Amniotic epithelial cell-derived cholangiocytes in experimental cholestatic ductal hyperplasia.

AIM: Bile duct paucity, ductopenia, is a feature of end-stage chronic cholangiopathies such as primary biliary cirrhosis. The limited proliferative ability of cholangiocytes after specific injury is thought to be the principal cause of ductopenia, although the detailed mechanisms involved are unclear. It has been reported that human amniotic epithelial cells (AEC) express differentiation markers of hepatic parenchymal cells, suggesting a resemblance of AEC to hepatic progenitor cells. The aim of the present study was to develop a mouse model of experimental cholestasis to assess the capability of mouse AEC to trans-differentiate into cholangiocytes. METHODS: Enhanced green fluorescent protein (EGFP)-transgenic C57BL/6 pregnant female mice were used as the source of AEC. At 11.5 gestational days, 1 x 10(5) AEC were isolated from EGFP-transgenic mouse embryos and transferred into C57BL/6 mice. Chronic cholestasis was induced by 0.1%alpha-naphthylisothiocyanate (ANIT) feeding immediately after the transfer of AEC. The proliferation of cholangiocytes in the livers was assessed morphologically and immunohistochemically (cytokeratin 7; CK7). The proliferative activity was also quantified immunohistochemically by proliferating cell nuclear antigen (PCNA) protein expression. EGFP of transferred AEC was confirmed by fluorescent laser microscopy and immunofluorescent staining for EGFP. Also, Notch2 and Hes1 expression was evaluated to examine the roles of the differentiation markers in this process. RESULTS: Marked proliferation of cholangiocytes was observed in ANIT-fed mice confirmed by quantitative CK7 (3-4 fold vs control) and PCNA (11-20 fold vs control) staining. EGFP and CK7 double positive cells in interlobular bile ducts were confirmed in the livers of AEC-transferred recipients. Positivity of EGFP was further confirmed by the immunofluorescent staining for EGFP. Moreover, both Notch2 and Hes1 expression was confirmed in the proliferative bile duct in this model. CONCLUSIONS: Significant ductular proliferation was observed in ANIT-fed mice. EGFP-positive cholangiocytes were confirmed in this chronic cholestasis model. AEC transfer was able to contribute to the repopulating of proliferating cholangiocytes under cholestasis, suggesting AEC might be a candidate cell source for stem cell administration in future clinical applications to re-model interlobular bile ducts.

Complete remission of a case of hepatocellular carcinoma with tumor invasion in inferior vena cava and with pulmonary metastasis successfully treated with repeated arterial infusion chemotherapy.

We report the case of a patient having hepatocellular carcinoma with tumor invasion to the inferior vena cava and with multiple pulmonary metastases who was treated with repeated one-shot administration of epirubicin, cisplatin, and mitomycin C by hepatic artery and bronchial artery, which led to complete remission. A 72-year-old woman was diagnosed with infiltrative hepatocellular carcinoma with Vv3, multiple intrahepatic metastases, and multiple pulmonary metastases associated with compensated liver cirrhosis. One-shot infusion of epirubicin, cisplatin, and mitomycin C was performed through proper hepatic artery and bronchial artery for twice at eight weeks of intervals. Pulmonary metastases disappeared and intrahepatic lesions indicated marked shrinkage leaving a scar-like lesion with decreases in tumor markers. After six months and 20 months, tumor markers indicated increasing tendency but no evident recurrence was found by computed tomography or hepatic arteriography. One-shot infusion of the same regimens through proper hepatic artery was performed and tumor markers decreased to normal levels. After 14 months of the last therapy, no evidence of recurrence has been found on image analysis or in tumor markers. This arterial infusion therapy is well tolerated for the patients with compensated liver cirrhosis and might be promising for the effective treatment of advanced hepatocellular carcinoma with pulmonary metastases.

The model for end-stage liver disease score is useful for predicting economic outcomes in adult cases of living donor liver transplantation.

BACKGROUND: The model for end-stage liver disease (MELD) is useful for assessing the recipients of liver transplants, namely, deceased-donor transplantation. The application of MELD for living donor liver transplantation (LDLT) is under investigation. Thus, the aim of this study was to analyze the impact of the MELD score in LDLT in Japan. METHODS: Seventeen adult cases of LDLT during 2001 to 2005 were enrolled. Indications for LDLT were primary biliary cirrhosis, seven; liver cirrhosis, two; hepatocellular carcinoma (HCC), three; metabolic liver disease, one; primary sclerosing cholangitis, two; Caroli's disease, one; and biliary atresia, one. Total medical charges during the operative periods were retrospectively evaluated. The united network of organ sharing (UNOS) modified was obtained using preoperative clinical data. RESULTS: The average medical expense of the 17 cases was approximately 97,901 US dollar. The UNOS-modified MELD score was 22.1. A statistically significant positive correlation was found between the MELD score and medical expense (P = 0.0086, rho = 0.657), and between the MELD score and the length of stay in the intensive care unit (ICU) (P = 0.0396, rho = 0.515). The cause of the liver disease leading to transplantation was not related to MELD score, medical expense, or length of ICU stay. CONCLUSIONS: Although not originally designed for the application to LDLT, the MELD score is useful for predicting medical expenses in LDLT. Similar to those of deceased-donor liver transplantation, the disadvantage of high medical expenses associated with a high MELD score allow consideration of an earlier elective operation in suitable cases.

Differential expressions of aquaporin proteins in human cholestatic liver diseases.

UNLABELLED: Aquaporins (AQPs) are the channel forming membranous proteins involved in the biliary physiological homeostasis. Recently, we have reported the heterogeneous expression of AQPs in intrahepatic biliary epithelial cells or cholangiocytes in mice. However, the involvements of AQPs in hepatobiliary disorder are still unclear. Thus, we hypothesized that the AQP protein expressions are altered in human cholestatic disorders. METHODS: The AQP expressions of the immortalized human cholangiocytes cell line (H69) were assessed by immunoblotting. The expression of AQPs in liver biopsy specimens from various human cholestatic diseases as well as viral hepatitis were evaluated immunohistochemically. The degrees of staining were classified into four grades by comparison with staining intensity from controls. RESULTS: AQP1 expression, predominantly membranous, was confirmed by immunoblotting analysis. However, the other subtypes of AQP expression were not detected. In human pathological tissues, AQP1 expression by interlobular bile ducts was similar to normal and viral hepatitis, although this expression was attenuated according to bile duct injuries in PBC. On the contrary, the AQP1 expression by proliferating bile ductile (equivalent for small cholangiocytes) was enhanced. In intrahepatic cholestasis, AQP1 expressions were diminished, which was further associated with the aberrant expressions by periportal hepatocytes. CONCLUSIONS: AQP1 was expressed intensely in smaller proliferating bile ducts in human cholestatic liver disease. Also, the AQP1 expression was decreased in injured duct cells undergoing degeneration in PBC. The AQP1 expression was decreased in intrahepatic cholestasis probably due to negative feed back of the decreased bile flow. The role of AQP expression profiles may help the understanding of the pathogenesis of human cholestatic liver diseases.

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B.

AIM: To study the mechanisms of hyporesponsiveness of HBV-specific CD4+ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS: Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4+CD25+ cells. RESULTS: Level of mRNAs for T-bet, IL-12R beta2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4+CD25highCTLA-4+ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4+ cells and CD4+CD25+ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAg-specific TH1 antibody compared with control antibody (P < 0.01, 0.34% +/- 0.12% vs 0.15% +/- 0.04%). Deletion of CD4+CD25+ T cells increased the amount of HBcAg-specific TH1 antibody when compared with lymphocytes reconstituted using regulatory T cells (P < 0.01, 0.03% +/- 0.02% vs 0.18% +/- 0.05%). CONCLUSION: The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

Sustained viral response of a case of acute hepatitis C virus infection via needle-stick injury.

A 29-year-old nurse with a hepatitis C virus (HCV) infection caused by needle-stick injury was treated with interferon-beta starting about one year after the onset of acute hepatitis. The patient developed acute hepatitis C with symptoms of general fatigues, jaundice, and ascites 4 wk after the needle-stick injury. When these symptoms were presented, the patient was pregnant by artificial insemination. She hoped to continue her pregnancy. After delivery, biochemical liver enzyme returned to normal levels. Nevertheless, HCV RNA was positive and the pathological finding indicated a progression to chronicity. The genotype was 1b with low viral load. Daily intravenous injection of interferon-beta at the dosage of six million units was started and continued for eight weeks. HCV was eradicated without severe adverse effects. In acute hepatitis C, delaying therapy is considered to reduce the efficacy but interferon-beta therapy is one of the useful treatments for hepatitis C infection in chronic phase.

A case of acute hepatitis with positive autoantibodies who actually had hepatitis E virus infection.

Hepatitis E virus (HEV) is one of the major causative agents of acute hepatitis in many developing countries. On the other hand, recent intensive investigation has revealed the existence of non-imported cases in industrialized countries. We encountered a sporadic patient with hepatitis E in 1999, who had had no recent travel abroad. He was a 67-year-old Japanese, and his laboratory data were negative for serum markers of hepatitis A, B and C virus infection and positive anti-nuclear antibody, anti-smooth muscle antibody and high level of serum immunoglobulin G. He scored as probable autoimmune hepatitis (AIH) with the scoring system by the international AIH group. He was given a diagnosis of acute cryptogenic hepatitis including acute onset AIH in those days, but the recent retrospective examination with frozen stocked serum revealed his exact diagnosis. In conclusion, we must take HEV infection into consideration for the diagnosis of cryptogenic acute hepatitis even in the developed countries, and some patients with hepatitis E could demonstrate positive for autoantibodies similar to clinical features of AIH. This case demonstrated the needs for further studies about clinical feature of acute hepatitis E virus infection.

Lack of common mutation in the alfa-subunit of the mitochondrial trifunctional protein and the polymorphism of CYP2E1 in three Japanese women with acute fatty liver of pregnancy/HELLP syndrome.

BACKGROUND:: Acute fatty liver of pregnancy (AFLP) and the HELLP syndrome are the serious disorders during pregnancy. The aim of this study is to clarify the prevalence of common mutation in the alpha-subunit of the mitochondrial tri-functional protein: hydroxyacyl-CoA dehydrogenase (LCHAD)/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase as well as to determine the correlation with the polymorphism of microsomal cytochrome P4502E1 (CYP2E1) in these conditions. METHODS:: Genomic DNA was extracted from three patients with AFLP/the HELLP syndrome. Exon 15 of LCHAD and 5'-flanking lesion of CYP2E1 was amplified by PCR and analyzed by RFLP with either of Pst I or the combination of Pst I and Rsa I, respectively. RESULTS:: None of the patients demonstrated the 1528G-C mutation in LCHAD gene. All the patients had homozygous wild-type genotype (c1/c1) in the 5'-flanking lesion of CYP2E1. CONCLUSIONS:: Although limited size of study, our observations suggest the low incidence rate of LCHAD common mutation among Japanese patients with AFLP/HELLP syndrome. Moreover, all of the patients had wild-type genotype of CYP2E1 in this study. Considering with the fact that the neonates from these patients has been in good health for at least 6 years from birth, there might be diverse etiologic factors of Japanese patients with AFLP/HELLP syndrome other than reported genetic mutations.

Extracellular branched-chain amino acids, especially valine, regulate maturation and function of monocyte-derived dendritic cells.

The functions of dendritic cells (DCs) are impaired in patients with liver cirrhosis. It is well-known that cirrhotic patients show decreased levels of plasma branched-chain amino acids (BCAA). Although amino acids are associated with maintaining the cell structure and function in many organs, limited data are available regarding the role of amino acids including BCAA in the immune system. We aimed to investigate the roles of BCAA in the function of human monocyte-derived DCs (MoDC). CD14-positive monocytes (CD14 (+)) were isolated from PBMC from healthy volunteers and hepatitis C virus (HCV) cirrhotic patients. In medium deprived of BCAA or valine, monocytes were able to differentiate into immature, but not into mature, DCs and showed weak expression of CD83. The deprivation of leucine or isoleucine did not affect this process. The MoDC allostimulatory capacity was significantly decreased in medium deprived of BCAA or valine (p = 0.017, p = 0.012, Bonferroni's analysis, respectively). Annexin V(FITC)/propidium iodide staining showed that the DC yield and viability were not significantly different under any medium. Immunoblotting demonstrated that depletion of valine or leucine decreased phospho-S6 kinase expression. Valine increased dose-dependently the allostimulatory capacity and IL-12 production of MoDC from both healthy volunteers and HCV cirrhotic patients. An elevated extracellular concentration of valine could improve the DC function in cirrhotic patients. These data provide a rationale for nutrition therapy that could be beneficial to patients with cirrhosis.

Retrospective evaluation of tumor-mass-reduction therapy for the prognosis of recurrent hepatocellular carcinoma.

Although hepatocellular carcinoma (HCC) is the liver cancer that requires repeated treatments because of a high tendency for recurrence, few data have been available about whether repeated treatments, including those to reduce tumor mass, are effective in prolonging survival. We retrospectively analyzed the effectiveness of tumor-mass-reduction therapy for the prognosis of patients with recurrent HCC. To analyze the effectiveness of various modalities of therapies with a single criterion, we defined a tumor-mass-reduction grade (TMRG), which was retrospectively evaluated by dynamic CT or MRI. Grade A: no evident HCC remains untreated; Grade B1: more than 50% of lesions are treated; and Grade B2: less than 50% of lesions are treated. Subjects were stratified by Child-Pugh classification and the number of admissions for HCC treatment. In those classified as Child-Pugh A, a better survival rate was obtained, depending on the degree of TMRG from the first to the fifth admission (P < .01), suggesting that these patients are endurable for repeated therapies and benefit from the many sessions of treatment. In those classified as Child-Pugh B, on the second to the fifth admissions, survival rates showed statistical difference depending on the TMRG (P < .01), which may suggest that only a few sessions of treatment are meaningful. In those classified as Child-Pugh C, any number of mass-reduction treatment sessions did not improve the survival rate. In conclusion, repeated tumor-mass-reduction therapies for recurrent HCC are most beneficial in Child-Pugh A patients. Patients with Child-Pugh B who experience several recurrence episodes and any patients with Child-Pugh C may benefit more from modalities other than tumor-mass-reduction therapies.

Filopodia formation via a specific Eph family member and PI3K in immortalized cholangiocytes.

Biliary ducts are lined with epithelial cells, which consist of at least two types of cholangiocytes, small and large. In contrast to large cholangiocytes, which are involved in secretion, the role of small cholangiocytes has not been elucidated. To address this question, we analyzed the migration-based characteristics of these cells that may help to understand their functions in vivo. Interestingly, dibutyryl cAMP induced marked filopodia formation and cdc42 activation in the normal mouse cholangiocyte (NMC)-small cell line compared with the NMC-large cell line. Analysis of members of the ephrin (Eph)A family of guidance molecules revealed a distinct subcellular distribution of EphA5 and EphA8 members: EphA8 was equally expressed by both cell types and localized subcellularly in peripheral cell membranes, whereas EphA5 was expressed predominantly in NMC-S and localized to filopodia. Moreover, cAMP-inducible filopodia formation in these cells was abrogated using EphA5 short interfering RNA. Finally, we found that the Rho family GTPase cdc42 was activated in a manner dependent on EphA5. Wortmannin, a specific inhibitor of phosphotidylinositol 3-kinase (PI3K), abolished the activation of cdc42 dependent on EphA5, suggesting the involvement of PI3K in the EphA5-cdc42 pathway. Together, our findings suggest a cAMP-EphA5-cdc42-dependent regulation of small cholangiocyte migration, which are anticipated to facilitate the understanding of the nature of cholangiocytes and to explain certain general aspects of cAMP-cdc42 activation signaling with regard to cell morphogenesis.

Lack of evidence that bone marrow cells contribute to cholangiocyte repopulation during experimental cholestatic ductal hyperplasia.

BACKGROUND: Ductopenia is observed in end-stage human cholestatic diseases. The limited capability of cholangiocytes for proliferation is suggested to be the principal reason. Recently, bone marrow cells (BMCs) have been reported to behave as hepatic stem cells; however, their capability to differentiate into cholangiocytes in cholestasis remains unclear. METHODS: Normal mice were lethally irradiated to suppress the proliferation of self-BMCs; thereafter, the BMCs from enhanced green fluorescent protein (EGFP)-transgenic mice were transferred to recipients. Chronic cholestasis was induced by 0.1%alpha-naphtylisothiocyanate (ANIT) feeding. The proliferation of cholangiocytes and oval cells was assessed morphologically and immunohistchemically (cytokeratin-7 (CK-7), A6). Proliferative activity (proliferating cell nuclear antigen (PCNA) protein expression), hepatic growth factor (HGF) receptor (c-Met), stem cell factor receptor (c-kit), Notch2 and Hes1 expression were also evaluated. RESULTS: Marked cholangiocyte proliferation was observed in ANIT-fed mice. However, no EGFP/CK-7 double positive cells were identified in any of the liver specimens after BMCs transfer (Tx). In hepatic parenchyma, there were scattered EGFP-positive cells, although none of them were positive for CK-7. CONCLUSIONS: In spite of the significant ductular proliferations after ANIT feeding, no EGFP-positive cholangiocytes were confirmed by any other means in this chronic cholestasis model. Thus, different from hepatocytes, BMCs Tx seems not to contribute to the differentiation of cholangiocytes. Future studies are feasible to clarify the origin of proliferative cholangiocytes observed in this chronic cholestatic ductular hyperplasia model.

Sustained clinical improvement of a patient with decompensated hepatitis B virus-related cirrhosis after treatment with lamivudine monotherapy.

Hepatitis B virus (HBV) infection, which causes liver cirrhosis and hepatocellular carcinoma, remains a major health problem in Asian countries. Recent development of vaccine for prevention is reported to be successful in reducing the size of chronically infected carriers, although the standard medical therapies have not been established up to now. In this report, we encountered a patient with decompensated HBV-related cirrhosis who exhibited the dramatic improvements after antiviral therapy. The patient was a 50-year-old woman. Previous conventional medical treatments were not effective for this patient, thus this patient had been referred to our hospital. However, the administration of lamivudine, a reverse transcriptase inhibitor, for 23 months dramatically improved her liver severity. During this period, no drug resistant mutant HBV emerged, and the serum HBV-DNA level was continuously suppressed. These virological responses were also maintained even after the antiviral therapy was discontinued. Moreover, both hepatitis B surface antigen and e antigen were observed to have disappeared in this patient. The administration of lamivudine to patients with HBV-related cirrhosis, like our present case, should be considered as an initial medical therapeutic option, especially in countries where liver transplantation is not reliably available.

Living related liver transplantation for acute fulminant hepatitis B: experience from two possible hyper-acute cases.

Fulminant hepatic failure, which is represented by fulminant hepatitis, is fatal in most cases unless prompt liver transplantation is performed. Even if liver transplantation is performed, irreversible neurological damage is often complicated. In this case report, we describe two cases of fulminant hepatitis induced by acute hepatitis B virus infection, both of which were successfully rescued by living related liver transplantation without significant complications. The case 1 was a 45-year-old Japanese male. He complained general malaise and anorexia. His local physician diagnosed him as acute hepatitis B, and referred to our hospital. Due to severe coagulopathy, plasma exchange was performed 3 times. However, his hepatic coma progressed rapidly along with rapid decrease of both his direct/indirect bilirubin (D/T) ratio and serum blood urea nitrogen (BUN) levels. Living related liver transplantation was performed under the diagnosis of acute fulminant hepatitis B. The case 2 was a 34-year-old Japanese male. His complaints were fever and skin rush. He was referred to our hospital under the diagnosis of acute hepatitis B. On the second day after admission, he developed grade II hepatic coma, which deteriorated into grade III in spite of intensive therapy including plasma exchange. He also demonstrated rapid decrease of both D/T ratio and serum BUN level. Living related liver transplantation was performed on the next day. Both cases recovered without any evidence of neurological sequelae. In general, it is extremely difficult to rescue fulminant hepatitis by conservative treatments, particularly in cases with rapid progression. Although emergency liver transplantation may be an only option to rescue in such a case, living related liver transplantation has an advantage in view of urgent organ donation over cadeveric transplantation.

Stimulation of alpha2-adrenergic receptor inhibits cholangiocarcinoma growth through modulation of Raf-1 and B-Raf activities.

Growth factor signaling, mediated by the mitogen-activated protein kinase (MAPK) cascade, induces cell mitosis. Adenosine 3',5'-monophosphate (cAMP) may inhibit or stimulate mitosis (depending on the cell type) through the activation of MAPK and Raf proteins. Among Raf proteins, Raf-1 and B-Raf differentially regulate mitosis. Our aims were to evaluate the role and mechanisms of action of the alpha(2)-adrenergic agonist UK14,304 in the regulation of growth of the human cholangiocarcinoma cell line Mz-ChA-1. Immunocytochemistry and immunoblotting for alpha(2A)-, alpha(2B)-, or alpha(2C)-adrenergic receptor subtypes showed positive reaction in Mz-ChA-1 cells. We found that physiological concentrations of UK14,304 increased cAMP levels and inhibited proliferation and MAPK activity in Mz-ChA-1 cells. Mz-ChA-1 cells expressed Raf-1 and B-Raf. Epidermal growth factor (EGF) immediately and transiently stimulated Raf-1 activity, whereas B-Raf activity was increased with prolonged EGF stimulation. EGF-stimulated Raf-1 and B-Raf activities were both inhibited by UK14,304. UK14,304 did not affect Ras activity. In Mz-ChA-1 cells, alpha(2)-adrenoreceptor stimulation causes up-regulation of cAMP, which inhibits EGF-induced MAPK activity through an acute increase of Raf-1 and sustained activation of B-Raf. In conclusion, because alpha(2)-AR inhibition of growth occurred downstream of Ras, adrenergic stimulation or other stimulants of cAMP may overcome the Ras mutations and offer a new therapeutic approach for patients with cholangiocarcinoma.

Evaluation of differential gene expression by microarray analysis in small and large cholangiocytes isolated from normal mice.

AIMS: We have shown that large and small cholangiocytes, which reside primarily in large and small intrahepatic bile ducts, respectively, have different functions and responses to injuries. However, there are no systematic studies of the molecular differences between small and large cholangiocytes, which would explain cholangiocyte heterogeneity. To evaluate the differential gene expression between small and large cholangiocytes, microarray analysis was performed. METHODS: Primary cultures of small and large cholangiocytes were isolated from normal mice (BALB/c), and immortalized by the introduction of the SV40 large T antigen gene. After cloning, small and large cholangiocyte cell lines were established. Their characteristic features were confirmed by electron microscopy (EM) and measurement of transepithelial electrical resistance (TER), and secretin-stimulated cAMP levels. Isolated total RNAs were hybridized with microarrays (Atlas Glass Array Mouse 1.0 and 3.8), which detects 4850 cDNA expressions. After hybridization, the fluorescent signals were scanned by a GenePix fluorescent scanner and analyzed using ArrayGauge software. RESULTS: EM, TER and secretin-stimulated cAMP synthesis are consistent with the concept that small and large immortalized cholangiocytes originate from small and large ducts, respectively. When a cut-off value at the expression signal difference of 3.0 times was employed, 230 cDNAs among 4850 cDNAs (4.74%) were differentially expressed between small and large cholangiocytes. Of these 230 cDNAs, aquaporin 8, IL-2 receptor beta chain and caspase 9 were more strongly expressed by large cholangiocytes. CONCLUSIONS: Microarray successfully displayed characteristic differential cDNA expression between small and large cholangiocytes. This technique provides molecular information, which further supports our hypothesis that small and large bile ducts have different functions.

Tauroursodeoxycholate inhibits human cholangiocarcinoma growth via Ca2+-, PKC-, and MAPK-dependent pathways.

Tauroursodeoxychate (TUDCA) is used for the treatment of cholangiopathies including primary sclerosing cholangitis, which is considered the primary risk factor for cholangiocarcinoma. The effect of TUDCA on cholangiocarcinoma growth is unknown. We evaluated the role of TUDCA in the regulation of growth of the cholangiocarcinoma cell line Mz-ChA-1. TUDCA inhibited the growth of Mz-ChA-1 cells in concentration- and time-dependent manners. TUDCA inhibition of cholangiocarcinoma growth was blocked by BAPTA-AM, an intracellular Ca(2+) concentration ([Ca(2+)](i)) chelator, and H7, a PKC-alpha inhibitor. TUDCA increased [Ca(2+)](i) and membrane translocation of the Ca(2+)-dependent PKC-alpha in Mz-ChA-1 cells. TUDCA inhibited the activity of MAPK, and this inhibitory effect of TUDCA was abrogated by BAPTA-AM and H7. TUDCA did not alter the activity of Raf-1 and B-Raf and the phosphorylation of MAPK p38 and JNK/stress-activated protein kinase. TUDCA inhibits Mz-ChA-1 growth through a signal-transduction pathway involving MAPK p42/44 and PKC-alpha but independent from Raf proteins and MAPK p38 and JNK/stress-activated protein kinases. TUDCA may be important for the treatment of cholangiocarcinoma.