Biliary Cancer Cell Lines Research Articles

Generation of a biliary tract cancer cell line atlas reveals molecular subtypes and therapeutic targets.

Biliary tract cancers (BTCs) are a group of deadly malignancies encompassing intrahepatic and extrahepatic cholangiocarcinoma, gallbladder carcinoma, and ampullary carcinoma. Here, we present the integrative analysis of 63 BTC cell lines via multi-omics clustering and genome- scale CRISPR screens, providing a platform to illuminate BTC biology and inform therapeutic development. We identify dependencies broadly enriched in BTC compared to other cancers as well as dependencies selective to the anatomic subtypes. Notably, cholangiocarcinoma cell lines are stratified into distinct lineage subtypes based on biliary or dual biliary/hepatocyte marker signatures, associated with dependency on specific lineage survival factors. Transcriptional analysis of patient specimens demonstrates the prognostic significance of these lineage subtypes. Additionally, we delineate strategies to enhance targeted therapies or to overcome resistance in cell lines with key driver gene mutations. Furthermore, clustering based on dependencies and proteomics data elucidates unexpected functional relationships, including a BTC subgroup with partial squamous differentiation. Thus, this cell line atlas reveals potential therapeutic targets in molecularly defined BTCs, unveils biologically distinct disease subtypes, and offers a vital resource for BTC research.

Photodynamic Therapy Using a Novel Phosphorus Tetraphenylporphyrin Induces an Anticancer Effect via Bax/Bcl-xL-related Mitochondrial Apoptosis in Biliary Cancer Cells.

Photodynamic therapy (PDT) uses photosensitizer activation by light of a specific wavelength, and is a promising treatment for various cancers; however, the detailed mechanism of PDT remains unclear. Therefore, we investigated the anticancer effect of PDT using a novel phosphorus tetraphenylporphyrin (Ptpp) in combination with light emitting diodes (Ptpp-PDT) in the NOZ human biliary cancer cell line. Cell viability and apoptosis were examined by MTT assay, flow cytometry and TUNEL assay for 24 hr after Ptpp-PDT. MitoTracker and JC-1 were used as markers of mitochondrial localization and membrane potential. The levels of mitochondrial oxidative phosphorylation (OXPHOS) complexes, Bcl-2 family proteins, cytochrome c and cleaved caspase-3 were examined by western blotting and immunohistochemistry. The results revealed that Ptpp localized to mitochondria, and that Ptpp-PDT efficiently decreased cell viability in a dose- and time-dependent manner. JC-1 and OXPHOS complexes decreased, but apoptotic cells increased from 6 to 24 hr after Ptpp-PDT. A decrease in Bcl-xL and increases in Bax, cytochrome c and cleaved caspase-3 were also found from 6 to 24 hr after Ptpp-PDT. Based on these results, we conclude that Ptpp-PDT induces anticancer effects via the mitochondrial apoptotic pathway by altering the Bax/Bcl-xL ratio, and could be an effective treatment for human biliary cancer.

Promising Gene Therapy Using an Adenovirus Vector Carrying REIC/Dkk-3 Gene for the Treatment of Biliary Cancer.

We previously demonstrated that the reduced expression in immortalized cells (REIC)/dikkopf-3 (Dkk-3) gene was downregulated in various malignant tumors, and that an adenovirus vector carrying the REIC/Dkk-3 gene, termed Ad-REIC induced cancer-selective apoptosis in pancreatic cancer and hepatocellular carcinoma. In this study, we examined the therapeutic effects of Ad-REIC in biliary cancer using a second- generation Ad-REIC (Ad-SGE-REIC). Human biliary cancer cell lines (G-415, TFK-1) were used in this study. The cell viability and apoptotic effect of Ad-SGE-REIC were assessed in vitro using an MTT assay and Hoechst staining. The anti-tumor effect in vivo was assessed in a mouse xenograft model. We also assessed the therapeutic effects of Ad-SGE-REIC therapy with cisplatin. Cell signaling was assessed by Western blotting. Ad-SGE-REIC reduced cell viability, and induced apoptosis in biliary cancer cell lines via the activation of the c-Jun N-terminal kinase pathway. Ad-SGE-REIC also inhibited tumor growth in a mouse xenograft model. This effect was further enhanced in combination with cisplatin. Ad-SGE-REIC induced apoptosis and inhibited tumor growth in biliary cancer cells. REIC/Dkk-3 gene therapy using Ad-SGE-REIC is an attractive therapeutic tool for biliary cancer.

5-Aminolevulinic-acid-mediated Photodynamic Diagnosis Enhances the Detection of Peritoneal Metastases in Biliary Tract Cancer in Mice.

Previous studies on the accuracy of 5-aminolevulinic-acid-mediated photodynamic diagnosis (5-ALA PDD) have been reported for various cancers and brain surgery. However, biliary tract cancer is rare. Therefore, 5-ALA PDD has not been fully evaluated in biliary tract cancers. Small biliary tract cancer lesions such as peritoneal dissemination, liver metastases, and lymph node metastases are negative prognosticators in patients with biliary cancer. The purpose of this exploratory study was to determine if 5-ALA PDD could detect small biliary tract cancer lesions in murine models of biliary cancers. Biliary cancer cell lines (TFK-1, HuCCT-1, G415, HuH28, SSP25, RBE, KKU055 and KKU100) and Normal human dermal fibroblast cells were used to evaluate protoporphyrin IX (PpIX) accumulation in vitro. Subcutaneous tumor mice were established using two cell lines (TFK-1 and HuCCT-1). 5-ALA (250 mg/kg) was administered intraperitoneally, and fluorescent 5ALA-PDD was performed 3 h later to evaluate tumoral PpIX accumulation. A murine peritoneal disseminated nodule model was established by intraperitoneal injection of TFK-1 cells. Four weeks later, 5-ALA was administered intraperitoneally, and 5-ALA-PDD was performed 3 h post administration to evaluate PpIX accumulation in the disseminated nodules. The presence of tumor cells in tumors and nodules was confirmed by haematoxylin and eosin staining. Compared TO non-cancerous cell lines, PpIX accumulation was increased in biliary tract cancer cell lines. PpIX accumulation led to a strong fluorescent signal in all subcutaneous tumors. In the murine model of peritoneal dissemination, microdisseminated nodules (<1 mm) that could not be detected under white light were clearly visible using 5-ALA-PDD. 5-ALA PDD was useful for diagnosis of biliary tract cancer and detection of small peritoneal metastatic lesions in murine models of biliary cancers. Clinical studies and applications of 5-ALA PDD for biliary tract cancer are expected in the future.

Photodynamic therapy of human biliary cancer cell line using combination of phosphorus porphyrins and light emitting diode

A series of phosphorus porphyrin complexes ([(RO)2P(tpp)]Cl, tpp = tetraphenylporphyrinato group, R = −(CH2CH2O)m(CH2)nH; 1a: m = 2, n = 2; 1b: m = 2, n = 4; 1c: m = 2, n = 6; 1d: m = 3, n = 6) were used for the photodynamic therapy (PDT) of human biliary cancer cell line (NOZ) when exposed to the irradiation of light emitting diodes (LEDs). A Dulbecco’s modified Eagle’s medium (DMEM) containing NOZ cells (2000 cell well−1) and 1 (0–100 nM) was introduced into a 96-well microplate and incubated for 24 h to accumulate 1 into the NOZ cells and to multiply the NOZ cells until the cell number reached 104 cells well−1. After replacing the DMEM medium containing 1 with a fresh DMEM medium without 1, the plates were irradiated for 30 min at 610 nm. After incubation was performed for 24 h in dark conditions, the cell viability of the NOZ cells was determined using the MTT assay. The half maximum inhibitory concentrations 50 (IC50) of 1a–1d were found to be in the range of 33.7–58.7 nM for NOZ. These IC50 values for the NOZ were one hundredth the IC50 value (7.57 μM) for mono-l-aspartyl chlorin e6 (laserphyrin®). Thus, it was found that the PDT activity of 1a–1d was much higher than the mono-l-aspartyl chlorin e6. Similarly, IC50 vales of 1a–1d for HeLa cells were found to be 27.8–52.5 nM. This showed that 1a–1d had high photodynamic activity in cancer cells. At the same time, it was speculated that an LED is a useful light source for deactivating the cancer cells because it can excite the sensitizers with peak width in their absorption spectra using the light of the specified wave length with band width of 10–20 nm; LEDs provide a homogeneous light distribution for the target cells.

Fluorescence In Situ Hybridization with the UroVysion Kit for the Detection of Biliary Cancer in Chinese Patients.

Conventional biliary brush via ERCP has low clinical detection for biliary malignancy. Therefore, new approaches are needed to facilitate diagnosis. We therefore explored the application of fluorescent in situ hybrization (FISH) using a UroVysion kit for the detection of malignancy in the bile duct. Genetic alterations of target chromosomes such as aneuploidy in Chinese biliary cancer cell lines and tissues were measured using a UroVysion kit. The diagnostic value of the FISH assay was assessed by probing 27 brush samples of biliary cytology and control routine cytology (RC) samples. The gold standard was established by the pathology or clinical outcomes at the 12-month follow-up. Aneuploidy is commonly found in cell lines and tissues of biliary cancers, but not in normal cells or tissues. Here we probed for aneuploidy in clinical biliary brush specimens obtained by ERCP using FISH and a UroVysion kit. The sensitivity, specificity, and positive and negative predictive values for biliary malignancy were found to be 50%, 100%, 100% and 31.3%, respectively. The sensitivity, specificity, and positive and negative predictive values by RC were found to be 22.7%, 100%, 100% and 22.7%, respectively. In combination with RC, FISH increased the diagnostic sensitivity to 63.6% although this difference was not found to be statistically significant. Aneuploidy is frequently present in bile duct carcinomas. Here we found that the FISH assay is useful for the detection of Chinese biliary cancers.

Clinical implications of APEX1 and Jagged1 as chemoresistance factors in biliary tract cancer

PurposeBiliary cancer is a highly malignant neoplasm with poor prognosis and most patients need to undergo palliative chemotherapy, however major clinical problem associated with the use of chemotherapy is chemoresistance. So far, we aimed at investigating clinical implications of apurinic/apyrimidinic endodeoxyribonuclease 1 (APEX1) and Jagged1 as chemoresistance factors in biliary tract cancer.MethodsWe used 5 human biliary tract cancer cell lines (SNU-245, SNU-308, SNU-478, SNU-1079, and SNU-1196), and investigated the chemosensitivity of APEX1 and Jagged1 through 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and Western blot. Alternately, the 10 patients of advanced biliary cancer consist of 2 group according to the chemotherapy response examined by immunohistochemistry using APEX1 and Jagged1 antibody, and protein expression level was scored for staining intensity and percent positive cell.ResultsThe result of MTT assay after APEX1 knockdown showed that strong coexpression of APEX1 and Jagged1 cell line (SNU-245, SNU-1079, and SNU-1196) showed a greater decrease in IC50 of chemotherapeutic agent (5-fluorouracil, gemcitabine and cisplatin). The Western blot analysis of APEX1 and Jagged1 expression in biliary cancer cell lines after APEX1 knockdown definitively demonstrated decreased Jagged1 expression. The APEX1 and Jagged1expression level of immunohistochemistry represented that chemorefractory patients had higher than chemoresponsive patients.ConclusionThese results demonstrate that simultaneous high expression of APEX1 and Jagged1 is associated with chemoresistance in biliary cancer and suggest that is a potential therapeutic target for chemoresistance in advanced biliary cancer.

Abstract 259: Novel biliary tract cancer stem cell biomarker TAGLN2 regulates migration via c-met signaling cascades in biliary tract cancer cells.

Abstract Background/Aims : Biliary tract cancer is one of the lethal malignancy. Most biliary tract cancer is diagnosed at advanced stages and have poor prognosis. Currently, there are no standard biomarkers and chemotherapeutic agents for biliary tract cancers. In order to find the biomarkers and therapeutic targets for biliary tract cancer, we have focused on the cancer stem cells (CSCs) with tumor initiating/self renewing/metastatic capacity and chemo/radio-resistance. In this study, we investigated the function of biliary tract CSC related biomarker TALGN2 discovered from comparision of cDNA microarray between sphere and adherent cell populations of biliary tract cancer cell lines. Methods : Analysis using cDNA microarray between sphere and adherent cell populations and selection of CSC specific biomarkers were performed. Among them, we selected TAGLN2 as a biliary CSC related gene. TAGLN2 expression was confirmed by RT-PCR, western blot and immunohistochemistry. To evaluate the function of TAGLN2 in biliary carcinogenesis, we performed targeted knockdown via siRNA in SNU1196 biliary tract cancer cell line. Results : The expression of stemness and c-met signaling cascades were up-regulated in sphere cells compared with adherent cells. TAGLN2 was also up-regulated in sphere cells. TAGLN2 was highly expressed in most of biliary tract cancer cell lines and biliary tract cancer tissues. TAGLN2 knockdown cells showed decreased cell migration and increased chemosentitivity compared with mock cells. In addition, we found that c-met signaling cascades including PI3K and MAPK were inactivated in TAGLN2 knockdown cells. Conclusions : These results suggest that biliary tract CSC biomarker TAGLN2 is implicated in biliary tract cancer progression and TAGLN2 may be a potential biomarker and therapeutic target for biliary tract cancer. Citation Format: Soo Been Park, Sun A Kim, Si Young Song. Novel biliary tract cancer stem cell biomarker TAGLN2 regulates migration via c-met signaling cascades in biliary tract cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 259. doi:10.1158/1538-7445.AM2013-259

Abstract 934: MEK162, an allosteric MEK inhibitor promotes apoptosis and in vivo antitumor activity against human biliary cancer cell lines.

Abstract Biliary cancer (BC), or cholangiocarcinoma, is derived from the malignant transformation of epithelial cells in the bile ducts. The disease usually presents at advanced stages and is almost uniformly deadly. Current therapeutic regimens have limited efficacy for treatment of BC, thus the need for developing novel and effective targeted strategies is a priority. This malignancy is characterized by deregulated PI3 kinase/Akt signaling, upregulated mitogen-activated protein kinase (MAPK) pathway and an autocrine IL-6/STAT3 signal transduction feedback loop, which can also drive immunosuppression. A recent report showed clinical responses for BC patients receiving the MEK inhibitor AZD244, which promoted more extensive pre-clinical studies to validate the effect of MEK inhibition and importance of this pathway in BC. In the present study, MEK162, a MEK inhibitor, was used to assess the growth inhibitory, pro-apoptotic, and immunomodulatory effects in BC. In vitro studies in a panel of human BC cell lines (HuCCT1, HuH28, MzCha1) revealed that MEK162 significantly inhibited cell growth and induced apoptosis in a concentration-dependent manner in all three cell lines. Immunoblot data confirmed decreased phosphorylated ERK (pERK) in the HuCCT1 BC cell line following a 24 hour treatment with MEK162. MEK inhibition was also associated with clinically-relevant immunomodulatory effects. First, culture supernatants from human BC cells displayed significant reductions in IL-6 protein by ELISA after treatment with MEK162 at varying concentrations. Second, MEK162 inhibited in vitro generation of human myeloid derived suppressor cells from peripheral blood mononuclear cells cultured with IL-6 and GM-CSF. Finally, in vivo studies in athymic mice bearing HuCCT1 xenografts showed that daily oral administration of MEK162 (30 mg/kg in 1% (w/v) carboxymethylcellulose, 0.5% (v/v) Tween 80) was well-tolerated and resulted in significant growth inhibition as compared to vehicle-treated controls (p &amp;lt; 0.0001). Immunohistochemical analysis of tumor xenografts revealed significant decreases in both Ki67 and pERK staining in the MEK162-treated mice as compared to controls (p = 0.0163 and 0.0132,respectively). Together, these data suggest that MEK inhibition represents a promising therapeutic approach with potential for direct antitumor activity and immunomodulation in BC. Citation Format: Jennifer Yang, Thomas A. Mace, Gergory S. Young, Patrice A. Lee, Kaitlin E. Keenan, Zheng Che, Tonios Bekaii-saab, Gregory B. Lesinski. MEK162, an allosteric MEK inhibitor promotes apoptosis and in vivo antitumor activity against human biliary cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 934. doi:10.1158/1538-7445.AM2013-934

Methylated Bone Morphogenetic Protein 3 (BMP3) Gene: Evaluation of Tumor Suppressor Function and Biomarker Potential in Biliary Cancer.

BackgroundAlthough cholangiocarcinoma (CC) is an uncommon and highly lethal malignancy, early detection enables the application of potentially curative therapies and improves survival. Consequently, tools to improve the early diagnosis of CC are urgently needed. During a screen for genes epigenetically suppressed by methylation in CC that might serve as methylation markers for CC, we found that the BMP3 gene is methylated in CC cell lines, but the potential diagnostic value and the function of BMP3 in CC are unknown.MethodsWe aimed to quantitatively assess BMP3 methylation in resected CC tumor specimens using methylation specific PCR and evaluate the tumor suppressor role of BMP3 in biliary cancer cell lines in comparison to an immortalized normal cholangiocyte cell line. Expression of BMP3 was quantified by mRNA levels before and after treatment with 5-Aza-2’-deoxycytidine and trichostatin A. After transfection with a BMP3-containing plasmid, cell viability was measured using the bromodeoxyuridine incorporation assay and apoptosis quantified by caspase assay.ResultsIn primary CC tumor tissue specimens significantly more methylated BMP3 copies were found when compared to matched benign bile duct epithelium from the same patient, with high specificity. BMP3 expression was absent in cell lines with BMP3 methylation; this suppression of BMP3 expression was reversed by treatment with a DNA demethylating agent and histone de-acetylase inhibitor. Transfection of a BMP3-expressing construct into a BMP3-negative biliary cancer cell line restored BMP3 mRNA expression and reduced cell proliferation and cell viability while increasing the rate of apoptosis.ConclusionThese findings strongly support a tumor suppressor role for BMP3 in CC and suggest that BMP3 methylation may be a new biomarker for early detection of CCs. of the peptidome are also involved.

Preclinical study of MEK1/2 inhibitor AZD6244 combined with gemcitabine for treatment of biliary cancer.

240 Background: MEK1/2 is an integral component of the Ras/Raf/MEK/ERK signaling pathway, implicated in uncontrolled cell proliferation and cell survival, a key hallmark of cancer. AZD6244, a novel inhibitor of MEK1/2, is currently completing Phase II clinical trials in biliary cancer, with modest antitumor activity observed as monotherapy. Gemcitabine is a cytotoxic drug commonly used in biliary cancer therapy but many patients showed early resistance. In this preclinical study, we investigated the sequence-dependent antitumor effects of AZD6244 combined with gemcitabine in biliary cancer models. Methods: Two biliary cancer cell lines (EGI-1 and TFK-1) were used. In vitro the effects of single drug or three combination protocols(concurrently; AZD6244 followed by GEM or Gem followed by AZD6244) on cell proliferation, DNA synthesis, and cell cycle distribution were evaluated by MTS, clonogenic assay, EdU uptake and flow cytometry. Drug interactions were analyzed by Chou-Talaly method. In vivo, 4 tumor models subcutaneously xenografted in SCID mice from the two cell lines and 2 human patients were set up to compare the therapeutic effects of different sequence-scheduled combinations. Results: AZD6244 caused G1-S cell cycle arrest in biliary cancer cells in vitro and in vivo, and this effect is correlated with the MEK/ERK signaling pathway blocking. Synchronized progression of the population through S phase were observed in 15h after removal of AZD6244 in cell culture or 48h after final dose of acute AZD6244 treatment in vivo. Antagonistic or additive effects was observed in vitro when combination were given as concurrently(CI=2.03~2.46) or Gem followed by AZD6244(CI=1.34~1.78). In contrast, a synergistic antiproliferative activity was obtained when AZD6244 was given first followed by a drug-free interval before Gem treatment (CI=0.53~0.69). In vivo, the best therapeutic effects were obtained with the sequence of AZD6244 followed by Gem, compared with concurrent or reverse sequence. Conclusions: This study provides a sound rationale for a Phase II trial of a potentially synergistic sequence of MEK inhibitor AZD6244 followed by gemcitabine in patients with advanced biliary cancer.

Protection against Cholestasis and Ischemia in vitro by Ursodeoxycholyl Lysophosphatidylethanolamide (UDCA-LPE) is Associated with Upregulation of Anti-apoptotic Proteins

Introduction: An increase of toxic bile acids such as glycochenodeoxycholic acid (GCDCA) frequently occurs in an early phase of liver transplantation. Ischemia during transplantation can cause hepatic cholestasis and damage of intrahepatic biliary epithelial cells. Both stresses contribute to the failure in liver transplantation. We aim to test the effectiveness of UDCA-LPE in inhibition of apoptosis induced by GCDCA and ischemia. Methods: Mouse hepatocytes were isolated and treated with 50µM GCDCA for 15h. As an experimental model of ischemia, CoCl2 at 500–750µM was used to treat the intrahepatic biliary epithelial cancer cell line MzChA1 for 24h. Protein expression of pro- and anti-apoptosis proteins and signalling molecules was measured by Western blot. The effect of UDCA-LPE on protein turnover was studied by immunoprecipitation and immunoblotting. Caspase 3 and 8 activities were measured by luminescence spectrometry. Results: In mouse hepatocytes and MzChA1, apoptosis was induced as measured by cleaved PARP–1, caspase 3 and 8 proteins and activities. Co-treatment with UDCA-LPE markedly inhibited GCDCA- and CoCl2-induced apoptosis. Expression of anti-apoptotic protein Mcl–1, cIAP2 and cFlip was decreased with CoCl2 treatment, and UDCA-LPE co-treatment prevented this decrease. By measuring ubiquitinated proteins, we found that UDCA-LPE inhibited the degradation of Mcl–1, but not of cIAP2 and cFlip proteins. This inhibition by UDCA-LPE was concomitant with phosphorylation of GSK–3α/β, which is a known mediator for Mcl–1 stabilization. Conclusion: The effect of UDCA-LPE on inhibition of Mcl–1 ubiquitination via phosphorylation of GSK–3α/β is one of the mechanisms for UDCA-LPE protection against apoptosis during bile acid and ischemic stresses. Further work is needed to investigate transcriptional control of cFlip and cIAP2 proteins by UDCA-LPE. UDCA-LPE may thus be used as an agent to prevent complications associated with liver transplantation.

Loss of RUNX3 expression by histone deacetylation is associated with biliary tract carcinogenesis

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region frequently inactivated through hypermethylation, histone modulation, and other processes in various human tumors. In this study, to elucidate a causal link between RUNX3 expression and biliary tract cancer, we investigated 17 human biliary cancer specimens. In addition, to examine roles of RUNX3 in biliary tract cancer, we restored silenced RUNX3 in the human biliary cancer cell line Mz-ChA-2 using a histone deacetylase inhibitor. Thirteen of 17 human cancer specimens exhibited suppressed RUNX3 expression compared with normal biliary ducts. Moreover, the decreased RUNX3 expression was related to a lower accumulation of acetylated histone H3 associated with RUNX3. In in vitro experiments, vorinostat, a member of a new class of highly potent histone deacetylase inhibitors, restored RUNX3 expression in Mz-ChA-2 cells. Furthermore, vorinostat-induced RUNX3 significantly enhanced p21 expression and growth inhibition of Mz-ChA-2 cells through restoration of TGF-β signaling. These data suggest the significance of histone deacetylation-associated suppression of RUNX3 expression in biliary tract carcinogenesis. Furthermore, vorinostat might hold promise for treating biliary tract cancer through enhancement of TGF-β signaling by restoration of RUNX3.

Comparative characterization of the efficiency and cellular pharmacokinetics of Foscan®- and Foslip®-based photodynamic treatment in human biliary tract cancer cell lines

Due to the poor prognosis and limited management options for perihilar cholangiocarcinoma (CC) the development of alternatives for treatment is an important topic. Photodynamic therapy (PDT) with porfimer as palliative or neoadjuvant endoscopic treatment of non-resectable perihilar CC has improved quality of life and survival time, but cannot eradicate the primary tumors because of inadequate tumoricidal depth (4 mm only around the tumor stenoses). The use of meta-tetrahydroxyphenyl chlorin (mTHPC) and photoactivation at higher wavelengths (650-660 nm) provides high tumoricidal depth (10 mm) for PDT of pancreatic cancer and should yield similar tumoricidal depth in CC. This study investigates the photodynamic characteristics of mTHPC in solvent-based formulation (Foscan) and in liposomal (water soluble) formulation (Foslip) in an in vitro model system consisting of two biliary cancer cell lines (GBC, gall bladder cancer and BDC, bile duct cancer cells). Dark toxicity, photodynamic efficiency, time-dependent uptake and retention and intracellular localization of Foscan and Foslip were studied. The results prove mTHPC as a potent photosensitizing agent with high phototoxic potential in biliary cancer cells as a concentration of 600 ng ml(-1) and irradiation with 1.5 J cm(-2) (660 +/- 10 nm) is sufficient for about 90% cell killing. Addition of foetal bovine serum (FBS) to the incubation medium and analysis of the uptake and phototoxic properties reveals that both photosensitizer formulations bind to serum protein fractions, i.e. no difference between Foscan and Foslip can be found in the presence of FBS. Laser scanning fluorescence microscopy indicates a similar pattern of perinuclear localization of both sensitizers. This study demonstrates the potential of mTHPC for treatment of bile duct malignancies and provides evidence that Foslip is an equivalent water-soluble formulation of mTHPC that should ease intravenous application and thus clinical use of mTHPC.

E1A, E1B Double-Restricted Adenovirus with RGD-Fiber Modification Exhibits Enhanced Oncolysis for CAR–Deficient Biliary Cancers

Cancers of biliary system represent highly malignant diseases of dismal prognosis. We have previously introduced AxdAdB3, an E1A, E1B double-restricted oncolytic adenovirus, which showed excellent oncolytic efficacy for approximately half of the biliary cancer lines with an enhanced safety to normal cells. The purpose of this study was to evaluate whether RGD-fiber modification (AxdAdB3-F/RGD), which enables integrin-dependent infection, can improve the infectivity and efficacy of AxdAdB3 for biliary cancers. Expressions of adenoviral receptors, coxsackievirus adenovirus receptor (CAR) and integrins (alpha(v)beta(3) and alpha(v)beta(5)), were compared with the level of infectivity of LacZ-expressing replication-defective adenoviruses with wild-type fibers or RGD-modified fibers in a panel of biliary cancer cell lines in vitro. Viral replication and cytotoxicity in vitro of AxdAdB3-F/RGD, a novel E1A, E1B double-restricted replication-selective adenovirus with RGD-modified fibers, were compared with those of its parent virus, AxdAdB3, in various biliary cancer cells and in normal cells. In vivo antitumor effects of these oncolytic viruses were compared in a xenograft tumor model. Expression of CAR significantly correlated with the adenovirus infectivity, whereas integrin alpha(v)beta(5) was abundantly expressed in almost all biliary cancer cells. Whereas AxdAdB3 effectively replicated and lysed only the biliary cancer cells with a preserved expression of CAR, AxdAdB3-F/RGD exhibited efficient replication and potent oncolysis in both CAR-positive and CAR-negative biliary cancer cells. AxdAdB3-F/RGD showed attenuated replication and little cytopathy in human normal cells (i.e., hepatocytes, WI-38 cells) as well as AxdAdB3. Furthermore, in nude mice with s.c. xenografts of CAR-deficient human biliary cancer, i.t. AxdAdB3-F/RGD therapy caused a marked inhibition of tumor growth. The RGD-fiber modification strategy enhanced the infectivity, replication, and oncolytic effects of the E1A, E1B double-restricted oncolytic adenovirus for CAR-deficient biliary cancers. In addition, it preserved the merit of excellent safety of the double-restricted virus for normal cells. These results suggest a potential use of this agent for the treatment of biliary cancers.

Restoration of RUNX3 enhances transforming growth factor‐β‐dependent p21 expression in a biliary tract cancer cell line

RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)-beta signaling in biliary tract cancer, we transfected Mz-ChA-2 cells, which do not express RUNX3 but have intact TGF-beta type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz-ChA-2/RUNX3 clones and one control clone were obtained. Although TGF-beta1 only slightly inhibited growth of the control cells, growth inhibition and TGF-beta-dependent G(1) arrest were significantly enhanced in the RUNX3-transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF-beta1-induced p21 expression in the control clone was strongly enhanced in the RUNX3-transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF-beta-dependent induction of p21 was reduced in the RUNX3-transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3-transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF-beta-induced expression of p21 and the resulting induction of TGF-beta-dependent G(1) arrest.

Dual mitogen-activated protein kinase and epidermal growth factor receptor inhibition in biliary and pancreatic cancer

This study aimed to develop rational combinations of targeted agents against biliary and pancreatic cancers. To this end, we compared the global gene expression profile of biliary cancer cell lines with different degrees of sensibility to the epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib using the Affymetrix U133A microarray platform. A set of 32 genes, including genes involved in signal transduction pathways, cell cycle regulation, and angiogenesis, was highly overexpressed in resistant cells. Five of these genes encoded proteins in the Ras/Raf/mitogen-activated protein kinase (MAPK) pathway, a finding that was confirmed by Western blot and immunohistochemistry. Gefitinib failed to inhibit the MAPK pathway in resistant cell lines. Based on these data, we explored the activity of dual treatment with gefitinib in combination with CI-1040, a MAPK inhibitor. This strategy effectively resulted in inhibition of the MAPK signaling pathway and exerted antitumor effects in vitro and in vivo in tumors resistant to each of the agents alone. To further confirm these results, we tested the combined treatment in four tumor xenografts generated from patients with resected pancreatic cancer. Combined treatment was more effective than either single agent alone in this model. This study illustrates the value of global analysis of gene expression to rationally design combinations of mechanistic-based drugs. In addition, the data support the efficacy of combined epidermal growth factor receptor and MAPK inhibitors in biliary and pancreatic cancers, providing the basis to test this combination in the clinic.

Detection of Human Aspartyl (Asparaginyl) Beta-Hydroxylase and Homeobox B7 mRNA in Brush Cytology Specimens from Patients with Bile Duct Cancer

The diagnosis of bile duct cancer is hampered by the low sensitivity of intraductal brush cytology and forceps biopsy. In the present study real-time reverse transcription polymerase chain reaction (RT-PCR) assays for the detection of human aspartyl (asparaginyl) beta-hydroxylase (HAAH) and homeobox B7 (HoxB7) mRNA from intraductal brush cytology specimens were established. Both markers are overexpressed in biliary cancer cell lines and possibly involved in the pathogenesis of bile duct cancer. RT-PCR assays were validated for detection limit, in-assay variability, and inter-assay variability. Target gene expression was determined in brush cytology specimens from 16 patients with biliary strictures (11 with histologically proven cholangiocarcinomas and five with benign biliary strictures). The assay was quick (about 3 h), highly sensitive (with detection limits between 3 and 106 molecules), and reproducible (maximum in-assay variability 10.3 %, maximum inter-assay variability 11.8 %). The sensitivity of routine brush cytology alone was 36 % (four of 11 cases), with 100 % specificity. A combination with detection of HoxB7 and HAAH mRNA increased the overall diagnostic sensitivity to 82 %. Detection of these markers using the RT-PCR assays from brush cytology specimens described here may prove to be a useful additional tool for the diagnosis of bile duct carcinoma.

Interleukin-2 gene-encoded stromal cells inhibit the growth of metastatic cholangiocarcinomas

To demonstrate bone marrow stromal cells (BMSCs) can be used as an attractive target for genetic modification in the treatment of malignant diseases. Using a hamster model of biliary cancer, we investigated the therapeutic effects of interleukin-2 (IL-2) gene-modified BMSCs. Syrian golden hamsters were injected via the femoral vein with 5 x 10(5) cells of the KIGB-5 biliary cancer cell line (n=20). One week later, the hamsters were injected intraperitoneally with BMSCs containing Ad/hIL-2 and Ad/ Delta E1, unmodified BMSCs, or RPMI only (control) and observed for 12 wk (n=5 /each group). All hamsters treated with BMSCs containing Ad/hIL-2 survived with no evidence of the disease during this period. In contrast, hamsters in the other three groups showed disseminated metastases involving the lungs as early as 4 wk. Ad/IL-2 therapy is effective in the treatment of biliary cancer.

Homozygous deletions of methylthioadenosine phosphorylase in human biliary tract cancers

The p16(INK4A)/CDKN2A gene on chromosome 9p21 is a site of frequent allelic loss in human cancers, and in a subset of cases, homozygous deletions at this locus encompass the telomeric methylthioadenosine phosphorylase (MTAP) gene. The MTAP gene product is the principal enzyme involved in purine synthesis via the salvage pathway, such that MTAP-negative cancers are solely dependent on de novo purine synthesis mechanisms. Inhibitors of the de novo pathway can then be used to selectively blockade purine synthesis in cancer cells while causing minimal collateral damage to normal cells. In this study, we determine that 10 of 28 (35%) biliary tract cancers show complete lack of Mtap protein expression. In vitro analysis using a selective inhibitor of the de novo purine synthesis pathway, L-alanosine, shows robust growth inhibition in MTAP-negative biliary cancer cell lines CAK-1 and GBD-1 accompanied by striking depletion of intracellular ATP and failure to rescue this depletion via addition of exogenous methylthioadenosine, the principal substrate of the MTAP gene product; in contrast, no significant effects were observed in MTAP-expressing HuCCT1 and SNU308 cell lines. Colony formation studies confirmed that L-alanosine reduced both number and size of CAK-1 colonies in soft agar assays. Knockdown of Mtap protein by RNA interference in L-alanosine-resistant HuCCT1 cells conferred sensitivity to this agent, confirming that intracellular Mtap protein levels determine response to L-alanosine. Inhibitors of de novo purine synthesis can be a potential mechanism-based strategy for treatment of biliary tract cancers, one third of which show complete loss of MTAP function.