Predicting IDH mutation status of intrahepatic cholangiocarcinomas based on contrast-enhanced CT features.

OBJECTIVES: To explore the difference in contrast-enhanced computed tomography (CT) features of intrahepatic cholangiocarcinomas (ICCs) with different isocitrate dehydrogenase (IDH) mutation status. METHODS: Clinicopathological and contrast-enhanced CT features of 78 patients with 78 ICCs were retrospectively analysed and compared based on IDH mutation status. RESULTS: There were 11 ICCs with IDH mutation (11/78, 14.1%) and 67 ICCs without IDH mutation (67/78, 85.9%). IDH-mutated ICCs showed intratumoral artery more often than IDH-wild ICCs (p = 0.023). Most ICCs with IDH mutation showed rim and internal enhancement (10/11, 90.9%), while ICCs without IDH mutation often appeared diffuse (26/67, 38.8%) or with no enhancement (4/67, 6.0%) in the arterial phase (p = 0.009). IDH-mutated ICCs showed significantly higher CT values, enhancement degrees and enhancement ratios in arterial and portal venous phases than IDH-wild ICCs (all p < 0.05). The CT value of tumours in the portal venous phase performed best in distinguishing ICCs with and without IDH mutation, with an area under the curve of 0.798 (p = 0.002). CONCLUSIONS: ICCs with and without IDH mutation differed significantly in arterial enhancement mode, and the tumour enhancement degree on multiphase contrast-enhanced CT was helpful in predicting IDH mutation status. KEY POINTS: • IDH mutation occurred frequently in ICCs. • ICCs with and without IDH mutation differed significantly in arterial enhancement mode. • ICCs with IDH mutation enhanced more than those without IDH mutation. • Enhancement ratio and tumour CT value can predict IDH mutation status.

Three-dimensional structural analysis reveals a Cdk5-mediated kinase cascade regulating hepatic biliary network branching in zebrafish.

The intrahepatic biliary network is a highly branched three-dimensional network lined by biliary epithelial cells, but how its branching patterns are precisely established is not clear. We designed a new computer-based algorithm that quantitatively computes the structural differences of the three-dimensional networks. Utilizing the algorithm, we showed that inhibition of Cyclin-dependent kinase 5 (Cdk5) led to reduced branching in the intrahepatic biliary network in zebrafish. Further, we identified a previously unappreciated downstream kinase cascade regulated by Cdk5. Pharmacological manipulations of this downstream kinase cascade produced a crowded branching defect in the intrahepatic biliary network and influenced actin dynamics in biliary epithelial cells. We generated larvae carrying a mutation in cdk5 regulatory subunit 1a (cdk5r1a), an essential activator of Cdk5. cdk5r1a mutant larvae show similar branching defects as those observed in Cdk5 inhibitor-treated larvae. A small-molecule compound that interferes with the downstream kinase cascade rescued the mutant phenotype. These results provide new insights into branching morphogenesis of the intrahepatic biliary network.

Enzyme-Histochemistry Technique for Visualizing the Dipeptidyl-Peptidase IV (DPP-IV) Activity in the Liver Biliary Tree.

Dipeptidyl-peptidase IV is an enzyme involved in a lot of biochemical processes, where it modifies a number of regulatory proteins by removing the terminal peptides by hydrolysis. Here we describe a histochemical method to demonstrate with accuracy and precision its in situ activity on cryostatic section of Wistar rat liver by means of a simultaneous azo-coupling method.

Prognostic Value of C-met Expression in Cholangiocarcinoma.

AIM: To explore the relationship of clinicopathological features and the proteins of C-met expression in the prognosis of cholangiocarcinoma. METHODS: Clinical data and the completed follow-up information of patients with cholangiocarcinoma who underwent cholangiocarcinoma operation from January 2004 to December 2010 were analyzed retrospectively. The relationship of clinicopathological features and C-met in the prognosis of the patients was analyzed. RESULTS: Patients with high expression of C-met had significantly shorter overall survival than those with low expression of C-met, the difference being statistically significant (P = .003). Patients with high C-met expression had significantly shorter disease-free survival time than those with low expression of C-met, the difference being statistically significant (P = .009). By COX multivariate analysis, high C-met expression in tumor tissues was an independent risk factor in predicting overall survival and disease-free survival for patients with cholangiocarcinoma (P = .038, .048, relative risk = 1.390, 1.427). CONCLUSION: Patients with high C-met expression in cancer tissues had shorter disease-free survival and overall survival. High expression of C-met is an independent risk factor for overall survival and disease-free survival.

A trial to find appropriate animal models of dichloropropane-induced cholangiocarcinoma based on the hepatic distribution of glutathione S-transferases.

OBJECTIVES: It has been reported that 1,2-Dichloropropane (DCP) induced cholangiocarcinoma (CCA) in offset color proof-printing workers. However, exposure to DCP by inhalation or gavage for 2 year did not induce CCA in mice and rats. The present study mapped the hepatic distribution of GST, which is known to activate dihalogenated alkanes, and proliferative and fibrotic changes in bile ducts in various species to find the most appropriate animal model of DCP-induced CCA. METHODS: First, 12 each of C57BL/6J mice, Balb/cA mice, F344 rats, Syrian hamsters, and guinea pigs were divided into four equal groups and exposed to DCP at 0, 300, 1,000, or 3,000 ppm 8 hours/day for 7 days. Second, 32 Balb/cA mice and 32 Syrian hamsters were each divided into four equal groups and exposed to DCP at 0, 200, 400, and 800 ppm 6 hours/day for 14 days. After the last exposure, the animals were decapitated, and the livers were dissected out for histopathological evaluation. Immunostaining was conducted to determine the distribution of GSTT1, GSTM1, and GSTPi, as well as the expression of proliferation marker Ki67. RESULTS: GSTT1, GSTM1, and GSTPi were expressed in both hepatocytes and bile duct cells in all control and exposed animals. There was no clear difference in the expression of Ki67 between the exposed groups and the control. No fibrotic changes were observed in any species or strains examined. CONCLUSIONS: Expression of GSTT1 or other GST isozymes might not explain the difference in sensitivity of hepatocytes and the bile duct to DCP between humans and rodents.

High Expression of Matrix Metalloproteinase-11 indicates Poor Prognosis in Human Cholangiocarcinoma.

BACKGROUND: Cholangiocarcinoma (CHCA) is serious public health problem in Thailand, especially in the northeastern and northern regions. CHCA is known as one of the most aggressive malignant tumors associated with local invasion and a high rate of metastasis. A crucial step in the invasion process is the proteolytic degradation of the extracellular matrix (ECM) and basal membranes, for which several studies have shown a critical role played by matrix metalloproteinase-11 (MMP-11). OBJECTIVE: This study aim to detect MMP-11 expression in CHCA specimens and any correlation with survival time. MATERIALS AND METHODS: A retrospective analysis was conducted of 30 patients with CHCA in Rajvithi hospital, who had undergone immunohistochemical staining of MMP-11. Relationships between clinicopathological data and MMP-11 expression in CHCA specimens were analyzed by the χ2 test or Fisher's exact test. The estimated survival and the survival differences were analyzed by the Kaplan-Meier method and the log-rank test, respectively. RESULTS: MMP-11 expression was found in 15 specimens (50%). The overall mean survival time is 237.0 days (95% CI 135.4-338.5, SD 271.9). Specimens with a positive MMP-11 had an average survival time of 136.7 days (95%CI 50.3-223.1, SD 156.0). Survival differences was signficant for the positive and negative MMP-11(p=0.022), but not well differentiated tumor and moderate to poor differentiated tumor (p=0.755), CA19-9 level of >1,000 and <1,000 (p=0.488), and between advanced and non-advanced staging (p=0.388). CONCLUSIONS: The positive MMP-11 expression indicates poor prognosis in CHCA specimens.

Omega-3 Polyunsaturated Fatty Acids Upregulate 15-PGDH Expression in Cholangiocarcinoma Cells by Inhibiting miR-26a/b Expression.

Prostaglandin E2 (PGE2) is a proinflammatory lipid mediator that promotes cancer growth. The 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes oxidation of the 15(S)-hydroxyl group of PGE2, leading to its inactivation. Therefore, 15-PGDH induction may offer a strategy to treat cancers that are driven by PGE2, such as human cholangiocarcinoma. Here, we report that omega-3 polyunsaturated fatty acids (ω-3 PUFA) upregulate 15-PGDH expression by inhibiting miR-26a and miR-26b, thereby contributing to ω-3 PUFA-induced inhibition of human cholangiocarcinoma cell growth. Treatment of human cholangiocarcinoma cells (CCLP1 and TFK-1) with ω-3 PUFA (DHA) or transfection of these cells with the Fat-1 gene (encoding Caenorhabditis elegans desaturase, which converts ω-6 PUFA to ω-3 PUFA) significantly increased 15-PGDH enzymes levels, but with little effect on the activity of the 15-PGDH gene promoter. Mechanistic investigations revealed that this increase in 15-PGDH levels in cells was mediated by a reduction in the expression of miR-26a and miR-26b, which target 15-PGDH mRNA and inhibit 15-PGDH translation. These findings were extended by the demonstration that overexpressing miR-26a or miR-26b decreased 15-PGDH protein levels, reversed ω-3 PUFA-induced accumulation of 15-PGDH protein, and prevented ω-3 PUFA-induced inhibition of cholangiocarcinoma cell growth. We further observed that ω-3 PUFA suppressed miR-26a and miR-26b by inhibiting c-myc, a transcription factor that regulates miR-26a/b. Accordingly, c-myc overexpression enhanced expression of miR-26a/b and ablated the ability of ω-3 PUFA to inhibit cell growth. Taken together, our results reveal a novel mechanism for ω-3 PUFA-induced expression of 15-PGDH in human cholangiocarcinoma and provide a preclinical rationale for the evaluation of ω-3 PUFA in treatment of this malignancy.

ß-escin reverses multidrug resistance through inhibition of the GSK3ß/ß-catenin pathway in cholangiocarcinoma.

AIM: To develop a safe and effective agent for cholangiocarcinoma (CCA) chemotherapy. METHODS: A drug combination experiment was conducted to determine the effects of ß-escin in combination with chemotherapy on CCA cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay was performed to determine the effects of ß-escin and common chemotherapeutics on the proliferation of human CCA cells (QBC939, Sk-ChA-1, and MZ-ChA-1). Immunocytochemistry was used to detect the expression of P-glycoprotein (P-gp) protein. Luciferase reporter assay was used to detect the activation of the Wnt/ß-catenin pathway. The protein levels of P-gp, pS9-GSK3ß, pT216-GSK3ß, GSK3ß, ß-catenin, and p-ß-catenin were further confirmed by western blotting. RESULTS: The drug sensitivity of QBC939 and QBC939/5-fluorouracil (5-FU) cells to 5-FU, vincristine sulfate (VCR), or mitomycin C was significantly enhanced by ß-escin compared with either agent alone (P<0.05). In addition, the combination of ß-escin (20 µmol/L) with 5-FU and VCR was synergic with a combination index<1. Further investigation found that the mRNA and protein expression of P-gp was down-regulated by ß-escin. Moreover, ß-escin induced GSK3ß phosphorylation at Tyr-216 and dephosphorylation at Ser-9, resulting in phosphorylation and degradation of ß-catenin. Interestingly, activation of the GSK3ß/ß-catenin pathway induced by Wnt3a resulted in up-regulation of P-gp, which was effectively abolished by ß-escin, indicating that ß-escin down-regulated P-gp expression in a GSK3ß-dependent manner. CONCLUSION: ß-escin was a potent reverser of P-gp-dependent multidrug resistance, with said effect likely being achieved via inhibition of the GSK3ß/ß-catenin pathway and thus suggesting a promising strategy of developing combination drugs for CCA.

IDH mutations in liver cell plasticity and biliary cancer.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive cancer associated with the bile ducts within the liver. These tumors are characterized by frequent gain-of-function mutations in the isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) genes-that are also common in subsets of neural, haematopoietic and bone tumors, but rare or absent in the other types of gastrointestinal malignancy. Mutant IDH acts through a novel mechanism of oncogenesis, producing high levels of the metabolite 2-hydroxyglutarate, which interferes with the function of α-ketoglutarate-dependent enzymes that regulate diverse cellular processes including histone demethylation and DNA modification. Recently, we used in vitro stem cell systems and genetically engineered mouse models (GEMMs) to demonstrate that mutant IDH promotes ICC formation by blocking hepatocyte differentiation and increasing pools of hepatic progenitors that are susceptible to additional oncogenic hits leading to ICC. We found that silencing of HNF4A-encoding a master transcriptional regulator of hepatocyte identity and quiescence-was critical to mutant IDH-mediated inhibition of liver differentiation. In line with these findings, human ICC with IDH mutations are characterized by a hepatic progenitor cell transcriptional signature suggesting that they are a distinct ICC subtype as compared to IDH wild type tumors. The role of mutant IDH in controlling hepatic differentiation state suggests the potential of newly developed inhibitors of the mutant enzyme as a form of differentiation therapy in a solid tumor.

High expression of matrix metalloproteinase-9 indicates poor prognosis in human hilar cholangiocarcinoma.

High expression of matrix metalloproteinase-9 (MMP-9) was found to be correlated with tumor progression and poor prognosis in a variety of carcinomas. However, few studies have investigated the role of MMP-9 in human hilar cholangiocarcinoma. In this study, a total of 58 patients with hilar cholangiocarcinoma who underwent curative resection were included in this study. The expression of MMP-9 was analyzed by immunohistochemistry using the streptavidin peroxidase complex method. The correlation of MMP-9 overexpression with clinicopathological features and survival time of patients was investigated. The results showed that MMP-9 overexpression was prominent in cancer cells and mainly localized in the cytoplasm. MMP-9 overexpression was observed in 46.5% tumors, which showed no correlation with clinicopathological parameters. Patients with high MMP-9 expression had a significantly poorer overall survival rate than those with negative or low MMP-9 expression (P = 0.038). Multivariate analysis confirmed that MMP-9 overexpression was an independent prognostic factor (P = 0.007). In conclusion, overexpression of MMP-9 is a valuable independent prognostic indicator in hilar cholangiocarcinoma.

Transforming growth factor-ß1-induced epithelial-mesenchymal transition generates ALDH-positive cells with stem cell properties in cholangiocarcinoma.

Epithelial-mesenchymal transition (EMT) is a major factor that facilitates the invasiveness and metastasis of cancer. Recent studies have demonstrated that EMT plays a key role in generating cancer stem cells (CSCs). This study aimed to investigate the effect of EMT on CSCs that were identified as positive for aldehyde dehydrogenase (ALDH) in cholangiocarcinoma (CCA). We demonstrated that transforming growth factor-ß1 (TGF-ß1)-induced EMT in the human cholangiocarcinoma (CCA) cell line, TFK-1, resulted in the acquisition of mesenchymal traits, as well as the expression of ALDH, which was accompanied by decreased sensitivity to the chemotherapeutic agent, 5-fluorouracil. ALDH-positive cells isolated from TFK-1 cells had higher proliferation potential in vitro and tumourigenic ability in vivo. They also expressed mesenchymal markers. Moreover, the expression levels of TGF-ß1 and ALDH1 were correlated with poor prognosis in patients. We conclude that ALDH acts as a marker for CSCs in CCA, and TGF-ß1-induced EMT is involved in the generation of CSCs. These findings offer a new tool for the study of CCA stem cells and illustrate a direct link between EMT and the gain of stem-cell properties.

Mutant IDH inhibits HNF-4α to block hepatocyte differentiation and promote biliary cancer.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer. Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation. However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs, cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

Saturated free fatty acids induce cholangiocyte lipoapoptosis.

UNLABELLED: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. CONCLUSION: Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

Overexpression of membrane metalloendopeptidase inhibits substance P stimulation of cholangiocarcinoma growth.

Substance P (SP) promotes cholangiocyte growth during cholestasis by activating its receptor, NK1R. SP is a proteolytic product of tachykinin (Tac1) and is deactivated by membrane metalloendopeptidase (MME). This study aimed to evaluate the functional role of SP in the regulation of cholangiocarcinoma (CCA) growth. NK1R, Tac1, and MME expression and SP secretion were assessed in human CCA cells and nonmalignant cholangiocytes. The proliferative effects of SP (in the absence/presence of the NK1R inhibitor, L-733,060) and of L-733,060 were evaluated. In vivo, the effect of L-733,060 treatment or MME overexpression on tumor growth was evaluated by using a xenograft model of CCA in nu/nu nude mice. The expression of Tac1, MME, NK1R, PCNA, CK-19, and VEGF-A was analyzed in the resulting tumors. Human CCA cell lines had increased expression of Tac1 and NK1R, along with reduced levels of MME compared with nonmalignant cholangiocytes, resulting in a subsequent increase in SP secretion. SP treatment increased CCA cell proliferation in vitro, which was blocked by L-733,060. Treatment with L-733,060 alone inhibited CCA proliferation in vitro and in vivo. Xenograft tumors derived from MME-overexpressed human Mz-ChA-1 CCA cells had a slower growth rate than those derived from control cells. Expression of PCNA, CK-19, and VEGF-A decreased, whereas MME expression increased in the xenograft tumors treated with L-733,060 or MME-overexpressed xenograft tumors compared with controls. The study suggests that SP secreted by CCA promotes CCA growth via autocrine pathway. Blockade of SP secretion and NK1R signaling may be important for the management of CCA.

Activating mutations in PTPN3 promote cholangiocarcinoma cell proliferation and migration and are associated with tumor recurrence in patients.

BACKGROUND & AIMS: The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched for mutations in human ICC tumor samples and investigated how they affect ICC cell function. METHODS: We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). RESULTS: Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3. Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3(L232R) and PTPN3(L384H), which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. CONCLUSIONS: Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3. Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.

TTF-1 and Napsin-A are expressed in a subset of cholangiocarcinomas arising from the gallbladder and hepatic ducts: continued caveats for utilization of immunohistochemistry panels.

Thyroid transcription factor-1 (TTF-1) and Napsin-A (NapA) are frequently used to classify a tumor of unknown origin as lung or thyroid primary. Although recent studies have shown that nuclear TTF-1 positivity occasionally occurs in adenocarcinoma of nonpulmonary or thyroid origin dependent upon the antibody clone, TTF-1 has been reported as negative or infrequently positive in tumors of biliary origin. On the basis of an index case of cholangiocarcinoma expressing TTF-1, we were prompted to study TTF-1 and NapA positivity in cholangiocarcinoma. Archived paraffin-embedded tissue blocks from liver, gallbladder, and pancreato-biliary resections were chosen for cholangiocarcinoma (n=33) and non-neoplastic intrahepatic and extrahepatic biliary epithelium control tissue (n=26). Immunohistochemical analysis for TTF-1 and NapA was performed and graded for intensity and quantity. TTF-1 was negative in control biliary tissue but positive in 27.2% of cholangiocarcinomas. All TTF-1-positive cases (n=9) were extrahepatic (P=0.01), and most arose from the upper biliary tract (gallbladder and hepatic ducts). TTF-1 positivity was associated with age 60 years and above (P=0.01) but not with sex. Three TTF-1-positive cases were also NapA positive. NapA staining showed apical granular staining of the adjacent non-neoplastic epithelium in 6 cases (18.1%). In summary, 47.4% of extrahepatic cholangiocarcinoma expressed TTF-1, 33.3% of which coexpressed NapA. Cholangiocarcinoma should be considered in the differential when evaluating a TTF-1-positive tumor of unknown primary. As TTF-1 and NapA are not known for biliary system development or detected in non-neoplastic biliary epithelium, the significance of this "pulmonary" phenotype in a subset of extrahepatic cholangiocarcinoma is unknown and needs further investigation.

JNK inhibitor SP600125 enhances TGF-ß-induced apoptosis of RBE human cholangiocarcinoma cells in a Smad-dependent manner.

Transforming growth factor ß (TGF-ß) signaling is pivotal for the progression of specific types of tumors at certain stages. However, the mechanism by which TGF-ß is regulated by other factors remains unclear. In this study, the involvement of SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), in TGF-ß-induced apoptosis of the RBE human cholangiocarcinoma cell line was investigated. Exogenous TGF-ß1 activated Smad and non­Smad signaling pathways, including the JNK pathway in RBE cells, and induced apoptosis, which was inhibited by knockdown of Smad4 expression. SP600125 increased the TGF-ß1­induced phosphorylation of Smad2 and Smad3, which enhanced the TGF-ß1­induced transcriptional response and apoptosis in RBE cells. The effect of SP600125 on the transcriptional response and apoptosis was reduced by knockdown of Smad4 expression. In addition, TGF-ß1­induced apoptosis was abrogated using the pan-caspase inhibitor Z­VAD-fmk. SP600125 promoted the TGF-ß1­induced caspase cleavage, while knockdown of Smad4 expression counteracted this effect. These results indicate that SP600125 enhances TGF-ß-induced apoptosis of RBE cells through a Smad­dependent pathway that involves Smad­dependent caspase activation. SP600125 is hypothesized to be an ideal therapeutic candidate for treating human cholangiocarcinoma.