Treatment Of CCA Research Articles

Myofibroblast-derived PDGF-BB promotes hedgehog survival signaling in cholangiocarcinoma cells

Cholangiocarcinoma (CCA) cells paradoxically express the death ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and, therefore, are dependent upon potent survival signals to circumvent TRAIL cytotoxicity. CCAs are also highly desmoplastic cancers with a tumor microenvironment rich in myofibroblasts (MFBs). Herein, we examine a role for MFB-derived CCA survival signals. We employed human KMCH-1, KMBC, HuCCT-1, TFK-1, and Mz-ChA-1 CCA cells, as well as human primary hepatic stellate and myofibroblastic LX-2 cells, for these studies. In vivo experiments were conducted using a syngeneic rat orthotopic CCA model. Coculturing CCA cells with myofibroblastic human primary hepatic stellate cells or LX-2 cells significantly decreased TRAIL-induced apoptosis in CCA cells, a cytoprotective effect abrogated by neutralizing platelet-derived growth factor (PDGF)-BB antiserum. Cytoprotection by PDGF-BB was dependent upon Hedgehog (Hh) signaling, because it was abolished by the smoothened (SMO; the transducer of Hh signaling) inhibitor, cyclopamine. PDGF-BB induced cyclic adenosine monophosphate-dependent protein kinase-dependent trafficking of SMO to the plasma membrane, resulting in glioma-associated oncogene (GLI)2 nuclear translocation and activation of a consensus GLI reporter gene-based luciferase assay. A genome-wide messenger RNA expression analysis identified 67 target genes to be commonly up- (50 genes) or down-regulated (17 genes) by both Sonic hedgehog and PDGF-BB in a cyclopamine-dependent manner in CCA cells. Finally, in a rodent CCA in vivo model, cyclopamine administration increased apoptosis in CCA cells, resulting in tumor suppression. MFB-derived PDGF-BB protects CCA cells from TRAIL cytotoxicity by a Hh-signaling-dependent process. These results have therapeutical implications for the treatment of human CCA.

Abstract 4794: Monoamine oxidase A expression correlates with cholangiocarcinoma tumor grade and survival and is suppressed via IL-6-driven hypermethylation of the promoter

Abstract The metabolism of dopamine and serotonin is dysregulated in cholangiocarcinoma (CCA), which has subsequent growth promoting effects. Monoamine oxidase A (MAOA), the enzyme responsible for the degradation of serotonin and dopamine, is switched off in CCA although the mechanism by which this occurs is unknown. MAOA transcriptional activity can be suppressed by hypermethylation. IL-6 is known to be overproduced in CCA and can control hypermethylation events. The aims of this study were to 1) Correlate MAOA expression with pathophysiological parameters of CCA; 2) determine if IL-6 drivenhypermethylation of the MAOA promoter is involved in the suppression of expression. Methods: MAOA expression was assessed in biopsy samples from 84 patients with hilar CCA, or non-malignant controls by immunohistochemistry and realtime PCR and Kaplan-Meier survival analysis and Log Rank test performed. Hypermethylation of the MAOA promoter was assessed in four CCA cell lines previously shown to have suppressed MAOA expression, in two non-malignant cholangiocyte lines (H69 and HIBEC), and in human tumor samples using bisulfite modification followed by pyrosequencing analysis. Mz-ChA-1 and H69 cells were treated with a DNA methyltransferase inhibitor (5-aza; 10μM) for 4 days to inhibit the hypermethylation followed by qPCR assessment of MAOA expression. In parallel, Mz-ChA-1 cells were treated with a neutralizing IL-6 antibody (1μg/mL) for 4 days, or IL-6 expression was knocked down by stable transfection of IL-6 shRNA plasmids and MAOA expression was assessed by qPCR. Results: MAOA expression has a negative correlation with tumor differentiation (correlation coefficient: -2.68; P=0.014<0.05(2-tailed)). Furthermore there was a distinct correlation between MAOA expression and total survival (10.8 ± 0.9 months for low MAOA expression vs 27.2 ± 4.3 months for moderate or strong expression group, P<0.01). The MAOA promoter region was hypermethylated in CCA cells, but not H69 or HIBEC and treatment with 5-aza increased MAOA expression in Mz-ChA-1 cells, but not H69 cholangiocytes. Suppression of IL-6 expression either by neutralizing antibodies or by specific knock down of expression (to 3% of the parental cell line) reversed the hypermethylation and increased the subsequent MAOA expression. Conclusions: MAOA expression correlates with both differentiation grade and overall survival of CCA patients. Aberrant IL-6 expression seen in CCA cells may drive the suppression of MAOA expression by regulating promoter methylation and subsequent MAOA gene expression. Agents that may restore MAOA expression may have therapeutic value in the treatment of CCA. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4794. doi:10.1158/1538-7445.AM2011-4794

The novel cannabinoid receptor GPR55, inhibits cholangiocarcinoma growth

Anandamide (AEA) inhibits cholangiocarcinoma (CCA) growth via stabilization of lipid rafts, and the activation of death receptors. GPR55 has been identified as a putative cannabinoid receptor that may be activated by AEA, although its effects on CCA proliferation are unknown. Our aims were to evaluate the effects of GPR55 activation on CCA growth and the downstream signaling cascade. GPR55 was expressed in CCA cells and biopsy samples to a similar level as the non-malignant counterparts. The GPR55 agonists O-1602 or AEA reduced viability in vitro and tumor growth in vivo. Activation of GPR55 by AEA or O-1602 activated JNK, which was required for their antiproliferative actions. Pretreatment with the lipid raft disruptors prevented these antiproliferative effects. Lipid rafts were isolated from CCA cells treated with AEA or O-1602 and immunoblotting of the resulting fractions reveals both GPR55 and Fas are recruited into lipid rafts after AEA and O-1602. Transfection of CCA cells with FADD shRNA prevented the antiproliferative effects of GPR55 receptor activation. Activation of GPR55 exerts antiproliferative effects on CCA via the activation of a JNK-mediated pathway, resulting in the recruitment of Fas into lipid raft membrane structures. Specific targeting of GPR55 may prove to be useful in designing adjunct therapies for the treatment of CCA. This work was supported by NIH K01 and R03 awards.

Cholangiocarcinoma — Current Treatment Options

The treatment for cholangiocarcinoma (CCA) remains a challenge because of the aggressive nature of the disease and the absence of effective treatments besides surgical resection (HR) and liver transplantation (LT). In intrahepatic CCA, HR remains the treatment of choice whereas with concomitant liver disease such as cirrhosis or primary sclerosing cholangitis (PSC), LT is the only option. Hilar CCA or Klatskin tumours have in recent decades been managed with a more aggressive surgical approach to achieve R0 resection. This approach usually involves preoperative portal embolisation, followed by liver resection – sometimes extensive and even with portal vein resection. The recent protocols that combine preoperative neoadjuvant chemoirridation and LT show promising results that need to be confirmed. The development of diagnostic modalities (tumour markers, cytology and radiology) are of the utmost importance to identify these patients at an early stage to preserve radical surgery possible. Cholangiocarcinoma (CCA) is a malignant disease of the epithelial cells in the intra- and extrahepatic bile ducts. While still a rare malignant disease, CCA is the second most common primary malignancy of the liver. The incidence is increasing; especially the incidence of intrahepatic CCA (1). The treatment of CCA is challenging as it is usually difficult to diagnose when radical surgical treatment, resection (HR) or liver transplantation (LT) is possible. The lack of effective medical treatment makes a radical surgical resection or hepatectomy the only therapeutic option. Most of the CCAs are unresectable at presentation and the prognosis for these patients is dismal.

Mesothelin as a Potential Therapeutic Target in Human Cholangiocarcinoma

Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are the two most common primary liver cancers, yet there have been no significant advances in effective therapeutics. Mesothelin has been reported as a new therapeutic target in various types of cancer. Here, we investigated the expression of mesothelin in liver cancer and its potential role as a novel therapeutic target for immunotherapy. HCC and CCA specimens were examined by immunohistochemistry for mesothelin expression. Protein expression was assessed by immunoblotting and flow cytometry. The SS1P immunotoxin targeting mesothelin was evaluated in the well-established CCA cell lines HuCCT1, HuH-28, KMBC, KMCH, Mz-ChA-1 and OZ. We showed strong immunochemical mesothelin staining in 33% of the surgically resected CCA specimens and 3 of 6 CCA cell lines (OZ, KMBC and KMCH). No mesothelin staining was found in HCC or normal liver tissue. Mesothelin was primarily localized to the cellular plasma membrane and the mature form (molecular weight, ~40 kDa) was expressed at a high level in CCA tissues. Moreover, 22% of CCA specimens had a high mesothelin expression level which was comparable to the CCA cell line models. Interestingly, SS1P showed very high and specific growth inhibition when added to mesothelin-expressing CCA cells with IC(50) values ranging from 0.5 to 11 ng/mL. Mesothelin is overexpressed in one-third of CCA tissues. SS1P targeting mesothelin reveals a remarkable single agent activity against CCA in vitro. These findings indicate a potential for SS1P in the immunotherapeutic treatment of CCA.

Dicoumarol enhances gemcitabine-induced cytotoxicity in high NQO1-expressing cholangiocarcinoma cells

To investigate whether dicoumarol, a potent inhibitor of NAD(P)H quinone oxidoreductase-1 (NQO1), potentiates gemcitabine to induce cytotoxicity in cholangiocarcinoma cells (CCA) and the role of reactive oxygen generation in sensitizing the cells. Four human cell lines with different NQO1 activity were used; the human CCA cell lines, KKU-100, KKU-OCA17, KKU-M214, and Chang liver cells. NQO1 activity and mRNA expression were determined. The cells were pretreated with dicoumarol at relevant concentrations before treatment with gemcitabine. Cytotoxicity was determined by staining with fluorescent dyes. Oxidant formation was examined by assay of cellular glutathione levels and reactive oxygen species production by using dihydrofluorescein diacetate. Measurement of mitochondrial transmembrane potential was performed by using JC-1 fluorescent probe. Western blotting analysis was performed to determine levels of survival related proteins. Dicoumarol markedly enhanced the cytotoxicity of gemcitabine in KKU-100 and KKU-OCA17, the high NQO1 activity and mRNA expressing cells, but not in the other cells with low NQO1 activity. Dicoumarol induced a marked decrease in cellular redox of glutathione in KKU-100 cells, in contrast to KKU-M214 cells. Dicoumarol at concentrations that inhibited NQO1 activity did not alter mitochondrial transmembrane potential and production of reactive oxygen species. Gemcitabine alone induced activation of NF-kappaB and Bcl-(XL) protein expression. However, gemcitabine and dicoumarol combination induced increased p53 and decreased Bcl-(XL) levels in KKU-100, but not in KKU-M214 cells. NQO1 may be important in sensitizing cells to anticancer drugs and inhibition of NQO1 may be a strategy for the treatment of CCA.

Current Surgical Management of Hilar and Intrahepatic Cholangiocarcinoma: The Role of Resection and Orthotopic Liver Transplantation

Cholangiocarcinoma (CCA) is a rare but devastating malignancy that presents late, is notoriously difficult to diagnose, and is associated with a high mortality. Surgical resection is the only chance for cure or long-term survival. The treatment of CCA has remained challenging because of the lack of effective adjuvant therapy, aggressive nature of the disease, and critical location of the tumor in close proximity to vital structures such as the hepatic artery and the portal vein. Moreover, the operative approach is dictated by the location of the tumor and the presence of underlying liver disease. During the past 4 decades, the operative management of CCA has evolved from a treatment modality that primarily aimed at palliation to curative intent with an aggressive surgical approach to R0 resection and total hepatectomy followed by orthotopic liver transplantation.

Cholangiocarcinoma in Primary Sclerosing Cholangitis

Cholangiocarcinoma (CCA) is an aggressive and nearly always fatal tumor of the biliary tract. This review explores risk factors, epidemiology, current diagnostic approaches, and treatment of CCA arising in patients with primary sclerosing cholangitis (PSC). We review latest recommendations about screening strategies to enable the early detection of CCA in PSC, using CA 19-9 and ultrasound imaging, as well as fluorescent in situ hybridization techniques to enhance the accuracy of biliary cytology. We also review the emerging role of liver transplantation.

S1584 Carbon Monoxide Reduces Cholestatic Liver Injury Induced By Bile Duct Ligation

stained with a commercially available kit and co-localization with RhoA was determined by confocal microscopy. RESULTS: Simvastatin significantly decreased cell number in all of the six CCA lines (40 to 80%; 5 μM for 48 hrs) and HiBEC as compared to untreated cells. To distinguish whether the decrease was due to apoptosis or cell cycle arrest, cell proliferation was assessed by BrdU uptake. Simvastatin (5 μM for 4-6 hrs) induced a significant increase in apoptosis in Mz-ChA-1, but not BrdU uptake. However, simvastatin while not triggering apoptosis in HiBEC did reduce BrdU uptake or proliferation in HiBEC. Pretreatment of MzChA-1 with Mev, FPP and GPP did not reverse the effects of simvastatin on Mz-ChA-1 cell viability suggesting that simvastatin-induced apoptosis is not heavily dependent upon HMGCoA reductase inhibition. In contrast, cholesterol reversed simvastatin-induced apoptosis indicating involvement of lipid rafts. Simvastatin disrupts lipid rafts in Mz-ChA-1 (present in higher levels than in HiBEC) resulting in a loss of RhoA colocalization and the potential disruption of a RhoA dependent cell survival pathway. In fact, disruption of lipid rafts with MBCD or inhibition of the upstream activator of RhoA, GGTase, with GGTI-298 also reduced Mz-ChA-1 viability to that achieved by simvastatin alone. CONCLUSION: Targeting of lipid rafts and RhoA may represent a new target for the treatment of CCA.

Overexpression of vitamin D receptor indicates a good prognosis for cholangiocarcinoma

Up-regulation of vitamin D receptor (VDR) expression has been shown in several tumors and is thought to represent an important endogenous response to tumor progression. The authors aimed to verify the expression of VDR and its clinical significance in histologically proven cholangiocarcinoma (CCA). The antiproliferative activity of vitamin D3 on CCA cell lines was explored. The immunohistochemistry of 111 paraffin-embedded CCA tissues showed that VDR expression gradually increased during CCA development. Normal bile duct epithelium rarely expresses VDR, whereas more than 74% of CCA tissues showed positive VDR staining, of which 40% were high. Approximately 80%-90%of CCA patients with papillary and well differentiated adenocarcinomas had positive VDR expression in tumor tissues, whereas 39% positive VDR expression was found in those with poorly differentiated CCAs (P < .001). Expression of VDR was shown to be compatible with an overall favorable prognosis for CCA. Treatment with 1,25(OH)(2)D(3), an active metabolite of vitamin D3, in the CCA cell lines with high expression of VDR significantly reduced cell proliferation in a dose-dependent manner. The effect was not demonstrated in the CCA cell lines that had lower VDR expression. These data indicated an active role for VDR in mediating the antiproliferative effects of 1,25(OH)(2)D(3) in CCA cell lines. VDR expression may constitute an important prerequisite for using vitamin D and/or its analogs in the treatment of CCA. Investigation of a mechanism by which VDR and its ligand mediate these processes is needed to provide the basis for the potential use of this hormone and its derivatives in the prevention and treatment of CCA.

Reply

Our success with liver transplantation for hilar cholangiocarcinoma (CCA) is indeed attributable to neoadjuvant therapy, careful patient selection, and timely transplantation. Neoadjuvant therapy is a critical component of our success. Results with transplantation for both known and incidental CCA (in patients transplanted for primary sclerosing cholangitis) without neoadjuvant therapy are poor (Transplantation 2000;69:1633–1637, Ann Surg 2004;239:265–271, Liver Transplantation 2005;12:1412–1416). We presume that neoadjuvant therapy prevents disease progression while awaiting transplantation and tumor dissemination during the staging and transplant operations. Patient selection and operative staging are of paramount importance. We are aware of several patients with regional lymph node or intrahepatic metastases and one with an incisional biopsy of the primary tumor that received neoadjuvant therapy and liver transplantation at other centers; all died from recurrent disease. Until we have effective therapy for systemic disease, efforts to apply this treatment to patients with advanced disease are doomed to fail. We have also observed tumor seeding along endoscopic ultrasound (EUS)-guided and percutaneous needle aspiration tracts. EUS is helpful to exclude patients with regional lymph node metastases, but transperitoneal aspiration or biopsy of the primary tumor should be avoided in transplant candidates. Widespread necrosis of the hilar bile ducts and vasculopathy are commonly observed in the explanted livers subjected to neoadjuvant therapy. Resection is already a challenging procedure, and attempts to perform a hepaticojejunostomy or an arterial reconstruction after high-dose neoadjuvant therapy are high-risk endeavors. Wider application of this strategy for the treatment of CCA—especially for patients with potentially resectable disease—will require validation of our experience by other centers. We and our colleagues are committed to helping other centers adopt this protocol or similar strategies involving neoadjuvant therapy and liver transplantation for patients with CCA. Transplantation for cholangiocarcinoma: Advance or supply-demand dilemma?GastroenterologyVol. 130Issue 7PreviewRea DJ, Heimbach JK, Rosen CB, Haddock MG, Alberts SR, Kremers WK, Gores GJ, Nagorney DM (Divisions of Gastroenterologic & General Surgery, Transplantation Surgery, Radiation Oncology, Medical Oncology, and Gastroenterology, Hepatology and Nutrition, Mayo Clinic College of Medicine, Rochester, Minnesota). Liver transplantation with neoadjuvant chemoradiation is more effective than resection for hilar cholangiocarcinoma. Ann Surg 2005;242:451–461. Full-Text PDF

Effect of all-trans retinoic acid on tissue dynamics of choriocarcinoma cell lines: an organotypic model

Background: All-trans retinoic acid (ATRA) is a natural vitamin A derivative that has a profound effect on the regulation of cell growth, differentiation and death. Aim: To investigate the tissue...

Drug sensitivity and drug resistance profiles of human intrahepatic cholangiocarcinoma cell lines

To study the effect of a number of chemotherapeutic drugs on five human intrahepatic cholangiocarcinoma (CCA) cell lines. The expressions of genes that have been proposed to influence the resistance of chemotherapeutic drugs including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), glutathione-S-transferase P1 (GSTP1), multidrug resistance protein (MDR1) and multidrug resistance-associated proteins (MRPs) were also determined. Five human CCA cell lines (KKU-100, KKU-M055, KKU-M156, KKU-M214 and KKU-OCA17) were treated with various chemotherapeutic drugs and growth inhibition was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Semi-quantitative levels of gene expression were determined by a reverse transcriptase polymerase chain reaction (RT-PCR). Results of IC(50) values and the ratios of gene expression were analyzed by linear regression to predict their relationship. Among five CCA cell lines, KKU-M055 was the most sensitive cell line towards all chemotherapeutic drugs investigated, particularly taxane derivatives with IC(50) values of 0.02-3 nmol/L, whereas KKU-100 was apparently the least sensitive cell line. When compared to other chemotherapeutic agents, doxorubicin and pirarubicin showed the lowest IC(50) values (<5 mumol/L) in all five CCA cell lines. Results from RT-PCR showed that TS, MRP1, MRP3 and GSTP1 were highly expressed in these five CCA cell lines while DPD and MRP2 were only moderately expressed. It should be noted that MDR1 expression was detected only in KKU-OCA17 cell lines. A strong correlation was only found between the level of MRP3 expression and the IC(50) values of etoposide, doxorubicin and pirarubicin (r = 0.86-0.98, P<0.05). Sensitivity to chemotherapeutic agents is not associated with the histological type of CCA. Choosing of the appropriate chemotherapeutic regimen for the treatment of CCA requires knowledge of drug sensitivity. MRP3 was correlated with resistance of CCA cell lines to etoposide, doxorubicin and pirarubicin, whereas other chemotherapeutic drugs showed no association. The role of this multidrug resistance-associated protein, MRP3, in chemotherapeutic resistance in CCA patients needs to be further investigated.

Immuno-modulating effects of CCA in C3H/He mice

CCA {disodium 4-chloro-2, 2'-imminodibenzoate} is a new compound synthesized and developed by Chugai Pharmaceutical Co., Ltd. CCA has been suggested to have an immuno modulating effect in animals. In this paper, we presented the effect of CCA on the PFC and RFC (rosette forming cell) response for SRBC in vivo. We have already reported that the immunoresponse in C3H/He mice decreased with the age. In this experiment, we used aged C3H/He mice and found that the number of PFC and RFC in the aged C3H/He mice was increased by the treatment of CCA. This results suggest that the decreased immunoresponse in the mice was recovered to normal level by CCA treatment.

Effects of CCA on the production of plaque forming cells in murine spleen

Effects of CCA (disodium 4-chloro-2, 2'-iminobenzoate) on the production of plaque forming cells (PFC) in murine spleens were investigated and the following findings and results were obtained in this paper. (1) CCA had almost no effect on anti SRBC PFC responses at optimal experimental conditions in normal animals but had at some suboptimal condi-tions. (2) In T cell-deficient nude mice, CCA increased the percent of Thy-1+ cells in the spleen and enhanced the product ion of PFC to T-dependent antigen such as SRBC. CCA also showed an enhancing effect on the production of PFC to T-independent antigen, LPS. (3) Induction of suppressor T cells by Con A in the spleen of NZB/W F1 mice in which the activity of these cells are known to be decreased was enhanced by the treatment of CCA. (4) From the above findings. CCA seems to affect both helper and suppressor T cells and B cells. 5 Such a multiple action of CCA on lymphocytes populations may explain the mechanism by which CCA does not affect the normal immune responses but does the hyper-or hypo-immune responses resulting from the deficiency and the impainment of a specific lymphocyte population as observed in the case of nude mice or NZB/W F1 mice.