5-fluorouracil (5-FU), vincristine sulfate (VCR), cisplatin (CDDP), mitomycin C (MMC), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), caspase-3 inhibitor (z-VAD-fmk), G418, BMS453 and CoCl2 were all purchased from Sigma-Aldrich (St. Louis, MO, USA). The RPMI-1640 and fetal bovine serum (FBS) were all purchased from Gibco BRL (Grand Island, NY). The 100 U/ml penicillin and bicinchoninic acid protein assay reagent were purchased from Wuhan Boster (Wuhan, China). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ab9485) and RARβ (ab198557) antibodies were purchased from Abcam (Cambridge, UK). The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining apoptosis detection kit and TdT-mediated dUTP nick end labeling (TUNEL) kit were obtained from Roche Diagnostics (Basel, Switzerland). Lipofectamine 2000 was purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA., USA). The Caspase-3 assay kit was from R&D Systems, Inc. (Minneapolis, MN, USA). All of other chemicals and reagents were the highest quality and purchased from standard commercial sources.

Analysis of RARβ expression in clinical CCA tissues was performed in agreement with the ethical guidelines of the 1975 Declaration of Helsinki, and was approved by the Institute Research Ethics Committee at The First Affiliated Hospital of Xiamen University. Between 2009–2013, 33 CCA samples were collected from patients without metastatic disease that had not received pre-operative treatment. Tumor tissues were fixed in 10% formalin and then paraffin-embedded for immunohistochemical (IHC) analysis and routine histological characterization.

The human CCA cell line QBC939 was a kind gift from Professor Shu-Guang Wang from Southwest Hospital, the Third Military Medical University (Chongqing, China). The CCA cell lines Sk-ChA-1, MZ-ChA-1 and Hccc9810 were a kind gift from Professor Chun-Dong Yu laboratory of Xiamen University (Xiamen, China). HIBEpiC human intrahepatic biliary epithelial cells were purchased from Cell Bank of the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All four human CCA cell lines and HIBEpiC cells were all cultured in RPMI-1640 supplemented with 10% FBS and 100 U/ml penicillin in a humidified chamber at 37°C in 5% CO₂. RARβ cDNA was cloned into the expression vector pBoBi as described previously (15). The RARβ expression vector (4 μg) was stably transfected into QBC939 cells (1×10⁶) to produce pBoBi-RARβ using 10 μl Lipofectamine 2000, according to the manufacturer's instructions. Positive selection of stable transfectants was achieved by supplementing complete medium with 400 mg/ml of G418. Stable control vector QBC939 transfectants are defined as pBoBi-Ctrl from herein.

To determine cell viability in response to treatment, an MTT assay was used. Briefly, QBC939 cells were seeded onto 96-well culture plates at a density of 5×10³ cells/well and grown in complete RPMI-1640 culture medium. Following overnight incubation, cells were treated with a variety of agents (60 μM) including 5-FU, CDDP, VCR and MMC for 48 h. MTT (5 mg/ml) was added to each well and incubated at 37°C for 4 h, before the resulting formazan crystals were dissolved in dimethyl sulfoxide. Absorbance was read at 490 nm using a microplate reader (Model 680; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Cells were cultured in a 35 mm dish until 70–80% confluence was reached. They were then transferred to a hypoxic incubator with a humidified atmosphere that was flushed with ≤0.1% O₂, 5% CO₂ and 95% N₂, followed by incubation at 37°C for 12 h.

Cells were seeded onto 6-well culture plates at a density of 2×10⁵ cells/well and grown in complete RPMI-1640 culture medium overnight. Then the medium was removed and washed with PBS, and added the RPMI 1640 medium without serum. The duration of serum starvation was determined according to the experimental requirements.

In vivo drug sensitivity experiments were divided into two groups: RARβ (pBoBi-RARβ) and control (pBoBi-Ctrl). Female specific pathogen-free BALB/c nude mice (Shanghai Laboratory Animal Center, Shanghai, China) (age, 4–5 weeks) were injected subcutaneously with 100 μl cells (2×10⁶). Mice were treated with 70 mg/kg/d 5-FU at day 8 post-transplantation. Tumor volumes were determined using the following formula: (AxB²)/2, where A is the largest diameter and B is the perpendicular diameter. After 35 days from injection, mice were sacrificed by inhalation of carbon dioxide. All animal procedures were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Xiamen University.

mRNA expression levels were determined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein expression was measured using a western blotting assay. Both analyses were performed as described previously (15).

IHC was performed to detect RARβ expression in CCA tissues as described previously (16). Human CCA tissue sections were immunostained with an antibody against RARβ (1:100). IHC scoring was conducted by two independent pathologists. The number of positively-stained cells per 1,000 cells in randomly selected fields was recorded, and the mean number from a minimum of five slides was calculated. Staining intensity was categorized as low (<50%) or high (>50%).

Apoptosis was evaluated using an annexin V-FITC/PI double-staining assay and a TUNEL assay. The annexin V-FITC/PI double-staining assay was performed according to the manufacturer's instructions (Roche Diagnostics) and analyzed using a flow cytometer (FACSCabilur; BD Biosciences, Franklin Lakes, CA, USA). TUNEL staining was performed using an in situ cell death detection kit according to the manufacturer's instructions (Roche Diagnostics).

Caspase-3 activity was determined using the caspase-3 assay kit (R&D Systems Inc., Minneapolis, MN, USA) according to the manufacturer's instructions. Briefly, cells were lysed and centrifuged at 10,000 × g for 20 min at 4°C to obtain supernatants. The protein concentration of sample supernatants was determined using a bicinchoninic acid protein assay reagent before they were added to a dithiothreitol and caspase-3 substrate reaction mixture and incubated for 2 h at 37°C. Absorbance was measured at 405 nm using a microplate reader (Model 680; Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± standard error from a minimum of three independent experiments. Student's t-test or one-way analysis of variance was conducted using the SPSS 13.0 software package (SPSS, Inc., Chicago, IL, USA). Dunn test was used if there was statistical significance after one-way analysis. P<0.05 was considered to indicate a statistically significant difference.

IHC was performed to investigate RARβ protein expression in a set of 33 paraffin-embedded human CCA tissues. A total of 28/33 (84.8%) tissues were found to exhibit low RARβ protein expression (Fig. 1A), only 5 tissues exhibited high RARβ protein expression (Fig. 1B). In addition, RARβ mRNA and protein expression levels in four CCA cell lines (QBC939, SK-ChA-1, MZ-ChA-1 and Hccc9810 cells) were found to be significantly lower than the HBd Epi normal bile duct epithelial cell line (Fig. 2A). RARβ mRNA and protein levels were markedly reduced within QBC939 cells. Notably, the proportion of surviving QBC939 cells following exposure to three commonly used chemotherapeutic agents including 5-FU, VCR and MMC, was found to be significantly higher compared with the remaining three CCA cell lines (Fig. 2B). These results give rise to the hypothesis that the observed resistance of CCA cells to common therapeutic agents, may be associated with silenced RARβ expression.

In order to investigate the role of RARβ in the therapeutic response of CCA cells, RARβ expression vectors were transfected into QBC939 cells prior to treatment with chemotherapeutic agents. As shown in Fig. 3A, an increase in RARβ mRNA and protein levels was observed within stably RARβ-transfected cells (pBoBi-QBC939) compared with control (pBoBi-Ctrl) cells. Cell survival analysis results indicated that the sensitivity of pBoBi-QBC939 cells to common chemotherapeutic agents, including 5-FU, CDDP, VCR and MMC, was significantly enhanced following RARβ upregulation compared with pBoBi-Ctrl cells (Fig. 3B). Furthermore, the proportion of apoptotic pBoBi-QBC939 cells generated in response to 5-FU, CDDP, VCR and MMC, was found to be significantly higher than pBoBi-Ctrl cells (Fig. 3C). These in vitro findings suggest that upregulation of RARβ enhances the sensitivity of CCA cells to chemotherapeutic agents.

5-FU is a common chemotherapeutic agent used for the treatment of hepatobiliary tumors. Therefore, this agent was selected to investigate the contribution of RARβ upregulation in the sensitivity to chemotherapeutic treatment in the xenografted QBC939 CCA tumors. As shown in Fig. 4A, the rate of tumor growth inhibition in xenografted pBoBi-RARβ tumors following treatment with 5-FU, was significantly higher than in control pBoBi-Ctrl tumors. A significant difference in tumor growth inhibition was apparent from day 20 and increased further during late tumor growth (Fig. 4B). Moreover, analysis of apoptosis in xenografted CCA tumor tissues using the TUNEL assay was consistent with the results of the in vitro assays, and indicated that the number of apoptotic cells induced by 5-FU in xenografted pBoBi-RARβ tumors was significantly higher than control pBoBi-Ctrl tumors (P=0.0035; Fig. 4C). These in vivo findings provide additional evidence that RARβ upregulation enhances the sensitivity of CCA cells to chemotherapeutic agents.

Taking the above results into consideration, the observed increase in the sensitivity of CCA cells to chemotherapeutic agents following RARβ upregulation may render CCA cells more susceptible to apoptosis. Hence, the expression levels of genes associated with apoptosis following RARβ upregulation were investigated. Results from RT-qPCR assays showed that the expression of proapoptotic genes, including bax, bak and bim, were increased >4 fold in pBoBi-RARβ QBC939 cells, while the expression of antiapoptotic genes, including bcl-2, bcl-xL and mcl-1, were all significantly decreased compared with pBoBi-Ctrl cells (Fig. 5A). To investigate these findings further, the association between RARβ expression and caspase-3 activity was explored. Treatment of pBoBi-RARβ cells with the RARβ-activator, BMS453, resulted in a 3.5-fold increase in caspase-3 activity compared with pBoBi-Ctrl cells, which exhibited a 1.8-fold increase. The observed increase in caspase-3 activity was further enhanced when cells were cultured under hypoxic conditions or following serum starvation. By contrast, the activity of caspase-3 was significantly decreased in both pBoBi-RARβ and pBoBi-Ctrl cells after treatment with the caspase-3 inhibitor z-VAD-fmk, even when cells were cultured under hypoxic conditions or following serum starvation (Fig. 5B). Furthermore, apoptosis induced by 5-FU, hypoxia or serum starvation was enhanced upon upregulation or activation of RARβ (Fig. 5C). These results indicate that the susceptibility of CCA cells to caspase-dependent apoptosis may be enhanced by the upregulation of RARβ.