KMBC cells in a dose-dependent manner. When the concentration of HT reached 75 μM, the apoptosis rate of KMBC cells was significantly higher than that of untreated cells (P<0.05). Furthermore, 150 μM HT resulted in a highly significant difference in the rate of apoptosis between the HT-treated (75 μM) and untreated cells (P<0.05). Similarly, HT induced the apoptosis of TFK-1 and GBC-SD cells in a dose-dependent manner (Fig. 3A). The corresponding histogram from the flow cytometric analysis showed that the apoptosis rates of KMBC cells were 0.94, 35.3 and 80% when these cells were treated with HT at concentrations of 0, 75 and 150 μM, respectively (Fig. 3B). Following treatment with these same concentrations of HT, the apoptosis rates of TFK-1 cells were 4.52, 9.01 and 78.69%, respectively (Fig. 3B), and the rates of GBC-SD cells were 0.66, 9.8 and 28.51%, respectively (Fig. 3B).
Morphological changes in these cell lines were observed by DAPI staining. In the TFK-1 cells, HT treatment induced marked apoptosis-related morphological alterations, including cell shrinkage and granular apoptotic body formation (Fig. 3C and D) (P<0.05), and similar results were obtained for KMBC and GBC-SD cells (data not shown).
To further evaluate whether HT inhibits cell viability by inducing apoptosis, we collected cells after exposure to HT (75 or 150 μM) and examined changes in the levels of intracellular signaling proteins by western blotting. The expression of caspase-3, caspase-9 and PARP is a hallmark of cells undergoing apoptosis. Our results showed that the levels of cleaved PARP, Bax, cleaved caspase-3 and cleaved caspase-9 increased, whereas the levels of PARP and Bcl-2 were decreased, compared to the levels detected in the non-HT-treated controls (Fig. 3E). Moreover, the general caspase inhibitor z-VAD-fmk partially blocked the HT-induced cell death of CCA cells (Fig. 3F and G) (P<0.05).
HT inhibits in vivo tumor growth and induces apoptosis
To explore the role of HT in tumor proliferation in vivo, we examined the ability of HT to suppress the growth of TFK-1 xenografts in nude mice. TFK-1 cell-derived xenograft tumors were allowed to develop and grow to a size of 0.5 cm3, at which time HT (500 mg/kg/day) was administered by intraperitoneal injection every day for 3 weeks. In the xenograft model, the mice were sacrificed following 3 weeks of HT treatment and their tumors were excised. HT-treated animals demonstrated a significantly reduced tumor size and weight as compared to these values in the controls (Fig. 4A and B) (P<0.05). In summary, the tumors in the control group grew continuously during the experimental period, whereas the tumor growth in HT-treated mice was markedly inhibited (Fig. 4A and B) (P<0.05). However, there was no apparent change in liver weight, spleen weight (Fig. 4D), or body weight (Fig. 4C), indicating that HT is a potential therapeutic agent for CCA cancers and is relatively non-toxic to mice. Ki-67 staining for cell proliferation was also performed in these xenografts, and the relative number of Ki-67-positive tumor cells was lower in tumors from mice treated with HT when compared to this number in the controls (Fig. 4E) (P<0.05). Regarding apoptosis, as shown in the representative images, xenografts from the HT-treated groups showed a marked increase (P<0.05) in TUNEL-positive cells when compared to the controls (Fig. 4E); moreover, quantification of the TUNEL-stained samples showed 2- to 3-fold increases (P<0.05) in the number of TUNEL-positive cells in the HT-treated groups as compared to the controls (Fig. 4E). Thus, the Ki-67- and TUNEL-based quantifications from HT-treated CCA xenografts indicated the presence of fewer Ki-67-positive cells and greater numbers of apoptotic cells (Fig. 4E). Western blot analysis of subcutaneous tumors excised at 3 weeks following HT treatment indicated decreased protein expression levels for caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, cyclin B1 and p-cdc2 (Tyr15) and a decreased Bcl-2/Bax ratio as compared to the controls (Fig. 4F). These results suggest that HT significantly inhibits CCA and human gallbladder cancer cell growth and induces apoptosis in vivo.
Discussion
CCAs are a heterogeneous group of tumors that arise from cholangiocytes and are associated with poor patient prognosis. Unfortunately, in controlled clinical trials, no treatment with repeatable benefits has been identified, and treatment outcome remains poor for various reasons, including a high rate of tumor recurrence, drug resistance and toxicity, leading to poor 5-year survival rates (1,2). Thus, we sought to investigate a novel therapy for this disease. In the present study, we found that HT possesses great potential as a promising anti-CCA therapeutic agent. We investigated the inhibitory effect of HT on CCA cell lines and transplanted tumors in mice, and we identified several meaningful changes in the levels of proteins involved in survival and apoptosis of CCA in vivo and in vitro.
In the present study, we observed that HT induced cell apoptosis via the mitochondrial cell death pathway, an important apoptosis pathway. Bcl-2, Bax, caspase-9, caspase-3 and PARP are crucial signaling proteins in this pathway, and Bcl-2 and Bax play an important role in the mitochondrial outer membrane to mediate cell death by apoptosis. Bcl-2 demonstrates anti-apoptotic activity, whereas Bax has proapoptotic activity (28). When cytochrome c is released into the cytosol, it activates caspase-9, together with Apaf-1. Caspase-9 then activates caspase-3, which leads to the cleavage or degradation of several important factors downstream, including PARP (29). In the present study, HT-treated CCA cells expressed significantly lower levels of Bcl-2 yet increased levels of Bax in comparison to the control cells (30). The activation of cleaved caspase-9 and cleaved caspase-3 is followed by the cleavage of PARP, which is an important marker in the process of apoptosis (31). However, even pretreatment of cells with 50 mM z-VAD-fmk could not completely block HT-induced CCA cell death, which suggests that there are multiple mechanisms, involving both caspase-dependent and caspase-independent pathways, underlying the proapoptotic effect of HT on CCA cell lines. Based on DAPI nuclear staining, we also observed a marked morphological manifestation of apoptosis in CCA cells.
ERK is known to be involved in the promotion of cellular proliferation and is generally upregulated in many types of human cancer cells (32–35). In the context of human CCA tumor formation, it is well documented that ERK1/2 is constitutively activated in CCA cell lines, suggesting that activation of the ERK pathway may be linked to the proliferation of CCA cells and their progression into the state of uncontrolled growth associated with CCA formation. Moreover, certain phenolic compounds, including kaempferol (36), apigenin (37) and luteolin (38), have been reported to modulate ERK1/2. Thus, we investigated whether the effect of HT on CCA cells is associated with ERK inhibition, and we found that this effect was associated with ERK inhibition in an HT dose- and time-dependent manner. Western blot analyses of the TFK-1, KMBC and GBC-SD cell lines indicated significantly decreased ERK phosphorylation following HT (75 or 150 μM) treatment. Therefore, we believe that HT may regulate cell growth via the ERK pathway in CCAs, highlighting a potential mechanism for HT activity, which may be used as a therapeutic agent for the treatment of CCA progression. However, the precise mechanism underlying this effect of HT remains unknown. Therefore, our next phase of research will examine the effect of HT-activated ERK on the inhibition of CCA cells.
Han et al(25) showed that HT stimulated G2/M phase cell cycle arrest in human breast cancer MCF-7 cells. Moreover, Corona et al(26) reported that the inhibitory effect of HT on human colon adenocarcinoma cell proliferation was associated with G2/M phase cell cycle arrest and increased G2/M checkpoint protein levels. The results of the present study demonstrated that 75 μM HT induced G2/M phase cell cycle arrest in all 3 selected CCA cell lines. In addition, our assessment of the cell cycle-related protein levels revealed that following treatment with 75 μM HT, the cyclin B1 protein level was markedly decreased, along with a decrease in Tyr15 phosphorylation and an increase in Thr161 phosphorylation, both of which are a prerequisite for activation of cdc2 kinase at the G2/M phase.
In the present study, we observed marked suppression of tumor growth in the mouse xenografts following HT treatment, as a significant reduction in the relative tumor volume was noted in the HT-treated animals as compared to the untreated controls. In addition, a clear suppression of proliferation was observed based on the results of the TUNEL assay, and immunostaining for Ki-67 showed that there was an increased number of apoptotic cells in the HT-treated animals. However, further studies are needed to confirm and extend these finding before HT can be developed as an effective therapy for CCAs.
In summary, the results of our experiments revealed that HT potently inhibited CCA and human gallbladder cancer cell proliferation and induced apoptosis in vivo and in vitro. Moreover, we found that there were clear changes in the levels of proteins involved in survival and apoptosis in CCA cells, particularly those involved in the ERK1/2 signaling pathway. In addition, G2/M arrest of CCA cells was also observed. More importantly, to the best of our knowledge, the present study was the first to observe the anticancer effect of HT in CCA xenografts, as our in vivo experiments revealed that tumor growth was significantly suppressed after HT treatment. The precise molecular mechanisms for the HT-mediated inhibition of CCA cell growth and promotion of apoptosis remain poorly understood, and this will constitute the aim of our next phase of research. The present results provide experimental data supporting the clinical use of HT for the treatment of CCAs, and HT may, therefore, offer a novel therapeutic strategy for CCA.