Biliary Cancer Cell Research Articles

The Potential of Narrow-Band Imaging as a Novel Light Source for Photodynamic Therapy for Superficial Cancers via Endoscopes.

Photodynamic therapy (PDT) has advanced through the utilization of photosensitizers and specific-wavelength light (≥ 600 nm). However, the widespread adoption of PDT is still impeded by high equipment costs and stringent laser safety requirements. Porphyrins, crucial in PDT, have another absorbance peak of blue light (λ = 380 - 500 nm). This peak corresponds to the wavelength of narrow-band imaging (NBI) (λ = 390 - 445 nm), an image-enhancement technology integrated into endoscopes by Olympus Medical Systems. The study aimed to investigate the potential of widely adopted NBI as a PDT light source for superficial cancers via endoscopes. Esophageal and biliary cancers were selected for investigation. Human esophageal cancer cell lines (KYSE30, KYSE70, KYSE170) and cholangiocarcinoma cell lines (HuCCT-1, KKU-213) were subjected to verteporfin-mediated PDT under NBI light (λ = 390 - 445 nm). Assessments included spectrometry, crystal violet staining, and fluorescein imaging of singlet oxygen generation and apoptosis. Verteporfin exhibited a peak (λ = 436 nm) consistent with the NBI spectrum, suggesting compatibility with NBI light. NBI light significantly inhibited the growth of esophageal and biliary cancer cells. The half-maximum effective concentration (EC50) values (5 J/cm2) for KYSE30, KYSE70, KYSE170, HuCCT-1, and KKU-213 were calculated as 2.78 ± 0.37µM, 1.76 ± 1.20 µM, 0.77 ± 0.16 µM, 0.65 ± 0.18 µM, and 0.32 ± 0.04 µM, respectively. Verteporfin accumulation in mitochondria, coupled with singlet oxygen generation and observed apoptotic changes, suggests effective PDT under NBI light. NBI is a promising PDT light source for superficial cancers via endoscopes.

Immunotherapy in Gastrointestinal Cancers.

Immunotherapy has revolutionised cancer treatment over the past decade. Long-term durable responses can be achieved in some cancer patient populations that were previously facing terminal disease. In this chapter, we summarise current phase 3 clinical trial evidence for the use of immunotherapy in gastrointestinal cancers (oesophageal squamous cell carcinoma, oesophago-gastric adenocarcinoma, pancreatic cancer, biliary cancer, hepatocellular carcinoma, colorectal cancer, and squamous cell cancer of the anus). We discuss meaningful biomarkers used in clinical trials to select patients most likely to benefit from immunotherapy, such as mismatch-repair deficiency (MMRd)/microsatellite instability (MSI) and programmed-death-ligand-1 (PD-L1) immunohistochemistry (IHC) expression. Clinical questions are arising regarding the role of immunotherapy in the adjuvant/perioperative setting, optimal timing of surgery in patients who respond to immunotherapy, and toxicities specific to patients with gastrointestinal malignancies. We outline the current landscape and future horizon of immunotherapy in gastrointestinal cancers, such as strategies to increase effectiveness of checkpoint blockade through combinations with other checkpoint inhibitors, cytotoxic chemotherapy, targeted agents, radiotherapy, CAR-T therapy, and cancer vaccines.

Simultaneous activation of Kras-Akt and Notch pathways induces extrahepatic biliary cancer via the mTORC1 pathway.

Biliary tract cancer (BTC) has poor prognosis. The Notch receptor is aberrantly expressed in extrahepatic cholangiocarcinoma (eCCA). However, the role of Notch signaling in the initiation and progression of eCCA and gallbladder (GB) cancer remains unknown. Therefore, we investigated the functional role of Notch signaling during tumorigenesis of the extrahepatic bile duct (EHBD) and GB. Activation of Notch signaling and oncogenic Kras resulted in the development of biliary intraepithelial neoplasia (BilINs) in the EHBD and GB, which were premalignant lesions that progressed to adenocarcinoma in mice. The expression of genes involved in the mTORC1 pathway was increased in biliary spheroids from Hnf1b-CreERT2; KrasLSL-G12D ; Rosa26LSL-NotchIC mice and inhibition of the mTORC1 pathway suppressed spheroid growth. Additionally, simultaneous activation of the PI3K-AKT and Notch pathways in EHBD and GB induced biliary cancer development in mice. Consistent with this, we observed a significant correlation between activated NOTCH1 and phosphorylated Ribosomal Protein S6 (p-S6) expression in human eCCA. Furthermore, inhibition of the mTORC1 pathway suppressed the growth of Notch-activated human biliary cancer cells in vitro and in vivo. Mechanistically, the Kras/Notch-Myc axis activated mTORC1 through TSC2 phosphorylation in mutant biliary spheroids. These data indicate that inhibition of the mTORC1 pathway could be an effective treatment strategy for Notch-activated human eCCA. © 2023 The Pathological Society of Great Britain and Ireland.

Sorafenib Chemosensitization by Caryophyllane Sesquiterpenes in Liver, Biliary, and Pancreatic Cancer Cells: The Role of STAT3/ABC Transporter Axis.

A combination of anticancer drugs and chemosensitizing agents has been approached as a promising strategy to potentiate chemotherapy and reduce toxicity in aggressive and chemoresistant cancers, like hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and pancreatic ductal adenocarcinoma (PDAC). In the present study, the ability of caryophyllane sesquiterpenes to potentiate sorafenib efficacy was studied in HCC, CCA, and PDAC cell models, focusing on the modulation of STAT3 signaling and ABC transporters; tolerability studies in normal cells were also performed. Results showed that the combination of sorafenib and caryophyllane sesquiterpenes synergized the anticancer drug, especially in pancreatic Bx-PC3 adenocarcinoma cells; a similar trend, although with lower efficacy, was found for the standard ABC transporter inhibitors. Synergistic effects were associated with a modulation of MDR1 (or Pgp) and MRP transporters, both at gene and protein level; moreover, activation of STAT3 cascade and cell migration appeared significantly affected, suggesting that the STAT3/ABC-transporters axis finely regulated efficacy and chemoresistance to sorafenib, thus appearing as a suitable target to overcome drawbacks of sorafenib-based chemotherapy in hepato-biliary-pancreatic cancers. Present findings strengthen the interest in caryophyllane sesquiterpenes as chemosensitizing and chemopreventive agents and contribute to clarifying drug resistance mechanisms in HCC, CCA, and PDAC cancers and to developing possible novel therapeutic strategies.

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

Background and AimsChanges in single microRNA (miRNA) expression have been associated with chemo‐resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy.Approach and ResultsHigh‐throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA‐sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR‐CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide–treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo‐resistant CCLP cells. MIR1249 modulated the chemotherapy‐induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt‐regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild‐type models showed only stable disease over treatment.ConclusionsMIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.

Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

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.

Piperlongumine induces autophagy in biliary cancer cells via�reactive oxygen species-activated Erk signaling pathway

Biliary cancer (BC) is an aggressive neoplasm with high mortality. BC can be categorized into three groups: Intrahepatic cholangiocarcinoma (CCA; also known as bile duct cancer), extrahepatic cholangiocarcinoma and gallbladder cancer. Due to its heterogeneity and aggressiveness, the response to current chemotherapy and radiotherapy methods in patients with BC is poor. Therefore, there is an urgent requirement to develop drugs to treat BC. Piperlongumine (PL), a naturally occurring small molecule isolated from Piper longum L., exhibits anticancer activity by inducing reactive oxygen species (ROS) production. In the present study, the effects of PL on cell proliferation, cell cycle, apoptosis and autophagy in BC cells were investigated. PL induced BC cell death in a concentration- and time-dependent manner by inducing ROS production. PL induced cell cycle arrest in CCA cells (HuCCT-1) and gallbladder cancer cells (OCUG-1) cells, but with distinct cell cycle distribution profiles. PL caused G2/M cell cycle arrest in HuCCT-1 cells, and G0/G1 cell cycle arrest in OCUG-1 cells. PL induced apoptosis and autophagy; PL treatment induced accumulation of LC3-II in a concentration- and time-dependent manner. The Erk signaling pathway appeared to be involved in autophagy induction. Application of the ROS scavenger, N-acetyl-l-cysteine, to BC cells attenuated the cell death, cell cycle arrest, apoptosis and autophagy induced by PL treatment. These findings indicated that PL may be a potential agent for BC treatment in the future.

Abstract 2940: Chemosensitization of cholangiocarcinoma by guadecitabine

Abstract Cholangiocarcinoma (CC) originates from biliary epithelial cells and is one of the most common hepatobiliary cancers second only to hepatocellular carcinoma. CC patients have poor long-term prognosis and have limited treatment options. Currently, a combination of gemcitabine and cisplatin is used for systemic treatment of CC patients. There are several tumor suppressor genes that are epigenetically inactivated in CC including p16INK4a, RASSF1A and SEMA3B. Guadecitabine is a next-generation hypomethylating agent that has never been tested in biliary cancer cells. It is a dinucleotide of decitabine and deoxyguanosine that is resistant to degradation by cytidine deaminase. Subcutaneous guadecitabine results in a longer window of exposure than IV decitabine in the clinic. The purpose of this study is to examine the effect of this agent alone and in combination with gemcitabine or cisplatin. We found that guadecitabine inhibits cell proliferation of CC cell lines, SK-ChA-1 and Mz-ChA-1, in a dose-dependent manner starting at 125 nM with concomitant inhibition of DNMT1 protein expression. Additionally, there is a dose-dependent induction of PARP cleavage and increase in expression of semaphorin 3B which is highly methylated in CC starting at doses of 125 nM. Sequential treatment with 250 nM guadecitabine for 48 hours and 20 nM gemcitabine or 1 µM cisplatin for 24 hours further inhibits cell proliferation of CC cells compared to either single agent treatments. Furthermore, western blot analysis of sequentially treated cell shows that DNMT1 is inhibited more by combining guadecitabine and gemcitabine than with either single agents or by combining guadecitabine with cisplatin. We also noted induction of PARP cleavage and increase in semaphorin 3B expression in the SGI-110 and gemcitabine combination treatment than in any other condition tested. In a xenograft model of SK-ChA-1 cells, sequential treatment with 2 mg/kg guadecitabine s.c. daily for 1 week and 15 mg/kg gemcitabine i.p. twice a week for 2 weeks significantly reduced tumor growth as compared to single agent therapy. In contrast to the other conditions, this effect continued for up to 18 days after the last treatment. In fact, the sequential treatment reduced tumor growth by 78% and 72% compared to single agent treatments of SGI-110 and gemcitabine, respectively. Furthermore the sequential combination also increased mRNA expression of p16INK4a, RASSF1A and SEMA3B compared to other treatments. However, guadecitabine did not sensitize SK-ChA-1 tumors when combined with 2 weeks of daily 2 mg/kg cisplatin s.c. treatments. In conclusion, guadecitabine is a potent demethylating agent for CC cells that can inhibit tumor growth by itself as well as sensitize the tumor to chemotherapy such as gemcitabine through epigenetic priming. This novel combination is a promising therapy for CC and may represent a significant improvement to current treatment strategies. Citation Format: Oliver Surriga, Gary K. Schwartz. Chemosensitization of cholangiocarcinoma by guadecitabine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2940.

Mechanisms of MAFG Dysregulation in Cholestatic Liver Injury and Development of Liver Cancer

MAF bZIP transcription factor G (MAFG) is activated by the farnesoid X receptor to repress bile acid synthesis. However, expression of MAFG increases during cholestatic liver injury in mice and in cholangiocarcinomas. MAFG interacts directly with methionine adenosyltransferase α1 (MATα1) and other transcription factors at the E-box element to repress transcription. We studied mechanisms of MAFG up-regulation in cholestatic tissues and the pathways by which S-adenosylmethionine (SAMe) and ursodeoxycholic acid (UDCA) prevent the increase in MAFG expression. We also investigated whether obeticholic acid (OCA), an farnesoid X receptor agonist, affects MAFG expression and how it contributes to tumor growth in mice. We obtained 7 human cholangiocarcinoma specimens and adjacent non-tumor tissues from patients that underwent surgical resection in California and 113 hepatocellular carcinoma (HCC) specimens and adjacent non-tumor tissues from China, along with clinical data from patients. Tissues were analyzed by immunohistochemistry. MAT1A, MAT2A, c-MYC, and MAFG were overexpressed or knocked down with small interfering RNAs in MzChA-1, KMCH, Hep3B, and HepG2 cells; some cells were incubated with lithocholic acid (LCA, which causes the same changes in gene expression observed during chronic cholestatic liver injury in mice), SAMe, UDCA (100 μM), or farnesoid X receptor agonists. MAFG expression and promoter activity were measured using real-time polymerase chain reaction, immunoblot, and transient transfection. We performed electrophoretic mobility shift, and chromatin immunoprecipitation assays to study proteins that occupy promoter regions. We studied mice with bile-duct ligation, orthotopic cholangiocarcinomas, cholestasis-induced cholangiocarcinoma, diethylnitrosamine-induced liver tumors, and xenograft tumors. LCA activated expression of MAFG in HepG2 and MzChA-1 cells, which required the activator protein-1, nuclear factor-κB, and E-box sites in the MAFG promoter. LCA reduced expression of MAT1A but increased expression of MAT2A in cells. Overexpression of MAT2A increased activity of the MAFG promoter, whereas knockdown of MAT2A reduced it. MAT1A and MAT2A had opposite effects on the activator protein-1, nuclear factor-κB, and E-box-mediated promoter activity. Expression of MAFG and MAT2A increased, and expression of MAT1A decreased, in diethylnitrosamine-induced liver tumors in mice. SAMe and UDCA had shared and distinct mechanisms of preventing LCA-mediated increased expression of MAFG. OCA increased expression of MAFG, MAT2A, and c-MYC, but reduced expression of MAT1A. Incubation of human liver and biliary cancer cells lines with OCA promoted their proliferation; in nude mice given OCA, xenograft tumors were larger than in mice given vehicle. Levels of MAFG were increased in human HCC and cholangiocarcinoma tissues compared with non-tumor tissues. High levels of MAFG in HCC samples correlated with hepatitis B, vascular invasion, and shorter survival times of patients. Expression of MAFG increases in cells and tissues with cholestasis, as well as in human cholangiocarcinoma and HCC specimens; high expression levels correlate with tumor progression and reduced survival time. SAMe and UDCA reduce expression of MAFG in response to cholestasis, by shared and distinct mechanisms. OCA induces MAFG expression, cancer cell proliferation, and growth of xenograft tumors in mice.

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.

EBP50 inhibits the migration and invasion of human breast cancer cells via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway.

The scaffold protein ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50, also known as NHERF1 or NHERF) inhibits epidermal growth factor (EGF)-induced breast cancer cell proliferation after 3 days by blocking EGF receptor (EGFR) phosphorylation. The loss of EBP50 stimulates EGFR activity and induces the appearance of epithelial-to-mesenchymal transition phenotypic features in biliary cancer cells. However, the involvement of EBP50 in breast cancer migration and invasion remains unknown. We report that EBP50 inhibits the migration and invasion of breast cancer cells by inhibiting the phosphorylation of LIN-11, Isl1, and MEC-3 protein domain kinase, as well as cofilin. This phosphorylation is a critical step in cofilin recycling and actin polymerization mediating cytoskeletal rearrangement. Additionally, EGF-induced phosphorylation of Akt and mTOR was suppressed by upregulation of EBP50. Our results indicate that EBP50 is significantly involved in breast cancer invasion/metastasis via LIMK/cofilin and the PI3K/Akt/mTOR/MMP signaling pathway.

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.

Altered Expression of Cellular Bcl-2 in the Progression of Hamster Cholangiocarcinogenesis

Bcl-2 is an intracytoplasmic and membrane-associated apoptosis suppressor, and its overexpression is closely associated with survival of malignant tumors, in particular their aggressive behavior and poor prognosis. The role of Bcl-2 is, however, still controversial in cholangiocarcinogenesis because of the discrepancies in the expression of the protein. In the present study, alteration in the expression of Bcl-2 in cholangiocarcinogenesis was investigated by studying the immunoreactivities of this protein in normal, hyperplastic bile ducts with or without dysplastic changes, and neoplastic bile duct cells from a hamster cholangiocarcinoma (ChC) model. Cytoplasmic staining, which reflects high-Bcl-2 immunoreactivity, was negative to very weak in normal and hyperplastic bile ducts without dysplastic changes, while hyperplastic bile ducts with dysplasia indicated heterogeneously strong expression. On the other hand, most of the neoplastic cells of invasive cholangiocarcinomas were negative to weak as much as the level of normal bile ducts. The results suggest that the antiapoptotic factor Bcl-2 plays a limited role in the survival of highly proliferative, potentially dysplastic bile duct cells. However, the role of Bcl-2 in biliary cancer cells was not significant.