OR Oncology Reports 1021-335X 1791-2431 D.A. Spandidos 10.3892/or.2017.5921 or-38-04-2489 Articles Chaetocin induces cell cycle arrest and apoptosis by regulating the ROS-mediated ASK-1/JNK signaling pathways HeJingliang 1 2 * ChenXiaoxun 3 * LiBowei 4 ZhouWenjie 1 XiaoJinfeng 1 HeKe 1 ZhangJinqian 5 XiangGuoan 1 Department of General Surgery, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong 510317, P.R. China Department of Postgraduate Studies, Guangdong Medical University, Zhanjiang, Guangdong 524023, P.R. China Department of Gastrointestinal Surgery, The Guigang City People's Hospital, Guigang, Guangxi 537100, P.R. China The Third Clinical Medical College of Southern Medical University, Guangzhou, Guangdong 510317, P.R. China Department of Laboratory Medicine, Guangdong Second Provincial General Hospital, Guangzhou, Guangdong 510317, P.R. China Correspondence to: Professor Guoan Xiang, Department of General Surgery, Guangdong Second Provincial General Hospital, 466 Xingang Middle Road, Guangzhou, Guangdong 510317, P.R. China, E-mail: bbqsnowhjl@hotmail.com Professor Jinqian Zhang, Department of Laboratory Medicine, Guangdong Second Provincial General Hospital, 466 Xingang Middle Road, Guangzhou, Guangdong 510317, P.R. China, E-mail: jingwanghou@163.com

Contributed equally

 042017 24082017 38 4 2489 2497 05032017 07082017 Copyright © 2017, Spandidos Publications 2017

The present study demonstrated that chaetocin, a natural small-molecule product produced by Chaetomium fungal species and a potential anticancer agent, inhibited the viability and invasive ability of the human intrahepatic cholangiocarcinoma cell line CCLP-1 in vivo and in vitro as revealed by CCK-8 and Transwell invasion assays and mouse xenograft tumor experiments. As determined using flow cytometry and intracellular ROS assays, chaetocin was found to induce cell cycle arrest and oxidative stress, leading to CCLP-1 cell apoptosis. Cell apoptosis can be initiated via different apoptotic signaling pathways under oxidative stress. As determined by western blot analysis, expression levels of the apoptosis signal-regulating kinase 1 (ASK-1) signalosome and its downstream c-Jun N-terminal kinase (JNK) signaling pathway were increased under oxidative stress stimulation. These findings indicate that chaetocin arrests the cell cycle and induces apoptosis by regulating the reactive oxygen species-mediated ASK-1/JNK signaling pathways.

 chaetocin intrahepatic cholangiocarcinoma cell viability apoptosis reactive oxygen species cell cycle ASK-1 JNKs Introduction

Chaetocin, a natural small-molecule product produced by Chaetomium fungal species (1), has repeatedly been reported to be a promising anticancer agent over the last several years. It has been reported that chaetocin is an inhibitor of lysine-specific histone methyltransferases (HMTs), which are the key enzymes that mediate epigenetic control of gene expression. Chaetocin is also an inhibitor of the redox enzyme thioredoxin reductase, as it competes with thioredoxin for binding to thioredoxin reductase, and thus induces cellular oxidative stress which can eradicate tumor cells (2). Chaetocin has been shown to inhibit the viability of various types of cancer, including melanoma, ovarian and non-small cell lung cancer (3). However, the effects of chaetocin on human intrahepatic cholangiocarcinoma (ICC) and the related mechanisms have not yet been reported.

Human cholangiocarcinoma is an epithelial cell malignancy arising from varying locations within the biliary system, and is the most common primary malignancy of the biliary tract (4). It can be classified into two major categories: ICC and ductal cholangiocarcinoma. ICC has received increased attention as various studies have shown marked increases in the morbidity and mortality rates of ICC in recent years (5). ICC is characterized by insidious development, late onset of symptoms, high recurrence rates after surgical resection, and limited treatment options for the vast majority of patients. Moreover, many ICC cases are resistant to traditional chemotherapeutics due to the desmoplastic character of the cancer, the complex tumor microenvironment and rich genetic heterogeneity (6,7); this creates further challenges for clinical treatment. Therefore, effective therapeutic strategies for the treatment of ICC with minimal side-effects are urgently required.

Induction of apoptosis is considered to be one of the most effective antitumor strategies. Apoptosis is a type of cell suicide that is regulated by a series of complex signaling pathways. Intrinsic or external stimuli induce apoptosis. Oxidative stress, characterized by high concentrations of intracellular reactive oxygen species (ROS), is reported to be one of the intrinsic inducers of apoptosis (8,9).

ROS, which are mainly produced in the mitochondria, have been reported to induce DNA sequence changes (rearrangements, deletions, mutations and gene amplifications) and cell apoptosis (10,11). ROS arrest the cell cycle and activate different apoptotic pathways, including the apoptosis signal-regulating kinase (ASK)/c-Jun N-terminal kinase (JNK) pathways (12–14). Previous findings have revealed that chaetocin increases the level of ROS and induces cell apoptosis (15). ROS, which can be produced in response to various types of cytotoxic stressors, activate ASK-1 directly and, thus, activate JNKs downstream (16). Activated JNKs then directly or indirectly activate apoptotic signaling pathways, and this ultimately results in cell apoptosis (17). In the present study, the effect of chaetocin on human ICC and the associated mechanisms were investigated.

 Materials and methods <sec> <title>Cell culture and reagents

The human ICC cell lines TFK-1 and CCLP-1 (acquired from the University of Pittsburgh, Pittsburgh, PA, USA) and RBE and SSP-25 (obtained from Piken University, Japan) were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS). A normal human intrahepatic bile duct cell line HIBEC (ScienCell Research Laboratories, San Diego, CA, USA) was cultured in the same way as mentioned above. HuCCT1 cells were cultured in Dulbeccos modified Eagles medium (DMEM) (HyClone, Logan, UT, USA) containing 10% FBS. All cell lines were supplemented with 100 U/ml of penicillin and 100 µg/ml of streptomycin and cultured at 37°C with 5% CO2. The cells were split every 4 days and some of the logarithmically growing cells were used for all experiments as described below. Chaetocin (11076 no. 13156; Cayman Chemical Co., Ann Arbor, MI, USA), SP600125 (no. s1460; Selleck Chemicals, Houston, TX, USA) and N-acetyl-L-cysteine (NAC) (no. 194603; MP Biomedicals, Solon, OH, USA) were dissolved in dimethyl sulfoxide for usage.

 Cell viability analysis

Cell viability was determined by Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). In brief, cells were digested and cultured in 96-well plates (5×103 cells/well) for 24 h. Then, the cells were placed in fresh medium (10% fetal) with different concentrations of chaetocin (0, 50, 100, 150 and 200 nM) for 24, 48 and 72 h, respectively. After incubation for the above times, we replaced each well with CCK-8 at a final concentration of 10% to co-culture for another 1 h. Cell viability was determined by its optical density (OD) measured at 450 nm of absorbance using a microplate reader (Mithras LB 940; Berthold Technologies, Bad Wildbad, Germany). In some experiments, NAC (5 mM) or SP600125 (50 nM) was used for pretreatment for 1 h before incubation with chaetocin. Cell viability = (trial group OD - blank group OD)/(control group OD - blank group OD) × 100%.

 Transwell invasion assay

Cell invasion potential was determined using a Transwell chamber (NY14831; Corning Inc., Corning, New York, NY, USA). CCLP-1 cells (5×104) in serum-free RPMI-1640 medium with different concentrations of chaetocin (0, 100 or 200 nM) were cultured in the upper chamber, which was coated with Matrigel (356234; Corning Inc.). The bottom chambers contained 600 µl medium with 10% FBS. Following 24 h of incubation at 37°C with 5% CO2, the cells were fixed using 4% paraformaldehyde and stained with crystal violet at room temperature for 30 min. The cells that invaded to the bottom side of the membrane were photographed at ×100 with a microscope (IX71; Olympus, Tokyo, Japan). After that, the bottom membrane with crystal violet was eluted with 33% acetic acid for 30 min and the OD of the acetic acid was determined at an absorbance rate of 570 nm using a microplate reader.

 Wright-Giemsa staining

On 6-well plates, 1.0×105 cells/well were seeded and incubated as described above. Solutions of chaetocin (0, 100 and 200 nM) with 10% serum-medium were added to each well, and then incubated for another 48 h. After being fixed with methanol and washed with phosphate-buffered saline (PBS), the cells, stained with Giemsa staining (Xiangya, China), were observed and photographed using an optical inverted microscope at ×200 (IX71; Olympus, Tokyo, Japan).

 Flow cytometry

The CCLP-1 cells (15×104/well) were cultured in a 6-well plate as described above. After co-culturing with chaetocin for 48 h, the cells were harvested, and then treated using an Annexin V-FITC apoptosis detection kit and propidium iodide (PI) (BioLegend, Inc., San Diego, CA, USA) according to the manufacturers protocol. For cell cycle analysis, the cells were cultured with chaetocin for 24 and 48 h, and then fixed with alcohol at 4°C. After 12 h of fixation, the cells were stained with PI (GBC BIO™ Technologies, Guangzhou, China). Finally, stained cells were analyzed using flow cytometry with FACSCalibur (Becton-Dickinson, Franklin Lakes, NJ, USA) and FlowJo 7.6.1 software.

 Intracellular ROS measurement

The intracellular ROS level was determined using an ROS detection kit (KeyGen Biotech Co., Ltd., Nanjing, China) using 2′-7′-dichlorodihydrofluorescein diacetate (DCFH-DA). In brief, after culturing with chaetocin for 24 h in 6 well-plates, the CCLP-1 cells were washed twice using PBS, and then stained with DCFH-DA in the dark for 30 min. Cells were then washed and resuspended in PBS to detect ROS accumulation by flow cytometry with FACSCalibur and analyzed using FlowJo 7.6.1 software.

 Western blot analysis

The CCLP-1 cells were treated with different concentrations of chaetocin for 48 h in the presence or absence of SP600125 or NAC. The cells were harvested, and the total proteins were extracted using the protein extraction kit (KeyGen Biotech Co., Ltd.). The proteins were separated on SDS-polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). Antibodies including phospho-ASK-1, phospho-JNK (Cell Signaling Technology, Beverly, MA, USA), phospho-p53, caspase-3, Bcl-2, GAPDH and α-tubulin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used to detected protein. After being washed, the membranes were incubated with homologous secondary antibody for 1 h. The signals were detected with chemiluminescent substrate and photographed using chemiluminescence immunoassay (Tanon 5200; Tanon Science & Technology Co., Ltd., Shanghai, China).

 In vivo experiment

Twelve nude mice were purchased from the Institute of Laboratory Animal Sciences (Southern Medical University) and used for the xenograft model. The mice were housed in controlled conditions of temperature and humidity with a 12 h light/dark cycle. The experiment was initiated with 6 week-old mice weighing 20–25 g. CCLP-1 cell suspension in PBS (2×106 cells/ml) was subcutaneously injected into the right flanks of the nude mice. When the mouse tumors reached 3–8 mm in size, the experiment was initiated as the 1st day. Chaetocin was formulated in 3% physiological saline. The nude mice were then randomly divided into 2 groups and were intraperitoneally injected either with chaetocin (0.3 mg/kg body weight) or vehicle once every three days for 6 times and terminated on the 18th day. The tumor dimensions were measured using Vernier calipers once every three days for the entire life span of the mice. Tumor volumes were calculated using the formula: a2x b/2 (a is the width and b is the length of the tumor in mm). The mice were sacrificed on the 30th day and the tumor mass from each mouse was dissected and weighed. The experimental mice were treated according to the standards supported by the Animal Protection Committee of Southern Medical University.

 Statistical analysis

As specified, every experiment was performed at least three times in triplicate, and the results are presented as means ± standard errors (SDs). Statistical analysis was performed by one-way ANOVA or by Student's t-test with SPSS version 18.0 (SPSS, Inc., Chicago, IL, US), and the results were considered statistically significant at P<0.05.

 Results <sec> <title>Chaetocin reduces the viability and invasive ability of the ICC cells

In order to investigate the effects of chaetocin on ICC, the viability of different human ICC cell lines was analyzed using a CCK-8 kit, and the invasive ability of the CCLP-1 cells was determined using a Transwell invasion assay. As shown in Fig. 1A-C, chaetocin reduced the viability of all ICC cell lines in a dose- and time-dependent manner. A significant reduction in cell viability was observed when cells were treated with 100 nM chaetocin for 48 h. In addition, the viability of the normal human intrahepatic bile duct HIBEC cell line was reduced in a concentration- and time-dependent manner, but HIBEC sensitivity to chaetocin was lower than that of the cancer cell lines.

Additionally, the invasive ability of the CCLP-1 cell line was reduced by chaetocin. Considering that the reduced ICC viability by chaetocin was significant at 48 h, we decided to observe the results of the Transwell assay after culturing cells with chaetocin for 24 h. Under microscopic observation (magnification, ×100), the number of cells that invaded through the membrane in the control group was markedly higher than that in the chaetocin-treated group. Quantification by OD detection confirmed the distinction between the control group and chaetocin-treated group, indicating that chaetocin inhibited the invasion of CCLP-1 cells (Fig. 1D and E).

 Chaetocin causes cell cycle arrest in the G2/M phase

To investigate the effect of chaetocin on cell cycle distribution, the cell cycle of CCLP-1 cells was analyzed by flow cytometry, using the PI staining method, following treatment with chaetocin. The results showed that every phase had no statistical difference at 24 h (Fig. 2B). Yet, the number of cells in the S phase was significantly decreased and that in the G2/M phase was increased following chaetocin treatment at 48 h. The changes were statistically significant. These results indicated that chaetocin caused cell cycle arrest in the G2/M phase, inhibiting proper DNA replication (Fig. 2A and C).

 Chaetocin induces CCLP-1 cell apoptosis

As the previous experiments revealed, chaetocin exerted an inhibitory effect on ICC cell viability and invasion. We applied Wright-Giemsa staining and flow cytometry was performed to examine whether chaetocin induces CCLP-1 cell apoptosis. Under optical microscopic observation, control group CCLP-1 cells were plump and densely populated in the visual field. However, the cells treated with chaetocin were shriveled and flat with reduced numbers in each visual field. In order to clearly observe the cell morphology, Giemsa was used to stain the cells. Compared with the normal morphology of the control cells, the cells of the chaetocin-treated group exhibited morphological characteristics of apoptosis, including nuclear pyknosis, sublobe, fragmented shapes, fringe collection and apoptotic body formation (Fig. 3A). The number of apoptotic bodies was observably increased with the increasing concentration of chaetocin. To further ascertain the effects of chaetocin on the apoptosis of the CCLP-1 cells, apoptosis was detected using Annexin V-FITC and PI staining. The results (Fig. 3B and C) revealed that the rate of cell apoptosis was increased in a dose-dependent manner, which was more evident at the early apoptotic stage.

 Chaetocin induces oxidative stress in the CCLP-1 cells

The intracellular ROS generation in CCLP-1 cells was measured using DCFH-DA. The flow cytometric analysis showed that the chaetocin-treated cells (at high concentrations) had significantly higher levels of ROS than the levels noted in the control cells. However, when cells were cultured with NAC and chaetocin (100 nM), the intracellular ROS level was less than that noted in the chaetocin-treated (100 nM) group (Fig. 4A and C). In addition, a low concentration of chaetocin did not have a significant effect on the ROS level in the cells, which may be the result of the short incubation time. The results suggest that chaetocin promotes the generation of intracellular ROS, leading to oxidative stress.

 The ASK-1/JNK pathway is involved in CCLP-1 cell apoptosis

ASK-1 is a member of the mitogen-activated protein kinase (MAPK) family that can be activated by oxidative stress. Activated ASK-1 has been reported to activate JNK proteins via phosphorylation. Therefore, the expression of ASK-1/JNK was determined using western blot analysis. The results showed that chaetocin activated ASK-1 and its downstream proteins JNK and p53 in a dose-dependent manner. In addition, the expression level of Bcl-2 was downregulated in a dose-dependent manner. By contrast, following chaetocin treatment, the level of caspase-3 exhibited no obvious change compared with the control (Fig. 5A-C). Furthermore, pretreatment with NAC suppressed the chaetocin-induced activation of ASK-1/JNK, which indicated that ROS have a vital role in the chaetocin-induced activation of these proteins (Fig. 6B-a). In another experiment, pretreatment with SP600125 (a JNK inhibitor) attenuated the chaetocin-induced expression of p53 (Fig. 6B-b).

 Chaetocin inhibits the growth of CCLP-1 xenograft tumors in vivo

To detect the antitumor activity of chaetocin in vivo, human CCLP-1 cholangiocarcinoma xenografts were established. The results (Fig. 7A-C) showed that the xenografts of the control group grew rapidly, but growth was reduced by chaetocin treatment in vivo. Additionally, the average weight of the tumors in the control group was higher than that of the chaetocin-treated group. However, there was no statistically significant difference in tumor weight between the two groups. The difference in tumor weight may have increased if the duration of the experiment had been extended (Fig. 7C). Furthermore, at the time of sacrifice, the average tumor volume of the control group was significantly higher than that of the chaetocin-treated group. Therefore, the results indicated that chaetocin inhibited tumor cell proliferation, although complete regression of the tumor was not observed. Additionally, the average body weight of the chaetocin-treated mice was significantly higher at the beginning of the experiment compared with the weight of the mice at sacrifice. However, this difference was not observed in the control group (Fig. 7D).

 Discussion

ICC is a treatment-resistant primary liver cancer with increasing incidence and mortality rates observed worldwide in recent years (18). For the majority of patients with advanced ICC, there is no effective or standard first-line chemotherapy (19). Therefore, it is urgent to identify effective drugs to treat ICC that have minimal side-effects. In our previous preliminary drug screening trials, chaetocin was identified to effectively reduce the viability of RBE cell lines at low doses (20), indicating that chaetocin may effectively inhibit the growth of cancer cells with few side-effects. In vitro experiments in the present study confirmed that chaetocin reduced the viability of ICC cell lines in a dose- and time-dependent manner (Fig. 1A-C). In addition, the Transwell chamber assay demonstrated that chaetocin reduced the invasion of CCLP-1 cells. We hypothesized that chaetocin may have the same effect on other ICC cell lines. Our in vivo xenograft tumor model results (Fig. 7A-C) also confirmed that chaetocin inhibited ICC tumor growth in mice. The in vitro and in vivo experiments clearly showed that chaetocin reduced ICC cell proliferation, but also reduced the viability of HIBECs, a normal human intrahepatic bile duct cell line, although HIBEC cells were less sensitive to chaetocin than the cancer cell lines. In vivo, the bdt weight of the mice tended to be decreased following chaetocin treatment. The average body weight change between the early and late stages of the experiment was statistically significant, whereas the body weights of the control group were unchanged (Fig. 7D). Considering these results, the reduced body weight in the chaetocin-treated group may represent a side-effect of chaetocin treatment, which should be noted with due attention. Thus, it is necessary to study the molecular mechanisms that mediate the effects of chaetocin and identify drugs that could be used in combination with chaetocin to potentially reduce the required dose of chaetocin and lessen the associated side-effects.

Cell cycle arrest is a major target of tumor therapy (21). The uncontrolled proliferation of tumor cells is due to overexpression of cyclins or the inactivation of critical cyclin-dependent kinases, which makes tumor cells unable to stop at predetermined points of the cell cycle (22,23). This means that arrest of the cell cycle can inhibit cancer cell proliferation. In the present study, the results showed that every phase in the CCLP-1 cell cycle had no change at 24 h. The results at 48 h showed that the percentage of CCLP-1 cells in the S phase was decreased and that the percentage of cells in the G2/M phase was significantly increased. This indicates that chaetocin was able to arrest the cell cycle in the G2/M phase and decrease DNA replication to inhibit CCLP-1 cell proliferation. Oxidative stress affects the cell cycle by affecting the expression of cyclins (24,25). Considering that the ROS level was not influenced under a low concentration of chaetocin (Fig. 4A), every phase of the cell cycle of the CCLP-1 cells may not have been influenced at 24 h. This assumption requires validation in further experiments.

Apoptosis, a fundamental process essential for the development and maintenance of tissue homeostasis, is also a major mechanism used to kill cancer cells (26). Inducing apoptosis is now considered as one of the most effective strategies for cancer treatment. In the present study, flow cytometry and observed morphological changes preliminarily indicated that chaetocin induced the apoptosis of CCLP-1 cells (Fig. 3A-C). Additionally, expression of p53 (an executor of apoptosis) was increased by chaetocin (Fig. 5A). This suggests that apoptosis is one of the mechanisms influenced by chaetocin resulting in reduced ICC cell viability.

Considering that pretreatment with NAC partially abrogated the effect of chaetocin on the viability of CCLP-1 cells (Fig. 4B), we aimed to determine whether oxidative stress underlied the chaetocin-induced apoptosis in CCLP-1 cells. ROS, an indicator of oxidative stress, are produced during normal cellular processes and are present in normal and cancer cells. At certain concentrations, ROS are required as critical signaling molecules involved in cell survival and proliferation (27). However, oxidative stress occurs when excessive ROS levels overwhelm the cellular antioxidant system, either through an increase in ROS concentration or a decrease in the cellular antioxidant capacity. Oxidative stress induces cell apoptosis and DNA damage (28,29). The present study showed that the ROS level was higher in the chaetocin-treated group than that in the control group. These results indicate that chaetocin can increase the ROS level and thereby induce oxidative stress in the CCLP-1 cells.

Oxidative stress is an initial signal that can induce cell apoptosis (30). ASK-1 is one of the proteins most sensitive to oxidative stress. It is well known that various types of cytotoxic stressors activate ASK-1 by producing excessive ROS, and thus induce apoptosis (17). Under normal conditions, ASK-1 is inactivated via binding with thioredoxin. ROS can oxidize thioredoxin and dissociate it from ASK-1. Therefore, when oxidative stress occurs, ASK-1 becomes activated via dissociation from thioredoxin and oligomerization into the ASK-1 complex (31,32). The ASK-1 complex phosphorylates itself and induces the activation of JNKs (12,33). JNKs also participate in the regulation of various cellular processes, including cell survival, proliferation, differentiation and cell death (34). A previous study demonstrated that chaetocin inhibited energy production and glucose metabolism in glioma cells in an ROS-JNK-dependent manner (35). Additionally, JNKs are widely reported to have a close association with ASK-1; therefore, we hypothesized that, as JNKs are downstream of ASK-1, they may be involved in the activation of cell apoptosis (36). The potential role of JNKs in chaetocin-induced apoptosis was investigated. As expected, a CCK-8 assay (Fig. 6A) showed that SP600125 (a JNK inhibitor) partially abrogated the effect of chaetocin on ICC cells, and western blotting showed that the chaetocin-induced expression of p53 (a tumor-suppressor gene) was reduced following pretreatment with SP600125 (Fig. 6B-b). Furthermore, in our experiments, the expression levels of phosphorylated ASK-1 and JNKs were increased by chaetocin treatment (Fig. 5A) and decreased by co-treatment with chaetocin and NAC (Fig. 6B-a). This demonstrated that ROS activation of ASK-1/JNK is involved in chaetocin-induced apoptosis of CCLP-1 cells. Following activation of JNKs, apoptosis is mediated by two different signaling pathways: direct and indirect. In the direct pathway, JNKs inhibit Bcl-2, an anti-apoptotic protein, by phosphorylation at Ser-70 (37). In the indirect pathway, JNKs phosphorylate and transactivate other transcription factors, such as p53 (17,38). As expected, our results showed that inhibition of JNKs decreased p53 phosphorylation (Figs. 5A and 6B-b).

In conclusion, chaetocin suppressed ICC cell viability and invasion in vitro and tumor growth in vivo. Furthermore, chaetocin caused CCLP-1 cell apoptosis, cell cycle arrest and activated the ASK-1/JNK signaling pathways associated with oxidative stress. In addition, chaetocin reduced the viability of a normal bile duct cell line. These results may provide an experimental basis with which to identify new combinatorial drugs that could be used to reduce the required dosage of chaetocin.

 Acknowledgements

The present study was supported by grants from the Natural Science Foundation of China (no. 81641110), the Guangdong Province Natural Science Foundation (no. 2015A030313725), and the Guangdong Science Province and Technology Program projects (2012B031800411).

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Effects of chaetocin on cell viability and invasion. (A-C) Different ICC cell lines CCLP-1, HuCCT1, RBE, TFK-1 and SSP-25, and normal human intrahepatic bile duct cell line HIBEC were treated with chaetocin at various concentrations (0, 50, 100, 150 and 200 nM) for 24, 48 and 72 h, and then cell viability was detected using CCK-8; *P<0.05 compared with the control group. (D and E) CCLP-1 cells were treated with various concentrations of chaetocin (0, 100 and 200 nM) for 24 h. The cells that invaded to the bottom side of the membrane were observed and photographed at a magnification of ×100 via a contrast microscope. Then, the bottom membrane was dyed with crystal violet and eluted with acetic acid and the OD of the acetic acid was detected by a microplate reader; *P<0.05 compared with the control.

Effects of chaetocin on cell cycle distribution of the CCLP-1 cells. (A) Representative histograms of DNA content in the CCLP-1 cells incubated with chaetocin (0, 50, 100 and 200 nM) for 48 h. (B and C) Histograms display the percentage of cell cycle distribution at 24 or 48 h; *P<0.05 compared with the control group.

Effects of chaetocin on the apoptosis of CCLP-1 cells. (A) Cell morphology of CCLP-1 cells following treatment with chaetocin (0, 100 and 200 nM) for 48 h and staining with Giemsa and photographed at a magnification of ×200 via a contrast microscope. Dotted arrows indicate normal CCLP-1 cells. Dotted circles indicate a change in morphology of the CCLP-1 cells. Solid arrows indicate cell nuclear pyknosis, sublobe, fragment shape (morphological characteristics of apoptosis). (B) The apoptotic ratio of the CCLP-1 cells following treatment with chaetocin (0, 50, 100 and 200 nM) at 48 h was detected using flow cytometry. (C) Histograms display the percentage of apoptotic cells; *P<0.05 compared with the control group.

Effect of chaetocin on ROS production in the CCLP-1 cells. (A) The CCLP-1 cells were cultured with chaetocin (0, 50, 100 and 200 nM) (a-d) or 100 nM chaetocin in the present of 5 mM NAC (f) for 24 h. Then, the cells were stained with DCFH-DA and the percentage of DCF fluorescence was detected by flow cytometry. The proportion of cells with bright fluorescence was calculated. (B) The cell lines CCLP-1 and HuCCT1 were cultured with chaetocin (Ch) (100 nM), NAC (5 mM) or both (Ch + NAC) for 48 h, respectively. The viability of the cells was detected via CCK-8; *P<0.05 compared with the control group; #P<0.05 compared with the cells treated with chaetocin. (C) The histograms show the percentage of DCF fluorescence (mean ± SD) compared with the control group; *P<0.05 compared with the control group; #P<0.05, chaetocin (100 nM)-treated group compared with the chaetocin and NAC-treated group.

Effect of chaetocin on the protein expression in the CCLP-1 cells. (A-C) Expression changes and relative quantification of proteins were detected by western blotting; 0, 50, 100 and 200 nM are the doses of chaetocin used; *P<0.05 compared with the control group.

(A) The cell lines CCLP-1 and HuCCT1 were cultured with chaetocin (Ch) (100 nM), SP600125 (50 nM) or both (Ch+SP) for 48 h, respectively. The viability of cells was detected via CCK-8; *P<0.05 compared with the control group; #P<0.05 compared with the cells treated with chaetocin. (B) CCLP-1 cells were treated with chaetocin (100 nM) in the presence or absence of NAC (5 mM) or SP600125 (50 nM) for 48 h. Protein levels of (a) p-ASK-1, p-JNKs and (b) p-p53 were examined by western blotting: 0, control group; 1, chaetocin-treated group; 2, chaetocin-treated and NAC pretreated group; 3, chaetocin and SP600125 treated group.

The antitumor effect of chaetocin on xenografts of CCLP-1 in vivo. CCLP-1 cells were subcutaneously injected into the rear flanks of nude mice. After the inoculation, mice were randomly grouped and subjected to different treatments as indicated: chaetocin-treated group, intraperitoneally injected with chaetocin (0.3 mg/kg body weight); control group, intraperitoneally injected with vehicle. Mice were sacrificed on the 30th day. (A and B) The image and histograms depict the differences between the two groups in terms of average tumor weight on the 30th day. (C) The curve represents the mean tumor volume/group ± SD (n=6) at different times during the entire experiment. Arrows indicate the times (day) that the mice were injected with either chaetocin or vehicle; *P<0.05, chaetocin-treated group compared with the control group on the 30th day. (D) The curve represents the mean mouse body weight/group ± SD (n=6) at different times during the entire experiment; *P<0.05, weight of initiation compared with weight of termination in the chaetocin-treated group.